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# CHAPTER 5 INTEGRATED HEALTH EF FECTS OF LEAD EXPOSURE

5.1 Introduction
This chapter reviews, summarizes, and integrates the evidence for the broad spectrum of 1 

### health effects associated with exposur e to Pb. The chapter begins ( Section 5.2 ) with a 2 

### discussion of the evidence for the modes of action that mediate the health effects of Pb, 3 

### including those modes of action that are shared by all of the health effects evaluated in 4 

### thi s ISA and those modes of action that are specific for particular endpoints. Subsequent 5 

### sections comprise evaluations of the epidemiologic and toxicological evidence for the 6 

### health effects of Pb exposure on major outcome categories such as nervous system ef fects 7 

### ( Section 5.3 ), cardiovascular effects ( Section 5.4 ), renal effects ( Section 5.5 ), immune 8 

### effects ( Section 5.6 ), effects on heme synthesis and red blood cell function ( Section 5.7 ), 9 

### and reproductive and developmental effe cts ( Section 5.8 ). Section 5.9 provides reviews 10 

### of the evidence for the effects of Pb on other noncancer health outcomes, for which the 11 

### cumulative bodies of evidence are smaller, including those related to the hepatic system 12 

### ( Section 5.9.1 ), gastrointestinal system ( Section 5.9.2 ), endocrine system ( Section 5.9.3 ), 13 

### bone and teeth ( Section 5.9.4 ), ocular health ( Section 5.9.5 ), and respiratory system 14 

### ( Section 5.9.6 ). Chapter 5 concludes with a discussion of the evidence for Pb effects on 15 

### cancer ( Section 5.10 ). 16 

### Individual sections for major outcome categories ( e.g., nervous system, cardiovascular, 17 

### renal) begin with a brief summary of conclusions from the 2006 Pb AQCD followed by 18



### an evaluation of recent evidence that is intended to build upon evidence from previous 19 

### reviews. Within each of these sections, results are organized by endpoint ( e.g., cognitive 20 

### function, behavior, neurodegenerative diseases) then by specif ic scientific disci pline 21 

### ( i.e., epidemiology, toxicology). Sections for each of the major outcome categories 22 

### ( e.g., nervous system, cardiovascular, renal effects) conclude with an integrated summary 23 

### of the evaluation of evidence and a conclusion regarding causality. Based up on the 24 

### framework ( described in the Preamble) , a determination of causality was made for a 25 

### broad outcome category ( i.e., nervous system effects) by evaluating the coherence of 26 

### evidence across disciplines and across a spectrum of related endpoints. Each disc ussion 27 

### leading up to the causal determination characterizes the evidence on which the causal 28 

### judgment was based, including the strength of evidence for the individual endpoints 29 

### within the major outcome category. 30February 2012 5

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## 5.2 Modes of Action

5.2.1 Introduction

The diverse hea lth effects of Pb are dependent on multiple factors, including the 1 

### concentration and duration of exposure, the particular Pb compounds constituting the 2 

### exposu re, and which tissues are affect ed. A mode of action (MOA) is the common set of 3 

### biochemical, physi ological, or behavioral responses ( i.e., empirically observable 4 

### precursor steps) that can cumulatively result in the formation of negative health 5 

### outcomes. Although the effects of Pb appear to be mediated through multiple modes of 6 

### action, alteration of cel lular ion status (including disruption of calcium homeostasis, 7 

### altered ion transport mechanisms, and perturbed protein function through displacement of 8 

### metal cofactors) seems to be the major unifying mode of action underlying all subsequent 9 

### modes of action ( Figure 5

- 1 ). This section draws information from all of the subsequent 10 

### health effects sections in Chapter 5 , and identifies the major modes of action operating at 11 

### the molecular, cellular, and tissue/organ level. In turn, the individual health effect 12 

### sections bridge these effects to those observ ed on the organismal level. Each of the 13 

### individual health effect sections include s a more detailed description of the mechanisms 14 

### specific to the individual health effect. Accordingly, this section differs in structure and 15 

### content from other health effects sections as it does not primarily focus on the literature 16 

### published since the 2006 Pb AQCD , but rather incorporates recent information with older 17 

### studies (which represent the current state of the science ) on the possible modes of action 18 

### of Pb. 19February 2012 5

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– Do Not Cite or Quote Note: The sub sections where these MOAs are discussed are indicated in parentheses. 

### Figure 5
- 1 Schematic representation of the relationships between the various MOAs by which Pb exerts its health effects.

5.2.2 Alte red Ion Status
Physiologically

- relevant metal ions ( e.g., Ca, Mg, Zn, Fe) are known to have a multitude 1 

### of functions in biological systems, including roles as charge carriers, intermediates in 2 

### enzymatically

- catalyzed reactions, and as structural elements in the proper maintenance 3 

### of tertiary protein conformations ( Garza et al., 2006 )

. It is through disruption of these 4 

### biological functions that Pb effects its negative actions, ultimately interfering with such 5 

### tightly regulated processes as c ell signaling, intracellular ion homeostasis, ion transport, 6 

### energy metabolism, and enzymatic function. 7



### 5.2.2.1 Disruption of Ca 2+ 

### Homeostasis
Calcium (Ca 2+ 

### ) is one of the most important carriers of cell signals and regulates virtually 8 

### all aspects of cell function , including energy metabolism, signal transduction, hormonal 9 

### regulation, cellular motility, and apoptosis ( Carafoli, 2005 )

. Ca 2+ 

### homeostasis is 10 

### maintained through a tightly regulated balance of cellular transport and intracellular 11 

### storage ( Pentyala et al., 2010 )

. Disruption of Ca 2+ 

### homeostasis by Pb has been observed 12 

### in a number of different cell types and cell

- free environments, indicating that this is a 13 

### major mode of action for Pb

- induced toxicity on a cellular level. 14February 2012 5

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### Ca 2+ 

### homeostasis is particularly important in bone cells, as the skeletal system serves as 1 

### the major dynamic reservoir of Ca 2+ 

### in the b ody ( Wiemann et al., 1999 ; Long et al., 2 

### 1992 )

. Bone cells also are unique in that they can exist in a microenvironment that is high 3 

### in both Ca 2+ 

### and Pb concentrations, potenti ally increasing their relative susceptibility to 4 

### Pb

- induced toxicity ( Long et al., 1992 )

. A series of studies from the laboratory of Long, 5 

### Dowd, and Rosen have indicated that exposure of cultured osteoblastic bone cells t o Pb 6 

### disrupts intracellular Ca 2+ 

### levels ([Ca 2+ 

### ] i 

### ). Exposure of osteoblasts to 1, 5, or 25 μ M Pb 7 

### for 40

- 300 minute s resulted in prolonged increases in [Ca 2+ 

### ] i 

### of 36, 50 and 120% over 8 

### baseline, respectively ( Schann e et al., 1997 ; Schanne et al., 1989 )

. Long et al. ( 1992 ) 9 

### observed that exposure of osteoblasts to either 400 ng parathyroid hormone (PTH)/ mL 10 

### culture for 1 hour or 25 μ M Pb for 20 hours increased [Ca 2+ 

### ] i 

### . Pretreatment of P b

11 

### exposed cells with PTH increased [Ca 2+ 

### ] i 

### above concentrations observed in either single 12 

### exposure, indicating that Pb may disrupt the ability of bone cells to respond to normal 13 

### hormonal control. A similar additive increase in [Ca 2+ 

### ] i 

### was also observed when bone 14 

### cells were co

- treated with e pidermal growth factor (EGF) plus Pb , versus Pb alone ( Long 15 

### and Rosen, 1992 )

. Pb

- induced increases in [Ca 2+ 

### ] i 

### were blocked by a protein kinase C 16 

### (PKC) inhibitor, indicating that PKC activation may serve as the mechanism by which Pb 17 

### perturbs [Ca 2+ 

### ] i 

### ( Schanne et al., 1997 )

. Schirrmacher et al. ( 1998 ) also observed 18 

### alterations in Ca 2+ 

### homeostasis in osteoblasts exposed to 5 μ M Pb for 50 minute s due to 19 

### potential disruption of Ca 2+



### - ATPases. However, Wiemann et al. ( 1999 ) demonstrated that 20 

### exposure to 5 or 12.5 μ M Pb inhibited the Ca 2 +



### - release

- activated calcium influx of Ca 2+ 21 

### independent ly of any inhibitory effect on Ca 2+



### - ATPases. 22


### Ca 2+ 

### homeostasis has also been shown to be disturbed in erythrocytes exposed to Pb 23 

### ( Quintanar

- Escorza et al., 2010 ; Quintanar

- Escorza et al., 2007 ; Shin et al., 2007 )

. In 24 

### blood samples taken from Pb

- exposed workers (mean [SD] blood Pb level: 74.4 25 

### [21.9] μ g/dL), the [Ca 2+ 

### ] i 

### was approximately 2.5

- fold higher than that seen in nonexposed 26 

### workers (mean [SD] blood Pb level: 9.9 [2] μ g/dL) ( Quintanar

- Escorza et al., 2007 )

. The 27 

### increase in [Ca 2+ 

### ] i 

### was associated with higher osmotic fragility and modifications in 28 

### erythrocyte shape. In a separate investiga tion, erythrocytes from 10 healthy volunteers 29 

### were exposed to Pb at concentrations of 0.2 to 6.0 μ M for 24 or 120 hours, concentration

30 

### related increases in [Ca 2+ 

### ] i 

### were observed across all concentrations for both durations of 31 

### exposure ( Quintanar

- Escorza et al., 2010 )

. Subsequent exposures of erythrocy tes to either 32 

### 0.4 or 4.0 μ M Pb [ corresponding to 10 or 80 μ g/dL in exposed workers ( Quintanar

33 

### Escorza et al., 2007 ) ] for 12

- 120 hours resulted in duration

- related increases with 34 

### durations >12 hours. Osmotic fragility (measured as percent hemolysis) was increased in 35 

### erythrocytes exposed to 0.4 μ M Pb for 24 hours. Co

- incubation with a vitamin E analog 36 

### mitigated these effe cts, indicating that the increase in [Ca 2+ 

### ] i 

### is dependent on the 37 

### oxidative state of the erythrocytes. Shin et al. ( 2007 ) observed that incubation of human 38 

### erythrocytes with 5 μ M Pb for 1 hour resulted in a 30

- fold increase in [Ca 2+ 

### ] i 

### in vitro, 39February 2012 5

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### inducing the pro

- coagulant activity of exposed erythrocytes. Induction of pro

- coagulant 1 

### activity in erythrocytes could lead to thromb us formation and negatively contribute to 2 

### overall cardiovascular health, whereas increased osmotic fragility could substantially 3 

### reduce erythrocyte life span and ultimately lead to anemic conditions. 4 

### Similar to effects seen in erythrocytes, Pb has been obs erved to interfere with Ca 2+ 5 

### homeostasis in platelets and white blood cells. Dowd and Gupta ( 1991 ) observed that 6 

### 1 μ M Pb (for 3.5 hours) was the lowest exposure concentration to result in increases in 7 

### [Ca 2+ 

### ] i 

### in human platelets. The observed increase in Ca 2+ 

### levels was attributed to the 8 

### increased influx of external Ca 2+ 

### , possibly through receptor

- operated Ca 2+ 

### channels. In 9 

### mouse splenic lymphocytes, 1 μ M Pb was the lowest exposure concentration found to 10 

### increase [Ca 2+ 

### ] i, 

### with incubation periods of 10 minute s or greater ( Li et al., 2008c )

. These 11 

### inc reases in Ca 2+ 

### appeared to be reversible as [Ca 2+ 

### ] i 

### returned to baseline after one hour. 12 

### Pretreatment with a calmodulin antagonist slightly mitigated the effects of Pb exposure, 13 

### indicating a role for calmodulin in disruption of Ca 2+ 

### homeostasis in lymphocy tes. In rat 14 

### tail arteries exposed to 1.2 μ M Pb

- acetate for 1 hour, intracellular stores of Ca 2+ 

### increased 15 

### over controls, possibly through increased transmembrane influx of Ca 2+ 

### ( Piccinini et al., 16 

### 1977 )

. 17 

### Exposure of the microsomal fraction of rat brain cells to as little as 0.25 μ M Pb for 18 

### 2 minute s resulted in increased release of Ca 2+ 

### into the media ( Pentyala et al., 2010 )

. 19 

### Further, Pb exposure also decreased the activity of the microsomal Ca 2+



### - ATPase, thus 20 

### decreasing the sequestration of Ca 2+ 

### into microsomes. The results of this study suggest 21 

### that disruption of microsomal release and re

- uptake of Ca 2+ 

### may alter Ca 2+ 

### homeo stasis, 22 

### ultimately leading to altered signal transduction and neuronal dysfunction. However, 23 

### Ferguson et al. ( 200 0 ) observed that [Ca 2+ 

### ] i 

### was decreased in rat hippocampal neurons in 24 

### response to exposure to 0.1 μ M Pb for 1

- 48 hours, although the observed decreases were 25 

### not time

- dependent. The decrease in [Ca 2+ 

### ] i 

### was shown to be due to increased efflux of 26 

### Ca 2+ 

### out o f the neuron via a calmodulin

- regulated mechanism, possibly through 27 

### stimulated Ca 2+ 

### efflux via Ca 2+



### - ATPase. 28



### 5.2.2.2 Disruption of Ion Transport Mechanisms
As described above, deregulation of Ca 2+ 

### homeostasis results in negative effects in 29 

### multiple organ systems. U nder normal conditions in the life cycle of most cells, cytosolic 30 

### concentrations of free Ca 2+ 

### fluctuate around 100

- 200 nM and Ca 2+ 

### that has entered the 31 

### cell must be removed in order to maintain normal homeostatic concentrations ( Carafoli, 32 

### 2005 ) . An important component of the maintenance of Ca 2+ 

### homeostasis is 33 

### transmembrane transport of Ca ions via Ca 2+



### - ATPase and voltage

- sensitive gates 34February 2012 5

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### ( Carafoli, 2005 )

. Pb has been shown to disrupt the normal movement of Ca 2+ 

### ions, as 1 

### well as other physiologically important ions through interactions with these transport 2 

### mechanisms. 3 

### Multiple studies have reported the effects of Pb e xposure on Na +



### - K +



### - ATPase, Ca 2+



### - 4 

### ATPase, and Mg 2+



### - ATPases in animal models. Decreases in the activity of all three 5 

### ATPases were observed in the kidneys and livers of rats exposed to 750 ppm Pb in 6 

### drinking water for 11 weeks (mean [SD] blood Pb level: 55.6 [6 .3] μ g/dL) ( Kharoubi et 7 

### al., 2008a ) and in erythrocytes of rats exposed to 0.2% Pb in drinking water for 5 weeks 8 

### (mean [SD] blood Pb level: 97.56 [11.8] μ g/dL) ( Sivaprasad et al., 2003 )

. Increases in 9 

### lipid peroxidation were seen in both studies, and the decrements in ATPase activities may 10 

### be explained by generation of free radicals in Pb

- exposed animals. A decrease in the 11 

### activity of Na +



### - K +



### - ATPase was observed in rabbit kidney membranes e xposed to 0.01 to 12 

### 10 μ M Pb, possibly due to Pb inhibiting the hydrolytic cleavage of phosphorylated 13 

### intermediates in the K

- related branch of the pump ( Gramigni et al., 2009 )

. Similar 14 

### decreases in Na +



### - K +



### - ATPase activity were observed in synaptosomes isolated from rats 15 

### exposed to 200 ppm Pb in drinking water for 3 months (blood Pb level: 37.8 μ g/dL) 16 

### ( Rafalowska et al., 1996 ) or 15 mg / kg Pb injected (i.p.) for 7 days (blood Pb level: 17 

### 112.5 μ g/dL) ( Struzynska et al., 1997a )

. Inhibition of Na +



### - K +



### - ATPase activity was also 18 

### observed in primary cerebellar granule neuronal cultures obtained from rats pre

- and 19 

### postnatally exposed to Pb (0.1 % Pb

- acetate in dams drinking water, resulting in blood Pb 20 

### level of 4 μ g/dL) ( B aranowska

- Bosiacka et al., 2011a )

. The activity of Ca 2+



### - ATPase in 21 

### the sarcoplasmic reticulum of rabbits exposed to 0.01 μ M Pb was similarly decreased 22 

### ( Hechtenberg and Beyersmann, 1991 )

. The inhibitory effect of Pb was diminished in the 23 

### presence of high MgATP concentrations. The activity of generic ATPa se was reported to 24 

### be altered in the testes of rats exposed to 300 ppm Pb

- acetate gestationally, and in 25 

### drinking water after weaning to the age of 6, 8, 10, or 12 weeks ( Liu et al., 2008 )

. In 26 

### pregnant r ats fed a Pb

- depleted (20 ± 5 μ g/kg) or control (1 mg /kg) diet during gestation 27 

### and lactation, no difference was observed in the activity of Na +



### - K +



### - ATPase and Ca 2+



### - 28 

### Mg 2+



### - ATPase in the parental generation ( Eder et al., 1990 )

. However, the offspring 29 

### (exposed via placental and lactational transfer of Pb) of Pb

- depleted rats displayed 30 

### decreased activities in both enzymes compared with offspring of rats with higher Pb 31 

### exposures. A similar increase in the Na +



### - K +



### - ATPase activity was observed in rats 32 

### exposed (i.p.) to 20 mg /kg Pb for 14 consecutive days ( Jehan and Motlag, 1995 )

. Co

33 

### exposure of Pb with zinc and copper greatly attenuated the increase in ATPase activity. 34 

### Although the precise me chanism was not investigated, Navarro

- Moreno et al. ( 2009 ) 35 

### reported that Ca 2+ 

### uptake was d iminished in proximal renal tubule cells in rats chronically 36 

### exposed to 500 ppm Pb in drinking water for 7 months (mean [SD] blood Pb level: 43.0 37 

### [7.6] μ g/dL). 38February 2012 5

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### In vitro studies of ATPase activities in human erythrocyte ghosts have also shown that Pb 1 

### affect s the transport of metal ions across membranes. Calderon

- Salinas et al. ( 1999b ) 2 

### observed that 1

- 5x10 4 

### μ M Pb and Ca 2+ 

### were capable of inhibiting the passive transport of 3 

### each other in human erythrocyte ghosts incubated with both cations. Subsequent 4 

### inhibition experiments indicated that both cations share the same electrogenic transport 5 

### pathway ( Sakuma et al., 1984 )

. Further study by this group ( Calderon

- Salinas et al., 6 

### 1999a ) demonstrated that Pb can noncompetitively block the transport of Ca 2+ 

### by 7 

### inh ibiting the activity of Ca 2+



### - Mg 2+



### - ATPase at concentrations of 1

- 5x10 3 

### μ M. Mas

- Oliva 8 

### ( 1989 ) demonstrated that the activity of Ca 2+



### - Mg 2+



### - ATPase in human erythrocyte ghosts 9 

### was inhibited by incubation with 0.1

- 100 μ M Pb. The inhibitory action was most likely 10 

### due to direct reaction with sulfhydryl groups on the ATPase at Pb concentrations greater 11 

### than 1 μ M, but due to the action of Pb on calmodulin at lower concentrations. Grabowska 12 

### and Guminska ( 1996 ) observed that 10 μ g/dL Pb was the lowest concentration to 13 

### decrease the activity of Na +



### - K +



### - ATPase in erythrocyte ghosts; activity of Ca 2+



### - Mg 2+



### - 14 

### ATPase was less sensitive to Pb exposure, and Mg 2+



### - ATPase activity was not affected. 15 

### In a study investigating ATPase activities in occupationally

- exposed workers in Nigeria, 16 

### Abam et al. ( 2008 ) observed that the activity of erythrocyte membrane

- bound Ca 2+



### - Mg 2+



### - 17 

### ATPase was decreased by roughly 50% in all occupational groups (range of mean [SD] 18 

### blood Pb level across nine occupational groups: 28.75 [11.31]

- 42.07 [12.01] μ g/dL) 19 

### compared to nonexposed controls (mean [SD] blood Pb level: 12.34 [2.44] in males and 20 

### 16.85 [6.01] μ g/dL in females). Higher membrane concentrations of Ca 2+ 

### and magnesium 21



### w ere also observed, indicating that Pb prevented the efflux of those cations from the cell, 22 

### most likely by substituting for those metals in the active site of the ATPase. In a study of 23 

### 247 mother

- newborn pairs, Campagna et al. ( 2000 ) observed that newborn (cord) blood 24 

### Pb (geometric mean [5 th , 95 th percentile]: 4.8 [2.8

- 9.2] μ g/dL) was negatively and 25 

### significantly associated with maternal blood Ca 2+ 

### pump activities; h owever, newbor n 26 

### (cord) blood Pb was not significantly associated with cord blood Ca 2+ 

### pump activities. 27 

### Newborn hair Pb (geometric mean [5 th , 95 th percentile]: 1.1 [0.1

- 8.0] μ g/g) was 28 

### negatively and significantly associated with both maternal and cord blood Ca 2+ 

### pump 29 

### activities. In a population of 81 newborns, Huel et al. ( 2008 ) found that newborn hair and 30 

### cord blood Pb levels (mean [SD] newborn hair Pb an d blood Pb levels: 1.22 [1.41] μ g/g 31 

### and 3.54 [1.72] μ g/dL) were negatively associated with Ca 2+



### - ATPase activity in plasma 32 

### membranes of erythrocytes isolated from cord blood; newborn hair Pb levels were more 33 

### strongly associated with cord Ca 2+ 

### pump activity than were cord blood Pb levels. 34 

### Pb has also been shown to disrupt cation transport mechanisms through direct action on 35 

### voltage

- sensitive cation channels. Audesirk and Audesirk ( 1993 , 1991 ) demonstrated that 36 

### extracellular free Pb inhibits the action of multiple voltage

- sensitive Ca 2+ 

### channels, with 37 

### free Pb IC 50 

### (half maximal inhibitory concentration) values of 0.7 μ M for L

- type 38February 2012 5

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### channels and 1.3 μ M for T

- type channels in neuroblastoma cells, and IC 50 

### values as low 1 

### as 0.03 μ M for L

- type channels in cultured hippocampal neurons. Sun and Suszkiw 2 

### ( 1995 ) co rroborated the inhibitory action of extracellular Pb on Ca 2+ 

### channels, 3 

### demonstrating an IC 50 

### value of 0.3 μ M in adrenal chromaffin cells. The observed 4 

### disruption of the Ca 2+ 

### channels most likely reflects competition between Pb and Ca 2+ 

### for 5 

### the extracellula r Ca 2+ 

### binding domain of the channel. Research by other laboratories 6 

### supported these findings: Pb inhibited the action of multiple Ca 2+ 

### channels in human 7 

### embryonic kidney cells transfected with L

- , N

- , and R

- type channels (IC 50 

### values of 8 

### 0.38 μ M, 1.31 μ M, and 0.10 μ M, respectively) ( Peng et al., 2002 ) and P

- type channels in 9 

### cultured hippocampal neurons at concentrations up to 3 μ M ( Ujihara et al., 1995 )

. 10 

### However, intracellular Pb was observed to enhance Ca 2+ 

### currents through attenuation of 11 

### the Ca 2+ 

### dependent deactivation of Ca 2+ 

### channels at an EC 50 

### value of 200 μ M, possibly 12 

### through blocking the intracellular Ca 2+ 

### binding domain, or through Ca 2+ 

### dependent 13 

### dephosphorylation of the channel ( Sun and Suszkiw, 1995 )

. Recently, Pb has also been 14 

### shown to enter cells (HEK293, HeLa, and PC12 cell lines) through store

- operated Ca 2+ 15 

### channels ( Chiu et al., 2009 ; Chang et al., 2008b )

. In particular, the Orai1

- STIM1 complex 16 

### was shown to be critical in the entry of Pb ions into cells, and increased Pb permeation 17 

### was directly related to decreased [Ca 2+ 

### ] i 

### concentrations at exposure concentrations as low 18 

### as 0.1 μ M. 19


### Pb also dis rupts the action of Ca 2+



### - dependent potassium channels. Alvarez et al. ( 1986 ) 20 

### observed that Pb promoted the efflux of potassium from inside

- out erythrocyte vesicles in 21 

### a concentration

- dependent manner at concentrations of 1

- 300 μ M, either thr ough action 22 

### on a Mg modulatory site or through direct interaction with the Ca 2+ 

### binding site. Fehlau 23 

### et al. ( 1989 ) also demonstrated Pb

- induced activation of the potassium channel in 24 

### erythrocytes. However, Pb only activated the potassium channels at concentrations below 25 

### 10 μ M; higher concentrations of Pb completely inhibited channel activity, indicating the 26 

### modulation of potassium permeability is due to alterations in channel gating. Silken et al. 27 

### ( 2001 ) observed that Pb activated potassium channels in erythrocytes from the marine 28 

### teleost Scorpaena porcu s in a concentration

- dependent manner after a 20

- minute 29 

### incubation; minor loss of potassium was seen at Pb concentrations of 1

- 2 μ M, whereas 30 

### exposure to 20

- 50 μ M Pb resulted in approximately 70% potassium loss. Competitive 31 

### and inhibitory binding assays sug gest that Pb directly activates potassium channels in 32 

### S. porcus
. 33



### Disruption of Neurotransmitter Release Pb has been shown to inhibit the evoked release of neurotransmitters by inhibiting Ca 2+ 34 

### transport through voltage

- sensitive channels in in vitro experim ents ( Cooper and Manalis, 35 

### 1984 ; Suszkiw et al., 1984 )

. However, in these same experiments, concentrations of Pb as 36February 2012 5

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### 5 μ M and higher were also observed to actu ally increase the spontaneous release of 1 

### neurotransmitters. Subsequent research by other groups affirmed that Pb demonstrates 2 

### Ca 2+



### - mimetic properties in enhancing neurotransmitter release from cells in the absence 3 

### of Ca 2+ 

### and Ca 2+



### - induced depolarization. T omsig and Suszkiw ( 1995 , 1993 ) reported that 4 

### Pb exposure induced the release of norepinephrine (NE) from bovine adrenal chromaffin 5 

### cells, and was considerably more potent at doing so than was Ca 2+ 

### (K 0.5 

### of 4.6x10 3 

### μ M for 6 

### Pb versus 2.4 μ M for Ca 2+ 

### ). Activation of PKC was observed to enhance the Pb

- induced 7 

### release of NE. West er rink and Vijverber g ( 2002 ) observed that Pb acted as a high affinity 8 

### substitute for Ca 2+ 

### , and triggered enhanced catecholamine release from PC12 cells at 9 

### 10 μ M in intact cells and 3x10 4 

### μ M in permeabilized cells. The suppressio n of Ca 2+



### - 10 

### induced evoked release of neurotransmitters combined with the ability of Pb to enhance 11 

### spontaneous releases could result in higher noise in the synaptic transmission of nerve 12 

### impulses in Pb

- exposed animals. In rats exposed to Pb at concentrations of 0.1

- 1.0% in 13 

### drinking water beginning at gestational day (GD)15

- 16 and continuing to 120 days 14 

### postnatal, decreases in total potassium

- stimulated hippocampal GABA release were seen 15 

### at exposure levels of 0.1

- 0.5% (range of mean [SD] blood Pb levels: 26.8 [1.3]

- 61.8 16 

### [2.9] μ g/dL) ( Lasley and Gilbert, 2002 )

. Ma ximal effects were observed at 0.2% Pb in 17 

### drinking water, but effects were less evident at 0.5%, and were absent at 1.0%. In the 18 

### absence of Ca 2+ 

### , potassium

- induced GABA release was increased with the two highest 19 

### exposure concentrations, suggesting a Pb

- ind uced enhancement of evoked release of 20 

### GABA. The authors suggest that this pattern of response indicates that Pb is a potent 21 

### suppressor of evoked release at low concentrations, but a Ca 2+ 

### mimic in regard to 22 

### independently evoking exocytosis and release at hi gher concentrations ( Lasley and 23 

### Gilbert, 2002 )

. Suszkiw ( 2004 ) reports that augmentation of spontaneous release of 24 

### neurotransmitters may involve Pb

- induced activation of CaMKII

- dependent 25 

### phospho rylation of synapsin I or direct activation of synaptotagmin I. Further, Suszkiw 26 

### ( 2004 ) suggests that unlike the intracellularly mediated effects of Pb on spontaneous 27 

### release of neurotransmitters, Pb

- induced inhibition of evoked transmitter releases is 28 

### largely due to extracellular blockage of the voltage

- sensitive Ca 2+ 

### channels. 29



### 5.2.2.3 Displacement of Metal Ions and Perturbed Protein Function

The binding of metal ions to proteins causes specific changes in protein shape, and the 30 

### specific cellular function of many proteins may be altered by conformational changes 31 

### ( Kirberger and Yang, 2008 )

. Metal binding sites on proteins are generally ion

- specific 32 

### and are influenced by multiple factors, including binding geometries, ligand preferences, 33 

### ionic radius, and metal coordination numbers ( Kirberger and Yang, 2008 ; Garza et al., 34 

### 2006 )

. The coordination chemistry that normally regulates metal

- protein binding makes 35February 2012 5

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### many proteins particularly susceptible to perturbation from Pb, as it is able to function 1 

### with flexible coordination numbers a nd can bind multiple ligands ( Kirberger and Yang, 2 

### 2008 ; Garza et al., 2006 )

. However, due to differences in its physical properties, Pb 3 

### induces abnormal conformational changes when it binds to proteins ( Kirberger and Yang, 4 

### 2008 ; Bitto et al., 2006 ; Garza et al., 2006 ; Magyar et al., 2005 ) , and these structural 5 

### changes elicit altered protein function. It is known that [Ca 2+ 

### ] i 

### is an important second 6 

### messenger in cell signaling pathwa ys, and operates by binding directly to and activating 7 

### proteins such as calmodulin and PKC ( Goldstein, 1993 )

. Alterations in the functions of 8 

### both of these proteins due to direct interaction with Pb have been well documented in the 9 

### literature. 10 

### PKC is a family of serine/threon ine protein kinases critical for cell signaling and 11 

### important for cellular processes, including growth and differentiation ( Goldstein, 1993 )

. 12 

### PKC contains a C2 Ca 2+



### - binding domain and requires the cation, as well as 13 

### diacylglycerol and phospholipids, for proper cellular activity ( Garza et al., 2006 )

. 14 

### Markovac and Goldstein ( 1988b ) observed that, in the absence of Ca 2+ 

### , exposure to 15 

### picomolar concentrations of Pb for 5 minute s directly activated PKC purified from rat 16 

### brains. Th e activation of PKC by Pb was more potent than was Ca 2+



### - dependent activation 17 

### by five orders of magnitude. Long et al. ( 1994 ) affirmed these findings, repo rting that Pb 18 

### had a K act 

### 4 , 800 times smaller than that of Ca 2+ 

### (5.5x10 - 5 

### μM versus 25 μ M, following a 19 

### 3 minute exposure). However, Ca 2+ 

### had a higher maximal activation of PKC than did Pb. 20 

### This possibly indicates the presence of multiple Ca 2+



### - binding sites on the protein, and 21 

### that Pb may bind the first site more efficiently than does Ca 2+ 

### , but not subsequent sites. 22 

### Tomsig and Suszkiw ( 1995 ) further demonstrated the ability of Pb to activate PKC at 23 

### picomolar concentrations in adrenal chromaffin c ells incubated with Pb for 10 minute s 24 

### but also reported that activation of PKC by Pb was only partial (approximately 40% of 25 

### the maximum activity induced by Ca 2+ 

### ) and tended to decrease at concentrations greater 26 

### than one nanomolar. 27 

### Contrary to the above fi ndings, Markovac and Goldstein ( 1988a ) observed that Pb and 28 

### Ca 2+ 

### activated PKC at equivalent concentrations and efficacies when broken cell 29 

### preparations of rat brain microvessels were incubated with either cation for 45 minute s. 30 

### However, when PKC activation was investigated in whole vessel preparations, no 31 

### activation was observed, but PKC did become redistributed from the cytosolic to the 32 

### particulate fraction. This suggests that Pb redistributes PKC at micromolar 33 

### concentrations, but does not activa te the protein in brain microvessels. In human 34 

### erythrocytes exposed to Pb

- acetate for 60 minute s, the amount of PKC found in 35 

### erythrocyte membranes and total PKC activity was increased at concentrations greater 36 

### than 0.1 μ M ( Belloni

- Olivi et al., 1996 )

. The observation that neither Ca 2+ 

### nor 37 

### diacylg l ycerol was increased due to exposure indicates that Pb

- induced activation of PKC 38February 2012 5

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– Do Not Cite or Quote 

### is due to direct interaction with the protein. Pb

- induced alterations in PKC have also been 1 

### observed in other tissues, including increased activity in rabbit mesenteric arteries at 2 

### picomolar concentrations of Pb ( Watts et al., 1995 ; Chai and Webb, 1988 ) and human 3 

### erythrocytes from Pb

- exposed workers (range of blood Pb levels: 5.4 to 69.3 μ g/dL) 4 

### ( Hwang et al., 2002 ) , and decreased activity in mouse macrophages and the rat brain 5 

### cortex at micromolar concentrati ons ( Murakami et al., 1993 ; Lison et al., 1990 )

. 6 

### Calmodulin is another important protein ess ential for proper Ca 2+



### - dependent cell 7 

### signaling. Calmodulin contains an “EF

- hand” Ca 2+ 

### binding domain, and is dependent on 8 

### the cation for proper activity ( Garza et al., 2006 )

. Calmodulin regulates events as diverse 9 

### as cellular structural in tegrity, gene expression, and maintenance of membrane potential 10 

### ( Vetter and Leclerc, 2003 ; Saimi and Kung, 2002 )

. Haberman n et al. ( 1983 ) observed 11 

### that exposure to Pb altered nu merous cellular functions of calmodulin, including 12 

### activation of calmodulin

- dependent phosphodiesterase activity after 10 minute s 13 

### incubation (minimal activation at 0.1 μ M, EC 50 

### = 0.5

- 1.0 μ M), stimulation of brain 14 

### membrane phosphorylation at Pb concentrations greater than 0.4 μ M after 1 minute 15 

### incubation, and increased binding of calmodulin to brain membranes at Pb concentrations 16 

### greater than 1 μ M after 10 minute s incubation. Haberman n et al. ( 1983 ) reported that the 17 

### affinity of Pb for Ca 2+



### - binding sites on calmodulin was approximate to that of Ca 2+ 

### itself 18 

### (K d 

### ~ 20 μ M), whereas Richardt et al. ( 1986 ) observed that Pb w as slightly more potent 19 

### than was Ca 2+ 

### at binding calmodulin (IC 50 

### = 11 and 26 μ M, respectively). Both studies 20 

### indicated that Pb was much more effective at binding calmodulin than was any other 21 

### metal cation investigated ( e.g., mercury, cadmium, iron). Kern et al. ( 2000 ) observed that 22 

### Pb was more potent in binding to, and affecting conformational changes in, calmodulin 23 

### compared to Ca 2+ 

### (EC 50 

### values of 4

- 5.5x10 - 4 

### μ M (threshold = 1x10 - 4 

### μ M) and 0.45

- 24 

### 0.5 μ M (threshold = 0.1 μ M), respectively). Pb, in the absence of Ca 2+ 

### , was also observed 25 

### to activate calmodulin

- dependent cyclic nucleotide phosphodiesterase activity at much 26 

### lower concentrations compared to Ca 2+ 

### (EC 50 

### value 4.3x10 - 4 

### μ M [thr eshold = 3x10 - 4 

### μ M 27 

### versus EC 50 

### 1.2x10 - 3 

### μ M (threshold = 0.2 μ M; 50 minute incubation]). When incubated 28 

### with physiological concentrations of Ca 2+ 

### , Pb induced phosphodiesterase activity at 29 

### concentrations as low as 5x10 - 5 

### μ M. Pb activated calcineurin, a phosp hatase with 30 

### widespread distribution in the brain and immune system, at threshold concentrations as 31 

### low as 2x10 - 5 

### μ M in the presence of Ca 2+ 

### (incubation time = 30 minute s), but inhibited its 32 

### activity at concentrations greater than 2x10 - 4 

### μ M ( Kern and Audesirk, 2000 )

. Thus, 33 

### picomolar concentrations of intracellular Pb appear to amplify the activity of calmodulin 34 

### and thus can be expected to alter intracellular Ca 2+ 

### signaling in exposed cells ( Kern et al., 35 

### 2000 )

. Mas

- Oliva ( 1989 ) observed that low

- exposure (<1 μ M, 20 minute i ncubation) 36 

### stimulatory effects of Pb exposure on the activity of Ca 2+



### - Mg 2+



### - ATPase was due to Pb 37 

### binding to calmodulin and subsequent activation of the ion pore. Ferguson et al. ( 2000 ) 38February 2012 5

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### observed that exposure of rat hippocampal neurons to Pb for 1 to 48 hours resulted in 1 

### increased activation of a calmodulin

- dependent Ca 2+ 

### extrusion mechanism. 2


### Pb has also been observ ed to alter the activity of other proteins that rely on Ca 2+ 

### binding 3 

### for normal cellular function. Osteocalcin is a matrix protein important in bone resorption, 4 

### osteoclast differentiation, and bone growth and has three Ca 2+



### - binding sites ( Dowd et al., 5 

### 2001 )

. Incubation of osteocalcin in solution with Ca 2+ 

### and Pb resulted in the competitive 6 

### displacement of Ca 2+ 

### by Pb ( Dowd et al., 1994 )

. Pb was found to bind to osteocalcin 7 

### more than 1000

- times m ore tightly than was Ca 2+ 

### (K d 

### = 1.6x10 - 2 

### μ M versus 7 μ M, 8 

### respectively), and analysis with nuclear magnetic resonance (NMR) indicated that Pb 9 

### induced similar, though slightly different, secondary structures in osteocalcin, compared 10 

### to Ca 2+ 

### . The authors hypo thesized that the observed difference in Pb

- bound osteocalcin 11 

### structure may explain previous findings in the literature that Pb exposure reduced 12 

### osteocalcin adsorption to hydroxyapatite ( Dowd et al., 1994 )

. Further research by this 13 

### group also found that Pb bound osteocalcin approximately 10,000

- times more tightly than 14 

### did Ca 2+ 

### (K d 

### = 0.085 μ M versus 1.25 × 10 3 

### μ M, respectively) ( Dowd et al., 2001 )

. 15 

### However, the authors reported that Pb exposure act ually caused increased hydroxyapatite 16 

### adsorption at concentrations 2

- 3 orders of magnitude lower than that seen with Ca 2+



### . 17 

### Additionally, Pb can displace Ca 2+ 

### in numerous other Ca 2+



### - binding proteins important in 18 

### muscle contractions, renal Ca 2+ 

### transport and neurotransmission, including troponin C, 19 

### parvalbumin, CaBP I and II, phospholipase A 2 

### , and synapto tagmin I, at concentrations as 20 

### low as the nanomolar range ( Bouton et al., 2001 ; Osterode and Ulberth, 2000 ; Richardt et 21 

### al., 1986 )

. 22 

### Pb can displace metal cations other than Ca 2+ 

### that are requisite for protein function. One 23 

### of the most researched targe ts for molecular toxicity of Pb is the second enzyme in the 24 

### heme synthetic pathway, aminolevulinic acid dehydratase (ALAD). ALAD contains four 25 

### zinc

- binding sites and all four need to be occupied to confer full enzymatic activity 26 

### ( Simons, 1995 )

. ALAD has been identified as the major protein binding target for Pb in 27 

### human erythrocytes ( Bergdahl et al., 1997a ) , and exposure to Pb results in inhibition of 28 

### th e enzyme in the erythrocytes of Pb

- exposed workers and adolescents (blood Pb levels 29 

### >10 μ g/dL) ( Ahamed et al., 2006 ; Ademuyiwa et al., 2005b ) , in human erythrocytes 30 

### exposed to Pb for 60 minute s (K i 

### = 7x10 4 

### μ M) ( Simons, 1995 ) , and in rats exposed to 31 

### 25 mg /kg Pb once a week for 4 weeks (mean [SD] blood Pb level: 6.56 [0.98] μ g/dL) 32 

### ( Lee et al., 2005 )

. Additional experiments indicated that lower concentrations of zinc 33 

### result in greater inhibition of enzyme activity by Pb, suggesting a competitive inhibition 34 

### between zinc and Pb at a single site ( Simons, 1995 )

. 35


### Zinc

- binding domains are also found in transcription factors and proteins necessary for 36 

### gene expression, including GATA proteins and transcription factors TFIIIA, Sp1, and 37February 2012 5

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### Erg

- 1 ( Ghering et al., 2005 ; Huang et al., 2004 ; Reddy and Zawia, 2000 ; Han as et al., 1 

### 1999 ; Zawia et al., 1998 )

. Pb was found to form tight complexes with the cysteine 2



### res idues in GATA proteins (Pb stability constant (β Pb 

### ) = 6.4 × 10 9 

### M - 1 

### for single zinc 3 

### fingers and β 2 Pb2 

### = 6.4 × 10 19 

### M - 2 

### ), and was able to displace bound zinc from the protein 4 

### under physiologically relevant conditions ( Ghering et al., 2005 )

. Once Pb was bound to 5 

### GATA proteins, they displayed decreased ability to bind t o DNA (Pb concentrations ≥ 6 

### 1.25 μ M) and activate transcription (Pb concentration = 1x10 6 

### μ M). Pb at a minimum 7 

### concentration of 10 μ M also binds to the zinc domain of TFIIIA, inhibiting its ability to 8 

### bind DNA at concentrations ( Huang et al., 2004 ; Hanas et al., 1999 )

. Huang et al. ( 2004 ) 9 

### also reported that exposure to Pb caused the dissociation of TFIIIA

- DNA adducts and 10 

### using NMR spectroscopy, found that altered TFIIIA activity was the result of a Pb

11 

### induced abnormal protein conformation. 12 

### Pb exp osure modulated the DNA

- binding profiles of the transcription factors Sp1 and 13 

### Erg

- 1 in rat pups exposed to 0.2% Pb

- acetate via lactation, resulting i n a shift in DNA

14 

### binding toward early development ( i.e., the first week following birth) ( Reddy and Zawia, 15 

### 2000 ; Zawia et al., 1998 )

. The shifts in Sp1 DNA

- binding profiles were shown to be 16 

### associated with abnormal expression of genes related to myelin formation 17 

### ( Section 5.2.7.5 ). Further mechanistic research utilizing a synthetic peptide containing a 18 

### zinc finger motif demonstrated that Pb can bind the histidine and cysteine residues of the 19 

### zinc finger motif, thus displacing zinc and resulting in an increase in the DNA

- binding 20 

### efficiency of the synthetic peptide ( Razmiafshari et al., 2001 ; Razmiafshari and Zawia, 21 

### 2000 )

. However, in DNA

- binding assays utilizing recombinant Sp1 (which has three zinc 22 

### finger motifs, opposed to only one in the synthetic peptide) , 37 μ M Pb was the lowest 23 

### concentration observed to abolish the DNA

- binding capabilities of Sp1 ( Razmiafshar i 24 

### and Zawia, 2000 )

. 25 

### Pb has also been reported to competitively inhibit Mg binding and thus inhibit the 26 

### activities of adenine and hypoxanthine/guanine phosphoribosyltransferase in erythrocyte 27 

### lysates of rats exposed to 0.1% Pb in drinking water for 9 mon ths (mean [SD] blood Pb 28 

### level: 7.01 [1.64] μ g/dL) and in human erythrocyte lysates exposed to 0.1 μ M Pb for as 29 

### little as 5 minute s ( Baranowska

- Bosiacka et al., 2009 ) , and cGMP phosphodiesterase at 30 

### picomolar concentrations in homogenized bovine retinas ( Srivastava et al., 1995 )

. Pb was 31 

### also reported to inhibit pyrimidine 5’

- nucleotidase through competitive inhibition of 32 

### magnesium binding, resulting in conformational changes and improper amino acid 33 

### positioning in the active site ( Bitto et al., 2006 )

. 34 

### In summary, Pb has the ability to displace metal cations from the active sites of multiple 35 

### enzymes and proteins, and thus to alter the functions of those proteins in occupationally 36 

### expose d humans with blood Pb levels of 5.4

- 69.3 μ g/dL, in adult rodents with blood Pb 37February 2012 5

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### levels of 6.5 μ g/dL (exposure 4 weeks), in suckling rats exposed to 0.2% Pb via lac t ation, 1 

### and in cell

- free and cellular in vitro experiments conducted at exposure concentratio ns 2 

### rangi ng from micromoles to picomoles . These alterations in protein function have 3 

### implications for numerous cellular and physiological processes, including cell signaling, 4 

### growth and differentiation, gene expression, energy metabolism, and biosynthetic 5 

### p athways. Table 5

- 1 provides a list of enzymes and proteins whose function may be 6 

### perturbed by Pb exposure. 7



### Table 5
- 1 Enzymes and proteins potentially affected by exposure to Pb and the metal cation cofactors necessary for their proper physiological activity
Metalloprotein/Enzyme Direction of Action a Metal Cation; Reference

Enzymes Aminolevulinic acid dehydratase ↓ Zn; Simons ( 1995 ) Ferrochelatase ↓ Fe (2Fe

- 2S Cluster); Crooks et al. ( 2010 ) Superoxide dismutase ↓↑ Mn, Cu, Zn, Fe; Antonyuk et al. ( 2009 ) , Borgstahl et al. ( 1992 ) Catalase ↓↑ Fe (Heme); Putnam et al. ( 2000 ) Glutathione peroxidase ↓↑ Se; Rotruck et al. ( 1973 ) Guanylate c yclase ↓ Fe (Heme); Boerrigter and Burnett ( 2009 ) cGMP phosphodiesterase ↓ Mg, Zn; Ke ( 2004 ) NAD synthase ↓ Mg; Hara et al. ( 2003 ) NAD(P)H oxidase ↑ Ca; Leseney et al. ( 1999 ) Pyrimidine 5’

- nucleotidase ↓ Mg, Ca; Bitto et al. ( 2006 ) , Amici et al. ( 1997 ) , Paglia and Valentine ( 1975 ) Erythrocyte phosphoribosyltransferase ↓ Mg (Mn, Ca, Co, Ni, Zn); Deng et al. ( 2010 ) , Arnold and Kelley ( 1978 )

Ion Channels/ Transport ATPase ↓↑ Ca, Mg, Na

- K; Technische Universitat Braunschweig ( 2011 ) Mitochondrial transmembrane pore ↑ Ca; He et al. ( 2000 ) Calcium

- depend ent potassium channel ↑ Ca; Silkin et al. ( 2001 ) , Alvarez et al. ( 1986 )

Signal Transduction Protein kinase C ↓↑ Ca; Garza et al. ( 2006 ) Calmodulin ↑ Ca; Garza et al. ( 2006 ) Pb Binding Metallothionein ↑ Zn, Cu; Yu et al. ( 2009 ) DNA Binding GATA transcriptional factors ↓ Zn; Hanas et al. ( 1999 ) , Huang et al. ( 2004 )

a ↑ indicates increased activity; ↓ indicates decreased activity; ↓↑ indicates activity can be alternatively increased or decre ased.February 2012 5

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### 5.2.2.4 Mitochondrial Abnormality

Alterations in mitochondrial func tion, including disruptions in ion transport, 1 

### ultrastructural changes, altered energy metabolism, and perturbed enzyme activities due 2 

### to Pb intoxication are well documented in the scientific literature. Exposure of rats to Pb 3 

### in feed (1% Pb for 4, 6, 8, 10 , 12, or 20 weeks) or drinking water (300 ppm for 8 weeks, 4 

### 500 ppm for 7 months, or 1% for 9 months) resulted in gross ultrastructural changes in 5 

### renal tubules and epididymal mitochondria characterized as a general swollen appearance 6 

### with frequent rupture of the outer membrane, distorted cristae, loss of cristae, frequent 7 

### inner compartment vacuolization, observation of small inclusion bodies, and fusion with 8 

### adjacent mitochondria ( Wang et al., 2010d ; Marchlewicz et al., 2009 ; Navarro

- Moreno et 9 

### al., 2009 ; Goyer, 1968 ; Goyer et al., 1968 )

. 10 

### Transmembrane mitochondrial ion transport mechanisms are perturbed by exposure to 11 

### Pb. Pb inhibits the uptake of Ca 2+ 

### into mitochondria ( Parr and Harris, 1976 ) , while 12 

### simultaneously stimulating the efflux of Ca 2+ 

### out of the organelle ( Simons, 1993a ) , thus 13 

### disrupting intracellular/mitochondrial Ca 2+ 

### homeostasis. Pb exposure has also been 14 

### shown to decrease the mitochondrial transmembrane potential in primary ce rebellar 15 

### granule neuronal cultures from rats exposed to 0.1% Pb in drinking water throughout 16 

### gestation and lactation ( Baranowska

- Bosiacka et al., 2011a ) , astroglia incubated with 0.1 17 

### or 1.0 μ M Pb for 14 days ( Legare et al., 1993 ) , proximal tubule cells exposed to 0.25, 0.5, 18 

### and 1.0 μ M for 12 hours ( Wang et al., 20 09c ) , and retinal rod photoreceptor cells 19 

### incubated with 0.01 to 10 μ M for 15 minute s ( He et al., 2000 )

. Further research indicated 20 

### that Pb

- induced mitochondrial swelling and decreased membrane poten tial is the result of 21 

### the opening of a mitochondrial transmembrane pore (MTP), possibly by directly binding 22 

### to the metal (Ca 2+ 

### )

- binding site on the matrix side of the pore ( Bragadin et al., 2007 ; He 23 

### et al., 2000 )

. Opening of the MTP is the first step of the mitochondrial

- regulated 24 

### apoptotic cascade pathway in many cells ( Rana, 2008 ; Lidsky and Schneider, 2003 )

. He 25 

### et al. ( 2000 ) additionally observed cytochrome c release from mitochondria, and caspase

26 

### 9 and

- 3 activation following exposure of rod cells to Pb. Induction of mitochondrially

27 

### regulated apoptosis via stimulation of the caspase cascade following exposure to Pb has 28 

### also been observed in rat oval cells ( Agarwal et al., 2009 )

. 29



### Altered Ener gy Metabolism Pb has been reported to alter normal cellular bioenergetics. In mitochondria isolated from 30 

### the kidneys of rats exposed to 1% Pb in feed for 6 weeks, the rate of oxygen uptake 31 

### during ADP

- activated (state 3) respiration was lower compared to co ntrols ( Goyer et al., 32 

### 1968 )

. The rate of ATP formation in exposed mitochondria was observed to be 33 

### approximately 50% that of control mitochondria. A decrease in state 3 respiration and 34February 2012 5

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### resp iratory control ratios (state 3/state 4 [succinate or pyruvate/malate

- activated]) was 1 

### also observed in kidney mitochondria from rats exposed continuously from conception to 2 

### six or nine months of age ( i.e., gestationally, lactationally, and via drinking wat er after 3 

### weaning) to 50 or 250 ppm Pb ( Fowler et al., 1980 )

. Pb

- induced decreases in ATP and 4 

### adenylate energy charge (AEC) were observed concurrently with increases in ADP, 5 

### AMP, and adenosine in rats exposed to 1% Pb in drinking water for 9 months 6 

### ( Marchlewicz et al. , 2009 )

. Similarly, ATP and AEC were decreased, and AMP 7 

### increased, in primary cerebellar granule neuronal cultures from rats exposed to 0.1% Pb 8 

### in drinking water throughout gestation and lactation ( Baranowska

- Bosiacka et al., 9 

### 2011a )

. One μ M Pb (48 hours) was the lowest concentration observed to decrease 10 

### cellular ATP levels in differentiated PC

- 12 cells, and these changes were correlated with 11 

### a Pb

- induced decrease in the expression of the voltage

- dependent anion chann el, which 12 

### maintains cellular ATP levels in neurons ( Prins et al., 2010 )

. Dowd et al. ( 1990 ) reported 13 

### that oxidative phosphorylation was decreased up to 74% after exposure o f osteoblasts to 14 

### 10 μ M Pb. Parr and Harris ( 1976 ) reported that Pb inhibited coupled and uncoupled 15 

### respiratory oxygen use in mitochondria, and that Pb prevented pyruvate, but not malate, 16 

### uptake. Mitochondrial levels of ATP were diminished after exposure, and the authors 17



### co mpared the effects of Pb on the energy supply to the actions of classic respiratory 18 

### inhibitors, low temperature , and chemical uncouplers. Bragadin et al. ( 1998 ) supp orted 19 

### this view by demonstrating that alkylated Pb compounds acted as a chemical uncoupler 20 

### of respiration by abolishing the proton gradient necessary for oxidative phosphorylation. 21 

### Further, the enzymatic activities of complex I and IV of the respiratory ch ain have been 22 

### shown to be decreased in the peroneous longus muscle of rats exposed to 250 ppm Pb or 23 

### 5 ppm thallium in drinking water for 90 days ( Méndez

- Armenta et al., 2011 )

. Contrary to 24 

### the above findings, Rafalowska et al. ( 1996 ) reported that, although ATP levels did 25 

### decrease, chronic exposure to Pb did not inhibit oxidative phosphorylation in the 26 

### synaptosomes of rats exposed to 200 ppm Pb in water for 3 months. Similar effects with 27 

### regard to the activity of the mitochondrial oxidative chain were observed in rats injected 28 

### with 15 mg/kg Pb i.p. daily for seven days, as reported by Struzynksa et al. ( 1997a ) , 29 

### although ATP levels were reported to increase after exposure to Pb. 30 

### Pb has also been shown to decrease glycolysis in osteoblasts exposed to 10 μ M Pb and in 31 

### human erythrocytes exposed to 30 μ g/dL Pb ( Grabowska and Guminska, 1996 ; Dowd et 32 

### al., 1990 )

. Contrary to these findings, Antonowicz et al. ( 1990 ) obser ved higher levels of 33 

### glycolytic enzymes in erythrocytes obtained from Pb workers directly exposed to Pb, 34 

### compared to controls exposed to lower concentrations of Pb (blood Pb levels: 82.1 versus 35 

### 39.9 μ g/dL), and suggested that Pb activated anaerobic glycoly sis. In vitro exposure of 36 

### human umbilical cord erythrocytes to 100

- 200 μ g/dL Pb for 20 hours was observed to 37 

### lower the cellular pools of adenine and guanine nucleotide pools, including NAD and 38 

### NADPH ( Baranowska

- Bosiacka and Hlynczak, 2003 )

. Thes e decreases in nucleotide 39February 2012 5

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### pools were accompanied by an increase in purine degradation products (adenosine, etc.). 1 

### Similar decreases in cellular nucleotide pools were observed when rats were exposed to 2 

### 1% Pb in drinking water for four weeks ( Baranowska

- Bosiacka and Hlyn czak, 2004 )

. In 3 

### erythrocytes, nucleotides are synthesized via salvage pathways such as the adenine 4 

### pathway, which requires adenine phosphoribosyltransferase. The activity of this enzyme 5 

### is inhibited by exposure to Pb in human and rat erythrocytes (see a bove for concentration 6 

### and duration) ( Baranowska

- Bosiacka et al., 2009 )

. 7 

### Disruptions in eryt hrocyte energy metabolism have been observed in workers 8 

### occupationally exposed to Pb. Nikolova and Kavaldzhieva ( 1991 ) reported higher ratios 9 

### of ATP/ADP in Pb

- exposed workers with an average duration of exposure of 8.4 years 10 

### (blood Pb not reported). Morita et al. ( 1997 ) evaluated the effect of Pb on NAD 11 

### synthetase in the erythrocytes of Pb

- exposed workers (mean [SD] blood Pb level: 34.6 12 

### [20.7] μ g/dL) and observed an apparent concentration

- dependent decrease in NAD 13 

### synthetase activity with increased blood Pb level. The blood Pb level associated with 14 

### 50% inhibition of NAD synthetase, which requires a magnesium cation for activity ( Hara 15 

### et a l., 2003 ) , was 43 μ g/dL. 16



### Altered Heme Synthesis Exposure to Pb is known to inhibit two key steps in the synthesis of heme: 17 

### porphobilinogen synthase ( i.e., δ

- aminolevulinic acid dehydratase), a cytoplasmic 18 

### enzyme requiring zinc for enzymatic activity tha t condenses two molecules of 19 

### aminolevulinic acid into porphobilinogen, and ferrochelatase, a mitochondrial iron

- sulfur 20 

### containing enzyme that incorporates Fe 2+ 

### into protoporphyrin IX to create heme. Farant 21 

### and Wigfield ( 1990 , 1987 ) observed that Pb inhibits the activity of porphobilinogen 22 

### synthase in rabbit and human erythrocytes, and that the effect on the enzyme was 23 

### dependent on the affinity for thiol gro ups at its active site. Taketani et al. ( 1985 ) 24 

### examined the activity of Pb on ferrochelatase in rat liver mitochondria and o bserved that 25 

### 10 μ M Pb (30 minute incubation) reduced NAD(P)H

- dependent heme synthesis by half 26 

### when ferric, but not ferrous, iron was used. Pb inhibits the insertion of Fe 2+ 

### into the 27 

### protoporphyrin ring and instead, Zn is inserted into the ring creating zin c protoporphyrin 28 

### (ZPP). While not directly measuring the activity of ferrochelatase, numerous studies have 29 

### shown that blood Pb levels are associated with increased erythrocyte ZPP levels in 30 

### humans (average blood Pb levels ranging from 21.92 to 53.63 μ g/dL) ( Mohammad et al., 31 

### 2008 ; Counter et al., 2007 ; Patil et al., 2006b ; Ademuyiwa et al., 2005b ) and animals 32 

### (blood Pb level: 24.7 μ g/dL) ( Rendón

- Ramirez et al., 2007 )

. 33February 2012 5

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### 5.2.3 Protein Binding
Evidence indicates that Pb binds to proteins within cells through interactions with side 1 

### group moieties ( e.g., thiol residues) and can potentially disr upt cellular function 2 

### (Secti ons 5.2.2.3 and 5.2.2.4 ). However, some proteins are also able to bind Pb and 3 

### protect against its negative effects through sequestration. The ability of Pb to bind 4 

### protei ns was first reported by Blackma n ( 1936 ) : Pb intoxication in children autopsy cases 5 

### was observed to induce th e formation of intranuclear inclusion bodies in the liver and 6 

### kidney. Since that time, further research has been conducted into characterizing the 7 

### composition of intr anuclear inclusion bodies and i dentifying specific Pb

- binding proteins. 8



### 5.2.3.1 Intranuclear and Cytoplasmic Inclusion Bodies
Goyer ( 1968 ) and Goyer et al. ( 1968 ) observed the formation of intranuclear inclusion 9 

### bodies in the renal tubules of rats fed 1% Pb in food for up to 20 weeks. The observation 10 

### of inclusion bodies was accompanied by altered mitochondrial structure and reduced 11 

### rates of oxidative phosphorylation. Pb has further been observed to form cytoplasmic 12 

### inclusion bodies preceding the formation of the intranuclear bodies, and to be 13 

### concentrated within the subsequently induced intranuclear inclusion bodies following i.p. 14 

### injection, drinking water, and dietary exposures ( Navarro

- Moreno et al., 2009 ; Oskarsson 15 

### and Fowler, 1985 ; Fowler et al., 1980 ; McLachlin et al., 1980 ; Choie and Richter, 1972 ; 16 

### Goyer et al., 1970a ; Goyer et al., 1970b )

. Inclusion bodies have also been observed in the 17 

### mitochondria of kidneys and the perinuclear space in the neuron s of rats exposed to 18 

### 500 ppm Pb

- acetate in drinking water for 60 days or 7 months ( Navarro

- Moreno et al., 19 

### 2009 ; Deveci, 2006 )

. Intranuclear and cytoplasmic inclusions have also been found in 20 

### organs other than the kidney, including liver, lung, and glial cells ( Singh et al., 1999 ; 21 

### Goyer and Rhyne, 1973 )

. Pb found within nuclei has also been shown to bind to the 22 

### nuclear membrane and histone fractions ( Sabbioni and Marafante, 1976 )

. 23 

### Upon denaturing intranuclear inclusion bodies with strong denaturing agents, Moore et 24 

### al. ( 1973 ) observed that proteins included in the bodies were rich in aspartic and glutamic 25 

### acid , glycine, and cysteine. Further work by Moore and Goyer ( 1974 ) characterized the 26 

### protein as a 27.5 kDa protein that migrates as a single band on acrylamide gel 27 

### electrophoresis. In contrast with the findings of Moore and Goyer, Shelton and Egle 28 

### ( 1982 ) identified a 32 kDa protein with an isoelectric point of 6.3 from the kidn eys of rats 29 

### exposed to 1% Pb

- acetate in feed or 0.75% in drinking water. This protein, dubbed 30 

### p32/6.3, was not found in control rats, indicating that the protein was induced by Pb 31 

### exposure. This finding was in agreement with studies that indicated formation of 32 

### intranuclear inclusion bodies required protein synthesis ( McLachlin et al., 1980 ; Choie et 33February 2012 5

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### al., 1975 )

. In addition to its presence in kidneys of Pb

- exposed animals, p32/6.3 has been 1 

### observed to be present and highly conserved in the brains of rats, mice, dogs, chickens, 2 

### and humans ( Egle and Shelton, 1986 )

. Exposure of neuroblastoma cells to 50 or 100 μ M 3 

### Pb glutamate for 1 or 3 days increased the abundance of p32/6.3 ( Klann and Shelton, 4 

### 1989 )

. Shelton et al. ( 1990 ) determined that p32/6.3 was enriched in the basal ganglia, 5 

### diencephalon, hippocampus, cerebellum, brainstem, spinal cord, and cerebral cortex, and 6 

### that it contained a high percentage of glycine, aspartic, and glutamic acid residues. 7 

### Selvin

- Testa et al. ( 1991 ) and Harry et al. ( 1996 ) reported that pre

- and post

- natal 8 

### exposure of rats to 0.2

- 1.0% Pb in drinking water increased the levels of another brain 9 

### protein, glial fibrillary acidic protein, in developing astrocytes and that t his increase may 10 

### be indicative of a demand for astrocytes to sequester Pb. 11



### 5.2.3.2 Cytosolic Lead Binding Proteins
Numerous studies have also identified cytosolic Pb

- binding proteins. Two binding 12 

### proteins, with molecular weights of 11.5 and 63 kDa, were identified by ( Oskarsson et 13 

### al., 1982 ) in the kidney postmitochondrial cytosolic f raction after injection with 50 mg 14 

### Pb. The two proteins were also found in the brain, but not the liver or lung. Mistry et al. 15 

### ( 1985 ) identified three proteins (MW = 11.5, 63, and >200 kDa) in rat kidney cytosol, 16 

### two of which, the 11.5 and 63 kDa proteins, were able to translocate into the nucleus. The 17 

### 11.5 kDa kidney protein was also able to reverse Pb binding to ALAD through chelation 18 

### of Pb and donation of a zinc cation to ALAD ( Goering and Fowler, 1985 , 198 4 )

. 19 

### Cadmium and zinc, but not Ca 2+ 

### or Fe, prevented the binding of Pb to the 63 and 11.5 20 

### kDa cytosolic proteins, which agree s with previous observations that cadmium is able to 21 

### reduce total kidney Pb and prevent the formation of intranuclear inclusion bodies ( Mistry 22 

### et al., 1986 ; Mahaffey et al., 1981 ; Mahaffey and Fowler, 1977 )

. Additional cytosolic Pb

23 

### binding proteins have been identified in the kidneys of Pb

- exposed rats and humans, 24 

### including the cleavage product of α2

- microglobulin, acyl

- CoA binding protein (MW = 9 25 

### kDa), and thymosin β4 (MW = 5 kDa) ( Smith et al., 1998 ; Fowler and DuVal, 1991 )

. 26 

### Cytosolic Pb

- binding proteins distinct from kidney proteins have also been identified in 27 

### the brain of exposed rats and human brain homogenates exposed in vitro ( Quintanilla

28 

### Vega et al., 1995 ; DuVal and Fowler, 1989 ; Goering et al., 1986 )

. One protein (MW = 29 

### 12 kDa) was shown to alleviate hepatic ALAD inhibition due to Pb exposure through 30 

### competitive binding with Pb and donation of zinc to ALAD. Cytosolic Pb

- binding 31 

### proteins have been shown to be high in glutamic acid, aspartic acid, and cysteine residues 32 

### ( Fowler et al., 1993 ; DuVal and Fowler, 1989 )

. Some evidence exists that cytosolic Pb

33 

### binding proteins directly target Pb and compartmentalize intracellular Pb as a protective 34



### measure a gainst toxicity ( Qian et al., 2005 ; Qian et al., 2000 )

. 35February 2012 5

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### 5.2.3.3 Erythrocytic Lead Binding Proteins
The majority (94%) of Pb in whole blood is found in erythrocytes ( Ong and Lee, 1980a )

. 1 

### Originally, the major Pb

- binding protein in erythrocy tes was identified as hemoglobin 2 

### ( Cohen et al., 2000 ; Lolin and O'Gorman, 1988 ; Ong and Lee, 1980a , b ; Raghavan and 3 

### Gonick, 1977 )

. However, B ergdahl et al. ( 1997b ) observed the pri ncipal Pb

- binding 4 

### protein to be 240 kDa and identified it as ALAD. Two smaller Pb

- binding proteins were 5 

### observed, but not identified (MW = 45 and <10 kDa). ALAD levels are inducible by Pb 6 

### exposure; the total concentration of the enzyme, but not the activit y, increases after 7 

### exposure in both exposed humans (blood Pb = 30

- 75 μ g/dL) and rats (Pb exposure = 25 8 

### mM in drinking water) ( Boudene et al., 1984 ; Fujita et al., 1982 ; Fujita et al., 1981 )

. 9 

### ALAD is a polymorphic gene with three isoforms: ALAD 1

- 1, ALAD 1

- 2, or ALAD 2

- 2. 10 

### C arriers of the ALAD

- 2 allele have been shown to have higher blood Pb levels than 11 

### carriers of the homozygous ALAD

- 1 allele ( Scinicariello et al., 2007 ; Zhao et al., 2007 ; 12 

### Kim et al., 2004 ; Pérez

- Bravo et al., 2004 ; Smith et al., 199 5a ; Wetmur, 1994 ; Wetmur et 13 

### al., 1991a ; Astrin et al., 1987 )

. Some newer studies, however, either observed lower 14 

### blood Pb levels in carriers of the ALAD

- 2 allele or no difference in Pb levels among the 15 

### different allele carriers ( Scinicariello et al., 2010 ; Krieg et al., 2009 ; Chen et al., 2008c ; 16 

### Chia et al., 2007 ; Chia et al., 2006 ; Wananukul et al., 2006 )

. 17


### The ALAD

- 2 protein binds Pb more tightly than the ALAD

- 1form: in workers carrying 18 

### the AL AD

- 2 gene, 84% of blood Pb was bound to ALAD versus 81% in carriers of the 19 

### ALAD

- 1 gene (p = 0.03) ( Bergdahl et al., 1997a )

. This higher affinity for Pb in ALAD

- 2 20 

### carriers may sequester Pb and prevent its bioavailability for reaction with other enzymes 21 

### or cellular component s. This is supported by the observation that carriers of the ALAD

- 2 22 

### gene have higher levels of hemoglobin ( Scinicariello et al., 2007 ) , decreased plasma 23 

### levulinic acid ( Schwartz et al., 1997b ) , decreased levels of zinc protoporphyrin 24 

### ( Scinicariello et al., 2007 ; Kim et al., 2004 ) , lower cortical bone Pb ( Smith et al., 1995b ) , 25 

### and lower amounts of DMSA

- chelatable Pb ( Scinicariello et al., 2007 ; Schwartz et al., 26 

### 2000a ; Schwartz et al., 1997a )

. However, the findings that ALAD

- 2 polymorphisms 27 

### reduced the bioavailability of Pb are somewhat equivocal. Wu et al. ( 2003a ) observed 28 

### that ALAD

- 2 carriers had lower blood Pb level (5.8 ± 4.2 μ g/dL) than carriers of the 29 

### ALAD

- 1 ge ne (blood Pb level = 6.2 ± 4.1 μ g/dL), and that ALAD

- 2 carriers 30 

### demonstrated decreased renal function at lower patellar Pb concentrations than those 31 

### observed to decrease renal function in ALAD

- 1 carriers. This potentially indicates that 32 

### ALAD

- 2 carriers hav e enhanced Pb bioavailability. Weaver et al. ( 2003b ) observed that 33 

### ALAD

- 2 polymorphisms were associated with higher DMSA

- chelatable Pb 34 

### concentrations, when normalized to creatinine levels. Further, Montenegro et al. ( 2006 ) 35 

### observed among individuals with ALAD 1

- 1 or ALAD 1

- 2/2

- 2 genotypes a significant 36February 2012 5

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### increase in the amount of Pb found in the plasma (0.44 μ g/L versus 0.89 μ g/L, 1 

### respectively) and in the % plasma/blood ratio (0.48% versus 1.45%, respectively). This 2 

### potentiall y suggests that individuals with the ALAD 1

- 2/2

- 2 genotype are at increased 3 

### risk of Pb

- induced health effects due to lower amounts of Pb sequestration by erythrocyte 4 

### ALAD, although this study did not specifically investigate the clinical implications of 5 

### AL AD polymorphisms. 6


### ALAD has the estimated capacity to bind Pb at 85 μ g/dL in erythrocytes and 40 μ g/dL in 7 

### whole blood ( Bergdahl et al., 1998 )

. The 45 kiloDalton (kDa) and < 10 kDa Pb

- binding 8 

### proteins bound approximately 12

- 26% and <1% of the blood Pb, respectively. At blood 9 

### Pb concentrations greater than 40 μ g/dL, greater binding to these components likely 10 

### would be observed. Bergdahl et al. ( 1998 ) tentatively identified the 45 kDa protein as 11 

### pyrimidine

- 5’

- nucleotidase and the <10 kDa protein as acyl

- CoA binding protein. Smith 12 

### et al. ( 1998 ) previously iden tified acyl

- CoA binding protein as a Pb

- binding protein 13 

### found in the kidney. 14 

### Studies also observed the presence of an inducible, low

- molecular weight (approximately 15 

### 10 kDa) Pb

- binding protein in workers occupationally exposed to Pb ( Gonick et al., 16 

### 1985 ; Raghavan et al., 1981 , 1980 ; Raghavan and Gonick, 1977 )

. The presence of this 17 

### low molecular weight protein seemed to have a protective effect as workers that exhibited 18 

### toxicity at low blood Pb concentrations were observed to have lowered expression of this 19 

### protein or low level s of Pb bound to it ( Raghavan et al., 1981 , 1980 )

. The presence of low 20 

### molecular weight Pb

- binding proteins in exposed workers was confirmed by Lolin and 21 

### O’Gorman ( 1988 ) and Church et al. ( 1993a , b )

. Further Lolin and O’Gorman ( 1988 ) 22 

### reported that the observed protein was only present when blood Pb levels were greater 23 

### than 39 μ g/dL, in agreement with the Pb

- binding capacity of ALAD, identified by 24 

### Bergdahl et al. ( 1998 )

. Xie et al. ( 1998 ) confirmed this, observing the presence of a 25 

### second low molecular weight protein with greater affinity than ALAD only at hig her 26 

### blood Pb levels. Church et al. ( 1993a , b ) observed the presence of a 6

- 7 kDa prote in in 27 

### the blood of 2 Pb workers (blood Pb >160 μ g/dL); approximately 67% of Pb was bound 28 

### to the protein in the blood of the asymptomatic worker, whereas only 22% of the Pb was 29 

### bound to it in the symptomatic worker. The reported protein was rich in cysteine residues 30 

### and tentatively identified as metallothionein. 31



### 5.2.3.4 Metallothionein

Metallothionein is a low

- molecular weight metal

- binding protein, most often zinc or 32 

### copper, which is rich in cysteine residues and plays an important role in the protection 33 

### against h eavy metal toxicity, trace element homeostasis, and scavenging free radicals ( Yu 34February 2012 5

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### et al., 2009 )

. Exposure to Pb

- acetate induces the production of Pb

- and Zn

1 

### metallothionein in mice exposed via i.p. or intravenous (i.v.) injection at 30 mg /kg 2 

### ( Maitani et al., 1986 ) , in mice exposed via i.p. injection at 300 μ mol/kg ( Yu et al., 2009 ) , 3 

### or in rats exposed via i.p. inj ection at 24 μ mol/100g ( Ikebuchi et al., 1986 )

. The induced 4 

### Pb

- metallothionein consisted of 28% half

- cysteine and reacted with an antibody for Zn

5 

### metallo thionein II ( Ikebuchi et al., 1986 )

. In contrast, exposure of rats to Pb via drinking 6 

### water (200 or 300 ppm ) failed to induce metallothionein in the kidn eys or intestines 7 

### ( Wang et al., 2009b ; Jamieson et al., 2007 )

. Goering and Fowler ( 1987a , b ) observed that 8 

### pretreatment of rats with zinc before injection with Pb resulted in Pb and zinc co

- eluting 9 

### with zinc

- thionein, and that zinc

- thionein I and II we re able to bind Pb in vitro ( Goering 10 

### and Fowler, 1987a , b )

. Further, Goering and Fowler ( 1987a ) found that kidney and liver 11 

### zinc

- thionein decreased binding of Pb to liver ALAD and was able to donate zinc to 12 

### ALAD, thus attenuating the inhibition of ALAD due to Pb exposure. These findings are 13 

### in agreement with Goering et al. ( 1986 ) an d DuVal and Fowler ( 1989 ) who demonstrated 14 

### that rat brain Pb

- binding proteins attenuated Pb

- induced inhibi tion of ALAD. 15 

### Metallothionein has been reported to be important in the amelioration of Pb

- induced 16 

### toxicity effects. Liu et al. ( 1991 ) reported that zinc

- metallothionein reduced Pb

- indu ced 17 

### membrane leakage and loss of potassium in cultured hepatocytes incubated with 600

- 18 

### 3,600 μ M Pb. Metallothionein

- null mice exposed to 1,000, 2,000, or 4,000 ppm Pb for 20 19 

### weeks suffered renal toxicity described as nephromegaly and decreased renal functio n 20 

### compared to Pb

- treated wild

- type mice ( Qu et al., 2002 )

. Interestingly, metallothionein

21 

### null mice were unable to form intranuclear inclusion bodies and accumulated less renal 22 

### Pb than did the wild

- type mice ( Qu et al., 2002 )

. Metallothionein levels were induced by 23 

### Pb exposure in non
- null mice. Exposure to Pb (1,000, 2,000, or 4,000 ppm ), both for 24 

### 104 weeks as adults and fr om GD8 to early adulthood, resulted in increased preneoplastic 25 

### lesions and carcinogenicity in the testes, bladder, and kidneys of metallothionein

- null rats 26 

### compared to wild type mice ( Tokar et al., 2010 ; Waalkes et al., 2004 )

. Inclusion bodies 27 

### were not observed in null mice. The authors concluded that metallothionein is important 28 

### in the formation of inclusion bodies and mitigation of Pb

- induced toxic effects, and that 29 

### those with polymorphisms in metallothionein coding ge nes may be at greater 30 

### susceptibility to Pb. In support of this theory, Chen et al. ( 2010a ) observed that Pb

31 

### exposed workers with a mutant met allothionein allele had higher blood Pb levels than did 32 

### carriers of the normal allele (24.17 and 21.27 versus 17.03 μ g/dL), and were more 33 

### susceptible to the effects of Pb on systolic BP and serum renal function parameters. 34February 2012 5

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### 5.2.4 Oxidative Stress

Oxidative stress occurs when free radicals or reactive oxygen species (ROS) exceed the 1 

### capacity of antioxidant defense mechanisms. This oxidative imbalance results in 2 

### uncontained ROS, such as superoxide (O 2 - 

### ), hydroxyl radical (OH • 

### ), and hydrogen 3 

### peroxide (H 2 

### O 2 

### ), which can attack and denature functional/structural molecules and, 4 

### thereby, promote tissue damage, cytotoxicity, and dysfunction. Pb has been shown to 5 

### cause oxidative damage to the heart, liver, kidney, reproductive organs, brain, and 6 

### erythrocytes, which may be responsible for a number of Pb

- induced health effects 7 

### ( Salawu et al., 2009 ; Shan et al., 2009 ; Vaziri, 2008b ; Gonick et al., 1997 ; Sandhir and 8 

### Gill, 1995 ; Khalil

- Manesh et al., 1994 ; Khalil

- Manesh et al., 1992b )

. The origin of ROS 9 

### produced after Pb exposure is likely a multipathway proce ss, resulting from oxidation of 10 

### δ

- aminolevulinic acid (ALA), membrane and lipid oxidation, nicotinamide adenine 11 

### dinucleotide phosphate (NAD(P)H) oxidase activation, and antioxidant enzyme 12 

### depletion, as discussed below. Some of these processes result from t he disruption of 13 

### functional metal ions within oxidative stress proteins, such as superoxide dismutase 14 

### (SOD), catalase (CAT), and glutathione peroxidase (GPx). Interestingly, Pb exposure in 15 

### many species of plants, invertebrates, and vertebrates discussed in the Ecological Effects 16 

### of Pb results in upregulation of antioxidant enzymes and increased lipid peroxidation 17 

### ( Chapter 7 ). Oxidative stress is a common mode of action for a number of other metals 18 

### ( e.g., Cd, Mn, As, Co, Cr) that are often found with Pb and by which possible interactions 19 

### with Pb have been suggested to occur ( Jomova and Valko, 2011 ; Jomova et al., 2011 ; 20 

### Matović et al., 2011 ; HaMai and Bondy, 2004 )

. Not all of these co

- occur r ing metals 21 

### directly produce ROS or redox cycle, but instead may suppress the free radical 22 

### scavenging ability of the organism thus leading to oxidative stress. 23



### 5.2.4.1 δ
- ALA Oxidation
The majority of Pb present in the blood accumulates in erythrocytes where it enters 24 

### through passive carrier

- mediated mechanisms including a vanadate

- sensitive Ca 2+ 

### pump. 25 

### Once Pb enters erythrocytes, it is predominantly found in the protein

- bound form, with 26 

### hemoglobin and δ

- ALAD both identified as targets ( Bergdahl et al., 1997a )

. Through its 27 

### sulfhydryl and metal ion disrupting properties, Pb incorporates with and inhibits a 28 

### number of enzymes in the heme biosynthetic process, including δ

- ALA synthetase, δ

29 

### ALAD, and ferrochelatase. Pb has been shown to be able to disrupt the zinc io ns requisite 30 

### for the activity of δ

- ALAD, the rate limiting step in heme synthesis, leading to enzyme 31 

### inhibition at picomolar concentrations ( Simons, 1995 )

. Additionally, low blood Pb levels 32 

### (mean: 7 μ g/dL) have been found to inhibit the activity of δ

- ALAD in humans, and the 33 

### lowest blood Pb level observed to be associated with lower δ

- ALAD activity in these 34February 2012 5

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### studies was 5 μ g/dL ( Ahamed et al., 2005 ; Sakai an d Morita, 1996 )

. A significant 1 

### negative correlation (r =

- 0.6) was found between blood Pb levels in adolescents (range 2 

### of blood Pb levels: 4

- 20 μ g/dL) and blood δ

- ALAD activity in adolescents ( Ahamed et 3 

### al., 2006 )

. This inhibition of δ

- ALAD results in the accumulation of δ

- ALA in blood and 4 

### urine, where δ

- ALA undergoes tautomerization and autoxidation. Oxidized δ

- ALA 5 

### generates ROS through re duction of ferricytochrome c and electron transfer from oxyHb, 6 

### metHb, and other ferric and ferrous complexes ( Hermes

- Lima et al., 1991 ; Monteiro et 7 

### al., 1991 )

. The autoxidation of δ

- ALA produces O 2 - 

### , OH • , H 2 

### O 2 

### , and an ALA radical 8 

### ( Monteiro et al., 1989 ; Monteiro et al., 1986 )

. 9



### 5.2.4.2 Membrane and Lipid Peroxidation
A large number of studies in humans and experimental animals have found that exposure 10 

### to Pb can lead to membrane and lipid peroxidation. It is possible that ROS produced from 11 

### δ

- ALA oxidation, as described above, interacts with and disrupts membrane lipids 12 

### ( Oteiza et al., 1995 ; Bechara et al., 1993 )

. Additionally, Pb has the capacit y to stimulate 13 

### ferrous ion initiated membrane lipid peroxidation serving as a catalyst for these events 14 

### ( Adonaylo and Oteiza, 1999 ; Quinlan e t al., 1988 )

. The extent of peroxidation of lipids 15 

### varies based on the number of dou ble bonds present in unsaturated fatty acids, since 16 

### double bonds weaken the C

- H bonds on the adjacent carbon, making H removal easier 17 

### ( Yiin and Lin, 1995 )

. After Pb exposure (4

- 12 μ g/dL in vitro, 24 hours), the production 18 

### of malondialdehyde (MDA), a marker of oxidative stress and lipid oxidation end product, 19 

### increased relative to the number of double bonds of the fatty acid. In the absence of Fe 2+


### , 20 

### Pb has not been shown to promote lipid peroxidation; however, it may accelerate 21 

### peroxidation by H 2 

### O 2 ( Quinlan et al., 1988 )

. 

### This could be due to altering membrane structure, 22 

### restricting phospholipid movement, and facilitating the propagation of peroxidation. 23 

### Pb induces changes in the fatty acid composition of a membrane, which could lead to 24 

### oxidative damage. Exposure to Pb (>62.5 ppm in drinking water, 3 weeks) in chicks 25 

### promoted an increase in arachidonic acid (AA, 20:4) as a percentage of total fatty acids, 26 

### and decreased the relative prop ortion of shorter chain fatty acids (linoleic acid, 18:2) 27 

### ( Lawton and Donaldson, 1991 )

. It is possible that Pb depressed the desaturation of 28 

### saturated fatty acids to the corresponding monoenoic fatty acids, while stimulating 29 

### elongation and desaturation of linoleic acid to AA. Sin ce fatty acid chain length and 30 

### unsaturation are related to the oxidative potential, changes in fatty acid membrane 31 

### composition may result in enhanced lipid peroxidation. In addition, changes in fatty 32 

### acids, thus membrane composition, can result in altered membrane fluidity ( Donaldson 33 

### and Knowles, 1993 )

. Changes in membrane fluidity will disturb the conformation of the 34February 2012 5

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### active sites of membrane associated enzymes, disrupt metabolic regulation, and alter 1 

### membrane permeability and function. 2


### A number of recent studies report increased measures of lipid peroxidation in various 3 

### organs, tissues, and species. Occupational Pb exposure resulting in elevated blood Pb 4 

### levels (means >8 μ g/dL) in various countries provides evidence of lipid peroxidation, 5 

### including increased plasma MDA levels ( Ergurhan

- Ilhan et al., 2008 ; Khan et al., 2008 ; 6 

### Mohammad et al., 2008 ; Quintanar

- Escorza et al., 2007 ; Patil et al., 2006a ; Patil et al., 7 

### 2006b )

. One study found a correlation between the MDA levels and blood Pb levels even 8 

### in the unexposed worke rs, although they had blood Pb levels higher than the mean blood 9 

### Pb level of the current U.S. population ( i.e., <12 μ g/ dL ) ( Quintanar

- Escorza et al., 2007 )

. 10 

### Other studies found evidence of increased lipid peroxidation among the general 11 

### population, including children, with elevated blood Pb levels (means >10 μ g/dL) 12 

### ( Ahamed et al., 2008 ; Ahamed et al., 2006 ; Jin et al., 2006 )

. In adolescents, Ahamed et 13 

### al. ( 2006 ) found a blood MDA levels to be positively correlated (r = 0.7) with blood Pb 14 

### levels ranging between 4

- 20 μ g/dL. Similar results have been shown after Pb exposure in 15 

### animal studies ( Abdel Moneim et al., 2011b ; Pandya et al., 2010 ; Dogru et al., 2008 ; Yu 16 

### et al., 2008 ; Adegbesan and Adenuga, 2007 ; Lee et al., 2005 )

. Enhanced lipid 17 

### peroxidatio n has been found in Pb treated (50 μ g, 1

- 4 hours) rat brain homogenates 18 

### ( Rehman et al., 1995 ) , rat proximal tubular cells (0.5

- 1 μ M, 12 hours) ( Wang et al., 19 

### 2011b ) , and in specific brain regions, hippocampus and cerebellum, after Pb exposure 20 

### (500 ppm , 8 weeks) to rats ( Bennet et al., 2007 )

. Overall, there was a correlation between 21 

### the blood Pb level and measures of lipid peroxidation often measured by MDA levels. 22 

### Interestingly, many species of plants, invertebrates, and vertebrates exhibit increased 23 

### lipid peroxidation with Pb exposure ( Sections 7.2.4 , 7.3.5 , and 7.3.12 ). The increase in 24 

### lipid peroxidation following Pb exposure observed acr oss species and kingdoms 25 

### demonstrate an evolutionarily conserved oxidative response following Pb exposure. 26



### 5.2.4.3 NAD(P)H Oxidase Activation
NAD(P)H oxidase is a membrane bound enzyme that requires Ca 2+ 

### in order to catalyze 27 

### the production of O 2 - 

### from NAD(P)H and molecular oxygen ( Leseney et al., 1999 )

. Two 28 

### studies provide evidence for increased activation of NAD(P)H oxidase that may 29 

### contribute to the production of ROS after P b exposure ( Ni et al., 2004 ; Vaziri et al., 30 

### 2003 )

. Vaziri et al. ( 2003 ) found increased protein expression of the NAD(P)H subunit 31 

### gp 91 

### phox in the brain, heart, and renal cortex of Pb treated rats (100 ppm in drinking 32 

### water, 12 weeks). This upregulation was present in Pb

- treated (1

- 10 ppm ) human 33 

### coronary artery endothelial cells, but not vascular smooth muscle cells (VSMC), which 34February 2012 5

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### do not express the protein ( Ni et al., 2004 )

. It is possible that NAD(P)H oxidase serves as 1 

### a potential source of ROS in cells that express this protein. 2



### 5.2.4.4 Antioxidant Enzyme Disruption
Oxidative stress can result not only from the increased production of ROS, but also from 3 

### the decreas ed activity of antioxidant defense enzymes. Pb has been shown to alter the 4 

### function of several antioxidant enzymes, including SOD, CAT, glucose

- 6

- phosphate 5 

### dehydrogenase (G6PD), and the enzymes involved in glutathione metabolism, GPx, 6 

### glutathione

- S

- transfe rase (GST), and glutathione reductase (GR). These changes in the 7 

### antioxidant defense system could be due to the high affinity of Pb for sulfhydryl groups 8 

### contained within proteins and its metal ion mimicry. However changes could also be a 9 

### consequence of in creased oxidative damage by Pb. 10 

### Studies of the effects of Pb on the activities of SOD and CAT give divergent results. 11 

### These metalloprotein enzymes require various essential trace elements for proper 12 

### structure and function, making them a target for Pb toxic ity. CAT is a heme containing 13 

### protein that requires iron ions to function ( Putnam et al., 2000 )

. SOD exists in multiple 14 

### isoforms that require copper and zinc (SOD1 and SOD3) ( Antonyuk et al., 2009 ) or 15 

### manganese (SOD2) ( Borgstahl et al., 1992 )

. A number of studies have found decreased 16 

### activity of these enzymes ( Pandya et al., 2010 ; Ergurhan

- Ilhan et al., 2008 ; Mohammad 17 

### et al., 2008 ; Yu et al., 2008 ; Patil et al., 2006a ; Patil et al., 2006b ; Conterato et al., In 18 

### Pre ss ) , whereas others have observed increased activity following Pb exposure ( Ahamed 19 

### et al., 2008 ; Lee et al., 2005 )

. Pb exposure (500 ppm , 1, 4, and 8 weeks) in ra ts showed 20 

### that organ SOD and CAT responded differently depending on the dose and tissue 21 

### investigated. Activity of SOD and CAT varied based on the brain region analyzed and 22 

### time of exposure ( Bennet et al., 2007 )

. Another study found that the brain had 23 

### consistently decreased SOD activity, irrespective of dose in prenatally exposed animals 24 

### (0.3 and 3.0 ppm , bloo d Pb level 20.4 and 24.5 μ g/dL); however hepatic SOD activity 25 

### increased at low level Pb administration and decreased after high level exposure 26 

### ( Uzbekov et al ., 2007 )

. It is possible that the increased SOD and CAT protein is due to 27 

### activation by ROS, while decreased enzyme activity is the result of metal ion substitution 28 

### by Pb causing enzyme inactivation. 29 

### Glutathione is a tripeptide antioxidant containing a cysteine with a reactive thiol group 30 

### that can act nonenzymatically as a direct antioxidant or as a cofactor in enzymatic 31 

### detoxification reactions by GST. Glutathione will donate an electron while in its reduced 32 

### state (GSH), which leads to conversion to the oxidized form, glutathione disulfide 33 

### (GSSG). Pb binds to the thiol and can both interfere with the antioxidant capacity of and 34February 2012 5

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### decrease levels of GSH. Short

- term administration of Pb in vitro (0.1 μ M) and 1 

### biomarkers of Pb exposure in humans (18 μ g/dL mean blood Pb level) have been 2 

### associated with decreased blood and organ GSH and cysteine content, which may be due 3 

### to increased GSH efflux from tissues ( Pandya et al., 2010 ; Pillai et al., 2010 ; Ahamed et 4 

### al., 2009 ; Ahamed et al., 2008 ; Flora et al., 2007 ; Ahamed et al., 2005 ; Chetty et al., 5 

### 2005 ; Nakagawa, 1991 , 1989 )

. Long

- term Pb exposure elicits a compensatory 6 

### upregulation in the biosynthesis of GSH in the attempt to overcome Pb toxicity, thus 7 

### often manifesting as an increase in Pb

- induced GSH ( Daggett et al., 1998 ; Corongiu and 8 

### Milia, 1982 ; Hsu, 1981 ; Conterato et al., In Press )

. However, other studies have found 9 

### that long

- term Pb exposure resulting in mean blood Pb levels between 6.6 and 22 μ g/dL, 10 

### causes the depletion of GSH ( Mohammad et al., 2008 ; Lee et al., 2005 ; Ercal et al., 11 

### 1996 )

. Thus, the time of exposure is important to consider when measuring GSH levels. 12


### Glutathione reductase is able to reduce GSSG back to GSH. Therefore, an increased 13 

### GSSG/GSH ratio is considered to be indicative of oxidative stress. Epidemiologic studies 14 

### have found higher blood Pb levels to be associated with increases in the GSSG/GSH ratio 15 

### ( Mohammad et al., 2008 ; Ercal et al., 1996 ; Sandhir and Gill, 1995 )

. In one study, this 16 

### association was observed in a population of children with a mean blood Pb level below 17 

### 10 μ g/dL ( Diouf et al., 2006 )

. Studies have found mixed effects on GR activation. GR 18 

### possesses a disulfide at its active site that is a target for inhibition by Pb. Studies in 19 

### animals and cells have reported decreased ( Bokara et al., 2009 ; Sandhir and Gill, 1995 ; 20 

### Sandhir et al., 1994 ) , increased ( Sobekova et al., 2009 ; Howard, 1974 ) , and no change 21 

### ( Hsu, 1981 ) in GR activity after Pb exposure. This could be because the effect of Pb on 22 

### GR varies depending on sex ( Sobekova et al., 2009 ) and o rgan or organ region ( Bokara et 23 

### al., 2009 )

. 24 

### GSH is used as a cofactor for peroxide reduction and detoxification of xenobiotics by the 25 

### enzymes GPx and GST. GPx requires selenium for peroxide decomposition ( Rotruck et 26 

### al., 1973 ) , whereas GST functions via a sulfhydryl group. By reducing the uptake of 27 

### selenium, depleting cellular GSH, and disrupting protein thiols, evidence indicates that 28 

### Pb decreases the activity of GPx and GST ( Pillai et al., 2010 ; Yu et al., 2008 ; Lee et al., 29 

### 2005 ; Nakagawa, 1991 ; Schrauzer, 1987 )

. Similar to other antioxidant enzymes, 30 

### compensatory upre gulation of these enzymes is described after Pb exposure in animals 31 

### and in Pb

- exposed workers (painters with a mean blood Pb level of 5.4 μ g/dL) ( Bokara et 32 

### al., 2009 ; Ergurhan

- Il han et al., 2008 ; Conterato et al., 2007 ; Daggett et al., 1998 ; 33 

### Conterato et al., In Press )

. However, in another study, these enzy mes were not able to 34 

### compensate for the increased Pb

- induced ROS, further contributing to the oxidative 35 

### environment ( Farmand et al., 2005 )

. 36February 2012 5

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### Recently, γ

- glutamyltran s ferase (GGT) within its normal range has been regarded as an 1 

### early and sensitive marker of oxidative stress. This may be because cellular GGT 2 

### metabolizes extracellular GSH to be used in intracellular GSH synthesis. Thus, cellular 3 

### GGT acts as an antioxidant enzyme by increasing the intracellular GSH pool. However, 4 

### the reasons for the association between GGT and oxidative stress have not been fully 5 

### realized ( Lee et al., 2004 )

. In one study, occupational Pb exposure (mean blood Pb level 6 

### of 29.1 μ g/dL) was associated with increased serum GGT levels ( Khan et al., 2008 )

. 7 

### Interestingly, higher blood Pb level was similarly associated with higher serum GGT 8 

### levels in a sample of the U.S. adult population (NHANES III) with lower blood Pb levels 9 

### ( Lee et al., 2006a )

. In this study of nonoccupationally

- exposed individuals, a 10 

### concentration

- dependent relationship was observed at blood Pb levels <7 μ g/dL. 11



### 5.2.4.5 Nitric Oxide Signali ng

• 

### NO, also known as endothelium

- derived relaxing factor, is a potent endogenous 12 

### signaling molecule involved in vasodilation. Short

- and long

- term Pb exposure in animals 13 

### decreases the biologically active • 

### NO, not through reduction in • 

### NO

- production capaci ty 14 

### ( Vaziri and Ding, 2001 ; Vaziri et al., 1999b ) , but as a result of inactivation and 15 

### sequestration of • 

### NO by ROS ( Malvezzi et al., 2001 ; Vaziri et al., 1999a )

. Endogenous 16

• 

### NO can interact with ROS, specifically O 2 - 

### , produced following exposure to Pb to form 17 

### the highly cytotoxic reactive nitrogen species, peroxynitrite (ONOO - 

### ). This re active 18 

### compound can damage cellular DNA and proteins, resulting in the formation of 19 

### nitrotyrosine among other products. Overabundance of nitrotyrosine in plasma and 20 

### tissues is present after exposure to Pb ( Vaziri et al., 1999a )

. • 

### NO is also produced by 21 

### macrophages in the defense against certain infectious agents, including bacteria. Studies 22 

### have indicated that Pb exposure can significantly reduce production of • 

### NO in immune 23 

### cells ( Pineda

- Zavaleta et al., 2004 ; Lee et al., 2001b ; Tian and Lawren ce, 1995 ) , possibly 24 

### leading to reduced host resistance ( Tian and Lawrence, 1996 )

. 25


### Production of • 

### NO is catalyzed by a family of enzymes called nitric oxide synthases 26 

### (NOS), including endothelial NOS (eNOS), neuronal NOS (nNOS) , and i nducible NOS 27 

### (iNOS), which require a heme prosthetic group and a zinc cation for enzymatic activity 28 

### ( Messerschmidt et al., 2001 )

. Paradoxically, the reduction in • 

### NO availability in vascular 29 

### tissue following Pb exposure is accompanied by statistically significant upregulation in 30 

### NOS isotypes ( Vaziri a nd Ding, 2001 ; Vaziri et al., 1999b ; Gonick et al., 1997 )

. A direct 31 

### inhibitory action of Pb on NOS enzymatic activity has been rejected ( Vaziri et al., 32 

### 1999b )

. Instead, the upregulation of NOS occurs as compensation for the decreased • 

### NO 33 

### resulting fro m ROS inactivation ( Vaziri et al., 2005 ; Vaziri and Ding, 2001 ; Vaziri and 34 

### Wang, 1999 )

. 35February 2012 5

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### Soluble Guanylate Synthase Many biological actions of • 

### NO, such as vasorelaxation, are mediated by cyclic guanosine 1 

### monophosphate (cGMP), which is produced by soluble guanylate cyclase (sGC) from the 2 

### substrate guanosine triphosphate. Soluble guanylate cyclase is a heterodimer requiring 3 

### one molecule of heme for enzymatic activity ( Boerrigter an d Burnett, 2009 )

. In VSMC, 4 

### sGC serves as the • 

### NO receptor. Marked reduction in plasma concentrations and urinary 5 

### excretion of cGMP is observed after Pb exposure to rats (5 ppm for 30 days and 100 ppm 6 

### for 12 months [resulting in a mean blood Pb level of 29.4 μ g/dL]) ( Marques et al., 2001 ; 7 

### Khalil

- Manesh et al., 1993b )

. In addition, Pb exposure downregulated the protein 8 

### abundance of sGC in vascular tissue ( Farmand et al., 2005 ; Courtois et al., 2003 ; 9 

### Marques et al., 2001 )

. This downregulation in sGC was prevented by antioxidant therapy 10 

### (ascorbic acid) suggesting that oxidative stress also plays a role in Pb

- induced 11 

### downregulation of sGC (no change in blood Pb level was observed after ascorbic acid 12 

### treatment) ( Marques et al., 2001 )

. 13



### 5.2.5 Inflammation
Misregulated inflammation represents one of the major hallmarks of Pb

- induced immune 14 

### effects. It is important to note that this can manifest in any tissue where immune cell 15 

### mobilization and tissue insult occurs. Enhanced inflammation and tissue damage occurs 16 

### through the modulation of inflammator y cell function and production of pro

17 

### inflammatory cytokines and metabolites. Overproduction of ROS and an apparent 18 

### depletion of antioxidant protective enzymes and factors ( e.g., selenium) accompany this 19 

### immunomodulation ( Chetty et al., 2005 )

. 20


### Traditional immune

- mediated inflammation can be seen with bronchial 21 

### hyperresponsiveness, asthma, and re spiratory infections associated with exposure to Pb. 22 

### But it is important to recognize that any tissue or organ can be affected by immune

23 

### mediated inflammatory dysfunction given the distribution of immune cells as both 24 

### permanent residents and infiltrating c ell populations ( Mudipalli, 2007 ; Carmignani et al., 25 

### 2000 )

. Pb spheres implanted in the brains of rats produced neu trophil

- driven 26 

### inflammation with apoptosis and indications of neurodegeneration ( Kibayashi et al., 27 

### 2010 )

. Pb also induces renal tubulointerstitial inflammation (mean blood Pb level of 28 

### 18 μ g/dL or 100 ppm exposure for 14 weeks) ( Rodriguez

- Iturbe et al., 2005 ; Ramesh et 29 

### al., 2001 ) , which has been coupled with activat ion of the redox sensitive nuclear 30 

### transcription factor kappa B (NFκB) and lymphocyte and macrophage infiltration in rats 31 

### (100 ppm for 14 weeks resulting in mean blood Pb levels ranging 23

- 27 μ g/dL,) ( Bravo 32 

### et al., 2007 )

. These events could be in response to the oxidative environment arising from 33February 2012 5

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### Pb exposure, since Pb

- induced inflammation and NFκB activation can be ameliorated by 1 

### antioxidant therapy ( Rodriguez

- Iturbe et al., 2004 )

. 2 

### Inflammation can be mediated by the production of chemical messengers such as 3 

### prostaglandins (PG). Pb exposure has been reported to increase arachidonic acid (AA) 4 

### metabolism, thus elevating the production of PGE 2 

### , PGF 2 

### , and thromboxane in 5 

### occupationally

- exposed humans (mean blood Pb level 48 μ g/dL) ( Cardenas et al., 1993 ) 6 

### and animal and cell models ( e.g., 0.01 μ M, 48 h) ( Chetty et al., 2005 ; Flohe et al., 2002 ; 7 

### Knowles and Donaldson, 1997 ; Lee and Battles, 1994 )

. Dietary Pb supplementation of 8 

### animals (500 ppm , 19 days) can increase the percentage of cell membrane AA, the 9 

### precursor of cyclooxygenase and lipoxygenase metabolism to PGs and leukotrienes 10 

### ( Knowles and Donaldson, 1990 )

. Additionally, Pb (1 μ M) may promote the release of 11 

### AA via activation of phospholipase A 2 

### , as shown in isolated VSMC ( Dorman and 12 

### Freeman, 2002 )

. 13 

### Inflammation may be the result of increased pro

- inflammatory signaling or may stimulate 14 

### these signaling pathways. Pb can elevate the expression of the pro

- inflammatory 15 

### transcription factors NFκB and activator protein

- 1 (AP

- 1), as well as the AP

- 1 16 

### component c

- Jun ( Korashy and El

- Kadi, 2008 ; Korashy and Ei

- Kadi, 2008 ; Bravo et al., 17 

### 2007 ; Ramesh et al., 1999 ; Pyatt et al., 1996 )

. Pb exposure (25 μ M) to dendritic cells 18 

### stimulated phosphorylation of the Erk/MAPK pathway, but not p38, STAT3 or 5, or 19 

### CREB ( Gao et al., 2007 ) 20



### 5.2.5.1 Cytokine Production

There are t hree modes by which Pb affects immune cytokine production. First, Pb can act 21 

### on macrophages to elevate the production of pro

- inflammatory cytokines such as TNF

- α 22 

### and interleukin (IL)

- 6 ( Cheng et al., 2006 ; Chen et al., 1999 ; Dentener et al., 1989 )

. This 23 

### can result in local tissue damage during the course of immune responses affecting such 24 

### targets as the liver. Second, when Pb acts on dendritic cells, it skews the ratio of IL

25 

### 12/IL

- 10 such that T

- derived lymphocyte helper (Th)1 responses are suppressed and Th2 26 

### responses are promoted ( Chen et al., 2004 ; Mille r et al., 1998 )

. Third, when acquired 27 

### immune responses occur following exposure to Pb, Th1 lymphocyte production of 28 

### cytokines is suppressed ( e.g., IFN

- γ) ( Lynes et al., 2006 ; Heo et al., 1996 )

. In contrast, 29 

### Th2 cytokines such as IL

- 4, IL

- 5, and IL

- 6 are elevated ( Gao et al., 2007 ; Kim and 30 

### Lawrence, 2000 )

. The combination of these three modes of cytokine changes induced by 31 

### Pb creates a hyperinflammatory state among innate immune cells and acquired immunity 32 

### is skewed toward Th2 responses. 33February 2012 5

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### Iavicoli et al. ( 2006a ) reported that low blood Pb concentrations produced significant 1 

### changes in cytokine levels in mice. At a low dietary Pb concentration (0.11 ppm , blood 2 

### Pb level of 1.6 μ g/dL), IL

- 2 and IFN

- γ were decreased compared to the controls 3 

### (0.02 ppm , 0.8 μ g/dL), indicating a suppressed Th1 response. As the dietary and blood Pb 4 

### concentrations increased (resulting in blood Pb levels 12

- 61 μ g/dL), a Th2 phenot ype 5 

### was observed with suppressed IFN

- γ and IL

- 2 and elevated IL

- 4 production. These 6 

### findings support the notion that the immune system is remarkably sensitive to Pb

- induced 7 

### functional alterations and that nonlinear effects may occur at low Pb exposures. TG F

- β 8 

### production is also altered by Pb exposure to cells (1 μ M, 3 days) ( Zuscik et al., 2007 )

. IL

9 

### 2 is one of the more variable cytokines with respect to Pb

- induced changes. Depending 10 

### upon the protocol it can be slightly elevated in production or unchanged. Recently, Gao et 11 

### al. ( 2007 ) found that Pb

- treated dendritic cells (25 μ M) promoted a slight but statistically 12 

### significant increase in IL

- 2 production among lymphocytes. Proinflammatory cytokines 13 

### have been measured in other organs and cell types after Pb exposure. Elevation of IL

- 1β 14 

### and TNF

- α were observed in the hippocampus after Pb exposure (15 ppm , i

. p
. , daily for 2 15 

### weeks, blood Pb level of 30.8 μ g/dL) and increased IL

- 6 was found in the forebrain 16 

### ( Struzynska et al., 2007 )

. 17


### Consistent with animal stud ies, epidemiologic studies also found higher concurrent blood 18 

### Pb levels in children and occupationally

- exposed adults to be associated with a shift 19 

### toward production of Th2 cytokines relative to Th1 cytokines. The evidence in children 20 

### was based on comparis ons of serum cytokine levels among groups with different blood 21 

### Pb levels without consideration of potential confounding factors. Among children ages 9 22 

### months to 6 years in Missouri, Lutz et al. ( 1999 ) found that children with concurrent 23 

### blood Pb levels 15

- 19 μ g/dL had higher serum levels of IL

- 4 and IgE ( Section 5.6.3 ) than 24 

### did children with lower blood Pb levels. These results were consistent with the mode of 25 

### action for IL

- 4 to activate B cells to induce B cell class switching to IgE. In another study 26 

### of children in grades 5 and 6 in Taiwan, investigators did not group children by blood Pb 27 

### levels but by potential for Pb exposures du e to age of home and location of residence 28 

### ( Hsiao et al., 2011 )

. Concurrent blood Pb levels did not differ by residence in old versus 29 

### new homes or by urban versus rural residence (means: 3.2

- 3.8 μ g/dL) but were higher 30 

### among children living near an oil refinery, in particular, among children with known 31 

### respiratory allergies (mean: 8.8 μ g/dL). This latter group of children also had the lowest 32 

### serum levels of IFN

- γ and highest levels of IL

- 4. There was no direct comparison of 33 

### cytokine levels between blood Pb level groups in the population overall; however, 34 

### cytokine levels were similar between healthy and allergy groups in the other Pb source 35 

### groups that had similar blood Pb levels. Thus, the differences in cytokine levels between 36 

### healthy and allergic children living near the oil refinery may have been influenced by 37 

### differences in their blood Pb levels. 38February 2012 5

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### Evidence of association between blood Pb levels and cytokine levels in 1 

### nonoccupationally

- exposed adults was equivocal. Among adult university students in 2 

### Incheon, Kor ea, Kim et al. ( 2007 ) found associations of concurrent blood Pb level with 3 

### serum levels of TNF

- α and IL

- 6 that were larger among male students with blood Pb 4 

### le vels 2.51

- 10.47 μ g/dL. Notably, the relative contributions of lower recent versus higher 5 

### past Pb exposures to these effects is not known. In models that adjusted for age, sex, 6 

### BMI, and smoking status, a 1 μ g/dL increase in blood Pb level was associated wit h a 7 

### 23% increase (95% CI: 4, 55%) in log of TNF

- α and a 26% increase in log of IL

- 6 (95% 8 

### CI: 0, 55%). The association between levels of blood Pb and plasma TNF

- α was greater 9 

### among men who were GSTM1 null or had the TNF

- α GG genotype. For the association 10 

### be tween blood Pb level and plasma IL

- 6, the effect estimate was slightly elevated in 11 

### TNF

- α GG genotype but not elevated in the GSTM1 positive group. The effects of Pb on 12 

### several physiological systems have been hypothesized to be mediated by the generation 13 

### of ROS ( Daggett et al., 1998 )

. Thus, the null variant of GSTM1, which is associated with 14 

### reduced elimination of ROS, may increase the risk of Pb

- associated immune effects. The 15 

### results for the TNF

- α polymorphism were difficult to interpret. The GG gen otype is 16 

### associated with lower expression of TNF

- α, and the literature is mixed with respect to 17 

### which variant increases risk of inflammation

- related conditions. Among adults in Italy, 18 

### blood Pb levels were not correlated with either Th2 or Th1 cytokine leve ls in men 19 

### ( Boscolo et al., 1999 ) or women ( Boscolo et al., 2000 ) 20 

### Results from studies of occupationally

- exposed adults also suggested that Pb exposure 21 

### may be associated with decreases in Th1 cytokines and increases in Th2 cytokines; 22 

### howev er, analyses were mostly limited to comparisons of levels among different 23 

### occupational groups with different mean blood Pb levels ( Di Lorenzo et al., 2007 ; 24 

### Valentino et al., 2007 ; Yucesoy et al., 1997a )

. The exception was a study of male f oundry 25 

### workers, pottery workers, and unexposed workers by Valentino et al. ( 2007 )

. Multiple 26 

### regression analyses were performed with age, BMI, smoking, and alcohol consumption 27 

### included as covariates. Although information on concentration

- response relationships 28 

### was not provided, higher blood Pb level was associated with higher IL

- 10 and TNF

- α. 29 

### Levels of IL

- 2, IL

- 6, and IL

- 10 also increased from the lowest to highest blood Pb group. 30 

### In contrast with most other studies, both exposed worker groups had lower IL

- 4 levels 31 

### compared with controls. In a similar analysis, DiLorenzo et al. ( 2007 ) separated exposed 32 

### workers into intermediate (9.1

- 29.4 μ g/dL) and high (29.4

- 81.1 μ g/dL) blood Pb level 33 

### groups, with unexposed workers comprising the low exposure group (blo od Pb levels 1

- 34 

### 11 μ g/dL). Mean TNF

- α levels showed a monotonic increase from the low to high blood 35 

### Pb group. Levels of granulocyte colony

- stimulating factor (G

- CSF) did not differ 36 

### between the intermediate and high blood Pb groups among the Pb recyclers; ho wever, G

37 

### CSF levels were higher in the Pb recyclers than in the unexposed controls. Furthermore, 38 

### among all subjects, blood Pb showed a strong, positive correlation with G

- CSF. Yucesoy 39February 2012 5

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### et al. ( 1997a ) found lower serum levels of the Th1 cytokines, IL

- 1β and IFN

- γ, in 1 

### workers ( mean blood Pb level of 59.4 μ g/dL) compared with controls (mean blood Pb 2 

### level of 4.8 μ g/dL); however levels of the Th2 cytokines, IL

- 2 and TNF

- α levels, were 3 

### similar between groups. As most occupationally

- exposed cohorts represent populations 4 

### highly expos ed to Pb, with mean blood Pb levels >22 μ g/dL, effects observed within 5 

### these cohorts may not be generalizable to the population as a whole. However, animal, 6 

### general population, and occupational studies suggest that exposure to Pb increases the 7 

### production o f pro

- inflammatory cytokines, skews the ratio of Th1 and Th2 cytokines to 8 

### favor Th2 responses, and suppresses lymphocyte cytokine production. 9



### 5.2.6 Endocrine Disruption

5.2.6.1 Hypothalamic
- Pituitary
- Gonadal Axis
Pb is a potent endocrine disrupting chemical found to be associated with reproductive and 10 

### developmental effects in both male and female animal models. Pb may act both at 11 

### multiple points along the hypothalamic

- pituitary

- gonadal (HPG) axis and directly at 12 

### gonadal sites. The HPG axis functions in a closely regulate d manner to produce 13 

### circulating sex steroids and growth factors required for normal growth and development. 14 

### Long

- term Pb exposure in animals has been shown to reduce serum levels of follicle

15 

### stimulating hormone (FSH), luteinizing hormone (LH), testosterone , and estradiol 16 

### ( Biswas and Ghosh, 2006 ; Rubio et al., 2006 )

. Similar changes in serum HPG hormones 17 

### have been observed after high

- level Pb exposure in animals, resulting in blood Pb levels 18


### > 20 μ g/dL ( Dearth et al., 2002 ; Ronis et al., 1998b ; Foster, 1992 ; Sokol and Berman, 19 

### 1991 )

. Increases in LH and FSH have been associated with increasing concurrent blood 20 

### Pb levels in adult women from the NHANES cohort ( Krieg, 2007 )

. The change in HPG 21 

### hormones likely occurs through the inhibition of LH secretion and the reduction in the 22 

### expression of the steroidogenic acute regulatory protein (StAR) ( Huang and Liu, 2004 ; 23 

### Srivastava et al., 2004 ; Huang et al., 2002 ; Ronis et al., 1996 )

. StAR expression is the 24 

### rate

- limiting step essential in maintaining gonadotropin

- stimulated steroidogenesis, 25 

### which results in the formation of testosterone and estradiol. Prenatal Pb exposure 26 

### (resulting in 3 μ g/dL bloo d Pb) was found to decrease basal StAR synthesis, but not 27 

### gonadotropin

- stimulated StAR synthesis, suggesting that Pb may not directly affect 28 

### ovarian responsiveness to gonadotropin stimulation ( Srivastava et al., 2004 )

. Instead, Pb 29 

### may act at the hypothalamic

- pituitary level to alter LH secretion, which is necessary to 30 

### drive StAR production and subsequent sex hormone synthesis. Release of LH and FSH 31 

### from the pituitary is controlled by gonado tropin

- releasing hormone (GnRH). Pb exposure 32 

### (10 μ M, 90 min) in rat brain median eminence cells can block GnRH release ( Bratton et 33February 2012 5

- 34 Draft

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### al., 1994 )

. Pb may also interfere with release of pituitary hormones through interference 1 

### with cation

- dependent secondary messenger systems , which mediate hormone release and 2 

### storage. 3 

### Endocrine disruption may also be a result of altered hormone binding to endocrine 4 

### receptors. Prenatal and postnatal Pb exposure (20 ppm ) to rats was able to decrease the 5 

### number of estrogen, LH, and FSH receptors found in the uterus or ovaries and receptor 6 

### binding affinity ( Wiebe et al., 1988 ; Wiebe and Barr, 1988 )

. Altered hormone binding 7 

### ability may be due to the ion binding properties of Pb, resulting in changes in receptor 8 

### tertiary structure that will disrupt ligand binding. In addition, Pb

- induc ed changes in 9 

### hormone levels that act as inducing agents for receptor synthesis may affect the number 10 

### of hormone receptors produced. 11 

### Some of these endocrine disrupting effects of Pb have been related to the generation of 12 

### ROS. Treatment with antioxidants i s able to counteract a number of the endocrine 13 

### disrupting effects of Pb, including apoptosis and decreased sperm motility and production 14 

### ( Salawu et al., 2009 ; Shan et al., 2009 ; Madhavi et al., 2007 ; Rubio et al., 2006 ; Wang et 15 

### al., 2006a ; Hsu et al., 1998a )

. Direct generation of ROS in epididymal spermatozoa was 16 

### observed after Pb exposure in rats (i.p. 20 or 50 ppm , 6 weeks) ( Hsu et al., 1998b )

. In 17 

### addition, lipid peroxidation has been observed in Pb

- exposed rats (i.p. 0.025 ppm , 15 18 

### days) ( Pandya et al., In Press )

. Lipid peroxidation in the seminal plasma was significantly 19 

### increased in a group of Pb

- exposed workers with high blood Pb levels (>40 μ g/dL) 20 

### ( Kasperc zyk et al., 2008 )

. 21 

### The liver is often associated with the HPG axis due in part to its production of insulin

22 

### like growth factor 1 (IGF

- 1). Children with increased blood Pb levels (>4 μ g/dL) 23 

### ( Huseman et al., 1992 ) and Pb

- exposed animals (blood Pb level of 14 μ g /dL) ( Pine et al., 24 

### 2006 ; Dearth et al., 2002 ) and gonada l cells (50 ppm Pb exposure) ( Kolesarova et al., 25 

### 2010 ) show a decrease in plasma IGF

- 1, which may be the result of decreased translation 26 

### or secretion of IGF

- 1 ( Dearth et al., 2002 )

. IGF

- 1 also induces LH

- releasing hormone 27 

### release, such that IGF

- 1 decrements may explain decreased LH and estradiol levels. IGF

28 

### 1 production is stimulated by growth hormone (GH) secreted from the pituitary gland and 29



### cou ld be the result of GH depletion. 30 

### A number of studies have revealed that Pb exposure affects the dynamics of growth. 31 

### Decreased growth after Pb exposure could be the result of Pb

- induced decreased GH 32 

### levels ( Berry et al., 2002 ; Camoratto et al., 1993 ; Huseman et al., 1992 ; Huseman et al., 33 

### 1987 )

. This decrease in GH could be a result of decreased release of GH releasing 34 

### hormone (GHRH) from the hypothalamus or disrupted GHRH binding to its receptor, 35 

### which has been reported in vitro after Pb treatment (IC 50 

### free Pb in solution 5.2x10 - 5 

### μ M, 36February 2012 5

- 35 Draft

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### 30 minute s) ( Lau et al., 1991 )

. GH secretion may also be altered from decreased 1 

### testost erone, a result of Pb exposure. 2



### 5.2.6.2 Hypothalamic
- Pituitary
- Thyroid Axis
The effects of Pb on the hypothalamic

- pituitary

- thyroid (HPT) axis are mixed. Pb 3 

### exposure impacts a variety of players in the thyroid hormone system. A number of human 4 

### studies (blood Pb l evels >7.3 μ g/dL) have shown that elevated blood Pb are associated 5 

### with lower thyroxine (T 4 

### ) and free T 4 

### levels without alteration in triiodothyronine (T 3 

### ), 6 

### suggesting that long

- term Pb exposure may depress thyroid function in workers ( Dundar 7 

### et al., 2006 ; Tuppurainen et al., 1988 ; Robins et al., 1983 )

. However, animal studies on 8 

### thyroid hormones have shown mixed results. Pb

- exposed cows (blood Pb levels 9 

### >51 μ g/dL) were reported to have an increase in plasma T 3 

### and T 4 

### levels ( Swarup et al., 10 

### 2007 ) , whereas mice and chickens manifested decreased serum T 3 

### concentrations after Pb 11 

### exposure accompanied by increased lipid peroxidation ( Chaurasia et al., 1998 ; Chaurasia 12 

### and Kar, 1997 )

. Decreased serum T 3 

### and increased l ipid peroxidation were both restored 13 

### by vitamin E treatment, suggesting the disruption of thyroid hormone homeostasis could 14 

### be a result of altered membrane architecture and oxidative stress; however, no data were 15 

### provided to exclude changes in Pb kinetics as the mechanism of protection ( Chaurasia 16 

### and Kar, 1997 )

. 17


### Decreased T 4 

### and T 3 

### may be the result of altered pituitary release of thyroid stimulating 18 

### hormone (TSH). However, several studies have reported higher TSH levels in high

- level 19 

### Pb

- exposed workers (blood Pb levels >39 μ g/dL) ( Lopez et al., 2000 ; Singh et al., 2000 ; 20 

### Gustafson et al., 1989 ) , which would result in increased T 4 

### levels. Overall, results on the 21 

### effects of Pb on the HPT axis are inconclusive. 22



### 5.2.7 Cell Death and Genotoxicity
A number of studies have attempted to characterize the genotoxicity of inorganic Pb in 23 

### human populations, laboratory animals, and cell cultures. Endpoints investigated include 24 

### DNA damage (single

- and double

- strand breaks, DNA

- adduct formation), mutagenicity, 25 

### clastogenicity (sister chromatid exchange, micronucleus formation, chromosomal 26 

### aberrations), and epigenetic changes (changes in gene expressio n, mitogenesis). It is 27 

### important to note that numerous studies have utilized exposure to Pb chromate to 28 

### investigate genotoxicity endpoints; some studies have specifically attributed the observed 29 

### increases in DNA damage and clastogenicity to the chromate io n while others have not. 30 

### Due to the uncertainty regarding whether observed genotoxic effects are due to chromate 31February 2012 5

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### or Pb in studies using this form of inorganic Pb, only studies utilizing other forms of 1 

### inorganic Pb ( e.g., Pb nitrate, acetate) are discussed below. 2



### 5.2.7.1 DNA Damage
A number of studies in human populations have observed associations between Pb 3 

### exposure and increased DNA damage, as measured as DNA strand breaks. Most of these 4 

### associations have been observed in occupationally

- exposed populations ( Grover et al., 5 

### 2010 ; Minozzo et al., 2010 ; Shaik and Jamil, 2009 ; Danadevi et al., 2003 ; Hengstler et 6 

### al., 2003 ; Palus et al., 2003 ; Fraca sso et al., 2002 ; de Restrepo et al., 2000 )

. It is 7 

### important to note that occupationally

- exposed adults have very high blood Pb levels, and 8 

### in one study ( de Restrepo et al., 2000 ) the association between blood Pb level and DNA 9 

### damage was observed only in workers with blood Pb levels greater than 120 μ g/dL. Also, 10 

### the stud ies were equivocal in regard to how blood Pb levels correlated with DNA 11 

### damage: Fracasso et al. ( 2002 ) observed that DNA damage increased with increasing 12 

### blood Pb levels (blood Pb levels, <25, 25

- 35, and >35 μ g/dL), whereas Pa lus et al. ( 2003 ) 13 

### (mean blood Pb level: 50.4 μ g/dL [range: 28.2

- 65.5 μ g/dL]) and Minozzo et al. ( 2010 ) 14 

### (mean [SD]: 5 9.43 μ g/dL [28.34]) observed no correlation. Lastly, Pb

- exposed workers 15 

### are also potentially exposed to other genotoxic materials, making it difficult to rule out 16 

### confounding co

- exposures. However, Hengstler et al. ( 2003 ) examined workers exposed 17 

### to Pb, cadmium, and cobalt and observed that neither blood (mean: 4.4 [IQR: 2.84

- 18 

### 13.6] μ g/dL) nor air Pb levels (mean: 3.0 [IQR: 1.6

- 50.0] μ g/m 3 

### ) were associated with 19 

### DNA damage when examined alone, but that blood Pb influenced the occurrence of 20 

### single strand DNA breaks when included in a multiple regression model along with 21 

### cadmium in air and blood and cobalt in air indicating lack of confounding. Two studies 22 

### were found that investigated Pb

- induced DNA damage resulting from nonoccupational 23 

### exposures. Mendez

- Gomez ( 2008 ) observed that children living at close and intermediate 24 

### distances to a Pb smelter had mean (range) blood Pb levels of 1 9.5 (11.3

- 49.2) and 28.6 25 

### (11.4

- 47.5) μ g/dL, respectively, compared to blood Pb level of 4.6 (0.1

- 8.7) μ g/dL for 26 

### children living distant to the smelter. DNA damage was increased in children living 27 

### nearest to the smelter, compared to the children at the inte rmediate distance, but was not 28 

### different from children living farthest away from the smelter. Multivariate analysis 29 

### (which considered children urinary As levels, highest in children farthest from the 30 

### smelter), revealed no statistically significant associat ions between DNA damage and 31 

### blood Pb level. Further, DNA repair ability was also observed to be unrelated to blood Pb 32 

### levels. Alternatively, Yanez et al. ( 2003 ) observed that children living close to a mining 33 

### complex (mean [range] blood Pb level: 11.6 [3.0 to 19.5] μ g/dL) did have higher levels 34February 2012 5

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– Do Not Cite or Quote 

### of DNA damage compared to control children who lived further away from the mining 1 

### facility (mean [range] blood Pb level: 8.3 [3.0

- 25.0] μ g/dL). 2 

### In mice given 0.7 to 89.6 mg /kg Pb nitrate by gavage for 24, 48, or 72 hours, or 1 or 2 3 

### weeks, single strand DNA breaks in white blood cells were observed but did not increase 4 

### with increasing concentration ( Devi et al., 2000 )

. The three highest concentrations had 5 

### responses which were similar in magnitude and were actually lower than the re sponses to 6 

### lower concentrations tested. In mice exposed to Pb (blood Pb level of 0.68 μg/dL) via 7 

### inhalation for up to 4 weeks, differential levels of DNA damage were observed in 8 

### different organ systems, with only the lung and the liver demonstrating statis tically 9 

### greater DNA damage compared to the respective organ controls after acute exposure 10 

### ( Valverde et al., 2002 )

. Statistically elevated levels of DNA damage were observed in the 11 

### kidneys, lungs, liver, brain, nasal cavity, bone marrow, and leukocytes of mice exposed 12 

### over a period of 4 weeks, although variability was high in all groups. The magnitude of 13 

### the DNA damage was characterized as weak and did not increase with increasing 14 

### durations of exposure. Xu et al. ( 2008 ) exposed mice to 10

- 100 mg /kg Pb

- acetate via 15 

### gavage for four weeks and observed a concentration

- dependent increase in DNA single 16 

### strand breaks in white blood cells that was statistically significant at 50 and 100 mg /kg. 17 

### The authors characterized the observed DNA damage as severe. Pb nitrate induced DNA 18 

### damage in primary spermatozoa in Pb

- exposed rats (blood Pb levels of 19.5 and 19 

### 21.9 μ g/dL) compared to control rats ( Nava

- Hernandez et al., 2009 )

. The level of DNA 20 

### damage was not concentration dependent and was comparable in both exposure groups. 21 

### Narayana and Al

- Bader ( 2011 ) observed no increase in DNA damage in the livers of rats 22 

### exposed to 0.5 or 1% Pb nitrate in drinking water for 60 days. Interestingly, although the 23 

### results were not statistically significant and highly variable within exposure groups, DNA 24 

### fragmentation appeared to be lower in the exposed animals. 25 

### Studies investigating Pb

- induced DNA damage in human cell cultures were 26 

### contradictory. Pb

- acetate did not induce DNA strand breaks in human HeLa cells when 27 

### exposed to 500 μ M Pb

- acetate for 20

- 25 hours or 100 μ M for 0.5

- 4 hours ( Hartwig et al., 28 

### 1990 ; Snyder and Lachmann, 198 9 )

. Pb nitrate, administered to lymphoma cells at 1000

- 29 

### 10,000 μ M for 6 hours, did not result in any DNA

- protein crosslinks ( Costa et al., 1 996 )

. 30 

### Pb

- acetate was observed by Woźniak and Blasiak ( 2003 ) to result in DNA single and 31 

### double strand breaks in primary human lymphocytes exposed to 1

- 100 μ M for 1 hour, 32 

### although the pattern of damage was peculiar. DNA damage was greater in cells exposed 33 

### to 1 or 10 μ M, compared to those exposed to 100 μ M. DNA

- protein crosslinks were only 34 

### observed in the 100 μ M exposure group, suggesting that the decreased strand breaks 35 

### observed in the high exposure group may be a result of increased crosslinking in this 36 

### group. Pasha Shai k et al. ( 2006 ) also observed DNA damage in human lymphocytes 37 

### exposed to 2 , 100

- 3 , 300 μ M Pb nitrate for 2 hours. Although there was a concentration

- 38February 2012 5

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### dependent increase in DNA dama ge from 2 , 100

- 3 , 300 μ M, no statistics were reported 1 

### and no unexposed control group was included making it difficult to interpret these results. 2 

### Gastaldo et al. ( 2007 ) observed that exposure of human endothelial cells to 1

- 1 , 000 μM 3 

### Pb nitrate for 24 hours resulted in a concentration

- dependent increase in DNA double 4 

### strand breaks. 5 

### Studies in animal cell lines were equally as ambiguous as th ose using human cell lines. 6 

### Zelikoff et al. ( 1988 ) and Roy and Rossman ( 1992 ) reported that Pb

- acetate 7 

### (concentration not stated and 1 , 000 μM, respectively) did not induce si ngle or double 8 

### DNA strand breaks or DNA

- protein or DNA

- DNA crosslinks in CHV79 cells. However, 9 

### both Xu et al. ( 2006 ) and Kermani et al. ( 2008 ) reported Pb

- acetate

- induced DNA 10 

### damage in P C12 cells exposed to 0.1, 1, or 10 μ M for 24 hours and in bone marrow 11 

### mesenchymal stem cells exposed to 60 μ M for 48 hours, respectively. Wedrychowski et 12 

### al. ( 1986 ) repo rted that DNA

- protein crosslinks were induced in a 13 

### concentration

- dependent manner in hepatoma cells exposed to 50

- 5 , 00 0 μ M Pb nitrate 14 

### for 4 hours. Pb

- acetate and Pb nitrate increased the incidence of nick translation in 15 

### CHV79 cells when a bacterial DNA pol ymerase was added. 16


### Pb

- acetate did not induce single strand DNA breaks in HeLa cells exposed to 500 μM for 17 

### 20

- 25 hours ( Hartwig et al., 1990 )

. However, exposure to both Pb

- acetate and UV light 18 

### resulted in increased persistence of UV

- induced strand breaks, compared to exposure to 19 

### UV light alone. Similar effects were seen in hamster V79 cells: UV

- induced mutation 20 

### rates and SCE frequency was exacerbated by co

- incubation with Pb

- acetat e . Taken 21 

### together, these data suggest that Pb

- acetate interferes with normal DNA repair 22 

### mechanisms triggered by UV exposure alone. Pb nitrate was observed to affect different 23 

### DNA double strand break repair pathways in human endothelial cells exposed to 100 μM 24 

### for 24 hours. Exposure to Pb inhibited nonhomologous end joining repair, but increased 25 

### two other repair pathways, MRE11

- dependent and Rad51

- related repair ( Gastaldo et al., 26 

### 2007 )

. Interestingly, in contrast to the above studies, exposure of lung carcinoma cells to 27 

### 100, 300, or 500 μM Pb

- acetate for 24 hours resulted in an increase in nucleotide excision 28 

### repair efficiency ( Li et al., 2008a )

. Roy and Rossman ( 1992 ) observed an increase in UV

29 

### induce d mutagenicity when CHV79 cells were co

- exposed to 400 μM Pb

- acetate (a 30 

### nonmutagenic concentration of Pb

- acetate ), indicating an inhibition of DNA repair. 31 

### Treatment of Chinese hamster ovary cells to 0.5

- 500 μM Pb

- acetate resulted in a 32 

### concentration

- depende nt accumulation of apurinic/apyrimidinic site incision activity, 33 

### indicating that DNA repair was diminished ( McNeill et al., 2007 )

. 34February 2012 5

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### 5.2.7.2 Mutagenicity
Only one human study was found that investigated Pb

- induced mutagenicity. Van 1 

### Larebeke et al. ( 2004 ) investigated the frequency of mutations in the hypoxanthine 2 

### phosphoribosyltransferase (HPRT) gene in Flemish women without occupational 3 

### exposures to Pb or a number of other heavy metals and organic contaminants. Higher 4 

### blood Pb level (range 1.6

- 5.2 μ g/dL) was associated with greater HPRT mutation 5 

### frequency in the total population. Also, women with high blood Pb levels ( i.e., greater 6 

### than the population median, not reported) demonstrated a greater mutation frequen cy 7 

### compared to women with lower blood Pb levels. 8


### Pb

- induced mutagenicity was investigated in four studies using human cell cultures. Ye 9 

### ( 1993 ) exposed human keratinocytes to 0.1

- 100 μ M/mL Pb

- acetate for 2

- 24 hours. This 10 

### study did not measure HPRT mutati ons directly, but rather measured the amount of 11 

### tritium incorporated into DNA as an indicator of mutation. In the presence of 6

- 12 

### thioguanine, tritium incorporation was increased in exposed cells, indicating weak 13 

### mutagenicity. Hwua and Yang ( 1998 ) reported that Pb

- acetate was not mutagenic in 14 

### human foreskin fibroblast s exposed to 500

- 2 , 000 μ M for 24 hours. Pb

- acetate remained 15 

### nonmutagenic in the presence of 3

- aminotriazole, a catalase inhibitor, indicating that 16 

### oxidative metabolism did not play a part in potential mutagenicity of Pb. Exposure to 17 

### Pb

- acetate alone did no t induce mutagenicity in lung carcinoma cells (100

- 500 μ M for 24 18 

### hours) or fibroblasts (300

- 500 μ M for 24 hours) ( Li et al., 2008a ; Wang et al., 2008c )

. 19 

### However, pretreatment with PKC inhibitors before Pb treatment did result in statistically 20 

### significant increases in mutagenicity in both cell lines. 21 

### Results from investigations into Pb

- induced mutagenicity using animal cell lines were as 22 

### equivocal as were the findings from human cell line studies, although the mixed find ings 23 

### may be reflective of specific Pb compounds used. Pb

- acetate was observed to be 24 

### nonmutagenic (HPRT assay) in Chinese hamster V79 cells exposed to 1

- 25 μ M of the 25 

### compound for 24 hours ( Hartwig et al., 1990 ) , but elicited a mutagenic response in 26 

### CHV79 cells (gpt assay) exposed to 1 , 700 μ M for 5 days ( Roy and Rossman, 1992 )

. 27 

### Pb

- acetate was observ ed to be nonmutagenic (HPRT assay) in Chinese hamster ovary 28 

### cells exposed to 5 μM for 6 hours ( McNeill et al., 2007 )

. The observation of mutagenicity 29 

### in the second study is complicated by the concurrent observation of severe cytotoxicity at 30 

### the sam e concentration. Pb nitrate was alternatively found to be nonmutagenic in CHV79 31 

### cells (gpt assay) exposed to 0.5

- 2 , 000 μ M for 5 days ( Roy and Rossman, 1992 ) , but 32 

### mutagenic in the same cell line (HPRT assay) exposed to 50

- 5 , 000 μ M for 5 days 33 

### ( Zelikoff et al., 1988 )

. However, mutagenicity was only observed at 500 μ M, and was 34 

### higher than that observed at higher concentrations. Pb sulfate was also observed to be 35 

### mutag enic in CHV79 cells (HPRT assay) exposed to 100

- 1 , 000 μ M for 24 hours, but as 36February 2012 5

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### with Pb nitrate, it was not concentration

- dependent ( Zelikoff et al., 1988 )

. Pb chloride 1 

### was the only Pb compound tested in animal cell lines that was consistently mutagenic: 2 

### three studies from the same laboratory observed concentration

- dependent mutagenicity in 3 

### the gpt assay in Chinese hamster ovary cells exposed to 0.1

- 1 μ M Pb chloride for one 4 

### hour ( Ariza and Williams, 1999 ; Ariza et al., 1998 ; Ariza and Williams, 1996 )

. 5



### 5.2.7.3 Clastogenicity
Clastogenicity is the ability of a compound to induce chromosomal damage, and is 6 

### commonly observed as sister chromatid exchange, micronuclei formation, or incidence of 7 

### chromosomal aberrations ( i.e., breaks or gaps in chromosomes). The potential for Pb to 8 

### be clastogenic has been investigated in numerous studies as described below. 9



### Sister Chromatid Exchange An association between blood Pb levels (means: 10.48

- 86.9 μ g/dL ) and sister chromatid 10



### exc hange (SCE) was observed in a number of occupational studies ( Wiwanitkit et al., 11 

### 2008 ; Duydu et al., 2005 ; Palus et al., 2003 ; Duydu et al., 2001 ; Pinto et al., 2000 ; 12 

### Bilban, 1998 ; Anwar and Kamal, 1988 ; Huang et al., 1988 )

. However, there are 13 

### numerous methodological issues that limit firm conclusions from being drawn. Most 14 

### notably, occupational co

- exposures to other genotoxic materials were possible, although 15 

### some studies excluded workers with exposures to known mutagens ( Pinto et al., 2000 ; 16 

### Huang et al., 1988 )

. In most studies that attempted to investigate the concentration

17 

### response relationship in workers, no association was observed between increasing blood 18 

### Pb levels and the number of SCE ( Palus et al., 2003 ; Duyd u et al., 2001 ; Pinto et al., 19 

### 2000 )

. However, Huang et al. ( 1988 ) did observe increased SCE in exposed workers in 20 

### the two highest blood Pb groups (52.1 and 86.9 μ g/dL), with a statistically significant 21 

### association observed in the 86.9 μ g/dL group. Pinto et al. ( 2000 ) did report an association 22 

### with duration of exposure (range of years exposed: 1.6

- 40). Two studies reported no 23 

### correlati on between occupational exposure to Pb and number of SCE ( Rajah and Ahuj a, 24 

### 1996 ; Rajah and Ahuja, 1995 )

. However, these two studies may have suffered from 25 

### limited statistical pow er to observe an effect as they included very small numbers of Pb

26 

### exposed workers. Mielzynska et al. ( 2006 ) found no association between blood Pb level 27 

### and SCEs in children in Poland. Children had an average blood Pb level of 7.69 μ g/dL 28 

### and 7.87 SCEs/cell. 29 

### Pb exposure has been observed to induce SCEs in multiple laboratory animal studies. In 30 

### mice exposed to up to 100 mg /kg Pb

- acetate i.p., Pb induced SCEs with 50 and 31 

### 100 mg /kg ( Fahmy, 1999 )

. Pb nitrate, also administered i.p. and induced the formation of 32February 2012 5

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### SCEs in a concentration

- dependent manner (10

- 40 mg / kg) in the bone marrow of 1 

### exposed mice ( Dhir et al., 1993 )

. Nayak et al. ( 1989b ) exposed pregnant mice to 100

- 2 

### 200 mg /kg Pb nitrate via i.v. injection and observed an increase in SCEs in dams at 150 3 

### and 200 mg /kg; no SCEs were observed in the fetuses. Tapisso et al. ( 2009 ) exposed rats 4 

### t o 21.5 mg /kg Pb

- acetate (1/10th the LD 50 

### ) via i.p. injection on alternating days for 11 or 5 

### 21 days, for a total of 5 or 10 exposures. Induction of SCEs in the bone marrow of 6 

### exposed rats was increased over controls in a statistically significant duration

- d ependent 7 

### manner. It is important to note that all three of these studies utilized an injection route of 8 

### exposure that may not be relevant to routes of exposure in the general population 9 

### ( e.g., air, drinking water exposure). 10


### Few studies were found that inv estigated SCE formation in human cell lines due to Pb 11 

### exposure. Statistically significant, concentration

- dependent increases in SCEs were 12 

### observed in human lymphocytes obtained from a single donor when incubated with 1, 5, 13 

### 10, or 50 μ M Pb nitrate ( Ustundag and Duydu, 2007 )

. Melatonin and N

- acetylcysteine 14 

### were reported to ameliorate these effects, indicating Pb may induce SCEs through 15 

### increased oxidative stress. Pb ch loride was also observed to increase SCE levels in 16 

### human lymphocytes exposed to 3 or 5 ppm ( Turkez et al., In Press )

. 17 

### Studies investigating SCE in rodent cells were more equivocal than those in human cells. 18 

### Pb sulfate, acetate, and nitrate were found not to induce SCE in Chinese hamster V79 19 

### cells ( Hartwig et al., 1990 ; Zelikoff et a l., 1988 )

. Both of these studies only examined 25

- 20 

### 30 cells per concen tration, reducing their power to detect Pb

- induced SCEs. Cai and 21 

### Arenaz ( 1998 ) , on the other hand, used 100 cells per treatmen t and observed that 22 

### exposure to 0.05

- 1 μ M Pb nitrate for 3

- 12 hours resulted in a weak, concentration

23 

### dependent increase in SCEs in Chinese hamster ovary cells. Lin et al. ( 1994 ) also 24 

### observed a concentration

- dependent increase in SCEs in Chinese hamster cells exposed 25 

### to 3

- 30 μ M Pb nitrate for 2 hours. 26



### Micronucleus Formation

Pb

- induced micronucleus formation was observed in numerous occupational studies 27 

### ( Grover et al., 2010 ; Khan et al., 2010b ; Minozz o et al., 2010 ; Shaik and Jamil, 2009 ; 28 

### Minozzo et al., 2004 ; Palus et al., 2003 ; Vaglenov et al., 2001 ; Pinto et al., 2000 ; Bilban, 29 

### 1998 ; Vaglenov et al., 1998 )

. The workers in the occupational studies generally had high 30 

### blood Pb levels (>20 μ g/dL) making comparisons to the general population difficult, 31 

### although Pinto et al. ( 2000 ) observed increased micronuclei in exposed workers with an 32 

### average blood Pb level of 10.48 μ g/dL compared with unexposed controls. In studies 33 

### investigating the correlation between blood Pb levels and micronucleus formation, no 34 

### association was observed ( Minozzo et al., 2010 ; Minozzo et al., 2004 ; Palus et al., 2003 ; 35February 2012 5

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### Pinto et al., 2000 ) , although Pinto et al. ( 2000 ) , Grover et al. ( 2010 ) , and Minozzo et al. 1 

### ( 2010 ) did report an association between micronuclei formation and duration of exposure. 2 

### Only one study was found that investigated micronucleus formation in a no nworker 3 

### population; Mielzynska et al. ( 2006 ) reported a statistically significant positive 4 

### correlation between blood Pb levels and micronuclei frequency in children in Poland. 5 

### Children, with a n average blood Pb level of 7.69 μ g/dL, were observed to have 4.44 6 

### micronucleated cells per 1 , 000 cells analyzed. Children with blood Pb levels greater than 7 

### 10 μ g/dL had significantly more micronucleated cells than did children with blood Pb 8 

### levels less th an 10 μ g/dL. 9 

### Micronucleus formation in response to Pb exposure has been observed in rodent animal 10 

### studies. Celik et al. ( 2005 ) observed that exposure of female rats to 140, 250, or 500 g/kg 11 

### Pb

- acetate once per week for 10 weeks resulted in statistically significantly increased 12 

### numbers of micronu cleated polychromatic erythrocytes (PCEs) compared to controls. 13 

### Similarly, Alghazal et al. ( 2008b ) exposed rats to 100 ppm Pb

- acetate daily for 125 days 14 

### and observed statistically significant increases in micronucleated PCEs in both sexes. 15 

### Tapisso et al. ( 2009 ) exposed rats to 21.5 mg /kg Pb

- acetate (1/10th the LD 50 

### ) via i.p. 16 

### injection on alternating days for 11 or 21 days, for a total of 5 or 10 exposures. Formation 17 

### of micronuclei in the bone marrow of e xposed rats was increased over that in controls in 18 

### a significant duration

- dependent manner. Two further studies investigated formation of 19 

### micronuclei in the bone marrow of exposed mice: Roy et al. ( 1992 ) exposed mice to 10 20 

### or 20 mg /kg Pb nitrate i.p. and observed a con centration

- dependent increase in 21 

### micronuclei, whereas Jagetia and Aruna ( 1998 ) observed an increase in micronuclei in 22 

### mice exposed to 0.625

- 80 mg /kg Pb nitrate i.p., though the increase was not 23 

### concentration

- dependent. Mice exposed to 1 g/L Pb

- acetate via drinking water, a more 24 

### environmen tally relevant route of exposure, for 90 days had statistically significant 25 

### increases in micronucleated PCEs ( Marques et al., 2006 )

. 26 

### A few studies were found that reported increased micronucleus formation in human cell 27 

### lines treated with Pb. Concentration

- dependent micronucleu s formation was observed in 28 

### human lymphocytes when exposed to either 1, 5, 10, or 50 μ M Pb nitrate or 3 or 5 ppm 29 

### Pb chloride ( Ustundag and Duydu, 2007 ; Turkez et al., In Press )

. Gastaldo et al. ( 2007 ) 30 

### also observed a dose

- dependent increase in micronuclei in human endothelial cells 31 

### exposed to 1

- 1 , 000 μ M Pb nitrate for 24 hours. Two animal cell culture studies 32 

### investigating micronuclei formation produced contrasting results. One study ob served 33 

### that micronuclei were not induced in Chinese hamster cells exposed to 3

- 30 μ M Pb 34 

### nitrate for 2 hours ( Lin et al., 1994 ) , whereas the other observed that Pb

- acetate induced a 35



### con centration

- dependent increase in Chinese hamster cells when administered at 0.03

- 36 

### 10 μ M for 18 hours ( Bonacker et al., 2005 )

. 37February 2012 5

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### Chromosomal Aberrations Chromosomal aberrations ( e.g., chromosome breaks, nucleoplasmic bridges, di

- and a

1 

### centric chromosomes, and rings) were exami ned in a number of occupational studies 2 

### ( Gro ver et al., 2010 ; Shaik and Jamil, 2009 ; Pinto et al., 2000 ; Bilban, 1998 ; De et al., 3 

### 1995 ; Huang et al., 1988 )

. Methodological limitations outlined in previous sections, 4 

### including potential for occupational co

- exposure to genotoxic substances and generally 5 

### high blood Pb levels (>20 μ g/dL) that limit the relevance of findings to the general 6 

### population, also pertain to the present findings. No correlation was observed between 7 

### increasing blood Pb level and the number of chromosomal aberrations, although an 8 

### associat ion was observed between duration of exposure and chromosomal damage 9 

### ( Grover et al., 2010 ; Pinto et al., 2000 )

. Other studies reported no association between 10



### occu pational exposure to Pb and chromosomal aberrations ( Anwar and Kamal, 1988 ; 11 

### Andreae, 1983 )

. Smejkalova ( 1990 ) observed greater chromosomal damage and 12 

### aberrations in children living in a heavily Pb

- contaminated area of Czechoslovakia 13 

### compared with children livi ng in an area with less contamination, although the difference 14 

### between the two areas was not statistically significant. Although blood Pb levels were 15 

### statistically significantly higher in children living in the Pb

- contaminated area than in 16 

### children living in the less contaminated area, they were generally comparable (low 30s 17 

### versus high 20s μg/dL, respectively), indicating there may not be enough of a dose 18 

### contrast to detect a significant difference in aberration rates. 19 

### The majority of animal studies inves tigating Pb

- induced genotoxicity focused on the 20 

### capacity of Pb to produce chromosomal damage. Fahmy ( 1999 ) exposed mice to 25

- 21 

### 400 mg /kg Pb

- acetate i.p., either as a single exposure or repeatedly for 3, 5, or 7 days. 22 

### Chromosomal damage was observed to increase in bone marrow cells (100

- 400 mg /kg) 23 

### and spermatocytes (50

- 400 mg /kg) in a concentration

- dependent manner after both 24 

### exposure regimens. Pb nitrate was also observed to produce concentration

- dependent 25 

### chromosomal damage in mice exposed i.p. to a single exposure of 5, 10, or 20 mg /kg 26 

### ( Dhir et al., 1992b )

. In a similar experiment, Dhir et al. ( 1990 ) exposed mice to 10, 20, or 27 

### 40 mg /kg Pb nitrate and saw an increase in chromosomal aberrations, although there was 28 

### no concentr at i on
- dependent response as the response was similar in all concentrations 29 

### tested. Nayak et al. ( 1989b ) exposed pregnant mice to 100

- 200 mg /kg Pb nitrate via i.v. 30 

### injection and observed no chromosomal gaps or breaks in dams or fetuses, but did report 31 

### some karyotypic chromosomal damage and weak aneuploidy at the low exposure. In a 32 

### similar experiment, low levels of chromosomal aberrations were observed in dams and 33 

### fetuses injected with 12.5

- 75 mg /kg Pb nitrate, but the re was no concentrat i on
- dependent 34 

### response reported and few cells were analyzed ( Nay ak et al., 1989a )

. In rats given 35 

### 2.5 mg /100 g Pb

- acetate i.p. daily for 5

- 15 days or 10

- 20 mg /100 g once and analyzed 36 

### after 15 days, Pb

- induced chromosomal aberrations were observed ( Chakraborty et al., 37February 2012 5

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### 1987 )

. The above studies all suffer from the use of a route of exposure that may not be 1 

### relev ant to human environmental exposures. However, studies utilizing drinking water or 2 

### dietary exposures also observed increases in chromosomal damage. Aboul

- Ela ( 2002 ) 3 

### exposed mice to 200 or 400 mg /kg Pb

- acetate by gavage for 5 days and reported that 4 

### chromosomal damage was present in the bone marrow cells and spermatocytes of animals 5 

### exposed to both exposure concentrations. Dhir et al. ( 1992a ) also observed a 6 

### concentration

- dependent increase in chromosomal damage in mice exposed via gavage, 7 

### albeit at much lower concentrations: either 5 or 10 mg /kg. Nehez et al. ( 2000 ) observed a 8 

### Pb

- induced increase in aneuploidy and percent of cells with damage after exposure to 9 

### 10 mg /kg administered by gavage 5 days a week for 4 weeks. In the only study that 10 

### investigated diet ary exposure, El

- Ashmawy et al. ( 2006 ) exposed mice to 0.5% 11 

### Pb

- acetate in feed, and observed an increase in abnormal cells and frequency of 12 

### chromosomal damage. 13 

### In the few studies that investigated the capacity of Pb to induce chromosomal damage in 14 

### human cell lines, Pb exposure did not induce chromosomal damage. Wi se et al. ( 2005 ; 15 

### 2004 ) observed that Pb glutamate was not mutagenic in human lung cells ex posed to 250

- 16 

### 2 , 000 μ M for 24 hours. Pasha Shaik et al. ( 2006 ) observed that Pb nitrate did not increase 17 

### chromosomal aberrations in primary lymphocytes (obtained from healthy vol unteers) 18 

### when incubated with 1 , 200 or 2 , 000 μ M for 2 hours. Studies utilizing animal cell lines 19 

### generally supported the finding of no Pb

- induced chromosomal damage in human cell 20 

### lines. Pb nitrate was found to induce no chromosomal damage in Chinese hamster ovary 21 

### cells exposed to 500

- 2 , 000 μ M for 24 hours ( Wise et al., 1994 ) , 3

- 30 μ M for 2 hours ( Lin 22 

### et al., 1994 ) , or 0.05

- 1 μ M for 3

- 12 hours ( Cai and Arenaz, 1998 )

. Wise et al. ( 1994 ) did 23 

### observe increased chromosomal damage in Chinese hamster ovary cells exposed to 24 

### 1 , 000 μ M Pb glutamate for 24 hours, but did not see any damage in cells exposed to 25 

### higher concentrations (up to 2 , 000 μ M). 26



### 5.2.7.4 Epigenetic Effects
Epigenetic effects are heritable changes in gene expression resulting without changes in 27 

### the underlying DNA sequ ence. A prime example of an epigenetic effect is the abnormal 28 

### methylation of DNA, which could lead to altered gene expression and cell proliferation 29 

### and differentiation. 30



### DNA Methylation A single i.v. injection of 75 μmol/kg Pb nitrate resulted in global h ypomethylation of 31 

### hepatic DNA in rats ( Kanduc et al., 1991 )

. The observed hypomethylation in the liver 32February 2012 5

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### wa s associated with an increase in cell proliferation. A few additional studies in humans 1 

### observed that higher bone Pb levels were associated with lower DNA methylation 2 

### patterns in adults and cord blood of newborns ( Wright et al., 2010 ; Pilsner et al., 2009 )

. 3 

### Changes in DNA methylation patterns could potentially lead to dysregulation of gene 4 

### expression and altered tissue differentiation. 5



### Mitogenesis Conflicting results have been reported regarding Pb

- induced effects on mitogenesis, with 6 

### both increased and decreased cell growth and mitogenesis. A discernable pattern of 7 

### effects is difficult to detect when analyzing effects across human, in vivo animal, and in 8 

### vitro studies. Only a few studies have investigated the potential epigenetic effects of Pb 9 

### exposure in human populations indirectly by examining mitogenesis or the induction of 10 

### cell proliferation, which can be a consequence of epigenetic changes . These studies 11 

### ( Minozzo et al., 2010 ; Minozzo et al., 2004 ; Rajah and Ahuja, 1995 ) reported that Pb 12 

### reduced mitogenesis in Pb

- exp osed workers (mean blood Pb levels: 35.4 μ g/dL, 13 

### 59.4 μ g/dL, and not reported, respectively). The observation of decreased cell division in 14 

### exposed workers may indicate that cells suffered DNA damage and died during division, 15 

### or that division was delayed to allow for DNA repair to occur. It is also possible th at Pb 16 

### exerts an aneugenic effect and arrests the cell cycle. 17 

### Many studies have investigated the ability of Pb to induce mitogenesis in animal models, 18 

### and have consistently shown that Pb nitrate can stimulate DNA synthesis and cell 19 

### proliferation in the liv er of animals exposed to 100 μ M/kg via i.v. injection ( Nakajima et 20 

### al., 1995 ; Coni et al., 1992 ; Ledda

- Columbano et al., 1992 ; Columbano et al., 1990 ; 21 

### Columbano et al., 1987 )

. Shinozuka et al. ( 1996 ) observed that Pb

- induced hepatocellular 22 

### proliferation was similar in magnitude to that induced by TNF

- α at 100 μ M/kg, and Pb 23 

### was observed to induce TNF

- α in g lial and nerve cells and NF

- κB, TNF

- α, and iNOS in 24 

### liver cells in exposed animals at 12.5 mg /kg and 100 μ M/kg, respectively ( Cheng et al., 25 

### 2002 ; Menegazzi et al., 1997 )

. In the only study that examined Pb exposure via 26 

### inhalation, exposure to 10,000 μ M Pb

- acetate for 4 weeks resulted in increased cellular 27 

### proliferation in the lungs ( Fortoul et al., 2005 )

. 28 

### A great amount of research has been conducted investigating the potential effects of Pb 29 

### on mitogenesis in human and animal cell culture s. In human cell cultures, Pb

- acetate 30 

### inhibited cell growth in hepatoma cells (0.1

- 100 μ M for 2

- 6 days) ( Heiman and Tonner, 31 

### 1995 ) and primary oligodendrocyte progenitor cells (1 μ M for 24 hours) ( Deng and 32 

### Poretz, 2002 ) but had no observable effects on growth in glioma cells (0.01

- 10 μ M for 33 

### 12

- 72 hours) ( Liu et al., 2000 )

. Pb glutamate had no effect on cell growth in human lung 34 

### cells, but did increase the mitotic index (250

- 1 , 000 μ M for 24 hours) ( Wise et al., 2005 )

. 35February 2012 5

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### The increase in the mitotic index was attributed to an arrest of the cell cycle at M

- phase, 1 

### and was not attributed to an actual increase of cell growth and proliferation. Gastaldo et 2 

### al. ( 2007 ) also reported S and G2 cell cycle arrests in human endothelial cells following 3 

### exposure to 100 μM Pb nitrate for 24 hours. Conflicting results with regard to DNA 4 

### synthesis were repo rted, with a concentration

- dependent inhibition of DNA synthesis 5 

### reported in hepatoma cells (1

- 100 μ M for 72 hours) ( Heiman and Tonner, 1995 ) , but an 6 

### induction of synthesis observed in astrocytoma cells (1

- 50 μ M for 24 hours) ( Lu et al., 7 

### 2002 )

. 8 

### In rat fibroblasts and epithelial cells, Pb

- acetate , chloride, o xide, and sulfate were all 9 

### observed to inhibit cell growth (10

- 1 , 000 μ M for 1

- 7 days and 0.078

- 320 μ M for 48 10 

### hours, respectively) ( Iavicoli et al., 2001 ; Apostoli et al., 2000 )

. Iavicoli et al. ( 2001 ) 11 

### observed that in addition to inhibiting cell growth in rat fibroblas ts, Pb

- acetate caused 12 

### GS/M and S

- phase arrest. Pb

- acetate decreased cell proliferation in mouse mesenchymal 13 

### stem cells when administered at 20

- 100 μM for 48 hours ( Kermani et al., 2008 )

. Pb 14 

### nitrate was alternatively reported to increase ( Lin et al., 1994 ) and decrease ( Cai and 15 

### Arenaz, 1998 ) the mitotic index in Chinese hamster ovary cells exposed to 1 μM Pb 16 

### nitrate. Lin et al. ( 1994 ) did not consider cell cycle arrest when measuring the mitotic 17 

### index and did not observe a decrease at higher concentrations; in fact, the highest 18 

### concentration tested, 30 μM, had a mitotic index equal to that in the untreated contr ol 19 

### cells. 20



### 5.2.7.5 Gene Expression
Two animal studies have investigated the ability of Pb to alter gene expression in regard 21 

### to phase I and II metabolizing enzymes. Suzuki et al. ( 1996 ) exposed rats to 100 μg/kg 22 

### Pb

- acetate or nitrate via i.p. injection and observed an induction of GST

- P with both Pb 23 

### compounds. T he induction of GST

- P by Pb was observed to occur on the transcriptional 24 

### level and to be dependent on the direct activation of the cis

- element GPEI enhancer. 25 

### Degawa et al. ( 1993 ) reported that i.v. exposure to 20, 50, or 100 μmol/kg Pb nitrate 26 

### selectively inhibited CYP1A2 levels. Pb was shown not to inhibit CYP1A2 by direct 27 

### enzyme inhibition, but rather to decrease the amount of CYP1A2 mRNA. In contrast, 28 

### Korashy and El Kadi ( 2004 ) observed that exposure of murine hepatoma cells to 10

- 100 29 

### μM Pb nitrate for 24 hours increased the amount of CYP1A1 mRNA while not 30 

### influenc ing the activity of the enzyme. NAD(P)H:quinone oxidoreductase and GST Ya 31 

### activities and mRNA levels were increased after exposure to Pb. Incubation of primary 32 

### human bronchial epithelial cells with 500 μg/L Pb

- acetate for 72 hours resulted in the 33 

### up
- regula tion of multiple genes associated with cytochrome P450 activity, glutathione 34February 2012 5

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### metabolism, the pentose phosphate pathway, and amino acid metabolism ( Glahn et al., 1 

### 2008 )

. 2 

### Additional animal studies provide further evidence that exposure to Pb compounds can 3 

### perturb gene expression. Zawia and Harry ( 1995 ) investigated whether the observed Pb

4 

### induced disruption of myelin formation in rat pups exposed postnatally was due to altered 5 

### gene expression. In pups exposed to 0.2% Pb

- acetate via lactation from postnatal day 6 

### (PND)1

- 20, the expression of proteolipid pro tein, a major structural constituent of 7 

### myelin, was statistically significantly elevated at PND20, compared to controls. The 8 

### expression of another structural element of myelin, myelin basic protein, was similarly 9 

### elevated in exposed animals, although not s ignificantly so. The expression of both genes 10 

### returned to control levels 5 days following the termination of exposure. These data 11 

### suggest that altered gene expression in structural myelin proteins due to Pb exposure may 12 

### be responsible for observed alterati ons in abnormal conduction of nerve impulses. Long 13 

### et al. ( 2011 ) investigated the Pb

- induced induction of ABCC5, an ATP

- binding cassette 14 

### transporter, in embryonic and adult zebrafish. In the initial in vitro portion of the study, 15 

### exposure of zebrafish fibroblasts to 20 μ M Pb nitrate for 24 hours significantly increased 16 

### the induction of ABBC5 mRNA 2.68

- fold over controls. Similar levels of induction were 17 

### observed when embryonic zebrafish w ere exposed to 5 μ M 24 to 96 hours; specifically, 18 

### induction of ABCC5 was seen in the livers of developing embryos. In adult fish, 19 

### induction of ABCC5 was observed in the brains, intestines, and kidneys of exposed fish, 20 

### but decreased in their livers. Inducti on of ABCC5 was observed to attenuate the toxicity 21 

### of Cd, but not Hg or As, in developing embryos, the attenuation of Pb

- induced toxicity 22 

### was not investigated. However, these findings indicate that increased expression of 23 

### ABCC5 due to heavy metal exposure may play a part in cellular defense mechanisms. 24



### 5.2.7.6 Apoptosis
Occupational exposure to Pb and induction of apoptosis was investigated in a few studies. 25 

### One study directly reported that exposure to Pb increased apoptosis compared to 26 

### nonexposed controls ( Minozzo et al., 2010 ) , whereas the others reported that two early 27 

### indicators of apoptosis, karyorrhexis and karyolysis, were elevated in exposed workers 28 

### ( Grover et al., 2010 ; Khan et al., 2010b )

. Pb nitrate was also observed to induce apoptosis 29 

### in the liver of exposed animals ( Columbano et al., 1996 ; Nakajima et al., 1995 )

. 30 

### Apoptosis was observed in rat fibroblasts exposed to Pb

- acetate and rat alveolar 31 

### macrophages exposed to Pb nitrate ( Iavicoli et al., 2001 ; Shabani and Rabbani, 2000 )

. 32 

### Observation of Pb

- induced apoptosis may represent the dysregulation of genetically

33 

### controlled cell processes and tissue homeostasis. 34February 2012 5

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### 5.2.8 Summary
The diverse health effects of Pb are mediated through multiple, interconnected modes o f 1 

### action. Each of the modes of action discussed has the potential to contribute to the 2 

### development of a number of Pb

- induced health effects ( Table 5

- 2 ). While this section 3 

### draws from older literature as well as newer lines of evidence, the inclusion of new 4 

### evidence does not qualitatively change the conclusions regarding individual modes of 5 

### action. However, the new evidence strengthens the conclusions. Evidence for the 6 

### majority of these modes of action is observed at low blood Pb levels in humans, between 7 

### 2 and 17 μ g/dL, with supporting evidence from in vitro assays. As many of these studies 8 

### are examining adults with likely higher past than current Pb exposures, uncertainty exists 9 

### as to the Pb exposure level, duration, and timing leading to these blood Pb levels 10 

### associated with these modes of action. These observable effect levels are reflecti ve of the 11 

### data and methods available and do not imply that these modes of action are not acting at 12 

### lower Pb exposure or blood Pb levels or that these concentrations represent the threshold 13 

### of the effect. The observable effect levels in hu mans, reported in Table 5

- 2 , are drawn 14 

### from the available data ; and , do not imply that theses modes of action are not acting at 15 

### lower exposure levels or that these doses represent the threshold of the effect. Also, the 16 

### data presented in this table does not inform regarding the exposure frequency and 17 

### duration required to elicit a particular MOA. 18February 2012 5

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### Table 5
- 2 MOAs, their related h ealth effects, and information on concentrations eliciting the MOAs

Mode of Action [Related Health Effects (ISA section)] Concentrations or doses (Conditions) a

Blood Pb Dose Altered Ion Status [All Health Effects of Pb] 3.5 μg/dL (mean in cord blood; association with cord blood Ca 2+

- ATPase pump activity) Huel et al. ( 2008 ) 0.00005 μM free Pb 2+ (In vitro; 30 minutes; calmodulin activation ass ay) Kern et al. ( 2000 )
Protein Binding [Renal ( 5.5 ), Heme Synthesis and RBC Function ( 5.7 )] 17.0 μg/dL (concurrent mean in adult workers with wildty pe metallothionein expression; increased BP susceptibility) Chen et al. ( 2010a ) 50 μM Pb glutamate (In vitro; 24 hours; increased nuclear p rotein in neurological cell) Klann and Shelton ( 1989 ) Oxidative Stress [All Heath Effects of Pb] 5.4 μg/dL (concurrent mean in adult male workers; decreased CAT activity in blood) Conterato et al. ( In Press ) 0.1 μM Pb

- acetate (In vitro; 48 hours; decreased cellular GSH in neuroblastoma cells) Chetty et al. ( 2005 ) Inflammation [Nervous System ( 5.3 ), Cardiovascular ( 5.4 ), Renal ( 5.5 ), Immune ( 5.6 ), Respiratory ( 5.6.4 ), Hepatic ( 5.9.1 )] 2.5 μg/dL (concurrent minimum in adult males; increased serum TNF

- α and blood WBC count) Kim et al. ( 2007 ) 0.01 μM Pb

- acetate (In vitro; 48 hours; increased cellular PGE 2 in neuroblastoma cells) Chetty et al. ( 2005 ) Endocrine Disruption [Reproductive and Developmental Effects ( 5.8 ), Endocrine System 5.9.3 ), Bone and Teeth ( 5.9.4 )] 1.7 μg/dL (concurrent minimum in women with both ovaries removed; increased serum FSH) Krieg ( 2007 ) 10 μM Pb nitrate (In vitro; 30 minutes; displaced GHRH binding to rat pituitary receptors) Lau et al. ( 1991 ) Cell Death/Genotoxicity [Cancer ( 5.10 ), Reproductive and Developmental Effects ( 5.8 ), Bone and Teeth ( 5.9.4 )] 3 .3 μg/dL (concurrent median in adult women; increased rate of HPRT mutation frequency) Van Larebeke et al. ( 2004 ) 0.03 μM Pb

- acetate (In vitro; 18 hours; increased formation of micronuclei) Bonacker et al. ( 2005 )

a This table provides examples of studies that report effects wit h low doses or concentration; they are not the full body of evidence used to characterize the weight of the evidence. In addition, the levels cited are reflective of the data and methods available and do not imply that these m odes of action are not acting at lower Pb exposure or blood Pb levels or that these doses represent the threshold of the effect. Additionally, the blood concentrati ons and doses (indicating Pb exposure concentrations from in vitro systems) refer to the concentrations and doses at which these modes of action were observed. While the individual modes of action are related back to specific health effects sections ( e.g., Nervous System, Cardiovascular), the concentrations and doses given should not be interpreted as levels at which those sp ecific health effects occur.


### The alteration of cellular ion status (including disruption of Ca 2+ 

### homeostasis, altered ion 1 

### transport mechanisms, and perturbed protein function through displacement of metal 2 

### cofactors) appears to be the major unifying mode o f action underlying all subsequent 3 

### modes of action ( Figure 5

- 1 ). Pb will interfere with endogenous Ca 2+ 

### homeostasis, 4 

### necessary as a cell signal carrie r mediating normal cellular functions. [Ca 2+ 

### ] i 

### has been 5 

### shown to increase after Pb exposure in a number of cell types including bone, 6 

### erythrocytes, brain cells, and white blood cells, due to the increased flux of extracellular 7 

### Ca 2+ 

### into the cell. This disr uption of ion transport is due in part to the alteration of the 8 

### activity of transport channels and proteins, such as Na +



### - K + 

### ATPase and voltage

- sensitive 9 

### Ca 2+ 

### channels. Pb can interfere with these proteins through direct competition between Pb 10 

### and the nativ e metals present in the protein metal binding domain or through disruption 11 

### of proteins important in Ca 2+



### - dependent cell signaling, such as PKC or calmodulin. 12February 2012 5

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### Disruption of ion transport not only leads to altered Ca 2+ 

### homeostasis, it can also result in 1 

### per turbed neurotransmitter function. Pb has been shown to be able to displace metal ions, 2 

### such as Zn, Mg, and Ca 2+ 

### , from proteins due to the flexible coordination number of Pb 3 

### and multiple ligand binding ability, leading to abnormal conformational changes to 4 

### proteins and altered protein function. Evidence for this metal ion displacement and 5 

### protein pertu r bation has been shown at picomolar concentrations of Pb. Additional effects 6 

### of altered cellular ion status are the inhibition of heme synthesis and decreased cellular 7 

### energy production due to perturbation of mitochondrial function. 8 

### Although Pb will bind to proteins within cells through interactions with side group 9 

### moieties, thus potentially disrupting cellular function, protein binding of Pb may 10 

### represent a mec hanism by which cells protect themselves against the toxic effects of Pb. 11 

### Intranuclear and intracytosolic inclusion body formation has been observed in the kidney, 12 

### liver, lung, and brain following Pb exposure. A number of unique Pb binding prote ins 13 

### have be en detected, constituting the observed inclusion bodies. The major Pb binding 14 

### protein in blood is ALAD with carriers of the ALAD

- 2 allele potentially exhibiting 15 

### higher Pb binding affinity. Additionally, metallothionein is a n important protein in the 16 

### format ion of inclusion bodies and mitigation of the toxic effects of Pb. 17 

### A second major mode of action of Pb is the development of oxidative stress, due in many 18 

### instances to the antagonism of normal metal ion functions. The origin of oxidative stress 19 

### produced af ter Pb exposure is likely a multipathway process, resulting from oxidation of 20 

### δ

- ALA, NAD(P)H oxidase activation, membrane and lipid peroxidation, and antioxidant 21 

### enzyme depletion. Through the inhibition of δ

- ALAD due to displacement of Zn, 22 

### accumulated δ

- AL A goes through an auto

- oxidation process to produce ROS. 23 

### Additionally, Pb can induce the production of ROS through the activation of NAD(P)H 24 

### oxidase. Pb

- induced ROS can interact with membrane lipids to cause a membrane and 25 

### lipid peroxidation cascade. Enhan ced lipid peroxidation can also result from Pb 26 

### potentiation of Fe 2+ 

### initiated lipid peroxidation and alteration of membrane composition 27 

### after Pb exposure. Increased Pb

- induced ROS will also sequester and inactivate 28 

### biologically active ·NO, leading to the i ncreased production of the toxic product 29 

### nitrotyrosine, increased compensatory NOS, and decreased sGC protein. Pb

- induced 30 

### oxidative stress not only results from increased ROS production but also through the 31 

### alteration and reduction in activity of the antio xidant defense enzymes. The biological 32 

### actions of a number of these enzymes are antagonized due to the displacement of the 33 

### protein functional metal ions by Pb. 34 

### In a number of organ systems Pb

- induced oxidative stress is accompanied by 35 

### misregulated inflamm ation. Pb exposure will modulate inflammatory cell function, 36 

### production of pro

- inflammatory cytokines and metabolites, inflammatory chemical 37February 2012 5

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### messengers, and pro

- inflammatory signaling cascades. Cytokine production is skewed 1 

### toward the production of pro

- inf lammatory cytokines like TNF

- α and IL

- 6 as well as 2 

### toward the promotion of a Th2 response and suppression of a Th1 response accompanied 3 

### by decreased production of related cytokines. 4 

### Evidence indicates that Pb is a potent endocrine disrupting chemical. Pb w ill disrupt the 5 

### HPG axis evidenced by a decrease in serum hormone levels, such as FSH, LH, 6 

### testosterone, and estradiol. Pb interacts with the hypothalamic

- pituitary level hormone 7 

### control causing a decrease in pituitary hormones, altered growth dynamics, in hibition of 8 

### LH secretion, and reduction in StAR protein. Pb has also been shown to alter hormone 9 

### receptor binding likely due to interference of metal cations in secondary messenger 10 

### systems and receptor ligand binding and through generation of ROS. Pb also may disrupt 11 

### the HPT axis by alteration of a number of thyroid hormones, possibly due to oxidative 12 

### stress. However, the results of these studies investigating HPT are mixed and require 13 

### further investigation. 14 

### The association of Pb with increased genotoxicity and cell death has been investigated in 15 

### humans, animals, and cell models. Occupational Pb exposure in humans has been 16 

### associated with increased DNA damage, however lower blood Pb and exposure levels 17 

### have been associated with these effects in experimental animals and cells. Results vary on 18 

### the effect of Pb on DNA repair activity, however a number of studies reported decreased 19 

### repair processes following Pb exposure. There is evidence of mutagenesis and 20 

### clastogenicity in highly

- exposed humans, however weak ev idence has been shown in 21 

### animals and cell based systems. Human occupational studies provide limited evidence for 22 

### micronucleus formation (blood Pb levels >10 μ g/dL), supported by Pb

- induced effects in 23 

### both animal and cell studies at higher exposure levels. Animal studies have also provided 24 

### evidence for Pb

- induced chromosomal aberrations. The observed increases in 25 

### clastogenicity may be the result of increased oxidative damage to DNA due to Pb 26 

### exposure, as co
- exposures with antioxidants ameliorate the observed toxicities. Limited 27 

### evidence of epigenetic effects is available, including DNA methylation, mitogenesis, and 28 

### gene expression. Pb may alter gene expression by displacing Zn from multiple 29 

### transcriptional factors, thus perturbing their normal cellular activi ties. Consistently 30 

### positive results have provided evidence of increased apoptosis following Pb exposure. 31 

### Similar to Pb, other polyvalent metal ions ( e.g., Cd, Cr, Be, Ba, Se, Sr, As, Al, Cu) have 32 

### demonstrated molecular mimicry and displacement of biological cation ( Garza et al., 33 

### 2006 )

. In this manner, these metal ions share with Pb a common central mode of action of 34 

### disruption of ion status. Specifically, these metals have been shown to disrupt cellular 35 

### processes as diverse as Ca 2+ 

### homeostasis, cell signaling, neurotransmitter release, cation 36 

### membrane channel function, protein

- DNA binding, and cellular membrane structure 37February 2012 5

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### ( Pentyala et al., 2010 ; Huang et al., 2004 ; Atchison, 2003 ; Jehan and Motlag, 1995 ; 1 

### Richardt et al., 1 986 ; Cooper and Manalis, 1984 ; Habermann et al., 1983 )

. Additionally, 2 

### presumably through their shared central mode of action, some of these metal ions also 3 

### display corresponding downstream modes of actions such as oxidative stress, apoptosis, 4 

### and genotoxicity ( Jomova and Valko, 2011 ; Jomova et al., 2011 ; Matović et al., 2011 ; 5 

### Agarwal et al., 2009 ; Méndez

- Gómez et al., 2008 ; Rana, 2 008 ; Hengstler et al., 2003 )

. 6 

### Overall, Pb

- induced health effects can occur through a number of interconnected modes 7 

### of action that generally originate with the alteration of ion status. 8



## 5.3 Nervous System Effects

5.3.1 Introduction
The 2006 Pb AQCD concluded that the “overall weight of the available evidence 9 

### provides clear substantiation of neurocognitive decrements being associated in young 10 

### children with blood

- Pb concentrations...” ( U.S. EPA, 2006b )

. This conclusion was based 11 

### on evidence from several prospective and cross

- sectional studies conducted in diverse 12 

### populations and after adjusting for potential confounding by socioeconomic status (SES), 13 

### parental intelligence, and caregiving environment. This association was substantiated in a 14 

### po oled analysis of children, 5 to 10 years of age, participating in seven prospective 15 

### studies (Boston, MA; Cincinnati, OH; Rochester, NY; Cleveland, OH; Mexico City, 16 

### Mexico; Port Pirie, Australia; and Kosovo, Yugoslavia) ( Lanphe ar et al., 2005 )

. 17 

### Associations between blood Pb levels and decrements in intelligence quotient (IQ), 18 

### mental development, memory, and other specific indices of cognitive function in children 19 

### ages 6 months to 17 years were most strongly indicated in child ren with population mean 20 

### blood Pb levels (measured at various lifestages) in the range of 5

- 10 μ g/dL; however, 21 

### several results indicated associations in groups of children (ages 2

- 10 years) with mean 22 

### blood Pb levels in the range of 3

- 5 μ g/dL ( Bellinger, 2008 ; Canfield, 2008 ; Hornung, 23 

### 2008 ; Rojo

- Tellez, 2008 )

. Based on fewer available studies, the 2006 Pb AQCD 24 

### described consistent associations of blood Pb levels with behavioral outcomes, including 25 

### inattention, and antisocial and delinquent behavior assessed in children ages 6 to 13 years 26 

### ( U.S. EPA, 2006b )

. 27 

### Toxicological studies provided coherence with similarly consistent findings for Pb

28 

### induced impairments in learning and behavior in rodents and monkeys ( U.S. EPA, 29 

### 2006b )

. In contrast with studies in children, Pb exposure was not found consistently to 30 

### affect memory of animals. Effects on learning were largely demonstrated as poorer 31 

### performance on Morris water maze and discrimination reversal tasks, and effects on 32February 2012 5

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### behavior w ere largely demonstrated as distractibility, impulsivity, and insensitivity to 1 

### changes in reinforcement. These effects in animals were predominantly found with Pb 2 

### exposures that resulted in blood Pb levels between 20

- 50 μ g/dL; however, some studies 3 

### observe d learning and behavioral impairments in animals with steady

- state blood Pb 4 

### levels of 10

- 15 μ g/dL ( Cory

- Slechta, 1994 ; Altmann et a l., 1993 ; Rice and Karpinski, 5 

### 1988 ; Gilbert and Rice, 1987 )

. Toxicological studies also provided biological plausibility 6 

### by characterizing modes of action for Pb

- indu ced nervous system effects. In particular, 7 

### toxicological evidence for Pb exposure interfering with neuronal metabolism at the 8 

### cellular and histological level ( e.g., synaptic architecture during development, 9 

### neurotransmitter release, glia, neurite outgrowth , the blood brain barrier, and oxidative 10 

### stress), provided biological plausibility for blood Pb levels in children being causally 11 

### associated with deficits in multiple functional domains such as cognitive function, motor 12 

### function, memory, mood, and behavior . Additional biological plausibility was provided 13 

### by associations observed of childhood blood Pb levels with changes indicative of 14 

### neuronal damage and altered brain physiology assessed in young adults using magnetic 15 

### resonance imaging techniques ( Yuan et al., 2006 ; Cecil et al., 2005 ; Meng et al., 2005 ; 16 

### Trope et al., 2001 )

. 17 

### A common finding across several epidemiologic studies of children was a supralinear 18 

### concentration

- response relationship between blood Pb level and neurocognitive deficits, 19 

### i.e., a larger decrement in neurocognitive function per unit increase in blood Pb levels in 20 

### children in the lower range of the population blood Pb level distribution ( Kordas et al., 21 

### 2006 ; Schnaas et al., 2006 ; Tellez

- Rojo et al., 2006 ; Lanphear et al., 2005 ; Rothenberg 22 

### and Rothenberg, 2005 ; Bellinger and Needleman, 2003 ; Canfield et al., 2003a )

. Most of 23 

### these epidemiologic results were based on the analysis of concurrent blood Pb levels and 24 

### a cut

- point of 10 μ g/dL to define lower and higher blood Pb levels. These findings were 25 

### corroborated by findings from analyses of the pooled cohort data indic ating that a 26 

### nonlinear relationship fit the data better than a linear relationship did ( Lanphear et al., 27 

### 2005 ; Rothenberg and Rothenberg, 2005 )

. Consisten t with epidemiologic findings, 28 

### toxicological studies observed nonlinear Pb concentration

- response relationships for 29 

### outcomes such as neuronal activation ( Lewis and Pitts, 2004 ) , neurogenesis ( Gilbert et 30 

### al., 2005 ) , and retinal responses ( Fox et al., 1991 ; Fox and Farber, 1988 ; Fox and Chu, 31 

### 1988 )

. 32


### Another area of focus included the comparison of various lifestages of Pb exposure in 33 

### terms of risk of neurodevelopmental deficits. Toxicological studies clearly demonstrated 34 

### that in utero with or without early postnatal exposure to Pb was an especially sensitive 35 

### windo w for Pb

- induced neurodevelopmental effects. Nonetheless, not all endpoints in 36 

### animal toxicology studies had a single defined window of sensitivity but instead were 37 

### shown to be affected by exposures at multiple periods during the lifespan of the 38February 2012 5

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### organism. Epidemiologic studies observed cognitive deficits in children ranging from 2 to 1 

### 10 years of age in association with prenatal, peak childhood, cumulative childhood, and 2 

### concurrent blood Pb levels. Although examined in few studies, tooth or bone Pb levels 3



### we re associated with cognitive and behavioral deficits in children and adolescents 4 

### ( Wasserman et al., 2003 ; Greene and Ernhart, 1993 ; B ellinger et al., 1991 ; Needleman et 5 

### al., 1979 ) , also pointing to an effect of cumulative childhood Pb exposure. Among studies 6 

### of children (ages 2

- 10 years) that examined blood Pb levels measured at multiple 7 

### lifestages, several found that concurrent blood Pb was associated with an equal or larger 8 

### decrement in IQ ( Chen et al., 2005 ; Lanphear et al., 2005 ; Wasserman et al., 1994 ; 9 

### Dietrich et al., 1993b ; Bellinger et al., 1992 )

. A common limitation of epidemiologic 10 

### studies of children was the high correlation among blood Pb levels at different ages, 11 

### making it difficult to ascertain which developmental periods of Pb expos ure were 12 

### associated with the greatest risk of neurodevelopmental decrements ( Lanphear et al., 13 

### 2005 )

. The issue of persistence of the neurodevelopmental effects of Pb exposure also 14 

### was considered, with some evidence suggesti ng that the associations of biomarkers of 15 

### early childhood Pb exposure ( e.g., deciduous tooth, blood at age 2 or 6 years) with 16 

### neurodevelopmental outcomes persisted into adolescence and young adulthood ( Ris et al., 17 

### 2004 ; Tong et al., 1996 ; Needleman et al., 1990 )

. Some studies in rats and monkeys also 18 

### demonstrated that the effects of in utero and early postnatal Pb exposures on 19 

### neurodevelopmental outcomes persisted into adulthood ( Cory

- Slechta, 1994 ; Altmann et 20 

### al., 1993 ; Rice and K arpinski, 1988 ; Gilbert and Rice, 1987 )

. 21 

### In epidemiologic studies of adults, a range of nervous system effects ( e.g., impaired 22 

### memory, attention, reaction time, visuomotor tasks and reasoning, alterations in visual or 23 

### brainstem evoked potentials, p ostural sway) were mostly clearly indicated in Pb

- exposed 24 

### workers with blood Pb levels in the range of 14 to 40 μg/dL ( Iwata et al., 2005 ; Bleecker 25 

### et al., 1997 ; Baker et al., 1979 ; Cantarow and Trumper, 1944 )

. In the limited literature 26 

### examining nonoccupationally

- exposed adults, the weight of evidence supported 27 

### associations of bone Pb levels with cognitive function ( Weisskopf et al., 2004 ; Wright et 28 

### al., 2003b ) but not concurrent blood Pb levels ( Krieg et al., 2005 ; Nordberg et al., 2000 ; 29 

### Payton et al., 1998 ; Muldoon et al., 1996 )

. These findings suggested that rather than 30 

### recent exposures, past or cumulative Pb exposures contributed to cognitive deficits in 31 

### nonoccupationally

- exposed adults. With regards to neurodegenerative diseases, whereas a 32 

### few toxicological studies de monstrated Pb

- induced amyloid plaques commonly 33 

### associated with Alzheimer’s disease pathophysiology ( Basha et al., 2005 ; Zawia and 34 

### Basha, 2005 ) , epidemiologic studies did not indicate that Pb exposure was associated 35 

### with Alzheimer's Disease in adults. Pb biomarker (blood or bone) levels were 36 

### inconsistently associated with amyotrophic lateral sclerosis (ALS) in adults in the g eneral 37 

### population; however, some case

- control studies found that history of occupational Pb 38 

### exposure was more prevalent among ALS cases than controls ( Kamel et al., 2002 ; 39February 2012 5

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### Chancellor et al., 1993 )

. Associations were reported for essential tremor and symptoms of 1 

### anxiety and depression, but each was examined in only a few studies. 2 

### As discussed throughout this section, recent epidemiologic and toxicological studies 3 

### continued to demonstrate associations of Pb exposure and biomarkers of Pb exposure 4 

### with nervous system effects. The weight of evidence continued to be derived from 5 

### associations observed of Pb exposure and blood Pb levels in young animals and children, 6 

### respectively, with cognitive function dec rements, inattention, and impulsivity. Expanding 7 

### upon the previous body of evidence, several recent studies in children found similar 8 

### associations with lower population mean (or quantile) blood Pb levels (1

- 5 μ g/dL). 9 

### Whereas previous evidence was inconsist ent, several new studies in children reported 10 

### associations between concurrent blood Pb levels and attention deficit hyperactivity 11 

### disorder (ADHD). Recent studies in adults focused primarily on cognitive function 12 

### decrements but also provided additional evid ence for Pb

- associated mood disorders, 13 

### ALS, Parkinson's Disease, and essential tremor. New toxicological studies expanded 14 

### evidence for the effects of prenatal and postnatal Pb exposure on learning, memory, and 15 

### attention and provided insight into the contri bution of social stress to this paradigm. New 16 

### or expanded areas of toxicological research related to Pb exposure included mood 17 

### disorders, neurofibrillary tangle formation, and adult dementia after early life Pb 18 

### exposures. Historically important areas of to xicological research were further expanded 19 

### with recent findings for Pb

- induced effects on neurotransmitters, synapses, glia, neurite 20 

### outgrowth, the blood brain barrier, and oxidative stress. The data detailed in the 21 

### subsequent sections continue to enhance the understanding of nervous system effects 22 

### associated with Pb exposure. 23



### 5.3.2 Cognitive Function and Learning

5.3.2.1 Epidemiologic Studies of Cognitive Function in Children
Epidemiologic studies have assessed global cognitive function most frequently by full

24 

### scale IQ (FSIQ) and its verbal and performance subscale components in children ages 3 25 

### to 17 years and by the Bayley Scales of Infant Development in children ages 6 months to 26 

### 3 years. These indices have strong psychometric properties and are among the most 27 

### rigorousl y standardized measures. A large body of evidence also comprises associations 28 

### of blood Pb levels with specific cognitive abilities, including memory and learning, 29 

### executive function, language, and visuospatial processing. These specific indices of 30 

### cognitiv e function are reflected in global measures of intelligence and also are more 31 

### comparable to tests of learning and memory in animals. Fewer studies have examined 32February 2012 5

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### academic performance and achievement; however, these outcomes may provide 1 

### information on the im pact of Pb exposure on life success. The epidemiologic evidence for 2 

### each of these categories of outcomes was evaluated separately in order of increasing 3 

### weight of evidence. Emphasis was placed on prospective studies with repeated 4 

### assessments of blood Pb le vels and cognitive function and on studies examining blood Pb 5 

### levels similar to those measured in contemporary U.S children ( i.e., less than 5 μ g/dL), 6 

### and younger children whose blood Pb levels are less influenced by higher past Pb 7 

### exposures. 8 

### Many factors have been shown to influence the intellectual abilities of children, including 9 

### parental SES, parental education, parental IQ, quality and stability of the caregiving 10 

### environment, nutritional status, and birth weight ( Nation and Gleaves, 2001 ; Wasserman 11 

### and Factor

- Litvak, 2001 )

. These and other influences on neurodevelopment often are 12 

### correlated with blood Pb levels. Thus, due to their association with both blood Pb level 13 

### and outcome, these other risk factors may potentially confound ( i.e., bias due to an 14 

### association with blood Pb level and causal association with the outcome) the associations 15 

### observed between blood Pb level and indices of cognitive function. In the evaluation of 16 

### the effect of Pb independent from the effects of the other variables, greater weight was 17 

### given to studies that accounted for potential confounding in the study design or in 18 

### statistical analyses. A detailed discussion of the collective weight of evidence for the 19 

### independent associations between Pb exposure and nervous system effects in relation to 20 

### the adequacy of control for confounding by other risk factors is located in Section 5. 3.11

. 21



### Full
- scale IQ in Children Several longit udinal cohort studies were initiated in the 1980s in order to address 22 

### limitations of cross

- sectional studies, including establishing a temporal association 23 

### between blood Pb levels and cognitive outcomes, examining the persistence of cognitive 24 

### deficits to o lder ages, and comparing risk estimates among blood Pb levels measured at 25 

### different lifestages. Moreover, cooperation among investigators to adopt similar 26 

### assessment protocols facilitated pooled and meta

- analyses and comparison of results 27 

### across population s that differed in the range of blood Pb levels, race/ethnicity, and SES. 28 

### Individual cohort studies in diverse populations were consistent in demonstrating 29 

### associations between higher blood Pb and lower FSIQ in populations of school

- aged 30 

### children with mean blood Pb levels in the range of 5 to 10 μ g/dL ( Schnaas et al., 2006 ; 31 

### Bellinger and Needleman, 2003 ; Canfield et al., 2003a ; Wasserman et al., 1997 ; Dietrich 32 

### et al., 1993a ; Baghurst et al., 1992 ; Bellinger et al., 1987 ) ( Figure 5

- 2 and Table 5

- 3 ). In 33 

### analyses restricted to children in the lower range of the blood Pb distribution ( e.g., < 34 

### 10 μ g/dL), associations were observed in groups of children with mean blood Pb levels 35 

### 3

- 4 μ g/dL ( Bellinger, 2008 ; Canfield, 2008 ; Hornung, 2008 )

. Across cohort studies, 36February 2012 5

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### associations were observed with concurrent, prenatal, early childho od ( e.g., age 2 years), 1 

### and childhood average blood Pb levels ( e.g., 0

- 8 year average) ( Figure 5

- 2 and Table 2 

### 5

- 3 ). These findings were substantiated in a pooled analysis of seven prospective studies 3 

### (Boston, MA; Cincinnati, OH; Rochester, NY; Cleveland, OH; Mexico City, Mexico; 4 

### Port Pirie, Australia ; and Kosovo, Yugoslavia) by Lanphear et al. ( 2005 ) as well as 5 

### multiple meta

- analyses that included both prospective and cross

- sectional studies 6 

### ( Pocock et al., 1994 ; Schwartz, 1994 ; Needleman and Gatsonis, 1990 )

. 7

Note: Studies generally are presented in ascending order of mean blood Pb level. To facilitate comparisons among effect estimates across studies with different distributions of blood Pb levels and model structures ( e.g., linear, log

- linear), effect estimates are standardized to a 1 μg/dL increase in blood Pb level within the 10th to 90th percentile interval. The percentiles are estimated using various methods and are only approximate values. Effect estimates are assumed to be linear within the 10th to 90th percentile interval of blood Pb level. The various tests used to measure FSIQ are scored on a similar scale (approximate ly 40

- 160). Black diamonds, blue circles, orange triangle, and gray squares represent associations with concurrent, earlier childhood, prenatal , and lifetime average blood Pb levels, respectively. 

### Figure 5
- 2 Associations of blood Pb levels with full
- scale IQ (FSIQ) among children. Study Kim et al. (2009) Chiodo et al. (2007) Chiodo et al. (2004) Jusko et al. (2011) Canfield et al. (2003) Bellinger et al. (1992) Kordas et al. (2011) Lanphear et al. (2005) Min et al. (2009) Min et al. (2009) Min et al. (2009) Schnaas et al. (2006) Kordas et al. (2011) Roy et al. (2011) Dietrich et al. (1993) Tong et al. (1996) Wasserman et al. (1997) Baghurst et al. (1992) Blood Pb Timing
Concurrent
Concurrent
Concurrent
Concurrent
Concurrent 2 yr Prenatal (cord)
Concurrent
Concurrent 4 yr 4 yr Prenatal (maternal)
Concurrent
Concurrent
Concurrent
0 to 11

- 13 yr avg 0 to 7 yr avg 0 to 8 yr avg B lood Pb Mean (SD) (μg/dL) 1.73 (0.80) 5.0 (3.0) 5.4 (3.3) 5.0 (3.3) 5.8 (4.1) 6.5 (4.9) 6.6 (3.3) 6.9 (1.2) 7.0 (4.1) 7.0 (4.1) 7.0 (4.1) 7.8 (geometric) 8.7 (4.4) 11.4 (5.4) 11.8 (6.3) (Age 5) 14.0 (1.2) geometric 16.2 (geometric) 16.6 (mean of 25th

- 50th) Blood Pb 10th
- 90th percentile (μg/dL) <1

- 2.8
2.1

- 8.7
2.3

- 9.5
1.9

- 9.0
2.1

- 10.7
2.2

- 12.3
1.2

- 2.4
3.4

- 25.4
3.0

- 12.1 3.0

- 12.1 3.0

- 12.1
3.2

- 19.1
1.4

- 2.7
5.8

- 18.4
5.5

- 19.7
12.7

- 18.1
6.0

- 44.2
10

- 27.5 FSIQ age (yr)
8

- 11
7
7.5
6
5
10
4
4.8

- 10
4 9 11
6

- 10
4
3

- 7
6.5
11

- 3
7
7

- 8

- 1.5

- 1

- 0.5 0 Change in full scale IQ per 1 μg/dL increase in the 10th to 90th percentile of blood Pb level (95% CI)February 2012 5

- 58 Draft

– Do Not Cite or Quote 

### Table 5
- 3 Additional characteristics and quantitative results for studies represented in Figure 5
- 2

Study Population/ Location Blood Pb Data (μg/dL) Statistical Analysis FSIQ Assessment a Effect Estimate (95% CI) b,c
Kim et al. ( 2009a ) 279 children in Seoul, Seongnam, Ulsan, and Yeoncheon, Korea, ages 8

- 11 yr in A pril

- December 2007 Concurrent Mean (SD): 1.73 (0.80) 10th

- 90th: < 1

- 2.8 Log linear regression model adjusted for age, sex, maternal education, paternal education, yearly income, maternal smoking during pregnancy, indirect smoking after birth, birth weight, maternal age KEDI

- WISC at ages 8

- 11 yr

- 0.62 (

- 1.05,

- 0.19)

Chiodo et al. ( 2007 ) 506 Afri can - American children in Detroit, MI area followed from birth (1989

- 1991) to age 7 yr. Large proportions of children with prenatal exposure to cocaine or marijuana Concurrent Mean (SD): 5.0 (3.0) 10th

- 90th: 2.1

- 8.7 Regression model adjusted for prenatal a lcohol and drug use, SES, Symptom Checklist (component of HOME score), maternal IQ WISC

- III at age 7 yr

- 0.19 (

- 0.30,

- 0.08) b

Chiodo et al. ( 2004 ) 246 African

- American children in Detroit, MI area followed from birth (not reported) to age 7 .5 yr. Large proportions of children with prenatal exposure to cocaine or marijuana Concurrent Mean (SD): 5.4 (3.3) 10th

- 90th: 2.3

- 9.5 Log linear regression model adjusted for SES, education, number of children <18 yr, HOME score, maternal vocabulary test score, sex, parity, family environment scale WISC

- III at age 7.5 yr

- 0.22 (

- 0.38,

- 0.05) b

Jusko et al. ( 2011 ) 194 children in Rochester, NY followed from age 6 mo (1994

- 1995) to age 6 yr. Concurrent Mean (SD): 5.0 (3.3) 10th

- 90th: 1.9

- 9.0 Linear regression model adjusted for sex, birth weight, transferrin saturation, maternal race, maternal IQ, maternal education, HOME score, family income, and maternal pr enatal smoking WPPSI

- R at age 6 yr

- 0.66 (

- 1.26,

- 0.06)

Canfield et al. ( 2003a ) 172 children in Rochester, NY born 1994

- 1995 followed from age 6 mo to age 5 yr. Concurrent Mean (SD): 5.8 (4.1) 10th

- 90th: 2.1

- 10.7 Linear regression model adjusted for sex, maternal race, prenatal smoking, maternal education, child iron status, household income, maternal IQ, HOME score, birth weight Stanford

- Binet at age 5 yr

- 0.61 (

- 0.99,

- 0.24)

Bellinger et al. ( 1992 ) 148 children in the Boston, MA area followed from birth (1979

- 1981) to age 15

- 17 yr. Early childhood (age 2 yr) Mean (SD): 6.5 (4.9) 10th

- 90th: 2.2

- 12.3 Linear regression model adjusted for HOME score (age 10 and 5 yr), child stress, race, maternal IQ, SES, sex, birth order, marital status WISC

- R at age 10 yr

- 0.58 (

- 0.99,

- 0.18)

Lanphear et al. ( 2005 ) 1 , 333 children pooled from Boston, Cincinnati, Cleveland, Mexico City, Port Pirie, Rochester, and Yugoslavia cohorts Concurrent Mean (SD): 6.9 (1.2) 10th

- 90th: 3.4

- 25.4 Log linear regression model adjusted for HOME score, birth weight, maternal IQ, maternal education FSIQ measured at ages 5

- 10 yr

- 0.25 (

- 0.34,

- 0.15)

Min et al. ( 2009 ) 267 primarily African

American children in the Cleveland, OH area followed from birth (1994

- 1996) to age 11 yr. Children were exposed prenatally to mu ltiple drugs. Age 4 yr Mean (range): 7.0 (1.3

- 23.8) 10th

- 90th: 3.0

- 12.1 Linear regression model adjusted for HOME score, caregiver's vocabulary test, sex, parity, maternal marital status, head circumference at birth WISC

- R at age 4 yr (concurrent) WISC

- R at age 9 yr WISC

- R at age 11 yr - 0.50 (

- 0.89,

- 0.11)

- 0.41 (

- 0.78,

- 0.04)

- 0.54 (

- 0.91,

- 0.17)

Schnaas et al. ( 2006 ) 150 children in Mexico City, Mexico followed from birth (1987

- 1992) to age 10 yr. Prenatal (maternal 28

- 36 weeks) Geometric mean (95% CI): 7.8 (2.5

- 24.5) 10th

- 90th: 3.2

- 19.1 Log linear mixed effects regression model adjusted for sex, SES, maternal IQ, HOME score, birth weight, postnatal blood Pb, random slope for subject McCarthy GCI at ages 6

- 10 yr

- 0.44 (

- 0.73,

- 0.15)February 2012 5

- 59 Draft

– Do Not Cite or Quote Study Population/ Location Blood Pb Data (μg/dL) Statistical Analysis FSIQ Assessment a Effect Estimate (95% CI) b,c
Kordas et al. ( 2011 ) 186 children in Mexico City, Mexico followed from birth (1994

- 1995) to age 4 yr Prenatal (cord) Mean (SD): 6.6 (3.3) 10th

- 90th: 1.2

- 2.4 Co ncurrent Mean (SD): 8.7 (4.4) 10th

- 90th: 1.4

- 2.7 Linear regression model adjusted for sex, birth weight, gestational age, maternal age, years of schooling, IQ, smoking status, marital status, crowding in home, type of floor in home McCarthy GCI at age 4 yr Prenatal Concurrent - 0.20 (

- 0.79, 0.39)

- 0.60 (

- 0.99,

- 0.21)

Roy et al. ( 2011 ) 717 children in Chennai, India ages 3

- 7 yr Concurrent Mean (SD): 11.4 (5.4) 10th

- 90th: 5.8

- 18.4 Log linear model adjusted for mid

- arm circumference, age, sex, family income, parental education, parental IQ, family size Binet

- Kamat at ages 3

- 7 yr

- 0.39 (

- 0.65,

- 0.13)

Dietrich et al. ( 1993a ) 253 children in Cincinnati, OH followed from birth (1979

- 1985) to age 20

- 23 yr. Concurrent NR Age 5 yr Mean (SD): 11.8 (6.3) 10th

- 90th: 5.5

- 19.7 Linear regression model ad justed for HOME score, maternal IQ, birth weight, birth length, child sex, maternal cigarette consumption during pregnancy WISC

- R at age 6.5 yr

- 0.33 (

- 0.60,

- 0.06)

Tong et al. ( 1996 ) 375 children in Port Pirie, Australia followed f rom birth (1979

- 1982) to age 11

- 13 yr. Lifetime avg Geometric mean (GSD): 14.0 (1.2) 10th

- 90th: 12.7

- 18.1 Regression model adjusted for sex, age, school grade, parental occupational prestige, HOME score, maternal IQ, family functioning score, parental smok ing, marital status, parental education, maternal age, birth weight, birth order, feeding method, breastfeeding duration, family size, life events, prolonged absences from school WISC

- R at age 11

- 13 yr

- 0.12 (

- 0.24,

- 0.003)

Wasserman et al. ( 1997 ) 290 children in Kosovo, Yugoslavia followed from birth (1985

- 1986) to age 10

- 12 yr. Lifetime avg Geometric mean: 16.2 10th

- 90th: 6.0

- 44.2 Generalized estimating equations with log

- transformed blood Pb adjusted for age, sex, sibship size, birth weight, language spoken in home, HOME score, maternal age, maternal education, maternal Raven score WISC

- III at ages 10

- 12 yr

- 0.20 (

- 0.28,

- 0.11)

Baghurst et al. ( 1992 ) 494 children in Port Pirie, Australia followed from birth (1979

- 1982) to age 11

- 13 yr. Lifetime avg Mean of 25

- 50th: 16.6 10th

- 90th: 10

- 27.5 Log linear regression model adjusted for sex, birth weight, birth order, feeding method, breastfeeding duration, parental education, maternal age, parental smoking, SES, quality of home environment, maternal IQ, parents living together WISC

- R at age 7

- 8 yr

- 0.20 (

- 0.40, 0.10)

a WISC = Wechsler Intell igence Scale for Children, WPPSI = Wechsler Preschool and Primary Scale of Intelligence, GCI = General Cognitive Index b Effect estimates are standardized to a 1 μg/dL increase in blood Pb level within the 10th to 90th percentile interval. Effect estimates are assumed to be linear within the 10th to 90th percentile interval of blood Pb level. The percentiles are estimated using various methods and are only approximate values.
c 95% CI was constructed using a standard error that was estimated for a p

- value of 0.01. Authors specified a p

- value of <0.01.


### The analysis pooling data from seven prospective studies included 1 , 333 children 1 

### ages 5

- 10 years of age with a median (5th

- 95th percentile) concurrent blood Pb level of 2 

### 9.7 μg/dL (2.5

- 33.2 μg/dL) ( Lanphear et al., 2005 )

. In multivariate models that adjusted 3 

### for study site, maternal IQ, Home Observation for the Measurement of Environment 4 

### (HOME) inventory (assessment of physical environment, parental responsivity, lea rning 5 

### stimulation, emotional climate, and family interactions), birth weight, and maternal 6 

### education, higher concurrent, peak, average lifetime, and early childhood blood Pb levels 7 

### were associated with lower FSIQ measured at age 5

- 10 years, with the larges t decrement 8 

### in FSIQ estimated for concurrent blood Pb level (

- 0.25 points [95% CI:

- 0.34,

- 0.15] per 9 

### 1 μg/dL increase in blood Pb level in the 10th to 90th percentile interval [2.2

- 12.3 μg/dL] 10February 2012 5

- 60 Draft

– Do Not Cite or Quote 

### of blood Pb level) 1 

### . Various models were investigated to charac terize the shape of the 1 

### blood Pb concentration

- response relationship. Consistent with findings from several 2 

### individual cohort studies, Lanphear et al. ( 2005 ) found that a log

- linear model best fit the 3 

### data, with a greater de crease in FSIQ estimated for an increase in concurrent blood Pb 4 

### from 2.4

- 10 μg/dL (3.9 points [95% CI: 5.3, 2.4]) than an increase from 10 to 20 μg/dL 5 

### (1.9 points [95% CI: 2.6, 1.2]). Among children with concurrent blood Pb less than 6 

### 10 μg/dL, the median b lood Pb level was 4.2 μg/dL ( Hornung, 2008 )

. 7 

### A key additional strength of the pooled analysis by Lanphear et al. ( 2005 ) was the 8 

### extensive examination of the potential for confounding by several factors related to SES 9 

### and the caregiving environment. Variables such as HOME score, birth weight, maternal 10 

### IQ, and maternal education were statistically significantly associated with FSIQ and were 11 

### included in the f inal model with blood Pb level. While a smaller decrement in FSIQ was 12 

### estimated for concurrent blood Pb level in this adjusted model than in the unadjusted 13 

### model (

- 0.43 points [95% CI:

- 0.53,

- 0.33] per 1 μg/dL increase in blood Pb level in the 14 

### 10th to 90t h percentile interval of blood Pb level), the adjusted blood Pb effect estimate 15 

### was nonetheless statistically significant. HOME score was not available in the Rochester 16 

### study; however, exclusion of data from that cohort resulted in a 3% less negative effec t 17 

### estimate, indicating the lack of a strong influence of HOME score alone on blood Pb 18 

### level

- IQ associations. Other variables such as child sex, tobacco exposure during 19 

### pregnancy, alcohol use during pregnancy, maternal age at delivery, marital status, and 20 

### b irth order were not statistically significantly associated with FSIQ and did not alter the 21 

### effect estimate for concurrent blood Pb level. The individual study populations 22 

### represented a wide range of SES, maternal education, and cultural backgrounds. 23 

### Sensit ivity analyses, in which one study was successively excluded, revealed that no 24 

### single study was responsible for driving the results. Per 1 μg/dL increase in blood Pb 25 

### level in the 10th to 90th percentile interval of blood Pb level, effect estimates excludin g 26 

### one study at a time ranged between

- 0.22 and

- 0.27, indicating the robustness of the 27 

### concurrent blood Pb level effect estimate despite between

- study variability in the 28 

### distributions of potential confounding factors. 29 

### The small number of studies published since the 2006 Pb AQCD continued to 30 

### demonstrate associations between higher blood Pb level (primarily concurrent) and lower 31 

### FSIQ in children between ages 3 and 11 years ( Figure 5

- 2 and Table 5

- 3 ). Similar to 32 

### studies reviewed in the 2006 Pb AQCD, most recent studies demonstrated associations 33 

### between blood Pb level and lower FSIQ in populations with mean blood Pb level between 34 

### 5 to 10 μg/dL. New results from the prospective cohorts were limited. Mazumdar et al. 35

1 To facilitate comparisons among effect estimates across studies with different distributions of blood Pb levels and model structures (e.g., linear, log

- linear), e ffect estimates are standardized to a 1 μg/dL increase in blood Pb level within the est imated 10th to 90th percentile interval

. Effect estimates are assumed to be linear within the 10th to 90th percentile interval of blood Pb level.February 2012 5

- 61 Draft

– Do Not Cite or Quote 

### ( 2011 ) reported on the follow

- up of the Boston cohort to age 28

- 30 years (55 out of the 1 

### original 249 enrolled at birth). Blood Pb levels measured at age 6 months, 4 years, 2 

### 10 years, and levels averaged over childhood were associated with decrements in FSIQ in 3 

### adults. The effect estimates were similar in magnitude for all childhood blo od Pb 4 

### measures, except for 6 month blood Pb level, which was associated with a smaller FSIQ 5 

### decrement. These findings indicated that the effect of childhood Pb exposures may persist 6 

### to adulthood. 7


### Jusko et al. ( 2011 ; 2008 ) affirmed the findings in the Rochester cohort previously 8 

### reported by Canfield et al. ( 2003a ) , who examined the cohort at age 5 years ( Figure 5

- 2 9 

### and Table 5

- 3 ). Jusko et al. ( 2011 ) examined the Rochester cohort at age 6 years and, 10 

### similar to Canfield et al. ( 2003a ) , found that a 1 μg/dL increase in concurrent blood Pb 11 

### level was associated with a 0.66

- point decrease (95% CI:

- 1.26,

- 0.06) in FSIQ. Model 12 

### covariates were the same as those analyzed by Canfield et al. ( 2 003a ) : sex, race, family 13 

### income, maternal education, race, prenatal smoking, birth weight, transferrin saturation, 14 

### maternal IQ, and HOME score. At both age 5 and 6 years, higher peak and lifetime 15 

### average blood Pb levels also were associated with lower F SIQ, and incremental increases 16 

### in blood Pb levels tended to be associated with larger FSIQ decrements in analyses 17 

### restricted to children with blood Pb levels less than 10 μg/dL ( Jusko et al., 2008 ; Canfield 18 

### et al., 2003a )

. Canfield et al. ( 2003a ) provided additional information on the extent of 19 

### confounding by SES

- and caregiving

- related variables. The effect estimate in the 20 

### covariate

- adjusted model was 40% smaller than it was in the model with concurrent 21 

### blood Pb level alone; however, the associati on with concurrent blood Pb level remained 22 

### statistically significant. 23 

### Additional evidence recently was provided for children in Mexico City, albeit in a 24 

### separate cohort of children born later with lower blood Pb levels at corresponding ages. 25 

### Among childre n born 1987

- 1992, Schnaas et al. ( 2006 ) previously reported larger Pb

26 

### associated decrements in FSIQ for prenatal maternal (28

- 36 weeks) blood Pb levels than 27 

### for concurrent blood Pb levels between ages 1 and 10 years. In contrast, Kordas et al. 28 

### ( 2011 ) found that concurrent blood Pb level was associated with a larger decrement in 29 

### FSIQ at age 4 years than was cord blood Pb level. Children in the lat ter study were born 30 

### between 1994 and 1995 and at age 4 years had a mean (SD) blood Pb level of 31 

### 8.7 (4.4) μg/dL. In Schnaas et al. ( 2006 ) , the geometric mean (95% CI) blood Pb level at 32 

### age 4 years was 10.3 (4.2, 20.5) μg/dL. It is not clear whether different temporal patterns 33 

### of Pb exposure may have contributed to the contrasting associations for pr enatal and 34 

### concurrent blood Pb levels in the two studies. 35


### Surkan et al. ( 2007 ) examined children from urban Boston, Massachusetts and rural 36 

### Farmington, Maine participating in a trial designed to assess the effect of a malgam dental 37February 2012 5

- 62 Draft

– Do Not Cite or Quote 

### fillings on child neurodevelopment. Prior to placement of amalgam fillings, blood Pb 1 

### levels were measured, and children were administered an extensive battery of 2 

### neuropsychological tests including tests of memory, learning, visual

- motor abili ty, 3 

### reading, reaction time. A strength of the study was that the range of concurrent blood Pb 4 

### level in this study, 1

- 10 μg/dL, was lower than that examined in previous studies. Thus, it 5 

### provided information on associations between blood Pb level and IQ dec rements within a 6 

### lower range of blood Pb levels. However, consistent with several previous studies, 7 

### Surkan et al. ( 2007 ) found lower FSIQ in the group of children with concurrent blood Pb 8 

### levels of 5 to 10 μg/dL. For example, adjusting for age, race/ethnicity, birth weight, SES, 9 

### and primary caregiver IQ, children with blood Pb levels 5

- 10 μg/dL had lower FSIQ 10 

### scores (

- 6.04 points [95% CI:

- 10.7,

- 1.36]) compared with children who had levels 11 

### 1

- 2 μg/dL (referent group). Children with blood Pb levels 2

- 5 μg/dL did not have FSIQ 12 

### scores (

- 0.12 [95% CI:

- 3.30, 3.06]) that differed from children with blood Pb levels 13 

### 1

- 2 μg/dL. Another strength of this study was the analysis of potential confounding by a 14 

### larger list of variable s than that included in the final models. Although HOME score was 15 

### not examined, several variables such as SES, caregiver education, parenting stress, 16 

### marital status of caregiver, and maternal utilization of prenatal or annual health care were 17 

### not significa ntly (p > 0.20) associated with child IQ either independently or in a model 18 

### with blood Pb level ( Surkan et al., 2007 )

. 19


### Other recent studies found blood Pb

- associated lower FSIQ in populations of children in 20 

### Asia with l ower blood Pb levels ( Kim et al., 2009b ; Zailina et al., 2008 ) than were 21 

### previously examined. Kim et al. ( 2009b ) examined children ages 8 to 11 years in Korea 22 

### (born mid

- to late

- 1990s) with a mean (range) concurrent blood Pb level of 1.73 23 

### (0.42

- 4.91) μg/dL. Children were tested using the Korean Educational Development 24 

### Institute

- WISC, which assesses vocabulary, arithmetic, picture arrangement, and block 25 

### design. In a log

- linear regression analy sis adjusted for age, sex, maternal and paternal 26 

### education, yearly income, prenatal smoking, postnatal environmental tobacco smoke 27 

### exposure, birth weight, and maternal age at birth, a 1 μg/dL higher concurrent blood Pb 28 

### level was associated with a 0.64

- poin t lower (95% CI:

- 1.05,

- 0.19) FSIQ within the 10th

29 

### 90th percentile interval of blood Pb level (< 1 to 2.8 μg/dL). The potential confounders 30 

### examined in this study were not found to have a strong influence on the findings as a 31 

### similar magnitude of effect w as estimated in a model that included only blood Pb level 32 

### (

- 0.73 points [95% CI:

- 1.19,

- 0.27] per 1 μg/dL increase in blood Pb level in the 10th to 33 

### 90th percentile interval). Although several important SES

- related confounders were 34 

### considered, there was no direct assessment of the home environment and the primary 35 

### caregiver IQ in this study, which are notable limitations. 36


### Kim et al. ( 2009b ) also examined effect modifi cation of the blood Pb

- FSIQ relationship 37 

### by concurrent blood manganese (Mn) levels. The mean (range) blood Mn level was 38February 2012 5

- 63 Draft

– Do Not Cite or Quote 

### 14.3 (5.3

- 29.02) μg/dL. Blood Pb and M n levels were not correlated (r = 0.03, p = 0.64). 1 

### To examine effect modification, children were d ivided into two groups: blood Mn level 2 

### above and below the median (14 μg/dL). Multivariate linear regression models predicting 3 

### FSIQ and verbal IQ (VIQ) used concurrent blood Pb level as the predictor variable in the 4 

### low and high Mn groups. As illustrated i n Figure 5

- 3, the associations of concurrent 5 

### blood Pb level with FSIQ and VIQ was larger in magnitude and statistically significant 6 

### for children in the high Mn group compared with children in the low Mn group (

- 0.65 7 

### points [95% CI:

- 1.26,

- 0.05] in high Mn group versus
- 0.50 points [95% CI:

- 1.23, 0.22] 8 

### in low Mn group per 1 μg/dL increase in blood Pb level in the 10th to 90th percentile 9 

### interval). However, higher blood Pb level was associated with lower FSIQ in children in 10 

### the low Mn group. 11

Source: Repri nted with permission of Elsevier Science, Kim et al. ( 2009b )

. Note: High and low Mn refer to levels above and below the median of 14 μg/dL, respectively. 

### Figure 5
- 3 Effect modification of the association between concurrent blood Pb level and FSIQ by blood Mn level.February 2012 5

- 64 Draft

– Do Not Cite or Quote 

### Higher concurrent blood Pb level also was associated with a lower IQ score (as assessed 1 

### by the McCarthy G eneral Cognitive Index) among children ages 6

- 8 years in Malaysia 2 

### with a mean blood Pb level of 3.69 μg/dL ( Zailina et al., 2008 )

. The concentr ation

3 

### response function was not reported; however, in a multivariate model, blood Pb level and 4 

### not potential confounding factors such as paternal education, maternal education, 5 

### household income, number of siblings, and birth order was reported to be signif icantly 6 

### associated with FSIQ. As with Kim et al. ( 2009b ) , a notable limitation was the lack of 7 

### consideration of confounding by the caregiving environment. 8


### Pb has lo ng been known to impact dopaminergic neurons, inhibit depolarization

- evoked 9 

### neurotransmitter release, and stimulate spontaneous neurotransmitter release 10 

### (Section 5.3.8.8 ). Further, dopaminergic activity is a key process in regulating cognitive 11 

### function. Thus, variants in dopamine

- related genes have the potential to act as effect 12 

### modifiers of Pb

- associated neu rodevelopmental effects. Recent studies in children that 13 

### examined effect modification by variants in dopamine genes produced contrasting results. 14 

### In a study of children at ages 2 and 4 years, Kordas et al. ( 2011 ) did not find differences 15 

### in association between concurrent blood Pb level and FSIQ by the Ta q A1/A1 dopamine 16 

### receptor 2 (DRD2) genotype, which is associated with reduced dopamine receptor 17 

### density. Roy et al. ( 2011 ) examined children of similar age (3

- 7 years) in Chennai, India 18 

### and found that among children with the Taq A1/A1 genotype, a 1 μg/dL higher blood Pb 19 

### level was associated with a 0.84

- point lower (95% CI:

- 1.66,

- 0.01) FSIQ within the 10th

20 

### 90th percentile interval of blood Pb level (5.8

- 18.3 μg/dL). The same increment in blood 21 

### Pb level was associated with a 0 .36

- point lower (95% CI:

- 0.76,

- 0.04) FSIQ in children 22 

### with the Taq A2/A2 higher receptor density genotype. Concurrent blood Pb level was 23 

### associated with FSIQ in both studies in analyses including all subjects. However, there 24 

### were many differences between study populations that may have contributed to 25 

### differences in effect modification by the DRD2 variant. Compared with the group in 26 

### India, the group in Mexico had a lower mean blood Pb level and lower mean FSIQ score. 27 

### Additionally, children in the Mexico Ci ty group with the Taq A1/A1 genotype had a 28 

### higher mean FSIQ score, whereas FSIQ scores were similar between Taq genotypes in 29 

### the children in India. 30 

### Other recent studies examined children in the U.S.; however, in these studies, there were 31 

### high proportions o f children with prenatal exposure to alcohol, tobacco, marijuana, or 32 

### cocaine that may limit the generalizability of findings. Chiodo et al. ( 2007 ) examined a 33 

### population of African American children (age 7 years, born 1989

- 1991) in the Detroit, 34 

### MI area with relatively low blood Pb level (mean [SD]: 5.0 [3.0]) but high prevalence of 35 

### prenatal exposure to co caine (38%) or marijuana (35%). In a detailed analysis of potential 36 

### confounding factors, investigators found that cocaine exposure did not meet the criterion 37 

### for inclusion in the model ( i.e., p > 0.10 for association with FSIQ). Prenatal marijuana 38February 2012 5

- 65 Draft

– Do Not Cite or Quote 

### exposure was associated with FSIQ (p < 0.10) and was included in a multivariate model 1 

### along with caretaker education, SES, HOME score, symptom checklist, number of 2 

### children in the home, maternal IQ, sex, and prenatal cigarette smoking. In this 3 

### multivariate model, higher blood Pb level was associated with lower FSIQ ( Figure 5

- 2 4 

### and Table 5

- 3 ). These findings supported an independent association of blood Pb level 5 

### despite the high prevalence of prenatal drug exposure in the study population. 6 

### Min and colleagues ( 2009 ) followed children in Cleveland, OH from age 4 to 11 years 7 

### who were exposed prenatally to alcohol (77%), cigarette smoking (61%), marijuana 8 

### (31%), or cocaine (51%). The study population was primarily African

- American (86%) 9 

### and of low SES (98%); 39% of mothers had not finished high school, and 86% were 10 

### unmarried at the time of enrollment. The mean (range) blood Pb level, measured at age 11 

### 4 years, was 7.0 μg/dL (1.3

- 23.8 ). Several lines of evidence indicated that prenatal drug 12 

### exposure did not heavily influence the association between blood Pb level and FSIQ. 13 

### Prenatal alcohol exposure was correlated with blood Pb level but was not statistically 14 

### significantly associated wi th IQ (p > 0.10) and did not change the blood Pb level effect 15 

### estimate by more than 10%. Prenatal cocaine exposure was associated with FSIQ at age 16 

### 9 years; however, blood Pb level at age 4 years remained associated with a decrement in 17 

### FSIQ at age 9 years a fter adjusting for cocaine exposure and several SES

- related factors 18 

### ( Table 5

- 3 ). Based on the examination of interaction terms, associations between b lood 19 

### Pb level and FSIQ were not statistically significantly different between drug

- exposed and 20



### - unexposed children. 21 

### Similar to previous studies with repeated assessments of cognitive function over time, 22 

### Min et al. ( 2009 ) found that the association between blood Pb level at age 4 years and 23 

### FSIQ persisted to older ages. Higher blood Pb level at age 4 years was associated with 24 

### similar magnitudes of decreme nts in FSIQ at ages 4, 9 and 11 years ( Figure 5

- 2 and 25 

### Table 5

- 3 ). Researchers also examined the shape of the concentration

- response function 26 

### using a restricted cubic spline function. Although the cubic spline term did not attain 27 

### statistical significance ( p = 0.19), qualitative analysis indicated that the association 28 

### between blood Pb level and FSIQ decrements persisted and was greater at lower levels 29 

### blood Pb (< 7 μg/dL). These findings were consistent with those from the pooled analysis 30 

### ( Lanphear et al., 2005 ) and other individual studies ( Tellez

- Rojo et al., 2006 )

. 31



### Bayley Scales of Infant Development The Bayley Scales of Infant Development are the most widely used tests of infant 32 

### intelligence. The Mental Development Index (MDI) is statistically analogous to IQ, 33 

### i.e., b oth scores have a population

- standardized mean of 100 and standard deviation of 34 

### 15. While MDI assesses general cognitive function in infants, it is important to note that 35February 2012 5

- 66 Draft

– Do Not Cite or Quote 

### MDI scores are not necessarily correlated with IQ scores measured later in childhood. 1 

### Associations between blood Pb levels and MDI scores were found in most of the 2 

### prospective cohort studies in children between age 6 months and 3 years and mean blood 3 

### Pb levels 5

- 10 μg/dL ( Table 5

- 4 ). Recent studies examined children who were born in the 4 

### mid

- 1990s to mid

- 2000s and continued to demonstrate associations between higher cord 5 

### and concurrent blood Pb level and lower MDI scores ( Table 5

- 4 ). Studies that found 6 

### associations with concurrent blood Pb levels also tended to find associations with 7 

### prenatal cord or maternal blood Pb levels. Recent stu dies provided new information on 8 

### effect modification by nutritional status, maternal self

- esteem, co

- exposure to Mn, and 9 

### genetic variants. While studies adjusted for multiple SES

- related variables including 10 

### maternal IQ and education, most did not consider confounding by the caregiving 11 

### environment. Concurrent and cord blood Pb levels were associated with MDI, adjusting 12 

### for HOME score in diverse cohorts ( Solon et al., 2008 ; Wasserman et al., 1992 ; Bellinger 13 

### et al., 1987 )

. In the Cleveland cohort, associations of cord and postnatal blood Pb levels 14 

### with MDI at ages 6 months to 2 years became null after adjusting for covariat es including 15 

### HOME score ( Ernhart et al., 1988 ; Ernhart et al., 1987 )

. In the analysis of the Cincinnati 16 

### cohort, HOME score was not significantly associated with blood Pb level or MDI and 17 

### thus, did not meet the criteria for model inclusion ( Dietrich et al., 1987a )

. Collectively, 18 

### evidence does not indicate that Pb

- associated MDI decrements are driven by confounding 19 

### by quality of the caregiving environment. 20February 2012 5

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– Do Not Cite or Quote 

### Table 5
- 4 Associations of blood Pb level with Bayley MDI in children ages 6 months to 3 years a

Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI) b Jedrychowski et al. ( 2009b ) 444 children born 2001

- 2004 followed prenatally to age 36 mo Krakow, Poland Prenatal (cord blood) Geometric mean (range): 1.29 (0.44

- 5) 10th

- 90th: 1.2

- 1.3 Log linear regression model adjusted for maternal education, birth order, prenatal smoking, sex, and within

- subject MDI correlation at age 12 mo at age 24 mo at a ge 36 mo - 1.9 (

- 3.8, 0.12)

- 2.6 (

- 5.0,

- 0.21)

- 2.3 (

- 4.3,

- 0.30)

Claus Henn et al. 455 children born 1997

- 2000 followed prenatally to age 36 mo Mexico City 12 month Mean (SD): 5.1 (2.6) 10th

- 90th: 2.5

- 8.4 Linear mixed effects regression adjusted for sex, hemoglobin, gestational age, maternal IQ, maternal education, blood Pb

- blood Mn interaction assessed ages 12 to 36 mo 12 mo blood Mn < 2.0 μg/dL 12 mo blood Mn 2.0

- 2.8 μg/dL 12 mo Mn > 2.8 μg/dL - 3.0 (

- 5.22,

- 0.78)

- 0.07 (

- 0.39, 0.25)

- 2.2 (0, 4.44)

Tellez

- Rojo et al. ( 2006 ) 294 children born 1997

- 1999 followed prenatally to age 24 mo Mexico City Same cohort as above Concurrent 12 month Mean (SD): 4.7 (2.9) 10th

- 90th: 1.9

- 8.2 24 month Mean (SD): 5.3 (4.1) 10th

- 90th: 2.1

- 10.7 Log linear regression model adjusted for sex, age, birth weight, maternal IQ, cohort at age 12 mo at age 24 mo - 0.26 (

- 0.79, 0.26)

- 0.89 (

- 1.32,

- 0.46)

Dietrich et al. ( 1993a ) 96 to 302 ch ildren born 1979

- 1984 followed prenatally to age 6 mo Cincinnati, OH Prenatal (cord) Mean (SD): 6.3 (4.5) 10th

- 90th: 2.3

- 11.7 Neonatal (10 day) Mean (SD): 4.6 (2.8) 10th

- 90th: 1.9

- 8.1 3 month Mean (SD): 5.9 (3.4) 10th

- 90th: 2.6

- 10.1 Linear regression model adjusted for birth weight, gestation, maternal age, race, sex, SES assessed at 6 mo Cord blood Pb Neonatal blood Pb 3

- month blood Pb - 0.66 (

- 1.4, 0.07)

- 3.49 (

- 6.0, 0.96)

- 0.48 (

- 1.0, 0.05)

Bellinger et al. ( 1987 ) 249 children born 1979

- 1981 followed prenatally to age 24 mo Prenatal (cord) Low: < 3 Medium: 6

- 7 High: ≥ 10 Regression model adjusted for maternal age, race, maternal IQ, maternal education, number of years of smoking, number of alcohol drinks per week in 3rd trimester of pregnancy, SES, HOME, sex, birthweight, gestational age, birth order assessed ages 6 to 24 mo High vs. low cord blood High vs. medium cord blood - 4.8 (

- 7.3,

- 2.3)

- 3.8 (

- 6.3,

- 1.3)

Surkan et al. ( 2008 ) 309 children ages 12

- 36 mo during 1996

- 2001 or 2004

- 2005 Mex ico City, Mexico Concurrent Mean (SD): 6.4 (4.3) 10th

- 90th: 2.0

- 12.4 Linear mixed effects regression model adjusted for sex, maternal age, maternal IQ, maternal education, parity, alcohol consumption, smoking, cohort, maternal self

- esteem assessed ages 12 to 36 mo All subjects High maternal self

- esteem Low maternal self

- esteem - 0.18 (

- 0.45, 0.09) 0.36 (

- 0.50, 1.2)

- 0.31 (

- 0.60,

- 0.02)

Pilsner et al. ( 2010 ) 255 children age 24 mo born 1994

- 1995 Mexico City, Mexico Prenatal (cord blood) Mean (SD): 6.7 (3.6) 10th

- 90th: 3.5

- 10.5 Linear regression model adjusted for maternal age, maternal IQ, marital status, parity, gestational age, inadequate folate intake, MTHFR genotype at age 24 mo

- 0.73 (

- 1.2,

- 0.23)February 2012 5

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– Do Not Cite or Quote Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI) b
Hu et al. ( 2006 ) 146 children born 1997

- 1999 followed prenatally to age 24 mo Mexico City, Mexico Prenatal (maternal blood Pb) in 1st trimester Mean (SD): 7.1 (5.1) 10th

- 90th: 2.5

- 13.1 3rd trimester Mean (SD): 6.9 (4.2) 10th

- 90th: 2.8

- 12.1 Cord blood Mean (SD): 6.2 (3.9) 10th

- 90th: 2.5

- 11.0 Concurrent Mean (SD): 4.8 (3.7) 10th

- 90th: 1.6

- 9.1 Log linear regression model adjusted for sex, maternal age, current weight, height

- for

- age Z score, maternal IQ, concurrent blood Pb (in models examining blood Pb at other lifestages) assessed at age 24 mo Prenatal 1st trimester Prenatal 3rd trimester Cord blood Concurrent - 0.64 (

- 1.3,

- 0.03)

- 0.38 (

- 1.0, 0.24)

- 0.06 (

- 0.82, 0.70)

- 0.23 (

- 0.92, 0.45)

Solon et al. ( 2008 ) 502 children ages 6

- 35 mo, born 1997

- 2004 Visayas, Philippines Concurrent Mean (SD): 7.1 (7.7) 10th

- 90th: 1.6

- 14.9 Two

- stage linear regression mod el to account for determinants of blood Pb (sex, roof materials, water source, breastfed for ≥ 4 months) and cognitive function (HOME score, maternal education, maternal smoking, born premature, region of residence) assessed ages 6 to 35 mo

- 3.32 (

- 5.02,

- 1.60)

Vimpani et al. ( 1985 ) 592 children followed prenatally to age 24 mo Port Pirie, Australia 20% subjects had 24

- month blood Pb levels > 30 Linear regression model adjusted for maternal age, paternal education, maternal education, paternal workplace, maternal workplace, parental relationship, maternal prenatal marital sta tus, child birth rank, mouthing activity, oxygen use at birth, apgar score, neonatal jaundice, size for gestational age, maternal IQ at age 24 mo Maternal avg prenatal blood Pb Cord blood Pb 6 mo blood Pb 24 mo blood Pb Lifetime avg - 0.64 0.03

- 0.40, p < 0.05

- 0.06

- 0.31, p < 0.05

Wasserman et al. ( 1992 ) 392 children followed prenatally to age 24 mo Kosovska Mitrovica and Pristina, Yugoslavia Concurrent Means: 35.5 (K. Mitrovica) 8.4 (Pristina) Log linear regression model adjusted for sex, birth order, birth weight, ethnic group, HOME, maternal education, maternal age, maternal IQ at age 24 mo Cord blood Pb 6 mo blood Pb 12 mo blood Pb 18 mo blood Pb 24 mo blood Pb Per tripling blood Pb

- 1.7, p = 0.12

- 1.1, p = 0.34

- 1.7, p = 0.17

- 1.8, p = 0.16

- 2.5, p = 0.03 MDI = Mental Development Index, MTHFR = methylenetetrahydrofolate reductase
a Studies are presented in order of increasing population mean blood Pb level. b Except where noted, e ffect estimates are standardized to a 1 μg/dL increase in blood Pb level within the 10th to 90th percentile interval of blood Pb level.


### In a population of children in Krakow, Poland with lower cord blood Pb levels (median 1 

### 1.23 μg/dL, 95% CI: 1.24, 1.34 μg/dL) than those previously examined, increasing cord 2 

### blood Pb level was associated with similar magnitudes of decrease in MDI at ages 12, 24, 3 

### and 36 months ( Jedrychowski et al., 2009b ) ( Table 5

- 4 ). Consistent with the hypothesis 4 

### that the developing male central nervous system may be more vulnerable than that of 5 

### females to environmental i nsults resulting in later behavioral problems ( Moffitt et al., 6 

### 2001 ) , investigators estimated a larger in MDI per unit increase in cord blood Pb level 7 

### among the 233 males than among the 223 females ( Figure 5

- 4 ). Although mean cord 8 

### blood Pb levels were similar between males (1.35 μg/dL) and females (1.41 μg/dL), the 9 

### mean 36

- month MDI score was lower among males than among females (101 and 105, 10 

### respectiv ely, p = 0.0001). 11February 2012 5

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– Do Not Cite or Quote Source: Reprinted with permission of Elsevier Science, Jedrychowski et al. ( 2009a )

. 

### Figure 5
- 4 Regression of fitted MDI score at 36 months on log
- transformed cord blood Pb level by sex.

Solon et al. ( 2008 ) examined children in the Philippines, ages 6 to 35 months. A key 1 

### strength of this study was the adjustment for HOME score as well as maternal education, 2 

### maternal smoking, premature birth, region of residence, and years of schooling of child. 3 

### Not only did investigators find an association between higher concurrent blood Pb level 4 

### and lower MDI score ( Table 5

- 4 ), but they also found effect modification by red blood 5 

### cell folate levels. Among children with folate levels less than or equal to 230 μg/mL, 6 

### blood Pb level had a marginal association with lowering MDI scores in the range of 0.80 7 

### to 2.44 points. Among children with higher folate levels, blood Pb level was not 8 

### estimated to have a negative marginal impact. These findings from Solon et al. ( 2008 ) 9 

### indicated that children with folate deficiencies may be at increased risk of Pb

- associated 10 

### decreases in cognitive function. The results were consistent with observations that higher 11 

### folate level is associated with lower blood Pb level since folate improves Pb excretion by 12 

### inhibiting the binding of Pb to blood elements. 13 

### Multiple studies in different Mexico City mother

- child pair cohorts recently reported 14 

### associations betwee n blood Pb levels ( e.g., maternal, cord blood, or child postnatal) and 15 

### decrements in Bayley MDI in children between age 12 and 36 months ( Claus Henn et al. ; 16 

### Pilsner et al., 2010 ; Surkan et al., 2008 ; Hu et al., 2006 ; Tellez

- Rojo et al., 2006 )

. Recent 17 

### studies extended findings from Tellez

- Rojo et al. ( 2006 ) with follow

- up of the same 18February 2012 5

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– Do Not Cite or Quote 

### cohort to age 3 years and observations of effect modification by maternal self

- esteem 1 

### ( Surkan et al., 2008 ) and blood Mn levels ( Claus Henn et al. )

. 2


### Claus Henn et al. examined i nteractions between blood Mn and Pb levels. Investigators 3 

### selected mid

- range (2.0

- 2.8 μg/dL) blood Mn levels as the reference group based on 4 

### previous observations that MDI scores were least affected by increases in blood Mn level 5 

### in this group. Among subje cts with age 12

- month blood Mn levels less than 2.0 μg/dL, a 6 

### 1 μg/dL higher age 12

- month blood Pb level was associated with a 3.0

- point (95% CI:

- 7 

### 5.2,

- 0.78) lower MDI. Among subjects with blood Mn levels greater than 2.8 μg/dL, a 8 

### 1 μg/dL higher age 12

- mon th blood Pb level was associated with a 2.2

- point (

- 4.4, 0) 9 

### lower MDI. Interactions were not found using age 24

- month blood Mn and Pb levels. 10 

### Effects estimates were similar in magnitude before and after adjustment for sex, 11 

### gestational age, hemoglobin, mate rnal IQ, maternal education, and visit. Findings in this 12 

### Mexico City cohort added to those from Kim et al. ( 2009b ) in older children that 13 

### associations between blood Pb level and cognitive function may be influenced by Mn co

14 

### exposure, and results from both studies indicated that blood Pb level was associated with 15 

### decrements in cognitive function with both lower and higher levels of blood Mn. 16 

### Biological plausibility fo r the interactive effects of Pb and Mn exposure is provided by 17 

### observations that Mn has similar modes of action and cellular targets as does Pb, 18 

### i.e., altering Ca +2 

### metabolism, inducing oxidative damage to neuronal cells, diminishing 19 

### dopamine transmission. Claus Henn et al. also indicated that Pb

- Mn co

- exposu re may 20 

### affect cognitive function in children as young as 12 to 36 months of age. 21


### Surkan et al. ( 2008 ) stratified data by the level of maternal self

- esteem as reported by 22 

### mothers. Higher age 24

- month blood Pb level was associated with lower MDI score 23 

### among children with mothers in the lowest three quartiles of self

- esteem but n ot among 24 

### children with mothers in the highest quartile of self

- esteem ( Table 5

- 4 ). Model covariates 25 

### included cohort, sex, maternal IQ, maternal age, maternal education, parity, maternal 26 

### smoking, maternal alcohol consumption, and maternal self

- esteem. While HOME score 27



### was not examined, maternal self

- esteem may incorporate many of the same aspects as 28 

### HOME score, including mother

- child interactions, educational interactions, and dietary 29 

### practices. These findings indicated that higher maternal psychosocial functioning 30 

### ( e.g., l ower stress, anxiety, and depression and higher self

- esteem) may contribute to 31 

### better caregiving, which in turn may improve neuropsychological functioning of the 32 

### child. The biological plausibility of these findings in children is well

- supported by 33 

### observat ions in animals unrelated to Pb that environmental enrichment reverses the 34 

### effects of early stress experiences on reactions such as depressed behavior, HPA 35 

### activation, and immunosuppressant ( Laviola et al., 2008 ; Lavi ola et al., 2004 ; Moreley

36 

### Fletcher et al., 2003 ; Francis et al., 2002 )

. With specific regards to Pb exposure, social 37 

### isolation or enrichment has been found to exacerbate or protect against, respectively, Pb 38February 2012 5

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– Do Not Cite or Quote 

### exposure
- induced learning impairments in ani mals (Section 5.3.2.2 ). It is worth 1 

### mentioning in this context that the potential programming effects of stress on childhood 2 

### health outcomes may oc cur at an even more fundamental level, i.e., through epigenetic 3 

### programming ( Dolinoy and Jirtle, 2008 )

. 4


### Hu et al. ( 2006 ) compared associations among prenatal blood Pb levels measured at 5 

### different stages of gestation among 146 mother

- child pairs meeting the following criteria: 6 

### born at 37 weeks or greater gestational age, at least one valid Pb measurement during 7 

### pregnancy, complete information on maternal age and IQ, and measurement of child 8 

### blood Pb level at 24 months when the 24

- month MDI was ascertained. Among blood Pb 9 

### levels measured at vari ous lifestages, 1st trimester maternal blood Pb level (whole or 10 

### plasma Pb) was associated with larger decreases in subsequent 24

- month MDI scores 11 

### compared with maternal 3rd trimester, cord, and child concurrent blood Pb ( Table 5

- 4 ). 12 

### These results were adjusted for sex, 24

- month blood Pb level, height

- for

- age Z score, 13 

### weight, maternal age, and maternal IQ. HOME score was not included in the final model; 14 

### however, investigators examined the potential for confounding by a larger list of 15 

### unspecified variables. Prenatal Pb exposure effects also were indicated in a study 16 

### conducted in Mexico City that examined FSIQ repeatedly during ages 6

- 10 years 17 

### ( Schnaas et al., 2006 )

. Third trimester (weeks 28

- 36) maternal blood Pb level, was 18 

### associated with larger d ecreases in FSIQ than were maternal blood Pb level at 19 

### 12

- 20 weeks, maternal blood Pb level at delivery, or concurrent child blood Pb levels 20 

### measured between ages 6 and 10 years. 21 

### In another recent study in Mexico City, Mexico, investigators found higher cor d blood Pb 22 

### level to be associated with a lower MDI score in children at age 24 months (

- 0.73 points 23 

### [95% CI:

- 1.2,

- 0.23] in score per 1 μg/dL increase in cord blood Pb level in the 10th

24 

### 90th percentile interval [3.5

- 10.5 μg/dL]) ( Pilsner et al., 2010 )

. Investigators additionally 25 

### examined effect modification by variants in the methylenetetrahydrofolate reductase 26 

### (MTHFR) gene. The MTHFR enzyme is involved in folate metabolism, which, in turn, is 27 

### involved in homocysteine methylation to the amino acid methionine. The transfer of 28 

### methyl groups that results from folate metabolism is important for biological processes 29 

### including Phase II detoxification reactions an d epigenetic regulation of gene expression. 30 

### The MTHFR gene has common functional variants, including the C677T SNP, which 31 

### produces an enzyme with lower metabolic activity and is associated with lower serum 32 

### folate levels ( Kordas et al., 2009 )

. Lower folate levels have been associated with higher 33 

### blood Pb levels. Although Pilsner et al. ( 2010 ) found that both cord blood Pb levels and 34 

### the MTHFR C677T allele were associated with lower child MDI score at age 24 months, 35 

### they did not find a statistically significant interaction between blood Pb level and the 36 

### MTHFR 677T allele. Results from stratified analyses were not reported, thus differences 37 

### in the magnitude of association between genotypes could not be compared. 38February 2012 5

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– Do Not Cite or Quote 

### Specific Indices of Cognitive Function in Children In addition to indices of global cognitive function, blood Pb levels also have been 1



### a ssociated with specific cognitive abilities, including memory, learning, visuospatial 2 

### processing, and other executive functions such as planning and problem solving in 3 

### children and adolescents ( Kordas et al., 2006 ; Tellez

- Rojo et al., 2006 ; Chiodo et al., 4 

### 2004 ; Ris et al., 2004 ; Canfield et al., 2003 b ; Lanphear et al., 2000 ; Bellinger and Stiles, 5 

### 1993 ; Dietrich et al., 1992 ; Bellinger et al., 1991 ; D ietrich et al., 1991 ; Needleman et al., 6 

### 1979 ) ( Figure 5

- 5 and Table 5

- 5 ). Studies often found associations with multiple indices 7 

### of cognitive f unction, and because several tests are interrelated, it is difficult to attribute 8 

### the effects of Pb exposure to a specific domain of cognitive function. Evidence that blood 9 

### Pb levels are associated with a spectrum of cognitive indices provides coherence fo r the 10 

### associations observed between blood Pb levels and FSIQ, a global measure of cognitive 11 

### function. Furthermore, these tests of learning and memory in humans are homologous to 12 

### tests in animals, and compared with that for FSIQ, evidence for these specific indices 13 

### may improve understanding of the coherence between findings in humans and animals 14 

### ( Rice, 1996 )

. These complex cognitive functions have been described to be mediated by 15 

### the actions of neurotransmitters dopamine and glutamate in the hippocampus, prefrontal 16 

### cortex, and ventral s triatum of the brain. In toxicological studies, Pb exposure has been 17 

### shown to disrupt the function of these systems, which provides biological plausibility for 18 

### associations observed between blood Pb levels and deficits in a range of executive 19 

### functions in children. 20 

### Studies published since the 2006 Pb AQCD added to the evidence for associations 21 

### between higher blood Pb level (concurrent most frequently examined) and lower 22 

### performance in specific indices of cognitive function ( Figure 5

- 5 and Table 5

- 5 ). The 23 

### weight of evidence indicated that decrements in specific cognitive indices were 24 

### associated with concurrent blood Pb level and in populations with mean blood Pb levels 25 

### between 5

- 10 μg/dL. Most studies considered potential confounding by multiple SES

26 

### related variables, and many found blood Pb

- associated decrements in cognitive function 27 

### in both unadjusted and covariate

- adjusted models ( Palaniappan et al., 2011 ; Chiodo et al., 28 

### 2007 ; Froehlich et al., 2007 )

. Many studies did not examine potential confounding by 29 

### HOME score. However, studies that did ( Min et al., 2009 ; Solon et al., 2008 ; Froehlich et 30 

### al., 2007 ; Canfield et al., 2004 ; Canfield et al. , 2003b ) and did not adjust for HOME score 31 

### ( Krieg et al., 2010 ; Jedrychowski et al., 2008 ; Surkan et al., 2007 ; Lanphear et al., 2000 ) 32 

### both found associations between blood Pb level and decrements in specific cognitive 33 

### indices.. Several st udies found associations after adjusting for different potential 34 

### confounding factors, including parental IQ or education and HOME score. The set of 35 

### covariates varied among the specific cognitive endpoints, and HOME score was not 36 

### associated with every cogni tive index. For example, only household income remained 37February 2012 5

- 73 Draft

– Do Not Cite or Quote 

### significantly associated with total errors in the spatial working memory task and was 1 

### included in the final model ( Froehlich et al., 2007 )

. Solon et al. ( 2008 ) found that in a 2 

### multivariable model with blood Pb level and several potential confounders, HOME score 3



### re mained significantly associated with verbal IQ in 5 year
- old children, whereas maternal 4 

### education, maternal smoking, and premature birth did not. Combined, these findings 5 

### support the independent association of blood Pb level with specific cognitive indices and 6 

### indicate that the potential confounding variables vary among endpoints and study 7 

### populations. 8 

### Several analyses of the Rochester cohort at ages 4 and 5 years indicated associations of 9 

### blood Pb level with various indices of cognitive function, including visual and working 10 

### memory, planning, and problem solving ( Froehlich et al., 2007 ; Canfield et al., 2004 ; 11 

### Canfield et al., 2003b )

. In particular, the associations observed with tests of memory and 12 

### rule learning and reversal provided strong coherence with observations of Pb

- induced 13 

### impaired performance in animals in homologous tests of the Morris Water Maze, the 14 

### delayed spatial alternation, and discrimination reversal learning (Section 5.3.2.2 ). 15 

### Compared with other prospective cohort studies, findings from the Rochester cohort 16 

### demonstrated associations between blood Pb level and d ecrements in executive functions 17 

### in younger children with a lower mean blood Pb level (6.5 and 6.0 μg/dL at ages 4 and 18 

### 5 years, respectively). An additional strength of these studies was the detailed analysis of 19 

### potential confounding by various demographic and SES

- related variables (including 20 

### HOME score) and other environmental exposures. Covariates were selected for specific 21 

### models based on their association with a particular test (p ≤ 0.20), and several models 22 

### adjusted for maternal IQ, maternal education, and HOME score. 23


### Canfield et al. ( 2003b ) reported associations of concurrent blood Pb level with various 24 

### parameters of the Shape School tasks, which test the ability of children to recall correctly 25 

### shapes and colors that are attributed to specific cartoon characters. Concurren t blood Pb 26 

### level was associated with color knowledge, shape knowledge, and number of correct 27 

### responses at age 4 years ( Figure 5

- 5 and Table 5

- 5 ). At age 5 years, higher lifetime 28 

### average blood Pb level (area under the curve calculation using repeat measurements 29 

### between age 6 months and 5 years) was associated with poorer performance o n multiple 30 

### tasks related to learning, working and spatial memory, and planning as assessed by the 31 

### Cambridge Neuropsychological Testing Automated Battery (CANTAB) in both 32 

### unadjusted and covariate

- adjusted analyses ( Canfield et al., 2004 )

. These memory tests in 33 

### ch ildren share homology with the Morris water maze test in animals in that both test the 34 

### ability of subjects to use efficient search strategies to identify target locations, retain 35 

### spatial information, and access remembered items in working memory. Both meas ure the 36 

### time to complete the task and the number of errors made. At age 4 and 5 years, 37 

### associations of concurrent blood Pb level with several indices of executive function were 38February 2012 5

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– Do Not Cite or Quote 

### robust to additional adjustment for attentiveness and child FSIQ, indicating th at higher 1 

### blood Pb level is associated with specific aspects of associative learning that are not 2 

### represented by attentiveness and general knowledge base. 3

a Standard error was estimated from p

- value.
b Sufficient data were not provided to calculate 95% C Is.

c Direction of effect estimate was changed to indicate that a negative estimate represents poorer performance and a positive estimate represents improved performance. Black diamonds, gray squares, and blue circles represent associations with concurre nt, lifetime average, and earlier childhood blood Pb levels Note: Regression coefficients were standardized to their standard deviation to facilitate comparisons among tests with differ ent scales. Studies generally are presented in order of increasing mean blood Pb level. Effect estimates are standardized to a 1 μg /dL increase in blood Pb level within the 10th

- 90th percentile interval. The percentiles are estimated using various methods and are only approximate values. Effect estimates are assumed to be linear within the 10th to 90th percentile interval of blood Pb level. 

### Figure 5
- 5 Standardized regression coefficients describing the associations of blood Pb levels with specific indices of cognitive function in children. Study Cho et al. (2010) Lanphear et al. (2000)

Kreig et al. (2010)

Chiodo et al. (2007)

Chiodo et al. (2004)

Froehlich et al. (2007)

Canfield et al. (2004)

Canfield et al. (2003b)

Solon et al. (2008)

Min et al. (2009)

Palaniappan et al. (2011) Blood Pb Mean

(SD) (μg/dL) 1.9 (0.67) 1.9 (geometric mean)

1.95 (0.16)

5.0 (3.0)

5.4 (3.3)

6.1 (4.9)

7.2 (3.6)

6.5

7.7 (7.7) 7.1 (5.1) 7.0 (range: 1.3

- 23.8)

11.5 (5.3) 10th
- 90th percentile interval (μg/dL)

1.2

- 2.8
1.74

- 2.06

1.69

- 2.19

2.1

- 8.87

2.3

- 9.5

1.9

- 11.7

3.5

- 11.8

Data not available

1.6

- 14.9

3.0

- 12.1

6.0

- 18.3 Outcome Color

- word score a Digit Span Math score Digit Span Reading score Math score
Arithmetic b Object assembly a Spatial span b Perseverative errors b Spatial memory a Rule learning a Spatial span a Nontarget errors c Total errors c Proportion correct Color knowledge
Verbal IQ Performance IQ Verbal comprehension, age 9 Verbal comprehension, age 11 Math score, age 9 Math score, age 11 Reading score, age 9 Reading score, age 11 Visuomotor composite ◄ ◄ ►

- 0.6

- 0.4

- 0.2 0 0.2 Change in standardized test score per 1 μg/dL increase in blood Pb level within the 10th
- 90th percentile interval (95% CI)February 2012 5

- 75 Draft

– Do Not Cite or Quote 

### Table 5
- 5 Additional characteristics and quantitative results for studies represented in Figure 5
- 5

Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI)a Cho et al. ( 2010 ) 667 children ages 8

- 11 yr in 2008 Five Korean cities Concurrent Mean (SD): 1.9 (0.67) 10th

- 90th: 1.2

- 2.8 Log linear regression model adjusted for age, sex, parental education, maternal IQ, child IQ, birth weight, urinary cotinine Color

- word score c using Stroop test at ages 8

- 11 yr 0 (

- 0.09, 0.08) b

Lanphear et al. ( 2000 ) 4 , 852 children ages 6

- 16 years U.S. NHA NES III (1991

- 1994) Concurrent Geometric mean: 1.9 (95% CI: 1.70, 2.10) 10th

- 90th: 1.74, 2.06 Linear regression model adjusted for sex, race/ethnicity, poverty index ratio, reference adult education, serum ferritin levels, serum cotinine levels Digit Span (WISC

- R) Math Score (WRAT

- R) at ages 6

- 16 yr - 0.02 (

- 0.03,

- 0.01)

- 0.007 (

- 0.01,

- 0.001)

Krieg et al. ( 2010 ) 842 children ages 12

- 16 yr U.S. NHANES III (1991

- 1994) Concurrent Mean (95% CI): 1.95 (1.63, 2.27) 10th

- 90th: 1.69

- 2.19 Log linear regression model adjusted for sex, caregiver education, family income, race

- ethnicity, test language Block design (WISC

- R) Digit span (WISC

- R) Reading score (WRAT

- R) Math score (WRAT

- R) assessed at ages 12

- 16 yr - 0.10 (

- 0.18,

- 0.02)

- 0.08 (

- 0.13,

- 0.04)

- 0.11 (

- 0.16,

- 0.05)

- 0.07 (

- 0.12,

- 0.01)

Chiodo et al. ( 2007 ) 506 African

- American children in Detroit, MI area followed from birth (1989

- 1991) to age 7 yr. Large proportions of children with prenatal exposure to cocaine or marijuana Concurrent Mean (SD): 5.0 (3.0) 10th

- 90th: 2.1

- 8.7 Regression model adjusted for SES, caretaker education, maternal IQ (all outcomes) Plus: age, HOME, Symptom Checklist (arithmet ic and mazes) Plus: sex, prenatal cigarettes/day, prenatal marijuana use (arithmetic) Arithmetic Mazes Object assembly WISC

- III at age 7 yr - 0.07, p > 0.05c

- 0.11 (

- 0.18,

- 0.04)

- 0.13 (

- 0.21,

- 0.05)

Chiodo et al. ( 2004 ) 246 African

- American children in Detroit, MI area followed from birth (not reported) to age 7.5 yr. Large proportions of children with prenatal exposure to cocaine or marijuana Concurrent Mean (SD): 5.4 (3.3) 10th

- 90th: 2.3

- 9.5 Log linear regression model adj usted for SES, maternal education, sex (all outcomes) Family Environmental Symptom (family functioning), number of children < 18 years, caregiver vocabulary, prenatal alcohol use (Perseverative errors) Prenatal cocaine use (conceptual level responses) HOME score, Symptom Checklist, caregiver age (Spatial span) Corsi Spatial Span WCST Perseverative errors WCST Conceptual Level Responses at age 7.5 yr - 0.10, p > 0.05 c,d

- 0.49, p > 0.05 c,d

- 0.77 (

- 1.5, 0) b

Froehlich et al. ( 2007 ) 174 children born 1994

- 1995 followed from birth to age 5 yr Rochester, NY Concurrent Mean (SD): 6.1 (4.9) 10th

- 90th: 1.9

- 11.7 Linear regression model adjusted for income (spatial memory); NICU, sex (rule learning); HOME score, maternal IQ, race (spatial span); or maternal IQ, transferrin saturation (problem solving) Spatial memory Rule learning and reversal Spatial span Problem solving using CANTAB at age 5 yr - 0.02 (

- 0.05. 0.01) b

- 0.03 (

- 0.06,

- 0.001) b

- 0.007 (

- 0.01, 0) b

- 0.04 (

- 0.09, 0.01) b

Canfield et al. ( 2004 ) 174 children 174 children born 1994

- 1995 followed from birth to age 5 yr Rochester, NY Lifetime avg Mean (SD): 7.2 (3.6) 10th

- 90th: 3.5

- 11.8 L inear regression model adjusted for maternal IQ, duration of breastfeeding, household income, maternal ethnicity, first prenatal visit (spatial span length), NICU admission, sex, spatial span length, spatial working memory problem (total nontarget errors, total errors), age at testing (nontarget errors), Maximum spatial span length Total nontarget errors Total errors Using CANTAB at age 5 yr - 0.17 (

- 0.25, 0.59) d

- 0.09 (

- 0.11,

- 0.06) d 0.04 (

- 0.07, 0.007) dFebruary 2012 5

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– Do Not Cite or Quote Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI)a Canfield et al. ( 2003b ) 170 children born 1994

- 1995 followed f rom age 6 mo to age 4 yr Rochester, NY Concurrent Mean: 6.5 10th

- 90th: data not available Linear mixed effects model adjusted for: Sex, gestational age, birth order, maternal IQ, maternal education, prenatal smoking (correct responses) Age, birth order, maternal IQ, maternal education, household income, marital status, HOME score, prenatal smoking (color knowledge) Age, maternal IQ, household income, marital status, HOME score, race (shape knowledge) Proportion correct responses Color knowledge Shape know ledge using Shape School Task at ages 4 and 4.5 yr - 0.017 (

- 0.027, 0.007)

- 0.074 (

- 0.101,

- 0.047)

- 0.057 (

- 0.094,

- 0.020)

Solon et al. ( 2008 ) 502 children ages 6

- 35 mo 377 children 3

- 5 yr, 2003

- 2004 Visayas, Philippines Concurrent Mean (SD): 7.1 (7.7) 10th

- 90th: 1.6

- 14.9 Two

- stage linear regression model to account for determinants of blood Pb (sex, roof materials, water source, breastfed for ≥ 4 months) and cognitive function (HOME score, maternal education, maternal smoking, born premature, region of residence) Verbal IQ Performance IQ using WPPSI

- III at ages 3

- 5 yr - 0.34 (

- 0.63,

- 0.05) b

- 0.45 (

- 2.37, 1.48) b

Min et al. ( 2009 ) 267 primarily African

American children in the Cleveland, OH area followed from birth (1994

- 1996) to age 11 yr. Children were exposed prenatally to multiple drugs. Age 4 yr Mean (range): 7.0 (1.3

- 23.8) 10th

- 90th: 3.0

- 12.1 Linear regression model adjusted for HOME score, caregiver's vocabulary test, sex, parity, maternal marital status, head circumference at birth Verbal comprehension age 9 yr Verbal comprehens ion age 11 yr Perceptual reasoning age 9 yr Perceptual reasoning age 11 yr Math at age 9 yr Math at age 11 yr Reading at age 9 yr Reading at age 9 yr Using WISC

- R and WJTA - 0.028 (

- 0.055, 0)

- 0.044 (

- 0.072,

- 0.015)

- 0.033 (

- 0.064,

- 0.003)

- 0.045 (

- 0.074,

- 0.016)

- 0.028 (

- 0.057, 0.002)

- 0.033 (

- 0.062,

- 0.004)

- 0.039 (

- 0.069,

- 0.009)

- 0.043 (

- 0.073,

- 0.014)

Palaniappan et al. ( 2011 ) 815 children ages 3

- 7 years in 2003

- 2006 in Chennai, India Concurrent Mean (SD): 11 .5 (5.3) 10th

- 90th: 6.0

- 18.3 Generalized estimating equation adjusted for sex, hemoglobin level, maternal education, parental education, average monthly income, clustering at school and class level Visuomotor composite Using WRAVMA at ages 3

- 7 yr - 0.017 (

- 0.029,

- 0.005)

Studies not included in figure due to u navailability of blood Pb
- cognitive function continuous effect estimates Jedrychowski et al. ( 2008 ) 452 children followed from birth (2001

- 2003) to age 6 mo Prenatal (cord) Mean (95% CI): 1.42 (1.35, 1.48) 10th

- 90th: 1.36

- 1.46 Logistic regression model adjusted for maternal education, parity, sex No maternal IQ Verbal recognition memory score < 52.2 Using FTII at 6 mo OR (95% CI) 1.47 (1.07, 2.01)

Surkan et al. ( 2007 ) 534 children ages 6

- 10 yr Boston, MA and Farmington, ME Concurrent Mean (SD): 2.2 (1.6) Analysis of covariance adjusted for child IQ, caregiver IQ, age, SES, race, birth weight WIAT at age 6

- 10 yr
Reading score Math score Visual memory score Perseveration errors Stroop color

- word interference blood Pb level 5

- 10 μg/dL vs. 1

- 2 μg/dL e

- 5.20 (

- 9.45,

- 0.95)

- 4.02 (

- 7.6,

- 0.43)

- 6.47 (

- 11.9,

- 1.1)

- 9.19 (

- 14.6,

- 3.7) d 0.75 (

- 1.6, 3.1) Kordas et al. ( 2006 ) 294 children with blood Pb levels < 1 0 μg/dL Concurrent Range: 2.1

- 10.0 Linear regression model adjusted for sex, age, hemoglobin, family possessions, forgetting homework, house ownership, crowding, maternal education, birth order, family structure, arsenic exposure, tester, school Visual memory span < 10 correct vs. ≥ 1 0 correct: 0.90 Stimulus discrimination < 20 correct vs. 20 correct OR (95% CI) 0.90 (0.74, 1.10)
0.85 (0.63, 1.13)February 2012 5

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– Do Not Cite or Quote Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI)a Bellinger and Stiles ( 1993 ) 148 children in the Boston, MA area followed from birth (1979

- 1981) to age 10 yr 57 month and concurrent blood Pb Exact levels NR, mean reported to be < 8 Linear regression model adjusted for HOME score, family stress, race, marital status Perseverative errors 57 month blood Pb Concurrent blood Pb
Percent conceptual responses 57 month blood Pb Concurrent blood Pb Using WCST at age 10 yr - 0.05 (

- 0.09,

- 0.01) d

- 0.05 (

- 0.11, 0.01) d

- 0.93 (

- 2.0, 0.13) NR

Bellinger et al. ( 1994a ) 79 children in th e Boston, MA area followed from birth (1979

- 1981) to age 19

- 20 yr Deciduous tooth Pb Q1: 2.9

- 5.9 μg/g Q2: 6.0

- 8.7 μg/g Q3: 8.8

- 19.8 μg/g Q4: 19.9

- 51.8 μg/g Regression model adjusted for parental IQ, sex, SES, current drug use, current alcohol use, current illicit drug use, maternal education, maternal age, birth order Stroop Color

- word test

WCST Number Correct Mean (SE) Q1: 103 (8.1), Q2: 116 (7.6), Q3: 127 (8.4), Q4: 125 (7.7) Q1: 70.5 (2.9), Q2: 72.7 (2.8), Q3: 74.5 (3.1), Q4: 71.4 (2.9) WISC = Wechsler Intelligence Scale for Children, WRAT = Wide Range Achievement Test, WCST = Wisconsin Card Sorting Test, CANTAB = Ca mbridge Neuropsychological Testing Automated Battery, WPPSI = Wechsler Preschool and Primary Scale of Intelligence, WRAVMA = Wide R ange of Visual Motor Ability, FTII = Fagan Test of Infant Intelligence, WIAT = Wechsler Individual Achievement Test
a Effect estimates are transformed to a z

- score and standardized to a 1 μg/dL increase in blood Pb level in the 10th

- 90th percentile interval

. b 95% CI was constructed using a standard error that was estimated from a p - value.
c Sufficient data were not provided to calculate 95% CI. d The direction of the effect estimate was changed such that a negative estimate represents poorer performance and a positive estimate represents better performance.
e Effect estimates compare test performance of children in higher blood Pb groups to children in lowest blood Pb group.


### A recent analysis of the Rochester cohort age 5 years extended previous findings with 1 

### observations that higher concurrent blood Pb level also was associated with lower 2 

### performance on many of the same tests executive function as assessed by CANTAB 3 

### ( Froehlich et al., 2007 ) ( Figure 5

- 5 and Table 5

- 5 ). In addition to spatial memory, 4 

### Froehlich et al. ( 2007 ) found associations with rule learning and reversal tasks. Pb 5 

### exposure of animals consistently has been found to induce impairments in homol ogous 6 

### tests of cognitive flexibility, including delayed spatial alternation tests (Section 5.3.2.2 ). 7 

### Both of these tests assess the ability of child ren and animals to complete a task according 8 

### to a change in rules or reinforcement. Impaired performance in both children and animals 9 

### is indicated by increased response errors, decreased percent of correct responses, and 10 

### perseverance ( i.e., animals repeate dly pressing the same lever without moving between 11 

### the two locations or children repeatedly selecting the wrong target despite a change in 12 

### rules or reinforcement. Some effect estimates describing the blood Pb

- cognitive function 13 

### association were attenuated after adjustment for covariates ( e.g., spatial span and 14 

### planning tasks), whereas others were magnified ( e.g., total trials in rule learning, total 15 

### errors in spatial working memory) ( Fr oehlich et al., 2007 )

. Further, despite attenuation, 16 

### several associations did not change in statistical significance ( Figure 5

- 5 and Table 5

- 5 ). 17


### Froehlich et al. ( 2007 ) also added to previous findings in the Rochester cohort by 18 

### indicating that associations wer e modified by sex and dopamine receptor (DRD4) genetic 19 

### variants. An increase in blood Pb level was associated with larger decrements in rule 20February 2012 5

- 78 Draft

– Do Not Cite or Quote 

### learning and reversal and spatial memory in children with the DRD4 exon III 7

- repeat 1 

### microsatellite (assessed using a blood Pb

- DRD4

- 7 interaction term, p = 0.042). The 2 

### biological plausibility for these findings is provided by observations that the DRD4

- 7 3 

### variant is associated with reduced dopamine

- induced signaling in downstream pathways 4 

### ( e.g., cyclic AMP), that Pb exp osure is found to impact dopaminergic activity 5 

### (Section 5.3.8.8 ), and that dopamine is a key neurotransmitter that regulates cognitive 6 

### processes. A ssociations of concurrent blood Pb level with rule learning and reversal also 7 

### were greater in boys. Additional support for these observed interactions was provided by 8 

### observations with children with the DRD4

- 7 variant and boys had poorer performance on 9 

### rul e learning and reversal tasks. 10 

### Several other studies found associations between higher blood Pb level and poorer 11 

### performance on rule learning and reversal tasks in children and young adults ( Surkan et 12 

### al., 2007 ; Kordas et al., 2006 ; Chiodo et al., 200 4 ; Bellinger et al., 1994a ; Bellinger and 13 

### Stiles, 1993 )

. Across studies, the ag e range of subjects was 6 to 20 years, and most 14 

### studies examined concurrent blood Pb levels. In the Boston cohort, age 4.5 year blood Pb 15 

### levels were associated with poorer cognitive flexibility at age 10 years ( Bellinger and 16 

### Stiles, 1993 ) , and deciduous tooth Pb (measured at ages 6

- 7 years) was associated with 17 

### poorer cognitive flexibility at ages 19

- 20 years ( Bellinger et al., 1994a )

. 18


### Blood Pb

- associated decrements in executive function in children with mean blood Pb 19 

### levels < 5 were not as clearly demonstrated. Among children in five Korean studies, ages 20 

### 8

- 11 years, blood Pb level was not associated with performance a color

- word association 21 

### test. In the study of childr en (ages 6

- 10 years) participating in NECAT, Surkan et al. 22 

### ( 2007 ) provided information on the blood Pb

- cognitive function concentration

- response 23 

### relationship. Children with concurrent blood Pb levels 3

- 4 μg/dL had low er scores 24 

### compared with children with blood Pb levels 1

- 2 μg/dL; however, only a few 25 

### associations were statistically significant ( e.g., digit span, memory, motor speed). In 26 

### analyses adjusted for caregiver IQ, age, SES, race, and birth weight, children with 27 

### concurrent blood Pb levels of 5 to 10 μg/dL had lower performance in a number of 28 

### executive functioning domains such as working memory, visuospatial skills, cognitive 29 

### flexibility, and ability to formulate, test, and adapt hypotheses ( Figure 5

- 5 and Table 5

- 5 ). 30 

### Compared with children with blood Pb levels of 1

- 2 μg/dL, children with blood Pb 31 

### levels 5

- 10 μg/dL had lower scores on the reading (5.2 points [95% CI: 0.95, 9.45]) and 32 

### math (4.0 points [95 % CI: 0.43, 7.6]) composites of the Wechsler Individual 33 

### Achievement Test. These decrements were robust to adjustment for child FSIQ. Thus, 34 

### these findings combined with those from the Rochester cohort ( Canfield et al., 2004 ; 35 

### Canfield et al., 2003b ) prov ide evidence that higher blood Pb level is associated with 36 

### decrements in specific cognitive functions that may not be represented by FSIQ, a test of 37 

### global function. 38February 2012 5

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– Do Not Cite or Quote 

### In other studies of populations with mean concurrent or cord blood Pb level < 3 μg/dL, 1 

### the contributions of likely higher past Pb exposures cannot be excluded. Also, these 2 

### studies did not have as extensive consideration for potential confounding. In infants in 3 

### Krakow, Poland with a mean cord blood Pb level of 1.42 μg/dL, higher cord blood Pb 4 

### le vel was associated with a lower score on a visual recognition memory test 5 

### ( Jedrychowski et al., 2008 ) ( Table 5

- 5 ). The cord blood Pb levels may have been 6 

### influenced by past Pb exposures of the mother mobilized from bone stores to the blood. 7 

### While investigators adjusted for maternal education, parity, and sex, they did not adjust 8 

### for other SES

- related variables such as maternal IQ or HOME score. 9 

### The influence of past Pb exposures also is uncertain in the NHANES analyses, which 10 

### included adolescent subjects born in the 1970s. Krieg et al. ( 2010 ) examined an older 11 

### subset of children (ages 12

- 16 years) from NHANES III (1988

- 1994) who were 12 

### previously examined in Lanphear et al. ( 2000 )

. Among all ages and older adolescents, 13 

### higher concurrent blood Pb level was associated with lower scores on block design, digit 14 

### span, reading score, and arithmetic tests ( Figure 5

- 5 and Table 5

- 5 ) ( Krieg et al., 2010 ; 15 

### Lanphear et al., 2000 )

. In analyses of quartiles of blood Pb level Lanphear et al. ( 2000 ) 16 

### additionally found a monotonic decrease in math and reading scores across increasing 17 

### quartiles in covariate

- adjusted models. Per unit increase in blood Pb level, the decrease in 18 

### arithmetic and reading scores were larger among children with blood Pb levels less than 19 

### 2.5 μg/dL than among all subjects, with statistically significant associations found for 20 

### children with blood Pb levels less than 5 μg/dL. While the NHANES results were 21 

### adjusted for sex, race/ethnicity, test language, and multiple SES

- related variables such as 22 

### family income and caregiver education, information on HOME score was unavailable. 23 

### Krieg et al. ( 2010 ) provided additional information on effect modification by vitamin D 24 

### receptor (VDR) variants. Although there were not differences in blood Pb lev els among 25 

### the various haplotypes of VDR, various polymorphisms and haplotypes modified the 26 

### association between blood Pb level and a range of neurocognitive tests. The VDR 27 

### regulates calcium absorption and metabolism, and effect modification by VDR variants is 28 

### consistent with the well

- established mode of action of Pb in mimicking calcium in shared 29 

### transport and metabolic pathways. However, several inconsistencies were observed by 30 

### Krieg et al. ( 2010 ) in that a particular variant was associated with a lower Pb

- associated 31 

### decrement in some indices but a greater Pb

- associated decrement in other indices. Fo r 32 

### example, among children ages 12

- 16 years, the VDR rs2239185 CC genotype was 33 

### associated with the largest blood Pb

- associated decrease in digit span score and reading 34 

### score. The decreases in digit span score (95% CI) per 1 μg/dL increase in concurrent 35 

### bloo d Pb level were

- 1.5 points (

- 2.2,

- 0.71) for the CC genotype and

- 0.26 points (

- 0.99, 36 

### 0.46) for the TT genotype. Conversely, the TT genotype was associated with the greatest 37 

### Pb

- associated decrease in arithmetic score. The decreases (95% CIs) in math score per 38February 2012 5

- 80 Draft

– Do Not Cite or Quote 

### 1 μg/dL increase in concurrent blood Pb level were

- 8.4 points (

- 11.5,

- 5.2) for the TT 1 

### genotype and

- 2.7 points (

- 10.1, 4.6) for the CC genotype. Effect modification by VDR 2 

### rs731236 was more consistent across cognitive tests, with larger blood Pb

- ass ociated 3 

### decrements in cognitive performance found in children with the CC genotype. It is 4 

### important to recognize that not all of these VDR variants have been linked to functional 5 

### changes. 6 

### Other recent studies conducted in diverse populations of children al so found associations 7 

### between higher blood Pb level and decrements in learning, memory, and visuospatial 8 

### skills; however, they had limited or no consideration of potential confounding by SES

9 

### related variables, which limits the implications of their finding s ( Nelson and Esp y, 2009 ; 10 

### Counter et al., 2008 ; Min et al., 2007 ; Vega

- Dienstmaier et al., 2006 )

. 11



### Academic Performance in Children

As described in the preceding sections, a preponderance of evidence demonstrates Pb

12 

### associated deficits in FSIQ and specific indices of cognitive function, including math, 13 

### reading, and vocabulary skills. Deficits in global cognitive function and the 14 

### aforementioned skills can lead to poorer academic achievement and school performance, 15 

### which may be more objective measures of abilities and skills and have important 16 

### implications for success later in life. Aptitude tests are used to predict future performance 17 

### of an individual on a task or test. Achievement tests and school performance, in 18 

### comparison, assess the actual knowledge of an individual in subject areas the individual 19 

### has studied and measure the acquired knowledge of that subject. Studies reviewed in the 20 

### 2006 Pb AQCD consist ently demonstrated associations of Pb biomarkers with measures 21 

### of academic achievement and performance including scores on math or vocabulary tests, 22 

### class rank, teacher assessment of academic functioning, and high school completion. 23 

### Several studies found t hat blood or dentin Pb levels measured at an early age (ages 24 

### 2

- 8 years) were associated with academic performance at older ages (ages 8

- 18 years), 25 

### suggesting the effect of early exposure to Pb may be persistent ( Leviton et al., 1993 ; 26 

### Bellinger et al., 1992 ; Needleman et al., 1990 )

. Several studies found associations with 27 

### concurrent blood Pb levels ( Kordas et al., 2006 ; Wang et al., 2002a ; Al

- Saleh et al., 2001 ; 28 

### Lanphear et al., 2000 )

. Among recent studies, academic performance was examined less 29 

### frequently; however, findings were consistent with the extant body of evidence. 30 

### The longitudinal study by Bellinger et al. ( 1992 ) was particularly noteworthy for 31 

### examining associations of blood Pb level at several ages from the prenatal period to age 32 

### 10 years with Kaufman Test of Educational Achievement (KTEA) scores at age 10 ye ars 33 

### in the Boston cohort. While blood Pb levels at several ages were associated with lower 34 

### academic achievement scores when in a model alone, only blood Pb level at age 2 years 35February 2012 5

- 81 Draft

– Do Not Cite or Quote 

### showed a statistically significant association with lower predicted academic ac hievement 1 

### after adjusting for HOME score, child stress, maternal age, race, maternal IQ, SES, sex, 2 

### birth order, marital status, and number of residence changes. These covariates were 3 

### selected because they were associated with blood Pb level and KTEA score, changed the 4 

### blood Pb effect estimate by more than 10%, or because they were recognized to be 5 

### important predictors of cognitive development ( e.g., maternal IQ, birth order, sex). Age 6 

### 2 year blood Pb level was associated with a similar magnitude of decrease in the KTEA 7 

### battery composite score in univariate and multivariate models. Additionally, the 8 

### association was robust to adjustment for FSIQ, indicating that blood Pb levels may be 9 

### associated with poorer performance on academic tasks not reflected in FSIQ. 10


### Miranda et al. ( 2007b ) and ( 2009 ) linked blood Pb surveillance data collected between 11 

### ages 0 and 5 years with end

- of

- grade (EOG) testing data for 8 , 600 fourth grade children 12 

### in seven of the largest counties in North Carolina and 57,678 children in the entire state, 13 

### respectively. A key strength of these analyses was the availability of individual

- level data 14 

### on blood Pb level and achievement score on a large number of children representative of 15 

### the North Carolina fourth grade population, which provided large numbers of children 16 

### with lower blood Pb levels (2

- 5 μg/dL). Thus, in this study, there was greater power to 17 

### discern differences in achievement sco res among children in the lower range of blood Pb 18 

### levels. However, due to the records

- based research design, investigators had a smaller set 19 

### of available potential confounding variables than those typically considered in analyses 20 

### of neurodevelopmental outc omes. In addition to adjusting for sex, race, school

- type, 21 

### school district, and age of blood Pb measurement, investigators adjusted for participation 22 

### in a free or reduced lunch program as a measure of SES, and parental education as a 23 

### measure of parental IQ . Miranda et al. ( 2007b ) additionally adjusted for daily use of a 24 

### computer as a measure of a stimulating home environment. Higher e arly childhood blood 25 

### Pb levels were associated with lower reading and math EOG scores (Figure 5

- 6 and 5

- 7), 26 

### and in both analyses, children with a blood Pb level of 2 μg/dL had lower EOG scores (p 27 

### ≤ 0.05) compared with children with a blood Pb level of 1 μg /dL. Further, across deciles 28 

### of blood Pb level, the decrease in EOG score generally was monotonic ( Figure 5

- 6 ). In 29 

### the statewide dataset, compared with children with an earlier (measured at some point 30 

### between birth and age 5 years) blood Pb level of 1 μg/dL, children with an earlier blood 31 

### Pb level of 2 μg/dL had a 0.30

- point lower (95% CI:

- 0.58,

- 0.01) reading EOG score 32 

### ( Miranda et al., 2009 )

. 33February 2012 5

- 82 Draft

– Do Not Cite or Quote Source: Reprinted with permission of Elsevier Science, Miranda et al. ( 2007b )

. Note: These results illustrate the decrease from a baseline score of 262.6 for a hypothetical referent white female, screened at 2 years of age, living in Wake County, NC, with parents with a high school education, not enroll ed in the school lunch program, and who does not use a computer every day ( i.e., model covariates = zero). 

### Figure 5
- 6 Associations between childhood blood Pb levels and fourth grade End
- of
- Grade (EOG) math scores.
While the linear regression analyses indicated fractional

- point decreases in EOG scores 1 

### per 1 μg/dL increases in blood Pb level, Miranda et al. ( 2009 ) additionally used quantile 2 

### regression to discern differential effects in various segments of the EOG distribution 3 

### ( i.e., what is the 10th percentile of EOG scores conditioned on early childhood blood Pb 4 

### levels). Compared with linear regression, quantile regression is more robust in response 5 

### to outliers and predicts outcomes at the top and bottom tails of the outcome distribution 6 

### rather than at the mean. With increasing blood Pb level, the lower tail of the EOG 7 

### distribution was stretched out more so than were the middle or upper portions of the 8 

### distribution. For example, an increase in blood Pb level from 1 to 5 μg/dL was associated 9 

### with a greater decrease in EOG score among children in the 5th percentile of EOG than in 10 

### children in the 95th percentile of EOG score ( Figure 5

- 7 ). These findings indicated that 11 

### children residing at the lowest performance regions of the EOG score distribution may be 12 

### more affected by Pb exposure. Similarly, using quantile regression, Miranda et al. ( 2009 ) 13 

### showed that while cumulative social risk (lower pa rental education, being enrolled in a 14 

### school lunch program) had a greater negative association with academic achievement in 15 

### these children, blood Pb level was independently associated with EOG score decrements 16 

### that were as large as 1 to 2 points. 17February 2012 5

- 83 Draft

– Do Not Cite or Quote Source: Reprinted with permission of Elsevier Science, Miranda et al. ( 2009 ) Note: Baseline score calculated for a hypothetical referent individual with a blood Pb level of 1 μg/dL, parents completed college, and not enrolled in the school lunch program ( i.e., model covaria tes = zero). 

### Figure 5
- 7 Greater reduction in EOG achievement test scores with increasing blood Pb level in lower percentiles of the test score distribution.

Similar to Miranda et al. ( 2009 ) , Chandramouli et al. ( 2009 ) observed associations 1 

### between early childhood blood Pb levels (age 30 months) and later academic 2 

### performance (Standard Assessment Tests [SAT] at age 7 years) among pa rticipants of the 3 

### Avon Longitudinal Study of Parents and Children conducted in the U.K. However, in this 4 

### study, decrements in SAT score were most clearly indicated in children with blood Pb 5 

### levels of 5 to 10 μg/dL. Compared with children with blood Pb leve ls 0

- 2 μg/dL, children 6 

### with blood Pb levels 5

- 10 μg/dL scored 0.51 points lower (95% CI:

- 0.82,

- 0.32) on the 7 

### reading test and 0.49 points lower (95% CI:

- 0.78,

- 0.31) on the math test, adjusting for 8 

### maternal education, home ownership, maternal smoking, ho me facilities score, parental 9 

### SES, family adversity index, and parenting attitudes. Children with blood Pb levels 10 

### 2

- 5 μg/dL did not consistently have lower scores compared with children with blood Pb 11 

### levels 0

- 2 μg/dL. While these aforementioned recent stud ies found associations for early 12 

### childhood blood Pb levels, unlike the longitudinal assessment by Bellinger et al. ( 1992 ) , 13 

### they did not have blood Pb measurements available at other lifestages. Therefore, recent 14 

### studies could not prov ide information on associations with blood Pb levels at other 15 

### lifestages. 16February 2012 5

- 84 Draft

– Do Not Cite or Quote 

### 5.3.2.2 Toxicological Studies of Cognition, Memory and Learning

As described in Section 5.3.2.1 , epidemiologic studies in children have consistently 1 

### demonstrated associations between blood Pb levels and decrements in a range of 2 

### executive functions related to memory, reasoning, problem solving, and verbal skills. 3 

### Studies in animals also demonstrate Pb

- induced decrements in various indices of 4 

### executive function, and several tests conducted in animals share homology with tests 5 

### conducted in humans. 6 

### Data on neurocognition and learning as well as other nervous system endpoints with 7 

### concentrat ion

- response data are shown in Figure 5

- 8 and accompanying Table 5

- 6 . These 8 

### data demonstrate that numerous studies in animal models have demonstrated that Pb 9 

### exposure produces a spectrum of changes in the central nervous system, including 10 

### deficits in CNS development and plasticity and altered homeosta sis of mediators of 11 

### cognitive function that manifest with deficits in memory, learning, and cognition. It is 12 

### thought that early life Pb exposure can permanently alter CNS development and other 13 

### pathways that contribute to these aforementioned memory and lea rning deficits, but the 14 

### animal toxicology data show that multiple sensitive developmental windows for Pb 15 

### exposure exist ( Rice and Gilbert, 1990 )

. As illustrated in Figure 5

- 8 and Table 5

- 6 , Pb

16 

### induced impairments in learning primarily previously were observed in animals with 17 

### blood Pb levels > 30 μg/dL. Several new studies added to the evidence for impaired 18 

### learning and memory in animals with lower blood Pb levels, 8

- 17 μg/dL ( Cory

- Slechta et 19 

### al., 2010 ; Li et al., 2009c ; Niu et al., 2009 ; Virgolini et al., 2008a ; Stangle et al., 2007 )

. In 20 

### these studies of animals with lower blood Pb levels, Pb exposures began early, during the 21 

### gestational or lactation period. 22

23February 2012 5

- 85 Draft

– Do Not Cite or Quote Dosimetric representation reported by blood Pb level. (ID corresponds to Table 5

- 6. ) 

### Figure 5
- 8 Nervous system summary array of toxicological outcomes after Pb exposure.February 2012 5

- 86 Draft

– Do Not Cite or Quote 

### Table 5
- 6 Summary of findings from neurotoxicological exposure
- response ar ray presented in Figure 5
- 8

Study ID in Figure 5
- 8 Reference Blood Pb Level (μg/dL) Outcome

1 Beaudin et al. ( 2007 ) 13 & 31 Behavior, neonate: Lactational Pb exposure, offspring deficient in Reward Omission testing. Physical development ; Postnatal Pb exposure (birth to 4 weeks of age): Pb

- dependent development of over

reactivity to reward omission and errors is reversible with chelation treatment.

2 Grant et al. ( Grant et al., 1980 ) 57 Behavior, neonate: chronic Pb exposure (drinking water) to dams and pups, Changed behavior, males.

3 Kishi et al. ( 1983 ) 59 & 186 Behavior, neonate: Pb exposure (oral gavage of pups) during lactational period, Changed emotional behavior, males and females. Motor function, neonate: Pb exposure (oral gavage of pups) during lactational period, motor function (rotarod performance) impaired, both sexes. Physical development; Pb exposure during lactation (oral gavage): Delayed development of righting reflex in male rats.

4 Overmann ( 1977 ) 33, 174 & 226 Behavior

- Pb exposure (oral gavage of pups) during lactation: aversive conditioning affected by Pb exposur e, male and females. Cognition

- Pb exposure (oral gavage of pups) during lactation: Response inhibition impaired, both sexes. Motor function

- Pb exposure (oral gavage of pups) during lactation: Increased motor activity and impaired motor coordination (rotarod), male and females.

5 Stangle et al. ( 2007 ) 13 & 31 Cognition; Developmental Pb exposure (PND1

- PND30): Impaired learning with visual discrimination task, heightened response to errors, both sexes. Motor function; Developmental Pb exposure (PND1

- PND30): Alcove latency and response latency significantly affected by Pb exposure, both sexes.

6 Gong & Evans ( 1997 ) 38 & 85 Cognition

- Adult male 21 day Pb exposure: Hyperactivity with Habituation to new cage environment. Morphology; 21 day Pb exposure to adult males: Marker of neuronal injury

- elevated hippocampal glial fibrillary acidic protein (GFAP). Physical development; Adult male rats (21 day Pb exposure): Neurotoxicity measured with brain glial fibrillary acidic protein (GFAP). 7 Rice ( 1990 ) 32 & 36 Cognition

- Chronic Pb exposure from birth: Spatial discrimination reversal task impairment, both sexes.

8 Li et al. ( 2009c ) 80 & 100 Cognition

- Gestational & lactational Pb exposure: Morris water maze performance impaired. Morphology; Gestational & lactational Pb exposure: Increased levels of inflammatory cytokines & exocytosis related proteins in brains of pups at weaning, both sexes. Oxidative stress

- gestational and lactational Pb exposure: Elevated hippocampal TNF levels in offspring, males and females.

9 Li et al. ( 2010b ) 80 & 102 Cognition

- Gestational & lactational Pb exposure: Morris water maze performance impaired. Morphology: Increased levels of Alzheimer disease

- associated proteins in mice with gestational and lactational Pb exposure, both sexes. Proliferation/diff/survival, gestational & lactational Pb exposure: Increased hippocampal expression of P

- tau and amyloid beta in male and female pups.

10 Cory

- Slechta et al. ( 2010 ) 10 & 13 Corticosterone: Lifetime Pb +/

- stress: Correlation between 9

- month old’s corticosterone level and frontal cortex dopamine levels in behaviorally tested female offspring. Stress: Corticosterone

- Lifetime Pb plus stress: Affects FI performance, dopamine and serotonin levels in female offspring. Stress: Corticosterone

- neurotransmitter

- Lifetime Pb exposure in female rats plus stress: Dopamine homeostasis affected.

11 Virgolini, Rossi

- George, Weston et al. ( 2008b ) 25 Corticosterone: Maternal Pb plus stress: Elevated corticosterone in male offspring with prenatal stress + offspring stress was further enhanced with Pb exposure. Stress: Corticosterone

- Maternal Pb plus stress: Affects FI performance. Neurotransmitter; Gestational and lactational Pb exposure: Induced DA and 5HT changes in rat offspring.February 2012 5

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– Do Not Cite or Quote Study ID in Figure 5
- 8 Reference Blood Pb Level (μg/dL) Outcome Stress induced neurotransmitter effects, Maternal Pb plus stress: 5

- HT and 5

- HIAA (5

- HT metabolite), and dopamine turnover were significantly affected in males.

12 Virgolini et al. ( 2005 ) 15 & 27 Corticosterone: Chronic Pb exposure from weaning: Pb exposure alone decreased basal plasma corticosterone leve ls at 5 months of age, males. Motor function: Chronic Pb exposure from weaning: Locomotor activity significantly decreased FI response rates & increased post

- reinforcement pause period in a concentration

- dependent manner, males. Stress: Corticosterone

- Chronic Pb plus stress: Affects neurotransmitters & FI performance

13 Virgolini, Rossi

- George, Lisek et al. ( 2008a ) 31 Corticosterone: Maternal Pb exposure (gestation and lactation) +/

- stress: Differential basal corticosterone levels between behavioral and non

- behavioral tested rats, females.

11 &/or 31 Stress: Corticosterone

- Maternal Pb plus stress: Affects FI performance, dopamine, serotonin, and NE levels. Motor function: Maternal Pb +/

- stress: Increased locomotor activity (run rate) with Pb and stress exposure.

31 Neurotransmitter; Gestational and lactational Pb exposure: Induced NE aberrations in adult rat offspring (both sexes). Stress induced motor function: Maternal Pb +/

- stress: Increased locomotor activity (run rate) with Pb and stress exposure. Stress induced neurotransmitter; Gestational and lactational Pb exposure + stress: Induced HVA (monoamine neurotransmitter metabolite) and NE aberrations in female adult rat offspring.

14 Hu et al. ( 2008b ) 4 & 12 Morphology; Gestational Pb exposure: Neurite out growth marker PSA

- NCAM decreased in rat pups, both sexes. Neurophysiology; gestation Pb exposure: decreased hippocampal sialyltransferase activity, both sexes. Physical development; t

- Gestational Pb exposure: Early brain synapse development impaired (hippocampal PSA

NCAM).

15 Wu et al. ( 2008a ) 19 & 26 Morphology: Elevated expression of Alzheimer’s disease

- related genes and Tc factors in aged brains of female monkeys (exposed to Pb as infants). Oxidative stress: Elevated oxidative DNA damage in aged brains of female monkeys (exposed to Pb as infants).
16 Tavakoli

- Nezhad et al. ( 2001 ) 18, 29, & 54 Morphology; 3 to 6 weeks of Postnatal (starting at PND22) Pb exposure in males: Decreased num ber of spontaneously active midbrain dopamine neurons.

17 Leasure et al. ( 2008 ) 10 & 42 Motor function; Mouse maternal (dam) Pb exposure: Induced decreased rotarod performance in offspring (1 year

old male offspring). Neurotransmitter; Mouse maternal (dam) Pb exposure: Affects 1 year old male offspring dopamine homeostasis, both sexes.
18 Fortune & Lurie ( 2009 ) 8 & 43 Neurotransmitter; Mouse maternal (dam) Pb exposure: Affects offspring superior olivary complex (auditory) neurotransmitters, both sexes.



### Memory and Learning
- Morris Water Maze Blood Pb levels have been associated with decrements in both verbal ( e.g., digit span) 1 

### and spatial memory in children ( e.g., spatial and visual memory span) ( Figure 5

- 5 and 2 

### Table 5

- 5 ). In the 2006 Pb AQCD, the results from different animal studies testing the 3 

### effect of Pb on memory were mixed with impaired memory observed in animals with 4 

### blood Pb levels 10

- 35 μg/dL but improved memory observed in other studies. Low

- dose 5 

### Pb was not found to affect short

- term memory, i.e., recall of a learned task. Mixed results 6 

### for memory also may be due to the fact that memory tests also test inattention. Memory 7 

### tests may give incorrect results when opportunities exist for impaired attention to 8 

### contribute to test results ( U.S. EPA, 2006b )

. In animals, spatial memory is tested most 9February 2012 5

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### comm only using the Morris water maze. The 2006 Pb AQCD reported deficits in the 1 

### Morris water maze performance with Pb exposure. New research continued to show Pb

2 

### induced impaired Morris water maze performance. The Morris water maze tests spatial 3 

### memory and lea rning by having a mouse swim and locate or remember the location of a 4 

### platform submerged in opaque water. The Morris water maze is homologous to the 5 

### spatial memory components of the CANTAB and WISC

- R, which test the ability of 6 

### subjects to recall correctly a sequence of locations ( Froehlich et al., 2007 ; Canfield et al., 7 

### 2004 ; Chiodo et al., 2004 )

. 8


### Li et al. ( 2009c ) examined Pb exposures from gestation through lactation. During this 9 

### period, dams received Pb

- acetate dissolved in drinking water (0.1%, 0.5%, and 1% with 10 

### corresponding blood Pb levels of 4, 8 and 10 μg/dL at postnatal day [PND]21). 11 

### Beginning at weaning, Pb

- exposed pups were subjected to Morris water maze 12 

### performance testing. Pb

- exposed pups had statistically signific ant increases in escape 13 

### latency and number of crossings of the platform area at 0.5% and 1% Pb

- acetate exposure 14 

### (blood Pb levels of 8 and 10 μg/dL, respectively), indicating impaired memory and 15 

### learning ( Li et al., 2009c )

. The pups in Li et al. ( 2009c ) were not separated by sex. Cao et 16 

### al. ( 2009 ) found that chronic administration of the supplement melatonin e xacerbated Pb

17 

### induced impairments in spatial memory and long

- term potentiation. Adult male Wistar 18 

### rats that had been exposed to Pb

- acetate (0.2%) from birth received melatonin (3 mg /kg) 19 

### from weaning via gastric gavage for 60 days. At this point (PND81

- 90), animals 20 

### performed in the Morris water maze, and LTP of the hippocampal dentate gyrus was 21 

### measured. 22 

### Another study found that various dietary supplements or methioninecholine concomitant 23 

### with Pb exposure in weanling males shortened the escape latency of Pb

- exposed pups to 24 

### resemble more closely the escape latency of control pups ( Fan et al., 2010 ; Fan et al., 25 

### 2009a )

. Zinc and methionine were effective dietary supplements in Fan et al. ( 2009a ) ; 26 

### glycine, taurine, vitamin C, vitamin B1, tyrosine had no effect on the Pb

- associated 27 

### Morris water maze results. These data on the effect of early life Pb exposure on learning 28 

### and memory in the Morris water maze affirm earlier findings including those by 29 

### K uhlmann et al. ( 1997 ) who used maternal Pb diet e xposure (gestation and lactation), 30 

### continuous Pb exposure (gestation through adulthood) or post

- weaning Pb exposure and 31 

### only found only significant impairments in the maternal and continuous exposure groups. 32 

### Another component of memory is working memory, w hich is the ability to temporarily 33 

### keep information in mind while using the information to perform a related or unrelated 34 

### task. The Morris water maze measures working memory in addition to learning. The 35 

### 2006 Pb AQCD reported that working memory as assessed by the Morris water maze was 36February 2012 5

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– Do Not Cite or Quote 

### significantly affected in chronic developmentally Pb

- exposed ( Pb

- acetate in feed 10 days 1 

### prior to mating through PND 21) female offspring at PND 21 ( Jett et al., 1997 )

. 2 

### Working memory also can be measured by testing delayed spatial alternation (DSA), 3 

### which specifically tests spatial reversal learning. With DSA, an animal receives rewards 4 

### based on alternating responses between two separate levers. In children, tests of spatial 5 

### reversal learning includes the rule learning and reversal components of the CANTAB, 6 

### Stroop Test, and WCST, and decrements in DSA have been associated with higher blood 7 

### Pb levels in children ( Figure 5

- 5 and Table 5

- 5 ). Studies detailed in ear lier Pb AQCDs 8 

### showed that Pb

- exposed animals had deficits under DSA testing ( Alber and Strupp, 1996 ; 9 

### Rice and Gilbert, 1990 ; Rice and Karpinski, 1988 ; Levin et al., 1987 ; Levin and 10 

### Bowman, 1986 )

. Studies in nonhuman primates showed Pb

- induced behavioral 11 

### impairment on DSA tasks ( Rice and Gilbert, 1990 ) across many exposure periods, 12 

### including early

- life and chronic Pb exposures. Specifically, there were multiple lifestages 13 

### during which Pb exposure induced deficits in this task. As in children, deficits in DSA 14 

### included increased response errors, decre ased percent of correct responses, and 15 

### perseverance at a task ( i.e., repeatedly pressing the same lever without moving between 16 

### the two locations). These observations have been consistently made in nonhuman 17 

### primates with continuous Pb exposure or juvenile t o adult exposure but less consistently 18 

### made in rats with juvenile only or juvenile to adult exposure. In fact, some rodent studies 19 

### showed increased accuracy in the delayed alternation trials ( Cory

- Slechta et al., 1991 )

. 20 

### Working memory is a subcategory of executive function or goal

- oriented problem 21 

### solving. Pb

- induced deficits in working memory may be one of many factors that 22 

### contribute to associa tions between blood Pb levels and inattention observed in humans 23 

### ( Min et al., 2007 ; Surkan et al., 2007 ; Chiodo et al., 2004 ; Schweitzer et al., 2000 ; Stiles 24 

### and Belli nger, 1993 )

. 25



### Learning
- Y Maze A recent study using the three

- branch radial Y

- maze showed Pb

- induced effects on 26 

### learning in rat offspring exposed during lactation and into adulthood ( Niu et al., 2009 )

. 27 

### The Y

- maze has a light at the end of each branch. The branch with the illuminated light is 28 

### a safe area whereas the other two branches are electrified and cause a mild electric shock 29 

### when entered . The Y

- maze test evaluates learning based on three criteria: 1) learning 30 

### days or th e number of days required to learn the maze (90% correctly); 2) the error 31 

### number (EN) or the number of Y

- maze runs required to learn the maze; and 3) the total 32 

### reaction time (TRT) or the total amount of time spent in the maze per test day. Wistar 33 

### Albino ra t pups were exposed to Pb

- acetate during lactation until the termination of the 34 

### experiment at 12 weeks of age (dam drinking water 300 mg /L, blood Pb level: 17 μg/dL 35 

### at 6 weeks of age. Rats were evaluated on the three Y

- maze endpoints at 6, 8, 10 and 36February 2012 5

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### 12 wee k s of age. ) Pb induced statistically significant impairments in learning, in 1 

### particular, at 8, 10 and 12 weeks of age. EN and TRT were significantly affected only at 2 

### 8 and 10 weeks, respectively. Hippocampal glutamate levels were also significantly 3 

### attenua ted in Pb

- exposed animals, and this is discussed in more detail in Section 5.3.8.4

. 4



### Learning
- Response Inhibition and Schedule
- Controlled Behavior Testing Response inhibition is another measure of executive function and is measured with 5 

### multiple tests of premature responses, decreased pause time between two scheduled 6 

### events, and increased perseverance. These tests include Differential Reinforcement of 7 

### Low Rates of Responding (DRL), Fixed Interval (FI) testing ( Table 5

- 7 ), FI with 8 

### Extinction (FI

- Ext) or Fixed Ratio (FR)

- FI, and Signal Detection with Distraction. 9 

### Multiple studies from the 2006 Pb AQCD as well as earlier literature showed that early 10 

### life Pb exp osure contributed to response inhibition across the spectrum of these 11 

### aforementioned tests. Monkeys with moderate blood Pb levels (11

- 13 μg/dL) learned the 12 

### DRL task more slowly but eventually acquired reinforcement rates equal to that in 13 

### controls. Newer da ta from female rats exposed to Pb ( Stangle et al ., 2007 ) continued to 14 

### show animals with premature responses after Pb exposure or response inhibition 15 

### decrements. These findings in animals are consistent with observations in children that 16 

### blood Pb levels are associated with poorer impulse control ( Figure 5

- 14 and Table 5

- 9 ). 17 

### In children, impulsiv ity is commonly assessed by having parents or teachers rate problem 18 

### behaviors such as trouble waiting, interrupting others, and responding at inappropriate 19 

### times. However, studies also have specifically tested response inhibition assessed using 20 

### the continu ous performance test, which similar to animal tests, measures reactions to a 21 

### stop signal (Section 5.3.3.1 ). 22 

### The 2006 Pb AQCD discussed learning or cognition as measured with schedule

23 

### controlled behaviors including FI and FR operant conditioning and found that FI response 24 

### rate was affected differentially with low

- level (as low as 11 μg/dL) and high

- level Pb 25 

### (peak levels of 115 μg/dL) exposures increas ing and decreasing FI response rate , 26 

### respectively . This nonlinear response was since further explored in recent work, much of 27 

### which also examined the interaction between stress and Pb exposure. 28February 2012 5

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### Table 5
- 7 Summary of effects of maternal and lifetime Pb exposure on FI performance

Pb (ppm) Maternal Pb b Lifetime Pb c
Overall rate a PRP a Overall rate a PRP a 0 ppm: 0

- PS No Significant Effect d No Significant Effect No Significant Effect No Significant Effect 0

- OS No Significant Effect *↓

- 23% No Significant Effect No Significant Effect 50 ppm: 50

- NS No Significant Effect No Significant Effect *↑ 95% No Significant Effect 50

- PS No Significant Effect No Significant Effect *↑ 79.2% *↓

- 42% 50

- OS *↑ 64.9% No Significant Effect *↑ 74.7% * ↓

- 39.3% 150 ppm: 150

- NS *↑ 42.4% *↓

- 30.3% No Significant Effect No Significant Effect 150

- PS No Significant Effect *↓

- 25.7% *↑ 90.7% *↓

- 44.7% 150

- OS *↑ 59.2% No Significant Effect *↑ 78.5% No Significant Effect Note: *Dam blood Pb levels ranged from 5

- 13 μg/dL over gestation and lactation; offspring blood Pb ranged from 7

- 13 μg/dL from early life time points out to ten months of age. Thus, this study demonstrates that lifetime Pb exposure with or without prenatal stress induced learning deficits in female mice. Mechanistically, these authors propose that associations of Pb and stress with learning deficits may be related to aberration s in corticosterone and dopamine.

a Based on calculation of group mean values acr oss session block post

- stress challenge for both maternal and lifetime Pb exposure studies. All ca lculations represent percent of 0

- NS control values; ↑ , represents increase; ↓, represents decrease. PRP = post

- reinforcement pause. b Data from Virgolini et al. ( 2005 )

. *Denotes significant effect versus 0ppm control ( p <0 .05).

c Data from current study [Rossi

- George et al. ( 2011 ) ] *D enotes sign ificant effect vs. 0ppm control ( p <0 .05). Source: Reprinted with permission of Elsevier Science, Rossi

- George et al. ( 2011 ) (Table 1).



### Learning Ability with Stress The combined paradigm of Pb exposure and stress experienced by a laboratory animal is 1 

### now being studied by multiple investigators who are focusing on the common pathway of 2 

### HPA axis alteration and altered brain neurotransmitter levels. These studies indicated 3 

### greater impairments in learning with Pb when combined with stress. These findings 4 

### provide support for the association between higher blood Pb level and lower MDI score 5 

### observed in children with mothers with low self

- esteem but not children with mothers 6 

### with high self

- esteem ( Surkan et al., 2008 ) ( Table 5

- 4 ). In children, maternal self

- esteem 7 

### has been linked with the ability to cope with stress and improved maternal

- child 8 

### interactions. As indicated in Figure 5

- 8 and Table 5

- 6 , Pb exposure has been shown to 9 

### increase corticosterone levels and exacerbate Pb

- induced dopamine release and learning 10 

### ability. Cory

- Slechta and colleagues have conducted multiple investigatio ns in this area. 11 

### Most recently, they showed enhanced learning deficits in female rat offspring following 12 

### lifetime Pb exposure combined with maternal restraint or prenatal stress ( Cory

- Slechta et 13 

### al., 2010 )

. This exp osure paradigm used dams who were exposed to Pb for 2 weeks prior 14February 2012 5

- 92 Draft

– Do Not Cite or Quote 

### to mating through lactation and pups from a mixed sex litter from their dams during the 1 

### aforementioned period and then via drinking water Pb (50 ppm ) through the remainder of 2 

### their lifetime. The resultant blood Pb levels of dams and pups ranged from 5 to 13 μg/dL. 3 

### In a separate investigation, Pb plus stress

- related outcomes were examined in female 4 

### offspring of dams who were exposed to Pb from 2 months prior to mating through 5 

### lactation, i.e., developmental Pb exposure (2 exposure groups: 50 or 150 ppb Pb

- acetate 6 

### in drinking water) ( Virgolini et al., 2008a )

. Dams were subject to restraint stress at 7 

### GD16

- 17. Compared with co ntrols, marked increases in response rates on FI performance 8 

### were found in the Pb plus stress female offspring, whose mean blood Pb level was 9 

### 11 μg/dL (50 ppb Pb

- acetate ). Because these animals did not show effects with maternal 10 

### stress or Pb exposure alone , the results indicated a potentiation of effects with Pb and 11 

### stress co

- exposures. 12 

### Similarly, lifetime Pb exposure (50 or 150 ppm , resulting in blood Pb levels of 11

- 16 and 13 

### 25

- 33 μg/dL, respectively) plus stress (maternal or offspring) also induced FI abe rrations 14 

### at the post

- reinforcement pause (PRP) period in female offspring ( Rossi

- George et al., 15 

### 2011 ) ( Table 5

- 7 ). Again, the results indicated a potentiation of effects. Within the FI 16 

### schedule, the PRP represents timing capacity or proper temporal discrimination. Namely, 17 

### the PRP is the period during wh ich the animal must wait or pause before depressing the 18 

### lever for a reward. In this case, Pb plus stress exposed animals started responding too 19 

### early due to a decreased pause or PRP interval. Aberrant FI performance in infants and 20 

### children has been used as a marker for impulsivity. Separately, overall FI response rate, a 21 

### hyperactive behavior, was significantly increased with Pb exposure alone and with 22 

### maternal or offspring stress at the 50 ppm exposure dose. At 150 ppm Pb, stress 23 

### (maternal or offspring) inc reased FI response rate but Pb alone had no effect on FI. 24 

### Biochemical analysis of possible mechanistic contributions to these aberrations revealed 25 

### alterations in frontal cortex norepinephrine, reductions in dopamine homeostasis in the 26 

### nucleus accumbens, an d enhancement of the striatal monoamine system. This study on 27 

### the effect of lifetime Pb exposure with or without stress on FI testing itself or during the 28 

### PRP component of FI testing further affirms Pb

- related learning deficits and provides 29 

### possible mechan istic explanations. 30 

### Pb exposure over various developmental windows has been shown to affect 31 

### corticosterone levels in rodents. These findings indicate that associations of Pb and stress 32 

### with learning deficits (FI testing in females) may be related to aberra tions in 33 

### corticosterone and dopamine. Maternal Pb exposure (150 ppm drinking water from 34 

### 2 months prior to mating through lactation with restraint stress as detailed above) induced 35 

### increased basal corticosterone in female and male offspring at 9 months of a ge; no 36 

### interactions of Pb and stress were observed in this model ( Cory

- Slechta et al., 2004 )

. By 37February 2012 5

- 93 Draft

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### 14 months of age, these offspring had reduced corticosterone concentrations versus 1



### c ontrol animals, indicating a possible acceleration of age

- related decreases in basal 2 

### corticosterone levels ( Cory

- Slechta et al., 2008 )

. Pb

- induced decreases in corticosterone 3 

### were enhanced with maternal stress. Postnatal exposure of male rodents to Pb (PND 4 

### 21

- 5 months of age) produced animals with s ignificant decrements in baseline 5 

### corticosterone; this effect produced a U

- shaped concentration

- response curve with 6 

### significant decrements in basal corticosterone levels in the 50 ppm exposure group versus 7 

### control ( Virgolini et al., 2005 )

. In summary, developmental (gestational and lactational) 8 

### and post

- weaning exposure to Pb induced changes in the HPA axis (corticosterone levels) 9 

### in both sexes that were dynamic as th e animal aged. 10 

### Mechanistic understanding of the cognitive deficits observed with Pb and/or stress 11 

### exposure was explored in a recent study. HPA hypofunction following dam Pb exposure 12 

### (pup gestational and lactational Pb exposure) with or without maternal st ress was 13 

### reported ( Rossi

- George et al., 2011 ; Virgolini et al., 2008a )

. This study used the same 14 

### mode l of developmental Pb exposure as is detailed in the preceding paragraph. Outcomes 15 

### were examined in both male and female offspring. At 2 months of age without stress, 16 

### basal corticosterone in females was significantly increased with 150 ppm Pb (resulting in 17 

### blood Pb level of 32 μg/dL at PND21) and 50 ppm Pb (resulting in blood Pb level of 18 

### 19 μg/dL) ( Figure 5

- 9 ). Pb plus stress attenuated the Pb

- induced el evations in 19 

### corticosterone to baseline levels ( Figure 5

- 9 ). At age 10 months, Pb and stress accelerated 20 

### the age

- dependent decrease in corticosterone le vels in females. In males, basal 21 

### corticosterone levels were not affected significantly by Pb and/or stress at 2 or 10 months 22 

### of age ( Figure 5

- 9 ). These authors also explored the function of the glucocorticoid 23 

### negative feedback loop using the dexamethasone suppression test and found that Pb 24 

### and/or maternal stress significantly impacted this negative feedback by increasing nuclear 25 

### glucocorticoid receptor l evels. This negative feedback loop was impacted more at the 26 

### lower dose (50 ppm versus 150 ppm Pb

- acetate ). As summarized in Table 5

- 7 , the results 27 

### indicate that lifetime Pb exposure when combined with stress can exacerbate impairments 28 

### in learning. The interaction between Pb and stress may be mediated via effects on 29 

### corticos terone and dopamine. 30February 2012 5

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– Do Not Cite or Quote Source: Rossi

- George et al. ( 2011 ) Note: *denotes significantly different from NS control; # denotes significantly different from corresponding Pb

- NS value; + differs from 50

- NS. (0, 50, 150 pp m ) and/or stress (PS [dam stress] or OS [PS followed by offspring stress]). 

### Figure 5
- 9 Mean basal corticosterone levels of female and male offspring exposed to lifetime Pb.
Another study examined the effects of Pb on the HPA axis but examined the interaction 1 

### with an outside stress administered using control vehicle injections ( Rossi

- George et al., 2 

### 2009 )

. The corticosterone response to this vehicle injection stress was prolonged in a 3 

### nonlinear concentration

- dependent manner in both sexes with the most profound effects 4 

### observed at the lower 50 ppm Pb dose. Maternal stress also prolonged the corticosterone 5 

### stress response to vehicle injection and enhanced the Pb effect in males. To test the 6 

### negative feedback of the HPA axis, exogenous dexamethasone (DEX) was administered 7 

### to suppress endogenous corticosterone. The DEX test revealed HPA axis hypofunction. 8 

### Specifically, Pb and Pb plus maternal stress initially reduced the ability of DEX to 9 

### suppress corticosterone. With time, the effect of DEX in males induced prolonged 10 

### corticosterone suppression or failure to return to baseline as was observed in control 11 

### animals. In summary, dam Pb exposure induced HPA negative feedback hypofunction in 12 

### both sexes of offspring with an inverse U concentration

- response function. 13 

### These studies of lifetime exposure ( 2011 ; Rossi

- George et al., 2009 ) reported different 14 

### basal stress hormone levels with Pb exposure. Males with lifetime Pb exposure had no 15 

### statistically significant corticosterone response to Pb exp osure; whereas males with dam 16 

### Pb exposure had statistically significant decreases in corticosterone at 5 months of age in 17February 2012 5

- 95 Draft

– Do Not Cite or Quote 

### the 50 ppm exposure group only (not in 150 ppm Pb exposure group). On the other hand, 1 

### females had concentration

- dependent corticostero ne responses to Pb exposure in both 2 

### exposure models (lifetime Pb exposure and dam Pb exposure). Maternal stress alone also 3 

### led to HPA axis negative feedback hypofunction. Pb plus maternal stress enhanced 4 

### negative feedback in males and attenuated this effec t in females. Pb exposure with or 5 

### without maternal stress prolonged the effect of DEX

- dependent corticosterone 6 

### suppression in males. These data together show that HPA axis alterations could provide a 7 

### link between the contribution of Pb and stress to health effects. 8


### Schedule

- controlled behavior is often measured using FI or FR testing. Because the FI 9 

### animals are regularly handled by laboratory personnel and participate in tests of 10 

### cognition, their baseline level of stress may be skewed from that of a labora tory animal 11 

### that constantly remains in a cage without daily handling. Because effects on the HPA axis 12 

### are of interest to Pb researchers, the baseline corticosterone levels of animals that have 13 

### participated in behavior testing (FI) and those who have not (N FI) have been compared. 14 

### Specifically, the corticosterone differences between FI and NFI animals after 15 

### developmental Pb exposure (dam

- only Pb exposure) have been measured. Virgolini et al. 16 

### ( 2008b ) found that basal corticosterone levels were significantly different between FI and 17 

### NFI animals. Also, the combination of dam Pb exposure with maternal stress was 18 

### explored in FI and NFI animals. At the baseline age of 4

- 5 months, NFI animals 19 

### displayed significant differences from FI animals. Pb expo sure with or without stress did 20 

### not induce differences in corticosterone levels in FI females. The corticosterone level of 21 

### male FIs was affected by Pb and stress exposure ( Virgolini et al., 2008b )

. In the FI males, 22 

### the 50 ppb Pb exposure group (50Pb) had decreased corticosterone versus control (no Pb 23 

### exposure) and the 150 ppb Pb exposure group (150Pb) had elevated corticosterone versus 24 

### control. Male NFI animals showed a U shaped concentration

- response curve with 50Pb 25 

### animals having signif icantly less corticosterone than did control or 150Pb animals. In the 26 

### NFI males, stress did not affect corticosterone levels or interact with the effect of Pb. NFI 27 

### females exposed to 150Pb had significantly elevated corticosterone versus control (no Pb 28 

### exp osure). These data demonstrate that behaviorally trained animals have altered HPA 29 

### axis and response to Pb exposure versus animals that are housed under conditions without 30 

### daily handling by caregivers. 31 

### Virgolini et al. ( 2008b ) also expanded evidence for Pb exposure

- stress interactions 32 

### through the examination of the effects additional intermittent stress as an adult. The 33 

### authors proposed that associations of Pb and stress with learning deficits (FI testing in 34 

### females) may be related t o aberrations in corticosterone and dopamine. Dam exposure to 35 

### Pb (50 or 150 ppm Pb

- acetate ) followed by intermittent stress (cold, novelty or restraint) 36 

### in offspring as adults induced statistically significant changes in FI response rate. 37 

### Females were more sensitive to the adult intermittent stressors at the higher dose of Pb 38February 2012 5

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### (150 ppm ) with statistically significant increases in FI response rate and decreased PRP, 1 

### i.e., increased impulsivity ( Figure 5

- 10 ). Males were more sensitive (decreased FI 2 

### response rate due to decreased run rate) to the restraint stress at the lower Pb dose 3 

### (50 ppm ). At the higher dose of Pb, males were more sensitive to the cold s tress 4 

### (increased FI response rate and increased run rate) ( Figure 5

- 11 ). Corticosterone levels 5 

### were examined in this study and showed concentration

- de pendent correlations with FI 6 

### outcomes in females but were independent of dose in males. 7 

### Another study examined female rats with lifetime Pb exposure combined with prenatal 8 

### stress and found enhanced learning deficits (drinking water 50 ppm Pb

- acetate , resul ting 9 

### in offspring blood Pb levels: 7

- 13 μg/dL) ( Cory

- Slechta et al., 2010 )

. Learning was 10 

### evaluated with multiple schedule of repeated learning (RL) and performance testing. RL 11 

### was impaired, but performance was not affected with Pb exposure. The Pb

- impaired RL 12 

### was further enhanced with prenatal stress. There were statistically sig nificant associations 13 

### between Pb/stress and corticosterone concentration, dopamine from the frontal cortex, 14 

### dopamine turnover in the nucleus accumbens, and total number of responses required to 15 

### learn a sequence. Also, Pb

- exposed offspring with and without maternal stress exposure 16 

### had statistically significant decreases in hippocampal nerve growth factor (NGF) versus 17 

### controls. Thus, this study demonstrated that lifetime Pb exposure with or without prenatal 18 

### stress induced learning deficits in female mice. 19February 2012 5

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– Do Not Cite or Quote Source: Reprinted with permission of Elsevier Science, Virgolini, Rossi

- George, Weston, et al. ( 2008b )

. 

### Figure 5
- 10 Changes in FI performance (FI overall performance, run rate, PRP) in female offspring with maternal Pb exposure plus various stressors (restraint, cold, novelty) in adulthood.February 2012 5

- 98 Draft

– Do Not Cite or Quote Source: Reprinted with permission of Elsevier Science, Virgolini, Rossi

- George, Weston, et al. ( 2008b )

. 

### Figure 5
- 11 Changes in FI performance (FI overall performance, run rate, PRP) in male offspring with maternal Pb exposure plus various stressors (restraint, cold, novelty) in adulthood.

Cognitive Flexibility Cognitive flexibility is a c omponent of executive function that measures the ability to 1 

### reallocate mental resources when situations change ( Monsell, 2003 )

. This flexibility is 2 

### assessed as the ability to alter behavioral responses according to the context of the 3 

### situation and incorporates the function of attention, working memory, and visual 4 

### processing. Discrimination reversal learning and concurrent random interval (RI

- RI) 5 

### sched uling are used to measure cognitive flexibility. The 2006 Pb AQCD reported 6 

### discrimination reversal learning deficits in monkeys with blood Pb levels of 11

- 20 μg/dL. 7 

### Rats also showed similar deficits but the authors attributed the changes to learning

- relate d 8 

### problems instead of cognitive flexibility ( Garavan et al., 2000 ; Hilson and Strupp, 1997 )

. 9 

### Interestingly, recent work has shown that N

- Methyl

- D

- aspartic acid or N

- Methyl

- D

10 

### aspartate (NMDA) r eceptors and D2

- like receptors, two well

- characterized targets of Pb, 11 

### are involved in discrimination reversal learning ( Herold, 2010 )

. Another test of cognitive 12 

### flexibility is called concurrent random interval (RI

- RI) scheduling in which depression on 13 

### two response levers is reinforced at different frequencies. The 2006 Pb AQCD reported 14 

### monkeys with Pb

- induced c ognitive flexibility impairment under RI

- RI ( Newland et al., 15 

### 1994 )

. Coherence for these observations of Pb

- induced deficits in cognitive flexibility is 16 

### provided by consistent evidence in animals and children for associations of Pb with 17February 2012 5

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### individual tests of inattention, memory, and visual processing. Studies in children als o 1 

### demonstrate associations between blood Pb levels and decrements in specific tests of 2 

### cognitive flexibility that are homologous to tests in animals such as discrimination 3 

### reversal learning, the WCST, and Stoop test ( Figure 5

- 5 and Table 5

- 5 ). While tests in 4 

### animals measure the ability to complete a task according to changes in reinforcement, 5 

### tests in humans assess the ability of subjects to complete a task according to changes in 6 

### rules. 7



### 5.3.2.3 Toxicological Studies on the Effects of Chelation
Earlier work in the animal toxicological literature has shown tha t succimer or chelation 8 

### treatment of Pb

- exposed lab animals was able to normalize various aberrant Pb

- induced 9 

### behaviors including activity level, habituation ( Gong and Evans, 1997 ) and forced

- swim 10 

### immob ility ( Stewart et al., 1996 )

. A more recent study examined the effect of succimer 11 

### treatment on various behavioral and cognitive outcomes in control and neonatally Pb

12 

### exposed f emale animals (PND 1

- 30 Pb

- acetate exposure, 300 ppm dam through lactation 13 

### and either 30 or 300 ppm pup water) by drinking water, generating a moderate Pb (m

- Pb) 14 

### exposure and a high Pb (h

- Pb) exposure group. Pb blood levels at PND52 in the control, 15



### m
- Pb, h

- Pb, m

- Pb+succi mer, and h

- Pb+succimer were 1.5; 12.6; 31; 2.8; and 8.5 μg/dL, 16 

### respectively. Brain P b levels at the same t ime for the same groups were 41 (control), 17 

### 1 ,040 (m

- Pb), 3 ,690 (h

- Pb); and 196 (m

- Pb+succimer) and 1 , 370 (h

- Pb+succimer) ng/g 18 

### dry we ight . Succimer treatment significantly attenuated the m

- Pb induced impaired 19 

### learning ability. Effects on arousal that were significantly affected in h

- Pb rats were 20 

### significantly attenuated with succimer treatment. Succimer treatment in the h

- Pb animals 21 

### only slightly improved learning ability but did not improve the impaired inhibitory 22 

### control ( Stangle et al., 2007 )

. These are important findings becau se they provide evidence 23 

### that certain neurobehavioral or cognitive impairments associated with Pb exposure appear 24 

### to be reversible with chelation therapy. 25 

### In another study, a 3

- week course of Pb

- acetate (PND 1

- 17, dam drinking water) plus or 26 

### minus succimer /chelator (PND 31

- 52) treatment was given to determine if succimer 27 

### could alleviate behavioral deficits in rats exposed to Pb for the first 4 weeks of life. Pb

28 

### exposed animals had altered reactivity and increased reward omission and errors. Pb

29 

### exposed anima ls receiving chelation treatment had normalized reactivity to reward 30 

### omission and errors ( Beaudin et al., 2007 )

. Pb

- induced behavioral abnormalities were 31 

### atten uated with chelation therapy. 32


### The interaction between Pb and methionine choline also has been examined ( Fa n et al., 33 

### 2009a )

. Pb

- exposed rats were supplemented with methionine choline to provide 34February 2012 5

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### information on its effect on Pb disposition in various tissues (blood, bone, brain) and its 1 

### contributions to neurocognitive or neurobehavioral changes. As a sulfur so urce, 2 

### methionine is a chelator and a free

- radical scavenger. Choline is important for cell 3 

### membranes and neurotransmitter synthesis ( Zeisel and Blusztajn, 1994 )

. In this model, 4 

### methionine choline attenuated Pb

- induced memory and learning deficits 5 

### (Section 5.3.2.2 ). Exposure of weanling male rats to Pb

- acetate in drinking water 6 

### (300 mg /L) through PND60 produced a blood Pb level of 60 μg/dL, bone Pb level of 7 

### 165 μg/g, and brain Pb level of 0.63 μg/g. Methionine choline supplementation 8 

### significantly attenuated blood and bone Pb levels but produced a nonsignificant 9 

### attenuation of brain Pb (0.51 μg/g) in rats that had significant improvements in learning 10 

### and memory (Section 5.3.2.2 ). Also, in another study, the metal chelators DP

- 109 and 11 

### DP

- 460 were neuroprotective for Pb

- related effects in the ALS mouse neurodegenerative 12 

### model or Tg(SOD1

- G93A) model ( Petri et al., 2007 )

. 13 

### In summary, succimer or chelation treatment a ppears to be able to restore Pb

- dependent 14 

### impairments of learning and arousal and be neuroprotective in a concentration

- dependent 15 

### fashion. In these studies, succimer use was more efficacious at lower doses of Pb 16 

### exposure. Chelation did not restore Pb

- impai red inhibitory control. Chelation with the 17 

### antioxidant supplement affected the disposition of Pb in various tissues, significantly 18 

### attenuating blood and bone Pb levels and nonsignificantly attenuating brain Pb. 19



### 5.3.2.4 Integrated Summary of Cognitive Function in Children
Results from recent epidemiologic studies and animals studies expand the strong existing 20 

### evidence base demonstrating that Pb exposure is associated with impaired cognitive 21 

### function in children. A large epidemiologic evidence base demonstrates asso ciations of 22 

### higher blood Pb level with lower FSIQ in school

- aged children, lower MDI scores in 23 

### children ages 6 months to 3 years, and lower scores on tests of specific cognitive 24 

### functions in children ages 6 months to age 16 years ( Figure 5

- 2 and Figure 5

- 5 ; Table 25 

### 5

- 3 , Table 5

- 4 , and Table 5

- 5 ). Blood Pb level has been associated with a range of 26 

### cognitive indices, including memory, verba l and math skills, and cognitive flexibility. 27 

### There was no clear indication that blood Pb level was more strongly associated with 28 

### performance in a particular domain of cognitive function. Studies in animals clearly 29 

### demonstrated impaired performance on Morr is Water Maze, the delayed spatial 30 

### alternation, and discrimination reversal learning with Pb exposure. These animal 31 

### observations provided strong coherence with associations in children with homologous 32 

### tests of spatial memory and rule learning and reversal ( Figure 5

- 5 and Table 5

- 5 ). Several 33 

### studies f ound that blood or dentin Pb levels measured at an early age (ages 2

- 8 years) 34 

### were associated with poorer academic performance at older ages (ages 8

- 18 years), 35February 2012 5

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### suggesting the effect of early exposure to Pb may be persistent and have important 1 

### implications for success later in life. 2 

### The weight of epidemiologic evidence for Pb

- associated cognitive function decrements 3 

### was provided by several prospective studies conducted in diverse populations that found 4 

### associations with blood Pb levels measured during vario us lifestages (concurrently, 5 

### prenatally (cord or maternal), earlier in childhood, and averaged over multiple years) and 6 

### had extensive evaluation of potential confounding variables. The association with FSIQ 7 

### was substantiated in a pooled analysis of childre n, 5 to 10 years of age, participating in 8 

### seven prospective studies (Boston, MA; Cincinnati, OH; Rochester, NY; Cleveland, OH; 9 

### Mexico City, Mexico; Port Pirie, Australia; and Kosovo, Yugoslavia) ( Lanphear et al., 10 

### 2005 )

. Several new studies added evidence for associations of FSIQ with concurrent 11 

### blood Pb level in populations with lower mean blood Pb levels. Previously, the weight of 12 

### evidence supported these associations in populations with mean blood Pb levels in the 13 

### rang e of 5

- 10 μg/dL. However, several new studies shifted the weight of evidence to 14 

### lower blood Pb levels (primarily concurrent), with populations means in the range of 15 

### 2

- 7 μg/dL ( 2011 ; Kim et al., 2009b ; Min et al., 2009 ; Zailina et al., 2008 ; Chiodo et al., 16 

### 2007 )

. Studies of specific cognitive indices and academic performance did not 17 

### consistently find decrem ents in performance in children with blood Pb levels < 5 μg/dL 18 

### ( Cho et al., 2010 ; Miranda et al., 2010 ; Chandramouli et al., 2009 ; Surkan et al., 2007 )

. 19 

### Several new toxicological studies added to the evidence for im paired learning and 20 

### memory in animals with lower blood Pb levels, 8

- 17 μg/dL ( Cory

- Slechta et al., 2010 ; Li 21 

### et al., 2009c ; Niu et al., 2009 ; Virgolini et al., 2008a ; Stangle et al., 2007 )

. A large body 22 

### of new evidence from Cory

- S lechta and colleagues demonstrated that lifetime Pb 23 

### exposure when combined with stress in animals exacerbated impairments in learning 24 

### ( 2011 ; Cory

- Slechta et al., 2010 ; Rossi

- George et al., 2009 ; Virgolini et al., 2008a )

. 25 

### Findings also indicated that these interactions potentially were mediated via effects on 26 

### corticosterone and dopamine. 27 

### With regards to important lifestages of Pb exposure, the weight of toxicological evidence 28 

### demonstrates impaired learn ing and memory in animals exposed to Pb gestationally with 29 

### or without early postnatal exposure. In particular, impairments in learning and memory 30 

### observed with lower blood Pb levels (8

- 17 μg/dL), were found with Pb exposures that 31 

### began during the gestation al or lactation period. The prospective epidemiologic studies 32 

### found decrements in cognitive function of children in association with concurrent, 33 

### prenatal (cord and maternal), early childhood, and cumulative average blood Pb levels. 34 

### However, collectively, b ased on the frequency of examination, the weight of evidence 35 

### demonstrates associations between cognitive function decrements and concurrent blood 36 

### Pb levels in children ages 4

- 10 years. Among studies that examined MDI in children ages 37 

### 6 months to 3 years, s everal found stronger associations of MDI with prenatal (maternal 38February 2012 5

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### or cord) blood Pb than with postnatal child blood Pb ( Hu et al., 2006 ; Bellinger et al., 1 

### 1987 ; Dietrich et al., 1987a ; Vimpani et al., 1985 )

. Child concurrent or lifetime average 2 

### blood Pb levels also were associated with MDI scores ( Claus Henn et al. ; Solon et al., 3 

### 2008 ; Surkan et al., 2007 ; Tellez

- Rojo et al., 2006 ; Wasserman et al., 1992 ; Vimpani et 4 

### al., 1985 )

. Thus, both postnatal child and maternal Pb exposures may contribute to lower 5 

### cognitive function in young children. It is important to note that in studies that examined 6 

### maternal pregnancy or cord blood Pb levels, there is greater uncertainty regarding the 7 

### relative contributions of maternal past and recent Pb exposures that contributed to 8 

### associations. The influence of past or cumulative maternal Pb exposures was supported 9 

### by observations that maternal patella Pb l evels were associated with MDI among children 10 

### at age 2 years in Mexico City ( Gomaa et al., 2002 )

. Nonetheless, results for prenatal 11 

### blood Pb levels and concurrent child blood Pb levels measured during specific postnatal 12 

### ages indicate that relatively short

- duration Pb exposures (< 1 year) influence the 13 

### cognitive d evelopment of children between birth and age 3 years. Short

- duration Pb 14 

### exposures may be especially important during early childhood because processes such as 15 

### neurogenesis and synaptic pruning are highly active during the first few years of life 16 

### ( Rice and Barone, 2000 ; Landrigan et al., 1999 )

. 17 

### Epidemiologic studies found in dependent associations of blood Pb level after adjusting 18 

### for multiple potential confounding factors, including parental IQ, SES, household 19 

### income, and HOME score. In most studies that provided unadjusted and adjusted effect 20 

### estimates, blood Pb level was fo und to be associated with a smaller but statistically 21 

### significant decrement in FSIQ after adjusting for potential confounding factors 22 

### ( Palaniappan et al., 2011 ; Kim et al., 2009b ; Chiodo et al., 2007 ; Froehlich et al., 2007 ; 23 

### Lanp hear et al., 2005 ; Canfield et al., 2003a )

. Analyses of associations of covariates with 24 

### blood Pb level and cognitive function indicated that the potential confounding variables 25 

### may vary across populations and endpoints. HOME score was not associated with every 26 

### cognitive index. Studies that adjusted for multiple SES

- related factors but did not 27 

### examine HOME score produced similar magnitudes of associations as did studies that 28 

### adjusted for HOME score ( Table 5

- 3 , Table 5

- 4 , to Table 5

- 5 ). Thus, confounding by 29 

### HOME score may be minimized when other correlated variables are accounted for in 30 

### analyses. Further, it is uncertain the extent to which HOME score alone may confound 31 

### blood Pb

- IQ associations as studies have not reported the magnitude of change in the 32 

### blood Pb effect estimate with just the addition of HOME score in the model. Thus, while 33 

### the caregiving environment can be an important confounder, the overall weight of 34 

### evidence indicates that it does not mitigate the strong findings linking higher blood Pb 35 

### levels with lower FSIQ in children. Prenatal drug exp osure also was not found to have a 36 

### large influence on the relationship between blood Pb level and FSIQ ( Min et al., 2009 ; 37 

### Chiodo et al., 2007 )

. That Pb exposure induces impairments in tests of learning and 38



### m emory in animals further that are directly homologous to tests conducted in children 39February 2012 5

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### further demonstrates that confounding by SES and HOME does not fully account for the 1 

### associations between blood Pb levels and cognitive function observed in children. 2


### The evidence from epidemiologic and toxicological studies linking Pb exposure with 3 

### decrements in multiple cognitive functional domains is strengthened by the well

4 

### characterized toxicological evidence for Pb exposure interfering with development of the 5 

### brain an d activity of neurochemical processes that mediate cognitive function 6 

### (Section 5.3.8 ). Pb has been shown to increase the permeability of the blood

- b rain barrier 7 

### and deposit in the target central nervous system. Pb has been shown to impair 8 

### neurogenesis, synaptic architecture, and neurite outgrowth. The high activity of these 9 

### processes during fetal and infant development provides biological plausibility for young 10 

### children being particularly at increased risk for Pb

- associated impairments in cognitive 11 

### function. Cognitive function is mediated by the cortical and subcortical structures of the 12 

### brain that integrate function in the hippocampus, prefrontal cort ex, and nucleus 13 

### accumbens using dopamine and glutamate as primary neurotransmitters. Experimental 14 

### studies have shown that Pb induces changes in dopamine release in these regions. 15 

### Numerous studies also have shown Pb

- induced changes in hippocampal function, 16 

### including changes in glutamate release, receptor binding, and long

- term potentiation. 17 

### Thus, several lines of toxicological evidence establish a neuroanatomical and 18 

### neurochemical basis for the effect of Pb on cognitive function 19



### 5.3.2.5 Epidemiologic Studies of Cognitive Function in Adults

Adults without Occupational Lead Exposures As described in the preceding section, Pb exposure of animals that begins in gestation 20 

### and lasts through the early postnatal period or for a lifetime has been shown to induce 21 

### learning impairments in adult animals. Less well characterized are learning impairments 22 

### in adult animals due to adult

- only Pb exposures. In contrast, epidemiologic studies have 23 

### examined cognitive performance in adults primarily in association with concurrently 24



### meas ured blood and bone Pb levels. For nonoccupationally

- exposed adults, the 2006 Pb 25 

### AQCD cited some evidence for associations of cognitive performance with bone Pb 26 

### levels but not blood Pb levels ( U.S. EPA, 2006b )

. Studies published since the 2006 Pb 27 

### AQCD continued to indicate that bone Pb levels were associated more consistently with 28 

### cognitive function in nonoccupationally

- exposed adults ( Table 5

- 8 ). Despite the large 29 

### number of available publications, i t is important to recognize that several studies are 30 

### variants on analyses in the same population ( e.g., NHANES or the Normative Aging 31 

### Study [ NAS]) and should not be considered as all independent assessments of the Pb

32 

### cognitive function relationship. Another point to consider is that although cross

- sectional 33February 2012 5

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### and longitudinal studies have been conducted, most examine the associations between 1 

### one measurement of cognitive function and one concurrent measurement of blood Pb 2 

### level. Such a design has the inherent weakness that temporality cannot be determined, 3 

### which limits the causal inference regarding the effects of Pb exposure. When bone Pb 4 

### level is used as the exposure measure, this issue is mitigated somewhat because bone Pb 5 

### is a biomarker that reflects many cumulative years of exposure. Additionally, with single 6 

### measurements of both blood and bone Pb level, it is difficult to characterize the specific 7 

### timing, duration, and level of Pb ex posure that contributed to the associations observed 8 

### with cognitive function. This uncertainty may apply particularly for assessments of blood 9 

### Pb levels, which are influenced both by current exposures and cumulative Pb stores in 10 

### bone that are mobilized dur ing bone remodeling compared with occupationally

- exposed 11 

### adults (Sections 4.3 and 4.7.3 ). 12 

### Several analyses of the large prospective Baltimore Memory Study and NAS have 13 

### contributed to greater understanding of the relationship between biomarkers of Pb and 14 

### cognitive effects in adul ts. In both cohorts, subjects were evaluated periodically with in

15 

### person clinical assessments and self

- administered questionnaires. Both studies were noted 16 

### for comparisons of associations between bone and blood Pb levels within the same cohort 17 

### and for thei r repeated assessments of Pb biomarker levels and cognitive function over 18 

### time. In particular, the repeated assessments permitted the examination of associations of 19 

### Pb biomarkers with changes in cognitive function over time, which allowed investigators 20 

### to establish temporality between Pb biomarker levels and subsequent changes in 21 

### cognitive function. The Baltimore Memory Study (BMS) included men and women, 22 

### 50

- 70 years of age, residing in Baltimore, MD. A total of 1 , 140 out of 2 , 351 (48.5%) 23 

### subjects participa ted from neighborhoods that represented a diversity of race and SES. Of 24 

### particular note, this study was unique in that it included a large proportion of African

25 

### Americans (n=395). In comparison, the NAS involved only men (original n = 2 , 280) 26 

### residing in th e Greater Boston area. Subjects primarily were white and at enrollment were 27 

### aged 21 to 80 years and had no current or past chronic medical conditions. Studies 28 

### differed with respect to the potential confounding variables analyzed. The most notable 29 

### differenc e was the inclusion of age, smoking and alcohol intake as covariates in the NAS 30 

### analyses . Despite differences in study population characteristics and potential 31 

### confounding variables considered, findings were similar between cohorts. 32 

### In the BMS, longitudina l analyses were conducted with repeat cognitive testing of study 33 

### subjects at approximately 14

- month intervals. Most subjects completed follow

- up; 91% 34 

### of the original cohort returned for a second round of testing and 83% for a third round 35 

### each ( Bandeen

- Roche et al., 2009 )

. An interquartile range higher tibia Pb level (12.7 36 

### μ g/g) was associated with a 0.019 units per year decrease in eye

- hand coordination z

37 

### score, adjusting for age, sex, interviewer, race and SES. Tibia Pb was associated with a 38February 2012 5

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### larger decrease among Africa n Americans than among whites ( Table 5

- 8 ). Tibia Pb levels 1 

### were weakly associated with other time

- related decreases in other indices of cognitive 2 

### func tion ( e.g., language, processing speed, executive function). 3


### Cross

- sectional analyses of the BMS included comparisons of the associations for blood 4 

### and bone Pb level. The 991 adults in Shih et al. ( 2006 ) had a m ean (SD) blood and tibia 5 

### Pb levels of 3.46 (2.23) μg/dL and 18.7 (11.2) μg/g, respectively, Whereas higher 6 

### concurrent blood and bone Pb level both were associated with poorer performance in the 7 

### domains of language, processing speed, eye

- hand coordination, executive function, 8 

### verbal memory and learning, visual memory, and visuoconstruction, associations with 9 

### tibia Pb level tended to be larger in magnitude (per SD increase) and statistically 10 

### significant ( Table 5

- 8 ). Tibia Pb levels were associated with worse performance on tests 11 

### in all domains in models adjusted for age, sex, testing technician, and presence of the 12 

### apolipoprotein (APO)E

- ε4 allele (potentia l risk factor for Alzheimer's Disease). The 13 

### magnitudes of associations were attenuated with additional adjustment for education, 14 

### race, and SES. In these fully

- adjusted models, higher tibia Pb levels were associated with 15 

### poorer performance in domains except language and processing speed, with a borderline 16 

### statistically significant association observed for visuoconstruction. In linear models, 17 

### visuoconstruction scores were 0.0044 SDs (95% CI:

- 0.0091, 0.0003) lower per 1 μg/g 18 

### bone higher tibia Pb level. Analys is of a quadratic term for tibia Pb indicated no evidence 19 

### of nonlinearity. 20


### Other cross

- sectional analyses indicated effect modification by race and neighborhood 21 

### psychosocial hazards. In contrast with the longitudinal results, r ace
- stratified analyses of 22 

### p ersistent effects in cross

- sectional analyses indicated that tibia Pb levels were associated 23 

### with greater decreases in performance on tests of eye

- hand coordination, executive 24 

### functioning, and verbal memory and learning among whites than among African 25 

### Amer icans ( Bandeen

- Ro che et al., 2009 ) ( Table 5

- 8 ). 26February 2012 5

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### Table 5
- 8 Associations of blood and bone Pb levels with cognitive function in adults a

Study Population/ Methods Cognitive Test Subgroup/ Model Blood Pb Effect Estimate (95% CI) b Bone Pb Effect Estimate (95% CI) b
Bandeen

Roche et al. ( 2009 ) 1 , 140 adults, ages 50

- 70 yr Baltimore Memory Study (BMS) cohort Baltimore, MD Marginal linear regression models adjusted for age, household wealth, education, race/ethnicity Longitudinal associations Eye/hand coordination African

- Americans White

Cross

- sectional associations Verbal memory/learning African

- Americans White

Executive Function African

- Americans White NOT EXAMINED Tibia Mean (SD): 18.8 (11.6) μg/g Per 12.7 μg/g (IQR) increase

- 0.032 (

- 0.052,

- 0.012) per year

- 0.009 (

- 0.024, 0.006) per year

0.006 (

- 0.09, 0.10)

- 0.076 (

- 0.15, 0.001)

0.009 (

- 0.05, 0.07)

- 0.07 (

- 0.12,

- 0.01) Shih et al. ( 2006 ) 985 adults, mean age: 59 yr BMS, Baltimore, MD Linear regression adjusted for: Model A: age, sex, technician, presence of APOE

- ε4 allele Model B: Model I, years of education, race/ethnicity, wealt h Raven’s Colored Progressive Matrices
Language
Eye

- hand coordination
Executive functioning

Visuoconstruction Model A Model B Model A Model B Model A Model B Model A Model B Concurrent blood Pb Mean (SD): 3.5 (2.2) μg/dL

- 0.013 (

- 0.064, 0.037)

- 0.004 (

- 0.044, 0.036)

- 0.024 (

- 0.0704, 0.022)

- 0.017 (

- 0.057, 0.024)

- 0.031 (

- 0.075, 0.012)

- 0.022 (
- 0.059, 0.015)

- 0.042 (
- 0.101, 0.017)

- 0.031 (

- 0.084, 0.021) Tibia Mean (SD): 18.7 (11.2) μg/g

- 0.0896 (
- 0.146,
- 0.034) 0.007 (

- 0.034, 0.047)

- 0.0896 (
- 0.134,
- 0.045)

- 0.034 (
- 0.067, 0)

- 0.0896 (
- 0.123,
- 0.056)

- 0.016 (

- 0.056, 0.025)

- 0.024 (

- 0.190, 0.141)

- 0.049 (
- 0.101, 0.002) TIBIA Pb STRONGER Glass et al. ( 2009 ) 1 , 001 adults, mean age 59 yr BMS, Baltimore, MD Multilevel hierarchical regression model adjusted for age, sex, race/ethnicity, education, testing technician, time of day Raven’s Colored Progressive Matrices Language
Eye

- hand coordination
Executive functioning

Visuoconstruction Middle NPH High NPH Middle NPH High NPH Middle NPH High NPH Middle NPH High NPH NOT EXAMINED Tibia Pb Mean (SD): 18.8(11.1) μg/g
0.011 (

- 0.089, 0.111) c

- 0.09 (

- 0.189,

- 0.011) c

- 0.04 (

- 0.133, 0.044) c

- 0.067 (

- 0.167, 0.033) c

- 0.022 (

- 0.111, 0.067) c

- 0.111 (

- 0.1998,

- 0.022) c

- 0.003 (

- 0.155, 0.149) c

- 0.007 (

- 0.189, 0.175) c Weisskopf et al. ( 2007a ) 1 , 089 males, mean age 68.7 yr; Normative Aging Study (NAS), Boston, MA Linear repeated measures analysis adjusted for age, age squared, education, smoking, alcohol intake, yr between bone Pb measurement and first cognitive test, yr between cognitive tests CERAD, Neurobehavioral Evaluation System, WIAS

- R, MMSE, VMI
Visuospatial, pattern comparison (+ = poorer performance) Executive function verbal fluency

Short

- term memory, word list NOT EXAMINED Mean (IQR): Tibia: 20 (15) μg/g Patella: 25 (20) μg/g
Estimates per IQR increase: Tibia: 0.79 (0.40 , 1.2) over time Patella: 0.73 (0.40, 1.2) over time

Tibia:

- 0.40 (

- 1.6, 0.80) over time Patella:

- 0.86 (

- 2.00, 0.30) over time

Tibia:

- 0.28 (

- 1.2, 0.60) over time Patella:

- 0.81 (

- 1.7, 0.05) over timeFebruary 2012 5

- 107 Draft

– Do Not Cite or Quote Study Population/ Methods Cognitive Test Subgroup/ Model Blood Pb Effect Estimate (95% CI) b Bone Pb Effect Estimate (95% CI) b Wang et al. ( 2007a ) 358 males, median ages: 67.2 yr (HFE wild

- type) 67.7 yr (HFE variant) Normative Aging Study, Boston, MA Linear regression adjusted for age, years of education, nonsmoker, former smoker, pack

- years, nondrinker, alcohol consumption, English as first language, computer experience, d iabetes MMSE HFE wildtype One HFE variant Two HFE variants NOT EXAMINED Median: 19 μg/g (Tibia), 23 μg/g (Patella)

- 0.20 (

- 1.0, 0.70) per year

- 1.40 (

- 3.3, 0.40) per year

- 6.3 (

- 10.4,

- 2.1) per year

Weuve et al. ( 2006 ) 720 males, ages ≥ 4 5 yr NAS, Boston, MA Linear mixed effects regression adjusted for smoking status, alcohol consumption, calorie adjusted calcium intake, regular energy expenditure on leisure time physical activity, diabetes MMSE score ALAD wildtype ALAD2 carrier
ALAD wildtype ALAD2 carrier Concurrent blood Pb Mean (IQR): 5.2 (3) μg/dL

- 0.013 (

- 0.053, 0.027)

- 0.087 (

- 0.180, 0.007) Median (IQR): Tibia: 19 (15) μg/g; Patella: 27 (21) μg/g

Tibia

- 0.03 (
- 0.14, 0.07)

- 0.11 (
- 0.30, 0.06) Patella

- 0.03 (
- 0.11, 0.04)

- 0.12 (
- 0.30, 0.06) Rajan et al. ( 2008 ) 720 males, ages ≥ 4 5 yr Normative Aging Study, Boston, MA Linear regression adjusted for blood Pb main effect, ALAD genotype, age at cognitive test, education, alcohol consumption, cumulative smoking, English as first language C ERAD, Neurobehavioral Evaluation System, WIAS

- R

Visuospatial, constructional praxis

Executive function verbal fluency
Verbal memory, word recall

Perceptual speed, mean latency Concurrent blood Pb Mean (SD): 5.3 (2.9) μg/dL (ALAD wildtype); 4.8 (2.7) μg/dL (ALAD2 carriers)

- 0.048 (

- 0.216, 0.120) d

- 0.028 (

- 0.2044, 0.148) d

0.003 (

- 0.168, 0.174) d

- 0.168 (
- 0.392, 0.056) d Mean (SD): Tibia: 21.9 (13.8) μg/g (ALAD wildtype), 21.2 (11.6) μg/g (ALAD2 carriers) Patella: 29.3 (19.1) μg/g (ALAD wildtype), 27.9 (17.3) μg/g (ALAD2 carriers)

Tibia:
- 0.216 (
- 0.419,
- 0.013) d Patella: 0.018 (

- 0.182, 0.218) d

Tibia:
- 0.089 (
- 0.292, 0.1143) d Patella:

- 0.018 (

- 0.218 0.1082) d
Tibia: 0.064 (

- 0.127, 0.254) d Patella: 0.127 (

- 0.728, 0.989) d

Tibia:

- 0.152 (

- 0.495, 0.191) d Patella:

- 0.146 (

- 0.40, 0.109) d TIBIA Pb STRONGER Weuve et al. ( 2009 ) 587 f emales, ages 47

- 74 yr Nurses’ Health Study, Boston, MA Generalized estimating equations adjusted for age, age - squared at Pb assessment, age at cognitive assessment, education, husband’s education, alcohol consumption, smoking status, physical activity, asp irin use, ibuprofen use, use of Vitamin E supplements, menopausal status and postmenopausal hormone use Telephone Interview for Cognitive Status and East Boston Memory Test
Composite cognitive score

Composite except letter fluency Concurrent blood Pb Mean (SD): 2.9 (1.9) μg/dL

- 0.015 (

- 0.068, 0.038)

0.015 (

- 0.070, 0.101) Mean (SD) Tibia Pb: 10.5 (9.7) μg/g Patella Pb: 12.6 (11.6) μg/g

Tibia:
- 0.039 (
- 0.087, 0.0097) Patella:

- 0.012 (

- 0.058, 0.035)

Tibia:
- 0.049 (
- 0.097, 0) Patella:

- 0.035 (

- 0. 081, 0.012)

TIBIA Pb STRONGERFebruary 2012 5

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– Do Not Cite or Quote Study Population/ Methods Cognitive Test Subgroup/ Model Blood Pb Effect Estimate (95% CI) b Bone Pb Effect Estimate (95% CI) b Krieg and Butler ( 2009 ) 2 , 823 adults, ages 20

- 59 yr, U.S. NHANES III (1991

- 1994) Log

- linear regression model adjusted for age, sex, education, family income, race - ethnicity, computer or video

- game familiarity, alcohol use w ithin the last 3 h, test language Neurobehavioral Evaluation System 2
Symbol Digit Substitution (mean total latency, sec) Serial digit learning score Ages 20

- 39 yr Ages 40

- 59 yr Ages 20

- 39 yr Ages 40

- 59 yr Concurrent Blood mean (SD): 2.88 (6.91) μg/dL

- 0.097 (

- 0.422, 0.228) e

- 0.290 (

- 0.601, 0.0207) e

- 0.117 (

- 0.463, 0.228) e 0.401 (

- 0.193, 0.995) e NOT EXAMINED

Krieg et al. ( 2009 ) 2 , 090 adults, ages 20

- 59 yr 1976 adults, ages ≥ 6 0 yr

U.S. NHANES III (1991

- 1994) Log linear regression model adjusted for sex, age, education, family income, race - ethnicity, computer or video game familiarity, alcohol use in the last 3 hrs, test language (20

- 59 yr) and sex, age, education, family income, race

- ethnicity, test language ( ≥ 6 0 yr) Neurobehavioral Evaluation System 2

Symbol Digit Substitution (mean total latency, sec)
Serial digit learning score

Word recall

Story recall Ages 20

- 59 yr ALAD GG ALAD CC/CG Ages 20

- 59 yr ALAD GG ALAD CC/CG Ages ≥ 6 0 yr ALAD GG ALAD CC/CG Ages ≥ 6 0 yr ALAD GG ALAD CC/CG Concurrent Blood Pb Mean (SD): 20

- 59 yr: 2.85 (7.31) μg/dL; ≥ 6 0 yr: 4.02 (3.56) μg/dL

- 0.132 (

- 0.358, 0.095) e

- 0.526 (

- 1.118, 0.066) e

- 0.022 (

- 0.526, 0.482) e 0.025 (

- 0.406, 0.456) e

- 0.075 (

- 0.285, 0.135) e 0.025 (

- 0.406, 0.456) e
0.085 (

- 0.0997, 0.271) e

- 0.466 (

- 1.072, 0.139) e NOT EXAMINEDFebruary 2012 5

- 109 Draft

– Do Not Cite or Quote Study Population/ Methods Cognitive Test Subgroup/ Model Blood Pb Effect Estimate (95% CI) b Bone Pb Effect Estimate (95% CI) b Krieg et al. ( 2010 ) 2 , 093 adults, ages 20

- 59 yr 1 , 799 adults, ages ≥ 6 0 yr U.S. NHANES III (1991

- 1994) Log linear regression model adjusted for sex, age, education, family income, race - ethnicity, computer or video game familiarity, alcohol use in the last three hours, test language (20

- 59 yr) and sex, age, education, family income, race

ethnicity, test language ( ≥ 6 0 yr) Neurobehavioral Evaluation System 2

Symbol Digit Substitution (mean total latency, sec)

Serial digit learning score (+ = poorer performance)

Word recall

Story recall Ages 20

- 59 yr VDR haplotype CC VDR haplotype CT VDR haplotype TC VDR haplotype TT Ages 20

- 59 yr VDR haplotype CC VDR haplotype CT VDR haplotype TC VDR haplotype TT Ages ≥ 6 0 yr VDR haplotype CC VDR haplotype CT VDR haplotype TC VDR haplotype TT Ages ≥ 6 0 yr VDR haplotype CC VDR haplotype CT VDR haplotype TC VDR haplotype TT Concurrent blood Pb Mean (SD): 20

- 59 yr: 2.85 (7.32) μg/dL; ≥ 6 0 yr: 4.02 (3.39) μg/dL

- 3.916 (

- 8.638, 0.805) e 0.139 (

- 0.278, 0.556) e

- 0.505 (

- 1.025, 0.015) e

- 0.695 (

- 0.783, 0.871) e

- 2.533 (

- 4.868,

- 0.198) e

- 0.322 (

- 0.922, 0.278) e 0.447 (0.542, 0.351) e 0.044 (

- 0.783, 0.871) e

- 0.766 (

- 1.817, 0.285)

- 0.085 (

- 0.40, 0.21)

- 0.034 (

- 0.471, 0.403)

- 0.095 (

- 0.895, 0.705)
0.146 (

- 1.674, 1.966) 0.003 (

- .193, 0.20) 0.034 (

- 0.322, 0.3899)

- 0.166 (

- 0.434, 0.102) NOT EXAMINED

Gao et al. ( 2008 ) 188 adults, mean age 69.2 yr Sichuan and Shandong Provinces, China ANCOVA adjusted for age, sex, education, BMI, APOE ε4 CERAD, CSID, IU story recall, Animal fluency test, IU token test Composite cognitive score Concurrent plasma Pb Mean (SD): 0.39 (0.63) μg/dL

- 0.006 (

- 0.016 , 0.004) NOT EXAMINEDFebruary 2012 5

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– Do Not Cite or Quote Study Population/ Methods Cognitive Test Subgroup/ Model Blood Pb Effect Estimate (95% CI) b Bone Pb Effect Estimate (95% CI) b
Van Wijngaarden et al. ( 2009 ) 47 adults, mean age 61.5 yr Rochester, NY Linear regression adjusted for age, gender, educational level, history of hypertension CANTAB and Montreal Cognitive Assessment

Delayed matching, % correct

Total trials NOT EXAMINED Mean (SD): Tibia: 2.0 (5.2) μg/g; Calcaneus: 6.1 (8.5) μg/g Calcaneus Lowest tertile: 87.56 f Medium tertile: 86.67 Highest Tertile: 80.67, p = 0.027 Tibia Lowest tertile: 85.42 f Medium tertile: 87.08 Highest tertile: 82.44, p = 0.25

Calc aneus Lowest tertile: 2.54 f Medium tertile: 2.61 Highest tertile: 2.72, p = 0.21 Tibia Lowest tertile: 2.62 f Medium tertile: 2.59 Highest tertile: 2.66

a Studies are presented by cohort then generally in the order of discussion in the text. b Effect estimates have been standardized to the standard deviation of the cognitive test scores and standardized to an SD or IQR incr ease in blood or bone Pb level.

c Effect estimates indicate interactions between Pb and category of neighborhood psychosocial hazard (NPH), with the lowest tertile of NPH serving as the reference group. d Effect estimates indicate interactions between Pb and ALAD genotype.

e The directions of effect estimates were changed to indicate a negative slope as a decrease in cognitive performanc e. f Results refer to mean cognitive function scores among tertiles of bone Pb.


### Similar to the BMS, in the NAS cohort, higher tibia Pb levels were associated with 1 

### decreases in cognitive performance over time ( Weisskopf et al., 2007a )

. Weisskopf et al. 2 

### ( 2007a ) expanded evidence by finding associations with patella Pb levels. Two 3 

### measurements of c ognitive function, collected approximately 3.5 years apart were 4 

### available for 60

- 70% of participants. Longitudinal analyses were conducted with repeated 5 

### measures plus a bone Pb

- time interaction term in order to estimate the association 6 

### between bone Pb level and decline in cognitive test score over time. Although bone Pb 7 

### levels were associated with increased response latency on a pattern comparison test, they 8 

### were associated with fewer errors on the same test. The authors proposed that this may be 9 

### related to slowing reaction time to improve accuracy. When the nine men with the 10 

### highest bone Pb levels were removed, the association with fewer errors was no longer 11 

### statistically significant. However, the authors did not indicate whether the point estimate 12 

### changed. In the analysis with patella Pb, Weisskopf et al. ( 2007a ) found a nonlinear 13 

### association, with latency times becoming worse over time ( i.e., larger values or slower 14 

### response time) up to approximately 60 μ g/g patella Pb, but the change over time levelin g 15 

### off at higher levels ( Figure 5

- 12 ). Below 60 μ g/g, a 20 μ g/g difference in patella Pb level 16 

### was associated with an increase in latency of approximately 0.15 ms. 17February 2012 5

- 111 Draft

– Do Not Cite or Quote Source: Reprinted with permission of Williams & Wilkins, Weisskopf et al. ( 2007a )

. Note: Models are ad justed for age, age squared, education, smoking, alcohol intake, years between bone Pb measurement and first cognitive test, and years between the cognitive tests. The 9 subjects with the highest patella Pb concentrations (>89 μg/g bone mineral) were remov ed. The estimate is indicated by the solid line and the 95% confidence interval by the dashed lines. Patella Pb concentrations of all individual subjects are indicated by short vertical lines on the abscissa. (reference = 0 at mean of patella Pb concentrat ion). 

### Figure 5
- 12 Nonlinear association between patella Pb level and the relative change over time in response latency on the pattern comparison test.
Longitudinal analysis of the NAS cohort also indicated effect modification by 1 

### hemochromatosis (HFE) gene variants ( Wang et al., 2007a )

. In models adjusted for 2 

### age, years of education, nonsmoker, former smoker, pack

- years, nondrinker, alcohol 3 

### consumption, English as first language, computer experience, and diabetes , an 4 

### int erquartile range higher tibia Pb level (15μg/g) was associated with a 0.22 point steeper 5 

### annual decline (95% CI:

- 0.39,

- 0.05) in MMSE score among men with either the H63D 6 

### and C282Y variant. The association was found to be nonlinear, with larger Pb

- associa ted 7 

### declines observed at higher tibia Pb levels ( Figure 5

- 13 ) . This difference was comparable 8 

### to the difference in MMSE score between men who were 4 y ears apart in age in their 9 

### study sample. Tibia Pb level was associated with a smaller decline in MMSE score in 10 

### men with only HFE wildtype alleles ( Figure 5

- 13 ). HFE variants, H63D and C282Y, are 11 

### associated with hemochromatosis, a disease characterized by higher iron body burden. 12 

### Higher iron body burden has been linked with lower Pb absorption, thus the apparent 13 

### interaction could be related to altered Pb toxicokinetics rather than some other direct 14 

### biological interaction. For example, the relation between tibia Pb level and Pb dose at the 15 

### biologically relevant site(s) could be shifted to the left among for HFE variant allele 16 

### carriers such that for the sam e tibia Pb level, Pb the dose at the relevant biological site(s) 17February 2012 5

- 112 Draft

– Do Not Cite or Quote 

### may be higher leading to a greater annual decline in MMSE score as indicated in Figure 1 

### 5

- 13 (solid curve). This implies that similar decreases in MMSE score would still be 2 

### found in wildtype HFE carriers only at higher tibia Pb levels. 3

4

Source: Wang et al. ( 2007a )

. Note: The lines indicate curvilinear trends estimated from the penalized spline met hod. Among HFE wild

- types, the association between tibia Pb and annual cognitive decline was nearly linear, but among variant allele carriers, the association tended to deviate from linearity (p = 0.08). The model was adjusted for age, years of education, nonsmoker, former smoker, pack

- years, nondrinker, alcohol consumption, English as first language, computer experience, and diabetes. 

### Figure 5
- 13 Exploration of nonlinear association of tibia Pb level with an nual rate of cognitive decline, by hemochromatosis (HFE) gene variant.
The 2006 Pb AQCD described associations of blood and tibia Pb levels with poorer 5 

### cognitive performance among 141 NAS men ( Payton et al., 1998 )

. Several new and 6 

### larger cross

- sectional NAS analyses corroborated previous findings for bone Pb but 7 

### generally indicated weak associations with concurrent blood Pb levels and only in groups 8 

### with specific genetic variants. In contrast with the longitudinal a nalyses, Weisskopf et al. 9 

### ( 2007a ) found that repeat measures of bone Pb levels were not consistently associated 10 

### with cognitive function in cross

- sectional analyses. In a study of Mini

- Mental State 11 

### Examination (MMSE) tests scores (test of general cognitive function) among 720 men 12 

### 45 years of age and older, higher concurrent blood Pb levels were associated with lower 13 

### MMSE scores among ALAD2 carriers ( Weuve et al., 2006 )

. A 3 μg/dL higher concurrent 14 

### blood Pb level (the interquartile range) was associated with a 0.26 point lower mean 15 

### MMSE score (95% CI:

- 0.54,

- 0.01) among ALAD2 carriers and a 0.04 point lower score 16 

### (95% CI:

- 0.16,

- 0.07) among noncarriers. A subsequent study did not find a consistent 17February 2012 5

- 113 Draft

– Do Not Cite or Quote 

### direction of modification of the association between blood or bone Pb levels and other 1 

### tests of cognitive function by ALAD genotype ( Rajan et al., 2008 )

. Nonetheless, Rajan et 2 

### al. ( 2008 ) found tibia Pb levels to be associated more strongly and consistently with 3 

### poorer cognitive performance compared with concurrent blood or patella Pb levels ( Table 4 

### 5

- 8 ). 5 

### Effect modification by stress was examined in both the NAS and BMS cohorts. In the 6 

### NAS cohort, higher bone Pb levels were associated with poorer cognitive function among 7 

### individua ls with higher individual

- level perceived stress ( Peters et al., 2007 )

. H igher tibia 8 

### Pb levels also were associated with larger decrements in cognitive performance among 9 

### BMS subjects living in neighborhoods with a greater number psychosocial hazards 10 

### ( e.g., number of violent crimes, emergency cal ls, off

- site liquor licenses) ( Glass et al., 11 

### 2009 ) ( Table 5

- 8 ). These observation of effect modification by environmental stress in 12 

### adult humans is supported by several observations of Pb

- stress interactions in impaired 13 

### learning and memory and adult animals with Pb exposures from gestation through post

14 

### weaning and lifetime Pb exposures (Section 5.3.2.2 ). 15


### Weuve et al. ( 2009 ) studied the association of blood and bone Pb levels with cognitive 16 

### function in a subset of 587 healthy women in the Boston, MA area participating in the 17 

### Nurses' Health Study. Blood and bone Pb levels were measured between the ages of 47 18 

### and 74 years, and the mean (SD) blood Pb level in this group was 2.9 (1.9) μg/dL 19 

### measured in samples collected an average of 5 years before cognitive testing. As in the 20 

### aforementi oned studies of adults, tibia and patella Pb levels were more consistently 21 

### associated with cognitive performance than was blood Pb levels ( Table 5

- 8 ). Contrary to 22 

### expectation, higher patella and tibia Pb levels were associated with higher scores on the 23 

### “f” naming test (naming words that begin with f). In separate models, the “f” naming test 24 

### was omitted from a composite index of all cognitive tests, and a one SD (10 μg/g bone) 25 

### higher tibia Pb level was associated with 0.051

- point lower (95% CI:

- 0.010,

- 0.003) 26 

### standardized composite score. A similar magnitude of decrease was estimated for an 27 

### increase in age of 3 years in these women. The magnitude of asso ciation was slightly 28 

### smaller for an SD unit increase in patella Pb level (

- 0.033 [95% CI:

- 0.080, 0.014). 29


### Several stud ies analyzed data from the U.S.

- representative NHANES III (1991

- 1994) 30 

### population of men and women and investigated effect modification by age and genetic 31 

### variants. Only blood Pb levels were available and were measured in samples collected 32 

### concurrently with cognitive testing. Krieg and Butler ( 2009 ) did not find blood Pb level 33 

### consistently to be associated with poorer performance on cognitive testing among 2 , 090 34 

### adults 20

- 59 years of age or among 1 , 796 adults 60 years of age and older. Because 35 

### different types and numbers of tests were conducted in the two age groups, it is difficult 36 

### to compare findings between age groups. In the subset of the population with genetic 37February 2012 5

- 114 Draft

– Do Not Cite or Quote 

### ana lysis, blood Pb

- cognitive function associations were not found to be modified by 1 

### ALAD genetic variants in the same direction ( Krieg et al., 2009 )

. Among adults ages 2 

### 20

- 59 years with the CC and CG ALAD genotype groups combined ( i.e., ALAD2 3 

### carriers), reaction time improved ( i.e., faster reaction time) by 38 ms per 10

- fold increase 4 

### in concurrent blood Pb level. In contrast, among ALAD2 carriers, higher blood Pb levels 5 

### were associated with poorer performance on a symbol

- digit substitution task. The 6 

### potential direction of effect modification by the ALAD2 genotype is not immediately 7 

### clear as the grea ter affinity of the ALAD2 enzyme subunit for Pb may increase 8 

### susceptibility to Pb

- associated health effects by increasing blood Pb levels or it may 9 

### diminish Pb

- associated health effects by decreasing Pb bioavailability by maintaining it 10 

### in a sequestered st ate in the bloodstream. Krieg et al. ( 2010 ) did find differences in the 11 

### association between con current blood Pb level and scores on a symbol

- digit substitution 12 

### test by the VDR variants, rs731236 and VDR rs2239185, and by the VDR haplotype. 13 

### Similar to observations in adolescent NHANES participants (Section 5.3.2.1 ), results 14 

### were not uniform across the various tests. However, blood Pb level generally was 15 

### associated with greater decrements in cognitive performance among adults with the CC 16 

### genotyp es of VDR variants. 17 

### Other studies with smaller numbers of subjects generally produced results consistent with 18 

### those from the larger studies above. A cross

- sectional study of 188 rural Chinese men and 19 

### women found a weak association between higher plasma Pb levels and a lower composite 20 

### cognitive score based on a battery of in

- person administered tests ( Gao et al., 2008 )

. It 21 

### should be noted, though, that Pb in plasma makes up a very small fraction of all Pb in 22 

### blood and is a different, and much less used, biomarker than Pb in whole blood. The 23 

### relevance of this Pb fraction is not entirely clear. Pb in plasma is not bound to 24 

### erythrocytes, as is about 99% of blood Pb. Thus, it has been postulated that plasma Pb 25 

### may be mo re toxicologically active ( Chuang et al., 2001 ; Hernandez

- Avila et al., 1998 )

. 26 

### In another cross

- sectional study of 47 men and women in Roch ester, NY (55

- 67 years of 27 

### age), subjects in the higher two tertiles of calcaneal bone (trabecular bone with higher 28 

### turnover rate than tibia) Pb level performed worse on delayed matching

- to

- sample and 29 

### paired associated learning tasks ( Van Wijngaarden et al., 2009 ) ( Table 5

- 8 ). In analyses 30 

### of tibia Pb levels, subjects in the highest tertile of tibia Pb level did not consistently 31 

### perform worse on cognitive tests ( Table 5

- 8 ). The exact calcaneal and tibia Pb levels in 32 

### tertiles were not reported. 33



### Adults with Occupational Lead Exposures The 2006 Pb AQCD concluded that in adults, blood Pb levels were associated with 34 

### cognitive function most consistently among those with occupational Pb exposures. These 35 

### findings were supported by results from a few recent studies of occupationally

- exposed 36February 2012 5

- 115 Draft

– Do Not Cite or Quote 

### adults. Dorsey et al. ( 2006 ) followed up on a cohort of Pb

- exposed workers in Korea with 1 

### a mean age of 43.4 years, on whom patella Pb measurements were made. This group 2 

### represented a typically highly

- exposed occupational group with an average blood Pb 3 

### level of 30.9 μg/dL. In this cohort, both blood and tibia Pb levels previously were found 4 

### to be associated with poorer performance on a battery of neurocognitive tests ( Schwartz 5 

### et al., 2005 ; Schwartz et al., 2001 )

. Dorsey et al. ( 2006 ) found higher patella Pb levels to 6 

### be associated with poorer manual dexterity, poorer sensory function, and greater 7 

### depression symptoms. These associations for patella Pb levels were not as strong as the 8 

### previously reported associations were with either blood or tibia Pb levels in this 9 

### occupational cohort. 10


### A follow

- up study of the original 1982 Lead Occupational Study was conducted 11 

### 2001

- 2004 with 83 of the original 288 Pb

- exposed workers and 51 of the original 181 12



### c ontrols ( Khalil et al., 2009b )

. Those originally in the exposed workers group had last 13 

### worked in a job with Pb exposure from 0.02 to 16 years (median: 6) prior to follow

- up 14 

### testing. While the follow

- up participation was somewhat low, participants did not di ffer 15 

### from nonparticipants on most baseline cognitive tests except for performing slightly 16 

### better on aspects of the grooved pegboard test and recall on a paired associates learning 17 

### task. This suggests that the follow

- up participation was not biased to poor performers. At 18 

### follow

- up, the former Pb

- exposed workers performed worse than did the controls in total 19 

### cognitive score and in the spatial and general intelligence domains (p <0.05). They also 20 

### performed worse in all other domains ( e.g., motor, executive, an d memory) although the 21 

### differences were not as large. A similar pattern was observed in analyses using tibia Pb 22 

### levels measured at the follow

- up visit to represent exposure. Weaker associations were 23 

### observed with concurrent blood Pb levels (median among th e exposed: 12 μg/dL)

. 24 

### Among the former Pb workers, higher tibia Pb levels were associated with a greater 25 

### decrease in total score and scores for spatial and executive domains between baseline and 26 

### follow

- up. Tibia Pb level were associated inversely with poor er performance in other 27 

### domains as well. As in nonoccupationally

- exposed adults, the stronger findings for tibia 28 

### Pb levels in former Pb

- exposed workers indicate stronger effects of higher past Pb 29 

### exposures than lower current exposures on cognitive function

. 30 

### Additional studies aimed to characterize factors that either mediate or modify the 31 

### association between Pb biomarkers and cognitive function. A study of 61 current Pb 32 

### smelter workers with a mean age of 40 years and blood Pb level of 29.1 μg/dL found that 33 

### both a working lifetime time

- weighted integrated blood Pb level (an index of cumulative 34 

### exposure) (p = 0.09) and tibia Pb level (p = 0.08) were associated with longer times to 35 

### complete the grooved pegboard test ( Bleecker et al., 2007a )

. Among 112 Pb smelter 36 

### workers, working lifetime time

- weighted integrated blood Pb level was associated with 37 

### poorer performance on attention and digit symbol tasks among those with low cognitive 38February 2012 5

- 116 Draft

– Do Not Cite or Quote 

### reserve (assessed by performance on the Wide Range Achievement Test

- R for reading) 1 

### ( Bleecker et al., 2007b )

. 2 

### Apolipoprotein E is a transport protein for cholesterol and lipoproteins. The gene appears 3 

### to regulate synapse formation (connections between neurons) and may be particularly 4



### c ritical in early childhood. A genetic variant, called the ApoE

- ε4 allele is a haplotype 5 

### between 2 exonic SNPs and is perhaps the most widely studied genetic variant with 6 

### respect to increasing risk of neurologic disease. ApoE

- ε4 carriers are at two

- fold 7 

### inc reased risk of developing Alzheimer’s disease, although the majority of such 8 

### individuals still do not develop the disease. Thus, it is biologically plausible that ApoE

- ε4 9 

### carriers may be biologically susceptible to cognitive dysfunction. A study of 10 

### occupat ionally

- exposed adults found that among individuals with at least one ApoE

- ε4 11 

### allele, blood Pb level was associated with poorer performance on digit symbol, pegboard 12 

### assembly, and complex reaction time tests ( Stewart et al., 2002 )

. 13


### Studies also indicated that Pb

- exposed workers may be at increased risk of motor 14 

### dysfunction. Among Pb smelter workers, working lifetime time

- weighted integrated 15 

### blood Pb level was associated with poorer mo tor performance ( p <0 .05). Iwata et al. 16 

### ( 2005 ) examined the cross

- sectional association between blood Pb level and aspects of 17 

### postural sway among 121 Pb

- exposed workers in Japan with blood Pb levels between 6 18 

### and 89 μg /dL (mean: 40 μg/dL). In multiple regression analyses adjusted for age, height, 19 

### and smoking and drinking status, higher blood Pb level was associated with greater 20 

### sagittal sway with eyes open (p <0.05) and eyes closed (p <0.01) and transversal sway 21 

### with ey es closed (p <0.05). The authors calculated a benchmark dose level ( Budtz

22 

### Jorgensen et al., 2001 ; NRC, 2000 ) of 14.3 μg/dL from a linear concentration

- response 23 

### model of their data. A supralinear concentration

- response function was found to fit the 24 

### data slightly better than was a linear function. 25



### Summary of Cogn itive Function in Adults In summary, among nonoccupationally

- exposed adults, there is weak evidence that 26 

### cognitive function is associated with concurrently measured blood Pb levels. The 27 

### strongest evidence was provided by NHANES analyses, in which concurren t blood Pb 28 

### levels were associated with lower cognitive function in particular age and genetic variant 29 

### subgroups ( Krieg et al., 2010 ; Krieg and Butler, 2009 ; Krieg et al., 2009 )

. These anal yses 30 

### did not have bone Pb measures for comparison. It is important to note that because bone 31 

### Pb is a major contributor to blood Pb levels, blood Pb level also can reflect to a large 32 

### extent longer term exposures, including higher past exposures, especially among adults 33 

### without occupational exposures. Thus, in the NHANES analyses of adults, it is difficult 34 

### to characterize the relative contributions of recent and past Pb exposures to the 35February 2012 5

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### associations observed between concurrent blood Pb level and cognitive fun ction. 1 

### Consistent with the conclusion of the 2006 Pb AQCD, several recent studies found 2 

### associations between bone Pb levels and cognitive function in adults ( Table 5

- 8 ). Much 3 

### of the evidence was provided by analyses of two cohorts: BMS and NAS. Recent 4 

### longitudinal analyses demonstrated that higher bone Pb levels measured at baseline were 5 

### associated with subsequent declines in cognitive function over 2

- t o 4

- year periods 6 

### ( Bandeen

- Roche et al., 2009 ; Weisskopf et al., 2007a )

. These findings suggested that 7 

### long

- term Pb exposure may contribute to ongoing declines in cogn itive function in adults. 8 

### Recent studies that analyzed both blood and bone Pb levels generally found stronger 9 

### associations for bone Pb levels, in particular tibia Pb levels, across the various cognitive 10 

### tests that were performed ( Table 5

- 8 ). The discrepant findings for blood and bone Pb 11 

### levels indicate that cumulative Pb exposure that likely included higher past exposures, 12 

### may be a better predictor of cognitive function in adul ts than is blood Pb level. Patella 13 

### and tibia Pb levels were examined in the NAS and Nurses' Health Study; however, 14 

### evidence did not consistently indicate that tibia Pb levels were more strongly associated 15 

### with decreases in cognitive performance ( Weuve et al., 2009 ; Weisskopf et al., 2007a )

. 16 

### Additional support for the effects of cumulative or past Pb exposure is provided by 17 

### analyses of the Boston prospective cohort as adults. Deciduous tooth Pb was associated 18 

### with decrements in specific cognitive indices a t ages 19

- 20 years ( Bellinger et al., 1994a ) , 19 

### and blood Pb levels measured at age 6 months, 4 years, 10 years, and levels averaged 20 

### over childhood were associated with decrements in FSIQ at ages 28

- 30 years ( Mazum dar 21 

### et al., 2011 )

. 22 

### Although based on limited examination, there is some indication that certain variants in 23 

### HFE, ALAD, or VDR genes modify the association between Pb and cognitive function in 24 

### nonoccupationally

- exposed adults; however, results were unifo rm across the various 25 

### cognitive tests performed. Aside from identifying populations potentially at increased 26 

### risk and elucidating underlying modes of action , such e ffect modification also serves to 27 

### strengthen the basic inference about associations between Pb biomarkers and cognitive 28 

### function. Specifically, when effect modification is identified, potential confounding 29 

### factors would have to vary by levels of the modifying factor, which is usually unlikely, 30 

### particularly when considering genotype. It also is im portant to bear in mind that it is not 31 

### always clear whether the observed effect modification reflects a change in the 32 

### toxicokinetics of Pb and therefore a change in dose at the biological site of action or a 33 

### direct biological interaction that increases the toxicity of Pb at a particular target organ or 34 

### tissue. 35 

### In contrast with nonoccupationally

- exposed adults, in adults with current occupational Pb 36 

### exposures, cognitive function was associated with both blood and bone Pb levels. These 37 

### findings indicate that among adults with occupational Pb exposures, both current and 38February 2012 5

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### cumulative exposures affect cognitive function. In the study of former Pb workers, blood 1 

### Pb levels (median: 12 μg/dL) and findings were more similar to those from 2 

### nonoccupationally

- exposed adult s. Among former Pb workers, tibia Pb levels were 3 

### associated more strongly with cognitive performance than were blood Pb levels ( Khalil et 4 

### al., 2009b )

. Thus, in the absence of higher current Pb exposures, cumulative Pb exposures 5 

### may have a greater effect on cognitive function in adults. 6



### 5.3.3 Behavioral Effects

5.3.3.1 Epidemiologic Studies of Behavioral Effects in Children
Noncognitive effects of Pb are more complex to study relative to IQ tests. There are 7 

### fewer objective tests of behavior, and existing tests do not have as strong psychometric 8 

### properties and are less rigorously standardized compared with IQ tests. In several studies, 9 

### behaviors are assessed frequently using teacher and/or parent ratings and thus are subject 10 

### to greater measurement error. However, domain

- spe cific neuropsychological assessments 11 

### are advantageous as they may provide greater insight into the underlying CNS damage 12 

### that may be associated with exposures ( e.g., structural, neural system, neurotransmitter) 13 

### ( White et al., 2009 )

. Several epidemiologic studies reviewed in the 2006 Pb AQCD 14 

### reported associations between blood Pb levels and a wide range of behavioral effects, 15 

### with the weight of evidence supporting associations with inattention and hyperactivity 16 

### and smaller bodies of evidence indicating associations with misconduct and delinquent 17 

### behaviors and withdrawn and depressive behaviors ( Bellinger and Rappaport, 2002 ; 18 

### Needleman et al., 2002 ; Dietrich et al., 2001 ; Burns et al., 1999 ; Wasserman et al., 1998 ; 19 

### Needleman et al., 1996 ; Bellinger et al., 1994a )

. Coherence was provided by similar 20 

### findings of inattention, impulsivity, and changes in social behavior in Pb

- exposed 21 

### animals (Section 5.3.3.2 ). Epidemiologic studies found that blood Pb levels were 22 

### associated with decrements in cognitive function and behavioral problems within the 23 

### same population of children, which demonstrates the strong relationship between the two 24 

### neurodevelopmental domains. The strong relationship between cognitive function and 25 

### behavior in children also is demonstrated by the fact that the schedule controlled behavior 26 

### tests in animals measure both memory and inattention. Thus, behavioral problems 27 

### associated with Pb e xposure may contribute to problems with learning, which may 28 

### progress to antisocial and delinquent behavior later in life. 29 

### Most previous epidemiologic studies found that blood or dentin Pb levels measured at an 30 

### early age ( e.g., 2

- 6 years of age) were associ ated with behavioral problems later in 31 

### childhood and early adulthood ( e.g., 7

- 22 years of age). Most studies examined 32February 2012 5

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### associations with blood Pb levels assessed at a single time point; however, even the 1 

### prospective studies with serial measurements of blood Pb levels, found a range of 2 

### behavioral problems to be associated with both prenatal and early childhood blood Pb 3 

### levels ( Dietrich et al., 2001 ) and lifetime average blood Pb levels ( Burns et al., 1999 )

. 4 

### Thus, evidence did not conclusively identify a single lifestage of Pb exposure that was 5 

### associated with the greatest risk of behavioral problems and misconduct. Recent studies 6 

### strengthened the evidence for associations of blood Pb levels with inattention and 7 

### aggression ( Figure 5

- 14 and Table 5

- 9 ). B ased on a smaller number of studies, new 8 

### evidence demonstrates associations of blood Pb levels with ADHD diagnosis and 9 

### diagnostic indices as well a s with conduct disorders ( Table 5

- 10 and Table 5

- 11 ). As with 10 

### cognitive function, the epidemiologic evidence for each category of outcomes was 11 

### evaluated se parately in order of increasing weight of evidence. Emphasis was placed on 12 

### prospective studies with repeated assessments of blood Pb levels and behavior, studies 13 

### assessing effects relevant to blood Pb levels in contemporary U.S. children ( i.e., less than 14 

### 5 μg/dL), and studies of younger children whose blood Pb levels are less influenced by 15 

### higher past Pb exposures. 16 

### Similar to cognitive function, associations between blood Pb levels and behavioral 17 

### outcomes may be potentially confounded by factors such as par ental SES, parental 18 

### education, parental IQ, quality and stability of the caregiving environment, and 19 

### nutritional status. Accordingly, in assessing whether blood Pb

- behavior associations were 20 

### independent of the effects of the other variables, greater weight was given to studies that 21 

### accounted for potential confounding in the study design or in statistical analyses. 22



### Inattention and Hyperactivity in Children Consistent with previous evidence, recent studies provided strong evidence that blood Pb 23 

### level were as sociated with various endpoints related to inattention, hyperactivity, and 24 

### impulsivity in children after adjusting for potential confounding by multiple SES

- related 25 

### variables and co

- exposures ( Figure 5

- 14 and Table 5

- 9 ). Evidence was equally consistent 26 

### for inattention assessed using teache r and parent ratings and objective tests that measure 27 

### sustained attention such as the continuous performance test (CPT). The associations 28 

### observed with CPT, in particular, provide strong coherence with findings in animals for 29 

### Pb

- induced impairments in homo logous tests of response inhibition in Schedule 30 

### Controlled Behavior Tests (Section 5.3.3.2 ). Both tests measure reactions to stop signals, 31 

### i.e., pre mature responses, reaction time. Most of the recent evidence is derived studies of 32



### non
- U.S. children. Whereas previous studies primarily examined associations with blood 33 

### Pb levels measured earlier in childhood, recent studies indicated associations with 34



### co ncurrent blood Pb levels. Further, many earlier studies of inattention and impulsivity 35 

### included children with higher blood Pb levels than those observed in contemporary 36February 2012 5

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### children. Recent studies provided new evidence of blood Pb

- associated inattention and 1 

### i mpulsivity in populations of children with mean blood Pb levels ranging from 2 to 2 

### 5 μg/dL ( Cho et al., 2010 ; Plusquellec et al., 2010 ; Chiodo et al., 2007 ; Plusquellec et al., 3 

### 2007 )

. 4 

### Previous results from prospective studies indicated that early childhood Pb exposures 5



### were ass ociated with attention deficits in adolescence and young adulthood ( Ris et al., 6 

### 2004 ; Bellinger et al., 1994a )

. Bellinger et al. ( 1994a ) found that compared with young 7 

### adults (ages 19

- 20 years) who had deciduous tooth Pb levels 2.9

- 5.9 ppm , adults with 8 

### deciduous tooth Pb leve ls > 19.9 ppm committed more errors on the CPT and performed 9 

### worse on Stroop and WCST, which measure the ability to shift focus and execute a 10 

### different task. In the CLS cohort, Ris et al. ( 2004 ) found increases in attention as 11 

### measured by CPT in association with prenatal maternal, 3

- 60 month average, and 12 

### 78 month blood Pb levels in adolescents ages 15

- 17 y ears, adjusting for maternal IQ, 13 

### SES, HOME, and adolescent marijuana use. Prenatal blood Pb level was associated with 14 

### slightly larger increases in attention, and larger associations were estimates for males 15 

### ( Figure 5

- 14 and Table 5

- 9 ). 16 

### Studies in children in Korea with relatively low blood Pb levels (mea ns < 3 μg/dL) both 17 

### demonstrated associations with measures of inattention ( Cho et al., 2010 ; Min et al., 18 

### 2007 )

. In a population of children ages 8

- 11 years, Cho et al. ( 2010 ) found relationships 19 

### of concurrent blood Pb levels with ADHD symptoms ( i.e., inattentiveness, hyperactivity, 20 

### and total score) rated by teachers and parents, with the association with teacher ADHD 21 

### rating attaining stati stical significance. In addition to the low blood Pb levels in this study 22 

### (mean: 1.9 μg/dL [range: 0.53

- 6.16]), a strength of this study was the comparison of 23 

### effect estimates with and without adjustment for potential confounders such as age, sex, 24 

### paternal education, maternal IQ, child IQ, city of residence, birth weight, and urinary 25 

### cotinine. In multivariate models, effect estimates decreased by 2 to 14%; however, 26 

### associations remained statistically significant. Mean ADHD ratings by teacher and 27 

### parents wer e similar (both 9.1); however, parental ratings had greater variability (SD: 28 

### 11.5 for parents and 8.6 for teachers), which may have contributed to differences in 29 

### association. Although higher blood Pb levels were associated with more errors 30 

### (responding to a nontarget) on the CPT test, they were not consistently associated other 31 

### indicators of inattention on the CPT or Stroop test ( Figure 5

- 14 and Table 5

- 9 ). Further, 32 

### effect estimates lost statistical significance when urinary cotinine was included in models. 33


### Cho et al. ( 2010 ) did not examine potential confounding by HOME score but they did 34 

### examine parental history of neuropsychiatric disease ( e.g., ADHD, learning disability, 35 

### depression, o bsessive

- compulsive disorder). Mean blood Pb levels were similar in 36 

### children with and without parental history of neuropsychiatric disease (1.80 and 37February 2012 5

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### 1.93 μg/dL, respectively, p = 0.32). Thus, the relationship between blood Pb level and 1 

### ADHD

- related symptom s in the child was not likely confounded by parental 2 

### neuropsychiatric disease. Results from this study also indicated that the relationship 3 

### between blood Pb level and ADHD

- related symptoms was independent of the 4 

### relationship between blood Pb level and IQ. Min et al. ( 2007 ) exam ined a population of 5 

### children (ages 7

- 16 years) in Korea with a mean concurrent blood Pb level of 2.9 μg/dL 6 

### and adjusted for maternal score on attention tests. Investigators found that a 1 μg/dL 7 

### increase in concurrent blood Pb level was associated with a 1 6.8 ms increase (95% CI:

- 8 

### 1.08, 0.06) in reaction time, a test of attention. Although this study did not consider 9 

### confounding by SES and caregiving environment, the findings was consistent with those 10 

### from Cho et al. ( 20 10 )

. 11 

### Similar results were obtained in a study of children, ages 8

- 12 years, in Romania, except 12 

### that concurrent blood Pb level was associated with similar magnitudes of increase in 13 

### inattention, hyperactivity, and impulsivity as assessed by both parents a nd teachers 14 

### ( Nicolescu et al., 2010 )

. Results for parent and teacher ratings were strengthened by 15 

### observations that blood Pb levels also were associated with increased false

- alarm rates in 16 

### responses to stop signals ( Figure 5

- 14 and Table 5

- 9 ). Blood Pb levels in this study also 17 

### were relatively low (median: 3.7 [95% CI: 1.7, 11.1]), and removing five children with 18 

### blood Pb levels at or above 10 μg/dL had minimal impact on observed associations. 19 

### Exposures to aluminum and mercury, other neurotoxic metals, also were examined, and 20 

### blood Pb level was associated with the largest, statistically significant increases in 21 

### inattention, impulsivity, and hyperactivity. 22 

### The aforementioned studies did not consider confounding by the caregiving environment, 23 

### i.e., HOME score. Studies that did adjust for HOME score, also found associations of 24 

### blood Pb leve l with inattention and hyperactivity ( Chandramouli et al., 2009 ; Chiodo et 25 

### al., 2007 )

. In their longitudinal study of children in the U.K., Chandramouli et al. ( 2009 ) 26 

### found association of higher blood Pb level at age 30 months with hyperreactivity at ages 27 

### 7 and 8 years; however, this association was observed primarily in children with blood Pb 28 

### levels greater than 10 μg/dL ( Table 5

- 9 ) and the strongest for ratings given by teachers. 29 

### Children with elevated blood Pb levels did not consistently have elevated odds of 30 

### inattention as assessed using stop signal tasks. Similar to Cho et al. ( 2010 ) , this 31 

### association was not influenced by the inclusion of child IQ in the model. Therefore, these 32 

### findings add support for increasing blood Pb levels having effects on behavior 33 

### independent of effects on cognitive function. Chiodo et al. ( 2007 ; 2004 ) found that 34 

### concurrent blood Pb level was associated with inattention in 7 year
- old children in 35 

### Detroit, MI, as assessed using teacher ratings and CPT ( Figure 5

- 14 and Table 5

- 9 ). 36 

### Potential confounders were selected based on the association of each with a particular 37 

### endpoint, thus model covariates varied among endpoints . HOME score was associated 38February 2012 5

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### with inattention teacher ratings ( Chiodo et al., 2007 ) but not with all C PT indices of 1 

### inattention ( 2007 ; Chiodo et al., 2004 )

. Bl ood Pb level was associated with inattention 2 

### alone and adjusted for covariates. These findings indicated that HOME score did not 3 

### account for all of the association between blood Pb level and behavioral outcomes. As 4 

### was discussed in Section 5.3.2.1 , this cohort comprised large proportions of children with 5 

### prenatal exposure to alcohol and drugs. While this may limit the generalizability of 6 

### findings, it is important to note that alcohol and drug use were not associated with every 7 

### inattention index or in each study. Blood Pb level remained associated with inattention in 8 

### a model that included alcohol and marijuana use along with other demographic and SES

9



### r elated variables. 10 

### A series of studies conducted in Inuit children living in northern Quebec, Canada 11 

### reported associations between blood Pb levels and measures of inattention after extensive 12 

### consideration of potential confounding variables ( Plusquellec et al., 2010 ; Plusquellec et 13 

### al., 2007 ; Fraser et al., 2006 )

. Plusquellec et al. ( 2007 ) found higher cord blood Pb level 14 

### to be associated with higher ratings by investigators for distractibility in completing 15 

### tasks, frenetic movement, and duration of off task behaviors at age 11 months ( Figure 16 

### 5

- 14 and Table 5

- 9 ). In the same cohort of children, concurrent blood Pb level but not 17 

### cord blood Pb level was associated with impulsivity, irritability, and duration of off task 18 

### behavior at ages 4

- 6 years ( Plusquellec et al., 2010 )

. Fraser et al. ( 2006 ) additionally 19 

### indicat ed that at ages 4

- 6 years, the relationship between concurrent blood Pb level and 20 

### motor function ( i.e., transversal sway, reaction time) may be mediated by the effects of 21 

### blood Pb level on an inattention/impulsivity index. These studies conducted in Inuit 22 

### children evaluated confounding by examining associations of demographic, SES, and co

23 

### exposures with outcomes independently and with blood Pb level in models. HOME score 24 

### was not associated with inattention or impulsivity. These findings further indicate tha t 25 

### confounding by HOME score cannot fully account for the collective body of evidence for 26 

### associations between blood Pb level and measures of inattention and impulsivity. 27 

### Consistent with findings in a group of children in Korea ( Cho et al., 2010 ) and children in 28 

### the U.K. ( Chandramouli et al., 2009 ) , Nigg et al. ( 2008 ) found that associations of blood 29 

### Pb level with a hyperactivity/impulsivity index in a group of U.S. children, ages 30 

### 8

- 17 years, were independent of associations with IQ. Whereas other studies assessed 31 

### direct and indirect effects of blood Pb level on behavioral outcomes by including IQ in 32 

### the model as a covariate, Nigg et al. ( 2008 ) specifically used regression

- based path 33 

### analysis, a more rigorous method to characterize the impact of one variable on the 34 

### association of another in the model after controlling for other previous variables. After 35 

### adjusting for sex and income, investigat ors found that concurrent blood Pb level had a 36 

### direct association with hyperactivity/impulsivity that was not completely mediated by the 37 

### blood Pb

- IQ association. Instead, the association between blood Pb level and IQ was 38February 2012 5

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### found to be mediated via the associ ation with hyperactivity/impulsivity. Based on 1 

### associations between hyperactivity/impulsivity slower stop signal reaction time and 2 

### response variability, the authors concluded that the association between blood Pb level 3 

### and hyperactivity/impulsivity was med iated via poorer response on the stop task. It is 4 

### important to note that other potential confounders including parental IQ and HOME score 5 

### were not examined 6


### Several other recent studies found associations between concurrent blood Pb levels and 7 

### inattention; however, their results should be interpreted with caution due to their limited 8 

### consideration of confounding by SES

- related variables ( Li et al., 2008d ) , their 9 

### examination of children living near Pb sources who likely have limited applicabil ity to 10 

### contemporary children in the U.S. general population ( Bao et al., 2009 ; Kordas et al., 11 

### 2007 ) , or both ( Liu et al., 2011a )

. 12February 2012 5

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– Do Not Cite or Quote Note: Test scores were standardized to their standard deviation to facilitate comparisons among tests with different scales. Studies generally are presented in order of increasing mean blood Pb level. Effect estimates are standardized to a 1 μg/dL increase in blood Pb level within the 10th

- 90th percentile interval. Bl ack diamonds, orange triangles, and blue circles represent associations with concurrent, prenatal (maternal), and earlier childhood blood Pb levels, respectively. 

### Figure 5
- 14 Associations of blood Pb levels w ith behavioral indices of inattention, hyperactivity, and impulsivity. Study
Nigg et al. (2008) Cho et al. (2010)

Plusquellec et al. (2007)

Chiodo et al. (2007)

Chiodo et al. (2004)

Fraser et al. (2006)

Plusquellec et al. (2010)

Kordas et al. (2007)

Ris et al. (2004) Mean (SD) blood Pb (μg/dL) 1.04 (0.54) 1.9 (0.67)

4.64 (3.54)

5.0 (3.0)

5.4 (3.3)

5.3 (4.9)

5.4 (5.0) 5.4 (5.0)

11.5 (6.1)

NR 10th
- 90th percentile
0.5

- 1.7 1.2

- 2.8

1.4

- 8.3

2.1

- 8.7

2.3

- 9.5

1.4

- 10.6

1.4

- 10.8 1.4

- 10.8

5.4

- 19.2

NR Blood Pb timing

Concurrent Concurrent

Prenatal

Concurrent

Concurrent

Concurrent

Concurrent

Concurrent

Prenatal (maternal) 3

- 60 mo. avg 78 mo. Outcome
Hyperactivity/Impulsivity ADHD rating scale, teacher ADHD rating scale, parent Comission Errors, CPT Response Time, CPT Word Reading Score

Distraction Off task duration

Hyperactivity Attention problems Comission Errors, CPT Omission Errors

Number of errors, CPT Comission Errors, CPT Reaction Time

Impulsivity/activity

Impulsivity Off task duration

Off

- task passive behavior

Inattention ►

- 0.5

- 0.3

- 0.1 0.1 0.3 0.5 Standardized Effect Estimate per 1 μg/dL increase in blood Pb level within the 10th
- 90th percentile intervalFebruary 2012 5

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### Table 5
- 9 Additional characteristics and quantitative results for studies presented in Figure 5
- 14

Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) a Nigg et al. ( 2008 ) 150 children ages 8

- 17 yr. Year of bi rth and location NR Concurrent Mean (SD) 8

- 11 yr: 1.04 (0.53) 12

- 17 yr: 1.03 (0.54) Regression

- based path analysis adjusted for sex and income. No parental IQ or education Hyperactivity/impulsivity using Teacher ADHD Rating Scale and Kiddie Schedule for Af fective Disorders and Schizophrenia at ages 8

- 17 yr. 0.21 (0, 0.42) b

Cho et al. ( 2010 ) 667 children ages 8

- 11 yr in 2008 Five Korean cities Concurrent Mean (SD): 1.9 (0.67) 10th

- 90th: 1.2

- 2.8 Log linear regression model adjusted for age, sex, parental education, maternal IQ, child IQ, city, birth weight, urinary cotinine ADHD rating scale, teacher ADHD rating scale, parent Korean ADHD Rating Scale IV
c om m ission errors, CPT Response time, CPT Word reading score, Stro op assessed at ages 8

- 11 yr. 0.042 (0.017, 0.067) 0.010 (

- 0.013, 0.033)
0.03 (

- 0.01, 0.07)

- 0.01 (

- 0.05, 0.03)

- 0.02 (

- 0.06, 0.02) c

Plusquellec et al. ( 2007 ) 148

- 164 children ages 11 mo born 1995

- 2002 Inuit communities Quebec, Canada Prenatal (cord) Mean (SD): 4.6 (3.5) 10th

- 90th: 1.4

- 8.3 Log linear regression model adjusted for delivery complication, home organization, low birth weight (distractibility) and sex, maternal anxiety, prenatal alcohol exposure (off task duration) Several more examined Distraction Off task duration using Bayley Behavioral Rating Scale at age 11 mo 0.06 (0.021, 0.098) 0.03 (

- 0.01, 0.073)

Chiodo et al. ( 2007 ) 506 African

- American children in Detroit, MI area followed from birth (1989

- 1991) to age 7 yr. Large proportions of children with prenatal exposure to cocaine or marijuana Concurrent Mean (SD): 5.0 (3.0) 10th

- 90th: 2.1

- 8.7 Linear regression model adjusted for child age, sex Several more examined Hyperactivity Attention problems using PROBS

- 14 and Conners' Teacher Rating Scale

- 39 at age 7 yr Comission Errors (%) Omission Errors (%) 0.13 (0.03, 0.23) b 0.13 (0.03, 0.23) b

- 0.08 d 0.18 (0.07, 0.29) c Chiodo et al. ( 2004 ) 246 African

- American children in Detroit, MI area followed from birth (not reported) to age 7.5 yr. Large proportions of children with prenatal exposure to cocaine or marijuana Concurrent Mean (SD): 5.4 (3.3) 10th

- 90th: 2.3

- 9.5 Log linear regression adjusted for SES, maternal vocabulary score (Number of errors and errors of com m ission); Number of children > 18 yr (Number of errors); Child age (errors of com m ission); Education, sex, prenatal cocaine exposure (reaction time ) Several more examined Number of errors Comission Errors Reaction time Using CPT at age 7.5 yr 0.35 (0, 0.69) b,c 0.05 (p > 0.05) d 0.25 (p > 0.05) d

Fraser et al. ( 2006 ) 90 children ages 5

- 6 yr born 1993

- 1996 Inuit communities Quebec, Canada Concurrent Mean (SD): 5.3 (4.9) 10th

- 90th: 1.4

- 10.6 Linear regression model adjusted for binge drinking in pregnancy Several more examined Impulsivity/activity using modified Infant Behavioral Rating Scale at ages 5

- 6 yr 0.25 (0. 04, 0.46)

Plusquellec et al. ( 2010 ) 95

- 98 children ages 5

- 6 yr born 1993

- 1996 Inuit communities Quebec, Canada Concurrent Mean (SD): 5.4 (5.0) 10th

- 90th: 1.4

- 10.8 Log linear regression model adjusted for birth weight, sex, parity, caregiver education (impulsivity) and birth weight, SES, child blood hemoglobin (off task duration) Several more examine d Impulsivity Off task duration using modified Infant Behavioral Rating Scale at ages 5

- 6 yr. 0.019 (0.001, 0.036) 0.02 (0, 0.039)February 2012 5

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– Do Not Cite or Quote Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) a
Kordas et al. ( 2007 ) 168 children ages 6

- 8 yr. born 1993

- 1995 Torreon, Mexico Residence near metal foundry Concurrent Mean (SD): 11.5 (6.1) 10th

- 90th: 5.4

- 19.2 Linear regression model adjusted for age, sex, SES, home ownership, crowding in home, maternal education, family structure, forgetting homework Off task passive behavior using instrument develope d by investigator at ages 6

- 8 yr 0.034 (0.005, 0.063)

Ris et al. ( 2004 ) 195 children, ages 15

- 17 years, born 1979

- 1985 NR Linear regression adjusted for sex, maternal IQ, HOME, SES, adolescent marijuana use Several more examined Inattention using CPT at ages 15

- 17 yr Prenatal (maternal) 3

- 60 mo avg 78 mo 0.16 (0.04, 0.27) 0.11 (0.04, 0.19) 0.12 (0.0 2, 0.22) Studies not included in figure because of categorical analysis of Pb biomarker or outcome : Chandramouli et al. ( 2009 ) 488 children ages 7

- 8 yr born 1991

- 1992 Avon, UK Age 30 m o Mean (SD): NR Linear regression model adjusted for maternal education, home ownership, maternal smoking, HOME score, maternal SES, family adversity index, parenting attitudes at 6 mo. Hyperactivity, teacher using Strengths and Difficulties Questionnaire at ages 7

- 8 yr

Selective inattention using Test of Everyday Attention for Children at ages 7

- 8 yr OR: 0.84 (0.47, 1.52) e blood Pb 2

- 5 vs. 0

- 2 μg/dL 1.25 (0.67, 2.33) e blood Pb 5

- 10 vs. 0

- 2 μg/dL 2.82 (1.08, 7.35) e blood Pb > 10 vs. 0

- 2 μg/dL OR: 0.97 (0.62, 1.52) e blood Pb 2

- 5 vs. 0

- 2 μg/dL 1.01 (0.64, 1.61) e blood Pb 5

- 10 vs. 0

- 2 μg/dL 0.88 (0.42,1.85) e blood Pb > 10 vs. 0

- 2 μg/dL Nicolescu et al. ( 2010 ) 83 children ages 8

- 12 yr born 1995

- 1999 Bucharest and Pantelimon, Romania Concurrent Median (IQR): 3.7 (2.6) 10th

- 90th: 2.0

- 8.5 Log linear regression model adjusted for city, sex, age, computer experience, handedness, eye problems, number of siblings, parental education, prenatal smoking, parental psychological/psychiatric problem Inattention Parent rating Inattention Teacher rating Premature Response using German versio n of Test battery for attention performance at ages 8

- 12 yr. 1.3% (

- 4.0, 6.9) f 4.3% (

- 0.02, 10.4) 8.3% (

- 0.02, 19.0)

Bellinger et al. ( 1994a ) 79 young adults, ages 19

- 20 yr, born 1970, Boston, MA area Deciduous tooth (age 5

- 8 yr) Q1: 2.9

- 5.9 ppm Q2: 6.0

- 8.7 ppm Q3: 8.8

- 19.8 ppm Q4: 19.9

- 51.8 ppm Regression model adjusted for parental IQ, sex, SES, current drug use, current alcohol use, current illicit drug use, maternal education, maternal age, birth order Several more exami ned Correct Responses on CPT Q1: 98.0 (1.0) g Q2: 97.6 (1.1) Q3: 96.9 (1.1) Q4: 94.6 (1.1)

a Effect estimates are standardized to a 1 μg/dL increase in blood Pb level within the 10th to 90th percentile interval and sta ndardized to the standard deviation of the test score to facilitate comparisons among tests that are scored on different scales. b Standard error was estimated from the reported p

- value.

c Direction of effect estimate was changed to indicate an improvement in performance.

d Sufficent data were not provided to calculate 95% CIs.

e Odds in higher quantile of blood Pb level compared to that in lowest quantile of blood Pb level.

f Results represent the change in false alarm rate.

g Results represent the mean (SD) score in each quartile.February 2012 5

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### Attention Deficit Hyperactivity Disorder in Children Studies of ADHD were included in the 2006 Pb AQCD; however, previous studies did 1 

### not conclusively demonstrate that blood Pb level was associated with ADHD itself. 2 

### Importantly, previously studies examining ADHD were fewer in number and limited by 3 

### smaller sample sizes ( Gi ttleman and Eskenazi, 1983 ; David et al., 1972 )

. Recent studies 4 

### consistently reported associations of blood Pb levels with ADHD in children. Coherence 5 

### and biological plausibility for these associations are provided by the consistent body of 6 

### epidemiologic and toxicological evidence indicating Pb

- associated increases in 7 

### inattention, hyperact ivity, and impulsivity. 8 

### Several recent studies reported associations between concurrent blood Pb level and 9 

### ADHD diagnosis or diagnostic indices in children between the ages of 8 and 17 years 10 

### ( Table 5

- 10 ). While none of the studies examined the potential for confounding by 11 

### HOME score, they did evaluate confounding by several other demographic and SES

12 

### related variables, as well as parental his tory of psychopathology, including ADHD ( Cho 13 

### et al., 2010 ; Nicolescu et al., 2010 )

. Recent studies also provided evidence of association 14 

### between blood Pb level and ADHD in populations born in the mid

- to late

- 1990s with 15 

### relatively low concurre nt blood Pb levels (means: 1.9 and 3.7 μg/dL) ( Cho et al., 2010 ; 16 

### Nicolescu et al., 2010 )

. However, the temporal trends in Pb exposure in these non
- U.S. 17 

### populations may have differed from those in U.S. children of the same age. 18February 2012 5

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### Table 5
- 10 Associations between blood Pb level and ADHD diagnosis or diagnostic indices in children

Study Population/Location Blood Pb Levels (μg/dL) a Statistical Analysis Outcome Effect Estimate (95% CI) b Nigg et al. ( 2008 ) 150 children ages 8

- 17 yr. Year of birth and location NR Concurrent Mean (SD) 8

- 11 yr: 1.04 (0.53) 12

- 17 yr: 1.03 (0.54) 10th

- 90th: 0.5

- 1.7 Three

- group analysis of covariance with sex and income. No parental IQ or education ADHD Dx by two licensed clinicians F(2,145) = 6.08, p = 0.04

Braun et al. ( 2006 ) 4 , 704 children ages 4

- 15 yr born 1984

- 1998 U.S. NHANES 1999

- 2002 Concurrent 3rd quintile: 1.1

- 1.3 Logistic regression model adjusted for postnatal ETS, prenatal ETS, age, sex, race, childcare attendance, health insurance coverage, ferritin levels ADHD Dx or medication use at age 4

- 15 yr 1.4 (0.4, 3.4) blood Pb 0.8

- 1.0 vs. <0.8 μg/dL c 2.1 (0.7, 6.8), blood Pb 1.1

- 1.3 vs. <0.8 μg/dL c 2.7 (0.9, 8.4), blood Pb 1.4

- 2.0 vs. <0.8 μg/dL c 4.1 (1.2, 14.0), blood Pb > 2.0 vs. <0.8 μg/dL c Froehlich et al. ( 2007 ) 2 , 588 children, ages 8

- 15 yr born 1986

- 1996 U.S. NHANES 2001

- 2004 Concurrent 2nd quartile: 0.9

- 1.3 Logistic regression model adjusted for current household ET S exposure, sex, age, race/ethnicity, income, preschool attendance, maternal age, birth weight, and interaction terms for Pb and prenatal ETS interaction ADHD Dx 8.1 (3.8, 18.7), blood Pb level > 2.0 μg/dL plus prenatal ETS exposure vs. blood Pb level <0.8 μg/dL and no prenatal ETS exposure c Cho et al. ( 2010 ) 667 children ages 8

- 11 yr born 1997

- 2000 Five Korean cities Concurrent Mean (SD): 1.9 (0.67) 10th

- 90th: 1.2

- 2.8 Log linear regression model adjusted for age, sex, parental education, maternal IQ, child IQ, city, birth weight, urinary cotinine Total ADHD rating, teacher Total ADHD rating, parent using Korean ADHD Rating Scale IV at ages 8

- 11 yr. 0.042 (0.017, 0.067) 0.010 (

- 0.013, 0.033)

Nicolescu et al. ( 2010 ) 83 children ages 8

- 12 yr born 1995

- 1999 Bucharest and Pantelimon, Romania Concurrent Median (IQR): 3.7 (2.6) 10th

- 90th: 1.8

- 7.1 Log linear regression model adjusted for city, sex, age, computer experience, handedness, eye problems, number of siblings, parental education, prenatal smoking, family psychopathology ADHD score, parent ADHD score, teacher using German version of Conner’s scales at ages 8

- 12 yr. OR: 1.04 (1.00, 1.10) OR: 1.06 (1.00, 1.12)

Roy et al. ( 2009a ) 756 children ages 3

- 7 yr tested 2005

- 2006 Chennai, India Concurrent Mean (SD): 11.4 (5.3) 10th

- 90th: 5.8

- 18.3 Log linear regression model adjusted for age, sex, hemoglobin, average monthly income, parental education, number of other children, clustering in school and classroom ADHD index z

- score using Conners’ ADHD/DSM

- IV Scales at ages 3

- 7 yr. 0.002 (0, 0.033)

Chen et al. ( 2007 ) 780 children in TLC trial followed ages 2

- 7 yr Ba ltimore, MD; Cincinnati, OH; Newark, NJ; Philadelphia, PA Children underwent chelation therapy Concurrent Mean (SD): 12.0 (5.2) 10th

- 90th: 6.5

- 18.7 Regression

- based path analysis adjusted for city, race, sex, language, parental education, parental employme nt, single parent, age at blood Pb measurement, caregiver IQ ADHD index Using Conners' Parent Rating Scale

- Revised at age 7 yr. 0.54 (

- 1.22, 2.30) Direct 0.90 (0.35, 1.45) Indirect

a Studies are presented in order of increasing quantile or population mean blood Pb level. b Except where noted, effect estimates represent regression coefficients. All effect estimates are standardized to a 1 μg/dL in crease in blood Pb level within the 10th

- 90th percentile interval.
c Odds ratio in higher quantile of blood Pb leve l, with children in the lowest quantile of blood Pb level serving as the reference group.


### In addition to finding associations of concurrent blood Pb level with inattention and 1 

### hyperactivity, Cho et al. ( 2010 ) found a statistically significant relationship with a total 2 

### ADHD index as rated by teachers in population of children ages 8

- 11 years in Korea. In a 3February 2012 5

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### multivariate model that included age, sex, paternal education, maternal IQ, child IQ, city 1 

### of residence, birth w eight, and urinary cotinine, the effect estimate decreased by 12%; 2 

### however, the association remained statistically significant ( Table 5

- 10 ). Neuropsyc hiatric 3 

### disease ( e.g., ADHD, learning disability, depression, obsessive

- compulsive disorder) in 4 

### parents was not associated with child blood Pb level, and thus, likely did not confound 5 

### the relationship between blood Pb level and ADHD index in the child. Results from this 6 

### study also indicated that the relationship between blood Pb level and ADHD index was 7 

### independent of the relationship between blood Pb level and IQ. 8


### Similar to Cho et al. ( 2010 ) , Nicolescu et al. ( 2010 ) found higher concurrent blood Pb 9 

### levels to be associated with greater inattention, impulsivity, and total ADHD score among 10 

### children in Romania, ages 8

- 12 years. As with individual symptoms, only concurrent 11 

### blood Pb level, and not aluminum or mercury, was associated with a higher total ADHD 12 

### rating (4% increase [95% CI: 0, 10] per 1 μg/dL increase in concurrent blood Pb level 13 

### within the 10th

- 90th percentile interval [1.8

- 7.1 μg/dL]). The association did not change 14 

### substantially in an analysis that excluded the 5 children with blood Pb levels at or above 15 

### 10 μg/dL. Adjustment for potential confounding by city, sex, age, computer experience, 16 

### handedness, eye problems, number of siblings, parental education, prenatal smoking 17 

### exposure, prenatal alcohol exposure, and parental history of psychological or psychiatric 18 

### problems resulted in a decrease in statistical significance for blood Pb level associations, 19 

### although investigators did not report whether the magnitude of association changed. It is 20 

### important to note that model covariates were selected a priori but not all were associated 21 

### wi th both blood Pb level and behavioral endpoint. Only sex, number of siblings, and 22 

### maternal education were reported to be significantly associated with both blood Pb level 23 

### and total ADHD rating. Parental history of psychological or psychiatric problems was 24 

### significantly correlated with parental but not teacher rating of ADHD total score; 25 

### however, ORs were fairly similar for ADHD score rated by teachers and parents. Despite 26 

### the potential for over

- adjustment in this study, higher blood Pb level was associated with 27 

### a higher ADHD total score rating. 28 

### A recent analysis of data from NHANES 1999

- 2002 found a relationship between higher 29 

### blood Pb level and greater odds of ADHD (parent

- report of a diagnosis of ADHD or use 30 

### of stimulant medication) among children ages 4

- 1 5 years ( Braun et al., 2006 )

. Strengths 31 

### of this study included the large sample representative of U.S. children (n = 4704) and the 32 

### low concurrent blood Pb levels at which ass ociations were observed. These authors 33 

### reported a monotonic increase in ORs from the lowest to highest quintile of blood Pb 34 

### level, adjusting for age, race, prenatal smoking exposure, smoker in the home, 35 

### preschool/child care attendance, health insurance cov erage, and ferritin levels ( Figure 36 

### 5

- 15 ). With children in the lowest quintile serving as the reference group (<0.8 μg/dL), 37 

### children in the fifth quint ile of concurrent blood Pb level (>2.0 μg/dL, maximum not 38February 2012 5

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### reported) had the highest prevalence of ADHD (OR: 4.1 [95% CI: 1.2, 14]). A similar 1 

### OR was estimated when children with blood Pb levels > 5.0 μg/dL were excluded from 2 

### the highest group. Children in the other three higher quintiles of blood Pb level (range: 3 

### 0.8

- 2.0 μg/dL) also had a higher prevalence of ADHD relative to the reference group 4 

### ( Table 5

- 10 ). 5

Source: Braun et al. ( 2006 ) Note: Adjusted for child age, gender, race/ethnicity, preschool attendance, serum ferritin, prenatal tobacco smoke exposur e, smoker in the household, and insurance status (p for trend = 0.012). Data from ages 4

- 15 years from NHANES 1999

- 2002 by quintile of concurrent blood Pb level. 

### Figure 5
- 15 Adjusted odds ratios for Attention Deficit Hyperactivity Disorder (ADHD) among U.S. children.

In the NHANES 2001

- 2004 dataset restricted to children ages 8

- 15 years, Froehlich and 6 

### colleagues ( 2009 ) found an interaction between prenatal tobacco smoke exposure 7 

### (maternal report) and concurrent blood Pb levels. Investigators found ADHD to be 8 

### independently associated with prenatal tobacco smoke exposure (OR: 2.4 [95% CI: 1.5, 9 

### 3.7]) and concurrent blood Pb levels (OR: 2.3 [95% CI: 1.5, 3.8]) in children with blood 10 

### Pb levels >1.3 μg/dL co mpared with children with blood Pb levels < 0.8 μg/dL. These 11 

### results were adjusted for current household smoke exposure, sex, age, race/ethnicity, 12 

### income, preschool attendance, maternal age, and birth weight. As in the younger 13 

### NHANES dataset, a similar OR was estimated when children with blood Pb levels > 14 

### 5.0 μg/dL were excluded from the highest tertile. The strongest association was observed 15 

### in children with both high blood Pb level and prenatal tobacco smoke exposure. 16 

### Compared to children in the lowest te rtile of blood Pb levels with no exposure to prenatal 17 

### tobacco smoke, children in the highest tertile of blood Pb level with exposure to prenatal 18 

### tobacco smoke had the highest prevalence of ADHD (OR: 8.1 [95% CI: 3.5, 18.7]). 19 

### Although ADHD was associated wi th low concurrent blood Pb levels (1.3

- 5 μg/dL), it is 20February 2012 5

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### important to acknowledge that the adolescents in both NHANES analyses were born in 1 

### the late 1980s and may have had higher Pb exposures earlier in childhood that 2 

### contributed to the observed associations

. 3 

### Consistent with findings in the NHANES population, Nigg et al. ( 2008 ) found an 4 

### association between concurrent blood Pb level and ADHD diagnosis among children 5 

### 8

- 17 years. Concurrent blood Pb levels in this population also were low, ranging between 6 

### 0.40 and 3.47 μg/dL. However, the examination of adolescents adds uncertainty 7 

### regarding the relative contributions of higher past Pb expo sures and current exposures to 8 

### the observed associations. A key strength of this study over other studies was the 9 

### verification of parental reports of ADHD by licensed clinicians using the same diagnostic 10 

### criteria. Although the results were adjusted for hou sehold income, other potential 11 

### confounders including parental IQ and HOME score were not examined. 12 

### Roy and colleagues ( 2009a ) examined associations between concurrent blood Pb level 13 

### and a range of behavioral problems in 756 children, ages 3

- 7 years, in Chennai, India. In 14 

### this population, the mean blood Pb level was highe r than that in most other studies (11.4 15 

### [SD: 5.3] μg/dL); however, this study demonstrated associations of concurrent blood Pb 16 

### level with inattention, hyperactivity, and a total ADHD index (assessed by teachers using 17 

### Conners’ ADHD/Diagnostic and Statistica l Manual for Mental Disorders) in younger 18 

### children compared with other studies. In generalized estimating equations, higher blood 19 

### Pb level was associated with a higher ADHD index score adjusting for age, sex, 20 

### hemoglobin, average monthly income, parental ed ucation, number of other children, and 21 

### clustering at school and classroom levels ( Table 5

- 10 ). In analyses that did not adjust for 22 

### potential confoundi ng variables, mean ADHD scores were similar in the first three 23 

### quartiles of blood Pb level and elevated in children with blood Pb levels > 18.71 μg/dL, 24 

### suggesting that in this population, associations may have been driven by children with the 25 

### highest blood Pb levels. 26 

### Whereas several studies indicated the direct effects of increasing blood Pb level on 27 

### inattention and hyperactivity indices, Chen et al. ( 2007 ) found stronger indirect effects of 28 

### blood Pb level on an ADHD index among children at age 5 years ( Table 5

- 10 ). Important 29 

### limitations of this study include the high blood Pb levels of children at ages 12 to 30 

### 33 months (20

- 44 μg/dL) that made them eligible for a randomiz ed controlled trial of 31 

### chelation. Because this study only enrolled children with Pb poisoning, it is difficult to 32 

### extend findings to children with lower blood Pb levels. Blood Pb levels of study subjects 33 

### remained high at age 5 years (mean: 12.0 μg/dL) and out the range of the current U.S. 34 

### general population of children. 35 

### In recent commentaries to studies reporting associations between blood Pb level and 36 

### ADHD in children, Brondum ( 2011 , 2007 ) asserted the need for studies to consider 37February 2012 5

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### confounding by parental history of ADHD. Given the highly heritable nature of ADHD, 1 

### parental history of ADHD is a strong risk factor for ADHD in children ( Faraone and 2 

### Doyle, 2001 )

. Therefore, the risk associated with parental history of ADHD may mask 3 

### the smaller magnitudes of ri sk associated with environmental exposures, including Pb. 4 

### However, little information is available on an association between ADHD in the parent 5 

### and blood Pb level in the child, thus, it is uncertain whether parental ADHD confounds 6 

### and fully accounts for th e associations observed between blood Pb level and ADHD in 7 

### children. It should be noted that for parental ADHD to be a confounder, parental Pb 8 

### levels would have to be highly associated with ADHD in the parent and with blood Pb 9 

### level in the child. Among chi ldren in Korea, Cho et al. ( 2010 ) found that parental history 10 

### of psychopathology (ADHD or several other conditions) was not associated with child 11 

### blood Pb level. Among children in Romania, parental history of psychop athology was 12 

### associated with a higher ADHD rating in the child as assessed by parents but not teachers 13 

### ( Nicolescu et al., 2010 )

. Higher blood Pb level was associated with higher ADHD ratings 14 

### in children in models with blood Pb level alone and in models that adjusted for parental 15 

### psychopathology plus other covariates. Further, associations of blood Pb level with 16 

### ADHD rating assessed by parents and teachers were similar in magnitude ( Table 5

- 10 ). 17 

### While available data are limited, they do not provide strong evidence that parental 18 

### psychopathology fully accounts for the associations observed between blood Pb level and 19 

### ADHD in children. 20



### Social Misconduct and Delinquent Behavior The 2006 Pb AQCD described several studies in whi ch higher levels of blood and bone 21 

### Pb were associated with higher frequency or risk of misconduct in children or delinquent 22 

### behavior in adolescents ( U.S. EPA, 2006b )

. Previous studies primarily indicated 23 

### associations with measure s of cumulative Pb exposure, including early childhood average 24 

### blood Pb level, lifetime average blood Pb level, or bone Pb level. Recent studies added to 25 

### the collective body of evidence by demonstrating associations for concurrent blood Pb 26 

### level and for a variety of conduct problems including aggression ( Chiodo et al., 2007 ) , 27 

### conduct disorder ( Braun et al., 2008 ) , oppositional defiant disorder ( Nigg et al., 2008 ) 28 

### and more serious behaviors such as criminal arrests ( Wright et al., 2008 ) ( Table 5

- 11 ). 29 

### Associations were found with early childhood and concurrent blood Pb levels and were 30 

### demonstrated in populations with lower blood Pb levels (means: 1

- 8 μg/dL) than those 31 

### examined in the 200 6 Pb AQCD. Nonetheless, the weight of cumulative evidence 32 

### supports associations in populations with mean blood Pb levels between 5 and 11 μg/dL. 33 

### However, because some studies examined outcomes in adolescents and young adults born 34 

### in the 1970s and 1908s, th ere is greater uncertainty regarding the level, timing, 35 

### frequency, and duration of Pb exposure that contributed to the observed associations. 36February 2012 5

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### Several other studies examined children and adolescents born in the 1990s, who likely 1 

### did not have as high histori cal Pb exposures earlier in childhood ( Chandramouli et al., 2 

### 2009 ; Braun et al., 2008 ; Chiodo et al., 2007 )

. 3



### Table 5
- 11 Associations between blood Pb level and misconduct and delinquent behavior in children and young adults a

Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) b
Blood Pb level as a continuous variable Chiodo et al. ( 2007 ) 506 African

- American children in Detroit, MI area followed from birth (1989

- 1991) to age 7 yr. Large proportions of children with prenatal exposure to cocaine or marijuana Concurrent Mean (SD): 5.0 (3.0) 10th

- 90th: 2.1

- 8.7 Linear regression model adjusted for HOME, child sex, current marijuana use (both outcomes) Caretaker education, alcohol us e during pregnancy, (delinquent behavior Age, caretaker psychopathology, maternal IQ (inappropriate behavior) Several more examined Delinquent behavior Inappropriate behavior using Achenbach Teacher Report Form at age 7 yr 0.09 (0, 0.18) c 0.09 (0, 0.18) c

Dietrich et al. ( 2001 ) 195 children followed from birth (1979

- 1985) to age 15

- 17 yr Cincinnati, OH 0

- 6 yr avg: NR Linear regression model adjusted for HOME score, parental IQ , current SES Delinquent behavior using the Self

- Report of Delinquent Behavior at ages 15

- 17 yr. 1.21 (1.08, 1.37)

Wright et al. ( 2008 ) 250 adults followed from birt h (1979

- 1985) to age 19

- 24 yr Cincinnati, OH Age 6 yr Mean (SD): 8.3 (4.8) 10th

- 90th: 3.9

- 14.5 Negative binomial regression models adjusted for maternal IQ, sex, SES, maternal education Several more examined Criminal arrests

Violent arrests assessed from county records at ages 19

- 24 yr RRs: 1.05 (1.00, 1.09) prenatal 1.05 (1.01, 1.09) 6 yr 1.01 (0.97, 1.05) 0

- 6 yr avg 1.06 (0.97, 1.15) prenatal 1.08 (1.03, 1.14) 6 yr 1.05 (1.01, 1.10) 0

- 6 yr avg Chen et al. ( 2007 ) 780 children participating in TLC trial followed between ages 2

- 7 yr Baltimore, MD; Cincinnati, OH; Newark, NJ; Philadelphia, PA Children underwent chelation therapy Concurrent Mean (SD): 12.0 (5.2) 10th

- 90th: 6.5

- 18.7 Regression

- based path analysis adjusted for city, race, sex, language, parental education, parental employment, single parent, age at blood Pb measurement, caregiver IQ Externalizing behavior, parent
Externalizing behavior, teacher using Behavior Assessment System for Children at age 7 yr. 1.024 (0.996, 1.053) Direct 1.008 ( 1.002, 1.014) Indirect 1.036 (1.003, 1.069) Direct 1.004 (0.998, 1.010) IndirectFebruary 2012 5

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– Do Not Cite or Quote Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) b Blood Pb level as a categorical variable Braun et al. ( 2008 ) Children ages 8

- 15 yr U.S. NHANES 2001

- 2004 2nd quartile: 0.8

- 1.0 Logistic regression with sample weights applied to produce national estimates, adjusted for oversampling of minorities and young children and adjusted for age, poverty income ratio, maternal age, sex, race, prenatal ETS, cotinine, blood Pb levels Conduct disorder Using Diagnostic Interview Schedule for Children

Caregiver Module at age 8

- 15 yr OR: 7.24 (1.06, 49.47), blood Pb level 0.8

- 1.0 μg/dL vs. bloo d Pb <0.8 μg/dL d 12.37 (2.37, 64.56), blood Pb level 1.1

- 1.4 μg/dL vs. blood Pb <0.8 μg/dL d 8.64 (1.87, 40.04), blood Pb level 1.5

- 10 μg/dL vs. blood Pb <0.8 μg/dL d Chandramouli et al. ( 2009 ) 488 children ages 7

- 8 yr born 1991

- 1992 Avon, UK Age 30 mo Mean (SD): NR Linear regression model adjusted for maternal education, home ownership, maternal smoking, HOME score, maternal SES, family adversity index, parenting attitudes at 6 mo. Antisocia l activities using Antisocial Behavior Interview at ages 7

- 8 yr OR: 0.93 (0.47, 1.83) d blood Pb 2

- 5 vs. 0

- 2 μg/dL 1.44 (0.73, 2.84) d blood Pb 5

- 10 vs. 0

- 2 μg/dL 2.90 (1.05, 8.03) d blood Pb > 10 vs. 0

- 2 μg/dL

a Results with continuous blood Pb are presented first followed by results with categorical blood Pb. Within each group, studies generally are presented in order of increasing mean blood Pb level. b Effect estimates are standardized to a 1 μg/dL increase in blood Pb level.
c 95% CI was estimated from a reported p

- value of 0.05. d Odds in higher quantile of blood Pb level compared to that in lowest quantile of blood Pb level.


### The consistency of association of Pb biomarker levels with social misconduct and 1 

### delinquent behavior was corroborated in a r ecent meta

- analysis ( Marcus et al., 2010 ) that 2 

### included 19 studies (those reviewed in the 2006 Pb AQCD plus several recent studies) 3 

### with a total of 8 , 561 children and adolescents (mean ages ranging from 3.5 years to 4 

### 18.4 years). Effect estimates were converted to Pearson correlation coefficients, and the 5 

### combined effect estimate was r = 0.19 (95% CI: 0.14, 0.23). The key finding of this study 6 

### was the robustness of associations to conside rations of heterogeneity in study design, 7 

### definition and assessment method of conduct problems, potential confounding variables 8 

### examined, and blood Pb levels. The major source of heterogeneity in effect estimates was 9 

### the biomarker of Pb examined. A larger magnitude of effect was estimated for hair Pb 10 

### levels compared with bone or blood Pb levels; however, similar effect sizes were 11 

### estimated for blood and bone Pb levels. Although the authors suggested that hair Pb may 12 

### be a better indicator of cumulative Pb ex posure compared to bone Pb levels, due to the 13 

### high turnover of bone in throughout childhood and into adolescence, an empirical basis 14 

### for interpreting hair Pb measurements in terms of body burden or exposure has not been 15 

### firmly established (Section 4.3.4.2 ). 16


### In the meta

- analysis, effect sizes did not differ significantly between longitudinal and 17



### cross
- sectional studies, among studies that examined dif ferent conduct problems 18 

### ( i.e., opposition defiance, delinquency, externalizing problems), or among studies that 19 

### assessed conduct disorders using self

- report, teachers report, or criminal records. 20 

### Controlling for covariates such as SES, birth weight, parent al IQ, and home environment 21 

### did not attenuate the relationship between blood Pb level and conduct problems. In 22February 2012 5

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### addition to strengthening the evidence for the independent associations of Pb biomarker 1 

### levels with conduct disorders, the results indicate that the lack of adjustment for any 2 

### particular covariate, including HOME score, does not warrant limiting inferences from a 3 

### particular study. 4 

### Studies of children ages 7

- 8 years found associations between blood Pb level and 5 

### misconduct adjusting for HOME score a nd several other potential confounding variables. 6 

### They examined different lifestages of blood Pb level and did not clearly indicate 7 

### associations between blood Pb level and misconduct with blood Pb levels less than 8 

### 5 μg/dL. In their longitudinal study of ch ildren in the U.K. who were born in the 1990s, 9 

### Chandramouli et al. ( 2009 ) found associations of age 30 month blood Pb level not only 10 

### with hyperreactivity but also with antisocial behavior at ag e 7

- 8 years. Results were 11 

### adjusted for maternal education, home ownership, maternal smoking, HOME score, 12 

### maternal SES, family adversity index, and parenting attitudes at 6 months. In analyses of 13 

### blood Pb level as a categorical variable, greater antisocial activity was most clearly 14 

### indicated in children with blood Pb levels greater than 10 μg/dL ( Table 5

- 11 ). Chiodo et 15 

### al. ( 2007 ) examined chi ldren, ages 7.5 years in Detroit, Michigan with a mean (SD) 16 

### concurrent blood Pb level of 5.0 (3.0) μg/dL. Higher blood Pb was associated with higher 17 

### teacher ratings of delinquent behaviors and inappropriate behaviors, adjusting for HOME 18 

### score and other var iables. The association with inappropriate behavior additionally 19 

### adjusted for caregiver psychopathology. As with cognitive function and inattention 20 

### outcomes, prenatal alcohol and drug exposure were not found to influence associations 21 

### for blood Pb level. Bl ood Pb level was associated with behavioral problems in unadjusted 22 

### and adjusted analyses, corroborating the independent associations for blood Pb level. 23


### In the U.S. NHANES 2001

- 2004 dataset of children ages 8

- 15 years (born in the 1990s), 24 

### Braun et al. ( 2008 ) analyzed blood Pb as a categorical variable and found higher 25 

### prevalence of conduct disorder with concurrent blood Pb levels in the range of 0.8 to 1.0 26 

### μg/dL, the lowest level among all studies examined. Compared with children with blood 27 

### Pb levels less than 0.8 μg/dL, the OR (95% CI) in children with blood Pb levels 0.8

- 1.0 28 

### μg/dL was 7.24 (1.06, 49.47). Odds rat ios also were elevated in children with blood Pb 29 

### levels higher than 1.0 μg/dL ( Table 5

- 11 ). The wide 95% CIs likely were due to the small 30 

### numbers of ca ses of conduct disorder, as assessed using DSM

- IV criteria. For example, 31 

### there were 22 cases of conduct disorder among children with blood Pb levels 0.8

- 1.0 32 

### μg/dL. Poisson regression models showed that children with blood Pb levels 0.8

- 1.0 33 

### μg/dL had 1.55 ( 95% CI: 1.09, 2.22) times as many conduct disorder symptoms as did 34 

### children with blood Pb levels < 0.8 μg/dL. Investigators had data available on a limited 35 

### set of potential confounders, and poverty income ratio was used to control for potential 36 

### confounding by SES. 37February 2012 5

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– Do Not Cite or Quote 

### While other recent studies supported associations between biomarkers of Pb exposure 1 

### and misconduct and delinquent behavior, they examined Pb levels in blood or tooth 2 

### samples collected in the 1980s when Pb exposures were much higher ( Fergusson et al., 3 

### 2008 ; Wright et al., 2008 )

. Although this evidence may be less informative as to the 4 

### relationship between current blood Pb levels and misconduct and delinquent behavior in 5 

### adolescents and young adults, it is important to note the consistency of findings among 6 

### populations with widely varying blood Pb levels and historical Pb exposures. 7 

### In the CLS cohort, prenatal and postnatal blood Pb levels previously were reported to be 8 

### associated with self

- and parent

- reported delinquent and social acts at ages 16

- 17 years 9 

### ( Dietrich et al., 2001 )

. Wright et al. ( 2008 ) extended these findings to include 10 

### associations of prenatal and postnatal blood Pb levels with criminal arrests (ascertained 11 

### from county records) at ages 19

- 24 years. Mean blood Pb levels were 8.3 μg/dL (range 12 

### 1

- 26) for the prenatal period (maternal blood), 13.4 μg/dL (range 4

- 37) for the average 13 

### between birth and age 6 years, and 8.3 μg/dL (r ange 2

- 33) at age 6 years. In models that 14 

### adjusted for maternal IQ, sex, SES score, and maternal education, the relative risks (RRs) 15 

### for total arrests per 1 μg/dL increment in blood Pb level were 1.07 (95% CI: 1.01, 1.13) 16 

### for prenatal blood Pb level, 1.01 (95% CI: 0.97, 1.05) for average childhood blood Pb 17 

### level, and 1.05 (95% CI: 1.01, 1.09) for blood Pb level at age 6 years. Blood Pb levels 18 

### measured during these lifestages also were associated with increased risk of violent 19 

### criminal arrests ( Table 5

- 11 ). Although interactions terms for blood Pb by sex were not 20 

### statistically significant, the attributable risk for males was considerably higher for males 21 

### ( 0.85 arrests/year [95% CI: 0.48, 1.47]) than for females (0.18 [95% CI: 0.09, 0.33]). 22 

### Results from the two CLS studies suggest that in addition to the prenatal blood Pb levels, 23 

### early childhood blood Pb levels may also predict criminal behavior in adulthood

. 24 

### However, it is important to note that in these CLS studies, concurrent blood Pb levels 25 

### were not analyzed. Therefore, these studies do not provide information on the potential 26 

### effects of more recent Pb exposures or differences in association between earli er and 27 

### more recent blood Pb levels. 28 

### A strength of Wright et al. ( 2008 ) was the detailed examination of potential confounding 29 

### by a large number of variables. All of the examined covariates were weakly correlated 30 

### with blood Pb levels (r = 0.24

- 0.35), thereby minimizing the potential for confounding. 31 

### Nonetheless, variables such as maternal IQ, maternal education, and SES were included 32 

### in the model because they were associa ted with arrests in the full multivariate model or 33 

### changed the blood Pb level by more than 10%. HOME score was similar between 34 

### subjects with and without criminal arrest records and did meet the criteria for inclusion in 35 

### the final model. 36February 2012 5

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### In the New Zealand cohort of children born in 1977, dentin Pb levels measured between 1 

### ages 6 and 8 years were associated with self

- reported and documented violent or property 2 

### convictions at ages 14

- 21 years ( Fergusson et al., 2008 )

. Similar to those from the meta

3 

### analysis, these findings pointed to an effect of cumulative childhood Pb exposure. 4 

### Analyses were adjusted for SES, parental criminal record, parental education, and 5 

### parental alcoholism, and although effects estimates decreased in adjusted models, 6 

### associations remained statistically significant. 7 

### Several studies of misconduct and delinquent behavior aimed to characterize whether 8 

### associations with biomarkers of Pb exposure were independent of effects on IQ and 9 

### educational attainment. Most studies found that associations of Pb biomarkers with 10 

### misconduct and delinquent behavior remained statistically significant in a model that 11 

### additionally adjusted for child IQ or educational attainment, indicating that Pb exposure 12 

### may have a direct effect on misconduct independent of its effect on IQ ( Chandramouli et 13 

### al., 2009 ; Fergusson et al., 2008 ; Burns et al., 1999 )

. It is important to note that because a 14 

### decrement in IQ may on the causal pathway to behavioral problems, including bo th IQ 15 

### and behavioral problems in the same model may result in an underestimate of the effect 16 

### on behavior. Chen et al. ( 2007 ) used path analysis to characterize the direct effects and 17 

### indirect effects (mediated through child IQ) of blood Pb level on externalizing problems 18 

### ( i.e., outbursts of behavior); however, results were inconclusive. A direct effect was 19 

### estimated for externalizing problems assessed by teachers and an indirect effect was 20 

### estimated for problems assessed by parents. These findings may have limited 21 

### applicabi lity to the general population given that the children in the study population had 22 

### been referred for chelation therapy at enrollment because of high blood levels, and it is 23 

### uncertain whether the observed associations were due to the residual effect of high blood 24 

### Pb levels (20

- 44 μg/dL) four years earlier. 25



### 5.3.3.2 Toxicological Studies of Behavior

Neurobehavioral Changes The effects of Pb on neurobehavioral changes in animals are well characterized with 26 

### various targeted sites including the prefrontal cerebral cortex , cerebellum, and 27 

### hippocampus; affected functions include cognition, execution of motor skills, and 28 

### memory/behavior. As discussed in earlier Pb AQCDs, young animals are especially 29 

### susceptible to the effects of Pb due to the ongoing development of the nervo us system 30 

### with greater Pb absorption and retention. Pb exposure has been documented to induce 31 

### neurobehavioral changes in exposed animals including effects on learning, social 32 

### behavior, memory, attention, motor function, locomotor ability and vocalization. At the 33February 2012 5

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### cellular level, Pb impairs axon and dendritic development and contributes to 1 

### neurochemical changes in proteins, membranes, redox/antioxidant balance, and 2 

### neurotransmission through a multitude of mechanisms, many of which involve the 3 

### capability of Pb to mimic calcium. Very early toxicological research on neurobehavioral 4 

### endpoints failed to capture the disposition of Pb and its resulting body burden or blood Pb 5 

### level and was thus difficult to use in risk assessment. 6 

### The 1986 Pb AQCD reported findings of Pb

- induced aberrant operant conditioning tasks 7 

### in rodents and non
- human primates (some with resulting blood Pb levels: 11 to 15 μg/dL) 8 

### as well as hyperactive or inappropriate Pb

- mediated responses ( U.S. EPA, 1986b )

. It was 9 

### ind icated that these effects were possibly of hippocampal origin and showed a curvilinear 10 

### response, decreasing at higher Pb doses possibly due to impairment of motor function 11 

### ( Ma et al., 1999 ; Crofton et al., 1980 )

. Pb exposure in laboratory animals resulted in 12 

### distractibility, reduced to adapt to changes in behavior, impaired ability to inhibit 13 

### inappropriate responses, and perseveration ( U.S. EPA, 2006b )

. Pb has been shown to 14 

### impair lear ning in Fixed Interval tasks (FI) as indicated by premature responses in the 15 

### absence of a fixed schedule of reinforcement or reward (Section 5.3.2.2 ). These findings 16 

### in animals are consistent with observations in children that blood Pb levels are associated 17 

### with poorer impulse control as rated by parents or teachers or assessed using the 18 

### continuous performance test ( Figure 5

- 14 and Table 5

- 9 ). Interresponse rates and overall 19 

### run rate are the two subcomponents of FI response rate. The 2006 Pb AQCD reported 20 

### consistent findings for Pb exposure (resulting in blood Pb levels: 58 to 94 μg/dL) 21 

### affecting FI response rates, by means of decreased interresponse times. Some studies 22 

### indicated decreased interresponse times in animals with blood Pb levels of 11 μg/dL 23 

### ( U.S. EPA, 2006b )

. Discrimination reversal has been shown to be especially sensitive to 24 

### Pb exposure. Spatial and non
- spatial discrimination reversal or reversal of a previously 25 

### learned habit was significantly affected after developmental Pb exposure and was 26 

### exacerbated with distracting stimuli. Repeat

- acqui sition testing revealed that these 27 

### deficits were likely not due to sensory or motor impairment at this dose. Together, the 28 

### data from the 2006 Pb AQCD showed that social behavior and learning in rodents and 29 

### nonhuman primates is significantly affected by Pb exposure that results in blood Pb 30 

### levels 15

- 40 μg/dL. 31 

### In the new literature, gestationally and early postnatally (gestation to PND10, G+P) Pb

32 

### exposed male mice (low and high dose Pb: 10 and 42 μg/dL blood Pb level at PND10, 33 

### respectively) were significantl y less active than were control mice, and low dose mice 34 

### were significantly less active than were high dose mice, demonstrating a nonlinear 35 

### concentration

- response relationship ( Leasure et al., 2008 )

. A similar nonlinear 36 

### concentration

- response relationship was observed for changes in corticosterone in male 37 

### mice exposed post

- weaning to Pb ( Virgolini et al., 2005 )

. Activity level of G+P Pb

- 38February 2012 5

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### exposed female mice versus controls was unaffected ( Leasure et al., 2008 )

. 1 

### Amphetamine

- induced motor activity was monitored in male and female G+P Pb

2 

### exposed mice at 1 year of age. Amphetamine

- induced activity of male low and high dose 3 

### G+P Pb

- exposed offspring was significantly elevated over that in controls; G+P Pb

4 

### exposed fema les had no change in sensitivity to amphetamine

- induced motor activity 5 

### ( Leasure et al., 2008 )

. 6 

### Herring gull chicks injected with a single i.p. bolus dose of Pb (100 mg /kg Pb

- acetate , a 7 

### dose created to be similar to that which wild herring gulls are exposed in the wild) on 8 

### PND2 were found to hav e neurobehavioral deficits and learning deficits. Pb

- exposed 9 

### chicks displayed multiple deficits related to impaired survival skills including decreased 10 

### time spent begging the parent for food, decreased accuracy at pecking for food in the 11 

### mouth of the paren t bird, decreased time spent in the shade (behavioral 12 

### thermoregulation), decreased learning in food location, decreased recognition of familiar 13 

### individuals (caretaker or sibling), and slower development of motor skills (treadmill test) 14 

### versus control birds ( Burger and Gochfeld, 2005 )

. The impaired thermoregulation with 15 

### Pb exposure agrees with earlier work in Pb

- exposed rat pups that also showed impaired 16 

### thermoregulatory behavior, i.e., impaired ultrasonic vocalization ( Davis, 1982 )

. These 17 

### studies in herring gull chicks demonstrate that a single dose of Pb early in life can induce 18 

### neurobehavioral deficits that affect survival skills. 19 

### Rhesus monkeys exposed to Pb in daily milk formula from PND8 to 1 o r 2 years of age 20 

### ( Pb

- acetate /50% glucose in 4 cc of commercial milk formula producing blood Pb of 21 

### 35

- 40 μg/dL) were assessed for tactile defensiveness using the Sensory Processing Scale 22 

### for Monkeys, an adaptation of laboratory observational measures of sen sory processing 23 

### for children ( Baranek and Berkson, 1994 )

. Tactile defensiveness in children is defined as 24 

### “feelings of discomfort and a desire to escape the situation when certain types of tactile 25 

### st imuli are experienced” ( Ayres, 1964 ) and is associated with emotional dysregu l ation, 26 

### inattention, and difficult social relations. Other reports have shown that tactile 27 

### defensiveness in monkey offspring is affected by prenatal stress alone without Pb 28 

### exposure ( Schneider et al., 2008 ) , a factor that has been shown to affect other Pb

- related 29 

### outcome s. 30



### Attention Epidemiologic studies consistently have reported associations between higher blood Pb 31 

### level and deficits in attention (inattention, distractibility, impulsivity, or ADHD) in 32 

### children (Section 5.3.3.1 ). Animal toxicological studies in the 2006 Pb AQCD detailed 33 

### attention deficits in animals undergoing various tests including FR/waiting

- for

- reward 34February 2012 5

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### testing and tests that employed signal detec tion with distraction, a test recording 1 

### omissions after exposure to an external distraction. 2 

### With FR testing, rats are trained on a FR/waiting

- for

- reward behavioral baseline, learning 3 

### to produce food delivery by pressing a lever 50 times. Rodents can earn additional food 4 

### by withholding lever presses by waiting; free food is given at increasing time intervals 5 

### after completion of the FR. Brockel and Cory

- Slechta ( 1998 ) exposed male Long

- Evans 6 

### rats to 0, 50, or 150 ppm Pb

- acetate in water from weaning, which produced respective 7 

### blood Pb levels of <5, 11, and 29 μg/dL after 3 months of exposure. After 40 days of 8 

### exposure, rats were trained on a FR schedule, and the higher dose animals had 9 

### significant ly more frequent response rates and resets of the waiting period than did the 10 

### low dose group and controls. In the waiting behavior component, wait time was 11 

### significantly lower in both treated groups compared to controls. The higher dose group 12 

### animals also had an increased number of reinforcers and a higher response to 13 

### reinforcement ratio than did low dose and controls. Mechanistic understanding of the 14 

### aforementioned FR deficits was studied using the same FR schedule with similar 15 

### postweaning dosing of 0, 50, and 150 ppm Pb that yielded respective blood Pb levels of 16 

### <5, 10, and 26 μg/dL after 3 and 7 months of exposure. Administration of dopamine 17 

### receptor antagonists attenuated Pb

- induced effects on FR schedule testing, suggested a 18 

### role for D2 receptors in Pb

- induced behavioral impairments. 19 

### Another study used a similar postweaning Pb exposure as that in earlier studies ( Cory

20 

### Slechta et al., 1998 ) to yield blood Pb levels of <5, 16, and 28 μg/dL and evaluate 21 

### sustained attention using t esting of signal detection during a distraction ( Brockel and 22 

### Cory

- Slechta, 1999 )

. Rats earned food rewards by discriminating correctly between a 23 

### target and distracter light. A 13 second time

- out was given for incorrect responses. Pb 24 

### exposure produced no deficits in attention with this testing. Further work since the 2006 25 

### Pb AQCD affirm ed these findings. Testing for signal detection with distraction showed 26 

### no effect after postnatal Pb exposure in female rats ( Stangle et al., 2007 )

. The two dose 27 

### groups (20 ppm or 300 ppm Pb

- acetate in drinking water with lactational, and drinking 28 

### water exposure, PND1

- PND30) yielded blood Pb levels o f 13 μg/dL and 31 μg/dL 29 

### ( Stangle et al., 2007 )

. Another recent study reported that impaired auditory threshold task 30 

### related behavioral testing was likely due to inattention in Pb

- exposed animals. The 31 

### inability of some of the monkeys to engage or focus on the task at hand yielded fewer 32 

### available measu rements in Pb

- exposed animals versus control animals ( Laughlin et al., 33 

### 2009 )

. These rhesus monkeys were exposed to Pb

- acetate gestationally (dam drinking 34 

### water, 3 months prior to mating) through age 5.5 months (weaning) and had resulting 35 

### bone Pb levels at 11 year s of 7 and 13 μg/dL for prenatal and postnatal groups, 36 

### respectively, and blood Pb levels during Pb exposure of 35 and 46 μg/dL, respectively. 37 

### Animals were tested at age 13 years when blood Pb levels had returned to baseline levels. 38February 2012 5

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### Thus, the inattention li terature demonstrates that impulsivity, waiting

- for

- reward 1 

### behavior, and sustained attention may all be affected by Pb with the first two possibly 2 

### affected to a greater extent than is sustained attention. 3



### 5.3.3.3 Epidemiologic Studies of Mood in Children
A majorit y of investigation of the effects of Pb on behavior in children has focused on 4 

### externalizing behaviors such as inattention, hyperactivity, aggression, and delinquency. 5 

### However, studies also have linked biomarkers of Pb exposure with internalizing 6 

### behaviors related to emotional dysfunction, including withdrawn behavior, depression, 7 

### fearfulness, and anxiety. These behaviors were assessed frequently in school

- aged 8 

### children using the Child Behavior Checklist, and as with inattention, hyperactivity, and 9 

### miscondu ct, often were rated by parents and/or teachers. 10 

### Several earlier results were provided by the prospective cohort studies in Boston, 11 

### Cincinnati, Port Pirie, Australia, and Yugoslavia. Collectively, these studies found higher 12 

### ratings for internalizing behavi ors in association with concurrent blood Pb level as well 13 

### as biomarkers of cumulative Pb exposure such as multiyear average blood, tooth, and 14 

### bone Pb levels ( Burns et al., 1999 ; Wasserman et al., 1998 ; Bellinger et al., 1994b ; 15 

### Dietrich et al., 1987b )

. While several studies demonstrated associations with biomarkers 16 

### of long

- term, cumulative exposures, results from the Cincinnati cohort provided support 17 

### for the effec ts of shorter

- duration exposures. Among CLS infants, blood Pb level at age 18 

### 3 months and average blood Pb level at age 6 months was associated with less positive 19 

### mood in white infants at age 6 months ( Dietrich et al., 1987b )

. In the Boston cohort, 20 

### subjects were examined at age 8 years and 19

- 20 years. Pb levels measured in deciduous 21 

### teeth but not cord blood were associated with a higher rating of inte rnalizing behaviors at 22 

### age 8 years but not in adulthood, indicating the lack of persistence of effects of early 23 

### exposure ( Bellinger et al., 1994a ; Bellinger et al., 1994b )

. Wasserman et al. ( 1998 ) also 24 

### examined blood Pb levels measured at various lifestages in the Yugoslavia cohort and 25 

### found that concurrent blood Pb was associated more strongly with anxious

- depressed and 26 

### withdrawn behaviors at age 3 years than were blood Pb levels measured prenatally or 27 

### between ages 6 months and 2 years. Thus, while biomarkers of Pb exposure were 28 

### consistently associated with poorer mood and emotional state in children, there was no 29 

### clear indication of differences in association among biomarkers measured at various 30 

### lifestage s . Associations between Pb biomarkers with mood also were consistently 31 

### reported in recent studies, with most studies examining concurrent blood Pb level ( Liu et 32 

### al., 2011a ; Bao et al., 2009 ; Roy et al., 2009b ; Chiodo et al., 2004 )

. However , several had 33 

### limited generalizability to the general population of U.S. children due to the examination 34February 2012 5

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### of children residing near Pb sources or high prevalence of prenatal alcohol or drug 1 

### exposure ( Liu et al., 2011a ; Bao et al., 2009 ; Chiodo et a l., 2004 )

. 2


### Associa tions of Pb biomarkers with mood and emotional state in children were observed 3 

### after adjusting for a wide range of potential confounding variables including parental 4 

### education and HOME score. Several studies that each adjusted for a different set of 5 

### covari ates found similar effect estimates in univariate and multivariate models ( Burns et 6 

### al., 1999 ; Wasserman et al., 1998 ; Bellinger et al., 1994b ) , supporting the robustness of 7 

### associations with Pb biomarkers. Associations between blo od Pb level and internalizing 8 

### behaviors also were observed in studies that adjusted for maternal psychopathology 9 

### ( Burns et al., 1999 ; Wasserman et al., 1998 )

. Thus, the evidence indic ates that 10 

### associations observed between Pb biomarkers and internalizing behaviors in children are 11 

### not driven by confounding by any particular measured variable. 12 

### In the collective body of evidence, associations between blood Pb level and internalizing 13 

### beha viors were observed in populations of school

- aged children with mean blood Pb 14 

### levels between 8 and 28 μg /dL. Mean blood Pb levels were not related to the age of the 15 

### study population. In the limited investigation of populations with mean blood Pb levels of 16 

### approximately 5 μg/dL, results were inconclusive. Chiodo et al. ( 2004 ) found an 17 

### association with internalizing behaviors in children (age 7 years) in Detroit, in whom the 18 

### prevalence of prenatal alcohol and drug use was high. Neither exposure was found to 19 

### influence associations with blood Pb level. Another study that examined Inuit children 20 

### (age 5 years) in Quebec, Canada, did not find an association between concurrent blood Pb 21 

### level and internalizing behaviors ( Plusquellec et al., 2010 )

. 22 

### A common observation across studies was finding that Pb biomarkers were associated 23 

### with multiple i ndices of neurodevelopmental function, i.e., FSIQ, executive function, 24 

### externalizing, and internalizing behaviors, within the same population. Whereas some 25 

### studies found stronger associations for externalizing behaviors than for internalizing 26 

### behaviors ( Plusquellec et al., 2010 ; Bellinger et al., 1994a ; Sciarillo et al., 1992 ) , most 27 

### did not find a clear difference in the strength of association ( Roy et al., 2009b ; C hiodo et 28 

### al., 2004 ; Wasserman et al., 1998 ; Bellinger et al., 1994b )

. In the Port Pirie population, 29 

### Burns et al. ( 1999 ) found that lifetime average blood Pb levels were associated with 30 

### externalizing behaviors more strongly in boys and with internalizing behaviors more 31 

### strongly in girls, indicating potential sex

- based differences. These findings demonstrating 32 

### Pb effects on a wide spectrum of cognitive and behavioral indices are not surprising 33 

### given that Pb exposure is linked to changes in the HPA axis and dopaminergic and 34 

### GABAergic systems , which are involved in mediating cognitive function, behavior, and 35 

### mood. Dietrich et al. ( 1987b ) provided support for Pb exposure affecting a wide range of 36 

### neurodevelopmental outcomes by characterizing the direct and indirect effects of Pb 37February 2012 5

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### using structural equations. The associations of prenatal Pb exposure (maternal and infant 1 

### day 10 blood) with poorer mood in infants aged 6 months were found to be mediated 2 

### through lower birth weight and/or shorter gestation. These results suggested that Pb may 3 

### exert its effects by impairing nervous system development. It is well known that the fetal 4 

### period is an active period for neuronal differentiation, dendritic branching, and 5 

### synaptogenesis. Thus, it is not surprising that prenatal Pb exposure effects on nervous 6 

### system development could result in a wide range of neurodevelopmental effects assessed 7 

### later in childhood. 8



### 5.3.3.4 Epidemiologic Studies of Mood and Psychiatric Effects in Adults
Examination of the potential effects of Pb exposure on mood and psychiatric outcomes in 9 

### adults has received far less attention than that in children and cognitive function in adults. 10 

### Nonetheless, evaluation of mood states is an integral p art of the neurocognitive test 11 

### battery of the World Health Organization (WHO), and it has been suggested that indices 12 

### of the Profile Of Mood States may be particularly sensitive to toxicant exposures 13 

### ( Johnson et al., 1987 )

. As with other nervous system endpoints in adults, several early 14 

### studies of Pb

- exposed workers (mean blood Pb levels ranging from 23.5 to 64.5 μg/dL) 15 

### found higher prevalence of symptoms related to mood disorders and anxiety among Pb

16 

### exposed workers than un exposed controls (mean blood Pb levels ranging from 17 

### 15

- 38 μg/dL) ( Schwartz et al., 2005 ; Maizlish et al., 1995 ; Parkinson et al., 1986 ; Baker 18 

### et al., 1985 ; Baker et al., 1984 ; Lilis et al., 1977 )

. 19 

### While compr ising a smaller body of evidence, studies of adults without occupational 20 

### exposures demonstrated associations of blood and bone Pb level with mood. Analyses of 21 

### men ages 48

- 70 years in the NAS indicated associations of both concurrent blood (mean: 22 

### 6.3 μg/dL [SD: 4.16]) and tibia (mean: 21.9 μg/g [SD: 13.5]) Pb levels with greater self

23 

### reported symptoms of depression and anxiety ( Rhodes et al., 2003 )

. As bone Pb is a 24 

### major contributor to blood Pb levels in adults without current occupational Pb exposure, 25 

### associations with both biomarkers may indicate effects of cumulative Pb exposure. In a 26 

### subsequent analysis of the same dataset, Rajan et al. ( 2007 ) found effect modification of 27 

### the associations for patella and tibia Pb levels by ALAD genotype, although results were 28 

### not in a consistent direction for a particular genotype. For a majority of the mood 29 

### symptoms c onsidered, tibia bone Pb levels were associated with larger ORs among men 30 

### with the ALAD 1

- 1 genotype. In contrast, ORs for associations between patella Pb levels 31 

### and several mood symptoms such as depression and positive symptom distress index 32 

### were larger a mong ALAD 1

- 2/2

- 2 carriers. In the NAS, inconsistent effect modification 33February 2012 5

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### by ALAD genotype also was observed for associations between tibia Pb levels and 1 

### cognitive performance ( Rajan et al., 2008 ) (Section 5.3.2.5 ). 2


### A cross

- sectional study of 1 , 987 adults age 20

- 39 years participating in NHANES 3 

### 1999

- 2004 was the largest study of mood and included both men and women ( Bouchard 4 

### et al., 2009 )

. However, only concurrent blood Pb levels were available for analysis. 5 

### Investigators assessed major depressive disorder (MD D), panic disorder and generalized 6 

### anxiety disorder (GAD) using the WHO Composite International Diagnostic Interview, 7 

### which follows criteria defined in DSM. Adults with concurrent blood Pb levels higher 8 

### than 0.7 μg/dL had higher prevalence of all three sym ptoms. Adults in the highest 9 

### quintile of blood Pb level (≥ 2.11 μg/dL) had the highest OR for MDD (OR: 2.32 [95% 10 

### CI: 1.13, 4.75]) and panic disorder (OR: 4.94 [95% CI: 1.32, 18.48]) compared with 11 

### adults with blood Pb levels < 0.7 μg/dL. For all endpoints, ORs were larger in analyses 12 

### excluding current smokers. While this study demonstrated associations with relatively 13 

### low concurrent blood Pb levels, it is important to note the uncertainty of the magnitude, 14 

### timing, frequency, and duration of Pb exposure that contributed to the observed 15 

### associations. 16 

### In analyses of cohorts in California and New England, Opler et al. ( 2008 ; 2004 ) reported 17 

### associations between higher prenatal levels of plasma δ

- ALA and subsequent diagnosis 18 

### of schizophrenia spectrum disorder (asce rtained using DSM

- IV criteria) in adolescence 19 

### and adulthood. In the absence of blood Pb levels, investigators measured δ

- ALA levels in 20 

### stored serum samples to serve as a surrogate for Pb exposure citing previous observations 21 

### of a high correlation (0.90) be tween δ

- ALA levels ≥ 9.05 ng/ mL and blood Pb levels ≥ 22 

### 15 μg/dL. In the California cohort, a δ

- ALA level ≥ 9.05 ng/ mL was associated with 23 

### schizophrenia spectrum disorder with an OR (95% CI) of 2.43 (0.99, 5.96), adjusting for 24 

### maternal age at delivery. In an alyses combining the California and New England cohorts, 25 

### a δ

- ALA level ≥ 9.05 ng/ mL was associated with schizophrenia spectrum disorder with 26 

### an OR (95% CI) of 1.92 (1.05, 3.52), adjusting for maternal age and education. A 27 

### covariate

- adjusted OR was not pres ented for the New England cohort alone, and it 28 

### appeared that the association in the combined cohorts was driven by that observed in the 29 

### California cohort. Studies in other populations with direct measurements of blood Pb 30 

### levels are warranted to characteriz e the potential effects of Pb exposure on schizophrenia. 31 

### These limited available data indicate associations with higher blood Pb levels (≥ 32 

### 15 μg/dL) in individuals born in the 1950s and 1960s who likely had higher early Pb 33 

### exposures. Thus, these findings m ay have limited relevance to current lower levels of Pb 34 

### exposure. 35February 2012 5

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### 5.3.3.5 Toxicological Studies of Mood, Emotional, and Psychotic Changes
As in epidemiologic studies, neurotoxicological studies often focus more on motor, 1 

### sensory, behavioral or cognitive outcomes and less on psychological pathologies. As 2 

### described in Section 5.3.3.3 above, epidemiologic studies indicating associations of Pb 3 

### biomarkers with mo od disorders in children and adults have grown in recent years. 4 

### Likewise, studies increasingly have found that developmental Pb exposure (gestation and 5 

### lactation) affects emotional state and mood disorder

- like behavior in adult animal 6 

### offspring. Offspring of Wistar rats treated with 10 mg of Pb

- acetate daily by gavage 7 

### during pregnancy (G) or pregnancy plus lactation (G+L) were tested in the open field test 8 

### and the forced swimming test also known as the Pors olt test ( de Souza Lisboa et al., 9 

### 2005 )

. Blood Pb levels in the pups at PND70 were 5

- 7 μg/dL. The open field test 10 

### monitors activity levels and movements of animals in three dimension and is a measure 11 

### of hyperactivity, emotion (grooming or freezing), and exploratory behavior (rearing). In 12 

### the forced swim test, animals are placed in a tank and monitored for helplessness, a 13 

### response common in animal models of depression. (G+L) Pb

- treated male rats had 14 

### increased emotionality with the open field test as indicated by decreased rearing and 15 

### increased immobility. (G+L) Pb

- exposed female offspring had a significantly increased 16 

### depressive phenotype in the forced swim test as indicated by increase d immobility 17 

### ( de Souza Lisboa et al., 2005 )

. It is interesting to note that this is one of many Pb

- induced 18 

### changes that seem to be sex

- specific wi th males showing increased emotionality and 19 

### females showing elevated depressive

- like symptoms ( Figure 5

- 16 ). In another study, 20 

### anxiety was assessed in offspring after G+L Pb exposure. Dams received Pb

- enriched 21 

### drinking water (2.84 mg /mL Pb

- acetate trihydrate) and produced pups with blood Pb 22 

### levels of 698 ng/g at PND25. Using the elevated plus maze test, Pb

- exposed animals 23 

### displayed no signs of anxiety ( Molina et al., 2011 )

. However, it is important to note the 24 

### high concentrations of Pb exposure used in this study. Another study found that female 25 

### pups exposed to Pb

- acetate (0.2% to pups PND1

- 30) had increased anxiety at PND30 as 26 

### measured by a decreased percentage of entries and time spent in the open arms of the 27 

### plus

- maze ( Fox et al., 2010 )

. 28 

### Depression may seem initially like an unexpected com orbidity for immune inflammatory 29 

### dysfunction, but many forms of depression are linked with the same cytokine imbalances 30 

### that occur with Pb

- induced innate immune dysfunction ( Maes ; Pace and Miller, 2009 )

. 31 

### Some researchers use sickness behavior and its associated malaise as a model for 32 

### depression. Sickness behavior is characterized by overall malaise, decreased food intake, 33 

### immobility and changes in core body temperature. Dyatlov and Lawrence ( 2002 ) 34 

### observed in mice that sickness behavior, which is due to an interaction of the immune 35 

### system and the CNS, was potentiated by Pb exposure (resulting in blood Pb level: 36February 2012 5

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### 17 μg/dL) and was correlated with depletions in specific thymic T

- cell populations 1 

### ( Figure 5

- 16 ). Pb exposure also potentiated the infection

- dependent elevation in IL

- 1β, a 2 

### cytokine that has been shown to inhibit hippocampal glutamate release in young but not 3 

### aged animals. Sickness behavior was induced with Listeria monocyt ogenes infection; Pb 4 

### exposure occurred from birth through lactation and was continued for a brief period after 5 

### weaning until the experiment was terminated. 6

Note: The blue headings provide four independent possible contributors to development of psychiat ric disorders in Pb

- exposed individuals. 

### Figure 5
- 16 Animal toxicology evidence of possible Pb
- induced contributors to the development of mood and psychotic disorders.
Schizophrenia is associated with a shortened lifespan in humans as reflected by increased 7 

### standardized mortality ratio ( McGrath et al., 2008 )

. An environmental origin of 8 

### schizophrenia was proposed years ago ( T suang, 2000 ) , and prenatal Pb exposure, 9 

### assessed using ALAD activity as a biomarker, was linked to schizophrenia in a group of 10 

### adolescents and young adults in California ( Opler et al., 2008 ; Opler et al., 2004 )

. 11 

### Because of these observations, the animal toxicological field is beginning to explore th e 12 

### mechanisms that may contribute to schizophrenia development and has proposed two 13 

### explanations. These are Pb

- induced NMDA receptor (NMDAR) hypofunction and Pb

14 

### induced decreases in hippocampal neurogenesis ( Figure 5

- 16 to Figure 5

- 18 ). Pb may 15 

### bind a divalent cation site in the NMDAR and allostericall y inhibit glycine binding 16 

### ( Hash emzadeh

- Gargari and Guilarte, 1999 ) ; human studies of patients with schizophrenia 17February 2012 5

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### have shown aberrations at this site ( Coyle and Tsai, 2004 )

. These findings are consistent 1 

### with the glutamatergic hypothesis of schizophrenia which purports that NMDAR 2 

### noncompetitive antagon ist use in patients with schizophrenia exacerbates their psychotic 3 

### symptoms and that administration of antagonist to non
- psychotic subjects can induce a 4 

### schizophrenic phenotype. The second mechanistic hypothesis for Pb

- associated 5 

### schizophrenia induction is decreased hippocampal degenerate gyrus (DG) neurogenesis, 6 

### which is seen in patients with schizophrenia ( Kempermann et al., 2008 ; Reif et al., 2006 ) , 7 

### in animal models of schizophrenia ( Maeda et a l., 2007 ) and in animal models with 8 

### developmental Pb exposure ( Verina et al., 2007 ; Jaako

- Movits et al., 2005 ) ( Figure 5

- 16 9 

### to Figure 5

- 18 ). Animal models of schizophrenia ( i.e., phencyclidine administration) 10 

### show decreased hippocampal DG neurogenesis that can be reversed by treatment with 11 

### clozapine, which is often used to treat schizophrenia ( Maeda et al., 2007 )

. These DG 12 

### pathways are also NMDAR

- dependent. Studies cited in this section are further detailed in 13 

### other sections of the ISA. Another study gives insight into pathways involved in Pb

14 

### induced changes in neurogenesis in the developing brain. Exposure of zebrafish embryos 15 

### to Pb (50

– 700 μ M Pb

- acetate in embryo medium fr om 0 to 6 days post hatch) caused 16 

### significant apoptosis (increased TUNEL positive brain cells) and impaired neurogenesis 17 

### as shown by decreased levels of gfap and huC in the brain; however two other genes 18 

### involved in neurogenesis, crestin and neurogenin1, w ere unaffected in brains of Pb 19 

### exposed embryos ( Dou and Zha ng, 2011 )

. Thus, toxicological studies indicate that Pb 20 

### exposure may result in mood disorders via behavioral, neurochemical, and ultrastructural 21 

### changes. 22February 2012 5

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### Figure 5
- 17 Schematic representation of the contribution of Pb exposure to the development of a phenotype consistent with schizophrenia. 

## Rodent Studies

Pb exposure and Schizophrenia

Associated in Human Cohorts (Epidemiologic) NMDA Receptor Hypofunction ( Neurochemical ) Glutamatergic hypothesis of schizophrenia 

## Human Populations or Cohorts

Decreased Hippocampal Neurogenesis

(Structural)

Pharmacologically

induced Schizophrenia in Animal models Pb exposure NMDAR AntagonismFebruary 2012 5

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– Do Not Cite or Quote Source: Reprinted with permission of Elsevier Science, Fox et al. ( 2010 ) Note: (A) Control; (B) Pb exposed light micrograph pictures of Brd

- U positive cells; (C) Counts of Brd

- U positive cells (proliferating cells); and (D) V olume of dentate gyrus. *p <0.05 vs. control. 

### Figure 5
- 18 Neurogenesis (production of new cells) in the rat dentate gyrus after postnatal Pb exposure.

5.3.3.6 Integrated Summary of Behavior and Mood
Epidemiologic studies in children demonstrate associations of higher blood Pb levels 1 

### with a range of behavioral problems, with the weight of evidence demonstrating 2 

### associations with inatt ention and hyperactivity as rated by parents or teachers and as 3 

### assessed using objective neuropsychological tests. Previous studies found associations 4 

### with early childhood blood or tooth Pb level ( i.e., ages 2

- 6 years), and recent studies 5 

### expanded evidence to include associations with concurrent blood Pb level. Recent 6 

### epidemiologic studies consistently found associations with inattention and hyperactivity 7 

### in children ages 1 to 12 years with mean concurrent blood Pb levels of 2 to 5 μ g/dL ( Cho 8 

### et al., 2010 ; Nicolescu et al., 2010 ; Plusquellec et al., 2010 ; Chiodo et al., 2007 ; 9 

### Plusquellec et al., 2007 ) , similar to those associated with cognitive function decrements. 10 

### The epidemiologic findings are strengthened by observ ations in animals of Pb

- induced 11February 2012 5

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### inattention and impulsivity in homologous tests of response inhibition ( e.g., DSA, FI, 1 

### DR). Tests in children such as CPT, Stroop, WCST share homology with tests in animals 2 

### such as DSA, DR, and FR tasks in that they all test the ability to inhibit inappropriate 3 

### responses, organize behavior in response to varying stimuli, and learn from the 4 

### consequences of previous actions. Inattention and impulsivity in animals are most clearly 5 

### indicated with gestational and early postnatal P b exposures that result in blood Pb levels 6 

### of 10 to 40 μ g/dL. These findings in children and animals for Pb

- associated dysfunction 7 

### in response inhibition and response variability may be explained by observations that Pb 8 

### affects dopaminergic neurons of the frontal striatum of the brain by altering dopamine 9 

### release and receptor density . The circuitry in this regions is thought to mediate response 10 

### inhibition. Whereas previous eviden ce was inconsistent, several recent epidemiologic 11 

### studies indicate association s between higher concurrent blood Pb level and higher 12 

### prevalence or incidence of ADHD diagnosis and its contributing diagnostic indices in 13 

### children ages 8

– 17 years ( Cho et al., 2010 ; Nicolescu et al., 2010 ; Roy et al., 2009a ; Nigg 14 

### et al., 2008 ; Braun et al., 2006 )

. The biological plausibility for as sociations with ADHD 15 

### is strongly supported by the large epidemiologic and toxicological evidence base 16 

### demonstrating Pb

- associated increases in inattention and impulsivity, both of which are 17 

### primary symptoms of ADHD. A smaller but equally consistent body of evidence 18 

### indicated associations of concurrent and early childhood blood Pb levels with social 19 

### misconduct in children and delinquent behaviors in adolescents and young adults 20 

### ( Chandramouli et al., 2009 ; Braun et al., 2008 ; Wright et al., 2008 ; Chiodo et al., 2007 )

. 21 

### Associations of blood Pb levels with ADHD, misconduct, and delinquency were 22 

### observed in populations of children with a wide range of blood Pb le vels, 1 to 11 μ g/dL, 23 

### all similar in the strength of evidence. 24 

### While mood and emotional state have been examined less frequently compared with 25 

### inattention and misconduct, several studies found associations of biomarkers of 26 

### cumulative Pb exposure ( i.e., toot h or childhood average blood Pb) and concurrent blood 27 

### Pb levels with parental or teacher reports of withdrawn behavior or depression in children 28 

### with mean blood Pb levels 8

- 28 μ g/dL (Section 5.3.3.3 ). These findings in children are 29 

### supported by a small body of toxicological studies in which prenatal plus lactational Pb 30 

### exposure resulted in depression

- like behavior in rodents. 31 

### Rather than examining ext ernalizing behaviors and criminal behavior, a small body of 32 

### studies of behavior in nonoccupationally

- exposed adults examined and found 33 

### associations of blood ( Bouchard et al., 2009 ) and tibia ( Rajan et al., 2008 ) Pb levels with 34 

### depression and anxiety symptoms. All of these studies used single assessments of Pb 35 

### biomarker levels and outcomes and analyzed associations in a cross

- sectional manner. 36 

### Thus, there is uncertainty regarding the critical level, timing, frequency, and duration of 37 

### Pb exposure associated with mood and psychiatric symptoms. The associations with bone 38February 2012 5

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### Pb levels indicate an effect of cumulative Pb exp osure. Concurrent blood Pb levels in 1 

### adults reflect both cumulative and recent exposure, and it is uncertain what are the 2 

### relative contributions of past versus recent Pb exposures to the observed associations. 3 

### Associations of Pb biomarkers with withdrawn b ehavior and anxiety in children and 4 

### adults may by explained by evidence for Pb

- induced changes in the HPA axis, 5 

### dopaminergic and GABA ergic CNS processes. These processed have been shown to 6 

### mediate anxiety and depression. While it may seem that Pb exposure affects different 7 

### behaviors in children and adults, it is important to acknowledge differences in the 8 

### evidence base. Studies of behavior in children and young adults have focused on 9 

### inattention and misconduct; studies of older adults did not examine extern alizing 10 

### behaviors or misconduct. Differential effects in children and adults also may be expected 11 

### given the predominance of different neurophysiological processes operating at different 12 

### ages, for example, neurogenesis and brain development in children and 13 

### neurodegeneration in adults. 14



### 5.3.4 Sensory Organ Function

5.3.4.1 Epidemiologic Studies of Sensory Organ Function in Children
Although not as widely examined as cognitive and behavioral outcomes, several studies 15 

### found associations of higher blood Pb level with higher he aring thresholds and poorer 16 

### auditory processing in children ( U.S. EPA, 2006b )

. Such evidence is limited largely to 17 

### studies described in the 2006 Pb AQCD. The prospective CLS with repeat measurements 18 

### of blood Pb prenatally to age 5 years provided information on potentially important 19 

### lifestages of exposure. In this cohort with higher blood Pb levels than those in 20 

### contemporary U.S. children (lifetime average mean: 17.4 [SD: 8.8] μ g/dL), poorer 21 

### auditory processing was associated with higher prenatal (maternal), neonatal (10

- day), 22 

### early childhood, and lifetime average blood Pb levels, with the strongest associations 23 

### observed for neonatal blood Pb level. A 1 μ g/dL higher neonatal blood Pb level was 24 

### associated with a 0.20

- point (p ≤ 0 .01) and 0.26

- point (p ≤ 0 .10) lower score on the total 25 

### and left ear Filtered Word test (indicative of incorrectly identified filtered or muffled 26 

### words), after adjusting for hearing screen, social class, HOME score, birth weight, 27 

### gestational age, obstetrical co mplications, alcohol consumption, and prenatal and 28 

### postnatal blood Pb levels ( Dietrich et al., 1992 )

. Overall, the findings pointed to an effect 29 

### of early Pb exposure during infancy. 30February 2012 5

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### Large U.S. studies, including NHANES II ( Schwartz and Otto, 1987 ) and the Hispanic 1 

### Health and Nutrition Examination Survey (HHANES) al so found associations between 2 

### higher blood Pb level and lower auditory function; however, their cross

- sectional design 3 

### precluded comparisons among various lifestages of exposure ( Schwartz and Otto, 1991 )

. 4 

### In these studies, concurrent blo od Pb level (median: 8 μ g/dL) from 6 to 18 μ g/dL was 5 

### associated with a 2

- db loss in hearing and an increase in the percentage (15%) of children 6 

### with a substandard hearing threshold (2,000 Hz). Higher concurrent blood Pb level also 7 

### was associated with higher hearing thresholds across several frequencies in a population 8 

### of children in Poland with similar blood Pb levels (median: 7.2 μ g/dL [range: 1.9 to 28]) 9 

### ( Osman et al., 1999 )

. In the HHANES and Polish studies, associations persisted in 10 

### analyses r estricted to subjects with concurrent blood Pb levels below 10 μ g/dL. 11 

### Mechanistic support for these observations in children was provided by Otto and 12 

### colleagues ( Otto and Fox, 1993 ; Otto et al., 1985b ) , who found associations of blood Pb 13 

### level with lower brainstem auditory evoked potentials in children. Rothen berg et al. 14 

### ( 1994b ) and Rothenberg et al. ( 2000 ) reported similar findings; however, the direction of 15 

### association differed between prenatal (maternal) and postnatal (ages 1

- 4 years) blood Pb 16 

### level. Postnatal blood Pb level was associated with lower interpeak intervals in auditory 17 

### evoked potentials at age 5

- 7 years. Prenatal maternal blood Pb level showed a biphasic 18 

### relationship, with a lower evoked potentials at blood Pb levels of 1

- 8 μ g/dL and higher 19 

### evoked potentials at blood Pb levels of 8

- 30 μ g/dL. Recent studies also aimed to identify 20 

### the locus in the auditory system where Pb may exert its effects on auditory function. 21 

### Investigation was limited to a population of children with hi gh blood Pb levels (means 33 22 

### and 37 μ g/dL) living in Pb glazing communities in Ecuador ( Buchanan et al., 2011 ; 23 

### Counter et al., 2011 )

. In these studies, concurrent blood Pb level was not correlated with 24 

### th e acoustic stapedius reflex ( Counter et al., 2011 ) or distortion product otoacoustic 25 

### emissions ( Buchanan et al., 2011 ) , indicating lack of effect on the auditory brainstem or 26 

### inner ear, respectively. Other loci were not examined. 27



### 5.3.4.2 Epidemiologic Studies of Sensory Organ Function in Adults
Studies of auditory function reviewed in the 2006 Pb AQCD provided consistent 28 

### evidence of association between blood Pb levels and changes in auditory evoked 29 

### brainstem potentials in occupationally

- exposed adults, but less consistent findings for 30 

### hearing thresholds ( U.S. EPA, 2006b )

. A few new studies of Pb workers found Pb

31 

### associated increases in hearing thresholds. A recent study provided new evidence in 32 

### nonoccupationally

- exposed adults for associations of tibia Pb levels with hearing loss. 33February 2012 5

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### Men in the NAS study were free of hearing loss at baseline and had hearing tested 1 

### repeatedly (median 5 observations per subject) o ver a median of 23 years ( Park et al., 2 

### 2010 )

. Higher tibia Pb level, measured up to 20 years after initial hearing testing was 3 

### associated with a faste r rate of increase in hearing threshold for frequencies of 1, 2, and 8 4 

### kHz and a pure tone average. Blood Pb was not examined in this study. In cross

- sectional 5 

### analyses adjusted for age, race, education, body mass index, pack

- years of cigarettes, 6 

### diabetes, hypertension and occupational noise (based on a job

- exposure estimate from 7 

### occupations), and presence of a noise notch (indicative of noise

- induced hearing loss), 8 

### higher patella bone Pb level (measured within 5 years of hearing test) was associated with 9



### a higher hearing thresholds for frequencies greater than 1 kHz. A 21 μg/g (IQR) increase 10 

### in patella bone Pb level was associated with pure tone average hearing loss with an OR of 11 

### 1.48 (95% CI: 1.14, 1.91) in adjusted analyses. Similar, but slightly weaker a ssociations 12 

### were found for tibia bone Pb levels. 13 

### With new investigation, the weight of evidence indicates that blood Pb levels are 14 

### associated increased hearing thresholds in adults with occupational Pb exposures. In a 15 

### study of 183 Pb workers with relatively low blood Pb levels, 1 to 18 μg/dL, higher blood 16 

### Pb level was correlated with higher hearing threshold at 4 kHz ( Forst et al., 1997 )

. Other 17 

### studies examined workers with much higher blood Pb levels. In a study of 220 Pb battery 18 

### workers with higher blood Pb levels (mean: 56.9 μg/dL [SD: 25.3]) ( Wu et al., 2000 ) , 19 

### although hearing impairment was associated with a measure of cumulative Pb exposure 20 

### based on years of work and ambient Pb measurements, no association was found with 21 

### blood Pb levels at the time o f hearing testing in analyses adjusted for age, sex, and 22 

### duration of employment. Another cross

- sectional study examined 259 steel plant workers 23 

### with no parental history of ear

- related problems, no congenital abnormalities, no 24 

### occupational organic solvent e xposure, and hearing loss difference no more than 15 dB 25 

### between both ears ( Hwang et al., 2009 )

. The participants had a mean (SD) concurrent 26 

### blood Pb level of 54.3 (34 .6) μg/dL. Average noise levels also were measured in work 27 

### areas and dichotomized at 80dB. In analyses adjusted for age and work area noise 28 

### (dichotomized at 80 dB), workers with blood Pb level ≥ 7 μg/dL had a statistically 29 

### significant higher prevalence (ra nge of ORs: 3.06 to 6.26) of hearing loss at frequencies 30 

### of 3, 4, 6, and 8 kHz compared to workers with blood Pb levels ≤ 4 μg/dL. 31 

### A hospital

- based case

- control study recruited workers referred for hearing testing 32 

### (average hearing thresholds above 25 dB) a s cases and workers with normal hearing 33 

### thresholds who were having occupational health examinations for other reasons as 34 

### controls ( Chuang et al., 2007 )

. The 121 cases had a geometric mean blood Pb level of 35 

### 10.7 μg/dL, and the 173 controls had a geometric mean blood Pb level of 3.9 μg/dL based 36 

### on measurements in samples collected at the time of the study. In models that adjusted for 37 

### age, smoking, alcohol c onsumption, years of noise exposure, as well as Mn, As, and Se 38February 2012 5

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### levels in blood, higher blood Pb levels were associated with a statistically significant 1 

### higher average hearing threshold (0.5

- 6 kHz). 2 

### In summary, together, the findings from NAS ( Park et al., 2010 ) and studies of Pb

3 

### exposed workers indicate that biomarkers of Pb exposure are associated with lower 4 

### auditory function in adults. Evidence for a ssociation with concurrent blood Pb levels is 5 

### provided studies of adults with current occupational Pb exposures. Because only bone Pb 6 

### levels measured after auditory testing were examined in the NAS study, further 7 

### investigation is required to characterize t he timing, level, frequency, and duration of Pb 8 

### exposure contributing to lower auditory function in adults without occupational 9 

### exposures. 10



### 5.3.4.3 Toxicological Studies of Sensory Organ Function
The 1986 and 2006 Pb AQCDs detailed the effects of Pb in animals on v ision, including 11 

### the retina and CNS visual processing areas, as well as the auditory system and described 12 

### possible or known mechanisms of action where available. Pb exposure effects on sensory 13 

### dysfunction and impaired sensory processing have been postulate d to contribute to Pb

14 

### associated effects on neurocognition and attention. In support of this hypothesis, Dietrich 15 

### et al. ( 1992 ) foun d that higher prenatal (maternal) and postnatal blood Pb levels were 16 

### associated with both lower performance on the screening test for auditory processing 17 

### disorders and lower cognitive function in 5 ye ar
- old CLS children (Section 5.3.4.1 ). New 18 

### research in this area expands upon the extant evidence by exploring sensory function in 19 

### animals with lower Pb exposures and blood Pb levels. 20



### Auditory Effects

The 2006 Pb AQCD discussed impaired auditory function in nonhuman primates exposed 21 

### to Pb from gestation through age 8

- 9 years (resulting in blood Pb levels 33

- 56 μ g/dL 22 

### during Pb exposure period). Brainstem auditory evoked responses (BAER), which are 23 

### used as a general test to assess neurological auditory function, revealed Pb

- related effects 24 

### that persisted even after Pb exposure had ceased and blood Pb levels had returned to 25 

### baseline. In Pb

- exposed animals (birth to age 13 years), half of the pure tone detectio n 26 

### thresholds were outside of the control range at certain frequencies ( Rice, 1997 )

. In 27 

### concordance with the data from developmentally Pb

- exposed laboratory animals, 28 

### elevated auditory thresholds, as measured with brainstem auditory evoked response 29 

### (BAER), have also been associated with blood Pb levels in children ( Rothenberg et al., 30 

### 2000 ; Rothenberg et al., 1994a )

. In addition to indicati ng hearing loss, BAER can 31 

### indicate impaired synaptic maturation and incomplete neuron axon myelination leading to 32February 2012 5

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### impaired neuronal conduction ( Schwartz and Otto, 1991 ; Gozdzik

- Zolnierkiewicz and 1 

### Moszyński, 1969 . Thus, the findings from Rice ( 1997 ) indicated that early life Pb 2 

### exposure impaired auditory function. The cochlear nerve in both developing and mature 3 

### humans appeared to be especially sensitive to the Pb insult. At low to moderate Pb 4 

### exposures, elevated thresholds and increased latencies were observed in brainstem 5 

### auditory evoked potentials. 6 

### In a recent study, Laughlin et al. ( 2009 ) studied rhesus monkeys exposed to Pb

- acetate 7 

### gestationally through age 5.5 months (dam drinking water, 3 months prior to mating until 8 

### weaning, resulting in bone Pb levels at 11 year s of 7 and 13 μ g/dL for prenatal and 9 

### postnatal groups, respectively and blood Pb levels during Pb exposure of 35 and 10 

### 46 μ g/dL, respectively) and conducted a uditory threshold testing and threshold task 11 

### testing at 13 years of age after blood Pb levels had returned to baseline. At birth, animals 12 

### were cross fostered, creating a control group, a prenatal Pb group, and a postnatal Pb 13 

### group; however, Pb exposed anim als were analyzed as a single group. Pb exposure 14 

### induced small elevations in auditory thresholds in animals. Auditory threshold task

15 

### related behavioral testing was also impaired in Pb

- exposed animals. This study has 16 

### multiple limitations which likely contri buted to its not finding statistically significant 17 

### aberrations. These limitation included limited power with the examination of 5 animals 18 

### per group, the inability of some of the monkeys to engage or focus on the task at hand 19 

### and thus had fewer available me asurements, differences between the sexes in inattention, 20 

### and mixing of the postnatal Pb and prenatal Pb animals into one group (Pb

- exposed 21 

### animals). 22 

### In summary, studies in nonhuman primates have shown lower BAER due to chronic Pb 23 

### exposure (gestation throu gh adulthood) that result in blood Pb levels in the range of 24 

### 33

- 56 μ g/dL. In concordance with these findings, previous epidemiologic studies 25 

### described lower BAER in children with higher prenatal maternal blood Pb levels and 26 

### concurrent blood Pb levels. Coll ectively, these findings provide support for the 27 

### associations observed between higher blood Pb levels and increased hearing thresholds 28 

### (concurrent blood Pb) and lower auditory processing in children (neonatal) 29 

### (Section 5.3.4.1 ). The animal evidence for long

- term Pb exposure effects provide 30 

### coherence with findings in adults with occupational Pb exposures for associations 31 

### between blood Pb levels and increasing hearing thresholds and in nonoccupationally

32 

### exposed adults, for associations with biomarkers of cumulative Pb exposure ( i.e., tibia 33 

### and patella Pb level) (Section 5.3.4.2 ). 34February 2012 5

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### Effects on Vision In toxicological studies, Pb has been shown to affect multiple aspects of the visual system 1 

### from the retina, to the sensory processing areas of the brain, to neurons involved in 2 

### vision. The selective action of Pb on retinal rod cells and bipolar cells is well documented 3 

### in earlier Pb AQCDs ( Fox et al., 1997 ; Fox and Sillman, 1979 ) and research in this area 4 

### continues to date. Pb exposure during perinatal development and adulthood has also been 5 

### shown to affect the visual cortex ( Costa and Fox, 1983 ) and subcortical neurons ( Cline et 6 

### al., 1996 )

. Extensive work in nonhuman primates with various Pb exposure paradigms 7 

### (development or lifetime Pb) showed sensory impairment, i.e., dysfunction of spatial and 8 

### temporal visual function ( Rice, 1998 )

. Environmentally

- relevant doses of Pb (10

- 3 

### μ M) 9 

### adminis tered to tadpoles inhibited the growth of developing neurons in the subcortical 10 

### retinotectal pathway, the main efferent from the retina ( Cline et al., 1996 )

. Functional 11 

### tests like electroretinograms (ERGs) show Pb

- related aberrations in children, rodents and 12 

### nonhuman primates. 13 

### The animal toxicological data show that developmental windows and the dose of Pb 14 

### contribute to the complex and variable effe cts of Pb with the retina. Table 5

- 12 15 

### summarizes Pb

- related effects in retinal ERG studies. Female rats exposed postnatally to 16 

### moderate and high levels of Pb (0.02 or 0.2% Pb

- acetate exposure in dam drinking water 17 

### from birth through weaning, resulting in weaning blood Pb levels of 19 and 59 μg/dL, 18 

### respectively) had subnormal sco topic ERGs (decreased A

- and B

- wave amplitudes) with 19 

### decreased sensitivity an d temporal resolution when assessed at 90 days of age ( Fox et al., 20 

### 1991 ) ( Table 5

- 12 ). Similar results were obtained in multiple studies conducted in in vitro 21 

### models ( Otto and Fox, 1993 ; Fox and Farber, 1988 ; Fox and Chu, 1988 )

. Monkeys 22 

### exposed to moderately high levels of Pb continuously from the prenatal period to age 23 

### 7 years (350 or 600 ppm Pb

- acetate , resulting in blood Pb levels of 40 and 50 μg/dL, 24 

### respectively) had persistently increased ma ximal retinal ERG amplitude (B

- wave only, 25 

### supernormality) and increased mean ERG latency when assessed 2 years after 26 

### termination of Pb exposure when blood Pb levels were <10 μg/dL ( Lilientha l et al., 1988 ) 27 

### ( Table 5

- 12 ). 28February 2012 5

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### Table 5
- 12 Summary of Pb
- related retinal ERG studies

Study Species Sex a Pb Exposure Protocol/Dose Maximal Blood Pb Level (μg/dL) ERG A bnormality Progenitor cell proliferation Retinal Cellular Apoptosis Retinal Dopamine Levels Retinal Cell Layer Thickness Fox et al. ( 2008 ) Long

- Evans Rat F Prenatal

- PND10 b DW Low 27 ppm Moderate, 55 ppm High, 109 ppm 12 24 46 Supernormal Supernormal

Subnormal Yes Yes No Not affected Not affected Yes Dose

- dependent ↓ Dose

- dependent ↓ Dose

- dependent ↓ ↑ ↑ ↓ Lilienthal et al., ( 1988 ) Rhesus Monkey M & F Pre

- and Post

- natal (lifetime), DW 350 ppm 600 ppm ~50 ~115 Supernormal Supernormal

— —

— —

— —

— Fox et al. ( 1997 ) Long

Evans Rat F PND1

- PND 21 0.02% DW 0.2% DW 19 59 Subnormal Subnormal — Yes Yes ↓ ↓ Rothenberg et al. ( 2002a ) Human children M & F Prenatal 1st trimester ≥ 10.5 Supernormal

— — — — Guguchkova et al. ( 1972 ) Human M Occupational Subnormal

— — — — Otto and Fox ( 1993 ) Human M Occupational Subnormal

— — — —

a F: Females; M: Males
b PND: postnatal day

“

— “ Denotes not measured.


### A recent study exposed female Long

- Evans rats to low (27 ppm ), moderate (55 ppm ), and 1 

### high (109 ppm ) levels of Pb

- acetate in drinking water beginning 2 weeks before mating, 2 

### throughout gestation, and until PND10 (G+P exposure) ( Fox et al., 2008 ) ( Table 5

- 12 ). 3 

### Blood Pb levels in G+P Pb

- exposed pups in the three groups were 10

- 12 μ g/dL (lower), 4 

### 21

- 24 μ g/dL (moderate), and 40

- 46 μ g/dL (higher). This developmental window in the 5 

### retina of the rat is equivalent to gestational human retinal development. Results of this 6 

### rodent study demonstrated persistent supernormal scotopic rod photorecept or
- mediated 7 

### ERGs (lower and moderate Pb exposure) similar to the associations observed between 8 

### ERG and prenatal maternal blood Pb levels ≥ 10.5 μg/dL in male and female children 9 

### ( Rothenberg et al., 2002a )

. Supernormal scotopic ERGs may be recor ded without other 10 

### overt optha logic al changes and are rarely seen in the clinical setting ( Terziivanov et al., 11 

### 1983 )

. Lower and moderate levels of G+P Pb exposure increased neurogenesis of rod 12 

### photoreceptors and rod bipolar cells without affecting Müller glial cells and s tatistically 13 

### significantly increased the number of rods in central and peripheral retina. Higher

- level 14 

### G+P Pb exposure (109 ppm , blood Pb level of 46 μ g/dL) or moderate to higher level 15 

### postnatal exposure (PND1

- 21, blood Pb levels 19 and 59 μ g/dL) statistic ally significantly 16 

### decreased the number of rods in central and peripheral retina, induced scotopic ERG 17 

### subnormality in adult rats ( Table 5

- 12 ), and sta tistically significantly decreased the retinal 18February 2012 5

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### Zn concentration. Pb exposure induced concentration

- dependent decreases in adult rat 1 

### retinal dopamine synthesis and utilization/release ( Fox et al., 2008 )

. Thus, the dose of Pb 2 

### and the exposure window produce complex int e ractions in the retina that differentially 3 

### affect retinal histology and functional tests, i.e., ERG as summarized in Table 5

- 12

. 4

Note: *p <0.05 Source: Fox et al. ( 2008 ) 

### Figure 5
- 19 Retinal a
- wave and b
- wave ERG amplitude in adult rodents after prenatal and early postnatal Pb exposure.
A recent study of adult zebrafish that were exposed as embryos (2 to 24 hours post

5 

### fertilization) to water containing 0.03 μM PbCl 2 

### showed fish with impaired response to 6 

### visual stimulation (visual response to a rotating bar) under low light conditions. These 7 

### zebrafish also failed to respond normally to mechanosensory stimulation (0.01 and 0.03 8 

### μM PbCl 2 

### ), showing a significantly impaired startle response. These data sho w that low

9 

### level developmental Pb exposure causes sensorimotor deficits in fish ( Rice et al., 2011 )

. 10 

### Another study evaluating visual spatial acuity in rhesus monkeys ( Laughlin et al., 2008 ) 11 

### exposed to Pb

- acetate postnatally (PND8

- 26 weeks of age via commerc ial milk formula, 12 

### achieving a target blood Pb of 35

- 40 μg/dL) found no effects of Pb exposure on spatial 13 

### acuity as assessed with modified the Teller preferential looking paradigm. 14 

### Mechanistic understanding of the effect of Pb on the visual system includes its capability 15 

### to displace divalent cations, act as an inhibitor of physiological enzymes, regulate cell 16 

### proliferation and apoptosis, impair and perturb normal neuroanatomy formation, 17 

### i.e., cytoarchitecture in the brain, and affect neurotransmitters. The e ffects of Pb on the 18 

### retina have been shown to by mediated by its capability to act as a cGMP 19 

### phosphodiesterase (PDE) inhibitor ( Srivastava et al., 1995 ; Fox and Farber, 1988 )

. The 20 

### drug sildenafil citrate, another cGMP PDE in hibitor, can also cause visual problems 21 

### including alterations in scotopic ERGs ( Laties and Zrenner, 2002 )

. With postnatal 22 

### exposure of animals or in vitro Pb exposure of isolated rods, Pb has been shown to induce 23 

### elevated cGMP which contributes to elevated rod calcium concentration ( Fox and Katz, 24 

### 1992 ) and subsequently to apoptotic cell death in a concentration

- dependent fashion. In 25February 2012 5

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### separate work in mice, low and moderate doses of Pb (27 or 55 ppm Pb

- acetate in dam 1 

### drinking water from gestation to PND10, resulting in blood Pb levels of 12 and 25 μg/dL, 2 

### respectively), induced greater rod and rod bipolar cell neurogenesis (proliferation) and 3 

### greater thickness and cell number of the outer and inner neuroblastic layers of the retina 4 

### ( Giddabasappa et al., 2011 ; Fox et al., 2008 )

. Ro dents with moderate dose G+P Pb 5 

### exposure (resulting in blood Pb level of 25 μ g/dL) had 27

- fold greater and prolonged 6 

### retinal progenitor cell proliferation ( Giddabasappa et al., 2011 ) ; at higher doses of Pb 7 

### (109 ppm Pb

- acetate , resulting in blood Pb levels of 56 μg/dL) there was no rod 8 

### neuroge nesis. Nitric oxide has been shown to regulate retinal progenitor cell proliferation 9 

### in chick embryos ( Magalhaes et al., 2006 )

. Thus, these authors postulated that impaired 10 

### NO production may contribute to aberrant retinal cell proliferation ( Giddabasappa et al., 11 

### 2011 )

. Pb exposure has been shown to impair NO synthase activity in other organs 12 

### (Section 5.2.4.5 ). Pb has been shown to affect a plethora of neurotransmitters in the brain 13 

### and it has recently been shown to affect neurotransmitters in the retina. In the 14 

### aforementioned model of G+P rodent Pb exposure through PND10, Pb decreased 15 

### dopamine (DA) synthesis and use in a concentration

- dependent manner ( Fox et al., 2008 ) 16 

### ( Figure 5

- 20 ). These new data provide further insight into retinal changes by showing 17 

### increased proliferation of Pb

- exposed retinal progenitor cells without changes in 18 

### apoptosis in G+P exposed rats ( Fox et al., 2008 )

. 19February 2012 5

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– Do Not Cite or Quote Note: *p <0.05 Source: Fox et al. ( 2008 ) 

### Figure 5
- 20 Retinal dopamine metabolism in adult control and gestationally lead
- exposed (GLE) rats.

As discussed in the 2006 Pb AQCD, Pb

- induc ed decreased Na/K+ ATPase activity have 1 

### been reported in vitro and in vivo. Also, structural changes from chronic Pb exposure 2 

### (birth to age 6) included cytoarchitectural changes in visual projection areas of the brain 3 

### of monkeys; maximum blood Pb level in the low and high dose group reached 20 μ g/dL 4 

### and 220 μ g/dL, respectively ( Reuhl et al., 1989 )

. Within the visual system, Pb has been 5 

### shown to affect multiple pathways including si gnaling, enzyme inhibition, 6 

### neurotransmitter levels, neuroanatomy formation, and cell proliferation and apoptosis. 7 

### In summary, developmental windows of exposure as well as exposure dose ( Table 5

- 12 ) 8 

### are important factors that contribute to Pb

- induced changes in retinal function. The retina 9 

### is a sensitive target for Pb

- induced altered histology and retinal function. Prenatal, 10 

### postnatal, lifetime or occupational exposures provide windows with different effects on 11 

### the retina, effects that are modified by dose of Pb. 12February 2012 5

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### 5.3.5 Motor Function

Some studies in children have assessed fine motor function, i.e., response speed, 1 

### dexterity, eye

- hand coordination, as part of a battery of cognitive function and behavioral 2 

### testing, and most have found associations with blood Pb level. Fewer studies have 3 

### examined gross motor function, i.e., postural balance, action trem or, agility but also have 4 

### found associations with blood Pb level. 5 

### Multiple studies were conducted in the CLS cohort at age 6 years and in adolescence at 6 

### ages 12 and 15

- 17 years. Assessments in younger children indicated associations of 7 

### concurrent and lifet ime average blood Pb levels but not prenatal blood Pb levels with 8 

### poorer visuomotor control and upper limb dexterity ( Dietrich et al., 1993b ) and poorer 9 

### postural balance ( Bhattacharya et al., 1995 )

. Associations with both fine and gross motor 10 

### function were adjusted for HOME sc ore and race. Additional covariates included 11 

### maternal IQ, SES, and sex for fine motor functions ( Dietrich et al., 1993b ) and height, 12 

### BMI, birth weight, bilateral ear infection, and foot area for postural balance 13 

### ( Bhattacharya et al., 1995 )

. Blood Pb levels were associated with fine and gross motor 14 

### function in unadjusted and adjusted analyses, indicating that bias from the measured 15 

### confounders was not driving the observed associations. Subsequent analyses in the CLS 16 

### demonstrated associations of earlier childhood average blood Pb levels (0

- 5 year average 17 

### or 78 month average) with poorer fine ( Ris et al., 2004 ) and gross motor functio n 18 

### ( Bhattacharya et al., 2006 ) assessed in adolescence. While these findings suggest the 19 

### persistence of Pb effects or early, long

- term exposure effects, it is important to note that 20 

### blo od Pb levels measured later in childhood or concurrently with motor function were not 21 

### examined. 22 

### Studies in other populations found associations of blood Pb level (primarily concurrent) 23 

### with poorer fine motor function in children ranging in age from 3 to 16 years 24 

### ( Palaniappan et al., 2011 ; Min et al., 2007 ; Wasserman et al., 2000 )

. Among childre n in 25 

### New England participating in NECAT (described in Section 5.3.2.1 ) Surkan et al. ( 2007 ) 26 

### found higher blood Pb levels to be associated with lower FSIQ but better fine motor 27 

### function as indicated by faster finger tapping speed. Gross motor function also was 28 

### assessed in other populations of children. In the Yugoslavian cohort, lifetime average 29 

### blood Pb level was not associated with gross motor function ( Wasserman et al., 2000 )

. 30 

### Concurrent blood Pb level was associated wi th greater sway oscillation, alternating arm 31 

### movements, and action tremor in a group of Inuit preschool children in Quebec, Canada 32 

### ( Despres et al., 2005 )

. Investigators considered potential confounding by several 33 

### variables including HOME score, maternal education and nutrient levels. In animal 34 

### studies, Pb exposure has shown mixed effe cts on endurance, balance and coordination as 35 

### measured by rotarod performance. G+P Pb

- exposed male mice had significantly shorter 36February 2012 5

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### mean latencies to fall from the rotarod compared with controls; females were unaffected. 1 

### Further, low dose G+P Pb

- exposed male mice had significantly poorer rotarod 2 

### performance ( i.e., fell off more quickly) than did high dose G+P male mice (resulting 3 

### blood Pb levels at PND10: 10 μ g/dL and 42 μ g/dL, respectively), indicative of a 4 

### nonlinear concentration

- dependent relationship ( Leasure et al., 2008 )

. Other rotarod 5 

### experim ents at higher doses of Pb exposure and at various speeds of rotarod rotation 6 

### yielded mixed results ( Kishi et al., 1983 ; Grant et al., 1980 ; Overmann, 1977 )

. 7 

### The collective body of evidence demonstrates that within the same population of 8 

### children, blood Pb levels are associated with decrements in cognitive function, attention, 9 

### and fine and gross motor function. Studies found associations with concurrent and 10 

### childhood average blood Pb levels mostly in populations with mean levels ranging from 11 

### 11 to 28 μ g/dL. Despres et al. ( 2005 ) found impaired gross motor function in a children 12 

### with a mean blood Pb level of 5 μ g/dL. Min et al. ( 2007 ) found impaired fine motor 13 

### function in children with a mean concurrent blood Pb level of 2.9 μ g/dL; however, the 14 

### results were not adjusted by SES

- related variables. motor skills are the result of the 15 

### coordina tion of complex cognitive and physical processes in the cortex, cerebellum, 16 

### vestibular systems, and visual system, and biological plausibility for the associations of 17 

### blood Pb levels with fine motor skills observed in children is provided by observations 18 

### t hat Pb exposure affects development and function of these systems. 19



### 5.3.6 Seizures in Animals
One neurological sign of high dose Pb exposure has been the development of epileptic 20 

### form activity or seizures in animals ( Krishnamoorthy et al., 1993 )

. However, earlier 21 

### studies in the animal toxicological literature explor ing the effects of Pb on seizure 22 

### activity and threshold showed mixed results. Pb

- acetate (250, 500, or 1 , 000 ppm for 30 23 

### days in drinking water to PND60 male Wistar rats, resulting in blood Pb levels of ~20, 24 

### 35, and 42 μ g/dL, respectively) significantly decreased the elapsed time required to 25 

### develop the first myoclonic jerk and tonic

- clonic seizure ( Arrieta et al., 2005 ) ; also, the 26 

### dose of pentylenetetrazol (PTZ) required to induce seizures was significantly decreased 27 

### across all Pb dose groups. Some studies showed no effect of Pb on kindled animals 28 

### ( Sc hwark et al., 1985 ; Alfano and Petit, 1981 )

. Other studies showed differential 29 

### susceptibility to convulsant

- inducing ag ents in developmentally Pb

- exposed rats ( Chen 30 

### and Chan, 2002 )

. Sprague Dawley rats were exposed to Pb

- acetate (0.2% w/v in drinking 31 

### water from PND1

- 25, followed by 25 days with no Pb exposure). Seizures were induced 32 

### at PND25 or PND50. At PND25, Pb exposure significantly decreased (PTZ)

- , picrotoxin 33 

### (PIC)

- , and strychnine (STRY)

- induced convulsion threshold s, but increased N

- methyl

34 

### D

- aspartate (NMDA) and 4

- aminopyridine (4

- AP)

- induced convulsion thresholds. At 35February 2012 5

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### PND50, the effects of PTZ, NMDA, and 4

- AP remained similar to those at PND25, but 1 

### PIC and STRY

- dependent convulsion thresholds were reversed and signi ficantly 2 

### increased ( Chen and Chan, 2002 )

. Chen and Chan ( 2002 ) hypothesized that this 3 

### differential effect may be due to selective effects on inhibitory and excitatory 4 

### neurotransmission as an effect of age an d blood Pb level. 5 

### Recent investigation expands on the work by Arrieta et al. ( 2005 ) by showing similar 6 

### effects in another rodent species, BALB/c mice. Adult male BALB/c mice were exposed 7 

### to Pb for 30 days via drinking water (resulting in blood Pb levels in control and 50, 100, 8 

### 200 and 400 ppm Pb groups of 0.02, 6, 11, 15 and 18 μ g/dL, respectively) ( Mesdaghinia 9 

### et al., 2010 )

. Exposure to 50 ppm Pb did not affect PTZ

- induced seizure threshold, but all 10 

### other doses significantly reduced the thresholds of face and forelimb clonus, myoclonic 11 

### twitch, running and bouncing clonus, and tonic hindlimb extension. These studies show 12 

### that Pb exposure may modulate seizure activity in animals. 13



### 5.3.7 Neurodegenerative Diseases

5.3.7.1 Epidemiologic Studies of Neurodegenerative Diseases in Adults
The 2006 Pb AQCD described several studies examining associations of blood and bone 14 

### Pb levels with neurodegenerative diseases such as Alzheimer’s disease and dementia. 15 

### Among NAS men, hi gher bone Pb levels were associated with lower MMSE scores 16 

### ( Weisskopf et al., 2004 ; Wright et al., 2003b ) , which indirectly pointed to a potential 17 

### association with dementia, given that the MMSE is widely used as a screen ing tool for 18 

### dementia. Overall, studies had sufficient limitations ( e.g., indirect assessment of 19 

### dementia, comparison of Alzheimer's Disease patients and healthy controls, lack of Pb 20 

### biomarker data), and findings were inconclusive ( U.S. EPA, 2006b )

. New studies on 21 

### dementia are not available to assess further th e associations with Pb biomarkers. 22 

### Similarly, new studies examining Alzheimer’s disease are not available, and as in 2006, 23 

### the evidence is still inconclusive regarding the association with Pb biomarkers. In 24 

### contrast, there has been additional investigation of ALS, Parkinson’s disease (PD), and 25 

### essential tremor, which is described below. 26



### Amyotrophic Lateral Sclerosis

Most studies of the association between Pb and ALS have relied on indirect methods of 27 

### assessing Pb exposure and overall, have produced inconsis tent results. Case

- control 28February 2012 5

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### studies that measured blood Pb levels produced contrasting results. A study of 16 ALS 1 

### cases (mean blood Pb level: 12.7 μ g/dL) and 39 controls (mean blood Pb level: 2 

### 10.8 μ g/dL) found a small difference in the mean concurrent blood Pb level ( Vinceti et 3 

### al., 1997 )

. Another case

- control study tha t examined concurrent blood and bone Pb levels 4 

### in a New England

- area population found higher odds of ALS among subjects with 5 

### concurrent blood Pb levels ≥ 3 μ g/dL ( e.g., OR: 14.3 [95% CI: 3.0, 69.3] for blood Pb 6 

### levels 3

- 4 μ g/dL) ( Kamel et al., 2002 )

. In analyses of tibia or patella Pb tertiles, subjects 7 

### in the highest two tertiles (≥ 10 μ g/g patella Pb and ≥ 8 μ g/g tibia Pb) had higher ALS 8 

### prevalence. For example, the ORs (95% CIs) for patella Pb levels 10

- 20 μ g/g and tibia Pb 9 

### levels 8

- 14 μ g/g were 2.1 (0.6, 7.4) and 1.6 (0.5, 5.6), respectively. Also in this 10 

### population, an estimate of cumulative Pb exposure based on occupational history was 11 

### found to be associated with ALS ( Kamel et al., 2002 )

. The stronger findings for blood Pb 12 

### level were surprising given that bone Pb level is a better biomarker of cumulative Pb 13 

### exposure. One explanation for these findings is that the association could be the result of 14



### reverse causa lity since the half

- life of blood Pb is only about 30 days, and blood was 15 

### collected from people who already had ALS. If, for example, reduced physical activity 16 

### among those with ALS led to more bone turnover, then more Pb would be released from 17 

### bones into c irculation leading to elevations in blood Pb levels among cases as a result of 18 

### effects of the disease. 19 

### Since the 2006 Pb AQCD, a few additional studies have been conducted with the same 20 

### New England

- area case
- control study population. Kamel et al. ( 2005 ) reported that the 21 

### association between blood Pb level and ALS was not modified by the ALAD genotype 22 

### ( Kamel et al., 2005 )

. Another report examined survival of ALS among 100 of the original 23 

### 110 ALS cases ( Kamel et al., 2008 )

. Higher tibia Pb levels were associated with longer 24 

### survival time. Findings were similar for patella and blood Pb levels, although they were 25 

### associated with smaller increases in survival time. These paradoxical findings raise the 26 

### concern that in a case

- con trol study of ALS, the association between bone Pb levels and 27 

### ALS may be biased because the case group may comprise more individuals with longer 28 

### survival time. Consequently, their bone Pb levels may be higher because they reflect a 29 

### longer period of cumulat ive exposure. On the other hand, the decreased mobility due to 30 

### the disease itself would tend to increase bone resorption and lower bone Pb levels over 31 

### time. This process might mitigate this effect for bone Pb but would tend to increase blood 32 

### Pb levels amon g cases. However, this was not observed in the one study that had bone 33 

### and blood Pb biomarkers ( Kamel et al., 2002 )

. Because the strongest findings for survival 34 

### were found for tibia Pb, it is unlikely that the findings were biased due to increased 35 

### survival of cases. 36


### Another case

- control study examined concurrent blood Pb levels and ALS among 184 37 

### cases (33 were either progressive muscular atrophy or primary lateral sclerosis, mean 38February 2012 5

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### blood Pb level: 2.41 μ g/dL) and 194 controls (mean blood Pb level: 1.76 μ g/dL) ( Fang et 1 

### al., 2010 )

. The cases were recruited from the National Registry of U.S. Veterans with 2 

### ALS, and controls were recruited from among U.S. Veterans without ALS and frequency 3 

### matched by age, gender, race, and past use of the Veterans Administration system for 4 

### health care. A doubling of blood Pb levels was associated with ALS with an OR (95% 5 

### CI) of 2.6 (1.9, 3.7). Associations did not differ substantially by indicators of bone 6 

### turnover but were slightly higher among ALAD 1

- 1 carriers. The association with b lood 7 

### Pb level was similar in analyses that excluded the progressive muscular atrophy and 8 

### primary lateral sclerosis cases. The similar results by degree of bone turnover suggest that 9 

### reverse causation is not likely driving the association between blood Pb l evel and ALS. 10 

### Whether other types of reverse causality are contributing, however, cannot be ruled out. 11 

### This study did not have measures of bone Pb and therefore could not assess the 12 

### association with biomarkers of cumulative Pb exposure. 13 

### In summary, studies have found associations of blood and bone Pb levels with ALS but in 14 

### relatively few different cohorts. The case

- control design of most studies and issues of 15 

### reverse causality and bias due to survival time make it difficult to draw firm conclusions. 16 

### Studies in additional cohorts using designs other than case

- control comparisons are 17 

### needed to address the limitations of the available studies and characterize better the 18 

### potential effects of Pb on ALS. 19



### Parkinson’s Disease A few previous studies, some ecological ( Rybicki et al., 1993 ; Aquilonius and Hartvig, 20 

### 1986 ) and some case

- control relying on questionnaire data or occupational history 21 

### ( Gulson et al., 1999 ; Gorell et al., 1997 ; Tanner et al., 1989 ) indicated associations 22 

### between exposure to heavy metals, particularly Pb, and risk of PD. Available evidence 23 

### was limited and far from conclusive. A recent large case

- control study (330 cases, 308 24 

### controls) recently reported on associations between biomarkers of Pb and PD in a 25 

### population with virtually no occupational exposures to Pb ( Weisskopf et al., 2010 )

. 26 

### Subjects in the highest quartile of tibia Pb level (>16.0 μ g/g) had higher odds of PD 27 

### compared to those in the lowest quartile (≤ 5 μ g/g) (OR: 1.91 [95% CI: 1.01, 3.60]). In 28 

### this study, cases an d controls were recruited from several different sources including 29 

### movement disorder clinics and community

- based cohorts, which could have introduced 30 

### some biases. However, when analyses were restricted to cases recruited from movement 31 

### disorder clinics and to their spouse, in

- law, or friend as controls, the results were even 32 

### stronger (OR: 3.21 [ 95% CI: 1.17, 8.83] ). Although the use of spouse, in

- law, and friend 33 

### controls can introduce bias, this is expected to be toward the null as these groups are 34 

### likely to share many exposures. 35February 2012 5

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### Manganese exposure has been associated with Parkinsonian symptoms and could 1 

### potentially confound associations between Pb and PD. Weisskopf et al. ( 2010 ) did not 2 

### adjust for Mn exposure in analyses. However, unlike a setting of occupational exposure 3 

### to Pb, general environment exposure to Pb is mu ch less likely to be correlated with 4 

### environmental Mn exposure. Thus, the likelihood of any associations with Pb being 5 

### confounded by co

- occurring Mn exposure is less likely. 6


### Coon et al. ( 2006 ) conducted a smaller case

- control study of 121 PD patients and 414 7 

### age
- , sex
- , and race

- , frequency

- matched controls all receiving health care services from 8 

### the Henry Ford Health System. S ubjects in the highest quartile of both tibia (OR: 1.62 9 

### [95% CI: 0.83, 3.17] for levels ≥ 15 μ g/g) and calcaneus (OR: 1.50 [95% CI: 0.75, 3.00] 10 

### for levels ≥ 25.29 μ g/g) bone Pb levels had higher odds of PD compared to those in the 11 

### lowest quartiles (0

- 5.91 μ g/g for tibia and 0

- 11.70 μ g/g for calcaneus). The highest OR 12 

### for PD was estimated for subjects in the highest quartile of whole

- body lifetime exposure 13 

### to Pb (≥ 80.81 μ g/g) estimated using PBPK modeling), compared to the lowest quartile of 14 

### exposure (0

- 40. 04 μ g/g) (OR: 2.27 [95% CI:1.13, 4.55] for levels ≥ 80.81 μ g/g). These 15 

### analyses did not adjust for Mn either; however, in this study it was not clear what the 16 

### extent of occupational exposure to Pb was among the participants. Thus, it is uncertain 17 

### whether t he observed associations were specifically related to Pb exposure or could have 18 

### resulted from co

- occurring Mn exposure. 19 

### In summary, a small number of recent studies expand on previous evidence by finding 20 

### associations of bone Pb levels, biomarkers of cumula tive Pb exposure, with PD in adults. 21 

### Nonetheless, additional investigation is warranted to establish the temporality between Pb 22 

### exposure and development of PD and to assess potential confounding by Mn exposure. 23



### Essential Tremor In a relatively small body o f literature, concurrent blood Pb levels have been consistently 24 

### associated with essential tremor, although studies have had relatively small sample sizes 25 

### and have produced imprecise effect estimates. The 2006 Pb AQCD described case

26 

### control studies that fou nd associations between concurrent blood Pb levels and essential 27 

### tremor in New York City metropolitan area populations ( Louis et al., 2005 ; Louis et al., 28 

### 2003 )

. In Louis et al. ( 2005 ) , the magnitude of association was larger among carriers of 29 

### an ALAD2 allele than among adults with only ALAD1 alleles. 30 

### Since 2006, Dogu et al. ( 2007 ) reported on a case

- control study of 105 essential tremor 31 

### cases fro m a movement disorder clinic in Turkey and 105 controls (69 spouses and 36 32 

### other relatives living in the same district). After adjusting for age, sex, education, 33 

### cigarette smoking, cigarette pack

- years, and alcohol use, a 1 μg/dL higher blood Pb level 34February 2012 5

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### (mea sured at the time of study recruitment) was associated with essential tremor with an 1 

### OR (95% CI) of 4.19 (2.59, 6.78). This OR was much larger than that obtained in the 2 

### New York area study (OR: 1.19 [95% CI: 1.03, 1.37]) ( Louis et al., 2003 )

. The 3 

### magnitude of association in Dogu et al. ( 2007 ) is even more striking because so many of 4 

### the cont rols were spouses who are expected to share many environmental exposures as 5 

### cases. Most of the essential tremor cases were retired at the time of the study, but past 6 

### occupational history was not reported. Occupational Pb exposures were less likely in the 7 

### N ew York area population. 8 

### In this small body of studies, associations were observed between blood Pb level and 9 

### essential tremor in adults. However, due to the case

- control design of studies, temporality 10 

### between exposure and development of essential tremor c annot be established. Further, the 11 

### level, timing, frequency, and duration of Pb exposure associated with PD is uncertain as 12 

### all studies examined blood Pb level at the time of study recruitment. Occupational 13 

### histories were not reported in these studies. Thu s, it is not clear what past exposure to Mn 14 

### may have contributed to the associations observed with blood Pb levels. 15



### 5.3.7.2 Toxicological Studies of Neurodegenerative Disease
Although epidemiologic studies have provided weak evidence for associations of blood 16 

### Pb level and ALS in adults, toxicological studies have found that Pb exposure induces 17 

### neurophysiologic changes consistent with ALS. For example, chronic Pb exposure 18 

### ( Pb

- acetate in drinking water at 200 ppm from weaning onward, resulting blood Pb level: 19 

### 27 μ g/dL) reduced astrocyte reactivity and induced increased survival time in the 20 

### superoxide dismutase transgenic (SOD1 Tg) mouse model of severe ALS ( Barbeito et al., 21 

### 2010 )

. In this model, Pb exposure did not significantly increase the onset of the ALS 22 

### disease but increased survival time in SOD1 Tg mice ( Barbeito et al., 2010 )

. This finding 23 

### does provide biological plausibility for the association observed between blood Pb level 24 

### and longer survival time in patients diagnose d with ALS ( Kamel et al., 2008 )

. In mice, 25 

### astrocyte of vascular endothelial growth factor (VEGF) also was examined to understand 26 

### its possible contribution to Pb effects on increasing survival time in ALS models. 27 

### Baseline levels VEGF were elevated in astrocytes from the ventral spinal cord of 28 

### untreated SOD1 Tg mice versus untreated nontransgenic animals. VEGF was not induced 29 

### in the astrocytes of Pb

- treated nontrans genic mice. Pb

- exposed SOD1 Tg mice had 30 

### significant elevations of astrocyte VEGF versus vehicle

- treated SOD1 Tg animals 31 

### ( Barbeito et al., 2010 )

. Nontransgenic animals exposed to Pb showed no elevation in 32 

### VEGF expression above that in nontransgenic vehicle

- treated animals ( Barbeito et al., 33 

### 2010 )

. Other research has suggested that ALS initiation is dependent on motor neuron 34 

### function and ALS progression is dependent on astrocyte and microglia function 35February 2012 5

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### ( Yamanaka et al., 2008 ; Boillee et al., 2006 )

. Thus, the aforementioned findings for Pb

1 

### induced effects on astrocytes provide a mechanistic explanation for Pb effects on ALS 2 

### progression in animals. 3 

### Others have reported that VEGF administration to the SOD1 Tg mice significantly 4 

### reduced glial reactivity, a marker or neuroinflammation ( Zheng et al., 2007 )

. Using a 5 

### cell

- b ased co

- culture system of neurons and astrocytes, Barbeito et al. ( 2010 ) found that 6 

### an up
- regulation of VEGF p roduction by astrocytes in the Pb

- exposed SOD1 Tg mice 7 

### was protective against motor neuron death in the SOD1 Tg cells ( Barbeito et al., 2010 )

. 8 

### Chronic Pb exposure in a mouse model of ALS was associated with increased survival 9 

### time and was correlated with higher spinal cord VEGF levels, making astrocytes less 10 

### cytotoxic to surrounding motor neurons ( Barbeito et al., 2010 )

. Also, in another study the 11 

### metal chelators DP

- 109 and DP

- 460 were neuroprotective in the ALS mouse m odel or 12 

### Tg(SOD1

- G93A) ( Petri et al., 2007 )

. 13 

### Improper activation of microglia and release of inflammatory cytokines and metabolites 14 

### can contribute to neurodegeneration ( Zhang et al., 2010b ; Qian and Flood, 2008 )

. These 15 

### two cell types are known to accumulate or sequester Pb in the nervous system. 16 

### Researchers have implicated dysfunctional astrocytes as playing an important role in the 17 

### chain of misregulated inflammation leading to neurodegenerative conditions ( Barbeito et 18 

### al., 2010 ; De Keyser et al., 2008 )

. 19



### Cell Death Pathways Earlier work has documented that Pb exposure can induce cell death or apoptosis in 20 

### various models including rat brain ( Tavakoli

- Nezhad et al., 2001 ) , retinal rod cells ( He et 21 

### al., 2003 ; He et al., 2000 ) , cerebellar neurons ( Oberto et al., 1996 ) , and PC12 cells 22 

### ( Sharifi and Mousavi, 2008 )

. These observations indicate that Pb

- induced cell apoptosis 23 

### may mediate its neurodegenerative effect s. A recent study reported that chronic (40 days) 24 

### Pb exposure induced hippocampal apoptosis in young (exposure starting at 2

- 4 weeks of 25 

### age) and adult (exposure starting at 12

- 14 weeks of age) male rats exposed to 500 ppm Pb 26 

### by drinking water (resulting in blood Pb levels of 98 μ g/dL); apoptosis was verified by 27 

### light and electron microscopy, and increased pro

- apoptotic Bax protein levels ( Sharifi et 28 

### al., 2010 )

. Another study followed the developmental profile of changes in various 29 

### apoptotic factors in specific brain regions of animals exposed to Pb

- acetate (0.2% dam 30 

### drinking water) during lactation. Male offspring blood Pb level at the end of lactation or 31 

### PND20 was 80 μ g/dL. The data showed that hippocampal mRNA for various apoptotic 32 

### factors including caspase

- 3, Bcl

- x and Brain

- derived neurotrophic factor (BDNF) was 33 

### significantly upregulated on PND12, PND15 and PND20. The cortex of these male pups 34 

### also showed upr egulation of Bcl

- x and BDNF on PND 15 and PND20 ( Chao et al., 35February 2012 5

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### 2007 )

. The cerebellum did not have elevated apoptotic mRNA levels in this model. Th is 1 

### study showed temporal and regional changes in activation of death protein message levels 2 

### in male offspring. 3 

### Pb exposure has also been shown to induce apoptosis during spinal cord development in 4 

### chicks exposed to 150 or 450 μ g in ovo Pb

- acetate exposure at embryonic day 3 or 5 and 5 

### visualized six days later; TUNEL positive cells were at significantly higher levels in Pb

6 

### exposed animals and were visualized in all layers of the developing spinal cords ( Müller 7 

### et al., In Press )

. Also, levels of glial fibrillary acidic protein (GFAP), a factor important in 8 

### neuronal migration and cellular differentiation during nervous system development, was 9 

### significantly attenuate d in spinal cords of Pb

- exposed chicks. Another recent study 10 

### examined Pb treatment animals (i.p. injections of 350 mg /kg, twice daily, blood Pb levels 11 

### at PND14 of 0.15 μg/dL in controls and 8.10 μg/dL in Pb

- treated animals) during 12 

### PND7

- 14, a known period o f sensitivity to NMDA antagonist

- dependent neuronal 13 

### apoptosis ( Ikonomidou et al., 1999 )

. Liu et al. ( 2010b ) examined apoptotic effects in 30 14 

### day

- old male rats that were exposed to Pb

- acetate 1x/d for 6 weeks via intragastric 15 

### infusion. Four dose groups: control, low (2), medium (20), and high (200 mg /kg BW) had 16 

### blood Pb levels of 1.0 to 7.5 μg/dL; 4.5 to 11 μg/dL; 9 to 42 μg/dL; and 48 to 73 μg/dL, 17 

### respectively. Liu et al. ( 2010b ) reported Pb

- induced apoptosis in the brain with 18 

### hippocampal XIAP (statistically significant at high dose only) and Smac (statistically 19 

### nonsignificant trend) downregulation and associated histopathology showing 20 

### hippocampal neuronal apoptosis (TUNEL positive staining, significant at all doses) at the 21 

### term ination of the 6 week treatment. In another study, Pb exposure (500 ppm Pb

- acetate 22 

### in drinking water for 8 weeks) of adult male rats induced regional

- specific changes in 23 

### br ain apoptotic proteins poly(ADP

- ribose ) polymerase, Bcl

- 2, caspase

- 3) with a greater 24 

### effect observed in the hippocampus and cerebellum and a lesser effect observed in the 25 

### brainstem and the frontal cortex ( Kiran Kumar et al., 2009 ) 26 

### Collectively, st udies have shown that Pb exposure induces neuronal apoptosis in animals 27 

### during various developmental windows: early postnatal and adulthood. The new data 28 

### continue to show that Pb exposure induced apoptosis in brains of animals. 29



### Lead
- Induced Neuronal Plaqu e Formation Epidemiologic studies have not provided compelling evidence that Pb exposure is 30 

### associated with Alzheimer's Disease in adults (Section 5.3.7.1 ); however, Pb exposure in 31 

### early life has been shown to promote Alzheimer’s Disease

- like pathologies in the brains 32 

### of aged adult animals. Alzheimer’s disease is characterized by amyloid

- beta peptide (A b) 33 

### accumulation, hyper

- phosphor y l ation of the tau protein, neuronal death and synaptic loss. 34 

### In the last decade, the developmental origins of adult health and disease (DoHAD) 35February 2012 5

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### paradigm and the similar Barker hypothesis have indicated that early life exposures can 1 

### result in aberrant adult outcomes. Bolin e t al. ( 2006 ) demonstrated the connection 2 

### between developmental exposure to Pb in the rat with early life programming and the 3 

### resulting inflammation

- associated DNA damage with neurodegenerative loss in the adult 4 

### brain. Wu and coll eagues ( 2008a ) had similar findings in a study using infantile exposure 5 

### to Pb in monkeys. The investigations reinforce the need to directly examine the long

- term 6 

### effects of developmental expo sure to toxicants rather than relying on adult exposure 7 

### alone to predict probable health risks from prenatal, neonatal or juvenile exposure 8 

### ( Dietert and Piepenbrink, 2006 )

. Mechanistically, some of these pathologies have been 9 

### associated with changes in the epigenome. 10 

### The fetal basis of amyloidogenesis has been examined extensively by the Zawia 11 

### laboratory in both rodents and nonhuman primates. Mechanistically, amyloid plaques 12 

### originate from the cleavage of the amyloid precursor protein (APP) to Ab, wh ich 13 

### comprises the plaque. In rodents exposed to Pb as neonates or as adults, neonatal Pb 14 

### exposure induced amyloidogenesis in the aged animal brains; adult exposure to Pb did 15 

### not contribute to plaque formation. Basha et al. ( 2005 ) exposed male rodents neonatally 16 

### via lactation to Pb (PND1

- PND20 exposure, dam drinking water Pb

- acetate 200 ppm , 17 

### resulting in pup PND20 blood Pb level of 46 μ g/dL and cortex 0.41 μ g/g wet weight of 18 

### tissue) a nd examined cortical APP gene expression over the lifetime. A bimodal response 19 

### was observed, with a significant increase in APP expression above that in control animals 20 

### first manifesting neonatally and second manifesting in old age (82 weeks of age) ( Basha 21 

### et al., 2005 )

. A concomitant bimodal response was observed in specificity protein 1 22 

### (Sp1), a transcription factor known to be related to APP expression. Ab, the amyloid 23 

### plaque consti tuent, was also significantly elevated in these aged animals developmentally 24 

### exposed to Pb. A subset of rodents exposed to Pb only as aged adults (18

- 20 weeks of 25 

### age) was unresponsive in APP or Sp1 expression or Ab production after Pb exposure, 26 

### indicating the developmental lifestage and not adult lifestage as the susceptible period for 27 

### Pb

- induced amyloidogenesis. The Zawia lab ( Wu et al., 2008a ) produced similar findings 28 

### for amyloid plaques in the brains of monkeys that were exposed to Pb as infants 29 

### (PND1

- PND400), i.e., significantly higher gene expression of APP, and Sp1 and 30 

### significantly higher protein expression of APP and Ab in aged female monkey cortex 31 

### tissue (23 year
- old Macaca fascicularis ) f rom a cohort of animals established in the 32 

### 1980s by Rice ( 1992 , 1990 )

. After weaning but with continued Pb exposure, the monkeys 33 

### had blood Pb levels of 19

- 26 μ g/dL. However, in old age when amyl oid plaques had 34 

### manifested, blood Pb levels and brain cortex Pb levels had returned to control or baseline 35 

### levels. Together, the rodent and nonhuman primate toxicological studies concur and show 36 

### that developmental Pb exposure induced elevations in neuronal plaque proteins in aged 37 

### animals. 38February 2012 5

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### Mechanistic understanding of Ab production and elimination after Pb exposure was 1 

### explored in human SH

- SY5Y neuroblastoma cells exposed to Pb concentrations of 0, 5, 2 

### 10, 20, and 50 μ M for 48 hours. These studies showed tha t Pb affected two separate 3 

### pathways to contribute to elevated Ab. Pb exposure induced both the overexpression of 4 

### APP and repression of neprilysin, a rate

- limiting enzyme involved in Ab metabolism or 5 

### removal ( Huang et al., 2011a )

. Further mechanistic understan ding of how Ab peptide 6 

### formation is affected by Pb exposure was examined by Behl et al. ( 2009 )

. The choroid 7 

### plexus is capable of removing beta

- amyloid peptides from the brain extracellular matrix; 8 

### however, Pb was show n to impair this function, possibly via the metalloendopeptidase, 9 

### insulin

- degrading enzyme (IDE), which metabolizes Ab ( Behl et al., 2009 )

. In another 10 

### study, the effect of Pb on transcription factors essential in the reg ulation of the 11 

### developing brain was explored. Pb exposure has been shown to perturb DNA binding of 12 

### transcription factors including SP1 at essential sites like zinc finger proteins. In Long

13 

### Evans hooded rat pups exposed to Pb during lactation, these Pb

- indu ced developmental 14 

### perturbations of SP1 DNA binding were found to be ameliorated by exogenous zinc 15 

### supplementation ( Basha et al., 2003 )

. 16 

### An additional study with developmental Pb exposure (gestational plus lactational, dam 17 

### drinking water solutions of 0.1%, 0.5% or 1%, blood Pb level 40, 80 a nd 1,00 μ g/dL) 18 

### showed that the hippocampus contained neurofibrillary changes as early as PND21. 19 

### These changes manifested with Tau hyper

- phosphorylation, and increased tau and beta 20 

### amyloid hippocampal protein levels in Pb

- exposed offspring ( Li et al., 2010b )

. 21 

### In summary, the multiple recent studies showed that developmental Pb exposure induced 22 

### si gnificant increases in neuronal plaque associated proteins, which is the pathology found 23 

### in humans with Alzheimer’s disease. Adult exposure to Pb did not generate this 24 

### neurofibrillary pathology, further demonstrating that early life Pb is a sensitive windo w 25 

### for Pb

- induced pathology including Ab

- peptide accumulation, activation of Ab

26 

### supporting transcription factors, as well as tau hyperphosphorylation. Epidemiologic 27 

### evidence for Pb

- associated Alzhemier's disease is weak, and although several study 28 

### design li mitations were noted (Section 5.3.7.1), the animal evidence indicates that 29 

### epidemiologic studies assessing concurrent bone or brain Pb levels or occupational Pb 30 

### exposure may not have examined the etiologically relevant exposure period. Notably, 31 

### animals wer e not behaviorally assessed for dementia. 32February 2012 5

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### 5.3.8 Modes of Action for Lead Nervous System Effects

5.3.8.1 Effects on Brain Physiology and Activity
A small body of available epidemiologic evidence with small sample sizes demonstrated 1 

### associations of Pb biomarkers with elec trophysiologic changes in the brains of young 2 

### adults as assessed by magnetic resonance imaging (MRI) or spectroscopy (MRS) ( Yuan 3 

### et al., 2006 ; Cecil et al., 2005 ; Meng et al., 2005 ; Trope et al., 2001 )

. By characterizing 4 

### underlying mechanisms by which Pb exposure may disrupt brain function, these studies 5 

### have provided biologically plausible evidence for the effects of Pb exposure on cognitive 6 

### and psychological and behavioral function observed in adults. 7 

### Findings such as lower levels of N

- acetylaspartate (NAA), creati ne (Cr), and choline are 8 

### linked to decreased neuronal density and alteration in myelin. Notably, Trope et al. 9 

### ( 2001 ) and Meng et al. ( 2005 ) reported that all subjects had normal MRIs with no 10 

### evidence of structural abnormalities. Thus, the clinical significance of the observed 11 

### physiological changes was unclear. Additionally, these studies compared subjects with 12 

### relatively high childhood blood levels (23

- 65 μ g/dL) to those with childhood blood Pb 13 

### levels <10 μ g/dL. Therefore, it is unclear whether physiological changes wo uld be 14 

### observed in association with lower blood Pb levels. Cecil et al. ( 2005 ) and Yuan et al. 15 

### ( 2006 ) conducted functional MRI in 42 adult (age s 20

- 23 years) participants from the 16 

### CLS cohort during a verb generation language task and found that childhood average 17 

### blood Pb level was associated with decreased activation in the left frontal gyrus and left 18 

### middle temporal gyrus, regions traditionally associated with semantic language function. 19 

### Although these findings were in adults, they were consistent with findings in the same 20 

### cohort of subjects that indicated associations of blood Pb level with other indices of 21 

### language skills in childhood. 22


### Since t he 2006 Pb AQCD, studies examining MRI data were largely limited to CLS 23 

### cohort participants as adults (ages 19

- 24), and recent results continue to support 24 

### associations of childhood blood Pb levels with physiological changes in the brain of 25 

### adults. These re cent studies expanded on previous studies by including larger sample 26 

### sizes, aiming to characterize important lifestages of Pb exposures, and evaluating 27 

### potential links between changes in brain activity and functional neurodevelopmental 28 

### deficits. Whereas pr evious CLS analyses focused on activity in specific regions of the 29 

### brain, Cecil et al. ( 2011 ) examine d brain metabolites in 159 subjects and found that 30 

### childhood average blood Pb levels were associated with lower levels of NAA and Cr in 31 

### the basal ganglia and lower levels of choline in white matter. These results were adjusted 32 

### for age and FSIQ. A recent an alysis of 31 men in the NAS cohort similarly reported an 33February 2012 5

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### association between biomarkers of cumulative, long

- term Pb exposure and changes in 1 

### brain metabolites in older adults. Weisskopf et al. ( 2007a ) found hig her tibia and patella 2 

### Pb levels to be associated with a higher myoinositol/Cr ratio, which may be indicative of 3 

### glial activation and is a signal reportedly found in the early stages of HIV

- related 4 

### dementia and Alzheimer’s disease. 5 

### Other studies in CLS youn g adults found that childhood average blood Pb levels were 6 

### associated with altered brain architecture. Brubaker et al. ( 2009 ) reported associations of 7 

### childhood average blood Pb levels with diffusion parameters that were indicative of less 8 

### organization of fibers throughout white matter. Results were adjusted for potential 9 

### confounding by variables such as maternal IQ, prenatal alcohol and tobacco exposure, 10 

### and adult marijuana use. In regions of the corona radiata, higher blood Pb levels were 11 

### associated with less myelination axonal integrity. In regions of the corpus callosum, 12 

### higher blood Pb levels were ass ociated with greater myelination and axonal integrity. The 13 

### differential impact on different neural elements may be related to the stage of myelination 14 

### development at various periods of exposure. 15 

### Another study of 157 CLS participants provided evidence of re gion

- specific reductions in 16 

### adult gray matter volume in association with childhood blood Pb levels ( Cecil et al., 17 

### 2008 )

. Th e most affected regions included frontal gray matter, specifically the anterior 18 

### cingulate cortex and the ventrolateral prefrontal cortex ( i.e., areas traditionally related to 19 

### executive functions, mood regulation, and decision

- making). Further, investigator s found 20 

### that fine motor factor scores positively correlated with gray matter volume in the 21 

### cerebellar hemispheres; adding blood Pb level as a variable to the model attenuated this 22 

### correlation. These findings suggested that MRI changes associated with blood Pb levels 23 

### may be indicative of decrements in cognitive function. The functional relevance of these 24 

### structural changes in the brain also is supported by observations from other studies that 25 

### link changes in brain architecture and activity with changes in co gnitive function 26 

### ( e.g., visuoconstruction, visual memory, eye

- hand coordination) ( Schwartz et al., 2007 ) 27 

### and behavior (impulsivity, aggression, violence) ( Yang et al., 2005 ; Raine et al., 2000 )

. 28



### In a subsequent comparison of blood Pb levels measured at various lifestages, Brubaker 29 

### et al. ( 2010 ) found that blood Pb levels at older ages were associated with greater losses 30 

### in gray matter volume than were childhood average or maximum blood Pb levels. Both 31 

### Cecil et al. ( 2008 ) and Brubaker et al. ( 2010 ) found that Pb

- associated reductions in gray 32 

### matter were more pronounced in CLS males than females. 33


### Studies of Pb

- workers also fo und associations of blood and bone Pb levels with changes 34 

### in brain structure and physiology, adding support for the effects of chronic Pb exposure. 35 

### Pb

- associated changes included white matter lesions, smaller brain volumes, less total 36 

### gray matter, and lowe r levels of brain metabolites such as NAA and Cr ( Hsieh et al., 37February 2012 5

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### 2009b ; Jiang et al., 2008 ; Bleecker et al., 2007a ; Stewa rt et al., 2006 )

. In a few of these 1 

### occupational groups, Pb

- associated brain changes were linked to poorer performance in 2 

### cognitive function tests ( Caffo et al., 2008 ; Bleecker et al., 2007a )

. 3 

### In summary, results in a few different pop ulations indicated associations of blood or bone 4 

### Pb levels with changes in brain structure and physiology as assessed by MRI or MRS. A 5 

### majority of evidence was derived from the CLS study. Several studies linked these 6 

### changes to functional changes in cognit ive performance or motor skills. While the 7 

### collective findings pointed to the effects of long

- term or past Pb exposures, it is important 8 

### to recognize that other lifestages of exposure, including recent adult, were not widely 9 

### examined. 10



### 5.3.8.2 Oxidative Stress

Pb h as been shown to induce oxidative stress in multiple animal models, and this 11 

### oxidative stress can contribute to DNA damage, which can be measured with the adduct 12 

### 8

- hydroxy

- 2’

- deoxyguanosine (8

- oxo
- dG). The contribution of reactive oxygen or 13 

### nitrogen specie s to these Pb induced changes was assayed by examining the ratio of 14 

### 8

- oxo
- dG to 2

- deoxyguanosine (2

- dG). 2

- dG is product of oxidative cleavage that can 15 

### generate 8

- oxo
- dG from a parent compound forming a DNA adduct during conditions of 16 

### nitrosative or oxidat ive stress. The 8

- oxo
- dG to 2

- dG ratio data from rodent male 17 

### offspring were similar to the amyloid data with significant biphasic elevations in 18 

### developmentally Pb

- exposed animals (0.2% Pb

- acetate in dam drinking water from 19 

### PND1

- 20) versus control, nonPb

- ex posed animals at early (PND5) and late life time 20 

### points (80 weeks of age) ( Bolin et al., 2006 )

. Activity of the base

- excision DNA repair 21 

### enzyme oxoguanine glycosylase or Ogg1 was unaffected by Pb exposure ( Bolin et al., 22 

### 2006 )

. Interestingly, similar findings were reported in a monkey study. The ratio of 23 

### 8

- oxo
- dG to 2

- dG in the brains of aged monkeys (23 years) after being exposed to Pb as 24 

### infants, was significantly elevated above that in controls ( Wu et al., 2008a )

. Several lines 25 

### of evidence indicate that oxidative stress is involved in neurodegenerative pathologies 26 

### including Alzheimer's disease; hydrogen peroxide

- induced oxidative stress has been 27 

### shown to induce intracellular accumulation of amyloid beta

- protein (Abeta) in human 28 

### neuroblastoma cells ( Misonou et al., 2000 )

. Similar to the amyloid findings in Pb

29 

### exposed animals the oxidative stress markers showed no significant changes above 30 

### baseline when animals were exposed to Pb only as aged adults ( Wu et al., 2008a ; Bolin et 31 

### al., 2006 )

. Thus, the data for biomarkers of oxidative stress concur with the 32 

### amyloidogenesis data with both dem onstrating kinetically similar biphasic significant 33 

### elevations in markers of oxidative stress and amyloidogenesis with early life Pb exposure 34 

### and an absence of effect with adult only exposure. 35February 2012 5

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### Because the brain has the highest energy demand and metabolism of any organ, energy 1 

### homeostasis is of utmost importance. Pb has been shown to inhibit various enzymes 2 

### involved in energy production or glucose metabolism including glyceraldehydes

- 3 3 

### phosphate dehydrogenase, hexokinase, pyruvate kinase, and succinate dehyd rogenase 4 

### ( Verma et al., 2005 ; Yun and Hoyer, 2000 ; Regunathan and Sundaresan, 1984 ; Sterling et 5 

### al., 1982 )

. Mitochondria p roduce ATP or energy through oxidative phosphorylation. 6 

### Aberrant mitochondrial function can decrease the energy pool and contribute to ROS 7 

### formation via electron transport chain disruption. ATP depletion can also affect synaptic 8 

### and extracellular neurotran smission. The mitochondrial Na/K ATPase is important in 9 

### maintaining the inner mitochondrial membrane potential (delta omega sub m) and the 10 

### functioning of the mitochondria. 11 

### To characterize the effect of Pb exposure on these mitochondrial parameters, brains from 12 

### the offspring of Pb

- exposed mice were collected at PND8 ( Baranowska

- Bosiacka et al., 13 

### 2011b )

. Cerebellar granular cells were harvested from PND 8 control and Pb

- exposed 14 

### animals (0.1% Pb

- acetate in dam drinking water, resulting in blood Pb levels of 4 μ g/dL 15 

### and cerebella Pb l evels of 7.2 μ g/g dry weight). These neuronal cells were cultured for 5 16 

### days in vitro, at which point various mitochondrial parameters were measured. With Pb 17 

### exposure, reactive oxygen species were significantly increased in both the cortical 18 

### granule cells and in the mitochondria. Intracellular ATP concentration and adenylate 19 

### energy charge values were significantly decreased in cells of Pb

- exposed mice versus 20 

### controls. Neuronal Na/K ATPase activity was significantly lower in cortical granule cells 21 

### from Pb

- ex posed mice versus cells from controls. Mitochondrial mass was unaffected 22 

### with Pb treatment, but mitochondrial membrane potential was significantly decreased 23 

### with Pb exposure. Pb

- exposed crayfish that were placed under hypoxic conditions 24 

### adapted to the situ ation by decreasing their metabolism ( Morris et al., 2005 ) and showed 25 

### mitochondrial changes consistent with those observed in granule cells ( Baranowska

26 

### Bosiacka et al., 2011b )

. These studies showed impaired mitochondrial function and 27 

### energy production in neuronal cells from mice with gestational and lactational Pb 28 

### exposure with concomitant increases in mitochondrial and cellular ROS production. 29


### The effects of co

- administration of flaxseed oil (FSO) with Pb

- acetate on oxidative stress 30 

### and neurotoxicity were examined in adult male Wistar albino rats ( Abdel Moneim et al. )

. 31 

### Animals were administered Pb

- acetate i.p. for 5 days (20mg/kg, resulting in blood Pb 32 

### level ~31 μ g/dL the day after the last Pb injection) or FSO (oral gavage 1000mg/kg body 33 

### weight for 5 days, 1 hour prior to Pb dosing, resulting in blood Pb level ~12 μ g/dL). 34 

### Administration of the polyunsaturated fatty acid FSO significantly attenuated the blood 35 

### Pb le vel of Pb

- exposed animals and control animals, indicating that FSO may alter Pb 36 

### toxicokinetics in animals. As would be expected with a lower blood Pb level, FSO+Pb 37 

### exposed animals had significant attenuations in Pb

- induced histological neuronal damage 38February 2012 5

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### and brain tissue DNA fragmentation. Pb exposure effects on indicators of oxidative stress 1 

### included decreased antioxidant GSH levels, elevated lipid peroxidation, and decreased 2 

### antioxidant enzyme activity in brain tissue. These Pb

- induced effects were attenuate d in 3 

### FSO+Pb exposed animals. These results indicated that FSO likely attenuates Pb

- related 4 

### neurotoxicological damage and oxidative stress via its action on altering Pb 5 

### toxicokinetics. 6 

### Green tea extract (GTE) was shown to be protective against Pb

- induced o xidative stress 7 

### in the brains of adult male rats (1.5% GTE +/

- 0.4% Pb

- acetate for 6 weeks) ( Hamed et 8 

### al., 2010 )

. GTE+Pb

- exposed animals had significantly less brain Pb than did Pb

- exposed 9 

### animals (1.2 versus 1.9 ppm ). Levels of pro

- oxidants or antioxidants including lipid 10 

### peroxides (LPO), nitric oxides (NO), total antioxidant capacity (TAC), glutathione 11 

### (GSH), glutathione

- S

- transferase (GST), and superoxide dismutase (SOD) were 12 

### measured in brain t issue homogenate 24 hours after the termination of Pb exposure. 13 

### Comparing the Pb

- exposed animals to controls, brain LPO was significantly elevated and 14 

### brain GSH, GST and NO were significantly decreased. GTE and Pb co

- exposure 15 

### significantly attenuated the P b

- related changes. This study reported a positive correlation 16 

### between whole blood Pb levels and brain tissue LPO levels and a negative correlation 17 

### between whole blood Pb and NO levels. The antioxidant quercetin (Que) rescued chronic 18 

### Pb exposure

- related (0. 2% Pb

- acetate in drinking water from birth to PND67, 30 mg /kg 19 

### BW Que for one week from PND60

- 67) impaired synaptic plasticity in adult male and 20 

### female Wistar rat dentate gyrus (DG) ( Hu et al., 2008a )

. Pb

- related impaired long

- term 21 

### potentiation, paired

- pulse reactions, and input/output functions were significantly 22 

### attenuated with Que treatment at PND67. Que treated animals had significantly less 23 

### hippocampal Pb than did the P b

- exposed animals and this decreased Pb brain burden 24 

### likely contributed to the attenuated response observed with Que exposure. 25 

### The vulnerability of the highly energetic brain tissue to stressors and cell death can be 26 

### exacerbated with an energy imbalance. B aranowska

- Bosiacka et al. ( 2011b ) s howed that 27 

### Pb exposure induced energy imbalances in immature rat brains exposed to Pb. Wistar rat 28 

### pups (PND15) of both sexes were injected daily for 2 weeks with Pb

- acetate (15mg/kg 29 

### BW, i.p., resulting in blood Pb levels of 3 and 30 μ g/dL, control and Pb

- e xposed, 30 

### respectively) and thereafter sacrificed for analysis of regional brain purines and 31 

### purinergic receptors. ATP and ADP were significantly decreased in various brain regions 32 

### with Pb exposure, with the cerebellum and hippocampus more strongly affected than the 33 

### forebrain cortex. Also, enhanced expression of the proinflammatory P2XR receptor was 34 

### observed in the glial fraction, indicating the astrocyte pool may be involved in the 35 

### pathological changes found in Pb

- exposed immature rat brains. 36February 2012 5

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### 5.3.8.3 Nitrosative Signaling and Nitrosative Stress
The nitric oxide system is increasingly being recognized as a signaling system in addition 1 

### to its more classical role as a marker of cellular stress. In studies of learning and memory 2 

### using the Morris water maze, hippocampa l changes in NO were noted in Pb

- exposed 3 

### animals after completion of the test. Pb exposure has been repeatedly shown to increase 4 

### the escape latency in Pb

- exposed animals (Section 5.3.2.2 ). Chetty ( 2001 ) initially 5 

### reported decreased hippocampal neuronal nitric oxide synthase (NOS) with p erinatal Pb 6 

### exposure. Namely, with repeated swim tests, control animals more quic kly found a 7 

### submerged platform ( i.e., escaped ) , than did Pb

- exposed animals. After either 4 or 8 

### 8 weeks of Pb exposure to weanling male rats (resulting in blood Pb level of 6.2 μ g/dL), 9 

### hippocampal NOS and NO were significantly decreased. Dietary supplementation with 10 

### taurine or glycine concomitant with 8 weeks of Pb exposure induced significant increases 11 

### in hippocampal NOS, whereas Pb plus dietary supplementation with vitamin C, 12 

### methionine, tyrosine, or vitamin B1 decreased hippocampal NOS. Supplementation also 13 

### changed Pb

- related effects on hippocampal NO. Pb

- induced NO increased with taurine 14 

### and decreased with vitamin C, tyrosine or glycine. Dietary supplementation with tyrosine, 15 

### methionine, or ascorbic acid after 4 weeks of Pb exposure in weanling males (4

- week 16 

### blood Pb level of 47.6 μ g/dL and 8

- week blood Pb level of 8.1 μ g/dL), induced 17 

### significant increases in NO. Zinc supplementation in this model had no effect on the NO 18 

### syste m. The investigators concluded various combinations of nutrients significantly 19 

### attenuate Pb

- related decreases in NO/NOS. Specifically, nutrients prevented (8 weeks Pb 20 

### plus concomitant exposure to methionine, zinc, ascorbic acid, and glycine) or restored 21 

### (4 weeks Pb exposure followed by 4 weeks nutrient exposure, taurine and thiamine) Pb

22 

### related decrements in NO/NOS concentrations ( Fan et al., 2009a )

. 23



### 5.3.8.4 Synaptic Changes
Previous toxicological studies point to an effect of developmental Pb exposure on 24 

### synapse development, which mechanistically may contribute to multiple Pb

- related 25 

### aberrant effects, including ch anges in long

- term potentiation (LTP) and facilitation. 26 

### Earlier work has shown that developmental Pb exposure results in altered density of 27 

### dendritic hippocampal spines ( Király and Jones, 1982 ; Petit and LeBoutillier, 1979 ) , 28 

### aberrant synapse e limination ( Lohmann and Bonhoeffer, 2008 ) , and abnormal long

- term 29 

### and short

- term plasticity ( MacDonald et al., 2006 )

. Newer research using the Drosophila 30 

### larval neuromuscular junction model has shown that compared with unexposed controls, 31 

### Pb

- exposed larvae had significant increases in intracellular calcium and significant delays 32 

### in calcium decays back to baseline levels at the pre

- synaptic neuronal bouton (as 33 

### stimulated with multiple action potentials, also called AP trains). Pb

- exposed larvae had 34February 2012 5

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### reduced activ ity of the plasma membrane calcium ATPase, which is responsible for 1 

### extravasations of calcium from the synaptic terminal ( He et al., 2009 )

. Intracellular 2 

### calcium in Pb exposed

- larvae was no different from that in controls under resting 3 

### conditions or in neurons with stimulation by a single action potential. Pb media 4 

### concentrations in these experiments were 100 or 250 μ M with the low

- dose body burden 5 

### (100 μ M) of Pb calculated to be 13

- 48 μ M per larvae. Facilitation of a neuronal terminal 6 

### is defined as the increase d capability to transmit an impulse down a nerve due to prior 7 

### excitation of the nerve. After stimulation of the axon, facilitation of the excitatory post

8 

### synaptic potential, which is dependent on residual terminal calcium, was significantly 9 

### elevated in Pb

- exposed larvae versus control ( He et al., 2009 )

. The data from this synapse 10 

### study demonstrate that developmental Pb exposure affected the plasma membrane 11 

### calcium ATPase, induced changes in the intracellular calcium levels during impulse 12 

### activation, and produced changes in facilitation of the neuronal ne tworks of Drosophil a. 13 

### Thus, the neuromuscular junction is a potential site of Pb interaction. 14 

### A study by Li et al. ( 2009c ) focused on inflammatory endpoints and synaptic changes 15 

### after gestational plus lactational dam drinking water Pb exposure (solutions of 0.1%, 16 

### 0.5% or 1%, resulting in offspring blood Pb levels of 40, 80 and 100 μ g/dL, respectively 17 

### at PND 21). Hippocampal TNF

- α was significantly elevated with Pb exposure and 18 

### proteins that comprise the SNARE complex were all changed with Pb exposure. The 19 

### SNARE complex of synaptic proteins includes SNAP

- 25, VAMP

- 2 and Syntaxin 1a and 20 

### is essential in exocytotic neurotransmitter release at the synapse ( Li et al., 2009c )

. Thus, 21 

### Li et al. ( 2009c ) found significant differences in hippocampal synaptic protein 22 

### composition and increased pro

- inflammatory cytokine levels in the brains of Pb

- exposed 23 

### offspring. 24 

### Neurotransmission is an energy

- dependent process as indicated by the presence of 25 

### calcium

- dependent ATP releases at the synaptic cleft. At the synapse, ATP is 26 

### metabolized by ecto nucleotidases. In heme synthesis, Pb is known to substitute for the 27 

### cation zinc in another nucleotidase, pyrimidine 5′

- nucleotidase, and thus, the nucleotidase 28 

### is used as a biomarker of Pb exposure. Acute exposure (96 hours) of male and female 29 

### zebrafish to Pb

- acetate (2 μ g/dL) in their water ind uced significant decreases in ATP 30 

### hydrolysis in brain tissue. This dose is deemed to be environmentally relevant. With 31 

### chronic exposure (30 days), Pb

- acetate promoted the inhibition of ATP, ADP and AMP 32 

### hydrolysis; these findings were consistent with findin gs in rodents ( Baranowska

33 

### Bosiacka et al., 2011b )

. The authors hypothesized that at 30 days, this Pb

- induced change 34 

### in nucleotide hydrolysis was likely due to post

- translational modification because 35 

### expression of enzymes responsible for the hydrolysis, NTPDase1 and 5′

- nucleotidase, 36 

### were unchanged ( Senger et al., 2006 )

. Thus, Pb has been shown to affect nucleotidase 37February 2012 5

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### activity in the central nervous system of zebrafish, possibly contributing to aberrant 1 

### neurotransmission. 2 

### Another enzyme important in synaptic transmission at cholinergic junctions in the CNS 3 

### and at neuromuscular junctions peripherally is acetylcholinesterase (AChE). After 24 4 

### hours of exposure to Pb

- acetate (2 μ g/dL water), AChE activity was significantly 5 

### inh ibited in zebrafish brain tissue. In Pb

- exposed fish, AChE activity returned to baseline 6 

### by 96 hours and maintained baseline activity after chronic exposure of 30 days. Thus, Pb 7 

### was shown also to affect synaptic homeostasis of AChE in the brains of zebrafi sh 8 

### ( Richetti et al., 2010 )

. 9 

### Pb has been shown to act as an antagonist of the NMDA receptor (NMDAR). The 10 

### NMDAR is essential for proper presynaptic neuronal activity and function. Primary 11 

### cult ures of mouse hippocampal cells were exposed to Pb (10 or 100 μ M solutions in 12 

### media) during the period of synaptogenesis ( Neal et al., 2010a )

. This exposure induced 13 

### the loss of two pro teins necessary for presynaptic vesicular release, synaptophysin (Syn) 14 

### and synaptobrevin (Syb), without affecting a similar protein synaptotagmin (Syt). This 15 

### deficit was found in both GABAergic and glutamatergic neurons. Pb also induced an 16 

### increase in numb er of presynaptic contact sites. But, these sites may be nonfunctional as 17 

### they lack the protein receptor complexes necessary for proper vesicular exocytosis. 18 

### Another factor involved in growth and signaling of presynaptic neurons is BDNF, which 19 

### is synthesiz ed and released by postsynaptic neurons. BDNF is regulated by the NMDAR 20 

### and acts in a retrograde fashion, participating in presynaptic maturation. In hippocampal 21 

### cells, both pro

- BDNF and BDNF release were significantly attenuated with Pb exposure 22 

### ( Neal et al., 2010a )

. Further, exogenous BDNF administration rescued the 23 

### aforementioned Pb

- related presynaptic effects. Thus, this cell culture model showed that 24 

### Pb

- related presynaptic aberrati ons are controlled by NMDAR

- dependent BDNF effects 25 

### on synaptic transmission. 26 

### Animals exposed to Pb postnatally (Wistar Albino rats, drinking water 300 mg /L 27 

### Pb

- acetate , resulting in blood Pb levels of 17 μ g/dL at 6 weeks of age), from birth 28 

### through lactatio n and through 12 weeks of age showed decreased learning ability and 29 

### decreased hippocampal glutamate at 6, 8, 10 and 12 weeks of age ( Niu et al., 2009 ) as 30 

### well as significant decrements in the hippocampal glutamate synthesis

- related enzymes 31 

### aspartate aminotransferase and alanine aminotransferase. 32



### 5.3.8.5 Blood Brain Barrier
Two barrier systems exist in the body to separate the brain or the central nervous system 33 

### from the blood. These two barriers are the blood brain barrier (BBB) and the blood 34February 2012 5

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### cerebrospinal fluid barrier (BCB). The BBB, formed by tight junctions at endothelia l 1 

### capillaries forming the zonulae occludens (occludins, claudins, and cytoplasmic proteins), 2 

### separates the brain from the blood and its oncotic and osmotic forces, allowing for 3 

### selective transport of materials across this barrier. 4 

### Pb exposure during variou s developmental windows has been shown to increase the 5 

### permeability of the BBB ( Dyatlov et al., 1998 ; Struzynska et al., 1997b ; Moorhouse et 6 

### al., 1988 ; Sundstrom et al., 1985 )

. Possibly due the underdevelopment of the BBB early 7 

### in life, prenatal and perinatal Pb exposure has been found to result in hi gher brain Pb 8 

### accumulation than have similar exposures later in life ( Moorhouse et al., 1988 )

. Studies 9 

### reviewed in earlier Pb AQCDs have shown that the chemical form of Pb and its 10 

### capability to interact with proteins and other blood components affects its capability to 11 

### penetrate the BBB ( U.S. EPA, 2006b )

. Pb also has been shown to compromise the 12 

### function of the BCB and decrease the CSF level of tra nsthyretin , a th y r oid binding 13 

### protein made in the choroid plexus. The choroid plexus and cerebral endothelial cells that 14 

### form the BBB and BCS tight junctions have been shown to accumulate Pb more than 15 

### other cell types and regions of the CNS do. Hypothyroid status can contribute to impaired 16 

### learning and IQ deficits ( Lazarus, 2005 )

. 17 

### Recent research with weanling rats exposed to Pb

- acetate via drinking water showed 18 

### leaky cerebral vasculature, an indication of a compromised BBB, as detected 19 

### histologicall y with lanth an um nitrate staining of the brain parenchyma. Cerebral 20 

### vasculature leakiness was ameliorated or resembled controls after iron supplementation. 21 

### These weanlings also had significant Pb

- induced decreases in the BBB tight junction 22 

### protein occludin in the hippocampus, brain cortex, and cerebellum that were rescued to 23 

### control levels with iron supplementation ( Wang et al., 2007b )

. These data demonstrate 24 

### that Pb induced a leaky BBB in weanling rats with asso ciated decreases in the junctional 25 

### protein occludin; dietary supplementation with iron ameliorated these Pb

- induced 26 

### impairments of the BBB in male rats. This loss of integrity at the junctional protein level 27 

### was affirmed with additional experiments using t he rat brain vascular endothelial cell line 28 

### RBE4, in which 10 μ M Pb

- acetate exposure for 2, 4, 8, 16 and 24 hours resulted in 29 

### decreases in junctional proteins occludin and claudin 5 as well as scaffold proteins ZO1 30 

### and ZO2 ( Balbuena et al., 2011 )

. Because expression of these junctional and scaffold 31 

### proteins did not show decrements, it was determined that these protein decrements were 32 

### due to post

- translational modifications. 33 

### A study examined the effects of Pb on transendothelial electrical resistance (TEER), a 34 

### marker of BBBB integrity, in an in vitro co

- culture system employing endothelial cells 35 

### (RBE4 or bovine brain microvascular endothelial cells) and astrocytes (primary Sprague

36 

### Dawley neona tal pup astrocytes, in utero day 21) as the barrier between Pb containing 37February 2012 5

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### exposure media and neurons. After 14 hours of exposure to Pb, TEER was significantly 1 

### impaired in a concentration

- dependent manner, with the lowest significant effects found 2 

### with 1 μ M Pb

- acetate exposure. Thus, Pb exposure was found to contribute to leakiness 3 

### of the BBB by decreasing the resistance across the junction. 4 

### Adult male rats exposed to Pb

- acetate in drinking water for 4 or 12 weeks (50 or 5 

### 500 ppm , resulting in blood Pb level s of 12 and 55 μ g/dL, respectively) were assessed by 6 

### diffusion weighted imaging for changes in apparent diffusion coefficent (ADC), a 7 

### measure of tissue water diffusivity that changes under pathological conditions like 8 

### cerebral edema. After 4 weeks of expos ure to 500 ppm Pb, the water ADC was 9 

### significantly increased in the hippocampus, mesencephalic reticular formation, and 10 

### cerebellum but unaffected in other brain areas. After 12 weeks of 500 ppm Pb exposure, 11 

### ADC was significantly increased in the corpus cal losum and caudate putamen. Exposure 12 

### to 50 ppm Pb for 12 weeks increased the ADC values in the cerebellum and 13 

### mesencephalic reticular formation. The brain areas with elevated ADC also showed 14 

### increased BBB permeability as measured with evans blue albumin com plex. These data 15 

### show that adult animals with chronic Pb exposure are susceptible to regional edema and 16 

### regional increased permeability of the BBB, even with low blood Pb levels (12 μ g/dL) 17 

### ( López

- Larrubia and Cauli, 2011 )

. 18



### 5.3.8.6 Cell Adhesion Molecules
Classic cell adhesion molecules including NCAM and the cadherins are junctional or cell 19 

### surface proteins that are critical for cell recognition and adhesion. Cell adhesion 20 

### molecules, particularly the cadherins, are calcium

- dependent and thus interaction from 21 

### competing cations like Pb can potentially contribute to nervous system barrie r function 22 

### disruption, tissue development dysregulation, immune dysfunction, and affect learning 23 

### and memory ( Prozialeck et al., 2002 )

. 24



### 5.3.8.7 G lial Effects
Astroglia and oligodendroglia are supporting cells in the nervous system that maintain the 25 

### extracellular space in the brain and provide support and nutrition to neurons via nutrient 26 

### transport, structural support to neurons, and myelination. Gl ial cells provide immune 27



### surv e il l ance in the brain or contribute to inflammation

- mediated pathologies. In the 28 

### central nervous system, Pb treatment of Wistar rats 15 mg /kg of Pb

- acetate , i.p.) during 29 

### early postnatal maturation was observed to produce chroni c glial activation with 30 

### coexisting features of inflammation and neurodegeneration ( Struzynska et al., 2007 )

. 31February 2012 5

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### Among the cytokines detected in the brains of these Pb

- treated rats were IL

- 1β, TNF

- α 1 

### and IL

- 6. Glial cells have been shown to serve as Pb sinks in the developing and mature 2 

### brain ( Tiffany

- Castiglioni et al., 1989 ) by sequestering Pb. This glial sequestration of Pb 3 

### has been shown to decrease brain glutamine concentrations at doses of 0.25 ± 1.0 μ M 4 

### Pb

- acetate via Pb

- rel ate d reduction in glutamine synthe tase activity in the astroglia; 5 

### astroglia take up glutamate after its release and convert it to glutamine. Pb has been 6 

### shown to induce hypomyelination and demyelination ( Coria et al., 1984 ) mediated 7 

### through the oligodendrocytes with younger animals being more susceptible to the effects 8 

### of Pb ( Tiffany

- Castiglioni et al., 1989 )

. Pb accumulation in young glial cells may 9 

### contribute to a lifelong exposure of neurons to Pb as Pb is released from the sink over 10 

### time. T hus, Pb accumulation in glial cells can contribute to continual damage of 11 

### surrounding neurons ( Holtzman et al., 1987 )

. 12



### Glial transmitters

To determine the contribution of the gliotransmitter serine to Pb

- mediated changes in 13 

### long

- term potentiation (LTP), Sun et al. ( 2007 ) exposed pups to Pb

- acetate in utero, 14 

### lactationally, through PND28 via drinking water and collected hippocampal sections. 15 

### CA1 section LTPs were examined using in vitro patch clamp monitoring. Chronic Pb 16 

### exposure impaired the magnitude of hippocampal LTPs, but the magnitude of LTDs was 17 

### restored with supplementation with D

- serine ( Sun et al., 2007 ) , which is known to be 18 

### regulated by the NMDAR ( Bear and Malenka, 1994 )

. The use of 7

- chlorokynurenic acid, 19 

### an antagonist of the glycine binding site of the N MDAR, which also is the binding site of 20 

### D

- serine, effectively abolished the rescue to LTP by D

- serine. NMDAR

- independent 21 

### LTP hippocampal neurotransmission, which was examined in slices of Pb

- exposed 22 

### mossy
- CA3 synapses, was not rescued by exogenous D

- serine supplementation. These 23 

### data indicate that glial transmission is affected with Pb exposure and that the NMDAR 24 

### may also be involved in this aberrant glial transmission. 25



### 5.3.8.8 Neurotransmitters
Pb has been shown to compete with calcium for common binding sites an d second 26 

### messenger activation. When Pb activates a calcium

- dependent system in the nervous 27 

### system, it can contribute to aberrant neurotransmitter regulation and release because this 28 

### system intimately relies on calcium signaling for its homeostasis. Pb also has been shown 29 

### to interfere with other physiological divalent cations. Pb

- related alterations in 30 

### neurotransmission are discussed in further detail below. 31February 2012 5

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### Monoamine Neurotransmitters and Stress Combined exposures of maternal stress and Pb exposure can syne rgistically enhance 1 

### behavioral and neurotoxic outcomes in offspring of exposed animals and can sometimes 2 

### potentiate an effect that would otherwise be sub

- threshold. Virgolini et al. ( 2008 a ) found 3 

### that CNS effects of developmental Pb exposure (50 or 150 ppm via drinking water, 4 

### 2 months prior to mating through lactation, resulting in blood Pb levels of 11 μ g/dL and 5 

### 35 μ g/dL, respectively) were enhanced with combined maternal and offspring stress. 6 

### Offspring neurotransmitter concentrations were significantly affected with Pb exposure, 7 

### but the most interesting findings were those of potentiated effects, i.e., effects that were 8 

### not observed with Pb exposure alone or stress alone. These potenti ated effects were only 9 

### observed when Pb was combined with stress (maternal [MS] and/or offspring stress 10 

### [OS]). Potentiation of serotonin (5HT) levels in females was significant in the frontal 11 

### cortex in females and in the nucleus accumbens (NAC) in the male offspring (50 and 12 

### 150 ppm Pb drinking water exposure) ( Cory

- Slechta et al., 2009 )

. Reg ional 5HT levels 13 

### were unaffected in offspring with Pb exposure alone. The concentration 14 

### of 5

- Hydroxyindoleacetic acid (5HIAA), the main metabolite of 5HT, was significantly 15 

### increased with Pb exposure alone in the striatum of male offspring with 150 ppb Pb 16 

### exposure alone; with the remaining Pb

- stress exposure combinations, Pb plus stress 17 

### potentiated striatal and frontal cortex 5HIAA in males. Potentiated 5HIAA levels in 18 

### females were significant in the NAC at both Pb doses; stress alone also significantly 19 

### inc reased 5HIAA levels in females with no Pb exposure. Pb

- induced changes in brain 20 

### neurochemistry with or without concomitant stress exposure are complex with 21 

### differences varying by brain region, neurotransmitter type and sex of the animal. 22



### Monoamine Neurotransmitters and Auditory Function The monoamine neurotransmitters include DA, 5HT, and norepinephrine (NE). Earlier 23 

### work has shown that perinatal Pb exposure of rats induced increased tyrosine 24 

### hydroxylase, increased DA and increased cerebral cortex c atecholamine 25 

### neurotransmission ( Devi et al., 2005 ; Leret et al., 2002 ; Bielarczyk et al., 1996 )

. Earlier 26 

### publications detailing important time windows of exposure, duration of exposure, and 27 

### dose of Pb indicated varying effects on monoamine transmitters. In more recent work, 28 

### these neurotransmitters, among others, have been implicated in Pb effects on auditory 29 

### function in the brainstem in various integration centers there including the lateral superior 30 

### olive (LSO), and the superior olivary complex (SOC). Among various functions, the SOC 31 

### is vital for sound detection in noisy settings. Low

- level Pb exposu re has been associated 32 

### with altered processing of auditory temporal signals in animal studies ( Lurie et al., 2006 ; 33 

### Finkelstein et al., 1998 )

. Blood Pb levels for control, very low Pb (VLPb) and low Pb 34 

### (LPb) exposure groups were 1.4, 8.0, and 42.2 μ g/dL, respectively. Developmental Pb 35February 2012 5

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### exposure from the formation of breeding pairs to PND21, which is at th e end of auditory 1 

### development in the mouse, led to significant decreases in immunostaining of LSO and 2 

### SOC brainstem sections for monoamine vesicular transporter VMAT2, a nd for 5HT and 3 

### dopamine beta

- hy d r oxylase (DbH), a marker for NE. Statistically signific ant alterations 4 

### in VMAT2 and DbH were found with both VLPb and LPb exposure; however, 5 

### decrements in 5HT were statistically significant only with VLPb. Immunostaining for TH 6 

### and transporters including VGLUT1, VGAT, VAChAT indicated that they were 7 

### unaffected by developmental Pb exposure. These data provide evidence that specific 8 

### regions of the brainstem involved in auditory integration are affected by developmental 9 

### Pb exposure via effects on the monoamine neurotransmitter system ( Fortune and Lurie, 10 

### 2009 )

. The Pb

- induced e ffects on the auditory portion of the brainstem at the level of the 11 

### monoamine system provide possible mechanistic understanding of the animal data 12 

### showing Pb

- induced impaired auditory processing. 13



### Dopamine The 2006 Pb AQCD detailed evidence for low

- dose Pb

- related decreased dopaminergic 14 

### cell activity in the substantia nigra and ventral segmental areas. Earlier studies with 15 

### moderate

- to high

- dose postnatal or adult Pb exposure have reported changes in DA 16 

### metabolism, as indicated by changes in DA and DOPAC, a DA metabolite. Given these 17 

### findings, a recent study measured DA and DOPAC in various brain regions of year
- old 18 

### male rodents to examine if GLE affected DA metabolism. Low

- and high

- dose GLE in 19 

### male rodents induced significant elevations in DOPAC concentrati on and the DOPAC to 20 

### DA ratio in the forebrain. In the forebrain, DA was significantly decreased in low

- dose 21 

### GLE males and significantly elevated in high

- dose GLE males compared to controls. In 22 

### the striatum, DOPAC was significantly elevated with both low

- a nd high

- dose GLE 23 

### males, but DA concentration was only significantly elevated in high

- dose GLE males. 24 

### The striatum ratio of DOPAC to DA was not significantly different from that in controls. 25 

### These new data expand upon the monoamine literature base in which indicates low 26 

### concentration perinatal Pb exposure of rats was found to induce increased sensitivity of 27 

### the dopamine receptors (D2 and D3) ( Gedeon et al., 2001 ; Cory

- Slechta et al., 1992 ) , 28 

### produce higher DA levels ( Devi et al., 2005 ; Leret et al., 2002 ) , and enhance 29 

### catecholamine neurotransmission in the cerebral cortex, cerebellum, and hippocampus 30 

### ( Devi et al., 2005 )

. 31 

### The interaction of DA and the nitric oxide system in the striatum was studied after 32 

### prenatal Pb exposure ( Nowak et al., 2008 )

. Blood Pb levels were not reported in this 33 

### study, but similarly treated Wistar rat pups had blood Pb levels at parturition in range of 34 

### 50

- 100 μ g/dL ( Grant et al., 1980 )

. 7

- nitroinidazole (7

- NI), a selective inhibitor of nNOS, 35 

### enhanced amphetamine

- evoked DA release in the rat striatum ( Nowak et al., 2008 )

. 36February 2012 5

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### Prenatal Pb exposure attenuated the facilitatory effect of 7

- NI on DA release in the 1 

### striatum. This interaction is ROS

- independent; using spin trap measurements, there were 2 

### no significant concentration changes in hydroxyl radical with Pb exposure ( Nowak et al., 3 

### 2008 )

. Thus, the neuronal NO system appears to be involved in specific aspects of Pb

4 

### related dopaminergic changes. 5



### Dopamine and Vision

In va rious experimental animal models, the loss of retinal DA or zinc is associated with 6 

### abnormal rod

- mediated scotopic ERGs. These effects may explain observations of Pb

7 

### associated retinal effects ( Rothenberg et al., 2002a ; Lilienthal et al., 1994 ; Lilienthal et 8 

### al., 1988 ; Alexander and Fishman, 1984 )

. In the human and animal toxicological 9 

### literature, prenatal maternal blood Pb level (humans), gestational + early postnatal Pb 10 

### exposure (rodents, low and moderate dose Pb only), or gestational continuing to lifetime 11 

### ( non
- human primate) Pb exposure has been associated with increased amplitude 12 

### (supernormality) of ERGs ( Rothenberg et al., 2002a ; Lilienthal et al., 1994 ; Lilienthal et 13 

### al., 1988 )

. In the animal toxicological literature, subnormality of the ERGs has been 14 

### observed with postnatal Pb exposure ( Fox et al., 2008 ; Otto and Fox, 1993 ; Fox et al., 15 

### 1991 ; Fox and Farber, 1988 ) and high

- dose developmental (prenatal + early postnatal, 16 

### rodent) Pb exposure ( Fox et al., 2008 )

. Producing results consistent with observations in 17 

### humans ( Rothenberg et al., 2002a ) , Fox et al. ( 2008 ) showed that low

- (LPb) and 18 

### moderate

- level (MPb) gestational+early postnatal Pb exposure in the rat, a period 19 

### equivalent to the human gestational retinal development period, produced supernormal 20 

### retinal ERGs. In children, sup ernormal ERGs were associated with prenatal Pb maternal 21 

### blood Pb levels >10.5 μ g /dL ( Rothenberg et al., 2002a )

. The animal data provide 22 

### mechanistic infor mation that may begin to explain these supernormal ERGs that are seen 23 

### in children and rodents, i.e., significant increases in retinal neurogenesis and significant 24 

### decreases in retinal DA use and dopamine turnover (DOPAC:DA ratio). High

- dose 25 

### developmental (rodent, gestational+early postnatal to PND10) Pb exposure (HPb) 26 

### produced significant subnormal retinal ERGs. Subnormal ERGs also were found in 27 

### occupationally

- exposed adults ( Otto and Fox, 1993 ; Guguchkova, 1972 )

. Female rats 28 

### were exposed to Pb

- acetate in drinking water from 2 weeks prior to mating throughout 29 

### gestation and lactation until PND 10, a period of developmental exposure that is 30 

### equivalent to gestational exposure in humans. Peak blood Pb levels in the offspring at 31 

### PND 1

- 10 were 12, 24, and 46 μ g/dL in the LPb, MPb, and HPb groups, respectively. LPb 32 

### and MPb gestational exposure induced increased cellularity or retinal thickness in the 33 

### outer nuclear layer, inner nuclear layer and total retina ( Leasure et al., 2008 )

. In 34 

### summary, the retina is affected by low

- dose Pb exposure and gestational Pb exposure, as 35 

### indicated by concentration

- dependent decreases in DA use and turnover. Inverted U

- 36February 2012 5

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### shaped Pb concentration

- response relationship were reported for retinal endpoints 1 

### including ERG and retinal thickness. 2



### NMDA

NMDA receptors (NMDAR) have been shown to contribute to synaptic plasticity, and Pb 3 

### exposure at different developmental stages has been shown to contribute to aberrations in 4 

### LTP or long term depression (LTD) in the hippocampus via reduced NMDA current, 5 

### among o ther mechanisms ( Liu et al., 2004 )

. The 2006 Pb AQCD indicated that Pb 6 

### induced decreases in stimulated glutamate release that affected LTP. Further, the 7 

### relationships between Pb exposure and decreased magnitude and increased threshold o f 8 

### the LTP in the hippocampus were found to be biphasic or nonlinear. NMDAR subtypes 9 

### have been shown to be significantly decreased with developmental Pb exposure ( Guilarte 10 

### and McGlothan, 1998 )

. Recent work examining dietary supplement use, found that Pb

11 

### related decreases in the gene expression and protein level of NMDAR subunit NR1 in 12 

### weanling male rats were rescued with methioninecholine co

- exposure ( Fan et al., 2010 )

. 13 

### Fan et al. ( 2010 ) found that Pb

- related suppression of the NMDAR subunits NR2A and 14 

### NR2B was not rescued with methioninecholin e treatment. Other recent mechanistic 15 

### studies found that pretreatment of primary fetal brain neuronal rat cultures with glutamic 16 

### acid, a NMDAR agonist, reversed Pb

- induced reductions in NMDAR subunits ( Xu and 17 

### Rajanna, 2006 ) whereas pretreatment with the NMDA antagonist MK

- 801 exacerbated 18 

### Pb

- induced NMDAR deficits ( Xu and Rajanna, 2006 )

. 19 

### Studies continue to show that Pb exposure affects neurogenesis or proliferation of new 20 

### cells in the hippocampus. Earlier work by Schneider et al. ( 2005 ) showed that postnatal 21 

### Pb exposure (30

- 35 days starting at PND25, 1 , 500 ppm Pb

- acetate in chow, resu lting in 22 

### blood Pb level of 20 μ g/dL) of male Lewis rats induced significant decrements in BrdU 23 

### incorporation (proliferation) at PND50

- 55. Recent publications affirm this original 24 

### finding with different sex of animals, dosing and exposure time windows. Post natal Pb 25 

### exposure to Wistar rat pups (0.2% Pb

- acetate from PND1

- 30, resulting in blood Pb levels 26 

### of 34 and 6.5 μ g/dL at PND21 and PND80, respectively) induced a statistically 27 

### significant decrement in the number of new cells (BrdU positive cells) in the dentate 28 

### gyrus at PND80 ( Fox et al., 2010 ) ( Figure 5

- 18 ). In another study, developmental/lifetime 29 

### Pb exposure (1 , 500 ppm Pb

- acetate in chow from 10 days before mating to termination of 30 

### experiment at PND50, resulting in blood Pb levels of 0.8 μ g/dL in controls and 26 μ g/dL 31 

### in Pb

- exposed animals) to female Long

- Evans rats induced significant decrements in 32 

### hippocampal granule cell neurogene sis or proliferation of new cells in adult rats ( Verina 33 

### et al., 2007 ) , outcomes that affect LTP, spatial learning, neuronal outgrowth, and possibly 34 

### mood disorders such as schizophrenia. NMDAR mediates the integration of new neurons 35 

### into existing neuronal pathways in the adult hip pocampal DG, which is important to 36February 2012 5

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### learning and memory. Lifetime Pb exposure (dam Pb

- acetate chow exposure 10 days 1 

### prior to mating through pregnancy to PND50 or PND78) induced significant decrements 2 

### in hippocampal granule cell neurogenesis or proliferation of new cells in adult rats. Also, 3 

### Pb

- exposed animals had significant decreases in brain volume in the stratum oriens (SO) 4 

### region of the hippocampus, specifically significant decreases in the mossy fiber terminals 5 

### of the SO. Pb

- exposed animals also showed a significant decrease in the length

- density of 6 

### immature or newly

- formed neuron in the outer portion of the DG. These findings show 7 

### that exposure to environmentally

- relevant doses of Pb induced significant aberrations in 8 

### adult hippocampus granule cell neur ogenesis and morphology, providing mechanistic 9 

### explanations for Pb

- induced neuronal aberrations. Guilarte et al. ( 2003 ) demonstrated that 10 

### Pb exposure of rats from an enriched environment was associated with reduced learning 11 

### impairment, increased expression of hippocampal NMDA receptor subunit 1, and 12 

### increased induction of brain derived neurotrophic factor mRNA ( Guilarte et al., 2003 )

. 13



### Glutamate Receptor Glutamate receptors including the ionotropic NMDAR and the metabotropic glutamate 14 

### receptors (mGluR) are known targets o f Pb toxicity with recent findings showing a role 15 

### for mGluR5 in learning and memory. In vitro (GD18 fetal rat cultures, 100 μ M, 1 μ M, 16 

### 0.01 μ M PbCl 2 

### in culture media) and in vivo studies (gestational and lactational 17 

### Pb

- acetate exposure; control, 0.05, 0.2, or 0.5% in dam drinking water, with respective 18 

### weanling blood Pb levels of 3, 18, 57, 186 μ g/dL) showed that Pb exposure induced 19 

### mGluR5 mRNA and protein decrements in a concentration

- dependent manner ( Xu et al., 20 

### 2009c )

. . The Pb

- related attenuation of mGlu5 expression may contribute to the effect of 21 

### Pb on LTP and LTD. 22



### 5.3.8.9 Neurite Outgrowth
The 2006 Pb AQCD reported that Pb decreased neurite outgrowth at 20μg/dL and not ed 23 

### that Pb interfered with neurite outgrowth via protein kinase mediated pathways 24 

### (MAPK/ERK); earlier work had documented decreased primary DA neuron outgrowth 25 

### with 0.001 μ M Pb exposure ( Lidsky and Schneider, 2004 )

. Recent studies have shown 26 

### that exposure of dams to low

- dose Pb (resulting in blood Pb level of 4 μ g/dL) 27 

### significantly decreased pup hippocampal neurite outgrowth (pup blood Pb level: 28 

### 12 μ g/dL) and reduced the expression of hippocampal polysialylated neural cell adhesion 29 

### molecule (PSA

- NCAM), NCAM, and sialy transferase ( Hu et al., 2008b )

. PSA

- NCAM is 30 

### transiently expressed in newly formed neurons during the period of neurite outgrowth 31 

### from emb r yrogenesis until the early postnatal period and is down

- regulated in the adults 32February 2012 5

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### except in areas known to exhibit synaptic plasticity ( Seki and Arai, 1993 )

. NCAM is 1 

### important for memory formation, plasticity and synapse formation, and early

- life Pb 2 

### exposure was found to affects its expression in laboratory rats. 3



### 5.3.8.10 Epigenetics
Many investigators are beginning to show that environmental chemical exposures are 4 

### associated with epigenetic changes. Air pollution exposure is being linked increasingly 5 

### with epigenetic changes ( Baccarelli and Bollati, 2009 ; Pavanello et al., 2009 ; Tarantini et 6 

### al., 2009 ; Bollati et al., 2007 )

. Epigenetic changes involve changes in DNA expression 7 

### without changes in the DNA sequence, and these changes may be heritable. Epigenetic 8 

### changes are mediated by histone modification, DNA methylation, miRNA changes, or 9 

### pathways that affect these proce sses. Differential epigenetic modification has the 10 

### potential to contribute to disease by silencing or activating genes in an aberrant manner. 11 

### Monozygotic twins are often used to study epigenetic changes, and a recent study 12 

### identified differential methylati on of a specific locus in twins discordant for 13 

### schizophrenia ( Dempster et al., 2011 ) ; Pb was not examined in this study. 14 

### DNA methyltransferases catalyze the transfer of a methyl group to DNA and are 15 

### important in epigenetics ( i.e., silencing of genes like tumor suppressors) and imprinting. 16 

### DNA methyltransferase activity was significantly decreased in cortical neurons from Pb

17 

### exposed monkeys (aged animals, blood Pb) and mouse brains (fetal cells exposed to Pb in 18 

### culture, 0.1 μ M Pb) ( Wu et al., 2008b )

. Changes in DNA methyltransferases (Dnm t1, 19 

### Dnmt3a) were noted in control primate brains as they aged and these changes were 20 

### further exacerbated by Pb exposure ( Bihaqi et al., 2011 )

. Another enzyme involved in 21 

### DNA methylation, methyl CpG binding protein 2 MECP2, showed a similar trend as the 22 

### Dnmts. Profiles of the histone modifying gene H34mc2 increases with age in control 23 

### animals. This age

- related increase is significantly attenuated in Pb

- exposed animals. The 24 

### cerebral cortex tissue used in this experiment was obtained from female primates who 25 

### had received 1.5 mg /kg • day Pb

- acetate via diet from birth until 400 days of age 26 

### (resulting in blood Pb levels 19

- 26 μ g/dL at age 400 days) ( Rice, 1990 )

. 27 

### Methyltransferases catalyze biological methylation reactions and are dependent on the 28 

### cofactor S

- adenosyl methionine (SAM) for this transfer to acceptor molecules. SAM 29 

### exposure after gestational and lactational Pb exposure (dam Pb

- acetate exposure to 30 

### 1 , 500 ppm Pb

- acetate followed by 20

- 22 days of daily 20 mg /kg BW SAM exposure) 31 

### improved hippocampal LTP and Morris water maze performance at PND 44

- 54. Thus, 32 

### the impaired cognition and synaptic plasticity induced by developmental Pb exposure 33 

### were attenuated wit h SAM treatment ( Cao et al., 2008 )

. 34February 2012 5

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### 5.3.8.11 Cholesterol and Lipid Homeostasis
Various pathological con ditions are associated with elevated plasma free fatty acids or 1 

### elevated cholesterol. Adult male rats exposed to Pb

- acetate (200, 300, or 400 ppm ) in 2 

### their drinking water for 12 weeks had increased cholesterogenesis and phospholipidosis 3 

### in brain tissue ( Ademuyiwa et al., 2009 )

. Pb

- induced changes in brain cholesterol showed 4 

### an inverse U concentration

- response relationship, with the largest increase in brain 5 

### cholesterol observed with 200 ppm Pb followed by 300 ppm Pb. Ani mals exposed to 6 

### 400 ppm Pb did not have significant changes in brain cholesterol. Mechanistically, Pb 7 

### exposure has been shown to depress the activity of cholesterol

- 7

- a
- hydroxylase, an 8 

### enzyme involved in bile acid biosynthesis ( K ojima et al., 2005 ) ; bile acids are the route 9 

### by which cholesterol is eliminated from the body. Pb exposure produced significant 10 

### increases in brain triglycerides with an 83% increase at 300 ppm and a 108% increase at 11 

### 400 ppm . At 200 ppm , Pb exposure ind uced a statistically nonsignificant decrease in 12 

### brain triglycerides. Pb exposure across all three dose groups induced significantly 13 

### increased brain phospholipids. Interestingly, plasma free fatty acids were significantly 14 

### elevated in a concentration

- depende nt manner; plasma triglycerides and cholesterol were 15 

### unaffected by Pb exposure. The molar ratio of brain cholesterol to phospholipids, an 16 

### indicator of membrane fluidity ( Abe et al., 2007 ) , was significantly increased at 200 and 17 

### 300 ppm Pb exposure indicating increased membrane fluidity. Brain Pb in all dose groups 18 

### was be low the limit of detection (0.1 ppm ). Blood Pb levels at 0, 200, 300, and 400 ppm 19 

### were 7, 41, 61, and 39 μ g/dL, respectively. In summary, based on limited examination, 20 

### Pb exposure significantly increased brain cholesterol, triglycerides, and phospholipids as 21 

### well as significantly increased plasma free fatty acids. These effects were sometimes 22 

### more prominent at lower doses of Pb. Future characterization of molecular and cellular 23 

### pathways affected by Pb exposure may bring insight to these Pb

- related changes in 24 

### phospholipidosis and cholesterogenesis. 25



### 5.3.9 Lifestage of Lead Exposure and Neurodevelopmental Deficits

Env ironmental exposures during critical lifestages can affect key physiological systems 26 

### that orchestrate plasticity ( Feinberg, 2007 )

. Exposure to environmental toxins during 27 

### prenatal and/or early postnatal devel opment may alter the normal course of 28 

### morphogenesis and maturation that occurs in utero and early in life, resulting in changes 29 

### that affect structure or function of the central nervous system via altered neuronal growth 30 

### and/or synaptogenesis/pruning struct ure ( Rice and Baro ne, 2000 ; Landrigan et al., 1999 )

. 31 

### Synaptic pruning , which is active throughout early childhood (ages 1

- 4 years), may 32 

### underlie the elevated risk of young children to environmental exposures. MRI studies 33 

### have provided understanding of brain development in normally developing children and 34February 2012 5

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### adults, ages 3

- 30 y ears ( Giedd et al., 2009 ; Lenroot and Giedd, 2006 )

. Total cerebral 1 

### volume was found to peak at age 10.5 and 14.5 years in females and males, respectively. 2 

### The volume of the cerebellum was found to peak 2 years after the cerebral volume peaks. 3 

### Lateral ventricular volume showed the most inter

- individual variation and was found to 4 

### increase throughout childhood and adolescence. White matter volume generally increased 5 

### throughout childhood and adolescence. Gray matter volume and associated structures 6 

### show inverted U

- shaped developmental trajectories with peak volumes found in late 7 

### childhood or early adolescence. Femal es generally had gray matter volume peaks 1 to 8 

### 3 years earlier than did males. Thus, the anatomical development of the brain during 9 

### childhood and adolescence is found to be a dynamic process with variation by age, brain 10 

### region, and sex. Observations that b rain development is active throughout childhood and 11 

### in adolescence indicates that Pb exposure may affect neurodevelopment throughout this 12 

### period. 13


### The elevated risk of Pb

- associated neurodevelopmental deficits in children is well 14 

### supported by findings in an imals that prenatal and postweaning Pb exposure alters brain 15 

### development via changes in synaptic architecture (Section 5.3.8.4 ) and neuronal 16 

### outgrowth (Section 5.3.8.9 ) and leads to impairments in memory and l earning 17 

### (Section 5.3.2.2 ) and emotional and depressive changes postnatally (Section 5.3.3.5 ). 18 

### Unlike other organ systems, the unidirectional nature of CNS development limits the 19 

### capability of the developing brain to compensate for cell loss, and environmentally

20 

### induced cell death can result in a permanent reduction in cell numbers ( Bayer, 1989 )

. 21 

### Hence, when normal development is alte red, the early effects may persist into adult life 22 

### even in the absence of current exposure, magnifying the public health impact. Supporting 23 

### evidence is provided by a few available new toxicological studies that find that Pb 24 

### exposure during neonatal develop ment but not in adulthood leads to neurodegenerative 25 

### amyloid plaque formation in the brains of aged ro dents and monkeys (Section 5.3.7.2 ). 26 

### With repe ated assessments of children prenatally to later childhood and early adulthood, 27 

### the prospective cohort studies have aimed to distinguish among neurodevelopmental 28 

### effects associated with blood Pb levels measured at different periods of development. In 29 

### the c ollective body of evidence, cognitive function decrements in children have been 30 

### associated with prenatal, early childhood, childhood average, and concurrent blood Pb 31 

### levels, without clear indication that the risk of neurodevelopmental decrements is greates t 32 

### for blood Pb levels measured at a particular lifestage. In these studies, the identification 33 

### of developmental periods when children are at increased risk of Pb

- associated 34 

### neurodevelopmental decrements has been complicated by the high degree of correlatio n 35 

### in the blood Pb levels of children over time and the confounding of age and peak blood 36 

### Pb levels ( Lanphear et al., 2005 ; Dietrich et al., 1993b ; Needleman et al., 1990 )

. 37February 2012 5

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– Do Not Cite or Quote 

### As described in detail in the 2006 Pb AQCD, several studies with varying lengths of 1 

### follow

- up demonstrated asso ciations of prenatal blood Pb levels (maternal and umbilical 2 

### cord) with neurodevelopmental deficits throughout childhood and into early adulthood 3 

### ( U.S. EPA, 2006b )

. Pb exposure during the prenatal lifestage may be associated with 4 

### increased risk of neurodevelopmental effects not only because of the nervous system 5 

### developmental processes that are active as described above but also because of factors 6 

### that result in elevated Pb exposures. Substantial fetal Pb exposure may occur from 7



### m obilization of maternal skeletal Pb stores that may be related to past Pb exposures 8 

### ( Gulson et al., 2003 ; Hu and H ernandez

- Avila, 2002 )

. Pb can cross the placenta to affec t 9 

### the developing fetal nervous system ( Rabinowitz, 1988 )

. Maternal and cord blood Pb 10 

### levels generally have been show n to be highly correlated, indicating that blood Pb levels 11 

### in a newborn infant reflects that of the mother ( Schell et al., 2003 )

. 12 

### Prenatal blood Pb levels (maternal and cord) were consistently associated with cognitive 13 

### function decrements and behavioral problems assessed betwe en infancy and age 3 years 14 

### ( Table 5

- 4 and Table 5

- 13 ). Among studies that had blood Pb measurements at both 15 

### lifestages, some found stronger associations for prenatal blood Pb levels ( Hu et al., 2006 ; 16 

### Bellinger et al., 1984 ) , and other found stronger associations for concurrent blood Pb 17 

### levels ( Wasserman et al., 1998 ; Wasserman et al., 1992 )

. Studies that found associations 18 

### with concurrent blood Pb levels also tend ed to find associations with prenatal cord or 19 

### maternal blood Pb levels. Thus, both postnatal child and maternal Pb exposures may 20 

### contribute to lower cognitive function in young children. Several studies found that 21 

### prenatal or neonatal blood Pb levels were associated with neurodevelopmental 22 

### decrements assessed neonatally (within 30 days) or early in infancy (within 3 months), 23 

### which indicated that relatively short durations of Pb exposure were associated with 24 

### neurodevelopmental decrements ( Shen e t al., 1998 ; Rothenberg et al., 1989 ; Dietrich et 25 

### al., 1987a ; Ernhart et al., 1986 )

. In the studies of neurodevelopmental effects in infancy, 26 

### prenatal blood Pb level may be serving as a surrogate of postnatal blood Pb levels as both 27 

### are expected to be highly correlated. 28February 2012 5

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– Do Not Cite or Quote 

### Table 5
- 13 Association s of cognitive function and behavioral outcomes with blood Pb levels measured at different lifestages a

Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) b Assessments in children up to age 3 years Rothenberg et al. ( 1989 ) 42 children follow ed prenatally to child age 30 days Mexico City, Mexico Maternal week 36 gestation mean (SD): 15.0 (6.4) Maternal at birth mean (SD): 15.5 (5.7) Regression model adjusted for smoking, single mother, problems in pregnancy, alcohol use in previous month, use of spinal block, gravidity, income Self

- quieting ability (regulation of state) at age 30 days Assessed using Newborn Brazelton Assessment System Prenatal:

- 0.091 (

- 0.18, 0)

Dietrich et al. ( 1986 ) 305 children followed prenatally to age 6 mo. in Cincinnati, OH Prenatal (maternal) mean (SD): 8.0 (3.8) Concurrent mean (SD): 5.9 (3.4) Log linear regression model adjusted for birth weight, gestation, sex Bayley MDI assessed at age 6 mo Prenatal:
- 0.6 (
- 1.1,
- 0.09) Concurrent:

- 0.23 (

- 0.58, 0.12)

Bellinger et al. ( 1987 ) 249 children followed from birth (1979

- 1981) to age 36 mo Boston area, MA Prenatal (cord blood) mean (SD): 6.6 (3.2) Regression and longitudinal analyses adjusted for the mother’s age, race, IQ, education, number of years of cigarette smoking, number of alcoholic drinks per week in the third trimester, mean family social class over the period of the study , quality of the care

- giving environment, infant’s sex, birth weight, gestational age, birth order Bayley MDI assessed at age 6, 12, 18, 24 mo Prenatal:
- 4.8 (
- 7.3,
- 2.3) , blood Pb levels ≥ 15 μ g/dL vs. blood Pb levels <3 c

Hu et al. ( 2006 ) 146 children born 1997

- 1999 follo wed prenatally to age 24 mo Mexico City, Mexico Prenatal (maternal 1st trimester) mean (range): 7.1 (1.5

- 43.6) Early childhood (12 mo) mean (SD): 5.2 (3.4) Concurrent mean (SD): 4.8 (3.7) Log linear regression model adjusted for concurrent blood Pb, sex, maternal age, current weight, height

- for

- age Z score, maternal IQ Bayley MDI assessed at age 24 mo Prenatal 1st trimester:
- 4.1 (
- 8.1,
- 0.17) Prenatal (avg):

- 3.5 (

- 7.7, 0.63) 12 month:

- 2.4 (

- 6.2, 1.49) Concurrent:

- 1.0 (

- 3.9, 1.9)

Gomaa et al. ( 2002 ) 197 children followed prenatally to age 24 mo Mexico City, Mexico Prenatal (cord blood) mean (SD): 6.7 (3.4) Log linear regression model adjusted for maternal IQ, maternal age, sex, parental education, marital status, breastfeeding duration, child hospitalization status Bayley MDI assessed at age 24 mo Pren atal:
- 2.1 (
- 3.9,
- 0.39)

Wasserman et al. ( 1992 ) 392 children followed prenatally to age 24 mo Kosovo, Yugoslavia (K. Mitrovica, Pristina) Prenatal (cord blood) mean (SD): 14.4 (10.4) Concurrent means: K. Mitrovica: 35.4, Pristina: 8.5 Log linear regression model adjusted for sex, birth order, birth weight, ethnic group, HOME score, years of maternal education, maternal age, maternal intelligence Bayley MDI assessed at age 24 mo Concurrent:
- 4.1 (
- 6.2,
- 2.0) Prenatal:

- 3.2 (

- 7.2, 0.86)

Jedrychowski et al. ( 2009b ) 444 children born 2001

- 2004 followed prenatally to age 36 mo Krakow, Poland Prenatal (cord blood) geometric mean (range): 1.29 (0.44

- 5) Linear regression model adjusted for maternal education, birth order, prenatal ETS, sex Bayley MDI assessed at age 36 mo Prenatal:
- 2.9 (
- 5.0,
- 0.75)

Cognitive function assessments at school age
Wasserman et al. ( 1994 ) 332 children followed prenatally to age 3

- 4 yr Kosovo, Yugoslavia (K. Mitrovica, Pristina) Prenatal (cord blood) mean (SD): 14.4 (10.4) Concurrent means: K. Mitrovica: 39.9 Pristina: 9.6 Log linear regression model adjusted for HOME score, maternal age, maternal intelligence, maternal education, language, birth weight, sex McCarthy GCI assessed at age 3

- 4 yr Concurrent:
- 4.1 (
- 6.2,
- 2.0) Prenatal:
- 3.2 (
- 5.1,
- 1.2)February 2012 5

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– Do Not Cite or Quote Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) b
Dietrich et al. ( 1992 ) 259 followed from birth (1979

- 1984) to age 5 yr Cincinnati, OH Prenatal (cord blood) mean (SD): 8.2 (3.8) Neonatal (10 days) mean (SD): 4.8 (3.3) Concurrent mean (SD): 11.9 (6.4) Linear regression model adjusted for fetal distress and growth, perinatal complications, postnatal indices of health and nutritional status, sociodemograph ic characteristics, HOME score Total FWS assessed using KABC at age 5 yr Neonatal:
- 0.38, p ≤ 0.01 d Prenatal:
- 0.25, p ≤ 0.01 d Concurrent:
- 0.19, p ≤ 0.01 d Lifetime avg:
- 0.16, p ≤ 0.01 d

Bellinger et al. ( 1991 ) 170 children followed from birth (1979

- 1981) to age 57 mo Boston area, MA Early childhood (24 mo) mean (SD): 6.8 (6.3) Early childhood tooth mean (SD): 2.8 (1.7) μg/g Concurrent mean (SD): 6.4 (4.1) Log linear regression model adjusted for family social class, maternal IQ, marital status, preschool attendance, HOME score, out of home care, number of residence changes, recent medication use, number of adults in household, sex, race, birth weight, birth order McCarthy GCI assessed at age 57 mo Early childhood blood:

- 3.0 (
- 5.7,
- 0.2) Early childhood tooth:

- 2.5 (

- 10.2, 5.2) Concurrent blood:

- 2.3 (

- 6.0, 1.4)

Dietrich et al. ( 1993b ) 245 children followed from birth (1979

- 1984) to age 6 yr Cincinnati, OH Prenatal (cord blood) mean (SD): 8.4 (3.8) Neonatal (10 days) mean (SD): 4.8 (3.1) Concurrent mean (SD): 10.1 (5.6) Linear regression model adjusted for obstetric complications, perinatal status, sex, social class, maternal intelligence, quality of rearing environment, earlier measures of neurobehavioral status Bruininks

- Oseretsky Test of Motor Proficiency assessed at age 6 yr Concurrent:

- 0.18 (
- 0.26,
- 0.10) Neonatal:

- 0.15 (

- 0.33, 0.03) Lifetime avg:
- 0.11 (
- 0.19,
- 0.03) Pr enatal:

- 0.04 (

- 0.20, 0.12)

Dietrich et al. ( 1993b ) 253 children followed from birth (1979

- 1985) to age 6.5 yr Cincinnati, OH Prenatal (cord blood) mean (SD): 8.3 (3.7) Neonatal (10 days) mean (SD): 5.0 (3.4) Concurrent mean (SD): 11.8 (6.3) Linear regression model adjusted for fetal distress and growth, perinatal complications, prenatal maternal substance abuse, postnatal indices of health and nutritional status, sociodemographic characteristics, maternal IQ, HOME score FSIQ assessed us ing WISC

- R at age 6.5 yr Concurrent:

- 0.33 (
- 0.60,
- 0.06) Lifetime avg:

- 0.13 (

- 0.35, 0.09) Neonatal:

- 0.03 (

- 0.42, 0.36) Prenatal: 0.15 (

- 0.26, 0.56)

Baghurst et al. ( 1992 ) 494 children followed from birth (1979

- 1982) to age 11

- 13 yr Port Pirie, Australia Prenatal mean of second quartile: 7.4 Early childhood (2 yr) mean of second quartile: 16.6 Lifetime avg mean of second quartile: 15.7 Log linear regression model adjusted for sex, birth weight, birth order, feeding method, breastfeeding du ration, parental education, maternal age, parental smoking, SES, quality of home environment, maternal IQ, parents living together FSIQ assessed using WISC

- R at age 7

- 8 yr Early childhood:

- 2.0 (
- 3.8,
- 0.21) Lifetime avg:

- 1.6 (

- 3.7, 0.52) Prenatal: 0.26 (

- 0.67, 1.5)

Lanphear et al. ( 2005 ) 1 , 333 children pooled from Boston, Cincinnati, Cleveland, Mexico City, Port Pirie, Rochester, and Yugoslavia cohorts Median (5th

- 95th) Early childhood: 12.7 (4.0

- 34.5) Peak: 18.0 (6.2

- 47.0) Lifetime avg: 12.4 (4.1

- 34.8) Concurrent: 9.7 (3.5

- 33.2) Log linear regression model adjusted for HOME score, birth weight, maternal IQ, maternal education FSIQ measured at ages 4.8

- 10 yr Concurrent:
- 0.23 (
- 0.32,
- 0.14) Peak:
- 0.20 (
- 0.29,
- 0.11) Lifetime avg:

- 0.15 (
- 0.22,
- 0.09) Early childhood:

- 0.14 (
- 0.23,
- 0.06)

Pocock et al. ( 1994 ) Meta

- analysis of 5 prospective (over 1 , 100 children and 14 cross

sectional studies (3 , 499 children) Early childhood (2 yr) range in means: 6.8

- 21.2 Meta

- analysis of combining effect estimates from individual studies FSIQ assessed using various tests at ages 5

- 10 yr Early childhood:

- 2.7 (
- 4.1,
- 1.2) Postnatal mean:

- 1.3 (2.9, 0.37) Around birth: 0.26 (

- 1.5, 2.0) Schnaas et al. ( 2006 ) 150 children followed from prenatally (1987

- 1992) to age 6

- 10 yr Mexico City, Mexico Prenatal (maternal 28

- 36 wk gestation): 7.8 (2.5, 24.6) Early childhood a vg (1

- 5 yr) mean (range): 9.8 (2.8

- 36.4) Later childhood avg (6

- 10 yr): 6.2 (2.2

- 18.6) Log linear mixed effects model adjusted for blood Pb levels at other lifestages, sex, birth weight, SES, maternal IQ, First FSIQ measurement FSIQ assessed using WISC

- R at ages 6

- 10 yr Prenatal:
- 3.9 (
- 6.5,
- 1.4) Early childhood avg: 0.10 (

- 3.9, 4.1) Later childhood avg: 0.17 (

- 1.4, 1.8)February 2012 5

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– Do Not Cite or Quote Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) b Bellinger et al. ( 1992 ) 148 children followed from birth (1979

- 1981) to age 15

- 17 yr Boston area, MA Prenatal: NR Early childhood (2 yr) mean (SD): 6.5 (4.9) Concurrent mean (SD): 2.9 (2.4) Linear regression model adjusted for HOME score (age 10 and 5), child stress, race, maternal IQ, SES, sex, birth order, marital status FSIQ assessed using WISC

- R at age 10 yr Early childhood:

- 0.58 (
- 0.99,
- 0.17) Prenatal:

- 0.48 (

- 5.7, 4.7), blood Pb >10 μg/dL vs. <3 μg/dL c Concurrent:

- 0.46 (

- 1.5, 0.56) Ris et al. ( 2004 ) 195 children in followed from birth (1979

- 1985) to age 15

- 17 yr Cincinnati, OH NR Linear regression model adjusted for maternal IQ, sex, and average total HOME score Learning/IQ composite assessed using WIS C

- III indices at age 15

- 17 yr Prenatal:

- 0.08 (

- 0.18, 0.03) E arly childhood, 6.5 yr:

- 0.08 (

- 0.17, 0.003) Early childhood avg:

- 0.03 (

- 0.18, 0.03) Behavioral assessments
Wasserman et al. ( 1998 ) 379 children followed prenatally to age 3 yr Kosovo, Yugoslavia (K. Mitrovica, Pristina) Prenatal mean (SD): 16.1 (2.6) Concurrent mean (SD): 25.8 (19.1) Hierarchical log linear regression analyses adjusted for town, sex, ethnicity, maternal education, HOME score Anxiety/depression assessed using Child Behavior Checklist at 3 yr Concurrent: 1.45 (0.04, 2.86) Prenatal: 1.16 (0.02, 2.3)

Leviton et al. ( 1993 ) 1 , 923 children followed from birth (1979

- 1980) to age 8 yr Boston area, MA Prenatal blood 2nd quartile: 4.8

- 6.3 Early childhood (tooth) second quartile: 2.0

- 2.9 μg/g Log linear regression model adjusted for single

- parent family, gestational age <37 wk, mother not a college graduate, self

- identification as black, o nly child, daycare during first 3 yr Hyperactivity assessed using Boston Teacher Questionnaire at age 8 yr Prenatal, girls: 0.26 (

- 0.69, 1.13 Early childhood, girls: 0.10 (

- 0.92, 1.1)

Bellinger et al. ( 1994a ) 1,782 children fo llowed from birth (1979

- 1980) to age 8 yr Boston area, MA Prenatal (cord blood) mean (SD): 6.8 (3.1) Early childhood (tooth) mean (SD): 3.4 (2.4) ppm Log linear regression analyses adjusted for prepregnant weight, race, delivery by cesarean section, marital status, paternal and maternal education, sex, birth weight, maternal smoking, prenatal care beginning after the first trimester, recipient of public assistance, number of children in family, child currently on medication Problem behaviors (t

- scores ) assessed using Teacher Report Form of the Child Behavior Profile at age 8 yr Early childhood: 1.8 (0.49, 3.1) P renatal:

- 0.31 (

- 1.7, 1.07)

Ris et al. ( 2004 ) 195 children in followed from birth (1979

- 1985) to age 15

- 17 yr Cincinnati, OH NR Linear regression model adjusted for maternal IQ, sex, and average total HOME score Inattention composite assessed using Continuous Performance Test at 15

- 17 yr Prenatal: 0.16 (0.04, 0.27) Early childhood, 6.5 yr: 0.12 (0.02, 0.22) Early childhood avg: 0.11 (0.03, 0.19)

Dietrich et al. ( 2001 ) 195 children follow ed from birth (born1979

- 1985) to age 15

- 17 yr Cincinnati, OH NR Linear regression model adjusted for birth weight, HOME score, SES, parental IQ Parental report of delinquent behavior at 15

- 17 yr Prenatal: 0.19 (0.02, 0.37) Early childhood, 6.5 yr: 0.13 (

- 0.01, 0.27) Ea rly childhood avg: 0.09 (

- 0.02, 0.20) MDI = Mental Developmental Index, ETS = Environmental tobacco smoke, HOME = Home Observation for Measurement of the Environme nt, GCI = General Cognitive Index, FWS = Filtered Word Test, KABC = Kaufman Assessment Battery of Children, FSIQ = Full

- scale IQ, WISC = Wechsler Intelligence Scale for Children, NR = Not reported
a Studies are organized by age of neurodevelopmental assessment. Effect estimates are presented in order of increasing magnitud e, with s tatistically significant results in bold. b Effect estimates are standardized to a 1 μg/dL increase in blood Pb level in analyses of blood Pb as a continuous variable.
c Effect estimate represent comparisons between children in different categories of blood Pb level, with children in the lower blood Pb category serving as the reference group. d Suffic i ent data were not provide d in order to calculate 95% CI.


### Prenatal and neonatal (10 days after birth) blood Pb levels also were associated with 1 

### cognitive function and behavioral outcomes in children examined at school

- age (ages 2 

### 4

- 17 years) ( Table 5

- 13 ). Concurrent blood Pb levels generally were estimated to have 3 

### similar or larger magnitudes of effect ( Wasserman et al., 1998 ; Wasserman et al., 1994 ; 4February 2012 5

- 195 Draft

– Do Not Cite or Quote 

### Dietrich et al., 1993a ; Dietrich et al., 1993b ; Bellinger et al., 1992 ; Dietrich et al., 1992 )

. 1 

### Studies conducted in the Cincinnati cohort examined diverse neurodevelopmental effects 2 

### and found that prenatal and neonatal blood Pb levels were associa ted with impairments in 3 

### behavior and auditory processing ( Ris et al., 2004 ; Dietrich et al., 2001 ; Dietrich et al., 4 

### 1992 ) but not cognitive function or motor function in children at age 6 years or in 5 

### adolescence ( Bhattacharya et al., 2006 ; Bhattacharya et al., 1995 ; Dietrich et al., 1993a ; 6 

### Dietrich et al., 1993b )

. These findings su ggest that the effects of prenatal Pb exposure 7 

### may vary among different populations and specific endpoints. 8 

### Early childhood blood Pb levels were associated with diverse neurodevelopmental effects 9 

### assessed later in childhood and into early adulthood in both recent ( Chandramouli et al., 10 

### 2009 ; Min et al., 2009 ; Miranda et al., 2009 ) and previous studies that did not compare 11 

### various lifestages of Pb exposure ( Yuan et al., 2006 ; Cecil et al., 2005 ; Tong et al., 2000 )

. 12 

### In a meta

- analysis of results from five cohort studies ( Pocock et al., 1994 ) , a larger 13 

### decrease in FSIQ was estimated for an increase in peak (around age 2 years) blood Pb 14 

### level than for blood Pb level measured around birth or after age 2 years ( Table 5

- 13 ). 15 

### This lag effect may be the result of a toxicological process in which some period of time 16 

### is required for past Pb exposure to affect CNS function. Alternatively, associations with 17 

### early childhood or peak blood Pb levels may reflect the greater reliability of 18 

### neurodevelopmental assessments at later ages when the processes mo dalities of children 19 

### are more highly differentiated. Early testing may lead to false negative results and fail to 20 

### identify a child who is at risk for later neurodevelopmental dysfunction. Further, due to 21 

### the correlation of blood Pb levels over time, it is difficult to assess whether early 22 

### childhood or peak blood Pb levels were serving as surrogates of concurrent or cumulative 23 

### blood Pb levels. 24 

### As presented in Table 5

- 13 , several studies of school

- aged children estimated larger 25 

### blood Pb

- associated decreases in neurodevelopmental function for concurrent or lifetime 26 

### average (range: 5 to 13 years) blood Pb levels than for blood Pb levels at other lifestages. 27 

### These findings were substantiated in the analysis pooling data from seven prospective 28 

### studies, in which concurrent, peak, average lifetime (5

- to 10

- year average), and age 29 

### 2 year blood Pb levels all were negatively associated with IQ measured between ages 5 30 

### and 10 years, with the larger magnitude of decrease associated with increases in 31 

### concurrent and peak blood Pb levels ( Lanphear et al., 2005 )

. Childhood average blood Pb 32 

### levels ( Lanphear et al., 2005 ; Dietrich et al., 1993a ; Dietrich et al., 1993b ) and deciduous 33 

### tooth Pb levels ( Bellinger et al., 1994a ) have been associated with neurodevelopmental 34 

### effects. While the ages of children varied among these studies as did the years over which 35 

### blood Pb levels were averaged, the results nonetheless indicate cumulative Pb exposure 36 

### over multiple years may contribute to neurodevelopmental effects in children. 37 

### Associations with concurrent blood Pb level also were demonstrated consistently in 38February 2012 5

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– Do Not Cite or Quote 

### studies without comparisons to blood Pb l e vels during other lifestages ( Figures and 1 

### Tables in Sections 5.3.2.1 and 5.3.3.1 ). 2 

### Some studies have aimed to improve the characterization of important lifestages of Pb 3 

### exposure by examining children in whom blood Pb levels are weakly correlated over time 4 

### ( i.e., children whose blood Pb level ran king changed over time) ( Hornung et al., 2009 ; 5 

### Schnaas et al., 2006 ; Chen et al., 2005 ; Tong et al., 1998 ; Bellinger et al., 1990 )

. 6 

### Collectively, the results did not conclusively demonstrate stronger findings for concurrent 7 

### blood Pb level. However, with the exception of Schnaas et al. ( 2006 ) , the results did not 8 

### preclude an association with concurrent blood Pb level. Schnaas et al. ( 2006 ) followed 9 

### children in Mexico City prenatally through age 10 years and found prenatal blood Pb 10 

### levels (maternal blood at 28

- 36 weeks of pregnancy) to be weakly correlated with 11 

### repe ated measures of blood Pb between ages 1 and 10 years (Pearson r ≤ 0.25). In a 12 

### mixed effects model that included prenatal and multiple postnatal blood Pb measures, 13 

### only prenatal blood Pb level was associated with a decrement in FSIQ ( Table 5

- 13 ). 14 

### Analysis of variance inflation factors indicated a lack of collinearity among the serial 15 

### blood Pb measures. 16 

### Tong et al. ( 1998 ) found that higher early

- life blood Pb level was associated with a larger 17 

### deficit in IQ ( Figure 5

- 21 and Table 5

- 14 ). As part of the Port Pirie, Australia cohort 18 

### study, investigators separately examined intellectual attainment in group s of children 19 

### with different degrees of decline in blood Pb levels between ages 2 and 11

- 13 years. 20 

### Although the mean blood Pb level in the study population declined overall from 21 

### 21.2 μ g/dL at age 2 years to 7.9 μ g/dL at age 11

- 13 years, the magnitude of de cline 22 

### varied among children. In comparisons of tertiles of change in blood Pb level between 23 

### age 2 and 11

- 13 years, investigators found that FSIQ at ages 2, 4, 7, and 11

- 13 years did 24 

### not significantly differ between children with the largest declines (>16 μ g/dL) in blood 25 

### Pb level and children with a smaller decline (<10 μ g/dL). These findings indicated an 26 

### influence of higher blood Pb levels early in life despite declines in blood Pb with age and 27 

### a persistence of Pb effects. The results do not preclude an ind ependent association with 28 

### concurrent blood Pb level. 29 

### In several different cohorts of U.S. children, larger decrements in cognitive function were 30 

### estimated for concurrent blood Pb levels with consideration of blood Pb levels measured 31 

### earlier in childhood ( Hornung et al., 2009 ; Chen et al., 2005 ; Bellinger et al., 1990 ) 32 

### ( Figure 5

- 21 and Table 5

- 14 ). In the Boston cohort, Bellinger et al. ( 1990 ) found that at 33 

### age 57 months, FSIQ, as assessed by McCarthy GCI, was similar between children with 34 

### higher (≥ 10 μ g/dL) and lower (<3 μ g/dL) prenatal cord blood Pb levels. Additionally, 35 

### higher concurrent blood Pb level (age 57 months) was asso ciated with the largest decline 36 

### in GCI score over time (score at age 57 months

- score at age 24 months) among children 37February 2012 5

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– Do Not Cite or Quote 

### with high prenatal blood Pb levels (≥ 10 μ g/dL), which pointed to a larger decrease in 1 

### FSIQ in children with both high early and concurren t blood Pb levels ( Figure 5

- 21 and 2 

### Table 5

- 14 ). The findings indicated that by age 5 years, children with higher pren atal 3 

### blood Pb levels appeared to recover the Pb

- associated decrements in cognitive function 4 

### unless concurrent blood Pb levels remained high. The investigators also demonstrated 5 

### that positive home and caregiving environment ( e.g., HOME score >52, higher SES , 6 

### higher maternal IQ) may also protect against decrements in cognitive function associated 7 

### with higher postnatal Pb exposures. Collectively, these results suggest that cognitive 8 

### development is not fixed early in childhood and can be affected negatively or positively 9 

### by postnatal influences. 10

a Values represent the ratio of blood Pb level at age 6 years to that at age 2 years.
b Results represent the decrease in FSIQ per 1 μg/dL increase in concurrent blood Pb level in children in different groups of prenatal cord blood Pb levels. FSIQ scores were standardized to their standard deviation. Effect estimates in blue represent associati ons for higher prenatal or early childhood blood Pb levels relative to concurrent blood Pb levels. Note: Effect estimates represen t associations between concurrent blood Pb level and cognitive function (standardized to standard deviation) in children. Studies are presented in order of increasing prenatal level. 

### Figure 5
- 21 Associations of cognitive function in children with different degrees of changes in blood Pb levels over time. Study

Tong et al. (1998) Bellinger et al. (1990)

Chen et al. (2005) Hornung et al. (2009) Age of FSIQ Assessment

11

- 13 yr 24 and 57 mo

7 yr. 6 yr.

age 2 to 11

- 13 age 2 to 11

- 13 age 2 to 11

- 13 prenatal < 3 b prenatal 3

- 10 b prenatal ≥ 10 b

Low 7 yr (< 7.2) High 7 yr (> 7.2) Low 7 yr (< 7.2) High 7 yr (> 7.2) Blood Pb (μg/dL) variable examined

< 10.2 decline 10.2

- 16.2 decline > 16.2 decline Concurrent, Concurrent, Concurrent,

Low 2 yr (< 24.9), Low 2 yr (< 24.9), High 2 yr. (> 24.9), High 2 yr. (> 24.9), 0.5 ratio age 6 to 2 yr a 2.0 ratio age 6 to 2 yr a

- 1

- 0.5 0 Change in FSIQ (95% CI)February 2012 5

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### Table 5
- 14 Additional characteristics and quantitative results for studies presented in Figure 5
- 21

Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) a Hornung et al. ( 2009 ) 462 children followed from birth (1979

- 1984) to age 6 yr Rochester, NY and Cinc innati, OH Geometric mean (5th

- 95th): Peak: 13.6 (4.6

- 34.4) Early childhood: 8.9 (3.0

- 23.8) Lifetime mean: 8.5 (3.0

- 22.1) Concurrent: 6.0 (1.9

- 17.9) Linear regression model adjusted for city, HOME score, birth weight, maternal IQ, maternal education FSIQ assessed using WISC

- R at age 6 yr 0.5 ratio of blood Pb level at age 6 to age 2: 0 (reference) 2.0 ratio of blood Pb level at age 6 to age 2 yr:

- 0.70 (

- 1.0,

- 0.40)

Bellinger et al. ( 1990 ) 170 children followed prenatally to age 57 mo Boston area, MA NR Log linear regression adjusted for HOME score, social class, maternal IQ, maternal age, sex, ethnicity Change in McCarthy GCI score (z

- score) between age 57 and 24 mo For concurrent blood Pb level b Prenatal <3 μg/dL:

- 0.16 (

- 0.43, 0.11) Prenatal 3

- 10 μg/dL:

- 0.14 (

- 0.57, 0.29) Prenatal ≥ 10 μg/dL:

- 0.46 (

- 0.81,

- 0.11) Tong et al. ( 1998 ) 375 children followed from birth (1979

- 1982) to age 11

- 13 yr Port Pirie, Australia Means: 21.2 (age 2 yr), 7.9 (age 11

- 13 yr) Log linear regression model adjusted for sex, birth weight, birth rank, feeding style, breastfeeding duration, maternal IQ, maternal age, SES, HOME score, parental smoking, parents living together. ANOVA to assess association of change in IQ with change in blood Pb across time intervals Change in cognitive function (z

- scores) using Bayley MDI at age 2 yr, McCarthy GCI at age 4 yr, WISC

- R at ages 7, and 11

- 13 yr <10.2 μg/dL decline: 0.03 (

- 0.15, 0.21) c 10.2

- 16.2 μg/dL decline: 0.04 (

- 0.15, 0.23) c >16.2 μg/dL decline:

- 0.01 (

- 0 .20, 0.18) c

Chen et al. ( 2005 ) 780 children participating in the TLC trial from age 12

- 33 mo to age 7 yr Baltimore, MD; Cincinnati, OH; Newark, NJ; Philadelphia, PA Children underwent chelation therapy Mean (SD): Age 2 yr: 26.2 (5.1) Age 5 yr: 12.0 (5.2) Age 7 yr: 8.0 (4.0) Low age 2 yr: <24.9 Low age 7 yr: < 6.2 Linear regression model adjusted for city, race, sex, language, parental education, parental employment, single parent, age at blood Pb measurement, caregiver IQ WISC

- III at age 7 yr Low age 2, Low age 7: 0 d Low age 2, High age 7:

- 0.27 (

- 0.48,

- 0.05) High age 2, Low age 7: 0 (

- 0.21, 0.20) High age 2, High age 7:

- 0.28 (

- 0.47,

- 0.10)

a Effect estimates represent the cognitive function score or change in score over time standardized to its standard deviation. b Effects are estimated for concurrent blood Pb level (continuous variable) in children in different categories of prenatal blood Pb level: <3 μg/dL, 3

- 10 μg/dL, and ≥ 10 μg/dL.
c Investigators estimated changes in IQ in groups of children with different degrees of decline in blood Pb levels over the stu dy period: children with <10.2 μg/dL decline, childre n with a 10.2

- 16.2 μg/dL decline, and children with >16.2 μg/dL. d Investigators compared IQs among children with different categories of blood Pb level early and later in childhood: low level s at age 2 (<11.4 μg/dL) and age 7 (<7.2 μg/dL), low levels at ag e 2 (<11.4 μg/dL) and high levels at age 7 (>7.2 μg/dL), high levels at age 2 (>11.4 μg/dL) and low levels at age 7 (<7.2 μg/dL), and high levels at age 2 (>11.4 μg/dL) and age 7 (>7.2 μg/dL). Cutoffs were based on the median blood Pb levels.


### Pooling the Cincinnati and Rochester cohorts (n = 397), Hornung et al. ( 2009 ) created a 1 

### new indicator of Pb exposure: the ratio of blood Pb level at 6 years of age to that at 2 

### 2 years of age. The greatest decrease in cogniti ve and behavioral development was 3 

### observed for children with blood Pb ratios greater than 1 (indicating an increase in blood 4 

### Pb level fr om 2 to 6 years of age) ( Figure 5

- 21 , Figure 5

- 22 , and Table 5

- 14 ). Presumably 5 

### areas under the curve would be similar among children with blood Pb level ratios of 1, 6February 2012 5

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– Do Not Cite or Quote 

### greater than 1, and less than 1, indicating that cumulative blood Pb levels would not be 1 

### pr edictive. 2 

### As part of the multicenter TLC, Chen et al. ( 2005 ) also found higher concurrent blood Pb 3 

### level (≥ median 7.2 μ g /dL) to be associated with lower IQ at age 7 years, regardless of 4 

### whether blood Pb level at age 2 years was low or high (less than or greater than the 5 

### median of 24.9 μ g/dL, respectively). Blood Pb levels at ages 2 and 7 years were weakly 6 

### correlated (r = 0. 27). It is important to note that children participating in TLC had 7 

### undergone chelation therapy due to high blood Pb levels (20

- 44 μ g/dL) at age 12 to 8 

### 33 months, and the findings may have limited application to the general population of 9 

### children currently living in the U.S. In fact, in all of the studies that examined weakly 10 

### correlated serial blood Pb measurements, blood Pb levels were higher than those 11 

### currently measured in U.S. children. Additionally, in several study populations, children 12 

### experienced lar ge decreases in blood Pb levels over time. It is unclear whether these 13 

### findings would apply to children in the U.S. who currently are within the same age range 14 

### and who would be expected to have smaller decreases in blood Pb levels over time. 15

Source: Horn ung et al. ( 2009 ) Note: All three patterns have an identical mean blood Pb level of 10 μg/dL. 

### Figure 5
- 22 Estimated IQ in combined Cinc innati and Rochester cohorts, for three patterns of blood Pb level levels from 1 through 6 years of age: peak at 2 years (blue diamonds), peak at 5 years (black triangles), and constant blood Pb level (white squares).February 2012 5

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### To conclude, in the collective body of epidemiologic evidence in children, it is difficult to 1 

### ascertain which lifestage of Pb exposure is associated with the greatest risk of Pb

2 

### associated neurodevelopmental effects. Associations have been observed with prenatal, 3 

### early

- childhood, lifetime aver age, and concurrent blood Pb levels as well as with 4 

### childhood tooth Pb levels. Comparisons among different lifestages of exposure is 5 

### complicated further by the fact that b lood Pb levels in children, although highly affected 6 

### by recent exposure, are also inf luenced by Pb stored in bone due to rapid growth

- related 7 

### bone turnover in children relative to adults. Thus, concurrent blood Pb level in children 8 

### also may reflect body burden (Section 4.3.4.6 ). Nonetheless, while the evidence indicates 9 

### that prenatal and e arly

- childhood blood Pb levels are associated with neurodevelopmental 10 

### deficits, subsequent exposures that are reflected in concurrent, cumulative blood Pb 11 

### levels or tooth Pb levels also are demonstrated to contribute to neurodevelopmental 12 

### deficits througho ut school

- age and into adolescence. With additional results from studies 13 

### described in Sections 5.3.2.1 and 5.3.3.1 , the weight of epidemiologic evidence supports 14 

### associations of concurrent blood Pb level with neurodevelopmental effects in children. 15 

### These findings are consistent with the understanding that the nervous system continues to 16 

### develop throughout childhood. 17



### 5.3.10 Examination of the Lead Concentration
- Response Relationship
With each successive Pb AQCD and supplement, epidemiologic and toxicological studies 18 

### find that progressively lower blood Pb levels are associated with cognitive deficits and 19 

### behavioral impairments. For example, among children, such effects were observed in 20 

### association with blood Pb levels in the range of 10

- 15 μ g/dL in the 1986 Addendum and 21 

### 1990 Supplement and 10 μ g/dL and lower in the 2006 Pb AQCD ( U.S. EPA, 2006b )

. 22 

### Furthermore, in the 2006 Pb AQCD, several individual studies, pooled analyses, and 23 

### meta

- analyses estimated a supralinear blood Pb concentration

- response relationship in 24 

### children, i.e., greater decrements in cognitive function per incremental increase in blood 25 

### Pb level among children in lower strata of blood Pb levels compared with children in 26 

### higher strata of blood Pb levels ( Figure 5

- 23 and Table 5

- 15 ). As lower concentrations of 27 

### Pb exposure are being used experimentally, the toxicological literature also has reported 28 

### nonlinear concentration

- response relationships for various endpoints, including those 29 

### coherent with cognitive function decrements observed in children. Also consistent with 30 

### the epidemiologic literature, some toxi cological studies have shown larger magnitudes of 31 

### effect (absolute effects) in lower Pb exposure groups (relative to control groups) than in 32 

### the higher exposure groups. 33 

### In epidemiologic studies, a supralinear concentration

- response relationship was observe d 34 

### for concurrent, early childhood, and lifetime average blood Pb levels. Most 35February 2012 5

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### epidemiologic studies used a blood Pb level of 10 μ g/dL to define lower and higher blood 1 

### Pb levels. Several of these observations were made in groups of children aged 2 to 2 

### 10 yea rs, and the mean blood Pb levels in the lower strata of blood Pb levels were in the 3 

### range of 3

- 5 μ g/dL ( Bellinger, 2008 ; Canfield, 2008 ; Hornung, 2008 ; Rojo

- Tellez, 2008 )

. 4 

### Except for the pooled analysis, lower strata of blood Pb levels comprised at least 55% of 5 

### the study population and consequen tly the concentration

- response relationships 6 

### calculated for these lower strata likely are not outliers comprised a minimum 55% of the 7 

### study population and consequently the concentration

- response relationships calculated for 8 

### these lower strata are not outli ers or unrepresentative of the overall study population. 9 

### Using data pooled from seven prospective studies, Lanphear et al. ( 2005 ) fit various types 10 

### of models to the data and observed that a cubic spline, log

- linear model, an d piece

- wise 11 

### linear model all supported a more negative concentration

- response relationship at 12 

### concurrent blood Pb levels <10 μg/dL. A linear model was found to be inadequate as the 13 

### polynomial terms for concurrent blood Pb were statistically significant. T hese findings 14 

### were corroborated in a separate analysis by Rothenberg and Rothenberg ( 2005 ) who 15 

### found that the log

- linear model fit the relationship between blood Pb level and IQ better 16 

### than a linear model did. 17


### A few studies demonstrated larger Pb

- associated decreases in cognitive function with 18 

### blood Pb levels < 5 μg/dL. Tellez

- Rojo et al. ( 2006 ) estimated a larger decrement in IQ 19 

### per unit increase in blood Pb level for children (age 2 years) with concurrent blood Pb 20 

### levels <5 μg/dL compared with children with levels 5

- 10 μ g/ dL, and > 10 μ g/dL ( Figure 21 

### 5

- 23 and Table 5

- 15 ). In an NHANES analysis 1989

- 1994, Lanphear et al. ( 2000 ) found 22 

### larger decrements in reading and math skills and memory per unit increase in blood Pb 23 

### level in children with concurrent blood Pb levels <2.5 μ g/dL compared with children 24 

### with levels <5 μ g/dL, <7,5 μg/dL, <10 μ g/dL, and all subjects. However, as the study 25 

### population included adolescents, the oldest of whom were 16 years of age and born 26 

### 1972

- 1978, higher Pb exposures earlier in childhood may have contributed to 27 

### associations. 28February 2012 5

- 202 Draft

– Do Not Cite or Quote Note: Studies are presented in order of increasing mean blood Pb level. Strata refer to peak blood Pb level measured in child at any point during follow up. FSIQ = full

- scale IQ, MDI = mental development index. Effect estimates are standardized to a 1 μ g/dL increase in blood Pb level. Black symbols represent effect estimates among all subjects or in the highest blood Pb stratum. Blue symbols represent effect estimates in lower blood Pb strata 

### Figure 5
- 23 Comparison of associations between blood Pb level and cognitive function among various blood Pb strata. Study Lanphear et al. (2000)

Bellinger and Needleman (2003)
Canfield et al. (2003) Tellez

- Rojo et al. (2006)

Jusko (2011)

Kordas et al. (2006) Lanphear et al. (2005)

Schwartz (1994) Blood Pb Timing
Concurrent

Peak

Lifetime avg Concurrent

Peak

Concurrent Concurrent

Early childhood Outcome Reading score

FSIQ

FSIQ Bayley MDI

FSIQ

Math score FSIQ

FSIQ Blood Pb stratum (μg/dL) All <10 <7.5 <5 <2.5

>10 <10

All <10 a ≥10 <10 5

- 10 <5

All < 10
all <10 ≥10 a <10 a ≥7.5 a <7.5 a

>15 ≤15 ◄

◄

◄ ► Sample size

4853 4681 4526 4043 2467

NR NR

172 101 90 294 101 193

174 96

532 293 1089 244 1230 103

NR NR

- 4

- 3

- 2

- 1 0 1 Change in Cognitive Function Score per 1 μg/dL increase in blood Pb level (95% CI)February 2012 5

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### Table 5
- 15 Additional characteristics and quantitative results for studies presented in Figure 5
- 23

Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Blood Pb stratum (μg/dL) Effect Estimate (95% CI) a Lanphear et al. ( 2000 ) 4 , 853 children ages 6

- 16 yr NHANES 1988

- 1994 Concurrent mean (SE): 1.9 (0.1) Linear regression model adjusted for sex, race/ethnicity, poverty index ratio, reference adult education level, serum ferritin level, serum cotinine level WRAT reading subtest at ages 6

- 16 yr All subjects <10 <7.5 <5 <2.5 - 0.70 (

- 1.03,

- 0.37)

- 0.89 (

- 1.52,

- 0.26)

- 1.06 (

- 1.82,

- 0.30)

- 1.06 (

- 2.00,

- 0.12)

- 1.28 (

- 3.20,

- 0.64) Tellez

- Rojo et al. ( 2006 ) 294 children followed from birth (1994

- 1995, 1997

- 1999) to age 2 yr Mexico City, Mexico Concurrent (age 2 yr) mean (SD): 4.28 (2.25) Linear regression model adjusted for sex, birth weight, maternal IQ Bayley MDI at age 2 yr ≥ 10 <10 5

- 10 <5 0.07 (

- 10, 9.2) b

- 1.04 (

- 1.8,

- 0.30) b

- 0.94 (

- 2.1, 0.2) b

- 1.71 (

- 3.0,

- 0.42) b Bellinger et al. ( 1992 ) Bellinger and Needleman ( 2003 ) 148 children followed from birth (1979

- 1981) to age 10 yr Boston area, MA Early childhood (age 2 yr) mean (SD): 6.5 (4.9) Linear regression model adjusted for HOME score (age 10 an d 5), child stress, race, maternal IQ, SES, sex, birth order, marital status WISC

- R at age 10 yr >10 <10 - 0.58 (

- 1.0,

- 0.2) b

- 1.56 (

- 2.9,

- 0.2) b

Canfield et al. ( 2003a ) 172 children born 1994

- 1995 followed from infancy to age 3

- 5 yr Rochester, NY Lifetime avg (3 or 5 yr) mean (SD): 7.4 (4.3) Mixed effects models adjusted for sex, maternal race, parental smoking, child iron status, maternal income, maternal IQ, HOME score Stanford

- Bine t at age 3 or 5 yr All <10 - 0.46 (

- 0.76,

- 0.15)

- 1.37 (

- 2.56,

- 0.17)

Lanphear et al. ( 2005 ) 1 , 333 children pooled from Boston, Cincinnati, Cleveland, Mexico City, Port Pirie, Rochester, and Yugoslavia cohorts Concurre nt Median (5th

- 95th) 9.7 (2.5

- 33.2) Linear regression model adjusted for HOME score, birth weight, maternal IQ, maternal education FSIQ measured at ages 4.8

- 10 yr ≥ 10 <10 ≥ 7.5 <7.5 - 0.13 (

- 2.3,

- 0.03)

- 0.80 (

- 1.74,

- 0.14)

- 0.16 (

- 2.4,

- 0.08)

- 2.94 (

- 5.16,

- 0.71)

Jusko et al. ( 2011 ) 194 children in Rochester, NY followed from age 6 mo (1994

- 1995) to age 6 yr. Peak Mean (SD): 11.4 (7.3) Linear regression model adjusted for sex, birth weight, transferrin saturation, maternal race, maternal IQ, maternal education, HOME score, family income, and maternal prenatal smoking WPPSI

- R at age 6 yr All < 10 - 0.19 (

- 0.46, 0.07)

- 1.66 (

- 3.1,

- 0.23)

Kordas et al. ( 2006 ) 602 children in 1st grade Torreon, Mexico Concurrent mean (SD): 11.4 (6.1) Linear regression model adjusted for sex, age, hemoglobin, family possessions, forgetting homework, house ownership, crowding, maternal education, birth order, family structure, arsenic exposure, tester, school Math achievement test in 1st grade all <10 - 0.17 (

- 0 .28,

- 0.06)

- 0.42 (

- 0.92, 0.08)

Schwartz ( 1994 ) Meta

- analysis of 7 studies with sample sizes 75

- 579 children Early childhood (2

- 3 yr) range in study means: 6.5

- 23 Meta

- analysis of combining effect estimates from individual studies FSIQ measured at school

- age Studies with mean >15 Studies with mean ≤ 1 5 - 2.32 (

- 3.10,

- 1.54)

- 3.23 (

- 5.70,

- 0.76)

a Effect estimates are standardized to a 1 μ g/dL increase in blood Pb level. b 95% CIs calculated from reported p

- value.



### Several studies also found a nonlinear blood Pb

- cognitive function relationship in 1 

### nonparametric regression analyses using lowess with smoothing parameters or using 2 

### splines ( Min et al., 2009 ; Jusko et al., 2008 ; Schnaas et al., 2006 )

. Min et al. ( 2009 ) 3 

### performed a formal test of nonlinearity using nonparametric regression by testing the 4 

### statistical significance of a restricted cubic spline term for blood Pb level. Al though the 5February 2012 5

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– Do Not Cite or Quote 

### term was not statistically significant, the concurrent blood Pb level

- FSIQ curve appeared 1 

### to be more negative at blood Pb levels < 7 μ g/dL. In a similar analysis in a Mexico City 2 

### cohort, Schnaas et al. ( 2006 ) found a more negative blood Pb

- FSIQ slope at concurrent 3 

### blood Pb levels < 6 μ g/dL and found a test of nonlinearity to be statistic ally significant. 4 

### Among children in India ages 3

- 7 years, Palaniappan et al. ( 2011 ) , most associations of 5 

### concurrent blood Pb level with various indices of executive function in nonparametric 6 

### regression analyses were found to be linear. 7 

### Studies of adults have not assessed widely whether the relationship between blood or 8 

### bone Pb level and cognitive performance is described better with a linear or nonlinear 9 

### function. In the various NHANES analyses, only log

- linear models were used to fit the 10 

### data ( Krieg et al., 2010 ; Krieg and Butler, 200 9 ; Krieg et al., 2009 )

. Nonlinearity in the 11 

### BMS and NAS cohorts was examined with the use of quadratic terms, penalized splines, 12 

### or visual inspection of bivariate plots ( Bandeen

- Roche et al., 2009 ; Weisskopf et al., 13 

### 2007a ; Shih et al., 2006 )

. There was some evidence fo r nonlinearity for some ( Figure 14 

### 5

- 12 and Figure 5

- 13 ) but not all cognitive tests or all subjects in the NAS cohort. For 15 

### example, Wang et al. ( 2007a ) found that among NAS men with an HFE variant, there 16 

### was a steeper Pb

- associated decline in MMSE score a t higher tibi a Pb levels (20

- 25 μg/g, 17 

### Figure 5

- 13 ). In the BMS cohort, observations of a statistically nonsignificant quadratic 18 

### term ( Shih et al., 2006 ) or spline ( Bandeen

- Roche et al., 2009 ) for tibia Pb indicated that 19 

### a linear model adequately fit the relationship between tibia Pb level and various tests of 20 

### cognitive performance. 21


### Attenuation of the concentration

- response rel ationships at higher exposure or dose levels 22 

### has been reported in the occupational literature, and explanations have included greater 23 

### exposure measurement error, competing risks, and saturation of biological mechanisms at 24 

### higher levels; larger proportions of at

- risk populations at lower exposure levels; and 25 

### variations in other risk factors among exposure levels ( Stayner et al., 2003 )

. Other 26 

### explanations for nonlinearity include differential activity of mechanisms at diff erent 27 

### exposure levels, confounding by omitted or misspecified variables, and the lower 28 

### incremental effect of Pb due to covarying risk factors such as low SES, poor caregiving 29 

### environment, and higher exposure to other environmental factors. 30


### The contribution of these factors to the supralinear relationship between blood Pb levels 31 

### and cognitive function in children has not been examined widely in epidemiologic studies 32 

### to date. However, in several different populations, higher blood Pb levels have been 33 

### measured in potentially at

- risk groups such as those with higher poverty, greater exposure 34 

### to tobacco smoke, lower parental education, and lower birth weight, which argues against 35 

### a larger proportion of at

- risk populations at lower blood Pb levels ( Lanphear et al., 2005 ; 36 

### Lanphear et al., 2000 )

. It has been suggested that in populations of low SES, poorer 37February 2012 5

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– Do Not Cite or Quote 

### caregiving environment, and greater social stress, the incremental effect of Pb exposure 1 

### may be attenuated due to the overwhelmingly larger effects of these other risk factors 2 

### ( Schwartz, 1994 )

. Several studies found statistically significant associations of these 3 

### sociodemographic risk factors with neurocognitive deficits, and Miranda et al. ( 2009 ) 4 

### found that indicato rs of SES ( i.e., parental education and enrollment in a free/reduced fee 5 

### lunch program) accounted for larger decrements in EOG scores than did blood Pb level 6 

### ( Figure 5

- 7 ). Few studies have compared blood Pb level effect estimates among groups in 7 

### different sociodemographic strata, and the limited data are mixed. Greater Pb

- associated 8 

### neurocognitive deficits we re reported in low

- SES groups by Bellinger et al. ( 1990 )

. In a 9 

### meta

- analysis of eight studies, Schwartz ( 1994 ) found a smaller decrement in IQ per 10 

### 1 μ g/dL increase in blood Pb level for studies in disadvantaged populations (

- 2.7 points 11 

### [95% CI:

- 5.3,

- 0.07]) than for studies in advantaged populations (

- 4.5 points [95% CI: 12



### - 5.6,

- 2.8]). It is important to note that blood Pb level is associated with deficits in 13 

### cognitive function in both higher and lower SES group s; however, it is unclear what 14 

### differences there are between groups in the decrement per unit increase in blood Pb and 15 

### whether these differences can explain the observed nonlinear concentration

- response 16 

### relationship. 17 

### Rothenberg and Rothenberg ( 2005 ) formally assessed the influence of residual 18 

### confounding on the nonlinear blood Pb

- FSIQ concentration

- response relationship by 19 

### comparing model fit between linear and spline transformations (df = 2) of covariates such 20 

### as maternal IQ, H OME score, and maternal education. Inclusion of covariates as spline 21 

### functions did not significantly improve model fit either with a linear blood Pb term or log 22 

### blood Pb term, which indicated that their inclusion as linear functions was adequate. 23 

### These fin dings demonstrate that the improved model fit with log

- specification of blood 24 

### Pb level was not due to residual confounding by covariates. 25


### Bowers and Beck ( 2006 ) postulated that a supralinear slope necessarily will be found 26 

### when modeling a relationship between a log

- normally distributed variable and a normally 27 

### distributed variable. However, as discussed in the 2006 Pb AQCD, this modeling strategy 28 

### was not employed in the epidemiologic analyses showing a supralinear concentration

29 

### response function. IQ scores generally were not forced into a normal distribution. Four of 30 

### the s even studies included in the pooled analysis by Lanphear et al. ( 2005 ) did not use 31 

### normalized IQ scores, and scores were not normalized in the pooled analysis ( Hornung et 32 

### al., 2006 )

. Further, a log

- linear model (a linear relationship between IQ and the log of 33 

### blood Pb) provided the best fit of the pooled data. 34 

### In support of the nonlinear associations between blood Pb levels and cognitive function 35 

### observed in children, toxicological studies provided some evidence of nonlinear 36 

### relationships between Pb exposure and effects related to impaired learning and memory 37February 2012 5

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### in animals. Multiple studies showed U

- or inverse U

- shaped curves with lower exposures 1 

### of Pb having different or often the opposite effect from higher exposures. Coherent with 2 

### epidemiologic findings, results summarized across multiple studies in multiple species 3 

### demonstrated that lower Pb exposures increased FI response rate s relative to controls and 4 

### higher Pb exposures decreased FI response rates ( Cory

- Slechta, 1994 )

. Consistent with 5 

### these findings, Rossi

- George et al. ( 2011 ) found that 50 ppm gestational plus lactational 6 

### Pb exposure when combined with stress increased FI responses of 2

- month old rats 7 

### whereas 150 ppm Pb exposure w ith stress did not affect FI responses. It is important to 8 

### note that the larger effects observed with lower Pb exposures were less consistently 9 

### observed with longer duration exposures ( e.g., 8

- 11 months) ( Rossi

- George et al., 2011 ; 10 

### Cory

- Slechta, 1990 )

. As discussed in Section 5.3.2.2, FI responses measure learning and 11 

### memory by reflecting the ability of animals to learn to respond to one schedule of 12 

### reinforcement and change responses according to a chan ge in reinforcement. 13 

### Toxicological studies provided additional support for nonlinear relationships between Pb 14 

### exposure and neurodevelopmental effects by finding that lower and higher Pb exposures 15 

### differentially activate mechanisms underlying such effects. Gilbert et al. ( 1999 ) found 16 

### reduced LTP with Pb exposures ranging between 0.1 and 0.5% but not 1.0%. LTP is one 17 

### indication of synaptic plasticity (Section 5.3.8.4) and is considered to contribute to 18 

### learni ng and memory. Likewise, glutamate release in the hippocampus was reduced in 19 

### animals with blood Pb levels 27

- 40 μ g/dL but not with blood Pb levels of 62

- 117 μ g/dL 20 

### ( Lasley and Gilbert, 2002 )

. Glutaminergic neurotransmission via its NMDA receptor has 21 

### been implicated in learning and memory (Section 5.3.8.8). 22 

### Dopam inergic neurotransmission is involved in many CNS processes including 23 

### cognition, behavior, and motor function. The shape of the Pb

- DA concentration

- response 24 

### relationship varied among toxicological studies. Some studies found that lower Pb 25 

### exposures (~ 50 p pm ) did not affect or increased DA activity relative to controls and 26 

### higher Pb exposure (109

- 250 ppm ) ( Leasure et al., 2008 ; Virgolini et al., 2005 ; Lewis and 27 

### Pitts, 2004 )

. However, compared with control and lower Pb exposures, higher Pb 28 

### exposures (109 or 150 ppm ) were found both to increase and impair DA activity ( Leasure 29 

### et al., 2008 ; Virgolini et al., 2005 )

. These differential responses of DA may be related to 30 

### the diverse actions of DA in different regions of the brain and on a range of CNS effects. 31 

### For e xample, the increased dopamine turnover with 50 ppm Pb exposure may explain the 32 

### greater spontaneous and amphetamine

- induced motor activity in males induced by 33 

### 50 ppm GLE ( Leasure et al., 2008 )

. 34 

### Lower and higher Pb exposures also were found to differentially affect calcineurin 35 

### enzyme activity; act ivity was inhibited by higher Pb exposure and stimulated by lower Pb 36 

### exposure ( Kern and Audesirk, 2000 )

. While calcineurin activity has been found to 37February 2012 5

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### mo dulate learning, LTP, and behavior in animals, studies have found lower calcineurin 1 

### activity to be associated with both impaired and improved effects related to learning 2 

### ( Zeng et al., 2001 )

. Thus, it is uncertain whether altered calcineurin activity contributes to 3 

### the nonlinear relationships observed between Pb exposure and learning. Nonetheless, 4 

### results indicated that lower and higher Pb exposures may have different modes of action. 5 

### At lower concentrations, Pb may displace calcium at its binding sites on calmodulin and 6 

### by acting as a calmodulin agonist at the catalytic A subunit of calcineurin, stimulate 7 

### calcineurin activity. At higher Pb exposure, Pb may bind directly to a separate calcium

8 

### b inding B subunit, override the calmodulin

- dependent effect and turn off the activity of 9 

### calcineurin. Similarly, Lasley and Gilbert (2002) also indicated that at higher 10 

### concentrations ( i.e., 1%), Pb stimulated gluatamate release by acting as a calcium 11 

### mimet ic. 12 

### While epidemiologic studies have not examined widely the shape of the concentration

13 

### response relationship for other nervous system effects, toxicological studies have found 14 

### nonlinear relationships for diverse outcomes. U

- shaped Pb concentration

- respons e 15 

### relationships were found for rotarod performance, amphetamine

- induced motor activity, 16 

### and latency to fall from rotarod ( Leasure et al., 2008 )

. Inverted U

- shaped Pb 17 

### concentration

- response relationships were found for histological parameters such as the 18 

### numbers of rod photoreceptors and bipolar c ells, activity level, and adult body weight 19 

### ( Leasure et al., 2008 ) as well as ERG wave amplitudes ( Fox et al., 2008 ) and 20 

### hippocampal neurogenesis ( Fox et al., 2008 ; Gilbert et al., 2005 )

. Additional evidence 21 

### points to differences in hormonal homeostasis by Pb exposure level. In male mic e with 22 

### chronic Pb exposure (PND21

- 9 months of age), basal corticosterone levels were 23 

### significantly lower in the 50 ppm exposure group than in the control or 150 ppm Pb 24 

### exposure group. 25 

### Sensory organ functions in animals also have shown to be differentially affected by lower 26 

### versus higher Pb exposure (developmental Pb exposurefrom gestation to PND10, pup 27 

### blood Pb levels 12, 24, and 46 μ g/dL. Inverted U

- shaped dose

- response curves have been 28 

### observed for rod photoreceptor numbers or neurogenesis ( Giddabasappa et al., 2011 ) and 29 

### retinal thickness ( Fox et al., 2010 )

. Thus, these dichotomous histological findings may 30 

### give insight to the complex sensory organ findings that vary by exposure window and 31 

### exposure dose (Section 5.3.4.3)

. 32 

### The supralinear concentration

- response relationship wi dely documented for Pb is 33 

### consistent with the lack of a threshold for Pb

- associated neurodevelopmental effects as a 34 

### smaller effect estimate would be expected at lower blood Pb levels if a threshold existed. 35 

### Schwartz ( 1994 ) explicitly assessed evidence for a threshold in data from the Boston 36 

### prospective cohort by regressing FSIQ and blood Pb level on po tential confounders 37February 2012 5

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### including age, race, maternal IQ, SES, and HOME score and fitting a nonparametric 1 

### smoothed curve to the residuals of both regression models (variation in FSIQ or blood Pb 2 

### level not explained by covariates). A 7

- point decrease in IQ was observed over the range 3 

### of blood Pb residuals below 0, which corresponds to the mean blood Pb level in the study 4 

### (6.5 μ g/dL). Thus, in the Boston study, the association between blood Pb level and FSIQ 5 

### was clearly demonstrated at blood Pb levels below 5 μ g/ dL. 6 

### An important limitation of previous studies in terms of characterizing the concentration

7 

### response relationship, in particular, identifying whether a threshold exists, was the limited 8 

### examination of associations in populations or subgroups with blood Pb levels more 9 

### comparable to the current U.S. population mean. While Schwartz ( 1994 ) did not find 10



### ev idence for a threshold in the Boston study data, the mean blood Pb in that population 11 

### was 6.5 μ g/dL, and 56% of subjects had a blood Pb level >5 μ g/dL. Recent studies 12 

### indicate a downward shift in the distribution of blood Pb levels. In the various NHANES 13



### a nalyses of children, a large proportion of children had blood Pb levels <1 μ g/dL, for 14 

### example, 50% of subjects in the 2001

- 2004 NHANES population ( Braun et al., 2008 )

. 15 

### More sensitive quantification methods have improved the detection limits, for example, 16 

### from 0.6 μ g/dL in 1999

- 2002 NHANES to 0.025 μ g/dL in 2003

- 2004 NHANES. In 17 

### analyses of children in multiple blood Pb quantiles below 1 μ g/dL, Braun et al. ( 2008 ) 18 

### found higher odds ratios for conduct disorder and ADHD a mong children with blood Pb 19 

### levels 0.8

- 1.0 μ g/dL (2nd quartile) compared with children with blood Pb levels 0.2

- 20 

### 0.7 μ g/dL (1st quartile). Despite the availability of large proportions of subjects at blood 21 

### Pb levels below 1 μ g/dL, the ability to discern a t hreshold for Pb

- associated 22 

### neurodevelopmental effects is limited due to the large proportions of adolescents in 23 

### NHANES analyses who were born the 1970s and whose higher past exposures may have 24 

### contributed to associations observed with concurrent blood Pb l evels. Nonetheless, 25 

### several recent studies reported associations between blood Pb levels and deficits in 26 

### cognitive and behavioral endpoints in children ages 8

- 11 years with mean or quantile 27 

### blood Pb levels <2 μ g/dL ( Cho et al., 2010 ; Kim et al., 2009b ; Miranda et al., 2009 )

. In 28 

### comparisons of various quantiles of blood Pb, Miranda et al. ( 2009 ) reported lower EOG 29 

### scores in children in North Carolina with blood Pb levels of 2 μ g/ dL compared with 30 

### children with blood Pb levels of 1 μ g/dL. Collectively, these new findings in children, do 31 

### not provide evidence for a threshold for the neurodevelopmental effects of Pb in the 32 

### range of blood Pb levels examined to date. 33 

### It is important to n ote, however, that the lack of a reference population with blood Pb 34 

### levels reflecting pre

- industrial Pb exposures limits the ability to identify a threshold. 35 

### Estimates of “background” blood Pb levels have been garnered from the analysis of 36 

### ancient bones in pre
- industrialized societies. These studies suggest that the level of Pb in 37February 2012 5

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### blood in preindustrial humans was approximately 0.016 μ g/dL ( Flegal and Smith, 1992 ) , 1 

### approximately 65

- fold lower than th at currently measured in U.S. populations and lower 2 

### than the levels at which neurodevelopmental effects have been observed (1 μ g/dL). Thus, 3 

### the current evidence does not preclude the possibility of a threshold for 4 

### neurodevelopmental effects in children exi sting in the large range of blood levels 5 

### between 1 μ g/dL and preindustrial “background” levels. 6



### T o conclude, several studies found a supralinear blood Pb

- cognitive function 7 

### concentration

- response relationship in children based on comparisons of effect esti mates 8 

### in lower and higher strata of blood Pb level and nonparametric regression. Supporting 9 

### evidence was provided by some toxicological studies that showed that lower Pb 10 

### exposures induced learning and memory impairments in animals compared to control 11 

### expos ures or higher Pb exposures. While explanations for this supralinear relationship 12 

### have not been well characterized by epidemiologic studies, recent toxicological studies 13 

### that examined lower concentrations of experimental Pb exposure suggest that 14 

### mechanisms may be differentially activated at lower and higher Pb exposures. 15


### Nonlinear concentration

- response relationships were found for effects such as LTP, 16 

### hippocampal glutamate release, and calcineurin expression. Observations of associations 17 

### between blood Pb levels and deficits in cognitive and behavioral endpoints in children 18 

### (ages 8

- 11 years) with mean or range of blood Pb levels ≤ 2 μg/dL ( Cho et al., 2010 ; Kim 19 

### et al., 2009b ; Miranda et al., 2009 ) do not provide evidence for a threshold for 20 

### neurodevelopmental effects of Pb in the range of blood Pb levels examined to date. 21



### 5.3.11 Confounding in Epidemiologic Studies of Nervous System Effects
In addition to Pb exposure, many factors influence cognitive function and behavior in 22 

### children, including parental IQ and education, SES of the family, quality of the 23 

### caregiving environment, and other environmental exposures ( Wasserman and Factor

24 

### Litvak, 2001 )

. These other risk factors often are correlated with blood, tooth, and bone Pb 25 

### levels, thus, a major challenge to observational studies examining as sociations of Pb 26 

### biomarker levels with cognitive and behavioral function in children has been the 27 

### assessment and control for potential confounding factors. By definition, a confounder is 28 

### associated with both the exposure and the outcome and consequently ha s the potential to 29 

### bias the association between the exposure of interest and the outcome. Epidemiologic 30 

### studies of Pb biomarkers in children have most commonly examined potential 31 

### confounding by quality of the caregiving environment ( i.e., HOME score), pare ntal IQ, 32 

### and SES

- related variables such as parental education, household income, and the 33 

### Hollingshead Four

- Factor Index of Social Position, which incorporates education and 34February 2012 5

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### income of both parents. A relatively smaller number of studies have considered 1 

### conf ounding by other environmental exposures, measures of parental substance abuse, 2 

### and psychopathology. Studies have varied with respect to the number of potential 3 

### confounders examined, with some studies considering multiple SES

- related variables and 4 

### other st udies focusing on a smaller set. It is important to note that the extent of 5 

### confounding by a particular variable likely is study

- specific, i.e., dependent on the 6 

### population examined. Thus, the impact of adjustment for specific covariates on the Pb 7 

### effect e stimate also is likely to be study

- specific. 8 

### Studies have used various methods to control for confounding, including examining a 9 

### homogeneous population with respect to SES, examining populations in which factors are 10 

### not correlated, conducting multivariate regression, characterizing the magnitude of 11 

### change in the blood Pb level effect estimate with adjustment for a covariate, and 12 

### examining associations in different strata of a covariate. While no single method is 13 

### without limitation, the consistency of findin gs among different methodologies 14 

### substantiates independent associations of blood Pb level with cognitive function 15 

### decrements and behavioral problems in children. The evidence derived from each of 16 

### these control strategies is discussed below. 17


### In the Boston P rospective Study, potential confounding by SES was largely controlled for 18 

### by study design. The study subjects were generally middle

- to upper

- middle

- class 19 

### children with married, college

- educated parents. Hence, the potential for confounding by 20 

### SES in this study was considerably less compared to other studies examining the same 21 

### associations. Yet, in this cohort, blood Pb levels measured prenatally, at age 2 years, and 22 

### integrated over ages 2 and 5 years were associated decrements in full

- scale IQ and 23 

### various measures of executive function ( Stiles and Bellinger, 1993 ) ; ( Bellinger et al., 24 

### 1992 ; Bellinger et al., 1990 )

. In some analyses of this cohort, larger effects were 25 

### estimated as compared with other studi es. 26 

### Studies also have demonstrated associations between blood Pb levels and cognitive 27 

### function in populations in which blood Pb levels were not associated with SES

- related 28 

### variables ( Factor

- Litvak et al., 1999 ; Bellinger et al., 1987 )

. In the Boston cohort, 29 

### parental education, social class, and HOME sco re were similar among low (<3 μ g/dL), 30 

### medium (6

- 7 μ g/dL), and high (≥ 10 μ g/dL) cord blood Pb level groups. Nonetheless, 31 

### adjusting for these and other demographic variables, Bellinger et al. ( 1987 ) found that 32 

### children in the high cord blood Pb group had a 4.8

- point lower Bayley MDI score at age 33 

### 24 months than did children in the low cord blood Pb group. In the Yugoslavia cohort, 34 

### blood Pb levels at age 4 years were higher in gro ups with higher maternal education, 35 

### maternal IQ, and HOME score in one city and were lower in another city. Among all 36 

### children, higher blood Pb level was associated with lower FSIQ and specific indices of 37February 2012 5

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### learning and memory and with greater depression and withdrawn behavior ( Factor

- Litvak 1 

### et al., 1999 )

. 2 

### The primary method used by epidemiologic studies to control for potential confounding 3 

### has been multivariate regression. This was the main method employed in recent studies 4 

### t hat examined children with blood Pb levels in the range of current U.S. levels. Some 5 

### studies selected a set of covariates based on a priori knowledge, whereas others selected 6 

### particular covariates based on their association with the outcome in a model with all 7 

### potential covariates and/or a greater than 10% change in the blood Pb level effect 8 

### estimate. Studies also varied in the number of variables included in models as potential 9 

### confounders. Some included multiple SES

- related variables, whereas others analy zed one 10 

### or two variables. Regardless of the method used to select model covariates or the number 11 

### of covariates included, studies consistently demonstrated associations of higher blood Pb 12 

### level with lower cognitive function and greater behavioral problems. This consistency 13 

### suggests that confounding by particular variables may be specific to the population 14 

### examined and that no single measured variable or set of variables fully accounts for the 15 

### associations observed with blood Pb levels and neurodevelopmental effects in children. 16 

### The consistency of associations across populations with different SES and across studies 17 

### examining different covariates was reinforced in pooled and meta

- analyses ( Marcus et 18 

### al., 2010 ; Lanphear et al., 2005 ; Schwartz, 1994 )

. Pooling data from seven interna tional 19 

### longitudinal cohorts, Lanphear et al. ( 2005 ) found similar blood Pb

- FSIQ effect estimates 20 

### (

- 2.6 to 8.6% difference) among models, each with one study omitted. These results 21 

### indicated that the pooled estimate is relati vely stable despite between

- study differences in 22 

### population characteristics, including SES. In a meta

- analysis, Schwartz ( 1994 ) found that 23 

### individual study blood Pb

- IQ effect estimates fell within a relatively narrow range despite 24 

### large differences among studies in the correlation between blood Pb level and SES. A 25 

### wider range of effect estimates would be expected if omitted SES factors confounded the 26 

### blood Pb level association. A recent meta

- analysis examined the association between 27 

### blood Pb level and conduct problems in older and recent studies of children ( Marcus et 28 

### al., 2010 )

. Among the studies included, adjustment for variables such as SES and home 29 

### environment did little to attenuate the association between Pb and conduct problems. 30 

### Among studies that provided both unadjusted and adjusted effect estimates, most 31 

### indicated statistically significant associations of blood Pb level with neurodevelopmental 32 

### outcomes in children before and after adjusting for potential confounders. Although 33 

### examples exist where blood Pb level was estimated to have a weaker, statistically 34 

### nonsignificant effect after adjustment for potential confounders ( Tong and Lu, 2001 ; 35 

### Ernhart et al., 1989 ) , most notably in multiple analyses of the Cleveland cohort, studies 36 

### also reported stronger blood Pb level associations in covariate

- adjusted models ( Canfield 37February 2012 5

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### et al., 2003b ; Factor

- Litvak et al., 1999 )

. In recent st udies with population mean blood Pb 1 

### levels between 5 and 10 μ g/dL, associations between blood Pb levels and cognitive 2 

### function ( e.g., FSIQ, executive function, learning, and memory) remained statistically 3 

### significant after adjusting for covariates ( Min et al., 2009 ; Chiodo et al., 2007 ; Froehlich 4 

### et al., 2007 ; Kordas et al., 2006 ; Schnaas et al., 2006 )

. Although most effect estimates 5 

### changed by 20

- 50% in multivariate models, they remained within the 95% CI of the 6 

### unadju sted estimate. In particular, Schnaas et al. ( 2006 ) and Froehlich et al. ( 2007 ) found 7 

### some adjusted effect estimates to be larger in magnitude compared with unadjusted 8 

### estimates. Although information was not provided on the magnitude of change in effect 9 

### estimates after adjustm ent for confounding, Chiodo et al. ( 2007 ) found statistically 10 

### significant associations between blood Pb level and behavioral problems 11 

### ( e.g., inattention, hyperactivity, social problems, impulsivity) in models with blood Pb 12 

### level alone and with covariates. These findings demonstrate that SES

- related and 13 

### demographic factors may partially account for but do not fully explain the associations 14 

### observed between higher blood Pb level and neurodevelopmental impairments. 15 

### A challenge to separating the effects of Pb exposure from those related to SES and 16 

### quality of the caregiving environment is the high correlation typically observed among 17 

### these measures. In such cases, it is difficult to know how much variation in the outcome 18 

### to attribute to each of the various risk factors ( Needleman and Bellinger, 2001 )

. For 19



### example, the high correlation between blood Pb level and SES may lead to an 20 

### underestimation of the Pb effect when SES is added to the model. A reduction in the 21 

### magnitude and statistical significance of the Pb effect estimate following adjustment for 22



### some measu re of SES may be the result of the misattribution of the variance in outcome 23 

### due to Pb to the variance due to SES. SES may be a proxy for Pb exposure rather than a 24 

### confounder of the association of interest. This misattribution may be exacerbated when 25



### sever al correlated variables are included in the same model ( i.e., overcontrol). 26 

### Instead of being a confounder, Pb may be on the causal pathway of the association 27 

### between social class and IQ. Lower social class in urban children is closely linked to 28 

### residence i n older housing in poor condition that, in turn, increases exposure of children 29 

### to environmental Pb and increases their risk of cognitive deficits ( Clark et al., 1985 )

. In 30 

### such cases, statistical adjustment for SES will lead to the underestimation of the Pb effect 31 

### ( Bellinger, 2004a )

. One extreme example of overcontrol of this nature can be found in the 32 

### New Zealand studies where investigators regularly adjusted for residence in older 33 

### wooden housing, which is associated with higher exposure to Pb paint and accumulated 34 

### dust and soil ( Fergusson et al., 1988a , b )

. However, it is worth noting that, even in the 35 

### models including this variable, Pb remained a statistically significant predictor of 36 

### intellectual and academic under

- attainment in the Christchurch Health St udy. Variables 37 

### related to SES have been shown to be effect modifiers in studies of Pb and child 38February 2012 5

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### development ( Tong et al., 2000 ; Bellinger et al., 1990 )

. That is, increases in blood Pb 1 

### level have been associated with larger decreases in cognitive function among children 2 

### from less advantaged ( e.g., lower SES, lower HOME score, lower maternal IQ) 3 

### household than among those from more advantaged households. Similarly, a larger 4 

### decrease in cognitive function in association with higher blood Pb level was found among 5 

### children with mothers with lower self

- esteem, an indicator of stress and less connection 6 

### with the child ( Surkan et al., 2008 )

. 7 

### In the collective body of epidemiologic evidence, higher blood Pb levels are consistently 8



### ass ociated with cognitive function decrements and behavioral problems in children, with 9 

### the weight of evidence indicating decrements in FSIQ and diverse measures of executive 10 

### function and inattention and hyperactivity, respectively. These associations have be en 11 

### observed in diverse populations in the U.S., Mexico, Europe, Asia, and Australia. 12 

### Associations have been observed across studies that use different methods of control for 13 

### confounding and adjust for different potential confounders. While no single method is 14 

### without limitation, the consistency of findings among different methodologies and sets of 15 

### covariates substantiates independent associations of blood Pb level with cognitive 16 

### function decrements and behavioral problems in children. In addition, Pb exposu re has 17 

### been extensively studied in animals that produce blood Pb levels in the range of those 18 

### examined in children. Experimental animal studies are not vulnerable to confounding by 19 

### such factors as social class and correlated environmental factors. Adding f urther support 20 

### for the independent associations of blood Pb levels with neurodevelopmental outcomes in 21 

### children is the coherence of findings in animal studies for Pb

- induced impairments in 22 

### tests of learning, inattention, and impulsivity, especially tests t hat are directly 23 

### homologous to those in children, i.e., spatial memory, rule learning and reversal, and 24 

### response inhibition. Further, an extensive body of toxicological evidence for Pb

- induced 25 

### changes in brain physiological processes that mediate cognition and behavior, including 26 

### changes in neurogenesis, synaptic pruning, and neurotransmitter function in the frontal 27 

### lobe and striatum of the brain provides biological plausibility for observations in 28 

### children. 29



### 5.3.12 Public Health Significance of Associations betwee n Lead Biomarkers and Neurodevelopmental Effects

As described in Section 5.3.2.1 , most studies found that a 1 μ g/dL increase in blood Pb 30 

### level was a ssociated with fractional decrements in FSIQ in school

- aged children ( Figure 31 

### 5

- 2 and Table 5

- 3 ) and Bayley MDI in infants ( Table 5

- 4 ). Similarly, a 1 μ g/dL increase 32 

### in blood Pb level typically was associated with decreases in specific cognitive abilities 33 

### ( Figure 5

- 5 and Table 5

- 5 ) and increases in inattention and hyperactivity (Section 5.3.3.1 , 34February 2012 5

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– Do Not Cite or Quote 

### Figure 5

- 14 and Table 5

- 9 ) on the order of less than 1 standard deviation. These findings 1 

### have generated discussion of the public significance of blood Pb level

- associated changes 2 

### in cognitive function and behavio r in children, specifically, whether these fractional 3 

### changes have consequences on the health and life

- success of individuals. The World 4 

### Health Organization definition of health is the following : “ Health is a state of complete 5 

### physical, mental and social well

- being and not merely the absence of disease or 6 

### infirmity ” ( WHO, 1948 )

. By this definition, even decrements in health status that are not 7 

### severe enough to meet diagnostic criteria might be undesirable if they reflect a decrement 8 

### in the well

- being of an individual. Deficits in health indices or life

- success may not be 9 

### observa ble except in aggregate, at the population level. The American Thoracic Society 10 

### discussed the need to consider the prevalence of exposures in the population and 11 

### exposure to other risk factors in evaluating whether shifts in the population

- level risk are 12 

### ad verse ( ATS, 2000 )

. It should also be noted that these deficits when measured in 13 

### children may set affected children on trajectories more prone toward lower educational 14 

### attainment and financial well

- being. Thus, early deficits in children may have lifetime 15 

### consequences. 16 

### It has been argued that fractional decrements in IQ points are meaningless given that 17 

### these Pb

- associated changes are within the 3

- to 4

- point standard error on a single test 18 

### ( i.e., the statistic that defines the ra nge within which the true value of an individual is 19 

### likely to lie) ( Kaufman, 2001 )

. However, this argument incorrectly assumes that 20 

### conclusions drawn from individ ual

- level data apply to populations. It is important to note 21 

### that evidence does not indicate that the standard error is nonrandom, i.e., biased in one 22 

### direction. In particular, evidence has not indicated that children with higher blood Pb 23 

### levels systematic ally test lower than their true IQ value and that children with lower 24 

### blood Pb levels test higher than their true IQ value. Thus, in a population of children, on a 25 

### given assessment, some children will test lower than their true value and others will test 26 

### h igher than their true value. In such cases, between

- group differences will be 27 

### measureable on a population basis. Error in the measurement of IQ in an individual will 28 

### contribute nondifferential error on a population

- level and bias the association to the nul l. 29


### The issue of individual

- level versus population

- level risk also pertains to the relevance of 30 

### the magnitude of decrease in cognitive function or increase in behavioral problems per 31 

### incremental increase in blood Pb level. Although fractional changes in I Q, memory, or 32 

### inattention may not be consequential for an individual, they may be consequential on a 33 

### population level, especially in the two tails of the distribution ( Bellinger, 2007 , 2004b )

. 34 

### Weiss ( 1990 ) predicted, on purely statistical grounds, that a downward shift of five points 35 

### in mean IQ, if the amount of dispersion in the distribution remained the same, should be 36 

### accompanied by a doub ling of the numbers of individuals with scores two or more 37 

### standard deviations below the mean and a reduction by half of the number of individuals 38February 2012 5

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### with scores two or more standard deviations above the mean. Thus, for an individual 1 

### functioning in the low ra nge of the IQ distribution, a Pb

- associated decline of several 2 

### points might be sufficient to drop that individual into the range associated with increase 3 

### risk of educational, vocational, and social failure. 4 

### This hypothesis is supported by several observat ions. In a recent study of fourth graders 5 

### across the entire state of North Carolina, Miranda et al. ( 2009 ) found that higher blood Pb 6 

### level measured once in each child between age 9 months and 3 years was associated with 7 

### larger decreases in EOG scores in lower distributio n of EOG scores ( Figure 5

- 7 ). 8 

### Needleman et al. ( 1982 ) found that a downward shift in the mean IQ value was associated 9 

### not only with a substantial increase in the percentage of individuals achieving very low 10 

### scores, but also with substantial decreases in percentages achieving very high scores. 11 

### Based on the study by Bellinger et al. ( 1987 ) examining a ssociations between blood Pb 12 

### level and IQ scores in children, Weiss ( 1988 ) discussed the shift in the population 13 

### distribution of IQ from a mean of 100 and a standard deviation of 15 to a mean of 95, a 14 

### 5% reduction. When the mean IQ level is 100, 2.3% of th e individuals in a given 15 

### population would score above 130. However, with the population distribution shift and 16 

### the resulting mean decline in IQ, only 0.99% of the individuals would score above 130. 17 

### Weiss ( 1988 ) stated that the implication of such a loss tr anscends the current 18 

### circumscribed definitions of risk. In a similar analysis presented in the 2006 Pb AQCD, 19 

### using a blood Pb

- IQ effect estimate of −0.9 points/ μ g/dL (based on the median of effect 20 

### estimates for blood Pb levels <10 μ g/dL), the fraction of t he population with an IQ level 21 

### less than 80 more than doubles from 9% with a blood Pb level of 0 μg/dL to 23% with a 22 

### blood Pb level of 10 μg/dL ( Figure 5

- 24 ). The proportion with an IQ level below 70, a 23 

### level often requiring community support to live ( WHO, 1992 ) increases from a little over 24 

### 2% with a blood Pb level of 0 μg/dL to about 8% with a blood Pb level of 10 μg/dL 25 

### ( Figure 5

- 24 ) ( U.S. EPA, 2006b )

. 26February 2012 5

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– Do Not Cite or Quote Source: U.S. EPA (2006) Pb AQCD 

### Figure 5
- 24 Effect of blood Pb level on the proportion of the population with IQ levels <70 and <80 points.
Evidence also has demonstrated larger changes in neurodevelopmental outcomes in 1 

### children with additional risk factors such as lower SES ( Tong et al., 2000 ; Bellinger et 2 

### al., 1990 ) , mothers with low self

- esteem ( Surkan et al., 2008 ) , or co
- exposures to 3 

### manganese ( Kim et al., 2009a )

. Moreover, interventions that shift the population mean, in 4 

### a beneficial direction, by an am ount that is without clinical consequence for an individual 5 

### have been shown to produce substantial decreases in the percentage of individuals with 6 

### values that are clinically significant ( Bellinger, 2007 , 2004 b )

. 7 

### Also supporting the public health significance of blood Pb level

- associated changes in 8 

### cognitive function and behavior are observations within the same cohorts that higher 9 

### blood Pb level are associated with decrements in IQ and measures of executive function 10 

### earlier in age and with lower academic performance, antisocial behavior, or delinquent 11 

### behavior assessed later in adolescence or in early adult hood ( Chandramouli et al., 2009 ; 12 

### Wright et al., 2008 ; Kordas et al., 2006 ; Canfield et al., 2004 ; Kordas et al., 2004 ; 13 

### Canfield et al., 2003b ; Dietrich et al., 2001 ; Dietrich et al., 1993a ; Bellinger et al., 1992 )

. 14February 2012 5

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### Thus, the blood Pb

- associated deficits measured in children may set affected children on 1 

### trajectories more prone toward lower educational attainment and life success. Studies also 2 

### found that higher blood Pb level is associated with measures of inattention and 3 

### hyperactivity as rated by teachers or parents and by objective tests and with ADHD 4 

### diagnosis or diagnostic indices ( Nicolescu et al., 2010 ; Roy et al., 2009a ; Nigg et al., 5 

### 2008 )

. These findings demonstrate t hat associations of blood Pb level with small changes 6 

### in a health index can be markers or indicators of other changes that are likely to have 7 

### occurred whose significance is more certain. 8



### 5.3.13 Summary and Causal Determination
Recent epidemiologic and toxicologic al studies substantiated the strong body of evidence 9 

### presented in the 2006 Pb AQCD for the role of Pb in nervous system effects in two 10 

### domains: cognitive function and behavior in children. Specifically, new studies of 11 

### cognitive function focused on indices of learning, memory, and measures of executive 12 

### function in children consistently showed Pb

- related impairments. Coherence for these 13 

### findings in children was provided by extensive toxicological evidence for Pb

- induced 14 

### impairments in homologous tests of lear ning and memory in juvenile rats and monkeys. 15 

### New epidemiologic studies of behavior focused on and found evidence for Pb

- related 16 

### effects on inattention and impulsivity in children. These findings were supported by a 17 

### large historical evidence base in juveni le animals. Consistent with these observations, 18 

### new evidence demonstrated associations with ADHD in children. Recent studies of adults 19 

### without current occupational Pb exposures continued to find associations between bone 20 

### Pb levels, a biomarker of cumulativ e Pb exposure, and poorer cognitive function. 21 

### Additional toxicological evidence for Pb

- induced inhibition of neurotransmitter release, 22 

### decline in synaptic plasticity, and decreases in the magnitude of LTP strengthened the 23 

### biological plausibility for Pb exp osure effects on decrements in both cognitive function 24 

### and behavior in children. This section presents a summary of the collective body of 25 

### evidence and identifies the new insights provided by recent studies. 26



### 5.3.13.1 Cognitive Function in Children
Epidemiologic stu dies provide robust evidence for higher blood Pb levels being 27 

### associated with lower FSIQ in children ages 3

- 11 years ( Figure 5

- 2 and Table 5

- 3 ), with 28 

### the strongest evidence demonstrated by the consistency of association in prospective 29 

### studies in diverse populations ( e.g., varying distributions of blood Pb levels, SES, 30 

### parental intel ligence, and quality of caregiving) and the persistence of associations after 31 

### adjustment for potential confounding by SES, parental intelligence, caregiving 32February 2012 5

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### environment, and other environmental exposures. Recent epidemiologic studies in 1 

### children focused on and found associations of concurrent blood Pb levels with specific 2 

### indices of cognitive function such as reading and verbal skills, memory, learning, and 3 

### visuospatial processing. Several new studies shifted the weight of evidence for 4 

### associations with cog nitive performance to lower blood Pb levels (primarily concurrent), 5 

### with populations means in the range of 2

- 7 μg/dL ( 20 11 ; Kim et al., 2009b ; Min et al., 6 

### 2009 ; Miranda et al., 2009 ; Zailina et al., 2008 ; Chiodo et al ., 2007 )

. Evidence clearly 7 

### indicates that prenatal cord and concurrent blood Pb levels are associated with lower 8 

### cognitive function in younger children ages 6 months to 3 years ( Table 5

- 4 ). Coherence 9 

### for findings in children is derived from extensive evidence in animals that gestational and 10 

### early postnatal Pb exposures resulted in impaired learning and memory, in particular, in 11 

### homologous tests of spatial memory and rule learning and reversal. Reflecting the 12 

### tendency to examine higher Pb exposures, toxicological studies most clearly 13 

### demonstrated neurodevelopmental effects with blood Pb levels of 20

- 40 μg/dL in 14 

### animals. However, s everal new studies added to the evidence for impaired learning and 15 

### memory in animals with lower blood Pb levels, 8

- 17 μg/dL ( Cory

- Slechta et al., 2010 ; Li 16 

### et al., 2009c ; Niu et al., 2 009 ; Virgolini et al., 2008a ; Stangle et al., 2007 )

. 17 

### The large body of evidence indicating associations between blood Pb levels and 18 

### decrements in the diverse set of indices related to learning, memory, and other executive 19 

### functions provides coherence with findings for FSIQ, a global measure of cognit ive 20 

### function that reflects the integration of these individual domains. Further, evidence for 21 

### effects on various diverse measures of cognitive function provides biological plausibility 22 

### for associations observed between blood Pb levels and factors that may be indicators of 23 

### life success, including the level of educational attainment and academic performance. 24



### 5.3.13.2 Behavior in C hildren
Epidemiologic studies in children demonstrate associations of higher blood Pb levels 25 

### with a range of behavioral problems, with the weight of evidence demonstrating 26 

### associations with inattention and hyperactivity as rated by parents or teachers and as 27 

### assessed using objective neuropsychological tests. Previous studies found associations 28 

### with early childhood blood or tooth Pb level ( i.e ., ages 2

- 6 years), and recent studies 29 

### expanded evidence to include associations with concurrent blood Pb level. Recent 30 

### epidemiologic studies consistently found associations with inattention and hyperactivity 31 

### in children ages 1 to 12 years with mean concur rent blood Pb levels of 2 to 5 μg/dL ( Cho 32 

### et al., 2010 ; Nicolescu et al., 2010 ; Plusquellec et al., 2010 ; Chiodo et al., 2007 ; 33 

### Plusquellec et al., 2007 ) , similar to those associated with cognitive function decrements. 34 

### The epidemiologic findings are strengthened by observations in animals of Pb

- induced 35February 2012 5

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### inattention and impulsivity in homologous tests of response inhibition. Such effects in 1 

### animals are most clearly indic ated with gestational and early postnatal Pb exposures that 2 

### result in blood Pb levels of 10 to 40 μg/dL. Whereas previous eviden ce was inconsistent, 3 

### several recent epidemiologic studies indicate associations between higher concurrent 4 

### blood Pb level and hig her prevalence or incidence of ADHD diagnosis and its 5 

### contributing diagnostic indices in children ages 8

– 17 years ( Cho et al., 2010 ; Nicolescu et 6 

### al., 2010 ; Roy et al., 2009a ; Nigg et al., 2008 ; Braun et al., 2006 )

. The biological 7 

### plausibility for associations with ADHD is strongly supported by the large epidemiologic 8 

### and toxicologica l evidence base demonstrating Pb

- associated increases in inattention and 9 

### impulsivity, both of which are primary symptoms of ADHD. A smaller but equally 10 

### consistent body of evidence indicated associations of concurrent and early childhood 11 

### blood Pb levels wit h social misconduct in children and delinquent behaviors in 12 

### adolescents and young adults ( Chandramouli et al., 2009 ; Braun et al., 2008 ; Wright et 13 

### al., 2008 ; Chiodo et al., 2007 )

. Associations of blood Pb levels with ADHD, misconduct, 14 

### and delinquency were observed in populations of children with a wide range of blood Pb 15 

### levels, 1 to 11 μg/dL, all similar in the strength of evidence. 16


### While the different behavi oral indices are examined, Pb exposure also was found to affect 17 

### behavior (decreased ability to escape predators or capture prey) in aquatic an d terrestrial 18 

### species (Sections 7.2 and 7.3 ). 19



### 5.3.13.3 Other Nervous System Effects in Children
A few new toxicological studies augmented the evidence for Pb

- related effects to the 20 

### visual system by demonstrating retinal changes in male rodent offspring in association 21 

### with lower blood Pb levels (<15 μg/dL) than previously examined (20 to > 100 μg/dL) 22 

### (Section 5.3.4.3 ). A small body of epidemiologic evidence together with a large historical 23 

### base of toxicological evidence indicated associations of Pb biomarkers or exposure with 24 

### impaired auditory function. Associations were found in children ranging from 4 to 25 

### 19 years in age and with mean concurrent blood Pb levels of 7

- 12 μg/dL Section 5.3.4.1 ). 26 

### Whi le mood and emotional state have been examined less frequently compared with 27 

### inattention and misconduct, several studies found associations of biomarkers of 28 

### cumulative Pb exposure ( i.e., tooth or childhood average blood Pb) and concurrent blood 29 

### Pb levels w ith parental or teacher reports of withdrawn behavior or depression in children 30 

### with mean blood Pb levels 8

- 28 μg/dL (Section 5.3.3.3 ). These findi ngs in children are 31 

### supported by a small body of toxicological studies in which prenatal plus lactational Pb 32 

### exposure resulted in depression

- like behavior in rodent models. Studies also reported 33 

### associations of early childhood average and concurrent blood Pb levels with lower fine 34 

### and gross motor function in children ages 3 to 17 years (Section 5.3.5 ). A common 35February 2012 5

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### observation across studies was finding t hat biomarkers of Pb exposure were associated 1 

### with decrements in multiple neurodevelopmental outcomes, including cognitive function, 2 

### externalizing behaviors, internalizing behaviors, and motor function, within the same 3 

### population. These findings indicate t hat Pb exposure affects a broad spectrum of 4 

### neurodevelopmental effects in children. 5



### 5.3.13.4 Factors that Modify Risk in Children

Several host and environmental factors were examined for their modification of 6 

### associations between blood Pb levels and nervous system effects in children. Although 7 

### each particular factor was examined in only one to two epidemiologic studies, it is 8 

### important to note that several findings are supported by a larger base of toxicological 9 

### evidence. Interactions of blood Pb levels with race/et hnicity and SES are not well 10 

### characterized. Most investigation focused on sex

- based differences. Cumulative 11 

### epidemiologic evidence does not conclusively demonstrate increased risk of males or 12 

### females for Pb

- associated cognitive function decrements. Animal studies continue to 13 

### demonstrate differential effects in males and females that vary depending on the 14 

### endpoint. 15 

### The weight of evidence from animal studies continues to support interactions between 16 

### developmental Pb exposure and stress. New animal studies fi nd a potentiating effect of 17 

### stress, whereby lower concentration Pb exposures impact behavior and memory with co

18 

### exposures to stress than with Pb exposures alone. In comparison, epidemiologic evidence 19 

### for such interactions is sparse; however, a recent study indicated that among children 20 

### with a positive social environment, as characterized by maternal self

- esteem, blood Pb 21 

### level is not associated with a decrease in cognitive function ( Surkan et al., 2008 )

. This 22 

### finding was consistent with a previous study in rats, in which Pb

- exposed animals reared 23 

### in cages with enriched environme nts ( i.e., toys) performed better in a test of spatial 24 

### learning and memory than did their Pb

- exposed littermates reared in traditional caging, 25


### Studies have examined modification of Pb

- associated cognitive function by genes that 26 

### affect Pb toxicokinetics an d/or function in neurophysiological and neurochemical 27 

### processes that mediate cognition. Most investigation has focused on ALAD variants but 28 

### has not consistently found to be in a consistent direction. Inconsistencies also have been 29 

### observed for VDR and dopa mine receptor variants. In addition to host factors, recent 30 

### studies suggested that associations between blood Pb levels and cognitive function in 31 

### children are greater with co

- exposures to environmental tobacco smoke ( Froehlich et al., 32 

### 2007 ) and manganese ( Claus Henn et al. ; Kim et al., 2009b )

. . 33February 2012 5

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### 5.3.13.5 Nervous System Effects in Adults
Among adults, associations of blood Pb l evel with the spectrum of nervous system effects 1 

### ( e.g., impairments in memory, attention, mood, balance, motor function) were most 2 

### consistently observed in occupationally

- exposed adults with blood Pb levels ≥ 14 μg/dL. 3 

### Evidence in adults without occupation al Pb exposures is derived primarily from cross

4 

### sectional analyses in a few different cohorts, thus the important magnitude, timing, 5 

### frequency, and duration of Pb exposures contributing to the observed associations are 6 

### uncertain. The weight of evidence dem onstrated associations of bone Pb levels and not 7 

### blood Pb levels with poorer cognitive performance, including declines in function over 8 

### time ( Bandeen

- Roche et al., 2009 ; Weisskopf et al., 2007a )

. These findings point to a 9 

### stronger effect of cumulative Pb exposure, including likely higher past exposures. One 10 

### explanation for the overall weaker body of evidence in adults may be that cognitive 11 

### reserve may compensate for th e effects of Pb exposure on learning new information. 12 

### Compensatory mechanisms may be overwhelmed with age, which may provide an 13 

### explanation for more consistent associations observed for higher tibia Pb levels, 14 

### representing higher long

- term or cumulative Pb exposure. 15 

### Based on a smaller body of epidemiologic studies, blood and bone Pb levels were 16 

### associated with essential tremor and PD, respectively, in adults (Section 5.3.7.1 ). 17 

### However, in these case

- control studies, it is difficult to establish temporality between Pb 18 

### exposure and disease. Support for epidemiologic findings for PD is provided by 19 

### toxicological evid ence for Pb

- induced decreased dopaminergic cell activity in the 20 

### substantia nigra, which contributes to the primary symptoms of Parkinson’s disease. 21 

### Whereas evidence for association with Alzheimer's Disease in adults is weak, 22 

### developmental but not adult

- only Pb exposures of monkeys (early postnatal, PND 1

- 400) 23 

### and rats (lactational) has been shown to induce formation of amyloid plaques, pathology 24 

### that underlies Alzheimer's Disease (Section 5.3.7.2 ). Thus, epidemiologic studies that 25 

### examined concurrent bone or brain Pb levels or occupational Pb exposure may not have 26 

### examined the etiologically relevant exposure period. 27 

### Rather than examining externalizing behaviors and criminal behavior, a small body of 28 

### studies of behavior in adults examined and found associations of blood ( Bouchard et al., 29 

### 2009 ) and tibia ( Rajan et al., 2008 ) Pb levels with depression and anxiety symptoms. It is 30 

### not surprising that Pb exposure may increase the risk of different nervous system 31 

### endpoints in children and adults given the predominance of different neurophysiological 32 

### processes operating at different ages, for example, neurogenesis and brain development 33 

### in children and neurodegeneration in adults. 34February 2012 5

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### 5.3.13.6 Neurophysiological and Neurochemical Changes
Extensive evidence from toxicological studies c learly provides the coherence and 1 

### biological plausibility for effects observed in epidemiologic and toxicological studies on 2 

### cognitive function, behavior, mood, and neurodegenerative conditions by characterizing 3 

### underlying mechanisms (Section 5.3.8 ). Dopamine plays a key role in cognitive functions 4 

### mediated by the prefrontal cortex and also motor functions mediated by the substantia 5 

### nigra. Thus, exten sive evidence for Pb

- induced dopaminergic changes in animals 6 

### provides mechanistic support for associations in humans between blood Pb levels and 7 

### cognitive deficits and in adults for associations with Parkinson’s Disease. Current 8 

### toxicological research has been expanded to document that early

- life Pb exposure can 9 

### contribute to neurodegeneration and neurofibrillary tangle formation in the aged brains of 10 

### animals. Pb has been shown to induce complex neurochemical changes in the brain that 11 

### differ by region of th e brain, neurotransmitter type, age and sex of the organism. These 12 

### changes remain aberrant over time and are dynamic in nature. Pb exposure affects 13 

### NMDA receptors, which may explain findings in animals and humans for Pb

- associated 14 

### symptoms of depression an d withdrawn behavior. Recent toxicological studies continue 15 

### to document Pb exposure effects on synapse formation, adhesion molecules, and 16 

### nitrosative stress. A new study of epigenetics details that Pb exposure affects methylation 17 

### patterns in rodent brains, which may provide a mechanism by which prenatal Pb exposure 18 

### leads to impaired neurodevelopmental function later in life. Biological plausibility also is 19 

### provided by a small body of evidence in young adults in which childhood blood Pb levels 20 

### are associated with altered structure and activity in regions of the brain (assessed by MRI 21 

### or MRS) that mediate cognitive processes and behavior. 22



### 5.3.13.7 Lifestages and Duration of Lead Exposure
Toxicological studies clearly demonstrate that in utero and early postnatal exposu re to Pb 23 

### results in impaired learning, memory, and behavior. This evidence is well supported by 24 

### knowledge that processes such as neurogenesis, synaptogenesis, and synaptic pruning are 25 

### most active during this developmental period. In epidemiologic studies r eviewed in the 26 

### 2006 Pb AQCD, biomarkers of prenatal, early life, concurrent, and cumulative Pb 27 

### exposures were associated with decrements in neurodevelopmental function in children, 28 

### with no clear indication that blood Pb levels measured at a particular life stage was more 29 

### strongly associated with neurodevelopmental effects (Section 5.3.9 ). Distinguishing 30 

### among the effects of Pb exposures at different lifestages is difficult in epidemiologic 31 

### studies due to the high correlations among blood Pb levels within children over time. 32 

### Recent studies in children primarily examined concurrent blood Pb levels but also found 33 

### association with prenatal cord ( Pilsner et al., 2010 ; Jedrychowski et al., 2009b ; 34February 2012 5

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### Plusquellec et al., 2007 ) and early childhood blood P b levels ( Chandramouli et al., 2009 ; 1 

### Min et al., 2009 ; Miranda et al., 2009 )

. Thus, while biomarkers of Pb exposure ac ross 2 

### lifestages were associated with neurodevelopmental decrements in children, the weight of 3 

### cumulative evidence demonstrated associations with concurrent blood Pb levels. In 4 

### adults, the weight of evidence supported associations of cumulative Pb exposure 5 

### ( i.e., tibia Pb) with poorer cognitive performance. 6 

### With regards to duration of exposure, several epidemiologic studies indicated that Pb 7 

### exposures of less than 1 year, represented by blood Pb levels prenatally (maternal or 8 

### cord) and at age 6, 12, or 24 months, were associated with lower cognitive function in 9 

### children ages 6 months to 3 years ( Table 5

- 4 ). Toxicological studies supported these 10 

### obser vations with effects observed with gestational and early postnatal Pb exposures. 11



### 5.3.13.8 The Lead Concentration
- Response Relationship
In the 2006 Pb AQCD, several individual epidemiologic studies, pooled analyses, and 12 

### meta

- analyses estimated a supralinear blood Pb concentration

- response relationship in 13 

### children, i.e., greater decrements in cognitive function per incremental increase in blood 14 

### Pb level or more negative slope among children in lower strata of blood Pb levels 15 

### compared with children in higher strata of blood Pb levels ( Figure 5

- 23 and Table 5

- 15 ). 16 

### Although a majority of epidemiologic evidence used a blood Pb level of 10 μg/dL to 17 

### define lower and higher blood Pb levels, some found that among children less than age 18 

### 10 years with mean concurrent blood Pb levels of 3 μg/dL, the blood Pb

- cognitive 19 

### funct ion slope was more negative with blood Pb levels <7.5 ( Lanphear et al., 2005 ) or 20 

### <5 μg/dL ( Tellez

- Rojo et al., 2006 )

. 21 

### While explanations for this supralinear relationship have not been well characterized by 22 

### epidemiologic studies, recent toxicological studies that examined lower concentrations of 23 

### experimental Pb exposure suggest that mechanisms may be differentially activated at 24 

### lower and higher Pb exposures. Multiple studies showed U

- or inverse U

- shaped curves 25 

### with lower Pb exposures having different levels or oft en the opposite effect from higher 26 

### exposures. Nonlinear concentration

- response relationships were found for effects such as 27 

### learning (Section 5.3.2.2 ), adult forebrain dopamine levels ( Leasur e et al., 2008 ) , and 28 

### neurogenesis ( Fox et al., 2008 )

. Senso ry organ parameters in animals, namely, the 29 

### numbers of rod photoreceptors and bipolar cells and ERG wave amplitudes also show 30 

### vastly different changes with low versus higher Pb exposure ( Table 5

- 12 ). 31 

### The examination of populations of children with large proportions of subjects at very low 32 

### blood Pb levels has improved the ability to discern a threshold for Pb

- associated effects 33 

### on cognitive function and b ehavior. Several recent epidemiologic studies reported 34February 2012 5

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### associations between blood Pb levels and deficits in cognitive and behavioral endpoints 1 

### in children (ages 8

- 11 years) with mean or range of blood Pb levels ≤ 2 μg/dL ( Cho et al., 2 

### 2010 ; Kim et al., 2009b ; Miranda et al., 2009 )

. Collectively, these new findings in 3 

### children do not provide evidence for a threshold for neurodevelopmental effects of Pb in 4 

### the ranges of blood Pb levels examined to date. 5



### 5.3.13.9 Evidence that Forms the Basis of the Causal Determination
In summary, recent findings strengthen epidemiologic and toxicological evidence 6 

### indicating that Pb exposure is associated with nervous system e ffects. The weight of 7 

### epidemiologic and toxicological evidence clearly supports associations of higher blood 8 

### Pb levels with lower cognitive function in children, i.e., full

- scale IQ and various 9 

### measures of learning and memory. In epidemiologic studies, the se associations were 10 

### substantiated in children ages 1 to 11 years and in populations with mean blood Pb levels 11 

### between 2 and 7 μg/dL. Observation of a supralinear concentration

- response relationship 12 

### and associations with mean (or quantile) blood Pb levels < 2 μg/dL do not provide 13 

### evidence for a threshold for the neurodevelopmental effects of Pb exposure. 14 

### Epidemiologic and toxicological evidence clearly demonstrates Pb

- associated increases 15 

### in behavioral problems, in particular, inattention and impulsivity. A ssociations are 16 

### substantiated in children ages 1 to 12 years with mean concurrent blood Pb levels of 2 to 17 

### 5 μg/dL. In animals, the weight of evidence demonstrates effects on cognition and 18 

### behavior with prenatal and early postnatal Pb exposures that resulte d in blood Pb levels 19 

### of 10 to 40 μg/dL. In epidemiologic studies, associations with cognitive function and 20 

### beha vior were observed after adjustment for a range of potential confounding variables, 21 

### but most commonly, parental IQ, parental education, and other SES

- related variables. In 22 

### children, the weight of evidence supports cognitive function decrements and behavioral 23 

### problems in association with concurrent blood Pb levels. Associations also are observed 24 

### with prenatal, early childhood, and childhood average blood Pb levels, thus uncertainty 25 

### remains regarding the lifestage of exposure within childhood that is associated with the 26 

### greatest risk. The weight of toxicological evidence demonstrates neurodevelopmental 27 

### effects with prenatal and early postnatal Pb expo sures that can have effects persisting to 28 

### adulthood. The biological plausibility for epidemiologic and toxicological findings for 29 

### effects on cognitive function and behavior is provided by evidence characterizing 30 

### underlying mechanisms, including Pb

- induced changes in neurogenesis, synaptogenesis 31 

### and synaptic pruning, long term potentiation, and neurotransmitter function. In adults, the 32 

### timing, level, frequency, and duration of Pb exposure implicated in nervous system 33 

### effects remain uncertain. Among occupatio nally

- exposed adults, a spectrum of nervous 34 

### system effects is associated with concurrent blood Pb level (≥ 14 μg/dL), which reflects 35February 2012 5

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### both current and cumulative exposure. However, in adults without occupational exposure, 1 

### cognitive performance is more stron gly associated with tibia Pb levels than blood Pb 2 

### levels, which indicates an effect of long

- term, cumulative Pb exposures. Based most 3 

### heavily on cognitive function decrements and inattention in children, the collective body 4 

### of evidence integrated across ep idemiologic and toxicological studies is sufficient to 5 

### conclude that there is a causal relationship between Pb exposures and nervous system 6 

### effects. 7



## 5.4 Cardiovascular Effects

5.4.1 Introduction
The 2006 Pb AQCD ( U.S. EPA, 2006b ) concluded that both epidemiologic and animal 8 

### toxicological studies support the relationship between increased Pb exposure and 9 

### increased cardiovascular effects, in particular, increased BP and increased incidence of 10 

### arterial hypertension. Although fewer in number, e pidemiologic studies demonstrated 11 

### associations of blood and bone Pb levels with other cardiovascular diseases (CVDs) in 12 

### adults , such as ischemic heart disease, cerebrovascular disease, peripheral vascular 13 

### disease, and CVD

- related mortality. As the cardiova scular and renal systems are 14 

### intimately linked, cardiovascular effects can arise secondarily to Pb

- induced renal injury 15 

### ( Section 5.5 ). Toxicological studies also provided compelling evidence supporting the 16 

### biological plausibility for Pb

- associated cardiovascular effects by characterizing a 17 

### number of the underlying mechanis ms by which Pb exposure can lead to human 18 

### cardiovascular health effects. Such studies demonstrated that the Pb content in heart 19 

### tissue of animals reflects the increases in blood Pb levels ( Lal et al., 1991 ) , indicating that 20 

### the cardiovascular morbidity associated with blood Pb levels may represent the effects of 21 

### the bioavailable Pb in the target tissue. The strongest evidence supported the role of 22 

### oxidative stress in the pathog enesis of Pb

- induced hypertension. Additionally, several 23 

### toxicological studies characterized other pathways or cellular, molecular, and tissue 24 

### events promoting the Pb

- induced increase in BP. These mechanisms included 25 

### inflammation, adrenergic and sympatheti c activation, renin

- angiotensin

- aldosterone 26 

### system (RAAS) activation, vasomodulator imbalance, and vascular cell dysfunction. 27 

### With regard to the concentration

- response relationship, meta

- analysis of human studies 28 

### found that each doubling of blood Pb level (between 1 and >40 μg/dL measured 29 

### concurrently in most studies) was associated with a 1 mmHg increase in systolic BP and 30 

### a 0.6 mmHg increase in diastolic BP ( Nawrot et al., 2002 )

. On a population

- wide basis, 31 

### the estimated effect size could translate into a clinically significant increase in the 32February 2012 5

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### segment of the population with the highest BP. In a moderately

- sized population, a 1 

### relatively small effect size thus has important health consequences for the r isk of 2 

### sequelae of increased BP, such as stroke, myocardial infarction, and sudden death. It was 3 

### also noted that most of the reviewed studies examining bone Pb levels, biomarkers of 4 

### cumulative Pb exposure, also showed increased BP ( Cheng et al., 2001 ; Hu et al., 1996b ) 5 

### or increased hypertension with increasing bone Pb level ( Lee et al., 2001a )

. Across 6 

### studies, over a range of bone Pb concentrations (< 1.0 to 96 μg/g), every 10 μg/g increase 7 

### in bone Pb was associated with increased odds ratios of hypertension between 1.28 and 8 

### 1.86. Studies observed an average increase in systolic BP of ~0.75 mmHg for every 10 9 

### μg/g increase in bone Pb concentration over a range of <1 to 52 μg/g. 10 

### With regard to etiologically

- relevant timing of Pb exposure, the weight of toxicological 11 

### evidence demonstrated increases in BP after long

- term (> 4 weeks) Pb exposure. In 12 

### epidemiologic studies, as cardiovascular outcomes were most often examined in cross

13 

### sectional studies with one or a limited number of Pb biomarker measurements, 14 

### uncertainty exists as to the specific Pb exposure level, timing, frequency, and duration 15 

### that contributed to the observed associations. While associations of adult bone Pb 16 

### (particularly tibia Pb) with health outcomes in adults are indicative of effects related to 17 

### past or cumulative exposures, interpretation of similar associations involving adult blood 18 

### Pb levels, especially those measured concurrently with o utcomes, are complicated by the 19 

### generally higher past exposures common in this population. Detailed interpretation of Pb 20 

### in blood and bone are provided in Sections 4.3 and 4.7.3 . Briefly, higher past Pb 21 

### exposures in adults increased their bone Pb stores which contribute to current blood Pb 22 

### leve ls through the normal process of bone remodeling, as well as during periods of 23 

### increased bone remodeling and loss ( e.g., osteoporosis and pregnancy). Due to the long 24 

### latency period for the development of increased BP and CVD, associations of 25 

### cardiovascular effects with low concurrent blood Pb levels ( e.g., population means 1.6

- 4 26 

### μg/dL) in adults may be influenced by higher past Pb exposures ( Section 4.4.1 ). 27 

### This section reviews the published studies pertaining to the cardiovascular effects of Pb 28 

### exposure in humans, experimental animals, isolated vascular tissues, and cultured 29 

### vascular cells. With the large and strong existing body of evidence serving a s the 30 

### foundation, emphasis was placed on studies published since the 2006 Pb AQCD ( U.S. 31 

### EPA, 2006b )

. Epidemiologic and toxicological studies continued to augment the evidence 32 

### for increases in BP and hypertension development assoc iated with long

- term Pb exposure 33 

### and expanded the evidence for the biological pathways of these effects. Epidemiologic 34 

### studies strengthened the evidence for associations between Pb biomarkers and 35 

### cardiovascular effects after adjusting for potential confoun ding factors such as age, diet, 36 

### alcohol use, BMI, comorbidities, and smoking. The epidemiologic evidence was 37February 2012 5

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### substantiated with results from several available prospective studies indicating 1 

### associations between Pb biomarkers and the incidence of cardiovasc ular health effects. 2



### 5.4.2 Blood Pressure and Hypertension

5.4.2.1 Epidemiology
The most commonly used indicator of cardiovascular morbidity was increased BP and its 3 

### derived index, hypertension. Hypertension in these studies was defined as diastolic and/or 4 

### systolic BP above certain cut

- points or use of anti

- hypertensive medicines. The BP cut

5 

### points employed have been historically established by reference to informed medical 6 

### opinion, and as medical knowledge has improved BP cut

- points defining hypertension 7 

### have been lowe red over time. Consequently, different studies using “hypertension” as a 8 

### cardiovascular outcome may have assigned different cut

- points, depending on the year 9 

### and location of the study and the individual investigator. All of the new studies in the 10 

### current r eview used the same criteria for hypertension ( e.g., systolic BP at or above 140, 11 

### diastolic BP at or above 90, or use of anti

- hypertensive medications). Studies in the 12 

### medical literature show that elevated BP is associated with increased risk of CVD 13 

### includ ing coronary disease, stroke, peripheral artery disease, and cardiac failure. 14 

### Coronary disease ( i.e., myocardial infarction, angina pectoris, sudden death) is the most 15 

### lethal sequela of hypertension ( Ingelsson et al., 2008 ; Chobanian et al., 2003 ; Pastor

16 

### Barriuso et al., 2003 ; Prospective Studies Collaboration, 2002 ; Kannel, 2000a , b ; Neaton 17 

### et al., 1995 )

. Several recent general population and occupational cohort and cross

18 

### sectional studies strengthened the evidence that blood and bone Pb level were associated 19 

### consistently with measures of BP ( Figure 5

- 25 and Table 5

- 16 ) as well as with the 20 

### prevalence and incidence of hypertension ( Figure 5

- 26 and Table 5

- 17 ). Further, recent 21



### s tudies expanded evidence by finding differences in association among racial/ethnic 22 

### groups, perceived stress, diet, and genetic variants and thus, identified populations 23 

### potentially at increased risk of Pb

- associated cardiovascular effects. 24



### In a cross

- secti onal analysis, Martin et al. ( 2006 ) examined the associations of concurrent 25 

### blood and tibia Pb levels with BP and hypertension in a com munity

- based study of older 26 

### adults (n = 964, age ranging from 50 to 70 years) in Baltimore, MD. A key strength of 27 

### this study was the extensive consideration of potential confounding variables. Four 28 

### models evaluated associations for BP and hypertension. The base model included age, 29 

### sex, BMI, sodium intake, potassium intake, total cholesterol, time of day, testing 30 

### technician, and hypertensive medication use. Other models added SES, race/ethnicity, or 31 

### both as covariates. Blood Pb but not tibia Pb level was a s trong predictor of BP in all 32February 2012 5

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### models; a 1 μ g/dL increase in concurrent blood Pb level was associated with an 1 

### approximately 1 mmHg increase in systolic BP and an approximately 0.5 mmHg increase 2 

### in diastolic BP. Tibia Pb but not blood Pb was associated with h ypertension in logistic 3 

### regression models. The authors applied propensity analysis to their models to better 4 

### account for the effect of other risk factors for hypertension such as race/ethnicity, age, 5 

### and SES that were strongly associated with tibia Pb leve l. The propensity score analysis 6 

### and model adjustment did not substantially change the numerical findings and 7 

### conclusions ( e.g., tibia Pb and hypertension were positively associated independently of 8 

### race/ethnicity and SES), indicating that neither SES nor race/ethnicity confounded the 9 

### association between tibia Pb level and hypertension. No evidence for effect modification 10 

### by race/ethnicity was found either. Martin et al. ( 2006 ) concluded that Pb in blood has an 11 

### acute effect on BP and that Pb contributes to hypertension risk as a function of 12 

### cumulative, chronic exposure (as represented as bone Pb in this population). While 13 

### different aspects of Pb exposure may contribute differentially to increases in BP and 14 

### hypertension, it is important to note that concurrent blood Pb levels in adults also reflect 15 

### cumulative Pb exposure. Thus, its association with BP may not reflect an acute effect but 16 

### may also reflect an effect of cumulative Pb exposure. 17February 2012 5

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– Do Not Cite or Quote a Pb distribution presents the median (IQR) that were estimated from the mean and SD assuming a normal distribution.
b Effect estimates were standardized to 1 μg/dL blood Pb or 10 μg/g bone Pb. Note: In general, results are categorized by specific BP parameter, then by Pb biomarker. For categories with multiple studie s, the order of the studies follows the order of discussion in the text. For associations of a 1 μg/dL increase in blood Pb level ( closed circles) or 10 μg/g increase in bone Pb (open circles) with systolic BP (SBP; blue), diastolic BP (DBP; red), and pulse pressure (PP; purple) in adults. 

### Figure 5
- 25 Concentration
- response relationships (95% CI).February 2012 5

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### Table 5
- 16 Additional characteristics and quantitative data for associations of blood and bone Pb with BP measures for studies presented in Figure 5
- 25

Study Population /Location Parameter Pb Data S tatistical Analysis Effect Estimate β (95% CI) Martin et al. ( 2006 ) 964 men and women, 50

- 70 yr, 40% African American, 55% White, 5% other, in Baltimore, MD BP Concurrent Mean

Blood Pb: Mean (SD): 3.5 (2.3) μg/dL African American: 3.4 (2.3) White: 3.5 (2.4) Tibia Pb: Mean (SD): 18.8 (12.4) μg/g African American: 21.5 (12.6) White: 16.7 (11.9) Multiple linear regression base model adjusted for age, sex, BMI, antihypertensive medication use, dietary sodium intake, dietary potassium intake, time of day, testing technician, serum total cholesterol. SES, race/ ethnicity also included in select model s that are presented in Figure 5

- 25 and tabulated here.) Blood Pb SBP: 1.05 (0.53,1.58) DBP; 0.53 (0.25, 0.81)

Tibia Pb: SBP: 0.07 (

- 0.05, 0.14) DBP: 0.05 (

- 0.02, 0.08)
mmHg per μg/dL blood Pb mmHg per μg/g bone Pb Glenn et al. ( 20 06 ) 575 Pb exposed workers, age 18

- 65 yr, in South Korea (10/1997

- 6/2001) BP Blood Pb mean (SD): Visit 1: 20.3 (9.6), Women Visit 2: 20.8 (10.8), Women Visit 3: 19.8 (10.7), Women Visit 1: 35.0 (13.5), Men Visit 2: 36.5 (14.2), Men Visit 3: 35.4 (15.9), Men
Tibia Pb, mean (SD): Visit 1: 28.2 (19.7), Women Visit 2: 22.8 (20.9), Women Visit 1: 41.7 (47.6), Men Visit 2: 37.1 (48.1), Men
Patella Pb, mean (SD): Visit 3 49.5 (38.5) Women Visit 3 87.7 (117.0) Multivariable models using GEE were used in longitudinal analyses. Models were adjusted for visit number, baseline age, baseline age squared, baseline lifetime alcohol consumption, baseline body mass index, sex, baseline BP lowering medication use, alcohol consumption, body mass index, sex, BP lowe ring medication use. Model 1 (short

- term) Blood Pb concurrent 0.08 (

- 0.01, 0.16) Blood Pb (longitudinal) 0.09 (0.01, 0.16)
Model 4: short and longer

term) Blood Pb concurrent 0.10 (0.01, 0.19) Blood Pb longitudinal: 0.09 (0.01, 0.16)
mmHg per 10 μg/dL blood Pb

Weaver et al. ( 2008 ) 652 current and former Pb workers in South Korea (12/1999

- 6/2001) BP Concurrent Blood Pb: Mean (SD): 30.9 (16.7) μg/dL

Concurrent Patella Pb: Mean (SD): 75.1 (101.1) μg/g Linear regression model adjusted for age, gender, BMI, diabetes, antihypertensive and analgesic medication use, Pb job duration, work status, tobacco and alcohol use SBP Patella Pb 0.0059 (

- 0.008, 0.02) a Blood Pb 0.1007 (0.02, 0.18) a
mmHg per 1 μg/dL blood Pb or 1 μg/g patella Pb Interaction between blood Pb/patella Pb with ALAD and vitamin D receptor polymorphisms not significant. Peters et al. ( 2007 ) 513 elderly men (mean 67 y) from NAS in Greater Boston, MA area BP Tibia Pb: mean (SD): 21.5 (13.4) μg/g

Patella Pb: Mean (SD): 31.5 (19.3) μg/g Logistic and linear regression models adjusted for age, age squared, sodium, potassium, and calcium intake, family history of hypertension, BMI, educational level, pack

- years o f smoking, alcohol consumption, and physical activity SBP Tibia Pb/ High Stress: 3.57 (0.39, 6.75) Low Stress: 0.21 (

- 1.70, 1.29) per SD increase in tibia Pb Patella Pb/ High Stress: 2.98 (

- 0.12, 6.08) per SD increase in tibia Pb Patella Pb/ Low Stress: NRFebruary 2012 5

- 231 Draft

– Do Not Cite or Quote Study Population /Location Parameter Pb Data S tatistical Analysis Effect Estimate β (95% CI) Scinicariello et al. ( 2010 ) 6,016 NHANES III (1988

- 1994) participants ≥ 17 yr BP Concurrent Blood Pb: Overall Mean (SE): 2.99 (0.09) μg/dL Non

- Hispanic Whites: 2.87 (0.09) Non

- Hispanic Blacks 3.59 (0.20) Mexican American 3.33 (0.11) Multivariable linear regression of log

transformed blood Pb level adjusted for age, sex, education, smoking status, alcohol intake, BMI, serum creatinine levels, serum calcium, glycosylated hemoglobin, and hematocrit SBP Non

- Hispanic whites: 1.05 (0.32, 1.78) Non

- Hispanic blacks: 2.55 (1.59, 3.51) Mexican Americans: 0.84 (

- 0.06, 1.74)

DBP Non

- Hispanic whites:

- 0.14 (

- 1.1, 0.82) Non

- Hispanic blacks: 1.99 (1.13, 2.85) Mexican Americans: 0.74 (

- 0.005, 1.48)
mmHg per unit increase in ln Blood Pb Significant interactions with blood Pb and ALAD observed in relation to SBP for non

- Hispanic whit es and non - Hispanic blacks Zhang et al. ( 2010a ) 619 older adult males (mean 67 yr) enrolled in the NAS in Greater Boston, MA area PP Wild type HFE Tibia Pb: Median (IQR):8(12

- 27) μg/g Patella Pb: Median (IQR):26(17

- 37) μg/g C282Y HFE Tibia Pb: Median (IQR):20 (14

- 27) μg/g Patella Pb: Median (IQR):25(17

- 37) μg/g H63D HFE Tibia Pb: Median (IQR):19(14

- 26) μg/g Patella Pb: Median (IQR):27(19

- 37)μg/g Linear mixed effects regression models with repeated measurements adjusted for age; education; alcohol intake; smoking; daily intakes of calcium, sod ium, and potassium; total calories; family history of hypertension; diabetes; height; heart rate; high

- density lipoprotein (HDL); total cholesterol:HDL ratio; and waist circumference PP mmHg per 13 μg/g Tibia Pb: Wild Type HFE: 0.38 (0,1.96) H63D HFE: 3.30 (0.16, 6.46) C282Y HFE: 0.89 (0, 5.24) Any HFE variant: 2.90 (0.31, 5.51)
mmHg per 19 μg/g Patella Pb: Wild Type HFE: 0.26 (0, 1.78) H63D HFE: 2.95 (0, 5.92) C282Y HFE: 0.55 (0, 1.66) Any HFE variant: 2.83 (0.32,5.37) Perlstein et al. ( 2007 ) 593 predominantly white men from NAS in Greater Boston, MA area (1991

- 1997) PP Blood Pb: Overall mean (SD): 6.12 (4.03) μg/dL Mean (SD) quintiles: Q1: 2.3 (0.8) μg/dL Q2: 3.9 (0.3) μg/dL Q3: 5.4 (0.5) μg/dL Q4: 7.4 (0.6) μg/dL Q5: 12.4 (4.4) μg/dL

Tibia Pb: Median: 19 μg/g Mean (SD) quintiles: Q1: 7.4 (3.2) μg/g Q2: 14.1 (1.4) μg/g Q3: 18.9 (1.4) μg/g Q4: 24.9 (2.2) μg/g Q5: 40.9 (14) μg/g BP association assessed using spearman correlation coefficients. PP association(adjusted mean difference) assessed using multiple linear regression model adjusted for age, height, race, heart ra te, waist circumference, diabetes, family history of hypertension, education level achieved, smoking, alcohol intake, fasting plasma glucose, and ratio of total cholesterol to HDL cholesterol PP 4.2 (1.9, 6.5) mmHg higher in men with tibia Pb > 19 μg/g (me dian) compared with men with tibia Pb < μg/g Blood Pb (mean difference): Q5:

- 1.49 (

– 4.93, 1.94) Q4:

- 1.39 (

– 4.94, 2.15), Q3:

- 2.56 (

– 5.78, 0.67) Q2:

- 4.37 (

– 7.88,

– 0.86) Q1: Referent group Tibia Pb (mean difference): Q5: 2.58 (

– 1.15, 6.33) Q4: 2.64 (

– 0.93, 6.21) Q3:

- 0.73 (

– 4.27, 2.82) Q2:

- 3.02 (

– 6.48, 0.44) Q1: Referent groupFebruary 2012 5

- 232 Draft

– Do Not Cite or Quote Study Population /Location Parameter Pb Data S tatistical Analysis Effect Estimate β (95% CI) Navas

- Acien et al. ( 2008 ) Meta

- analysis of studies using bone Pb as an exposure metric and BP as the outcome (8 studies) BP Inverse variance weighted random

effects meta

- analyses BP Po oled Estimates mmHg per 10 μg/g Tibia Pb Prospective/SBP 0.33 (

- 0.44, 1.11) X

- sectional SBP 0.26 (0.02, 0.50) X

- sectional DBP 0.02 (

- 0.15, 0.19) Hypertension per 10 μg/g patella Pb
x - Sectional hypertension OR: 1.04 (1.01, 1.07) Pooled Estimate hypertension OR: 1.04 (0.96, 1.12) Yazbeck et al. ( 2009 ) b 971 pregnant women, age 18

- 45 yr, in France BP Midpregnancy Blood Pb: PIH group mean (SD): 2.2 (1.4) No PIH group mean (SD): 1.9 (1.2) Multivariable lo gistic regression models adjusted for maternal age; cadmium, manganese, and selenium blood levels; hematocrit; parity; BMI; pregnancy weight gain; gestational diabetes; educational level; SES; geographic residence; and smoking status and alcohol consumptio n before and during pregnancy Log

- transformed blood Pb at mid

- pregnancy SBP: r = 0.08; p = 0.03 DBP: r = 0.07; p = 0.03 Significant correlations also observed after 24 weeks of gestation and after 36 weeks of gestation.

Elmarsafawy et al. ( 2006 ) b 471 elderly men (mean 67 yr) from NAS in Greater Boston, MA area BP Blood Pb: Mean (SD): 6.6 (4.3) μg/dL

Tibia Pb: Mean (SD): 21.6 (12.0) μg/g

Patella Pb: Mean (SD): 31.7 (18.3) μg/g Linear regression models adjusted for age, BMI, family history of hypertension, history of smoking, dietary sodium intake, and cumulative alcohol ingestion Tibia Pb High calcium group (>800 mg/d): SBP: 0.40.(0.11, 0.70)
Low calcium group (<800 mg/d): SBP: 0.19 (0.01, 0.3 7)
mmHg per μg/g tibia Pb

a 95% CIs estimated from given p

- value.
b References not included in Figure 5

- 25

.February 2012 5

- 233 Draft

– Do Not Cite or Quote Note: Studies are categorized by Pb biomarker. Within each category, studies generally are presented in order of discussion i n the text. ( a ) The outcomes plotted are hypertension prevalence with the exception of Yazbeck et al. ( 2009 ) which measured pregnancy induced hypertension and Peters et al. ( 2007 ) which measured hypertension incidence. ( b ) ALAD2 vs. 1 indicates comparison between ALAD 2 carriers ( e.g., ALAD1

- 2 and ALAD2

- 2) and ALAD 1 homozygotes ( e.g., ALAD1

- 1). (c) Effect estimates were standardized to a 1 μg/dL increase in blood Pb. (d) Effect estimates were standardized to a 10 μg/g increase in bone Pb. 

### Figure 5
- 26 Odds ratios (95% CI) for associations of blood and bone Pb with hypertension prevalence and incidence. ► Reference Martin et al. (2006)

Peters et al. (2007)

Elmarsafawy et al. (2006)

Yazbeck et al. (2009)

Muntner et al. (2005)

Scinicariello et al. (2010) age ≥ 17 y mn BLL= 2.99 μ g/dL

Park et al. (2009)
mn BLL= 3.52 μ g/dL Population

High Stress

High Stress Low Calcium
High Calcium

Pregnant Women

Non

- Hispanic Whites

Non

- Hispanic Blacks

Mexican Americans

Non

- Hispanic Whites

Non

- Hispanic Blacks

Mexican Americans

Overall White Men Black Men White Women Black Women Men <50 Men >50 Women <50 Women >50 Study Location Baltimore, MD

Boston, MA

NAS

France

NHANES III

NHANES III
1998

- 1994

NHANES III Blood Pb ( μ g/dL) 3.5 (2.3) Tibia Pb ( μ g/g) 18.8 (12.4) 21.5 (13.4) Patella Pb ( μ g/g) 31.5 (19.3) 6.6 (4.3) 21.6 (12.0) 31.7 (18.3) 6.6 (4.3) 21.6 (12.0) 31.7 (18.3)
1.2

- 1.7 1.71

- 2.30 >2.30 <1.06 1.06

- 1.63 1.63

- 2.47 >2.47 <1.06 1.06

- 1.63 1.63

- 2.47 >2.47 <1.06 1.06

- 1.63 1.63

- 2.47 >2.47 0.7

- 1.4 1.5

- 2.3 2.4

- 3.7 3.8

- 52.9 2.4

- 3.7 0.7

- 1.4 1.5

- 2.3 2.4

- 3.7 3.8

- 52.9 2.4

- 3.7 0.7

- 1.4 1.5

- 2.3 2.4

- 3.7 3.8

- 52.9 2.4

- 3.7 3.52 (0.10) Comparison per 2.5 μ g/dL

per 12.9 μ g/g per 11.6 μ g/g

per 17.1 μ g/g per 1 μ g/g per 1 μ g/g per 1 μ g/g per 1 μ g/g per 1 μ g/g per 1 μ g/g Reference Q2 v Q1 Q3 v Q1 Q4 v Q1 Reference Q2 v Q1 Q3 v Q1 Q4 v Q1 Reference Q2 v Q1 Q3 v Q1 Q4 v Q1 Reference Q2 v Q1 Q3 v Q1 Q4 v Q1 Reference Q2 v Q1 Q3 v Q1 Q4 v Q1 ALAD2 v 1 b Reference Q2 v Q1 Q3 v Q1 Q4 v Q1 ALAD2 v 1 b Reference Q2 v Q1 Q3 v Q1 Q4 v Q1 ALAD2 v 1 b Continuous per 0.75 μ g/dL increase

0 1 2 3 4 5 6 Odds Ratio (95% CI)February 2012 5

- 234 Draft

– Do Not Cite or Quote 

### Table 5
- 17 Additional characteristics and quantitative data for association s of blood and bone Pb with hypertension measures for results presented in Figure 5
- 26

Study Population/ Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI) Martin et al. ( 2006 ) 964 men and women, 50

- 70 y, 40% African American, 55% White, 5% other, in Baltimore, MD Hypertension (current use of antihypertensive medication, mean SBP ≥ 140 mmHg or DBP ≥ 90 mmHg) Blo od Pb: Mean (SD): 3.5 (2.3) μg/dL

Tibia Pb: Mean (SD): 18.8 (12.4) μg/g Logistic regression models adjusted for age, sex, BMI, antihypertensive medication use, dietary sodium intake, dietary potassium intake, time of day, testing technician, and serum hom ocysteine Blood Pb level OR=1.02 (0.87, 1.19) Tibia Pb OR=1.24 (1.05, 1.47) mmHg per μg/dL blood Pb mmHg per μg/g bone Pb

Weaver et al. ( 20 08 ) a 652 current and former Pb workers in South Korea (12/1999

- 6/2001) Hypertension (mean SBP ≥ 140 mmHg, DBP ≥ 90 mmHg; and/or use of antihypertensive medications; or physician diagnosis) Blood Pb: Mean (SD): 31.9 (14.8) μg/dL

Patella Pb: Mean (SD) : 37.5 (41.8) μg/g Logistic regression models adjusted for age, gender, BMI, diabetes, antihypertensive and analgesic medication use, Pb job duration, work status, tobacco and alcohol use None of the Pb exposure metrics examined were (blood, patella, and ln patella) were significantly associated with hypertension (results not reported)

Peters et al. ( 2007 ) 513 elderly men (mean 67 y) from NAS in Greater Boston, MA area Hypertension (mean SBP >140 mmHg, DBP >90 mmHg; or physician diagnosis) Tibia Pb: mean (SD): 21.5 (13. 4) μg/g

Patella Pb: Mean (SD): 31.5 (19.3) μg/g Cox proportional hazards models adjusted for age, age squared, sodium, potassium, and calcium intake, family history of hypertension, BMI, educational level, smoking, alcohol consumption, baseline SBP and DBP, and physical activity Hypertension Incidence High Stress RR=2.66 (1.43, 4.95) per SD increase in tibia Pb RR=2.64 (1.42, 4.92) per SD increase in patella Pb

Elmarsafawy et al. ( 2006 ) 471 elderly men (mean 67 y) from NAS in Greater Boston, MA area Hypertension (mean SBP ≥ 160 mmHg, DBP ≥ 95 mmHg; and/or physician diagnosis with current use of antihypertensive medications) Blood Pb: Mean (SD): 6.6 (4.3) μg/dL

Tibia Pb: Mean (SD): 21.6 (12.0) μg/g

Patella Pb: Mean (SD): 31.7 (18.3) μg/g Logistic regression models adjusted for age, BMI, family history of hypertension, history of smoking, dietary sodium intake, and cumulative alcohol ingestion Low calcium group (<800 mg/d): Blood Pb: 1.07 (1.00, 1.15) Tibia Pb: 1 .02 (1.00, 1.04) Patella Pb: 1.01 (1.00, 1.03)
High calcium group (>800 mg/d): Blood Pb: 1.03 (0.97, 1.11) Tibia Pb: 1.01 (0.97, 1.04) Patella Pb: 1.01 (0.99, 1.03) Per μg/dL blood Pb or μg/g tibia or patella Pb Yazbeck et al. ( 2009 ) 971 pregnant women, age 18

- 45 y, in France PIH (SBP ≥ 140 mmHg or DBP ≥ 90 mmHg after the 22nd wk of gestation) Blood Pb: PIH group mean (SD): 2.2 (1.4) μg/dL No PIH group mean (SD): 1.9 (1.2) μg/dL
Q1: <1.20 μg/dL Q2: 1.20

- 1.7 0 μg/dL Q3: 1.71

- 2.30 μg/dL Q4: >2.30 μg/dL Multivariable logistic regression models adjusted for maternal age, Cd, Mn, and Se blood levels, parity, hematocrit, BMI, gestational diabetes, educational levels, SES, geographic residence, and smoking status du ring pregnancy PIH Blood Pb OR=3.29 (1.11, 9.74) per 1 unit increase in log maternal blood Pb level
Q1: Reference group Q2: OR 1.84 (0.77, 4.41) Q3: OR=2.07 (0.83, 5.13) Q4: OR=2.56 (1.05, 6.22)February 2012 5

- 235 Draft

– Do Not Cite or Quote Study Population/ Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI) Muntner et al. ( 2005 ) 9,961 NHANES (1999

- 2002) participants Hypertension (current use of antihypertensive medication, SBP ≥ 140 mmHg, or DBP ≥ 90 mmHg) Concurrent Blood Pb: Overall Mean (CI): 1.64 (1.59

- 1.68) μg/dL quartile 1: <1.06 μg/dL, quartile 2: 1.06

- 1.63 μg/dL, quartile 3: 1.63

- 2.47 μg/dL, and quartile 4: ≥ 2.47 μg/dL Multivariable logi stic regression models adjusted for age, sex, diabetes mellitus, BMI, cigarette smoking, alcohol consumption, high school education, and health insurance status Adjusted OR Non

- Hispanic white: Q1: Reference group Q2 OR=1.12 (0.83, 1.50) Q3 OR=1.03 (0.78, 1.37) Q4 OR=1.10 (0.87, 1.41)

Non

- Hispanic black Q1: Reference group Q2 OR=1.03 (0.63, 1.67) Q3 OR=1.12 (0.77, 1.64) Q4 OR=1.44 (0.89, 2.32)

Mexican American Q1: Reference group Q2 OR=1.42 (0.75, 2.71) Q2 OR=1.48 (0.89, 2.48) Q3 OR=1.54 (0.99, 2.39) Significant trend (p=0.04) Scinicariello et al. ( 2010 ) 6,016 NHANES III (1988

- 1994) participants ≥ 17 y Hypertension (current use of antihypertensive medication, SBP ≥ 140 mmHg, or DBP ≥ 90 mmHg) Concurrent Blood Pb: Mean (SE): 2.99 (0.09) μg/dL Q1 0.7

- 1.4 μg/dL, Q2 1.5

- 2.3 μg/dL, Q3 2.4

- 3.7 μg/dL, Q4 3.8

- 52.9 μg/dL

Non

- Hispanic Whit es: 2.87 (0.09) Non

- Hispanic Blacks 3.59 (0.20) Mexican American 3.33 (0.11) Multivariable logistic regression model adjusted for race/ethnicity, age, sex, education, smoking status, alcohol intake, BMI, serum creatinine levels, serum calcium, glycosylated hemoglobin, and hematocrit Non

- Hispanic whites: Q1: Reference group Q2 POR=1.21 (0.66, 2.24) Q3 POR=1.57 (0.88, 2.80) Q4 POR=1.52 (0.80, 2.88) ALAD1

- 2/2

- 2: POR= 0.76 (0.17, 3.50) ALAD

- 1: Reference group

Non

- Hispanic blacks: Q1 Reference Q2 POR=1.83 (1.08 , 3.09) Q3 POR=2.38 (1.40, 4.06) Q4 POR=2.92 (1.58, 5.41) ALAD1

- 2/2

- 2: POR= 3.40 (0.05, 219.03) ALAD

- 1: Reference group

Mexican Americans: Q1 Reference Q2 POR=0.74 (0.24, 2.23) Q3 POR=1.43 (0.61, 3.38) Q4 POR=1.27 (0.59, 2.75) ALAD1

- 2/2

- 2: POR= 0.49 (0.08, 3.20) ALAD

- 1: Reference group
POR for hypertension with ALAD2 carriers across quartiles of blood Pb level also reported. ALAD2 carriers associated with hypertension in non

Hispanic whites.February 2012 5

- 236 Draft

– Do Not Cite or Quote Study Population/ Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI) Park et al. ( 2009a ) 12,500 NHANES III (1988

- 1994) participants Hypertension NHANES III Concurrent Blood Pb 3.52 (0.10)

White men <50 yr 4.02 (0.16) ≥ 50 yr 4.92 (0.18) Black men <50 yr 4.55 (0.15) ≥ 50 yr 7.57 (0.22) White women <50 yr 2.09 (0.07) ≥ 50 yr 3.53 (0.12) Black women <50 yr 2.52 (0.09) ≥ 50 yr 4.49 (0.16) Logistic regression models adjusted for age, education, smoking status, cigarette smoking, BMI, hematocrit, alcohol consumption, physical activity, antihypertensive medication use, and diagnosis of type

- 2 diabetes OR per SD (0.75 μg/dL) in log blood Pb: Overall: 1.12 (1.03, 1.23). White men: 1.06 (0.92, 1.22) Black men: 1.17 ( 0.98, 1.38) White women:1.16 (1.04, 1.29) Black women: 1.19 (1.04, 1.38) Men <50 yr: 0.98 (0.80, 1.22) Men > 50 yr: 1.20 (1.02, 1.41), Women <50 yr: 1.23 (1.04, 1.46), Women >50 yr:1.09 (0.94, 1.26).

a Not included in Figure 5

- 26 because OR data were not reported.


### In an occupational cohort in South Korea, Glenn et al. ( 2006 ) simultaneously modeled 1 

### multiple Pb dose measures of individuals collected repeatedly over four years of follow 2 

### up. Thus, through the assessment of cross

- sectional and longitudinal relationships with 3 

### BP, this study provided key insight on potentially i mportant time periods of Pb exposure. 4 

### The initial blood Pb level was used as a baseline covariate and the difference in blood Pb 5 

### level between visits was computed for each subsequent visit. The bone Pb measures were 6 

### used to indicate historical exposure and cumulative dose. Four models were specified: 7 

### Model 1 was conceptualized to reflect short

- term changes in BP associated with recent 8 

### dose; Model 2 to reflect longer

- term changes associated with cumulative dose controlling 9 

### for association of baseline BP with recent dose; Model 3 to reflect longer

- term changes 10 

### associated with cumulative dose controlling for cross

- sectional influence of cumulative 11 

### dose on baseline BP; and Model 4 to reflect both short

- term change with recent dose and 12 

### longer

- term change with cum ulative dose. Concurrent blood Pb and increases in blood Pb 13 

### between visits were associated with increases in systolic BP in Model 1 (short

- term dose) 14 

### and Model 4 (short

- and longer

- term dose). No association was observed between BP and 15 

### tibia Pb at baseline while higher tibia Pb was associated with a decrease in systolic BP in 16 

### each of the models. 17 

### These results indicate that circulating Pb ( e.g., blood Pb) may act continuously on 18 

### systolic BP and reduction in blood Pb may contribute to reductions in BP, while 19 

### cumulative Pb exposure (represented by bone Pb in this study) may contribute to 20 

### hypertension incidence by different mechanisms over longer time periods and in older 21 

### subjects. This analysis in relatively young subjects with a low prevalence of hypertension 22 

### suggests that at least one of the biological pathways that influences how systolic BP 23February 2012 5

- 237 Draft

– Do Not Cite or Quote 

### responds to Pb operates over a relatively rapid timeframe and may reflect an immediate 1 

### response to Pb at a biochemical site of action as a consequence of the biologically 2 

### available Pb circulating in blood. A persistent effect of cumulative doses over a lifetime 3 

### may occur via other mechanisms. This study was strengthened by the analysis of 4 

### associations between changes in blood Pb and changes in BP over time within individua l 5 

### subjects. Bone Pb level may exert influence on blood Pb levels and consequently on BP 6 

### in an aging population with prolonged Pb exposure. Thus, there is strength in the 7 

### conclusions drawn from this study that inform various short and long

- term exposure 8 

### rel ationships with increases in BP and hypertension. It is important to acknowledge the 9 

### uncertainty regarding the applicability of these findings regarding short

- term and long

10 

### term effects in Pb workers with relatively high current Pb exposures contributing t o blood 11 

### Pb levels (mean blood Pb levels over time: 20

- 37 μ g/dL) to adults in the U.S. general 12 

### population whose concurrent blood Pb levels are influenced more by Pb mobilized from 13 

### bone stores. 14 

### In a separate analysis of the same occupationally exposed group in year three of follow

15 

### up, Weaver et al. ( 2008 ) examined cross

- sectionally associations of concurrent patella Pb 16 

### and blood Pb level with sys tolic BP, diastolic BP, and hypertension and effect 17 

### modification by ALAD and vitamin D receptor (VDR) polymorphisms. None of the Pb 18 

### biomarkers were associated with diastolic BP. Patella Pb alone was not significantly 19 

### associated with systolic BP, while bloo d Pb, either alone or with patella Pb was 20 

### significantly associated with higher systolic BP. The patella Pb

- age and blood Pb

- age 21 

### interactions were not statistically significant. There were no significant associations of 22 

### blood Pb or patella Pb with hypertens ion status or effect modification by age or sex. 23 

### Further, interactions between polymorphisms of the VDR and of ALAD with blood Pb 24 

### and patella Pb on systolic BP were not statistically significant. Mean blood Pb level was 25 

### high (30.9 μ g/dL) compared to non
- oc cupational groups. 26


### Weaver et al. ( 2010 ) provided the results of further analysis of this Korean worker coh ort, 27 

### with a focus on determining the functional form of the concentration

- response 28 

### relationships. In a log linear model, the coefficient indicated that every doubling of blood 29 

### Pb level was associated with a systolic BP increase of 1.76 mmHg. The J test, a statistical 30 

### test for determining which, if either, of two functional forms of the same variable 31 

### provides a superior fit to data in non
- nested models ( Davidson and MacKinnon, 1981 ) , 32 

### returned a p

- value of 0.013 in favor of the natural log blood Pb level over the linear blood 33 

### Pb level specification. This analysis indicates that the systolic BP increase in this cohort 34 

### is better described as a logarithmic function of blood Pb level withi n the blood Pb level 35 

### range of the study than by a linear function. 36February 2012 5

- 238 Draft

– Do Not Cite or Quote 

### Several analyses in the NAS cohort of predominantly white older men in the greater 1 

### Boston area found associations of blood and bone Pb level with BP and hypertension and 2 

### indicated effect modification by calcium intake, perceived stress, and HFE gene variants. 3 

### In a cross

- sectional analysis, Perlstein et al. ( 2007 ) found a statistically significant 4 

### association between blood Pb and diastolic BP in adjusted models. The subjects in this 5 

### study had at least one bone Pb measurement during the years 1991

- 1997 and were not on 6 

### antihyperte nsive medication at the time of the measurement. While tibia Pb was not 7 

### significantly associated with BP, it was associated with pulse pressure (PP). Men with 8 

### tibia Pb above the median (19 μ g/g) had a higher mean PP (4.2 mmHg [95%CI: 1.9, 6.5]) 9 

### compared to men with tibia Pb below the median. The trend toward increasing PP with 10 

### increasing quintile of tibia Pb was statistically significant although none of the 11 

### confidence intervals for PP referenced to the lowest quintile of tibia Pb (< 7.4 μ g/g) 12 

### excluded the null value. 13


### Peters et al. ( 2007 ) exami ned cross

- sectionally the modification of the associations of 14 

### tibia and patella Pb with BP and hypertension by self

- reported stress (assessed by 15 

### questionnaire) in NAS men. High stress also has been linked with higher BP, potentially 16 

### via activation of sympa thetic pathways, ROS, and the HPA axis. Among all subjects, 17 

### higher bone Pb level was associated (statistically nonsignificant) with greater odds of 18 

### hypertension status and higher systolic BP. As indicated in Figure 5

- 27 , the association 19 

### between systolic BP and tibia Pb differed between those with high and low self

- reported 20 

### stress (β for tibia Pb x stress interaction = 3.77 [CI: 0.46, 7.09]) per SD increa se in tibia 21 

### Pb. Stress also was found to modify the patella Pb

- BP association (β for patella Pb x 22 

### stress interaction = 2.60 [CI:

- 0.95, 6.15] per SD increase in patella Pb). Neither bone, 23 

### self

- reported stress, nor their interaction was associated significa ntly with diastolic BP. 24 

### Peters et al. ( 200 7 ) also used Cox proportional hazards models to assess the interaction of 25 

### stress and bone Pb level in the development of hypertension among those free of 26 

### hypertension at baseline. The results of this analysis showed that increasing tibia and 27 

### patella Pb were associated with greater risk of developing hypertension among those with 28 

### high stress compared with those with lower perceived stress (RR of developing 29 

### hypertension among those with high stress: 2.66 [CI: 1.43, 4.95] per SD increase in tibia 30 

### Pb and 2.6 4 [CI: 1.42, 4.92] per SD increase in patella Pb). These results provide 31 

### evidence supporting adults with higher stress as a population at increased risk of Pb

32 

### associated cardiovascular effects. 33February 2012 5

- 239 Draft

– Do Not Cite or Quote Source: Peters et al. ( 2007 ) 

### Figure 5
- 27 The relationship between tibia Pb and estimated systolic BP (SBP) for those with high self
- reported stress versus those with low self
- reported stress.
Elmarsafawy et al. ( 2006 ) examined the modification of the r elationship between Pb and 1 

### hypertension risk by dietary calcium, with 467 subjects from the NAS. Responses on a 2 

### semi

- quantitative dietary frequency questionnaire with one

- year recall were used to 3 

### estimate calcium intake. Effect modification by calcium inta ke (dichotomized at 4 

### 800 mg /day) was examined using interaction terms in logistic regression models and by 5 

### conducting analyses stratified on the calcium variable. Increasing bone and blood Pb 6 

### increased the risk of hypertension, particularly among subjects with low dietary calcium. 7 

### Zhang et al. ( 2010a ) examined the effect of poly morphisms of the hemochromatosis gene 8 

### (HFE) on the relationship of bone Pb with PP in NAS men. Subjects had up to three PP 9 

### measurements during the 10 year study period. The overall results demonstrated a strong 10 

### relationship between bone Pb and PP in this s tudy, similar to an earlier cross

- sectional PP 11 

### study of many of the same subjects ( Perlstei n et al., 2007 )

. Zhang et al. ( 2010a ) extende d 12 

### these findings by demonstrating larger increases in PP per unit increase in tibia and 13 

### patella Pb level among those with the H63D variant compared to those with the wild

- type 14 

### or the C282Y variant. 15


### A small number of cross

- sectional studies examined and fou nd that blood Pb level was 16 

### associated with hypertension in pregnancy. Yazbeck et al. ( 2009 ) examined a 17 

### community

- based group of pregnant women in France and unlike most other studies, 18 

### adjusted for potential conf ounding by blood concentrations of cadmium, manganese, and 19 

### selenium. Pregnancy induced hypertension (PIH) was defined as systolic BP >140 mmHg 20 

### and/or diastolic BP >90 mmHg during at least two clinic visits after week 22 of gestation. 21 

### Patients with pre

- exis ting chronic hypertension were excluded. The mean (SD) blood Pb 22February 2012 5

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– Do Not Cite or Quote 

### levels measured during pregnancy were 2.2 (1.4 μ g/dL) in PIH cases and 1.9 (1.2) μ g/dL 1 

### in normotensive women. An association between blood Pb and PIH was observed (OR 2 

### 3.29 [95% CI: 1.11, 9.74] per unit increase in log

- transformed blood Pb level). Cadmium 3 

### and selenium concentrations were comparable between PIH and no PIH groups. 4 

### Adjustment for the metals slightly attenuated but did not eliminate the association 5 

### between blood Pb levels and the ri sk of PIH. Investigators observed no significant 6 

### interactions among blood Pb level, any of the other elements, and maternal characteristics 7 

### in predicting the risk of PIH. Interaction between blood selenium and Pb concentrations 8 

### was not significant, and the putative protection effects of selenium through antioxidative 9 

### properties were not found in this study. 10


### Wells et al. ( 2011b ) measured the relationship of cord blood Pb with BP in 285 women at 11 

### admission to the Johns Hopkins Hospital in Baltimore, MD, during labor and delivery. 12 

### Women with cord blood Pb levels in the highest quartile for the study group 13 

### (>0.96 μ g/dL) had significantly higher systolic and diastolic BP (upon admission and for 14 

### maximum BP) compared to women in the first quartile (<0.46 μ g/dL). The author s used 15 

### Benchmark Dose Software V2.1, developed by the EPA, to estimate the blood Pb level 16 

### (benchmark dose or BMD) and the associated lower confidence limit (BMDL) that was 17 

### associated with one standard deviation (SD) increase in BP. In this study group, one SD 18 

### is approximately equivalent to a 10% increase above the mean for the first quartile blood 19 

### Pb reference group. The BMD approach was used only as a means of quantifying the 20 

### relationship of blood Pb with BP in this population. This analysis indicated that the 95% 21 

### lower bound confidence limit on the maternal blood Pb level (estimated from cord blood 22 

### Pb levels) that was associated with a 1 SD increase in all blood pressure outcomes was 23 

### about 1.4 μg/dL. These reported results are similar to those reported in the 2006 Pb 24 

### AQCD as well as those found 25 years ago but with blood Pb level s in the more recent 25 

### study an order of magnitude lower. 26 

### New analyses using NHANES data continued to indicate associations of Pb biomarkers 27 

### with BP and hypertension. Muntner et al. ( 2005 ) previously used the NHANES 1999

- 28 

### 2002 data t o indicate that concurrent blood Pb levels were associated with hypertension, 29 

### peripheral artery disease (PAD), and chronic kidney disease. The PAD results are 30 

### discussed later in Section 5.4.3.4 , and chronic kidney disease results are discussed in 31 

### Section 5.5.2.2 . Blood Pb increased regularly w ith age (geometric means [95% CIs]: 32 

### 1.28 μ g/dL [1.23, 1.33] in the 18

- 39 age group to 2.32 μ g/dL [2.20, 2.44] in the 75 and 33 

### older age group). Associations were observed between concurrent blood Pb level and 34 

### hypertension across race/ethnicity groups with significant trends observed for 35



### non
- Hispanic blacks and Mexican Americans. 36February 2012 5

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### In the NHANES III 1 988

- 1994 population, Scinicariello et al. ( 2010 ) found a gene

1 

### environment interaction betwe en blood Pb level and ALAD genotype in relation to SBP 2 

### and DBP in a cross

- sectional analysis. These interactions varied across race/ethnicity 3 

### strata. The strongest associations were observed among non
- Hispanic blacks ( Figure 4 

### 5

- 25 , Table 5

- 16 ). A statistically significant interaction was observed betw een concurrent 5 

### blood Pb level and ALAD1

- 2/2

- 2b among non
- Hispanic whites and non
- Hispanic blacks. 6 

### Scinicariello et al. ( 2010 ) also found an interaction between ALAD genotype and blood 7 

### Pb level in the association with hypertension. Statistically significant associations 8 

### between concurrent blood Pb level and hypertension were observed among non
- Hi spanic 9 

### blacks and nonsignificant increases were observed among non
- Hispanic whites and 10 

### Mexican Americans (with the exception of Q2 association for Mexican Americans) 11 

### ( Figure 5

- 26 , Table 5

- 17 ). In addition, non
- Hispanic white ALAD2 carriers in the highest 12 

### blood Pb level quartile 3.8

- 52.9 μ g/dL) had a significantly higher association with 13 

### hypertension compared with ALAD1 homozygous individuals in the highest quartile of 14 

### blood Pb. In the same NHANES population, Park et al. ( 2009a ) predicted bone Pb levels 15 

### using a model developed with NAS data. Concurrent blood Pb was associated with 16 

### hypertension overall in the NHANES population, with larger associations observed 17 

### among black men and women as well as older adults Figure 5

- 26 , Table 5

- 17 ). 18 

### Associations also were obse rved with estimated bone Pb. 19



### 5.4.2.2 Toxicology
Studies on the effect of Pb (as blood Pb level) on systolic BP in unanesthetized adult rats 20 

### consistently reported an increase in BP with increasing blood Pb level as shown in Figure 21 

### 5

- 28 (results summarized in Table 5

- 18 ). An array of studies have provided evi dence that 22 

### long

- term Pb exposure (> 4 weeks), resulting in blood Pb levels below 10 μ g/dL can 23 

### result in the onset of hypertension (after a latency period) in experimental animals that 24 

### persists long after the cessation of Pb exposure ( U.S. EPA, 2006b )

. Tsao et al. ( 2000 ) 25 

### presented evidence for increased systolic and diastolic BP in rats with blood Pb levels 26 

### relevant to those in humans (mean [SD]: 2.15 [0.92] μ g/d L blood Pb; 140 [7] mmHg 27 

### systolic BP, 98 [7] mmHg diastolic BP) compared to untreated controls (mean [SD]: 0.05 28 

### [0.05] μ g/dL blood Pb; 127 [7] mmHg systolic BP, 88 [7] mmHg diastolic BP). As this 29 

### was the lowest Pb level tested, no evidence of a threshold w as evident. Further, a test for 30 

### linear trend revealed a statistically significant, positive trend for increasing BP with 31 

### increasing blood Pb levels up to 56 μ g/dL ( e.g., mean [SD]: 5.47 [2.1] μ g/dL blood Pb; 32 

### 143 [6] mmHg systolic BP, 97 [8] mmHg diastolic BP), with the effect leveling off at 33 

### higher blood Pb levels. 34February 2012 5

- 242 Draft

– Do Not Cite or Quote Note: Crosses represent standard error for blood Pb and BP measurements. If no crossbar is present, error results were not report ed. Arrows represent higher doses tested. 

### Figure 5
- 28 Changes in BP after Pb exposure (represented as blood Pb level) in unanesthetized adult rats across studies.February 2012 5

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### Table 5
- 18 Characteristics of studies of blood Pb with BP measures in animals presented in Figure 5
- 28

Reference a Lifestage; Sex Exposure Duration Exposure Level; Route Mean [SEM] b Blood Pb Level (μg/dL) n ΔSBP (mmHg; lowest blood Pb level compared with control) c Comments
Fiorim et al. ( 2011 ) Adult; M 7 days 4 μg/100 g followed by 0.05 μg/100 g daily; intramuscular 9.98 [1.7] 12 16

Nakhoul et al. ( 1992 ) Adult; M 8 weeks 100 ppm; drinking water 5.3 [3] 7 28 Spontaneously hypertensive rat model Chang et al. ( 2005 ) Adult; M 8 weeks 2% (20,000 ppm) then removal and measurements 1

- 7 mo after; drinking water Range: 4.5

– 83 5 13.8

Tsao et al. ( 2000 ) Adult 8 weeks 0.01

- 2% (100

- 20,000 ppm); drinking water Range of means: 2.15 [0.29]

– 85.76 [1.29] a 10 13

Rizzi et al. ( 2009 ) Adult; M 8 weeks 30

- 90 ppm; drinking water 7.6 [1.3], 19.3 [3.4] 11 13.3
Chang et al. ( 1997 ) Adult; M 8 weeks 0.5% (500 ppm); drinking water 29.1 [0.6] a 10 58
Heydari et al. ( 2006 ) Adult; M 12 weeks 100 ppm; drinking water 26.8 [2.2] 6 25.8 Bravo et al. ( 2007 ) Adult; M 14 weeks 100 ppm; drinking water 23.7 [1.9] a 12 30 Zhang et al. ( 2009a ) Adult; M 40 weeks 100 ppm; drinking water 28.4 [1.1] a 8

- 10 15.3

a Studies are presented in order of increasing duration of exposure. b Standard deviation converted to SEM.
c Difference in systolic BP (SBP) between group means not within one exposure group.


### Experimental animal studies continued to provide evidence that long

- term Pb exposure 1 

### results in sustained arterial hypertension after a latency period. Systolic BP increased in 2 

### rats after exposure to 90

- 1 0,000 ppm Pb (as Pb

- acetate in drinking water) for various 3 

### time periods that resulted in blood Pb levels between 19.3

- 240 μ g/dL ( Mohammad et al., 4 

### 20 10 ; Zhang et al., 2009a ; Badavi et al., 2008 ; Grizzo and Cordellini, 2008 ; Reza et al., 5 

### 2008 ; Bra vo et al., 2007 ; Vargas

- Robles et al., 2007 ; Heydari et al., 2006 ; Bagchi and 6 

### Preuss, 2005 )

. Past studies ha ve shown statistically significant elevations in BP in rats 7 

### with lower blood Pb levels. For example, long

- term Pb exposure to spontaneously 8 

### hypertensive rats (resulting in mean [SEM] blood Pb level: 5.3 [3] μ g/dL) led to 9 

### increased BP ( Nakhoul et al., 1992 )

. Consistent with measurements of systolic BP by tail

10 

### cuff plethysmography, Pb exposure (100 ppm for 14 weeks; mean blood Pb level: 11 

### 24 μ g/dL) also caused an increase in intra

- aortic mean arterial pressure ( B ravo et al., 12 

### 2007 )

. In a study that tested low levels of Pb exposure (30 ppm ; mean blood Pb level: 13 

### 7.6 μ g/dL), a statistically significant increase in systolic BP was not observed despite 14 

### elevated blood Pb level after 8 weeks of treatment. Nonetheless, there was a trend of 15 

### higher BP with higher blood Pb levels ( Rizzi et al., 2009 )

. 16February 2012 5

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### Studies found that Pb

- induced increased BP persisted long after cessation of Pb exposure. 1 

### Bagchi and Preuss ( 2005 ) found that elevated systolic BP was maintained for 210 days 2 

### after Pb exposure cessation. However, chelation therapy using Na 2 

### CaEDTA returned 3 

### syst olic BP to levels comparable to those in rats not treated with Pb ( Bagchi and Preuss, 4 

### 2005 )

. Chang et al. ( 2005 ) reported a partial reversibility of effect after cessation of Pb 5 

### exposure, where Pb

- induced elevated BP decreased but did not return to control levels 7 6 

### months post Pb exposure. After Pb exposure was removed, blood, heart, aorta, and 7 

### kidney Pb levels decreased quickly within the first three months ( Chang et al., 2005 )

. Pb

8 

### induced elevated systolic BP persisted for one month following Pb exposure cessation, 9 

### followed by obvious decreases in BP until 4 months after Pb exp osure cessation. Between 10 

### 4 and 7 months after Pb exposure cessation, the still

- elevated BP did not decrease further, 11 

### thus never returning to control BP levels. Decreases in BP were strongly correlated with 12 

### decreases in blood Pb level after exposure cessati on. 13 

### The aforementioned studies all assessed the relationship between long

- term exposure (> 4 14 

### weeks) of rats to Pb and measures of BP. However, recent research also investigated BP 15 

### elevation occurring after short

- term treatment with Pb (< 4 weeks). Studies found 16 

### increased systolic BP after 7 days of Pb treatment (daily injections resulting in mean 17 

### [SEM] blood Pb levels of 9.98 [1.7] μ g/dL) ( Fiorim et al., 2011 ) and after 2 weeks of Pb 18 

### exposure (100 ppm via drinking water) ( Sharifi et al., 2004 )

. A study utilizing intra

19 

### arterial pressure measurements found that a single high

- dose Pb injection (resultin g in 20 

### mean [SEM] blood Pb levels of 37 [1.7] μ g/dL) increased systolic arterial pressure after 21 

### only 60 minute s ( Simões et al., 2011 )

. These studies suggest that t here is the potential for 22 

### increase in BP following short

- term Pb treatment. It is possible that the increases in BP 23 

### following short

- and long

- term Pb exposures are occurring through separate mechanisms; 24 

### however, studies using both short

- and longer

- term Pb exposure have correlated increased 25 

### BP with an activation of the renin

- angiotensin system ( i.e., increase in angiotensin 26 

### converting enzyme (ACE) activity) ( Section 5.4.2.3 ). Several of these aforementioned 27 

### studies used the injection route of Pb administration, and the relevance of these bolus 28 

### doses over short periods of time to human routes of short

- term exposure is uncertain. 29 

### However, it is important to acknowledge that th e results were similar to those from the 30 

### study that examined short

- term exposure to Pb via drinking water, 31



### 5.4.2.3 Hypertension Modes of Action
The 2006 Pb AQCD examined a number of mechanisms leading to Pb

- induced 32 

### hypertension, including oxidative stress, hormon al and blood pressure regulatory system 33 

### dysfunction, vasomodulation, and cellular alterations. As described below, recent studies 34February 2012 5

- 245 Draft

– Do Not Cite or Quote 

### in experimental animals and cells further supported roles for these potential mechanisms 1 

### in mediating hypertension from Pb exp osure. 2



### Oxidative Stress Response
- Reactive Oxygen Species and Nitric Oxide Several studies discussed in the 2006 Pb AQCD demonstrated a role for oxidative stress 3 

### in the pathogenesis of Pb

- induced hypertension, mediated by the inactivation of nitric 4 

### oxide ( • 

### NO) and downregulation of soluble guanylate cyclase (sGC) ( Dursun et al., 2005 ; 5 

### Attri et al., 2003 ; Gonick et al., 1997 ; Vaziri et al., 1997 ; Khalil

- Manesh et al., 1994 ; 6 

### Khalil

- Manesh et al., 1993b )

. Pb

- induced reduction of bio logically active • 

### NO was found 7 

### not to be due to a reduction in • 

### NO

- production capacity ( Vaziri and Ding, 2001 ; Vaziri et 8 

### al., 1999b ) ; instead it was found to result from inactiv ation and sequestration of • 

### NO by 9 

### ROS ( Malvezzi et al., 2001 ; Vaziri et al., 1999a )

. Oxidative stress from Pb exposure in 10 

### animals may be due to upregulation of NAD(P)H oxidase ( Ni et al., 2004 ; Vaziri et al., 11 

### 2003 ) , induction of Fenton and Haber

- Weiss reactions ( Ding et al., 2001 ; Ding et al., 12 

### 2000 ) , and failure of the antioxidant enzymes, CAT and GPx, to compensate for the 13 

### increased ROS ( Farmand et al., 2005 ; Vaziri et al., 2003 )

. Many biological actions of 14

• 

### NO, such as vasorelaxation, are mediated by cGMP, which is produced by sGC from the 15 

### substrate GTP. Oxidative stress also has been found to play a role in Pb

- induced 16 

### downregulation of sGC ( Farmand et al., 2005 ; Courtois et al., 2003 ; Marques et al., 17 

### 2001 )

. The reduction of the vasodilator • 

### NO leads to increased vasoco nstriction and BP. 18


### Pb

- induced oxidative stress also has been found to induce renal tubulointerstitial 19 

### inflammation which plays a crucial role in models of hypertension ( Rodriguez

- Iturbe et 20 

### al., 2005 ; Rodriguez

- Iturbe et al., 2004 )

. Tubulointerstitial inflammation from treatment 21 

### with Pb has been coupled with activation of the redox sensitive NF

- κB ( Ramesh et al., 22 

### 2001 )

. Pb

- induced hypertension, inflammation, and NF

- κB activation can be ameliorated 23 

### by antioxidant therapy ( Rodriguez

- Iturbe et al., 2004 )

. There is mixed evidence to 24 

### suggest that Pb

- induced hypertension may also be promoted by activation of PKC leading 25 

### to enhanced vascular con tractility ( Valencia et al., 2001 ; Watts et al., 1995 )

. 26 

### Recent studies continued to provide evidence for the role of ROS and • 

### NO metabolism in 27 

### Pb

- induced hypertension and vascular disease. Increased systolic BP after Pb exposure 28 

### was accompa nied by increased superoxide (O 2 - 

### ) and O 2 - 

### positive cells ( Bravo et al., 2007 ; 29 

### Vargas

- Robles et al., 2007 ) , elevated urinary malondialdehyde (MDA, a measure of lipid 30 

### peroxidation) ( Bravo et al., 2007 ) , and increased 3

- nitrotyrosine ( Vargas

- Robles et al., 31 

### 2007 )

. Inhibition of NAD(P)H oxidase, an enzyme that generates O 2 - 

### and hydrogen 32 

### peroxide, was able to block Pb

- induced (1 ppm ) aortic contract ion 33 

### to 5

- hydroxytryptamine (5

- HT) ( Zhang et al. , 2005 )

. Increases in systolic BP, intra

- aortic 34 

### mean arterial pressure, and MDA after Pb exposure (100 ppm ; mean blood Pb level: 35February 2012 5

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– Do Not Cite or Quote 

### 23.7 μ g/dL) were also prevented by treatment with the immunosuppressant, 1 

### mycophenolate mofetil (MMF) (mean blood Pb level in MMF

- treated animals: 27 μ g/dL) 2 

### ( Bravo et al., 2007 )

. MMF has been shown to inhibit endothelial NAD(P)H oxidase, 3 

### which could explain how it decreases Pb

- induced increases in oxidative stress and BP. 4 

### MMF was not found to alter blood Pb levels of animals. Red grape seed extract and 5 

### ascorbic acid supplementation were also able to protect rats from Pb

- induced (100 ppm ) 6 

### increased BP and heart rate, perhaps through the antioxidant properties of the extract 7 

### ( Badavi et al., 2008 ) and vitamin ( Mohammad et al., 2010 )

. Red grape seed extract did 8 

### not alter th e accumulation of Pb in blood, indicating that its protective effect was not 9 

### mediated through altered Pb toxicokinetics; however, internal doses of Pb were not 10 

### measured in the vitamin C study to clarify the mechanism of action of vitamin C. Another 11 

### study f ound that the antioxidant, anti

- inflammatory chemical, curcumin, as well as 12 

### physical exercise training reversed Pb

- induced increases in serum creatinine kinase

- MB 13 

### (CK

- MB), low density lipoprotein (LDL), heart high

- sensitivity C

- reactive protein 14 

### (hs

- CRP), a nd MDA, and Pb

- induced decreases in serum total antioxidant capacity, high 15 

### density lipoprotein (HDL), and heart glutathione peroxidase (GPx); however, internal 16 

### doses of Pb were not measured to clarify the mechanism of action in this study ( Roshan 17 

### et al., 2011 )

. 18 

### Exposure to Pb can also affect the activity and levels of antioxidant enzymes. Male (♂) 19 

### and female (♀) rats exposed to Pb for 18 weeks (100

- 1 , 000 ppm ) had altered res ponses in 20 

### antioxidant enzymes in heart tissue ( Sobekova et al., 2009 ; Alghazal et al., 2008a )

. Pb 21 

### exposure in female rats increased the activity of car diac SOD, GST, GR, and GPx (> 22 

### 100 ppm ) and increased cardiac thiobarbituric acid reactive substances (TBARS, measure 23 

### of lipid peroxidation) (1 , 000 ppm ). Pb exposure in male rats did not affect the activity of 24 

### SOD or production of TBARS, however decreased t he activity of GST and GR 25 

### (>100 ppm ). Male and female rats also accumulated different amounts of Pb in the 26 

### cardiac tissue after similar exposure (♂ 100 ppm : 205% of control, 1 , 000 ppm : 379%; ♀ 27 

### 100 ppm : 246%, 1 , 000 ppm : 775%), which could explain the sex di fferences observed in 28 

### antioxidant enzyme responses. 29 

### Oxidative stress can trigger a cascade of events that promote cellular stress, renal 30 

### inflammation, and hypertension. As was shown previously ( Rodriguez

- Iturbe et al., 31 

### 2005 ) , Pb exposure can increase renal NF

- κB, which was associated with 32 

### tubulointerstitial damage and infiltr ation of lymphocytes and macrophages ( Bra vo et al., 33 

### 2007 )

. These events could also be ablated by MMF treatment, likely due to its anti

34 

### inflammatory and antioxidant properties. Pb also was found to induce inflammation in 35 

### human endothelial cells as a model for vessel intima hyperplasia ( Zeller et al., 2010 )

. The 36 

### pro
- inflammatory cytokine, interleukin (IL)

- 8 protein and mRNA were increased, 37 

### concentration

- and time

- dependently, after in vitro Pb exposure (5

- 50 μ M). Enhanced IL

- 38February 2012 5

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– Do Not Cite or Quote 

### 8 production was mediated through activation of the transcription factor Nrf2 (but not 1 

### NF

- κB, hypoxia inducible factor

- 1, or aryl hydrocarbon receptor), as shown through 2 

### increased nuclear translocation and Nrf2 cellular knockdown experiments. Additionally, 3



### m easures of endothelial stress, NQO1 and HO

- 1 protein, were induced by Pb exposure 4 

### ( Zeller et al., 2010 )

. Pb treatment (20 ppm , i.p., 3 days/week, 8 weeks) increased the 5 

### inflammatory markers hs

- CR P and CK

- MB in rat hearts ( Roshan et al., 2011 )

. 6 

### Oxidative stress affects vascular reactivity and tone through inactivation and 7 

### sequestration of • 

### NO, causing a reduction in biologically active • 

### NO. Recent studies 8 

### affirmed past conclusions on the interplay of ROS and • 

### NO metabolism in the 9 

### cardiovascular effects of Pb. Elevated systolic BP and altered vasorelaxation after Pb 10 

### exposure was accompanied by a decrease in total nitr ates and nitrites (NO X 

### ) ( Mohammad 11 

### et al., 2010 ; Zhang et al., 2007a ; Heydari et al., 2006 )

. Serum NO X 

### levels in Pb

- treated 12 

### rats remained depressed for 8 weeks and then reversed after 12 weeks, despite continued 13 

### elevation in systolic BP ( Heydari et al., 2006 )

. This return of serum NO X 

### levels to levels 14 

### similar in controls could be a result of compensatory increases in endothelial NOS 15 

### (eNOS) attempting to replenish an over

- sequestered • 

### NO supply. With this in mind, 16 

### studies showed increased eNOS protein expression after long

- term Pb exposure in kidney 17 

### ( Zhang et al., 2007a ) and isolated cultured aorta ( Vargas

- Robles et al., 2007 )

. No change 18 

### in inducible NOS was observed in isolated cultured aorta after 1 ppm Pb exposure ( Zhang 19 

### et al., 2007a )

. In contrast to long

- term exposure, Pb treatment over a short time period 20 

### (daily injections resulting in mean [SEM] blood Pb levels of 9.98 [1.7] μ g/dL) was found 21 

### to increase iNOS and phosphorylated eNOS protein ( Fiorim et al., 2011 ) which may 22 

### cause an increase in • 

### NO production and a short

- term increase in • 

### NO bioavailability. 23 

### This increase in • 

### NO bioavailability early after Pb exposure could be the imme diate 24 

### compensatory mechanism against the elevation in BP. 25

• 

### NO, also known as endothelium

- derived relaxing factor, is a potent endogenous 26 

### vasodilator. Toxicological studies continued to investigate the effects of Pb on 27

• 

### NO

- dependent vascular reactivity by using • 

### NO stimulating vasodilators, such as 28 

### acetylcholine (ACh) and sodium nitroprusside (SNP), and • 

### NO inhibiting 29 

### vasoconstrictors, such as L

- NAME. Studies provided mixed evidence; however, results 30 

### suggested that Pb disrupts the vasorelaxant response to • 

### NO in the aorta due to damage to 31 

### the endothelium. Pb exposure (1 ppm and 100 μ M, 1 hour) decreased ACh

- induced 32 

### vasorelaxation, which triggers the release of • 

### NO from the endothelial cell, in isolated rat 33 

### tail artery, suggesting damage to the endothelium ( Silveira et al., 2010 ; Zhang et al., 34 

### 2007a )

. In aortic rings of perinatally exposed rats (1 , 000 ppm through pregnancy and 35 

### lactation, mean blood Pb level: 58.7 μ g/dL), blocking NOS with L

- NAME abolished the 36 

### relaxant response evoked by ACh ( Grizzo and Cordellini, 2008 )

. Howev er, there was no 37 

### change observed in the relaxation response to ACh by Pb alone ( Fiorim et al., 2011 ; Rizzi 38February 2012 5

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– Do Not Cite or Quote 

### et al., 2009 ; Grizzo and Cordellini, 2008 )

. Conversely, Skoczynska and Stojek ( 2005 ) 1 

### found that Pb exposure (50 ppm ; blood Pb level 11.2 μ g/dL) enhanced • 

### NO

- mediated 2 

### vasodilation by ACh in rat mesenteric arteries, and NOS inhibition enhanced the ACh 3 

### relaxant response. A number of studies found that Pb exposure did not affect smooth 4 

### muscle integrity since SNP

- induced vasorelaxation, which is endoth elium independent, 5 

### was unchanged ( Fiorim et al., 2011 ; Silveira et al., 2010 ; Rizzi et al., 2009 ; Grizzo and 6 

### Cordellini, 2008 )

. 7

• 

### NO also was found to play a role in the interaction between Pb and the vasoconstrictor 8 

### response. Blocking NOS with L

- NAME or inhibiting iNOS specifically, which decreases 9

• 

### NO production, increased the contraction of aortic rings to the vasoconstrictor 10 

### phenylephrine (PHE) and Pb exposure potentiated this response ( Fiorim et al., 2011 )

. 11 

### Also, L

- NAME increased the Pb pressor response to PHE after perinatal Pb expos ure 12 

### (1 , 000 ppm through pregnancy and lactation, blood Pb level 58.7 μ g/dL) ( Grizzo and 13 

### Cordellini, 2008 )

. Conversely, in rat renal interlobar arteries, Pb exposure blunted the 14 

### increase in renal angiotensin II (AngII)

- mediated contraction from NOS inhibition by L

15 

### NAME ( Vargas

- Robles et al., 2007 )

. Treatment with the SOD mimetic t empol, which 16 

### would increase • 

### NO bioavailability, decreased, but did not e liminate, the Pb pressor 17 

### response ( Silveira e t al., 2010 )

. 18 

### In summary, recent studies continued to provide evidence for the role of ROS in Pb

19 

### induced hypertension and vascular disease by indicating Pb

- induced increases in ROS 20 

### and modulation of cardiovascular responses by antioxidant substances. Ad ditionally, 21 

### recent studies continued to show that Pb

- induced hypertension and vascular responses are 22 

### mediated primarily via inactivation of • 

### NO not via inhibition of • 

### NO production. 23



### Vascular Reactivity Alteration of the adrenergic system from Pb exposure, which can increase peripheral 24 

### vascular resistance, and thereby arterial pressure, may be one cause of Pb

- induced 25 

### hypertension. Pb exposure in animals can increase stimulation of the sympathetic nervous 26 

### system (SNS), as shown by increased plasma levels of norepinephrine (NE) and other 27 

### catecholamines ( Carmignani et al., 2000 ; Chang et al., 1997 ) , and decreased β adrenergic 28 

### receptor density and β agonist

- stimulated cAMP production in the aorta and heart ( Tsao 29 

### et al., 2000 ; Chang et al., 1997 )

. These stimulatory effects on the SNS paralleled the 30 

### effects of Pb on BP, cardiac contractility, and carotid blood f low. Pb

- induced elevations 31 

### in arterial pressure and heart rate were abrogated by ganglionic blockade ( Simões et al., 32 

### 2011 ; Lai et al., 2002 )

. Arterial pressure and heart rate gradually decreased 7 months 33 

### after Pb exposure cessation as did the Pb

- induced SNS alterations ( Chang et al., 2005 )

. 34February 2012 5

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– Do Not Cite or Quote 

### Increases in BP can be caused by activation of the SNS, which can lead to vascular 1 

### narrowing, in turn, resulting in increased total peripheral resistance. In this neural 2 

### mechanism, activation of the SNS leads to va soconstriction, whereas inhibition leads to 3 

### vasodilation. It has been suggested that Pb leads to increased vascular reactivity to 4 

### catecholamines ( i.e., epinephrine, NE, and dopamine), hormones of the SNS. Indeed, the 5 

### isolated mesenteric vessel bed from Pb

- treated rats (50 ppm with blood Pb level: 6 

### 11.2 μ g/dL, but not 100 ppm with blood Pb level: 17.3 μ g/dL) exhibited increased 7 

### reactivity to NE ( Skoczynska and Stojek, 2005 )

. However, in another study, 100 ppm Pb 8 

### did not affect the NE

- induced contractile response after 10 months of exposure ( Zhang et 9 

### al., 2009a ) , suggesting a small range of Pb doses affects pressor response to NE. 10 

### Catecholamines act primarily through the adrenergic and dopaminerg ic receptors. 11 

### Antagonists of α1

- adrenergic, α2

- adrenergic, β

- adrenergic, and dopamine D1 receptors 12 

### were found to abolish Pb

- induced aortic contraction ( Fazli

- Tabaei et al., 2006 ; Heydari et 13 

### al., 2006 )

. However, the α1

- adrenergic receptor agonist PHE induced aortic contra ctions 14 

### and these were enhanced by treatment with Pb (100 ppm ; blood Pb level: 26.8 μ g/dL), 15 

### indicating a specific role for the α1

- adrenergic receptor ( Silveira et al., 2010 ; Grizzo and 16 

### Cordellini, 2008 ; Heydari et al., 2006 )

. Removal of the endothelium blunted the PHE

17 

### induced contrac tion. Conversely, short

- term Pb exposure (7 days) decreased the 18 

### contractile response induced by PHE in rat aortas resulting in a decreased vascular 19 

### reactivity ( Fiorim et al., 2011 )

. This decrease may be playing a compensatory role in 20 

### attempting to correct the Pb

- induced BP elevation. Additionally, Pb blunted the 21 

### isoproterenol

- induced relaxation, supporting a role for the β

- adrenoc eptors ( Vassallo et 22 

### al., 2008 ; Heydari et al., 2006 )

. 23 

### Recently, there was mixed evidence for Pb disrupting vascular reactivity to other pressor 24 

### agents. Pb (1 ppm ) treatment of isolated rat thoracic aorta increased 5

- HT induced 25 

### contraction, which was endothelium dependent, but not due to 5

- HT2B receptor 26 

### expression ( Zhang et al., 2005 )

. Follow

- up of this study in whole animals found, on the 27 

### contrary, that Pb (100 ppm ; blood Pb level: 28.4 μ g/dL) decreased the maximum 28



### co ntractile response to 5

- HT, but did not affect 5

- HT plasma levels or 5

- HT2B receptor 29 

### expression ( Zhang et al., 2009a )

. In addition, Pb exposure (100 ppm , 12 weeks) increased 30 

### the renal vascular response to AngII in isolated perfused kidneys from Pb

- exposed rats 31 

### ( Vargas

- Robles et al., 2007 )

. 32 

### Studies continued to investigate the effects of Pb on • 

### NO

- dependent v ascular reactivity by 33 

### using • 

### NO stimulating vasodilators, such as ACh and SNP, and • 

### NO inhibiting 34 

### vasoconstrictors, such as L

- NAME. These studies were discussed in the preceding 35 

### section (Oxidative Stress Response). 36February 2012 5

- 250 Draft

– Do Not Cite or Quote 

### Renin
- Angiotensin
- Aldosterone and Kininer gic Systems The adrenergic system also affects the renin

- angiotensin

- aldosterone system (RAAS), 1 

### which is responsible for fluid homeostasis and BP regulation, and has been shown to be 2 

### affected by Pb exposure. A meta

- analysis found that Pb exposure (resultin g in blood Pb 3 

### levels: 30

- 40 μ g/dL) increased plasma renin activity and renal tissue renin in young but 4 

### not old rats ( Vander, 1988 )

. Exposure of experimental animals to Pb also induced 5 

### increases in plasma, aorta, heart, and kidney angiotensin converting enzyme (ACE) 6 

### activity; plasma ki ninase II, kininase I, and kall krein activities; and renal AngII positive 7 

### cells ( Rodriguez

- Iturbe et al., 2005 ; Sharifi et al., 2004 ; Carmignani et al., 1999 )

. ACE 8 

### activity declined over time while arterial pressure stayed elevated, suggesting that the 9 

### RAAS may be involved in the induction, but not the maintenance of Pb

- induced 10 

### hypertensi on in rats. 11 

### Recent studies continued to implicate the RAAS in the development of Pb

- induced 12 

### hypertension, especially during early exposure in young animals. AngII, a main player in 13 

### the RAAS, induces arteriolar vasoconstriction leading to increased BP. Pb e xposure 14 

### increased the vascular reactivity to AngII ( Vargas

- Robles et al., 2007 )

. Acute 15 

### (60 minute s) or short

- term (7 days) exposure of Pb to rats increased the plasma ACE 16 

### activity ( Fiorim et al., 2011 ; Simões et al., 2011 ) , and Fiorim et al. ( 2011 ) additionally 17 

### found this increase to be correlated with the Pb

- induced inc rease in systolic BP. 18 

### However, at these short time points there were no changes in the AngII receptors 1 or 2 19 

### protein levels or expression. Treatment with the AngII receptor (AT 1 

### R) blocker, l osartan, 20 

### or the ACE inhibitor, enalapril, blocked the Pb

- induced systolic BP increase ( Simões et 21 

### al., 2011 ) and decreased the PHE

- induced vasoconstrictor response in Pb

- treated aortas 22 

### ( Fiorim et al., 2011 )

. Similarly, treatment with Losartan resulted in a greater decrease in 23 

### systolic BP in highly Pb exposed rats (1% Pb, 40 days; blood Pb level >240 μ g /dL after 24 

### exposure, 12

- 13 μ g/dL after chelation after 1 year) compared to control rats that 25 

### continued into later periods of follow

- up (day 283) ( Bagchi and Preuss, 2005 )

. Increased 26 

### systolic BP after early exposure to Pb corresponded with i ncreased water intake, urine 27 

### output, potassium excretion, and decreased urinary sodium and urine osmolality. These 28 

### functional changes in renal behavior are consistent with the actions of a stimulated 29 

### RAAS. Lower level Pb (100 ppm , 14 weeks; range of blood Pb levels: 23.7

- 27 μ g/dL) 30 

### exposure increased renal cortical AngII content and the number of tubulointerstitial 31 

### AngII

- positive cells ( Bravo et al., 2007 )

. This heightened intrarenal angiotensin 32 

### corresponded with sodium retention and increased systolic BP and was ablated by the 33 

### anti

- inflammatory antioxidant, MMF. Sodium reabsorption is important for the 34 

### maintenance of BP, and Na + 

### transporters play a key role in this process. In other studies, 35 

### Pb exposure increased activity and levels of the α

- 1 subunit protein of Na +/ 

### K +

- 

### ATPase, 36 

### which plays a majo r role in Na + 

### reabsorption and is regulated by the RAAS ( Fiorim et al., 37February 2012 5

- 251 Draft

– Do Not Cite or Quote 

### 2011 ; Simões et al., 2011 )

. These studies point to the activation of the RAAS in the 1 

### course of Pb

- induced hypertension, particularly in the early stages of elevated BP. 2



### Vasomodulat ors The balance between production of vasodilators and vasoconstrictors is important in the 3 

### regulation of BP and cardiovascular function. The 2006 Pb AQCD reported that Pb did 4 

### not affect all vasomodulators in the same way. Urinary excretion of the vasocon strictor, 5 

### thromboxane (TXB 2 

### ), and the vasodilatory prostaglandin, 6

- keto

- PGF1α, were 6 

### unchanged in rats with Pb

- induced hypertension ( Gonick et al., 1998 )

. However, in vitro 7 

### Pb exposure promoted the release of the prostaglandin substrate, arachidonic acid, in 8 

### vascular smooth muscle cells (VSMCs) via activation of phospholipase A 2 

### ( Dorman and 9 

### Freeman, 2002 )

. 

### Plasma concentration and urinary excretion of the vasoconstrictive 10 

### peptide, endothelin (ET) 3 was increased after low (100 ppm ), but not high

- level 11 

### (5 , 000 ppm ) Pb exposure in rats ( Gonick et al., 1997 ; Khalil

- Manesh et al., 1994 ; Khalil

12 

### Manesh et al., 1993b )

. Antagonism of the ET receptor A blunted the downregulation of 13 

### sGC and cGMP production by Pb in isolated rat artery segments, suggesting that some of 14 

### the hypertensive effects of Pb exposure may be mediated through ET ( Courtois et al., 15 

### 2003 )

. Additionally, Pb

- exposed animals exhibited fluid retention and a 16



### co ncentration

- dependent decline in the vasodilator, atrial natriuretic factor (ANF) 17 

### ( Giridhar and Isom, 1990 )

. Results from these studies suggest that Pb may interfere with 18 

### the balance between vasodilators and vasoconstrictors that contribute to the complex 19 

### hormonal regulation of vascular contraction and BP. 20 

### The imbalance in vasomodulators is one explanation for the concentrati on
- dependent 21 

### vasoconstriction observed in animals after Pb exposure ( Valencia et al., 2001 ; Watts et 22 

### al., 1995 ; Piccinini et al., 1977 )

. Vasoconstriction after Pb exposure was not reported in 23 

### all studies ( Shelkovnikov and Gonick, 2001 ) and is likely varied depending on the type of 24 

### vessel used, the Pb concentration employed, and the animal species being studied. 25 

### Studies have reported Pb

- in duced attenuation of ACh

- and • 

### NO

- mediated vasodilation 26 

### ( Marques et al., 2001 ; Oishi et al., 1996 ) in some, but not all vascular tissues and in 27 

### some, but not all studies ( Purdy et al., 1997 )

. These effects have been variably attributed 28 

### to Pb

- mediated activation of PKC and direct action on the VSMCs through the Ca 2+ 29 

### mimetic properties of Pb among other possibilities ( Valencia et al., 2001 ; Watts et al., 30 

### 1995 ; Piccinini et al., 1977 )

. 31 

### A recent study investigated the role of the endothelial

- derived vasoconstrictor, ET

- 1, in 32 

### Pb

- induced hypertension. ET

- 1 from the endothelium acts on the ET A 

### - type receptors 33 

### located on the vascular smooth muscle layer and may be involved in vascular reactivity 34 

### by • 

### NO and COX derivatives. Pb exposure (1 ppm , 24 hours) to rat aortic segments 35February 2012 5

- 252 Draft

– Do Not Cite or Quote 

### decreased expression of sGC

- β1 subunit, an enzyme involved in • 

### NO

- induce d 1 

### vasodilation, and increased expression of COX

- 2 in an endothelium

- dependent manner 2 

### ( Molero et al., 2006 )

. Even though Pb treatment did not alter ET

- 1 or ET A 

### - type receptor 3 

### protein expression in this system, blocking the ET A 

### - type receptors partially reversed Pb

4 

### induced changes in sGC and COX

- 2 in vascular tissue. This study suggests that the 5 

### endothelium and ET

- 1 may contrib ute to Pb

- induced hypertension through activation of 6 

### ET A 

### - type receptors that alter expression of COX

- 2 and sGC

- β1 subunit, which affects 7

• 

### NO signaling. 8


### COX

- 2 blockade has been shown to prevent Pb

- induced downregulation of sGC 9 

### expression ( Courtois et al., 2003 )

. Inhibition of COX

- 2 also decreased the Pb

- induc ed 10 

### pressor response to ACh ( Grizzo and Cordellini, 20 08 ) and PHE ( Silveira et al., 2010 ) in 11 

### experimental animals. These studies suggest that Pb

- induced vascular reactivity may 12 

### depend on the participation of a COX

- derived vasoconstrictor, such as prostaglandins, 13 

### prostacyclins, or thromboxanes. 14 

### In summary, a small number of available recent studies continued to show that Pb 15 

### exposure affect vasomodulatory pathways that are important for the maintenance of 16 

### vascular tone; however, results indicated that not all vascular cell types are similarly 17 

### affected by Pb exposure. Further, effects appeared to vary according to the concentration 18 

### of Pb exposure. Pb exposure has been shown to interrupt baseline or endogenous • 

### NO

19 

### mediated vasodilation of vessels via alterations in PKC, sGC, VSMC, endothelial cells, 20 

### NADPH oxidase, and Ca +2 

### levels. Recent studies indicated that Pb exposure may affect 21 

### vascular reactivity by increasing COX

- 2 and COX

- 2

- dependent vasoconstrictors. Also, 22 

### the vasoconstrictor endothelin may contribute to Pb

- induced vasomodulation via similar 23 

### pathways as • 

### NO including sGC an d COX

- 2. 24



### 5.4.2.4 Summary of Blood Pressure and Hypertension
The 2006 Pb AQCD reported a clear association between higher blood Pb levels and 25 

### higher BP. The effect was modest, but robust, as determined by a meta

- analysis ( Nawrot 26 

### et al., 2002 ) of over 30 studies comprising over 40,000 adults ( Figure 5

- 29 ). In the meta

27 

### analysis, each doubling of concurrent blo od Pb was associated with a 1 mmHg increase 28 

### in systolic BP and a 0.6 mmHg increase in diastolic BP. Recent epidemiologic studies 29 

### supported this conclusion at lower concurrent blood Pb levels (in populations with mean 30 

### blood Pb levels < 2 μ g/dL) and added to the evidence base regarding populations 31 

### potentially at increased risk ( i.e., high stress, genetic variants) and regarding associations 32 

### of bone Pb levels with BP and hypertension in populations with mean bone Pb levels less 33 

### than 20 μ g/g. As these studies w ere mostly cross

- sectional in design and were conducted 34February 2012 5

- 253 Draft

– Do Not Cite or Quote 

### in adults whose concurrent blood Pb levels are influenced both by current Pb exposures 1 

### and past Pb exposures mobilized from bone, uncertainty exists over the Pb exposure 2 

### conditions that contributed to the associations observed between concurrent blood Pb 3 

### level with inc reased BP and hypertension ( Sections 4.3 and 4.7.3 ). 4 

### A recent study in an ethnically diverse community

- based cohort of wome n and men aged 5 

### 50

- 70 years of age found associations of both blood and tibia Pb levels with BP ( Martin 6 

### et al., 2006 )

. This study also af firmed findings from other studies by demonstrating that 7 

### with each increase of 1 μ g/dL concurrent blood Pb level, systolic BP increased 1 mmHg 8 

### and diastolic BP increased 0.5 mmHg and strengthened the evidence for an independent 9 

### association with blood Pb le vel through the extensive examination of potential 10 

### confounding. Additionally, recent epidemiologic studies provided evidence for 11 

### associations in an adult cohort between blood Pb and BP and hypertension with relatively 12 

### low blood Pb levels; a positive relati onship was found in the NHANES adult data (1999

- 13 

### 2002) with a geometric mean blood Pb level of 1.64 μ g/dL ( Muntner et al., 2005 )

. 14 

### However, as noted above, in adults, uncertainty exists regarding the magnitude, timing, 15 

### frequency, and duration of Pb exposure that contribute to the associations observed with 16 

### concurrent blood Pb levels. A new prospective study in Pb workers found independent 17 

### associations of both baseline blood Pb level and subsequent changes in blood Pb over 18 

### follow

- up with changes in BP over follow

- up and bone Pb level with hypertension 19 

### ( Glenn et al., 2006 )

. Although these Pb workers had higher current Pb exposure 20 

### compared with nonoccupationally

- exposed adults, the results indicated that different 21 

### mechanisms may mediate shorter

- term Pb

- associated increases in BP and longer

- t erm Pb

22 

### associated development of hypertension. 23 

### In concordance with epidemiologic evidence, collectively, the animal toxicological 24 

### studies providing blood Pb level and BP measurements reported higher BP with higher 25 

### blood Pb levels ( Figure 5

- 28 ). While the contribution of low concurrent blood Pb levels 26 

### to the findings is difficult to ascertain in adults, animal toxicological studies provide 27 

### support for low blood Pb level effects with increases in BP observed in groups of animals 28 

### with blood Pb levels as low as 2 μ g/dL ( Rizzi et al., 2009 ; Tsao et al., 2000 ; Nakhoul et 29 

### al., 1992 )

. However, the majority of animal toxicological studies s howing Pb

- induced 30 

### hypertension were conducted at higher Pb exposure levels that result in blood Pb levels > 31 

### 10 μ g/dL. In addition, new animal evidence suggests the potential for increased BP 32 

### following short

- term (4 weeks) Pb treatment that included injecte d boluses ( Fiorim et al., 33 

### 2011 ; Simões et al., 2011 ; Sharifi et al., 2004 )

. New studies also demonstrated partial 34 

### reversibility (not to levels in controls) of Pb

- induced elevations in BP following Pb 35 

### exposure cessation or chelation. 36February 2012 5

- 254 Draft

– Do Not Cite or Quote Source: Reprinted with permission of MacMillan Press, Nawrot et al. ( 2002 ) \ Study Key: C

- Caerphilly Study; HP

- Welsh Heart Program; W

- Whites; B

- Blacks; NI

- Non

- immigrants; I

- Immigrants; FW

- Foundry Workers; CS

- Civil Servants; P

- PheeCad (Public Health and Environmental Exposure to Cadmium) Study. Note: Circles represent individual groups and squares represent the combined association sizes. Open circles denote a nonsignificant association size that was assumed to be zero. 

### Figure 5
- 29 Change in systolic BP (SBP), in mmHg with 95% CI, associated with a doubling in the blood Pb concentration.
Epidemiologic studies continued to investigate the relationship between bone Pb and BP. 1 

### A recently published m eta

- analysis ( Figure 5

- 30 ) ( Navas

- Acien et al., 2008 ) included 2 

### several studies (three prospective, five cross

- sectional) which individually showed that 3 

### bone Pb level was associated with systolic BP but no t diastolic BP. In the cross

- sectional 4 

### studies, a pooled estimate indicated an increase in systolic BP of 0.26 mmHg (95% CI: 5 

### 0.02, 0.50) per 10 μ g/g tibia Pb. In the longitudinal studies, a 0.33 mmHg (95% CI:

- 0.44, 6 

### 1.11) increase was estimated per 10 μ g/g bone Pb. Most studies also reported associations 7 

### of bone Pb with hypertension. Pooled estimates of 1.04 (95% CI: 1.01, 1.07) per 10 μ g/g 8February 2012 5

- 255 Draft

– Do Not Cite or Quote 

### increase in tibia Pb and 1.04 (95% CI: 0.96, 1.12) per 10 μ g/g increase in patella Pb were 1 

### reported. 2

Source: Reprin ted with permission of Elsevier Publishers, Navas

- Acien et al. ( 2008 ) In the Normative Aging Study, Hu et al. ( 1996b ) reported the cross

- sectional association between bone Pb levels and the prevalence of hypertension and Cheng et al. ( 2001 ) reported the cross

- sectional association b etween bone Pb levels and systolic BP in study participants free of hypertension at baseline. Note: The studies are ordered by increasing mean bone Pb levels. The area of each square is proportional to the inverse of th e variance of the estimated change or log relative risk. Horizontal lines represent 95% confidence intervals. Diamonds represent summary estimates from inverse

- variance weighted random effects models. Because of the small number of studies, summary estimates are presented primarily for desc riptive purposes. RR indicates risk ratio. 

### Figure 5
- 30 Prospective and cross
- sectional increase in systolic BP (SBP) and diastolic BP (DBP) and relative risk of hypertension per 10 μ g/g increase in bone Pb levels.
A few recent epidemiologic studies also emphasized the potential interaction between 3 

### measures of long

- term Pb exposure, i.e., bone Pb levels, and factors such as chronic 4 

### stress and HFE genetic variants to moderate or modify the relationship of BP a nd 5 

### hypertension with Pb. For example, among NAS men, tibia Pb level was associated with 6 

### a larger risk of developing hypertension in an originally nonhypertensive group among 7 

### men with higher self

- reported stress ( Peters et al., 2007 )

. 8February 2012 5

- 256 Draft

– Do Not Cite or Quote 

### In addition to stress, recent epidemiologic studies investiga ted effect modification by 1 

### race/ethnicity and genetic variants. In the NHANES 1988

- 1994 population of adults, the 2 

### association of concurrent blood Pb with systolic BP was higher among Mexican 3 

### Americans. In the same NHANES population, the association between blood Pb level and 4 

### hypertension was higher among non
- hispanic Bl acks with the ALAD2 allele ( Figure 5

- 25 5 

### and Figure 5

- 26 for results) ( Scinicariello et al., 2010 )

. Additionally, the association 6 

### between blood Pb and PP was larger among NAS men with the HFE H63D variant 7 

### ( Figure 5

- 25 ). PP represents a good predictor of cardiovascular morbidity and mortality 8 

### and an indicator of arterial stiffness ( Zhang et al., 2010a )

. The aforementioned genes are 9 

### related to iron metabolism and have been linked with differences in Pb dist ribution in 10 

### blood and bone. Park et al. ( 2009b ) provided further evidence of variants in iron 11 

### metabolism genes impacting the associati on of bone Pb level s with QT interval changes 12 

### ( Table 5

- 19 for results). 13 

### Animal toxicological evidence continued to build on the evidence characterizin g the 14 

### mechanisms leading to these Pb

- induced cardiovascular alterations. Biological 15 

### plausibility for the consistent associations observed between blood and bone Pb and 16 

### cardiovascular effects is provided by enhanced understanding of Pb

- induced oxidative 17 

### str ess including • 

### NO inactivation, endothelial dysfunction leading to altered vascular 18 

### reactivity, activation of the RAAS, and vasomodulator imbalance. 19



### 5.4.3 Vascular Effects and Cardiotoxicity
Not only has Pb been shown to increase BP and alter vascular reactivity , but Pb can alter 20 

### cardiac function, initiate atherosclerosis, and increase cardiovascular mortality. Past 21 

### toxicological studies have reported that Pb can increase atheromatous plaque formation in 22 

### pigeons, increase arterial pressure, decrease heart rate an d blood flow, and alter cardiac 23 

### energy metabolism and conduction ( Prentice and Kopp, 1985 ; Revis et al., 1981 )

. A 24 

### limited number of available epidemiologic studies discussed in the 2006 Pb AQCD 25 

### provided eviden ce of associations of blood Pb level with ischemic heart disease (IHD) 26 

### and peripheral artery disease (PAD). 27



### 5.4.3.1 Effects on Vascular Cell Types
The endothelial layer is an important constituent of the blood vessel wall, which regulates 28 

### macromolecular permeabili ty, VSMC tone, tissue perfusion, and blood fluidity. Damage 29 

### to the endothelium is an initiating step in development of atherosclerosis, thrombosis, and 30 

### tissue injury. Given that epidemiologic and toxicological evidence suggests that long

- 31February 2012 5

- 257 Draft

– Do Not Cite or Quote 

### term Pb exposure i s associated with a number of these conditions, numerous 1 

### toxicological studies have investigated and found an effect of Pb on endothelial 2 

### dysfunction. A recent occupational study found that endothelial function assessed by 3 

### flow

- mediated dilatation was impa ired in highly Pb exposed workers (mean blood Pb 4 

### levels: 24.1 in workers versus 7.8 μ g/dL in unexposed controls) ( Poręba et al., 2010 )

. 5 

### The endothelial layer makes up only a small part of the vascular anatomy; the majority of 6 

### the vessel wall is composed of VSMCs, which work in concert with the endothelial cells 7 

### (EC) in contraction and relaxation of the vessel, local BP regulation, and atherosclerotic 8 

### plaque development. Since Pb has been shown repeatedly to result in hypertension and 9 

### vascular disease in experimental animals, studies continued to investigate and find an 10 

### effect of Pb on VSMCs. 11


### In in vi tro assays, Pb (50 μ M, 2 weeks) stimulated VSMC invasiveness in isolated human 12 

### arteries leading to the invasion of medial VSMC into the vessel intima and development 13 

### of intimal hyperplasia, a key step in atherosclerotic progression ( Zeller et al., 2010 )

. In 14 

### addition, treatment with Pb (50 μ M, 12 hours) promoted VSMC elastin expression and 15 

### increased arterial extracellular matrix in isolated human arteries. VSMC invasiveness was 16 

### also increased in cu lture by treatment with supernatant of Pb

- treated human EC (50 μ M), 17 

### suggesting that Pb

- exposed ECs secrete an activating compound. This compound was 18 

### confirmed to be IL

- 8. Pb exposure (5

- 50 μ M) was able to, in a concentration

- dependent 19 

### manner, increase IL

- 8 synthesis and secretion in human umbilical vein EC cultures 20 

### through activation of the transcription factor Nrf2. Neutralization of IL

- 8 could block 21 

### VSMC invasion and arterial intima thickening ( Zel ler et al., 2010 )

. This study provides 22 

### evidence that Pb exposure stimulates ECs to secrete IL

- 8 in an Nrf2

- dependent manner 23 

### that stimulates VSMC invasion from the vessel media to intima leading to a vascular 24 

### thickening and possibly atherogenesis. 25


### A numb er of CVDs, including atherosclerosis, are characterized by increased 26 

### inflammatory processes. Numerous studies have shown that Pb exposure is associated 27 

### with an inflammatory environment in vascular tissues of humans and animals as indicated 28 

### by higher level s of inflammatory mediators like prostaglandin E 2 

### (PGE 2 

### ). Human aortic 29 

### VSMCs treated with Pb (1 μ M, 1

- 12 hours) exhibited increased secretion of PGE 2 

### time

30 

### dependently through enhanced gene transcription ( Chang et al., 2011 )

. This was preceded 31 

### by a Pb

- induced increase in the gene expression of the rate limiting enzymes in the 32 

### regulation of prostaglandins, cytosolic phospholipase A 2 

### (cPLA 2 

### ) and COX

- 2. The 33 

### induction of these enzymes was mediate d by activation of ERK1/2, MEK1, and MEK2. 34 

### Further investigation into the entrance of Pb into the cell revealed that inhibition of the 35 

### store

- operated calcium channels (SOC) could only partially suppress cPLA 2 

### and COX 36 

### activation by Pb; however inhibition of epidermal growth factor receptor (EGFR) 37February 2012 5

- 258 Draft

– Do Not Cite or Quote 

### attenuated Pb

- induced PGE 2 

### secretion and activation of cPLA 2 

### and COX. A follow

- up to 1 

### this study found that Pb treatment (1μM) of a human epithelial cell line increased COX

- 2 2 

### gene expression, promoter activity, and p rotein ( Chou et al., 2011 )

. Inhibition of NF

- κB 3 

### decreased the Pb

- induced COX

- 2 activation; whereas EGFR inhibition blocked COX

- 2 4 

### upregulation and NF

- κB nuclear translocation. Overall these studies suggest that Pb can 5 

### induce pro

- inflammatory events in VSMC in the form of increased PGE 2 

### secretio n and 6 

### cPLA 2 

### and COX

- 2 expression through activation of EGFR via ERK1/2 and NF

- κB 7 

### pathways. 8 

### Damage to the endothelium is a hallmark event in the development of atherosclerosis. 9 

### Past studies have shown that Pb exposure results in de

- endothelialization, impai red 10 

### proliferation, and inhibition of endothelium repair processes after injury ( Fujiwara et al., 11 

### 1997 ; Ueda et al., 1997 ; Kaji et al., 1995 ; Kishimoto et al., 1995 )

. Howe ver, Pb exposure 12 

### was not found to lead to nonspecific cytotoxicity at low exposure levels (2

- 25 μ M) as 13 

### shown by the lack of release of lactate dehydrogenase (LDH) from Pb

- treated bovine 14 

### aortic EC ( Shinkai et al., 2010 )

. Instead, Pb induced specific cytotoxicity (caspase3/ 7 15 

### activation) through endoplasmic reticulum (ER) stress that was protected against by the 16 

### ER chaperones glucose

- regulated protein 78 (GRP78) and glucose

- regulated protein 94 17 

### (GRP94). GRP78 and GRP94 play key roles in the adaptive unfolded protein response 18 

### that serves as a marker of and acts to alleviate ER stress. Exposure of Pb to ECs induced 19 

### GRP78 and GRP94 gene (2

- 25 μ M) and protein (GRP78 [5

- 25 μ M] and GRP94 [10

- 20 

### 25 μ M]) expression through activation of the IRE1

- JNK

- AP

- 1 pathways ( Shinkai et al., 21 

### 2010 )

. This study sugge sts that the functional damage caused by Pb exposure to EC may 22 

### be partly attributed to induction of ER stress. 23



### 5.4.3.2 Cholesterol
As blood cholesterol rises so does the risk of coronary heart disease. Early occupational 24 

### studies ( Ademuyiwa et al., 2005a ; Bener et al., 2001a ; Kristal

- Boneh et al., 1999 ) 25 

### examining higher than current adult blood Pb levels reported higher total cholesterol 26 

### levels related to Pb exposure, but mixed results for HDL, LDL, and triglycerides. More 27 

### recently, Poreba et al. ( 2010 ) , in an occupational study, reported no significant 28 

### differences in parameters of lipid metabolism between Pb exposed workers (mean blood 29 

### Pb level: 25 μ g/dL) and unexposed individuals. Conversely, Kamal et al. ( 2011 ) reported 30 

### that occupational Pb exposure (mean blood Pb level: >40 μ g/dL) was associated with 31 

### higher levels of triglycerides, total cholesterol, and LDL, and decreased HDL

- C. Oth er 32 

### Pb studies adjusted models for total cholesterol to control for this coronary heart disease 33 

### risk factor. Higher mean total cholesterol with higher blood Pb levels has been reported 34 

### ( Menke et al., 2006 )

. In developing models to predict bone Pb levels, Park et al. ( 2009a ) 35February 2012 5

- 259 Draft

– Do Not Cite or Quote 

### noted that total and HDL cholesterol were selected as 2 of 18 predictors for the bone Pb 1 

### level model. Their findings suggested that higher Pb exposure may be associated with 2 

### higher total and HDL cholesterols. A recent study reported increased LDL and decreased 3 

### HDL in rats treated with Pb (20 ppm , i.p., 3 days/week, 8 weeks) ( Roshan et al., 2011 )

. 4 

### The major risk fac tor that lipids represent for heart disease make relating lipid levels to 5 

### Pb exposures an interesting but challenging hypothesis to test. 6



### 5.4.3.3 Heart Rate Variability
Pb has been shown not only to affect vascular contractility in animals, but also is 7 

### associated with cardiac contractility. Eum et al. ( 2011 ) and Park et al. ( 2009b ) followed 8 

### up a previous NAS report ( Cheng et al., 1998 ) , which found increasing duration of 9 

### corrected QT interval (QTc) with increasing bone Pb levels in men <65 years, but not in 10 

### men ≥ 65 years. Eum et al. ( 2011 ) prospectively examined the association between blood 11 

### and bone Pb levels and the development of electrocardiographic (ECG) conduction 12 

### abnormalities among 600 men who were free of ECG abnormalities at the baseline 13 

### assessment. A second ECG was obtained for 496 men 8.1 (SD = 3.1) years later on 14 

### average. Baseline Pb concentrations in blood (mean [SD]: 5.8 [3.6] μ g/dL), patella bon e 15 

### (mean [SD]: 30.3 [17.7] μ g/g), and tibia bone (mean [SD]: 21.6 [12.0] μ g/g) were similar 16 

### to those found in other samples from the general U.S. adult population and much lower 17 

### than those reported in occupationally exposed groups. Higher tibia Pb was assoc iated 18 

### with increases in QTc interval and QRSc duration. Compared with those in the lowest 19 

### tertile of baseline tibia Pb (< 16 μ g/g), participants in the highest tertile (> 23 μ g/g) had a 20 

### 7.94 msec (95% CI: 1.42, 14.45) greater increase in QTc interval and a 5.94 msec (95% 21 

### CI: 1.66, 10.22) greater increase in QRSc duration over 8 years after adjusting for 22 

### covariates. There were no statistically significant associations with patella or blood Pb 23 

### levels. These associations were observed in men with relatively lo w blood and bone Pb 24 

### concentrations who were free of cardiac conduction abnormalities at baseline and were 25 

### examined prospectively. Thus, they indicate that long

- term cumulative Pb exposure may 26 

### increase the risk of developing cardiac abnormalities. Uncertain ty exists as to the specific 27 

### Pb exposure level, timing, frequency, and duration contributing these associations 28 

### observed for tibia Pb levels. A recent occupational study reported lower HRV and 29 

### abnormal parameters of heart rate turbulence in Pb

- exposed work ers (mean blood Pb 30 

### levels: ~25 μ g/dL) compared to control subjects ( Poręba et al., 2011a )

. 31


### Park et al. ( 2009b ) examined whether poly morphisms in genes known t o alter iron 32 

### metabolism (HFE, transferrin [TF] C2, heme oxygenase

- 1 [HMOX

- 1]) modify the 33 

### association between Pb biomarker levels and the QT interval. Investigators examined 34 

### associations in data stratified on polymorphisms in the three genes. They also anal yzed 35February 2012 5

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### interaction models with cross

- product terms for genotype and the Pb biomarker. The 1 

### distributions of all genotypes but the HFE variant, H63D, were in Hardy

- Weinberg 2 

### equilibrium. Subjects homozygous for the other HFE variant, C282Y, had higher bone Pb 3 

### l evels and those homozygous for H63D and heterozygous for both C282Y and H63D had 4 

### lower bone Pb levels. The HMOX

- 1 L variant (longer repeats of GT, associated with 5 

### lower enzyme inducibility) alone, compared to the wild type, showed a statistically 6 

### significa nt interaction with tibia Pb (11.35 msec longer QTc interval for each 13 μ g/g 7 

### increase in bone Pb in L

- allele variants). No other gene variant alone showed different 8 

### Pb

- associated QTc intervals from those in wild types, either for tibia and patella Pb or f or 9 

### (linear) concurrent blood Pb. Lengthening of QTc with higher tibia and blood Pb was 10 

### more pronounced with an increase in the total number of gene variants, driven by a joint 11 

### effect between HFE variant and HMOX

- 1 L allele. There was a trend observed with 12 

### blood and tibia Pb

- associated QTc interval increasing with increasing number of gene 13 

### variants from 0 to 3. This study provided further evidence of gene variants modifying 14 

### associations of Pb biomarkers with cardiovascular effects. 15 

### The interaction of key ma rkers of the metabolic syndrome with bone Pb levels in 16 

### affecting HRV was investigated in a group of 413 older adults with patella Pb 17 

### measurements in the NAS ( Park et al., 2006 )

. Metabolic syndr ome was defined to include 18 

### three or more of the following: waist circumference >102 cm, hypertriglyceridemia 19 

### (>150 mg /dL), low HDL cholesterol (<40 mg /dL in men), high BP >130/85 mmHg, and 20 

### high fasting glucose (>110 mg /dL). Men using antihypertensive medic ation or diabetes 21 

### medications were counted as high BP or high fasting glucose, respectively. The strongest 22 

### relationships between patella Pb levels and lower HRV were observed among those with 23 

### three or more metabolic abnormalities. A trend was observed for larger patella Pb

24 

### associated decreases in HRV with increasing number of metabolic abnormalities. These 25 

### results suggest multiplicative effects of cumulative Pb exposure and metabolic 26 

### abnormalities on key predictors of CVD. Park et al. ( 2006 ) also reported the penalized 27 

### spline fits to bone Pb in models assessing only main effects of bone Pb. The optimal 28 

### degree of smoothing determined by the generalized cross

- validation criterion for all HRV 29 

### measures was 1, which indicated that the associations were nearly linear. The spline fits 30 

### and associated statistics showed that the bone Pb main effects on HRV measures were 31 

### linear. However, the relationship with LF/HF was linear with log(LF/HF). 32



### Increase d incidence of arrhythmia and atrioventricular conduction block was found in 33 

### rats after 12 weeks of Pb exposure (100 ppm ; mean blood Pb level 26.8 μ g/dL) ( Reza et 34 

### al., 2008 )

. Also, Pb exposure for 8 week s increased heart rate and systolic BP. These 35 

### increases corresponded with increased cardiac contractile force and prolonged ST 36 

### interval, without alteration in QRS duration or coronary flow. In contrast, another study 37 

### found that Pb (100 μ M) exposure, in a c oncentration

- dependent manner, reduced 38February 2012 5

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### myocardial contraction using rat right ventricular strips by reducing sarcolemmal Ca 2+ 1 

### influx and myosin ATPase activity ( Vassallo et al., 2008 )

. This study also found that Pb 2 

### exposure changed the response to inotropic agents and blunted the force produced during 3



### con traction. Conversely, past studies have found that Pb exposure increases intracellular 4 

### Ca 2+ 

### content ( Lal et al., 1991 ; Favalli et al., 1977 ; Piccinini et al., 1977 ) , which could 5 

### result in increased cardiac output and hypertension. 6



### 5.4.3.4 Peripheral Artery Disease
Peripheral artery disease (PAD) is an indicator of atherosclerosis and measured by the 7 

### ankle brachial index, which is the ratio of BP between the posterior tibia artery and the 8 

### brachial artery. PAD is typically defined as an ankle brachial index of less than 0.9. 9 

### Muntner et al. ( 2005 ) , whose results describing the association of blood Pb and 10 

### hypertension in the NHANES 1999

- 2002 data set for adults were discussed previously, 11 

### also examined the associ ation of blood Pb with PAD. The authors observed an increasing 12 

### trend in the odds of PAD with increasing concurrent blood Pb level. The OR for PAD 13 

### comparing the fourth quartile of blood Pb (> 2.47 μ g/dL) to the first quartile of blood Pb 14 

### (< 1.06 μ g/dL) was 1.92 (95% CI: 1.02, 3.61). These results are consistent with those 15 

### from a previous NHANES analysis conducted by Navas

- Acien et al. ( 2004 )

. 16



### Navas

- Acien et al. ( 2004 ) reported a trend of increasing OR for PAD with increasing 17 

### quartile of concurrent blood Pb or Cd in adults who were 40 years of a ge in the 1999

- 18 

### 2000 NHANES population. These authors tested both Pb and Cd in separate models, 19 

### tested the metals simultaneously, and tested the interaction between the metals. The 20 

### correlation coefficient between natural log Pb and natural log Cd was 0.32 ( p <0.001). 21 

### Although the interaction was not statistically significant, when blood Pb and blood Cd 22 

### were in the same model, the ORs were diminished slightly but both showed statistically 23 

### significant trends of increasing OR with increasing quartile of the met al. These results 24 

### indicate that blood Cd levels did not confound the association between blood Pb level and 25 

### PAD. In a subsequent analysis, Navas

- Acien et al. ( 2005 ) used the same 1999

- 2000 26 

### NHANES dataset, but constructed PAD models using a suite of urine metal 27 

### concentrations. Power was reduced in this study because only 659

- 736 subjects 28 

### (compared to 2,125) had spot urine metal tests in the data set. Urinary Cd, but not urinary 29 

### Pb, was consistently associated with PAD in all models. Associations also were observed 30 

### with urinary antimony and tungsten. Spot urine Pb measurements are less reliable 31 

### compared to blood Pb measurements. In Navas

- Acien et al. ( 2005 ) , the urinary Pb level 32 

### association with PAD was sensitive to adjustment for urinary creatinine, indicating that 33 

### spot urine Pb measurements are affected by differences in urine dilution. This finding 34February 2012 5

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### illustrates the limited reliability of spot urine Pb measurements compared to blood Pb 1 

### measurements. 2



### 5.4.3.5 Ischemic Heart Disease
A few studies discussed in the 2006 Pb AQCD indicated associations between Pb 3 

### biomarker levels and increased risk of cardiovascular outcomes associated with IHD, 4 

### including left ventricular hypertrophy ( Schwartz, 1991 ) and myocardial infarction 5 

### ( Gustavsson et al., 2001 )

. Recently, Jain et al. ( 2007 ) reported on the incidence of IHD 6 

### (physician confirmed MI, angina pectoris) among older adult males enrolled in the NAS 7 

### and followed during the period of Septembe r, 1991 to December, 2001. All subjects had 8 

### blood Pb and bone Pb measurements with no IHD at enrollment. Fatal and nonfatal cases 9 

### were combined for analysis. Baseline blood, tibia, and patella Pb levels were log

10 

### transformed. Blood Pb level and patella Pb l evel were associated with increased risk of 11 

### IHD over the 10

- year follow

- up period. When blood Pb and patella Pb were included 12 

### simultaneously in the model, each of their HRs was only moderately attenuated (HR: 13 

### 1.24 [95% CI: 0.80, 1.93] per SD increase in bl ood Pb and HR: 2.62 [95% CI: 0.99, 6.93] 14 

### per SD increase in patella Pb). When blood Pb and tibia Pb were included simultaneously 15 

### in the model, their risk estimates were only moderately attenuated (HR: 1.38 [95% CI: 16 

### 0.89, 2.13] per SD increase in blood Pb a nd HR: 1.55 [95% CI: 0.44, 5.53] per SD 17 

### increase in tibia Pb). These findings indicate that both blood and bone Pb levels 18 

### contribute independently to IHD incidence. 19 

### IHD, characterized by reduced blood supply to the heart, may result from increased 20 

### thrombosis. A recent animal study suggested that Pb exposure promotes a procoagulant 21 

### state that could contribute to thrombus formation ( Shin et al., 2007 )

. In a rat model of 22 

### venous thrombosis, Pb treatment (i.v. 25 mg /kg) resulted in increased thrombus 23 

### formation. Additionally, Pb treatment to human erythrocytes (red blood cells, RBCs) 24 

### increased coagulation at a dose of 5 μ M and thrombin generation in a concentration

25 

### dependent manner at doses from 2

- 5μM. This enhanced procoagulant activity in Pb

26 

### treated RBCs was the result of increased outer cell membrane phosphatidylserine (PS) 27 

### surfacing (human RBCs: 2

- 5 μ M; rat RBCs: 5 μ M). Sim ilar to these in vitro results, PS 28 

### externalization on erythrocytes was increased in Pb

- treated rats (i.v. 50

- 100 mg /kg, not 29 

### 25 mg /kg). Increased PS externalization was likely the result of increased intracellular 30 

### calcium (5 μ M Pb), enhanced scramblase acti vity (5

- 10 μ M Pb), inhibited flippase 31 

### activity (5

- 10 μ M Pb), and ATP depletion (1

- 5 μ M Pb) after Pb exposure ( Shin et al., 32 

### 2 007 )

. 33February 2012 5

- 26 3 Draft

– Do Not Cite or Quote 

### 5.4.3.6 Atherosclerosis
Epidemiologic and toxicological studies provide evidence for increased atherosclerosis 1 

### and intimal medial thickening (IMT) due to Pb exposure. The association between stroke 2 

### subtypes and severity of cerebral atherosclerosis was exa mined in relation to a single 3 

### concurrent blood Pb level and total 72

- hour urinary Pb level (body Pb store

- EDTA 4 

### mobilization test) in a cross

- sectional study of 153 patients (mean age 63.7 years) 5 

### receiving digital subtraction angiography in Chang Gung Memor ial Hospital in Taiwan 6 

### from 2002 to 2005 ( Lee et al., 2009 )

. In an analysis adjusted for age, sex, hypertension, 7 

### diabetes, triglyceride, uric acid, smoking, and alcohol consumption, a 1 μ g increase in 8 

### urine Pb was associated with ≥ 50% stenosis in the intracranial carotid system with an 9 

### OR (95% CI) of 1.02 (1.00, 1.03). Urine Pb was associated with greater stenosis in the 10 

### extracranial or vertebrobasilar systems. Blood Pb level was not associated with greater 11 

### stenosis in any region. As the development of atherosclerosis is a lifelong proc ess, body 12 

### Pb stores, analyzed by total 72

- hour urine Pb amount, may more strongly be associated 13 

### with atherosclerosis than are single blood Pb measurements. 14 

### A recent study correlated greater carotid artery IMT with higher concurrent serum Pb 15 

### levels (mean [ SD] 0.41 [0.38] μ g/dL) in hemodialysis patients ( Ari et al. )

. A few 16 

### available recent occupational studies also presented evidenc e for increased measures of 17 

### atherosclerosis in highly Pb exposed adult populations with mean blood Pb levels around 18 

### 25 μ g/dL. Poręba et al. ( 2011b ) reported increased local arterial stiffness and more 19 

### frequent left ventricular diastolic dysfunction in Pb

- exposed workers with hypertension 20 

### compared to nonexposed controls with hypertension. Occu pational exposure to Pb (mean 21 

### blood Pb levels: 24 μ g/dL in workers, 8.3 μ g/dL in nonexposed group) was also 22 

### associated with greater IMT and atherosclerotic plaque presentation, analyzed by Doppler 23 

### ultrasound ( Poreba et al., 2011 )

. 24


### Zeller et al. ( 2010 ) examined human radial and internal mammary arteries exposed to Pb 25 

### in culture and reported a concentration

- dependent increase in arterial intimal thickness 26 

### (statistically nonsignificant at 5 μ M Pb, significant at 50 μ M Pb, 2 week treatment) and 27 

### intimal extracellular matrix accumulation (50 μ M). Also, Pb promoted EC prolif eration 28 

### (5 and 50 μ M, 72 hours) and VSMC elastin expression (50 μ M, 12 hours), as discussed 29 

### above ( Section 5.4.3.1 ) ( Zeller et al., 2010 )

. Another study showed that Pb exposure 30 

### (100 ppm in drinking water for 10 months; mean blood Pb level 28.4 μ g/dL) of rats also 31 

### increased the aortic media thickness, media

- lumen ratio, and medial collagen co ntent 32 

### ( Zha ng et al., 2009a )

. These morphological changes to the vessel due to Pb exposure 33 

### indicate initiation of arteriosclerosis and could be the cause of decreased contractile 34 

### response of the vessel due to altered visco

- dynamic vessel properties. Alternatively, these 35 

### vascular changes could be an effect of Pb

- induced hypertension. 36February 2012 5

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### Table 5
- 19 Characteristics and quantitative data for associations of blood and bone Pb with other CVD measures in epidemiologic studies ordered as they appear in the text

Study Population/ Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI) a Heart rate variability
Eum et al. ( 2011 ) 600 men free of electrographic abnormalities at the time of baseline ECG from NAS in Greater Boston, MA area (496 with follow

- up ECG 8 years later) ECG con duction b (QTc, QRSc, JTc, QT prolongation, JT prolongation, IVCD c
, AVCD, Arrhythmia) Baseline Blood Pb: Mean (SD): 5.8 (3.6) μg/dL

Baseline Patella Pb: Mean (SD): 30.3 (17.7) μg/g

Baseline Tibia Pb: Mean (SD): 21.6 (12.0) μg/g Q1: <16 μg/g (n = 191) Q2: 16.0

- 23 μg/g (n = 208) Q3: >23 μg/g (n = 195) Repeated measures linear regression adjusted for age, education, smoking, BMI, albumin

- adjusted serum calcium, and diabetes status at baseline, and years between ECG tests and QT

- prolongation drugs at the tim e of ECG measurement. Tibia Pb: Adjusted 8

- year change (95% CI): QTc: Q2 vs

. Q1 (reference): 7.49 (1.22, 13.75) msec, Q3 vs. Q1: 7.94 (1.42, 14.45) msec p for trend = 0.03

QRSc: Q2 vs. Q1: 0.52 (

- 3.60, 4.65) msec Q3 vs. Q1: 5.94 (1.66, 10.22) msec p for trend = 0.005 No associations with patella or blood Pb Park et al. ( 2009b ) 613 men from NAS in Greater Boston, MA area (8/1991

- 12/ 1995) QTc b interval Baseline Blood Pb: Median (IQR): 5 (4

- 7) μg/dL

Baseline Patella Pb: Median (IQR): 26 (18

- 37) μg/g

Baseline Tibia Pb: Median (IQR): 19 (14

- 27) μg/g Linear regression models adjusted for age, BMI, smoking status, serum calcium, and diabetes. Per IQR (3 μg/dL) increase in blood Pb 1.3 (

- 0.76, 3.36) msec after 8

- year follow up
Per IQR (19 μg/g) increase in patella Pb 2.64 (0.13, 5.15) msec
Per IQR (13 μg/g) increase in tibia Pb 2.85 (0.29, 5.40) msec Park et al. ( 2006 ) 413 men from NAS in Greater Boston, MA area (11/14/2000

- 12/22/2004) HRV (SDNN, HF, HF norm , LF, LF norm , LF/HF) Baseline Patella Pb (measured within 6 mo of HRV: Median (IQR): 23.0 (15

- 34) μg/g Estimated a : Median (IQR): 16.3 (10.4

- 25.8) μg/g

Baseline Tibia Pb: Median (IQR): 19.0 (11

- 28) μg/g Log linear regression models adjusted for age, cigarette smoking, alcohol consumption, room temperature, season (model 2) BMI, fasting blood glucose, HDL ch olesterol, triglyceride, use of β

- blockers, calcium channel blockers, and/or ACE inhibitors Tibia Pb: Model 2 Change (95%CI) HF:

– 0.9 (

– 3.8, 2.1) normalized units (nu) LF: 0.9 (

– 2.0, 3.9) nu Log LF/HF: 3.3 (

– 10.7, 19.5) (%) Per 17 μg/g tibia Pb

Patella Pb: Model 2 Change (95%CI) HF:

– 0.6 (

– 3.1, 1.9) nu LF: 0.6 (

– 1.9, 3.1) nu Log LF/HF: 3.0 (

– 8.7, 16.2) (%) Per 15.4 μg/g patella Pb

Effect estimates were more pronounced among those with metabolic syndrome. Peripheral artery diseaseFebruary 2012 5

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– Do Not Cite or Quote Study Population/ Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI) a
Muntner et al. ( 2005 ) 9,961 NHANES (1999

- 2002) participants PAD Range Concurrent Blood Pb: Q1: <1.06 μg/dL, Q2: 1.06

- 1.63 μg/dL Q3: 1.63

- 2.47 μg/dL Q4: >2.47 μg/dL Logistic regression models adjusted for age, race/ethnicity, sex, diabetes mellitus, BMI, ciga rette smoking, alcohol consumption, high school education, health insurance status OR (95% CI): Q1: 1.00 (Reference), Q2: 1.00 (0.45, 2.22), Q3: 1.21 (0.66, 2.23), Q4: 1.92 (1.02, 3.61)
Navas

- Acien et al. ( 2005 ) 790 participants, age ≥ 40 yr, from NHANES (1999

- 2000) PAD Concurrent urinary Pb: Mean (10th

- 90th %): 0.79 μg/L (0.2

- 2.3) Logistic regression adjusted for the following: Model 1: age, sex, race, and education Model 2: covariates above plus smoking status Mod el 3:covariates above plus urinary creatinine Model 1: OR: 1.17 (0.81, 1.69) Model 2: OR: 1.17 (0.78, 1.76) Model 3: OR: 0.89 (0.45, 1.78)
Per IQR increase in urinary Pb
Array of metals in urine also evaluated. Ischemic Heart Disease
Jain et al. ( 2007 ) 837 men from NAS in Greater Boston, MA area (1991

- 20 01) IHD (MI or angina pectoris) Baseline Blood Pb Mean (SD): Non

- cases 6.2 (4.3) μg/dL; Cases 7.0 (3.8) μg/dL Baseline Patella Pb Mean (SD): Non

- cases 30.6 (19.7) μg/dL; Cases 36.8 (20.8) μg/dL
Baseline Tibia Pb Mean (SD): Non

- Cases 21.4 (13.6) μg/g; Cases 24.2 (15.9) μg/g

Cases: Blood Pb range: 1.0 to 20.0 μg/dL Patella Pb range: 5.0 to 101 μg/g Tibia Pb range:

- 5 to 75 μg/g Cox proportional hazards models adjusted for age, BMI, education, race, smoking status, pack

- years smoked, alcohol intake, hist ory of diabetes mellitus and hypertension, family history of hypertension, DBP, SBP, serum triglycerides, serum HDL, and total serum cholesterol Blood Pb level ≥ 5 μg/dL OR over 10

- year follow

- up: 1.73 (1.05, 2.87)
Ln blood Pb OR: 1.45 (1.01, 2.06) Ln patella Pb level OR: 2.64 (1.09, 6.37) Ln tibia Pb level OR: 1.84 (0.57, 5.90)

Per 1 SD increase in Pb biomarker

a Estimated patella Pb accounts for declining trend in patella Pb levels between analysis of bone Pb and HRV.
b Heart

- rate

- corrected QT inter val calculated by Bazett’s formula

c IVCD, intraventricular conduction defect; AVCD, atrioventricular conduction defect



### 5.4.3.7 Summary of Vascular Effects and Cardiotoxicity
There are a limited number of studies that investigate the associations between Pb 1 

### biomarkers and cardiovascular effects other than BP or hypertension ( Table 5

- 19 ). As 2 

### presented in Table 5

- 19 , these studies demonstrated associations between various 3 

### biomarkers of Pb exposure and clinical cardiovascular outcomes such as atherosclerosis, 4 

### IHD, PAD, and HRV occurrence in adult populations after adjusting for potential 5 

### confounding by variables such as age, sex, BMI, smoking, alcohol consumption, and 6 

### diabet es. In a limited body of studies, mixed evidence of association between 7 

### occupational exposure to Pb and altered cholesterol was reported. Studies of Pb

- induced 8 

### endothelial dysfunction, VMSC invasiveness, and inflammation in isolated vascular 9 

### tissues and ce lls provide mechanistic evidence to support the biological plausibility of 10 

### these other vascular effects and cardiotoxicity. A recent study provided evidence for the 11February 2012 5

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### interaction between biomarkers of Pb exposure and the HFE C282Y and HMOX

- 1 L 1 

### variant on the prolonged QT interval in nonoccupationally

- exposed older men ( Park et 2 

### al., 2009b )

. Also, in the NAS population, bon e Pb levels were associated with larger 3 

### decreases in HRV parameters among subjects identified as having metabolic 4 

### abnormalities ( Park et al., 2006 )

. These metabolic abnormalities, abdominal obe sity, 5 

### hypertriglyceridemia, low HDL cholesterol, high BP/medication use, or high fasting 6 

### glucose, have been shown to be associated with increased risk of cardiovascular events. 7 

### A prospective NAS study reported that higher baseline tibia Pb was associated w ith 8 

### increases in QTc interval and QRSc duration over an 8

- year follow

- up period ( Eum et al., 9 

### 2011 )

. Concurrent blood Pb levels (population means >2.5 μ g/dL) were associated with 10 

### greater odds of PAD in adults in NHANES analyses ( Muntner et al., 2005 ; Navas

- Acien 11 

### et al., 2004 )

. In addition, in the NAS cohort of older adult men, blood Pb (≥ 5 μ g/dL) and 12 

### patella Pb levels were associated with increased morbidity from IHD ( Jain et al., 2007 )

. 13 

### A recent study involving both human and toxicological studies elucidated mechanisms 14 

### for observed Pb

- mediated arterial IMT, an early event in Pb

- induced atherogenesis 15 

### ( Zeller et al., 2010 )

. Studies in isolated tissues and cells found that Pb stimulated the 16 

### synthesis and secretion of IL

- 8 in ECs, which was responsible for stimulating VSMC 17 

### invasio n into the vessel intimal layer. Pb treatment also increased extracellular matrix and 18 

### elastin, primary sites for lipid deposition in the vessel wall. Overall, the relatively 19 

### available few studies provide support for associations between Pb biomarkers and o ther 20 

### cardiovascular conditions, yet further research is warranted to understand these 21 

### relationships. 22



### 5.4.4 Cardiovascular Function and Blood Pressure in Children

5.4.4.1 Introduction
The study of cardiovascular function effects in relation to blood Pb levels in children 23 

### potentially offers unique information on several topics. First, by examining endpoints 24 

### predictive of future cardiovascular pathology, these studies may offer information on the 25 

### potential cardiovascular effects of Pb exposure in an understudied population. Second, 26 

### examination of cardiovascular changes that are antecedent to increased BP and changes 27 

### in other CVD

- related endpoints at later lifestage s may inform uncertainties in regards to 28 

### the time course of cardiovascular changes associated with Pb exposure. Finally, these 29 

### studies address gaps in knowledge regarding Pb exposure effects in populations with 30 

### mean blood Pb levels in the range of < 5 to 10 μ g/dL. 31February 2012 5

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### An advantage of examining the literature about the association between cardiovascular 1 

### effects and bloo d Pb levels in children is that the blood Pb levels of children in new 2 

### studies may better reflect their relatively recent Pb exposure and its effect on CVD than 3 

### blood Pb levels do in adults because of the uncertainties related to the effects of earlier 4 

### hig her Pb exposure levels. A possible disadvantage of studying the associations of blood 5 

### Pb levels and cardiovascular effects in children is the lower prevalence of such conditions 6 

### in children. For example, the prevalence for hypertension in children (9 to 10 years old) 7 

### ranges from to 2 to 5 percent ( Daniels, 2011 ; Steinthorsdottir et al., 2011 )

. The 8 

### prevalence of hypert ension increases with age. More than half of people aged 60 to 69 9 

### years have hypertension ( Chobanian et al., 2003 )

. Further, compensatory mechanisms in 10 

### children may be more active than in adults, and the cardiovascular tissue of the young 11 

### may be less susceptible to damage tha n that of adults. The lower disease prevalence may 12 

### make it more difficult to find a relationship between blood Pb level and cardiovascular 13 

### effects in child studies. Although studies in children may be limited with regard to study 14 

### power in assessing associa tions with present CVD pathology, they provide for the 15 

### examination of early function changes that are associated with subsequent CVD. 16 

### The limited numbers of studies published on children examined endpoints such as total 17 

### peripheral resistance (TPR), BP, and autonomic nervous system activation. These new 18 

### and earlier studies are presented in Table 5

- 20 . Multiple studies in New York State 19 

### evaluated two child cohorts born in the 1990s after Pb was removed from gasoline in the 20 

### U.S. with mean blood Pb levels of 4.62 and 1.01 μ g/dL ( Gump et al., 2 011 ; Gump et al., 21 

### 2009 ; Gump et al., 2007 ; Gump et al., 2005 )

. Zhang et al. ( In Press ) examined children in 22 

### Mexico City born from 1994 to 2003, when Pb was being taken out of gasoline in 23 

### Mexico ( Martinez et al., 2007 )

. The geometric mean cord and concurrent blood Pb levels 24 

### of the children in the Mexico Cit y cohort were 4.67 and 2.56 μ g/dL. 25February 2012 5

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### Table 5
- 20 Studies of Children Cardiovascular Endpoints and Pb Biomarkers Ordered as They Appear in the Text

Study Population/ Location Parameters Blood Pb Data a Statistical Analysis Effect Estimates/Results Gump et al. ( 2005 ) 122 children age 9.5 yr in Oswego, NY (born at a single h ospital in New York from 1991

- 94) SBP, TPR (total peripheral vascular resistance) Cord blood Pb: GM (GSD): 2.56 μg/dL (1.16)
Childhood (mean age of measurement: 2.6 yr) blood Pb: GM (GSD): 4.06 μg/dL (1.14) Multivariate linear regression models examine d the relationship of blood Pb with change in z

- score for outcome (post

- and pre

- stress). Potential confounders considered: HOME score, SES, birth weight, child BMI, child sex. Per 1 μg/dL increase in childhood blood Pb level, 0.088 (95% CI: 0.023, 0.153) dyne

- s/cm 5 change in TPR Per 1 μg/dL increase in cord blood Pb level, 12.16 (95% CI: 2.44, 21.88) mmHg higher SBP

Gump et al. ( 2007 ) 122 children a ge 9.5 yr in Oswego, NY SBP, TPR Childhood (mean ag e of measurement: 2.6 yr) blood Pb: GM (GSD): 4.06 μg/dL (1.14) Linear regression models adjusting for the same covariates as in Gump et al. ( 2005 )

. Separate models testing whether Pb is a mediator of SES associations,(Sobel test) and whether Pb moderates SES associations (Pb

- SES interaction). Blood Pb was a mediator of the SES

TPR relationship SES alone:

- 0.62 dyne

- s/cm 5 ( p <0 .05) SES with Blood Pb:

- 0.40 dyne

- s/cm 5 (p >0.10), change in R 2 attributable to SES:

- 55.3% Blood Pb was a potential moderator of the SES

- TPR relationship. Blood Pb × SES interaction: p = 0.07 .

Blood Pb was a moderator of SES

- SBP relationship Pb × SES interaction: p = 0.007 At blood Pb levels > 4 μg/dL, SES not significantly associated with SBP Gump et al. ( 2009 ) 122 children age 9.5 yr in Oswego, NY Salivary cortisol Cord blood Pb: GM (GSD): 2.56 μg/dL (1.16)
Childhood (mean ag e of measurement: 2.6 yr) blood Pb: GM (GSD): 4.06 μg/dL (1.14) Linear regression to examine whether blood Pb level mediates or moderates the relatio nship between SES and salivary cortisol as in Gump et al. ( 2007 ) Blood Pb was a mediator of the SES

cortisol association. SES was no longer significantly associated with co rtisol after adjusting for blood Pb level. R 2 for SES decreased by 40, 33, 50% for cortisol measured at 21, 40, and 60 min. Blood Pb was not a significant moderator of SES

- cortisol association. Blood Pb × SES interaction term was not statistically signific ant Gump et al. ( 2011 ) 140 children ages 9

- 11 yr SBP, TPR, HRV (heart rate variability) in response to acute stress (mirror tracing task) Concurrent blood Pb: GM: 1.01 μg/dL Quartiles: Q1: 0.14

- 0.68 μg/dL Q2: 0.69

- 0.93 μg/dL Q3: 0.94

- 1.20 μg/dL Q4: 1.21

- 3.76 μg/dL Outcomes were analyzed as continuous variables f or the pre - stress values or the change post

- and pre

- stress. Regression models were adjusted for sex, SES, BMI, and age. Blood Pb levels associated with autonomic and cardiovascular dysregulation in response to stress

– greater vascular resistance, reduced stroke volume, and cardiac output Change in SBP (mmHg) across quartiles: Q1: 5.30, Q2: 7.33, Q3: 7.07, Q4: 7.23, p for trend = 0.31 Change in TPR (%) across quartiles: Q1: 2.91, Q2: 8.18, Q3: 9.55, Q4: 9.51, p for trend = 0.03 Change in Stroke Volume (%) across quartiles: Q1: 2.23, Q2: 0.91, Q3:

- 3.47, Q4:

- 0.89, p for trend = 0.04February 2012 5

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– Do Not Cite or Quote Study Population/ Location Parameters Blood Pb Data a Statistical Analysis Effect Estimates/Results Zhang et al. ( In Press ) 457 mother child pairs in a birth cohort, born 1994 to 2003 in Mexico City. Children were e valuated 2008

- 2010 at ages 7

- 15 yr SBP Cord blood Pb: GM (GSD): 4.67 μg/dL (1.18) (N=323)

Concurrent blood Pb: GM (GSD) : 2.56 μg/dL (1.16) (N=367)
Maternal post

- partum bone Pb: Median (IQR): Tibia Pb: 9.3 (3.3, 16.1) μ g/g Patella Pb: 11.6 (4.5, 19.9) μ g/g Multiple regression models and generalized estimating equations (log linear for cord blood, linear for concurrent blood and maternal bone). The base model considered maternal education, birth weight, BMI, sex, and child concurrent age as covariates. Prenatal Pb exposure may be associated with higher BP in female offspring. Among girls, an IQR (13 μg/g) increase in ma ternal tibia Pb was associated with a 2.11 (95% CI: 0.69, 3.52) mmHg increase in SBP IQR (16 μg/g) increase in maternal patella Pb was associated with a 0.87 (95% CI:

- 0.75, 2.49) mmHg increase in SBP IQR (4 μg/dL) increase in cord blood Pb was associated with a 0.75 (95% CI:

- 1.13, 2.63) mmHg increase in SBP

Factor

- Litvak et al. ( 1999 ; 1996 ) 260 children ages 5.5 years old in K. Mitrovica and Pristina, Yugoslavia SBP Concurrent blood Pb range: 4.1 to 76.4 μg/dL Linear regression analysis. Potential confounders considered: gender, maternal education, birth weight, HOME score, and BMI. Per 1 μg/dL increase in concurrent blood Pb level, 0.05 (95% CI:

- 0.02, 0.13) mmHg higher SBP Blood Pb level at birth and cumulative blood Pb level were not as strongly associated with SBP at age 5.5 y r. Gerr et al. ( 2002 ) Young adults age 19

- 29 years, born 1965

- 1975, male and female; half of the subjects had grown up around an active Pb smelter in Silver Valley, Idaho BP While the concurrent mean blood Pb level was 3.15 μg/dL for the highest bone Pb category (> 10 μg/g), early childhood mean blood Pb levels in this group were s ubstantially elevated for all bone Pb level categories and were highest among participants in the highest bone Pb level category. The mean blood Pb level was 65 μg/dL among part icipants with bone Pb level >10 μg/g. Multiple linear regression models always included age, sex, height, BMI, current smoking status, frequency of alcohol consumption, current use of birth

- control medication, hemoglobin level, serum albumin, and income, regardless of significance levels. Both blood Pb (as a linear term) and bone Pb (a four category ordinal variable from <1 μg/g to >10 μg/g) were tested together. Group in highest quartile of tibia Pb level (> 10 μg/g) had 4.26 (95% CI: 1.36, 7.16) mmHg higher SBP and 2.80 (95% CI: 0.35, 5.25) mmHg higher DBP compared to the lowest tib ia Pb group (< 1 μg/g).

a Blood Pb data are estimates of geometric mean (GM) and geometric standard deviation (GSD) using the arithmetic mean and SD.



### 5.4.4.2 Cardiovascular Functioning in Children
The relationship between cardiovascular functioning (TPR, BP, stroke volume, and 1 

### cardiac output,) and blood Pb levels was examined by Gump et al. ( 2007 ; 2005 ) in a 2 

### cohort born at a single New York hospital. Higher early childhood Pb levels (average age 3 

### 2.6 years) were associated with greater TPR response to acute stress induce d by mirror 4 

### tracing on a computer at age 9.5 years as shown in Figure 5

- 31 . Testing blood Pb with 5 

### linear, quadratic, and cubic terms did not produce s ignificantly different Pb

- TPR 6 

### associations which the authors suggested showed effects that were concentration 7 

### dependent and, notably were not emergent at a specific exposure threshold. TPR 8 

### increased with increasing quartile of blood Pb level. A mediational analysis indicated that 9 

### Pb was a significant mediator of the SES

- TPR reactivity association; some evidence also 10 

### suggested moderation, whereby the inclusion of blood Pb into the model reduced the 11 

### effect estimate for SES. Biological plausibility for these o bservations in children is 12February 2012 5

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### provided by observations that Pb exposures increases TRP in toxicological studies and by 1 

### mechanistic evidence indicating that Pb

- induced changes in SNS activity may mediate 2 

### such effects ( Section 5.4.2.3 ). Additionally, higher blood Pb level measured at age 2.6 3 

### years was associated with a smaller stroke volume and cardiac output responses to a cute 4 

### stress at age 9.5 years. In a further analysis in this cohort, Gump et al. ( 2009 ) examined 5 

### the possibility that Pb may mediate an association between SES and c ortical responses to 6 

### acute stress. Elevated cortisol has been associated with hypertension ( Whitworth et al., 7 

### 2000 )

. Gump et al. ( 2009 ) found that lower family income was a ssociated with greater 8 

### cortisol levels following an acute stress task and that blood Pb was a mediator of this 9 

### association. 10

Source: Reprinted with permission of Elsevier ( Gump et al., 2005 ) 

### Figure 5
- 31 Children’s adjusted total peripheral resistance (dyn
- s/cm 5 

### ) responses to acute stress tasks, as a function of childhood Pb levels.
In a different cohort of 140 children 9 to 11 years of age recruited from local pediatrician 11 

### offices and from mailings to homes with children in this age group, Gump et al. ( 2011 ) 12 

### used a similar acute stress

- producing paradigm to that used in the previous studies to 13 

### examine the associations of concurrent blood Pb with cardiovascular responses. TPR 14 

### significantly increased in a concentration

- dependent relationship with blood Pb, with 15 

### most of the increase occurring between the first quartile blood Pb (0.14

- 0.68 μ g/dL) and 16 

### the second quartile blood Pb (0.69

- 0.93 μ g/dL). This result is co nsistent with those of 17 

### Gump et al. ( 2005 )

. Also, these new findings provided evidence of associations with 18 

### concurrent blood Pb levels and with lower blood Pb levels ( Gump et al., 2011 ) than were 19 

### previously examined in Gump et al. ( 2005 )

. 20February 2012 5

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### Studies examining HRV in adults and animal toxicology are discussed in Section 5.4.3.3

. 1 

### In Gump et al. ( 2011 ) , cardiac autonomic regulation decreased in a 2 

### concentration

- dependent manner with increasing concurrent blood Pb quartile, with the 3 

### largest change relative to the first quartile (0.14

- 0.68 μ g/dL) measured in the hig hest 4 

### blood Pb quartile (1.21

- 3.76 μ g/dL). Also, high frequency HRV, decreased more with 5 

### acute stress in the highest Pb quartile group (1.21

- 3.76 μ g/dL). In the earlier cohort, early 6 

### childhood (mean age at collection: 2.6 years) blood Pb level was associate d with reduced 7 

### stroke volume and cardiac output ( Gump et al., 2007 ; Gump et al., 2005 )

. In this new 8 

### study, Gump et al. ( 2011 ) found the same but for concur rent blood Pb level and at lower 9 

### blood Pb levels. 10



### 5.4.4.3 Blood Pressure in Children
Zhang et al. ( In Press ) conducted a longitudinal study that examined changes in BP in 11 

### 323 girls and boys aged 7 to 15 years old in a Mexico City cohort for a relationship with 12 

### maternal bone Pb measured one month post

- partum (a measure of cum ulative exposure 13 

### that could expose fetuses to Pb through accelerated mobilization of bone Pb during 14 

### pregnancy), and cord blood Pb at delivery. This was the first study to examine the 15 

### association of maternal bone Pb, as a marker of prenatal exposure, with o ffspring BP. 16 

### The model including both girls and boys (without adjustment for concurrent blood Pb) 17 

### showed no statistically significant association overall of any Pb biomarker with child BP. 18 

### A significant interaction was found between maternal tibia Pb and s ex, and in models 19 

### stratified by sex, maternal tibia Pb was associated with adjusted systolic and diastolic BP 20 

### in females, but not males. Maternal post

- partum median tibia Pb was 9.3 μ g/g (IQR: 3.3, 21 

### 16.1 μ g/g) with no significant differences between mothers of male and female offspring. 22 

### Suboptimal growth in utero is associated with accelerated weight gain in offspring during 23 

### childhood and greater risk of later hypertension ( Barker and Bagby, 2005 ; te Velde et al., 24 

### 2004 ; Barker et al., 1989 )

. These may represent biologically plausible mechanisms by 25 

### which prenatal Pb exposure may result in increased BP later in childhood as was 26 

### demonstrated in femal e offspring. The relationship between birth weight and Pb 27 

### biomarkers is discussed in Section 5.8.7

. 28 

### Gump et al. ( 2011 ; 2005 ) examined the relationship of blood Pb level with BP in their 29 

### two cohorts of contemporary children around age 10 years in New York State. Gump et 30 

### al. ( 2005 ) reported an association of cord blood levels with systo lic BP (12.16 mmHg 31 

### [95% CI: 2.44, 21.88] increase per 1 μ g/dL increase in cord blood Pb level). Gump et al. 32 

### ( 2011 ) found that with acute stress, children in higher quartiles of concurrent blood Pb 33 

### level (>0.69 μ g/dL) had larger increases in systolic BP. For example, children with blood 34 

### Pb levels between 1.21 and 3.76 μ g/dL had a 7.23 mmHg c hange, and children with 35February 2012 5

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### blood Pb levels between 0.14 and 0.68 μ g/dL had a 5.30 mmHg change. A linear trend 1 

### was not observed across quartiles. An interaction between long

- term perceived stress and 2 

### bone Pb levels in association with BP and hypertension also was reported in a study of 3 

### adults ( Peters et al., 2007 ) (described in Section 5.4.2.1 ). An earlier study ( Factor

- Litvak 4 

### et al., 1999 ; Factor

- Litvak et al., 1996 ) , of children with higher blood Pb level s ranging 5 

### from 4.1 to 76.4 μ g/dL found that a 1 μ g/dL increase in concurrent blood Pb was 6 

### associated with a 0.05 (95% CI:

- 0.02, 0.13) mmHg increase in systolic BP. An 7 

### additional study ( Gerr et al., 2002 ) reported that young adults (ages 19

- 29 years) in the 8 

### highest category of bone Pb levels (greater than 10 μ g/g), in whom the mean concurrent 9 

### blood Pb level was 65 μ g/dL, systolic BP was 4.26 mmHg higher compared with young 10 

### adults with bone Pb levels < 1 μ g/dL. 11 

### The pathogenesis of CVD has been hypothesized to begin in childhood ( Kapuku et al., 12 

### 2006 )

. Early markers observable in youth include increased blood pressure d uring stress, 13 

### reduced heart rate variability, increased IMT, and vascular endothelium dysfunction. 14 

### Kapuku et al. ( 2006 ) state that endothelial dysfunction is the center of the CVD 15 

### paradigm. The factors measured in childhood or as a cumulative burden since childhood 16 

### are predictors of outco mes in young adults who are still too young to experience coronary 17 

### events ( Li et al. , 2003 ) , and early

- life exposures may induce changes in arteries that 18 

### contribute to the development of atherosclerosis ( Raita kari et al., 2003 )

. Berenson et al. 19 

### ( 2002 ) observed that the effects of multiple risk factors on coronary atherosclerosis 20 

### support evaluation of cardiovascular risk in young people. Thus, evidence relating levels 21 

### of biomarkers of Pb exposure of child ren to cardiovascular function in the groups of 22 

### studies presented in the preceding text when combined with the evidence for the potential 23 

### pathogenesis of CVD starting in childhood that yield effects in adulthood provides 24 

### coherence with evidence in adults s upporting the effects of long

- term, cumulative Pb 25 

### exposures in the development of cardiovascular effects. 26 

### Few animal studies have examined the effect of Pb exposure during pregnancy and 27 

### lactation on BP in offspring and those that have used high levels of e xposure. Recently, 28 

### pups of Pb

- exposed dams (1 , 000 ppm through pregnancy and lactation) exhibited 29 

### increased blood Pb level (mean blood Pb level: 58.7 μ g/dL) and increased arterial 30 

### systolic BP after weaning ( Grizzo and Cordellini, 2008 ) suggesting a role for childhood 31 

### Pb exposure leading to adult dise ase. 32



### 5.4.4.4 Summary of Child Cardiovascular Studies
Studies have reported antecedent cardiovascular changes such as TPR responses to acute 33 

### stress tasks as a function of childhood blood Pb levels. Also, a study reported associations 34February 2012 5

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– Do Not Cite or Quote 

### with acute stress

- induced aut onomic and cardiovascular dysregulation responses. 1 

### Biomarkers of prenatal Pb exposure (maternal post

- partum patella and tibia) were related 2 

### to later higher BP. This may be related to intrauterine growth restriction and subsequent 3 

### accelerated weight gain in childhood and may indicate greater risk of hypertension later 4 

### in life. The results are not uniform with respect to the important lifestages of Pb exposure 5 

### and can differ by sex and other factors. Uncertainties in these studies may be related to 6 

### sample siz e, single measures of BP, variation of age of onset of puberty, and cross

7 

### sectional design. Some of these uncertainties result in random error, however, and result 8 

### in the attenuation of observed associations rather than the generation of spurious 9 

### associati ons. These study findings indicate that in children with mean blood Pb levels in 10 

### the range of < 5 to 10 μ g/dL, increasing blood Pb level may be associated with small 11 

### increases in BP and changes in the cardiovascular system that may be related to later 12 

### deve lopment of CVD. 13 

### Factors may limit the ability of studies to detect statistically significant Pb

- associated 14 

### changes with BP. The relatively young age of the subjects may have limited the ability of 15 

### these studies to detect significant BP effects (as opposed to early function effects) if 16 

### longer duration Pb exposure is necessary to produce the cardiovascular changes 17 

### considering the lower prevalence and strength of compensatory mechanisms in children. 18 

### There is uncertainty in the shape of the concentration

- respon se relationship to 19 

### cardiovascular endpoints at lower blood Pb levels since most studies modeled a linear 20 

### relationship. Several studies that compared linear and nonlinear relationships between 21 

### blood Pb level and decrements in cognitive function found a bett er fit for the nonlinear 22 

### relationship (See Section 5.3.10 ). 23 

### Cardiovascular endpoints other than baseline BP may be more sensitive outcomes by 24 

### which to measure Pb

- associated cardiovascular effects in very young children. The series 25 

### of studies by Gump et al. ( 2011 ; 2009 ; 2007 ; 2005 ) evaluating much smaller samples 26 

### than did the adult studies, was able to demonstrate statistically significant relationships of 27 

### blood Pb levels with cardiovascular outcomes such as TPR related to acute stress. It 28 

### suggests that the stress paradigm may be use ful to detect associations of blood Pb levels 29 

### with effects on the cardiovascular system of children. Selection of the appropriate 30 

### cardiovascular outcome in children is an important factor to consider in the design of 31 

### studies. Rather than using indicators o f already present cardiovascular problems, such as 32 

### BP, informative evaluation of cardiovascular changes that are antecedent to increased BP 33 

### and changes in other CVD

- related endpoints at later lifestage s may reduce uncertainties 34 

### regarding the time course of cardiovascular changes associated with early Pb exposure 35 

### where the relevance and persistence of these endpoints has been shown to be associated 36 

### with future pathology. 37February 2012 5

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### Overall this body of evidence, based on different cohorts, locations, and study designs, 1 

### begins to form a literature base that indicates there is a relationship between biomarkers 2 

### of Pb exposure and cardiovascular effects in children. One longitudinal study ties in 3 

### maternal bone Pb level, and cord and concurrent blood Pb level for the childre n. 4 

### Limitations exist in the studies. While blood pressure increases are more prevalent in 5 

### older adults than in children, BP increases have been related to higher blood Pb level in 6 

### older studies of children and young adults ( Gerr et al., 2002 ; Factor

- Litvak et al., 1999 ; 7 

### Factor

- Litvak et al., 1996 )

. The newer children studies provide information in 8 

### populations with mean blood Pb levels in the range of < 5 to 10 μ g/dL for BP and 9 

### antecedents for CVD such as increases in TPR and changes in cardiac autonomic 10 

### regulation. 11



### 5.4.5 Mortality
The 2006 Pb AQCD stated that collectively the then available evidence suggested an 12 

### effect of Pb on cardiovascular mortality in the general U.S. populati on but cautioned that 13 

### these findings should be replicated before these estimates for Pb

- induced cardiovascular 14 

### mortality could be used for quantitative risk assessment purposes ( U.S. EPA, 2006b )

. 15 

### Previous results involved NHANES II and III analyses that examined the association of 16 

### adult concurrent blood Pb with all cause and cause

- specific mortality ( Schober et al., 17 

### 2006 ; Lustberg and Silbergeld, 2002 )

. As blood Pb levels in adults reflect contributions 18 

### from both recent Pb exposure and mobilization of historic Pb from bone, it is uncle ar to 19 

### what extent recent, past, or cumulative Pb exposures contributed to the observed 20 

### associations. Given the decline in ambient air Pb concentrations and population blood Pb 21 

### levels, it is likely that study subjects had a much higher Pb exposure in their past than 22 

### during the study period. Using NHANES II (1976

- 1980) data, Lustberg and Silbergeld 23 

### ( 2002 ) found significant increases in all

- cause, circulatory and cancer mortality, 24 

### comparing adults with blood Pb levels (measured 12

- 16 yea rs before ascertainment of 25 

### vital status) of 20

- 29 μ g/dL to those with blood Pb levels less than 10 μ g/dL. Using 26 

### NHANES III data, Schober et al. ( 2006 ) found significant increased all

- cause mortality, 27 

### cardiovascular, and cancer mortality comparing adults with blood Pb levels (measured a 28 

### median of 8.8 years before ascertainment of vital status) from 5

- 9 μ g/dL and above 29 

### 10 μ g/dL to th ose with blood Pb levels less than 5 μ g/dL. 30 

### Several new studies substantially strengthen the evidence base for Pb

- associated 31 

### mortality. A further analysis of the NHANES III database by a different research group 32 

### using different methods addressed uncertaint ies from other earlier analyses by 33 

### considering a more extensive number of potential confounding factors and by 34 

### characterizing concentration

- response relationships. A few longitudinal prospective 35February 2012 5

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### studies in different cohorts conducted by different researche rs with different methods in 1 

### different parts of the U.S. provide coherence with the evidence base for blood Pb and add 2 

### new evidence for mortality associated with bone Pb levels. 3


### Menke et al. ( 2006 ) examined all

- cause and cause

- specific mortality using NHANES III 4 

### dat a. Subjects at least 18 years of age were followed up to 12 years after their blood Pb 5 

### was measured, and 1 , 661 deaths were identified. Those with baseline blood Pb levels 6 

### from 3.63 to 10 μ g/dL had significantly higher risks of all

- cause (HR: 1.25 [95% CI: 7 

### 1.04, 1.51]), cardiovascular (HR: 1.55 [95% CI: 1.08, 2.24]), MI (HR: 1.89 [95% CI: 8 

### 1.04, 3.43]), and stroke (HR: 2.51 [95% CI: 1.20, 2.26]) mortality compared to those with 9 

### baseline blood Pb levels less than 1.93 μ g/dL and increased risk of cancer mortali ty (HR: 10 

### 1.10 [95% CI: 0.82, 1.47]). Effect estimates adjusted for demographic characteristics 11 

### were robust to the additional adjustment for factors such as smoking, alcohol 12 

### consumption, diabetes, BMI, hypertension, and level of kidney function. The consiste ncy 13 

### of HRs across models with a varying number of control variables indicated little residual 14 

### confounding. Hazard ratios were not higher comparing adults with blood Pb levels from 15 

### 1.94 to 3.62 μ g/dL to those with blood Pb levels <1.93 μ g/dL. However, tests for linear 16 

### trend were statistically significant for all mortality outcomes except for cancer mortality. 17 

### Menke et al. ( 2006 ) evaluated several of the model covariates ( e.g., diabetes, 18 

### hypertension, and glomerular filtration rate [GFR]) in a subgroup analysis ( Figure 5

- 32 ). 19 

### The authors reported that there were no interactions between blood Pb and other adjusted 20 

### variables. In the previous NHANES III analysis of the association of blood Pb with 21 

### mortality, Schober et al. ( 2006 ) included participants greater than 40 years of age (N = 22 

### 9686) and adjusted for covariates including age, sex, ethnicity, and smoking rather than 23 

### the full suite of covariates evaluated by Menke et al. ( 2006 )

. Schober et al. ( 2006 ) 24 

### reported increased HRs comparing adults with blood Pb levels ≥ 10 μ g/dL to those with 25 

### blood Pb levels <5 μ g/dL for all

- cause (HR: 1.59 [95% CI: 1.28, 1.98]), CVD (HR: 1.55 26 

### [95% CI: 1.16, 2.07]), and cancer (HR: 1.69 [95% CI: 1.14, 2.52]) mortality and 27 

### generally statistically nonsignificant higher HRs comparing adults with blood Pb levels 28 

### from 5

- 9 μ g/dL to those with blood Pb levels <5 μ g/dL. The median follow

- up time 29 

### be tween measurement of blood Pb and death ascertainment was 8.55 years. 30

31February 2012 5

- 276 Draft

– Do Not Cite or Quote Source: Reprinted w ith permission of Lippincott Wil l iams & Wilkins, Menke et al. ( 2006 ) Note: Hazard ratios were calculated for a 3.4

- fold increase in blood Pb level with log

- blood Pb as a continuous variable. This increase corresponds to the difference between the 80th and 20th percentiles of the blood Pb distribution (4.92 μ g/dL versus 1.46 μ g/dL, respectively). 

### Figure 5
- 32 Multivariate adjus ted relative hazards of all
- cause and cardiovascular mortality.

Both Menke et al. ( 2006 ) and Schober et al. ( 2006 ) presented mortality curv es that plot 1 

### the HRs against blood Pb level. Figure 5

- 33 shows the mortality hazard ratio curves (not 2 

### absolute cases of mortality) for both stroke and MI reported by Menke et al. ( 2006 )

. 3 

### Nonlinear associations were modeled. The curves were fitted using predetermined 4 

### restricted quadratic splines with knots at the 10th perc entile (1.00 μ g/dL), the 50th 5 

### percentile (2.67 μ g/dL), and the 80th percentile (5.98 μ g/dL) blood Pb levels. The 6 

### authors did not explain the shape of the blood Pb

- mortality curves in detail; however, the 7 

### knots corresponded with the inflection points in the curve. In the tails of the blood Pb 8 

### distribution, hazard ratios decreased with increasing blood Pb level. However, hazard 9 

### ratios remained above 1 over most of the blood Pb distribution, and in the most heavily 10 

### populated portion of the blood Pb distributio n, hazard ratios increased with increasing 11 

### blood Pb level. Hazard ratios peaked for all outcomes at a blood Pb level of 12 

### approximately 6 μ g/dL. Lower concentration

- response functions at higher blood Pb 13February 2012 5

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### levels also have been found for blood Pb

- cognitive func tion relationships in children 1 

### ( Section 5.3.10 ). 2

Source: Reprinted with permission of Lippincott Wil l iams & Wilkins, Menke et al. ( 2006 ) Note: A histogram of blood Pb levels is superimposed in the background an d displayed on the right axis. 

### Figure 5
- 33 Multivariate
- adjusted relative hazard (left axis) of mortality associated with blood Pb level between 1 μ g/dL and 10 μ g/dL.

Schober et al. ( 2006 ) examined proportional hazard assumptions, tested for linear trend 3 

### across blood Pb tertiles, and evaluated log

- transformed continuous blood Pb level as 4



### a 5

- knot cubic spline (position of knots not reported). A statistically significant increasing 5 

### linear trend for mortality was observed across blood Pb tertiles. The results of the spline 6 

### fit of the continuous blood Pb level term to relative hazard of all cardiovascular diseases 7 

### reported by Schober et al. ( 2006 ) are shown in Figure 5

- 34 . In contrast to the curve 8 

### presented by Menke et al. ( 2006 ) , Schober et al. ( 2006 ) found the relative hazard axis and 9 

### the blood Pb axis largely to be linear (solid line). Dashed lines are 95% CIs. The hazard 10 

### ratio was fixed at 1.0 for the referent blood Pb level of 1.5 μ g/dL. In this study, hazard 11 

### ratios were less than one in the lower range of blood Pb levels. Despite differences in the 12February 2012 5

- 278 Draft

– Do Not Cite or Quote 

### age groups included, follow

- up time, categorization of blood Pb levels, and differences in 1 

### hazard ratio across the blood Pb range, results rep orted by Menke et al. ( 2006 ) and 2 

### Schober et al. ( 2006 ) are consistent in finding associations between higher blood Pb and 3 

### increased C VD mortality. 4

Source: Schober et al. ( 2006 ) Note: The solid line shows the fitted five

- knot spline relationship; the dashed lines are the point

- wise upper and lower 95% CIs. 

### Figure 5
- 34 Relative risk of all cause mortality for different blood Pb levels compared with referent level of 1.5 μ g/dL (12.5th percentile).

In addition to the NHANES analyses described above, studies of older adult males 5 

### ( Weisskopf et al., 2009 ) and older adult females ( Khalil, 2010 ; Khalil et al., 2 009a ) were 6 

### conducted recently. Weisskopf et al. ( 2009 ) used data from the NAS to determine th e 7 

### associations of blood, tibia, and patella Pb with mortality. The authors identified 241 8 

### deaths over an average observation period of 8.9 years (7 , 673 person

- years). The 9 

### strongest associations were observed between mortality and baseline patella Pb 10 

### concentration. Baseline tibia Pb levels were more weakly associated with CVD mortality. 11 

### Tibia bone Pb level is thought to reflect a longer cumulative expo sure period than is 12 

### patella bone Pb level because the residence time of Pb in trabecular bone is shorter than 13 

### that in cortical bone. IHD contributed most to the relationship between patella Pb and all 14 

### CVD death with an individual HR of 2.69 (95% CI: 1.42, 5.08). Although there was high 15 

### correlation between tibia and patella Pb (Pearson r = 0.77), compared with cortical bone 16 

### Pb, trabecular bone Pb may have more influence on circulating blood Pb level, and thus 17 

### local organ concentration of Pb, because of its s horter residence time in bone. In contrast 18February 2012 5

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– Do Not Cite or Quote 

### to the NHANES analyses, baseline blood Pb was not significantly related to 1 

### cardiovascular mortality in the NAS study. This discrepancy may be related to 2 

### differences in sample size and resulting power, modeling str ategies ( e.g., linear versus 3 

### log

- linear blood Pb level terms), or age range of the study populations. The duration of 4 

### follow

- up was similar across studies. The youngest subjects at baseline in the Weisskopf 5 

### et al. ( 2009 ) study were approximately 50

- 55 years old, compared to the youngest in the 6 

### Menke et al. ( 2006 ) and Schober et al. ( 2006 ) studies, who were 18 and 40 years, 7 

### respectively. Further, the blood Pb tertile analysis of Weisskopf et al. ( 2009 ) could have 8 

### been affected if the majority of a hypothesized nonlinear association was contained 9 

### largely in the lowest (reference) blood Pb tertile. 10 

### Weisskopf et al. ( 2009 ) also conducted a concentration

- response analysis. A linear trend 11 

### was observed for increasing HR across tertiles of both tibia and patella Pb l evels. The 12 

### linear relationship using tertile patella Pb was confirmed in other models in which 13 

### continuous patella Pb and nonlinear penalized spline terms (higher order terms) were not 14 

### statistically significant. The number of knots and their placement withi n the Pb variable, 15 

### which can influence these results, were determined by an iterative best fit procedure. 16 

### Concentration

- response relationships shown in Figure 5

- 35 were approximately linear for 17 

### patella Pb on the log HR scale for all CVD, but appeared nonlinear for IHD (p <0.10). 18 

### The peak HR is shown around 60 μ g/g, beyond which the HR tends to decrease. It is 19 

### important to note the wide confidence limits, which increase uncertainty at the lower and 20 

### upper bounds of patella Pb levels. 21February 2012 5

- 280 Draft

– Do Not Cite or Quote Source: Reprinted with permission of Lippincott Wil l iams & Wilkins, Weisskopf et al. ( 2009 ) Note: The reference logHR = 0 at the mean of patella Pb concentration. The estimates are indicated by the solid line and the 95% pointwise CIs by the das hed lines. The P values for significance of the nonlinear component for all

- cause, cardiovascular, and ischemic heart disease mortality were 0.42, 0.80 and 0.10 respectively. Patella Pb concentrations of all individual participa nts are indicated by short v ertical lines on the abscissa. Adjusted for age, education, smoking status, and pack

- years of smoking among participants without ischemic heart disease at baseline. 

### Figure 5
- 35 Associations between patella bone Pb level and the log of HR (logHR) for all
- cause, cardiovascular, and ischemic heart disease.
The association of adult blood Pb with mortality has also been examined among women 1 

### enrolled in the Study of Osteoporotic Fractures (SOF) ( Khalil et al., 2009a )

. This 2 

### prospective cohort (N = 533) enrolled female volunteers from two U.S. locations, 3 

### Baltimore, MD and Monongahela Valley, PA and followed women for an average of 12 4 

### y ears after blood Pb measurement. All

- cause mortality comparing women with blood Pb 5 

### levels >8 μ g/dL to those with blood Pb levels <8 μ g/dL was significantly increased (HR: 6 

### 1.59 [95% CI: 1.02, 2.49]). Combined cardiovascular disease mortality (HR: 1.78 [95% 7 

### CI: 0.92, 3.45]), coronary heart disease mortality (HR: 3.08 [95% CI: 1.23, 7.70]), but not 8 

### stroke mortality (HR: 1.13 [95% CI: 0.34, 3.81]) HR was increased among the women 9 

### enrolled in this study with blood Pb levels >8 μ g/dL. In addition, analyses of blo od Pb 10 

### tertiles and quintiles indicated that blood Pb

- mortality HRs were consistently elevated in 11 

### groups with blood Pb levels > 7 μ g/dL ( Khalil, 2010 )

. The findings for elevated mortality 12 

### HRs with the highest blood Pb levels are reinforced by the results displayed in Figure 13 

### 5

- 36 . The HR curve for all

- cause mortality is relatively flat over most of population 14 

### blood Pb distribution (represented by the blue dots) and increases only in the upper tail of 15 

### the blood Pb distribution where there are relatively few subjects ( i.e., fewer dots). 16February 2012 5

- 281 Draft

– Do Not Cite or Quote Source: Khalil et al. ( 2010 ) 

### Figure 5
- 36 Multivariate adjusted relative hazard (left axis) of mortality as a function of blood Pb levels between 1 μ g/dL and 15 μ g/dL.

5.4.5.1 Summary of Mortality
The mortality results in this review supported and expanded upon findings from the 2006 1 

### Pb AQCD ( U.S. EPA, 2006b ) , which included a few NHANES mortality studies 2 

### ( Schober et al., 2006 ; Lustberg and Silbergeld, 2002 )

. The recent NHANES mortality 3 

### study discussed above ( Menke et al., 2 006 ) addressed many of the limitations of the these 4 

### earlier studies, including control for a wider range of potential confounders, testing for 5 

### interactions with Pb, consideration of concentration

- response relationships, extensive 6 

### model evaluations, and examination of mortality from specific CVDs. Further, an 7 

### association with increased mortality was observed at lower mean population blood Pb 8 

### levels. The mean blood Pb level of the NHANES III population was 2.58 μ g/dL. The Pb 9 

### attributable risk of increased cardiovascular mortality in the NHANES III analysis of 10 

### Menke et al. ( 2006 ) increased with increasing blood Pb level over the most heavily 11 

### population portion of the blood Pb distribution and reached its maximum at blood Pb 12 

### levels between of 6 and 7 μ g/dL. It is import ant to note that the relative contributions of 13February 2012 5

- 282 Draft

– Do Not Cite or Quote 

### recent, past, and cumulative Pb exposure to associations observed with the baseline blood 1 

### Pb levels is uncertain. In addition, the first evidence that bone Pb, a metric of cumulative 2 

### Pb exposure, is associated with increased mortality was reported recently among NAS 3



### men ( Weisskopf et al., 2009 )

. 4 

### Quantitative differences in Pb

- asso ciated hazard for death between studies may be 5 

### influenced by age range of the study groups, follow up time to death, variation in model 6 

### adjustment, central tendency and range of the Pb biomarker levels, assumptions of 7 

### linearity in relationship with Pb biom arkers, and choice of Pb biomarker. Quantitative 8 

### differences in Pb

- associated mortality across NHANES II and NHANES III studies or 9 

### between different NHANES III analyses may be explained by the use of continuous or 10 

### ordered blood Pb terms and different data selection strategies. Further, studies using 11 

### ordered categories of blood Pb level may obtain different results, as the range of blood Pb 12 

### level represented in the reference category will affect the calculated coefficients of the 13 

### remaining percentiles or gro ups. 14 

### Specifically, Menke et al. ( 2006 ) is the strongest study presently published for estimating 15 

### the effects of Pb on cardiovascular disease

- related mortality. The study uses the 16 

### nationally representative NHANES III (1988

- 1994) sample of men and women. The 17 

### results provide confirmation of earlier published NHANES studies but address some of 18 

### the key weaknesses noted in those studies. For example, Menke et al. ( 2006 ) examined 19 

### potential confounding by a large number of factors, including hypertension and kidney 20 

### function. W eisskopf et al. ( 2009 ) is the first published mortality study using bone Pb as 21 

### an exposure index. The study is a prospective study with nearly 100% successful follow

22 

### up of deaths. This rigorous study found increased cardiovascular disease mortality in 23 

### association with patella bone Pb. The Khalil et al. ( 2010 ; 2009a ) SOF provides 24 

### supporting results in a different study cohort consisting of white females aged 65

- 87 25 

### years. Further, a number of prior studies have found association between accumulated Pb 26 

### reflected in bone Pb measurements and higher CVD morbidity (Se ctions 5.4.2.1 and 27 

### 5.4.3 ). This evidence base is augmented with new findings indicating that biomarkers of 28 

### longer

- term cumulative Pb exposure increases CVD mortality. The NAS and SOF 29 

### examine only men and women, respe ctively. However, the consistency of findings 30 

### between the two studies indicates that the results of either study may be applicable 31 

### widely. Despite the differences in design and methods across studies, with few exceptions 32 

### associations between higher levels of Pb biomarkers and higher risk of mortality were 33 

### consistently observed ( Figure 5

- 37 and Table 5

- 21 ). In studies that examined mortality 34 

### from specific CVD causes, MI, stroke, and IHD mortality, which are causes related to 35 

### higher BP and hypertension were all significantly elevated w ith higher Pb bioma rker 36 

### levels. 37February 2012 5

- 283 Draft

– Do Not Cite or Quote Note: Studies are presented in order of strength of study design and follow the order of discussion in the preceding text. Ha zard ratios represent the hazard in the higher blood or bone Pb group relative to that in the lowest blood or bone Pb group (refer e nce). 

### Figure 5
- 37 Hazard ratios for associations of blood Pb (closed markers) and bone Pb (open markers) with all
- cause mortality (black diamonds) and cardiovascular mortality (blue circles).February 2012 5

- 284 Draft

– Do Not Cite or Quote 

### Table 5
- 21 Additional characteristics and quantitative data for associations of blood and bone Pb with CVD mortality for studies presented in Figure 5
- 37

Study Population/ Location Parameter Pb Data Statistical Analys is Hazard Ratio or SMR (95% CI) M e nke et al. ( 2006 ) 13,946 adult participants of NHANES III , ≥ 17 yr (1988

- 1994) All cause and cause

- specific mortality (through 2000) CVD:ICD

- 9 390

- 434; ICD

- 10 I00

- I99), MI (ICD

- 9 410

- 414 and 429.2; ICD

- 10 I20

- I25), str oke (ICD

- 9 430

- 434 and 436

- 438; ICD

- 10 I60

- I69). Baseline Blood Pb (measured an average of 12 yr before mortality): Mean: 2.58 μg/dL Tertiles: <1.93 μg/dL, 1.94

- 3.62 μg/dL, ≥ 3.63 μg/dL Survey

- design adjusted Cox proportional hazard regression analysis ( up to 12 yr follow

- up) adjusted for Model 1: age, race/ethnicity, sex, Model 2: urban residence, cigarette smoking, alcohol consumption, education, physical activity, household income, menopausal status, BMI, CRP, total cholesterol, diabetes mellitus, Mode l 3: hypertension, GFR category All

- cause (3rd vs. 1st tertile): 1.25 (1.04, 1.51)
CVD (3rd vs. 1st): 1.55 (1.08, 2.24) MI (3rd vs. 1st):

1.89 (1.04, 3.43) Stroke (3rd vs. 1st):

2.51 (1.20, 5.26)
Cancer (3rd vs. 1st): 1.10 (0.82, 1.47) Schober et al. ( 2006 ) 9,686 adult participants of NHANES III, ≥ 40 yr All cause and cause

- specific mortality Ordered categorical blood Pb level, measured a median of 8.55 yr prior to death <5 μg/dL 5

- 9 μg/dL ≥ 10 μg/dL Survey

- design adjusted Cox proportional hazard adjusted for sex, age, race/ethnicity, smoking, education level All

- cause (2nd vs. 1st): 1.24 (1.05, 1.48) All

- cause (3rd vs. 1st): 1.59 (1.28, 1.98)
CVD (2nd vs. 1st): 1.20 (0.93, 1.55) CVD (3rd vs. 1st): 1.55 (1.16, 2.07)
Cancer (2nd vs. 1st): 1.44 (1.12, 1.86) Cancer (3rd vs. 1st): 1.69 (1.14, 2.52) Lustberg and Silbergeld ( 2002 ) 4,190 adult parti cipants of NHANES III, yr (1976

- 1980) All cause and cause

- specific mortality Categorical blood Pb level Mean: 14.0 (5.1) Median: 13 μg/dL <10 μg/dL (Reference) 10

- 19μg/dL 20

- 29 μg/dL Crude mortality RRs adjusted for age, sex, locatio n, education, race, income, smoking, BMI, exercise All

- cause (2nd vs. 1st): 1.40 (1.16

- 1.69) All

- cause (3rd vs. 1st): 2.02 (1.62

- 2.52)
Circulatory (2nd vs

. 1st): 1.27 (0.97

- 1.57) Circulatory (3rd vs. 1st): 1.74 (1.25

- 2.40)
Cancer (2nd vs

. 1st): 1.95 (1.28

- 2.98) Cancer (3rd vs. 1st): 2.89 (1.79

- 4.64)February 2012 5

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– Do Not Cite or Quote Study Population/ Location Parameter Pb Data Statistical Analys is Hazard Ratio or SMR (95% CI) Weisskopf et al. ( 2009 ) 868 men, >55 yr, 95% white, from NAS in Greate r Boston area, MA All cause and cause

- specific mortality Pb biomarkers collected an average of 8.9 years before death Blood Pb: Mean (SD): 5.6 (3.4) μg/dL

Patella Pb: Mean (SD): 31.2 (19.4) μg/g Tertiles: <22 μg/g, 22

- 35 μg/g, >35 μg/g

Tibia Pb: Mean (SD): 21.8 (13.6) μg/g Cox proportional hazard regression analysis adjusted for age, smoking, education. Additional models adjusted for alcohol intake, physical activity, BMI, total cholesterol, serum HDL, diabetes mellitus, race, and hypertension All

- caus e (3rd vs. 1st patella Pb tertile): 1.76 (0.95, 3.26)
All CVD (3rd vs. 1st tertile): 2.45 (1.07, 5.60) IHD (3rd vs. 1st):

8.37 (1.29, 54.4)
Cancer (3rd vs. 1st): 0.59 (0.21, 1.67)

After excluding 154 subjects with CVD and stroke at baseline: All

- cause (3rd vs. 1st): 2.52 (1.17

- 5.41) All CVD (3rd vs. 1st): 5.63 (1.73, 18.3)

All

- cause (3rd vs. 1st blood Pb tertile): 0.93 (0.59, 1.45)
All CVD (3rd vs. 1st): 0.99 (0.55, 1.78) IHD (3rd vs. 1st):

1.30 (0.54, 3.17) Khalil et al. ( 2009a ) 533 women, 65

- 87 yr, from Study of Osteoporotic Fractures cohort in Baltimore, MD and Monongahela Valley, PA All cause and cause

- specific mortality Blood Pb measured an average 12 (SD; 3) yr before death: Mean (SD; range): 5.3 (2.3; 1

- 21) μg/dL Cox proportional hazards regression analysis adjusted for age, clinic, BMI, education, smoking, alcohol intake, estrogen use, hypertension, total hip BMD, walking for exercise, and diabetes ≥ 8 μg/dL vs. <8 μg/dL

All cause: 1.59 (1.02, 2.49)
CVD: 1.78 (0.92, 3.45)

Coronary Heart Disease:

3.08 (1.23, 7.70) Stroke: 1.13 (0.34, 3.81)
Cancer: 1.64 (0.73, 3.71)February 2012 5

- 286 Draft

– Do Not Cite or Quote Study Population/ Location Parameter Pb Data Statistical Analys is Hazard Ratio or SMR (95% CI)

a Neuberger et al. ( 2009 ) Residents at or near Tar Creek Superfund site, Ottawa County, OK (exposed pop. 5,852, unexposed pop. 16,210) Cause

- specific mortality Not reported Standardized mortality ratio (SMR) based on 2000 U.S. Census data Hea rt disease: Both sexes:

114.1 (113.1, 115.2) Men:

118 (116.4, 119.6) Women:

111 (109.5, 112.5)

Stroke:

Both sexes:

121.6 (119.2, 123.9) Men:

146.7 (107.4, 195.7) Women:

106.5 (80.2, 138.6)

a Cocco et al. ( 2007 ) 933 male Pb smelter workers from Sardinia, Italy (1973

- 2003) All cause and cause

- specific mortality Not reported SMR All cause: 56 (46, 68)
CVD: 37 (25, 55)

a These r eferences not included in Figure 5

- 37 because they reported standardized mortality ratios.



### 5.4.6 Air Lead
- P articulate Matter Studies

5.4.6.1 Cardiovascular Morbidity
A relatively small number of studies used Pb measured in PM 10 

### and PM 2.5 

### ambient air 1 

### samples to represent Pb exposures. However, given that size distribution data for Pb

- PM 2 

### are fairly limited, it is difficult to assess the representativeness of these concentrations to 3 

### population exposure (Section 3.5.3). Moreover, data illustra ting the relationships of Pb

4 

### PM 10 

### and Pb

- PM 2.5 

### with blood Pb levels are lacking. A few available studies exposed 5 

### rats, dogs, or humans to concentrated ambient particles (CAPS) in which Pb and several 6 

### other components were measured. Consistent with epidemio logic studies of blood and 7 

### bone Pb and with studies of animals exposed to Pb, exposure to Pb

- containing CAPS 8 

### resulted in various changes related to increased vasoconstriction ( Urch et al., 2004 ; 9 

### Wellenius et al., 2003 ; Batalha et al., 2002 )

. Whereas studies of Pb biomarkers primarily 10 

### found cardiovascular effects with indicators of long

- term Pb exposure, studies of Pb

11 

### containing CAPS provide evidence for cardiovascular effects with short

- term exposure 12 

### (2

- 6 hours over multiple days) It is important to note that Urch et al. ( 2004 ) estimated the 13 

### Pb effect on brachial artery diameter based only on the ambient concentrations of Pb but 14 

### did not directly expose their young (mean age: 35 years, SD: 10), healthy adult 15 

### participants to Pb isolated from CAPS. 16February 2012 5

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– Do Not Cite or Quote 

### A U.S. time

- series study of almost 3 million pregnant women foun d that increa ses in 1 

### ambient Pb

- TSP concentrations were associated with increased odds of PIH assessed at 2 

### delivery ( Chen et al., 2006c )

. In contrast, epidemiologic studies provide weak evidence 3 

### for an association between short

- term changes (daily average) in am bient air 4 

### concentrations of Pb

- PM 2.5 

### and cardiovascular morbidity in adults. Some of these time

5 

### series studies analyzed Pb individually, whereas others applied source apportionment 6 

### techniques to analyze Pb as part of a group of correlated components. In a time

- series 7 

### study of 106 U.S. counties, Bell et al. ( 2009 ) found t hat an increase in lag 0 Pb

- PM 2.5 8 

### was associated with an increased risk of cardiovascular hospital admissions among adults 9 

### ages 65 years and older. Quantitative results were not presented; however, the 95% CI 10 

### was wide and included the null value. In this study, statistically significant associations 11 

### were observed for other PM metal components such as nickel, vanadium, and zinc. In the 12 

### absence of detailed data on correlations among components or results adjusted for 13 

### copollutants, it is difficult to exclude confounding by ambient air exposures to these other 14 

### components or copollutants. 15 

### To address correlations among PM chemical components, some studies applied source 16 

### apportionment techniques to group components into common source categories. In these 17



### source
- factor studies, it is not possible to attribute the observed association ( Sarnat et al., 18 

### 2008 ) or lack of association ( Andersen et al., 2007 ) specifically to Pb. 19



### 5.4.6.2 Mortality

Time

- series epidemiologic studies of ambient air Pb

- PM 2.5 

### reported positive associations 20 

### with mortality. Although limited in number, these studies indicated associations in 21 

### multiple cities across the U.S. In the Harvard Six Cities Study, Laden et al. ( 2000 ) found 22 

### a 1.16% (95% CI: 0.20, 2.9%) increased risk in all

- cause mortality per 461.4 ng/m 3 

### (5th

23 

### 95th percentile) increase in Pb

- PM 2.5 

### . In six California counties, Ostro et al. ( 2007 ) 24 

### found that a 5 ng/m 3 

### (interquartile range) increase in Pb

- PM 2.5 

### was associated with a 25 

### 1.89% (95% CI:

- 0.57, 4.40%) increased risk of cardiovascular mortality and a 1.74% 26 

### (95% CI: 0.24, 3.26%) increased risk of all

- c ause mortality during the cool season. The 27 

### limitations of air

- Pb studies were described in Section 5.4.6.1 above and also are relevant 28 

### to the interp retation of these findings for mortality. 29



### 5.4.7 Summary and Causal Determination
The 2006 Pb AQCD concluded that there was a relationship between higher blood Pb and 30 

### bone Pb and cardiovascular effects in adults, in particular increased BP and increased 31February 2012 5

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– Do Not Cite or Quote 

### incidence of hypertension ( U.S. EPA, 2006b )

. This conclusion was substantiated by the 1 

### coherence observed between epidemiologic and toxicological findings. The large 2 

### evidence base comprising epidemiologic studie s conducted in diverse populations using 3 

### different designs clearly demonstrated a positive association between blood Pb level and 4 

### BP. Meta

- analysis of these studies found that each doubling of concurrent blood Pb level 5 

### (between 1 and >40 μg/dL) was associa ted with a 1 mmHg increase in systolic BP and a 6 

### 0.6 mmHg increase in diastolic BP ( Nawrot et al., 2002 )

. In addition, most of the 7 

### reviewed studies using bone Pb levels also showed an association with B P, pointing to an 8 

### effect of cumulative Pb exposure. Similarly, toxicological studies provided evidence that 9 

### long

- term exposure to Pb (> 4 weeks) results in increased BP in experimental animals 10 

### that persists long after the cessation of Pb exposure. Also, an imal toxicological studies 11 

### provided mechanistic evidence to support the biological plausibility of Pb

- induced 12 

### hypertension, including Pb

- induced oxidative stress, activation of RAAS, altered 13 

### sympathetic activity, and vasomodulator imbalance. Studies in the 2006 Pb AQCD also 14 

### found associations between Pb biomarkers and other cardiovascular diseases such as 15 

### IHD, cerebrovascular disease, peripheral vascular disease, and cardiovascular disease 16 

### related mortality; however, the available evidence was limited. 17


### Bui lding on the strong body of evidence presented in the 2006 Pb AQCD, recent 18 

### epidemiologic and toxicological studies strengthened the evidence that long

- term Pb 19 

### exposure is associated with cardiovascular effects in adults with the largest body of 20 

### evidence de monstrating associations of Pb with increased BP and hypertension. Recent 21 

### epidemiologic studies addressed past uncertainties, including the potential for 22 

### confounding. A recent study in an ethnically diverse community

- based cohort of women 23 

### and men aged 50

- 7 0 years found hypertension risk to be associated with blood and tibia 24 

### Pb levels ( Martin et al., 2006 )

. These findings are consistent wit h those of other studies, 25 

### demonstrating that with each increase of 1 μ g/dL concurrent blood Pb level, systolic BP 26 

### increased 1 mmHg and diastolic BP increased 0.5 mmHg. Recent epidemiologic studies 27 

### in adults found associations with hypertension in populatio ns with relatively low mean 28 

### blood Pb levels. For example, a positive relationship was found in the nationally 29 

### representative NHANES III (1988

- 1994) where the geometric mean blood Pb level of the 30 

### population was 1.64 μ g/dL ( Muntner et al., 2005 )

. Despite the extensive evidence for 31 

### relatively low concurrent blood Pb levels, as these cardiovascular outcomes were most 32 

### often examined in adults that have been exposed to higher levels of Pb earlier in life, 33 

### uncertainty remains concerning the Pb exposure level, timing, frequency, and duration 34 

### contributing to the ob served associations. A new prospective study in Pb workers found 35 

### independent associations of both baseline blood Pb level and subsequent changes in 36 

### blood Pb over follow

- up with changes in BP over follow

- up and bone Pb level with 37 

### hypertension ( Glenn et al., 2006 )

. The results indicated that different mechanisms may 38February 2012 5

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– Do Not Cite or Quote 

### mediate shorter

- term Pb

- associated increases in BP and longer

- term Pb

- associated 1 

### development of hypertension. 2 

### Collectively, all animal toxicological studies providing blood Pb level and BP 3 

### measurements reported increases in BP with increasing blood Pb level ( Figure 5

- 29 ). 4 

### Importantly, an animal toxicological study provided support for increased BP following 5 

### long

- term Pb exposure resulting in low blood Pb levels with mean blood Pb levels as low 6 

### as 2 μ g/dL ( Tsao et al., 2000 )

. However, a majority of studies examined Pb exposures 7 

### that resulted in mean blood Pb levels > 10 μ g/dL. Thus, the weight of the evidence 8 

### demonstrated such effects in animal s with blood Pb levels >10 μ g/dL. New studies also 9 

### demonstrated only partial reversibility of Pb

- induced increased BP following Pb exposure 10 

### cessation or chelation and the possibility for short

- term Pb exposure

- induced increases in 11 

### BP. 12 

### The epidemiologic evi dence for a relationship between Pb exposure and cardiovascular 13 

### disease is strengthened by observations of cardiovascular effects in association with both 14 

### blood and bone Pb level after adjusting for multiple potential confounding factors, 15 

### including age, se x, BMI, antihypertensive medication, SES, race/ethnicity, alcohol 16 

### consumption, serum total cholesterol, smoking, educational level, diabetes, and measures 17 

### of renal function. The epidemiologic evidence also was substantiated by several available 18 

### prospective studies that found associations between biomarkers of Pb and cardiovascular 19 

### health endpoints. These studies inform the temporality of these relationship between 20 

### biomarkers of Pb exposure and cardiovascular morbidity ( e.g., HRV, IHD, BP, 21 

### hypertension) ( Eum et al., 2011 ; Jain et al., 2007 ; Peters et al., 2007 ; Glenn et al., 2006 ) 22 

### and mortality ( Khalil et al., 2009a ; Weisskopf et al., 2009 ; Menke et al., 2006 ; Schober et 23 

### al., 2006 )

. 24 

### Epidemiologic studies continued to demonstrate a relationship between bone Pb, which is 25 

### a metric of cumulative Pb exposure, and BP in adults. Studies that examined both blood 26 

### and bone Pb levels did not conclusively demonstrate a stronger association for either 27 

### blood or bone Pb ( Perlstein et al., 2007 ; Glenn et al., 2006 ; Martin et al., 2006 )

. In a 28



### r ecent meta

- analysis, Navas

- Acien et al. ( 2008 ) found that studies passing the detailed 29 

### inclusion criteria all showed a relationship between higher bone Pb levels and higher BP. 30 

### Also, all but one that characterized hypertension showed higher relative risks or odds 31 

### ratios asso ciated with higher bone Pb levels. Recent epidemiologic studies also 32 

### emphasized the interaction between bone Pb levels and factors that modify the 33 

### association with BP or hypertension, such as race/ethnicity, chronic stress and metabolic 34 

### syndrome. Bone Pb c oupled with high perceived stress was associated with an increased 35 

### risk of developing hypertension in an originally nonhypertensive group of adults ( Peters 36 

### et al., 2007 )

. Also, bone Pb level was associated with larger decreases in HRV (which has 37February 2012 5

- 290 Draft

– Do Not Cite or Quote 

### been associated with increased cardiovascular even ts) among adults with metabolic 1 

### syndrome, also associated with increased risk of cardiovascular events ( Park et al., 2006 )

. 2 

### The extensive epidemiologic evidence for bone Pb levels in concert wi th the animal 3 

### toxicological evidence for long

- term Pb exposure, support an effect of long

- term, 4 

### cumulative Pb exposure on cardiovascular morbidity. 5 

### Recent epidemiologic studies of adults investigated the interaction of Pb biomarkers with 6 

### genetic variants i n associations with cardiovascular effects. Evidence was presented for a 7 

### larger blood Pb

- associated increase in BP in carriers of the ALAD2 allele, which is 8 

### associated with greater binding affinity for Pb in the bloodstream ( Figure 5

- 26 for results) 9 

### ( Scinicariello et al., 2010 )

. Additionally, bone Pb concentration was associated with 10 

### larger increases in PP, which represents as a good predictor of cardiovascular morbidity 11 

### and mortality and an indicator of arterial stiffness, among adults with the HFE H63D 12 

### and/or C282Y variant ( Zhang et al., 2010a ) ( Figure 5

- 26 for results). Park et al. ( 2009b ) 13 

### provided furt her evidence of HFE and transferrin gene variants, related to iron 14 

### metabolism, impacting the associations of bone Pb levels with cardiovascular effects, 15 

### evaluated by QT interval changes. 16 

### Epidemiologic and toxicological evidence indicates that Pb exposure n ot only increases 17 

### BP and hypertension, but can contribute to the development of other cardiovascular 18 

### diseases in adults. However, fewer studies have been published compared to studies of 19 

### BP and hypertension. Both recent epidemiologic and toxicological stud ies provide 20 

### evidence in adults for blood Pb

- associated increased atherosclerosis, thrombosis, IHD, 21 

### PAD, arrhythmia, and cardiac contractility in populations with mean blood Pb levels 22 

### >2.5 μ g/dL ( Table 5

- 19 ). Further, animal toxicological evidence continued to build on the 23 

### evidence supporting the mechanisms leading to these cardiovascular system responses, as 24 

### well as Pb

- induced changes in BP and hypertens ion. Enhanced understanding of Pb

25 

### induced oxidative stress including • 

### NO inactivation, endothelial dysfunction leading to 26 

### altered vascular reactivity, activation of the RAAS, and vasomodulator imbalance 27 

### provides biological plausibility for the consistent a ssociations observed between higher 28 

### blood and bone Pb levels and greater cardiovascular effects. 29 

### Several studies in children reported associations of childhood (concurrent and measured 30 

### at an average age of 2.6 years) blood Pb levels with cardiovascular cha nges such as TPR 31 

### and autonomic and cardiovascular dysregulation in response to acute stress tasks 32 

### measured between ages 5 to 15 years ( Gump et al., 2011 ; 2009 ; Gump et al., 2007 )

. Also, 33 

### maternal bone and cord blood Pb levels, biomarkers of prenatal Pb exposure, were related 34 

### to higher BP in children ( Gump et al., 2005 ; Zhang et al., In Press )

. These study findings 35 

### of children add evidence that Pb exposure is associated with small increases in BP, and 36February 2012 5

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– Do Not Cite or Quote 

### that changes in the cardiovascular system precedent to later CVD are potentially 1 

### impac ted by early

- life Pb exposure. 2 

### New evidence extended the potential continuum of Pb

- related cardiovascular effects by 3 

### demonstrating associations between Pb biomarkers and both cardiovascular and all

- cause 4 

### mortality with follow

- up periods ranging between 8 a nd 12 years. All

- cause mortality 5 

### was increased with increasing blood Pb level. A recent analysis of the NHANES III 6 

### sample reported associations of adult blood Pb level with cardiovascular mortality, with 7 

### stronger associations observed with myocardial infar ction and stroke mortality ( Menke et 8 

### al., 2006 )

. These findings were supported by a community

- based cohort of women age 9 

### 65

- 87 years, in which higher effect estimates were observed for mortality from 10 

### cardiovascular disease and coronary heart disease ( Khalil et al., 2009a )

. Weisskopf et al. 11 

### ( 200 9 ) published the first mortality study using bone Pb as an exposure index. This 12 

### prospective study found that patella bone Pb levels were associated with increased 13 

### mortality from cardiovascular disease and IHD with hazard ratios of 5.6 and 8.4, 14 

### respectiv ely. 15 

### Changes in BP that have been associated with biomarkers of Pb exposure indicate a 16 

### modest change for an individual; however, these modest changes can have a substantial 17 

### implication at the population level. The reported effects represent a central tende ncy of 18 

### Pb

- induced cardiovascular effects among individuals; some individuals may differ in risk 19 

### and manifest effects that are greater in magnitude. For example, a small increase in BP 20 

### may shift the population distribution and result in considerable increas es in the 21 

### percentages of individuals with BP values that are clinically significant, i.e., an indication 22 

### of hypertension and medication use. Studies in the medical literature show that increasing 23 

### BP, even within the nonhypertensive range, is associated wit h increased rates of death 24 

### and cardiovascular disease, including coronary disease, stroke, PAD, and cardiac failure 25 

### ( Ingelsson et al., 2008 ; Chobanian et al., 2003 ; Pastor

- Barriuso et al., 2003 ; Prospective 26 

### Studies Collaboration, 2002 ; Kannel, 2000a , b ; Neaton et al., 1995 )

. 27 

### In summary, new studies evaluated in the current review supported and expanded upon 28 

### the strong body of evidence presented in the 2006 Pb AQCD that Pb exposure is 29 

### associated with cardiovascular health effects. The weight of both epidemiologic and 30 

### toxicological evidence continues to support a consistent relationship between P b 31 

### exposure and increased BP or hypertension development in adults. The epidemiologic 32 

### evidence is strengthened by several prospective studies that find associations between 33 

### biomarkers of Pb and BP and hypertension and by eff ect estimates that are observed a fter 34 

### adjustment for multiple potential confounding factors. The weight of epidemiologic 35 

### evidence supported associations in adults with mean concurrent blood Pb levels less than 36 

### 5 μ g/dL. As these outcomes in epidemiologic studies were most often observed in adults 37February 2012 5

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– Do Not Cite or Quote 

### with likely higher past than current Pb exposures, uncertainty exists as to the Pb exposure 1 

### level, timing, frequency, and duration contributing to the observed associations. Recent 2 

### epidemiologic studies found that bone Pb level, a metric of cumulat ive exposure, is 3 

### strongly related to hypertension risk in adults with mean bone Pb levels greater than 4 

### 20 μ g/g. However, uncertainties also exist as to the specific Pb exposure conditions that 5 

### contributed to the associations. The weight of animal evidence also demonstrates an 6 

### increase in BP after long

- term ( i.e., greater than 4 weeks) exposure to Pb. Whereas the 7 

### majority of studies examined and found increases in BP in animals with mean blood Pb 8 

### levels greater than 10 μ g/dL, a recent studies found elevated BP in animals with a mean 9 

### blood Pb level of 2 μ g/dL. By demonstrating Pb

- induced oxidative stress including • 

### NO 10 

### inactivation, endothelial dysfunction leading to altered vascular reactivity, activation of 11 

### the RAAS, and vasomodulator imbalance, toxicological studies have characterized the 12 

### modes of action of Pb and provided biological plausibility for the consistent associations 13 

### observed in epidemiologic studies between blood and bone Pb and cardiovascular effects. 14 

### These associations of Pb with cardiovascular morbidity observed in both epidemiologic 15 

### and toxicological studies support recent epidemiologic findings of increased Pb

16 

### associated cardiovascular mortality. Collectively, the evidence integrated across 17 

### epidemiologic and toxicological studies as well as ac ross the spectrum of other 18 

### cardiovascular endpoints examined is sufficient to conclude that there is a causal 19 

### relationship between Pb exposures and cardiovascular health effects
. 20



## 5.5 Renal Effects

5.5.1 Introduction
This section summarizes key findings with regard to effects of Pb on the kidney in animal 21 

### toxicology and epidemiologic studies. Findings summarized across epidemiologic and 22 

### toxicological studies indicate that chronic Pb exposure is associated with pathological 23 

### changes in the renal system such as proximal tubule (PT) cytomegaly, renal cell 24 

### apoptosis, mitochondrial dysfunction, aminoaciduria, increased electrolyte excretion, 25 

### ATPase dysfunction, oxidant redox imbalance, altered glomerular filtration rate (GFR), 26 

### chronic kidney disease (CKD) development, and a ltered • 

### NO homeostasis with ensuing 27 

### elevated BP. As several of these outcomes are most often observed in adults with likely 28 

### higher past Pb exposures, uncertainty exists as to the Pb exposure level, timing, 29 

### frequency, and duration contributing to the associ ations observed with blood or bone Pb 30 

### levels. 31February 2012 5

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### The cardiovascular and renal systems are intimately linked. Homeostatic control at the 1 

### kidney level functions to regulate water and electrolyte balance via filtration, re

2 

### absorption and excretion and is under tight hormonal control. Pb exposure damages the 3 

### kidneys and its vasculature and systemic hypertension ensues with effects on the 4 

### cardiovascular and renal systems ( Section 5.4 ). Chronic increases in vascular pressure 5 

### can contribute to glomerular and renal vasculature injury, which can lead to progressive 6 

### renal dysfunction and kidney failure. In this manner, Pb

- induced hypertension has been 7 

### noted as one cause of Pb

- induced renal disease. However, the relationship between BP 8 

### and renal function is more complicated. Not only does hypertension contribute to renal 9 

### dysfunction but damage to the kidneys can also cause increased BP. Long

- term control of 10 

### arterial pressure is affected by body fluid homeostasis which is regulated by the kidneys. 11 

### In examining the physiological definition of BP ( i.e., mean BP equates to cardiac output 12 

### multiplied by total peripheral resistance [TPR]) the role of the kidneys in BP regul ation is 13 

### highlighted. Cardiac output is driven by left ventricular and circulating blood volume. 14 

### TPR is driven by vasomodulation and electrolyte balance. Thus, it is possible to dissect 15 

### the causes of hypertension from features of primary kidney disease. In creased 16 

### extracellular fluid volume results in increased blood volume which enhances venous 17 

### return of blood to the heart and increases cardiac output. Increased cardiac output not 18 

### only directly increases BP, but also increases TPR due to a compensatory auto regulation 19 

### or vessel constriction. In addition, damage to the renal vasculature will alter the intra

20 

### renal vascular resistance thereby altering kidney function and affecting the balance 21 

### between renal function and BP. The interactions between these systems can lead to 22 

### further exacerbation of vascular and kidney dysfunction following Pb exposure. As 23 

### kidney dysfunction can increase BP and increased BP can lead to further damage to the 24 

### kidneys, Pb

- induced damage to both systems may result in a cycle of further increased 25 

### severity of disease. 26 

### In general, associations between bone Pb (particularly in the tibia) and health outcomes in 27 

### adults indicate chronic effects of cumulative Pb exposure. In adults without current 28 

### occupational Pb exposure, blood Pb level represe nts both recent and cumulative Pb 29 

### exposure. In particular, blood Pb level may represent cumulative exposure in 30 

### physiological circumstances of increased bone remodeling or loss ( e.g., osteoporosis and 31 

### pregnancy) when Pb from bone of adults contributes subst antially to blood Pb 32 

### concentrations. Blood Pb in children is also influenced by Pb stored in bone due to rapid 33 

### growth

- related bone turnover in children relative to adults. Thus, blood Pb in children is 34 

### also reflective of cumulative dose. Additional details on the interpretation of Pb in blood 35 

### and bone are provided in Section 4.3.5 . The toxicokinetics of Pb in blood and bone are 36 

### important consideration s in making inferences about etiologically

- relevant Pb exposures 37 

### that contributed to associations observed between blood and bone Pb levels and health 38 

### outcomes. 39February 2012 5

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### 5.5.1.1 Kidney Outcome Measures
The primary function of the kidneys is to filter waste from the body wh ile maintaining 1 

### appropriate levels of water and essential chemicals, such as electrolytes, in the body. 2 

### Therefore, the gold standard for kidney function assessment involves measurement of the 3 

### GFR through administration of an exogenous radionuclide or radio contrast marker 4 

### ( e.g., 125I

- iothalamate, iohexol) followed by timed sequential blood samples or, more 5 

### recently, kidney imaging, to assess clearance through the kidneys. This procedure is 6 

### invasive and time

- consuming. Therefore, serum levels of endogenous co mpounds are 7 

### routinely used to estimate GFR in large epidemiologic studies and clinical settings. 8 

### Creatinine is the most commonly measured endogenous compound; blood urea nitrogen 9 

### (BUN) has also been examined. Increased serum concentration or decreased kidn ey 10 

### clearance of these markers both indicate decreased kidney function. The main limitation 11 

### of endogenous compounds identified to date is that non
- kidney factors impact their serum 12 

### levels. Specifically, since creatinine is metabolized from creatine in muscl e, muscle mass 13 

### and diet affect serum levels resulting in variation in different population subgroups 14 

### ( e.g., women and children compared to men ) , that are unrelated to kidney function. 15 

### Measured creatinine clearance, involving measurement and comparison of creatinine in 16 

### both serum and urine, can address this problem. However, measured creatinine clearance 17 

### utilizes timed urine collections, tradition ally over a 24

- hour period, and the challenge of 18 

### complete urine collection over an extended time period makes compliance difficult. 19 

### Therefore equations to estimate kidney filtration that utilize serum creatinine but also 20 

### incorporate age, sex, race, and, i n some, weight (in an attempt to adjust for differences in 21 

### muscle mass), have been developed. Although these are imperfect surrogates for muscle 22 

### mass, such equations are currently the preferred outcome assessment method. 23 

### Traditionally, the Cockcroft

- Gault equation ( Cockcroft and Gault, 1976 ) , which estimates 24 

### creatini ne clearance, a GFR surrogate, has been used. In the last decade, the abbreviated 25 

### Modification of Diet in Kidney Disease (MDRD) Study equation ( Levey et al., 2000 ; 26 

### Levey et al., 1999 ) , which estimates GFR, has become the standard in the kidney 27 

### epidemiologic and clinical communities. With widespread use of the MDRD equation, it 28 

### became clear that the equa tion underestimates GFR at levels in the normal range. 29 

### Therefore, the CKD

- Epidemiology Collaboration (CKD

- EPI) equation was recently 30 

### developed to be more accurate in this range ( Levey et al., 2009 )

. This is a decided 31 

### advan tage in nephrotoxicant research since most participants in occupational and many 32 

### even in general population studies have GFRs in a range that is underestimated by the 33 

### MDRD equation. 34


### Both the MDRD and CKD

- EPI equations use serum creatinine. Due to the inabi lity to 35 

### adjust serum creatinine levels for muscle mass, alternative serum biomarkers have been 36February 2012 5

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### evaluated such as cystatin C, a cysteine protease inhibitor that is filtered, reabsorbed, and 1 

### catabolized in the kidney ( Fried, 2009 )

. It is produced and secreted by all nucleated cells 2 

### thus avoiding the muscle mass confounding that exists with serum creatinine ( Fried, 3 

### 2009 )

. However, recent research indicates that serum cystatin C varies by age, sex, and 4



### race ( Kottgen et al., 2008 )

. Thus, a cystatin C

- based eGFR equation was recently 5 

### developed that includes age, sex, and race ( Stevens et al., 2008 )

. 6 

### Most of the kidney outcome measures discussed above were developed for use in the 7 

### clinical setting. Unfortunately, they are insensitive for detection of early kidney damage, 8 

### as evidenced by the fact that serum creat inine remains normal after kidney donation. 9 

### Therefore, in the last two decades, the utility of early biological effect (EBE) markers as 10 

### indicators of preclinical kidney damage has been of interest. These can be categorized as 11 

### markers of function ( i.e., low molecular weight proteins that should be reabsorbed in the 12 

### PT such as β2

- microglobulin and retinol

- binding protein [RBP]); biochemical alteration 13 

### ( i.e., urinary eicosanoids such as prostaglandin E2, prostaglandin F2 alpha, 6

- keto

14 

### prostaglandin F 1 

### alpha, a nd thromboxane B2); and cytotoxicity ( e.g., N

- acetyl

- β

- D

15 

### glucosaminidase [NAG]) ( Cardenas et al., 1993 )

. Elevated levels may indicate an 16 

### increased risk for subsequent kidney dysfunction. However, most of these markers are 17 

### research tools only, and their prognostic value remains uncertain since prospective 18 

### studies of most of these markers in nephrotoxicant

- expo sed populations are quite limited 19 

### to date. Recently, microalbuminuria has been identified as a PT marker, not just 20 

### glomerular as previously thought ( Comper and Russo, 2009 )

. Kidney EBE markers are a 21 

### major recent focus for research in patients with acute kidney injury (AKI) and markers 22 

### such as neutrophil gelatinase

- associated lipocalin (NGAL) and kidney injury molecule

- 1 23 

### (Kim

- 1), developed in AKI research, may prove useful for chronic nephrotoxicant work 24 

### as well ( Ferguson et al., 2008 ; Devarajan, 2007 )

. 25



### 5.5.2 Nephrotoxicity and Renal Pathology

5.5.2.1 Toxicology
Figure 5

- 38 and Table 5

- 22 presents the recent animal toxicological data for studies 26 

### investigating the effects of Pb (as blood Pb level) on various measures of kidney health 27 

### and function. In animals, Pb has been found to induce changes in a wide range of 28 

### indicators of renal function. Most studies examined Pb exposure concentrations that 29 

### resulted in hig her blood Pb levels (> 20 μ g/dL) than those in the current U.S. general 30 

### population. As indicated in Figure 5

- 38 and Table 5

- 22 , toxicological information on 31 

### renal dysfunction with blood Pb levels < 10 μ g/dL) generally is not available. 32February 2012 5

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### Dysfunction in kidney function m easures, including urinary flow, ALP, microalbumin, 1 

### and NAG, was observed at blood Pb concentrations above 20 μ g/dL ( Wang et al., 2 

### 2010d )

. 3



### Figure 5
- 38 Concentration
- response representatio n of the effect of Pb on renal outcomes in animal toxicology studies. 1 10 100February 2012 5

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### Table 5
- 22 Additional characteristics for results of toxicological studies presented in Figure 5
- 38

Reference Species; Lifestage; Sex Pb Dose; Exposure Duration Blood Pb Level with Response (μg/dL) Outcome Wang et al. ( 2010d ) Rat; Adult; Female 300 ppm Pb

- acetate in drinking water; 8 weeks 20 Biomarker

- Aberrant NAG, GGT, β2

- microgobulin expression

Roncal et al

. ( 2007 ) Rat; Adult; Male 150 ppm Pb

- acetate in drinking water; 16 weeks with remnant kidney surgery at week 4 26 Inflammation

- Elevation in number of macrophages & marker MCP

- 1 in Pb exposed kidneys with remnant kidney surgery.

Navarro

- Moreno et al. ( 2009 ) Rat; Adult; Male 500 ppm Pb

- acetate in drinking water; 28 weeks 43 Oxidative s tress

- Increased kidney lipid peroxidation ( i.e., TBARS)
Wang et al. ( 2010d ) Rat; Adult; Female 300 ppm Pb

- acetate in drinking water; 8 weeks 20 Oxidative Stress

- Pb caused increased lipid peroxidation ( i.e., MDA production), elevated kidney antioxidant enzymes (SOD, GPx, CAT), and depleted GSH Massó

- González et al. ( 2009 ) Rat; Weanling pups 300 ppm Pb

- acetate in drinking water; GD1 to PND 21 23 Oxidative stress

- Elevated TBARS and catalase activity

Roncal et al. ( 2007 ) Rat; Adult; Male 150 ppm Pb

- acetate in drinking water; 16 weeks with remnant kidney surgery at week 4 26 Morphology

- Pb induced pre

- glomerular vascular disease of kidney ( i.e., sclerosis, fibrosis, peritubular capillary loss)

Navarro

- Moreno et al. ( 2009 ) Rat; Adult; Male 500 ppm Pb

- acetate in drinking water; 28 weeks 43 Morphology

- Electron micrography showed lumen reduction, microvilli loss, brush border loss, and mitochondrial damage Wang et al. ( 2010d ) Rat; Adult ; Female 300 ppm Pb

- acetate in drinking water; 8 weeks 20 Morphology

- Electron micrography showed Pb damages mitochondria, basement membrane, and brush border in kidney tissue. Some focal tubal necrosis observed. Massó

- González et al. ( 2009 ) Rat; Weanling pups 300 ppm Pb

- acetate in drinking water; GD1 to PND21 23 Morphology

- Pb elevated relative kidney weight at PND21

Navarro

- Moreno et al. ( 2009 ) Rat; Adult; Male 500 ppm Pb

- acetate in drinking water; 28 weeks 43 Kidney function

- Pb exposed males had elevated urinary pH and protein, and glucose and blood in the urine. Roncal et al. ( 2007 ) Rat; Adult; Male 150 ppm Pb

- acetate in drinking water; 16 weeks with remnant kidney surgery at week 4 26 Kidney function

- Remnant kidney surgery and Pb exposure induced decreased creatinine clearance and proteinuria.

Wang et al. ( 2010d ) Rat; Adult; Female 300 ppm Pb

- acetate in drinking water; 8 weeks 20 Kidney function

- Elevated urinary total protein, urinary albumin, and seru m urea nitrogen. Ademuyiwa et al. ( 2009 ) Rat; Adult 200, 300, and 400 ppm Pb

- acetate in drinking water; 12 weeks 39 and 61 Kidney Function

- Renal phospholipidosis and depletion of renal cholesterol after gestation al Pb exposure.



### Renal Function and Interstitial Fibrosis Past studies have shown that chronic continuous or repeated Pb exposure can result in 1 

### interstitial nephritis and focal or tubular atrophy. After an initial 3 months of Pb exposure 2 

### (in a longitudinal 12

- month exposure study to either 100 ppm [lower dose] or 5 , 000 ppm 3 

### [higher dose] Pb

- acetate in drinking water, male rats), elevated GFR, consistent with 4 

### hyperfiltration, and renal hypertrophy were observed; high dose animals also had 5 

### increase d NAG and GST ( Khalil

- Manesh et al., 1993a ; Khalil

- Manesh et al., 1992a ; 6 

### Khalil

- Manesh et al., 1992b )

. At 6 months of exposure, GFR decreased in the high dose 7 

### animals, albuminuria was present, and pathology ensued with focal tubular atrophy and 8February 2012 5

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### interstitial fibrosis formation. This p athology was persistent out to 12 months, and at 12 1 

### months glomeruli developed focal and segmental sclerosis. Similarly, GFR remained 2 

### decreased after 12 months of exposure in the high dose group. The toxicological evidence 3 

### for differences in GFR according to duration of Pb exposure, i.e., hyperfiltration with 3

- 4 

### month exposure versus decreased GFR with 6

- or 12

- month exposure, provide biological 5 

### plausibility for epidemiological studies that observed a similar phenomenon by age in 6 

### adults in association with P b biomarker levels. These duration

- dependent dichotomous 7 

### changes in GFR are consistent between the toxicological and epidemiologic literature. 8


### Biomarkers of Pb

- induced renal toxicity have been developed including the enzymes 9 

### lysosomal NAG, GST, brush bord er antigens (BB50, BBA, HF5), and Tamm

- Horsfall 10 

### protein. GST functions as a renal biomarker since renal ALAD is protected by the kidney 11 

### antioxidant GSH. Urinary NAG and GST levels were found to increase in rats after 3 12 

### months of high dose Pb exposure (resu lting in blood Pb level of 125 μ g/dL) in rats 13 

### ( Khalil

- Manesh et al., 1992a ) , whereas only urinary NAG was increased following low 14 

### dose Pb exposure (resulting in blood Pb level of 29 μ g/dL) in rats ( Khalil

- Manesh et al., 15 

### 1993a )

. An in vitro study also found an increase in NAG with high

- level Pb exposure 16 

### (50 μ g/dL) ( Dehpour et al., 1999 )

. Occupational studies found that urinary NAG 17 

### correlated best with recent blood Pb changes. 18 

### The renal effects of chronic Pb exposure as detailed above were partially rescued in rats 19 

### with chelation therapy such as DMSA ( Khalil

- Manesh et al., 1992b )

. Improvements 20 

### include increased GFR, decreased albuminuria, and decreased inclusion body numbers 21 

### but little change in tubulointerstitial scarring. Administration of an Indian herb to Pb

22 

### exposed mice, as is discussed in greater detail in the antioxidant section ( Section 5.5.5 ), 23 

### produced similar findings. There wa s a functional rescue however Pb

- induced pathology 24 

### remained ( Jayakumar et al., 2009 )

. Thus, administration of various compounds 25 

### (chelators, antioxidants) t o Pb

- exposed animals produced hemodynamic rescue. 26 

### Recent studies have corroborated the previously observed increase in serum creatinine 27 

### following Pb exposure in rats. Abdel Moneim et al. ( 2011b ) reported Pb

- induced (i.p. 28 

### 20 ppm , 5 days) incre ased serum creatinine accompanied by histological alterations in rat 29 

### kidneys. Berrahal et al. ( 2011 ) reported on the effects of age

- dependent exposure to Pb on 30 

### nephrotoxicity in male rats. Pups were exposed to Pb lactationally (as a result of dams 31 

### consuming water containing 50 ppm Pb

- acetate ) until weaning. Thereafter, the offspring 32 

### were exposed to the same solution from weaning (day 21) until sacrifice. Male pups were 33 

### sacrificed at age 40 days (puber ty; blood Pb level 12.7 μg/dL) and at age 65 days (post

34 

### puberty; blood Pb level 7.5 μg/dL). Serum creatinine was elevated at both 40 days and 65 35 

### days (0.54 and 0.60 mg /dL compared to control values of 0.45 mg /dL 36 

### [ p <0 .001]).Various parameters of Pb

- induced renal dysfunction are listed in Table 5

- 23 37February 2012 5

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### below. The elevated serum creatinine in the Pb

- exposed animals compared to controls 1 

### suggests that animals e xposed to low dosage ( i.e., 50 ppm ) Pb from birth may develop 2 

### renal abnormalities. However, the lack of measurements of GFR or renal pathology 3 

### weakens the conclusions. Other investigators have also shown that chronic Pb exposure 4 

### has detrimental effects on renal function. Pb

- exposed male rats (500 ppm Pb

- acetate in 5 

### drinking water for 7 months) had elevated urinary pH, proteinuria, as well as glucose and 6 

### blood in the urine ( Navarro

- Moreno et al., 2009 )

. 7



### Table 5
- 23 Indicators of renal damage in male rats exposed to 50 ppm Pb for 40 and 65 days, starting at parturition

Biomarker (Mean ± SD) PND40 Control PND40 Pb PND65 Control PND65 Pb
Blood Pb level (μg/dL) 1.8 ± 0.33 12.7±1.7 2.1±0.35 7.5±0.78

Plasma Creatinine (mg/L) 4.5±0.21 5.35±0.25 a 4.55±0.27 6.04±0.29 a

Plasma Urea (mg/L) 0.37±0.019 0.47±0.021 a 0.29±0.009 0.29±0.009

Plasma Uric Acid (mg/L) 7.51±0.44 7.65±0.32 9.39±0.82 5.91±0.53 a

a p <0.001 Source: Modified with permission of John Wiley & Sons, Berrahal et al. ( 2011 )


### Qiao et al. ( 2006 ) measured the effect of Pb on the expression of the renal fibrosis

- related 8 

### nuclear factor

- kappa B (NF

- κB), transforming growth factor (TGF

- β) and fibronectin in 9 

### Sprague

- Dawley rat kidney. Pb was administered at a dose of 0.5% Pb

- acetate , 10 

### continuously for ei ther one, two or three months. All growth factors increased by the end 11 

### of three months of treatment but only NF

- κB increased progressively at each time period. 12 

### These changes were hypothetically related to the development of Pb

- induced renal 13 

### fibrosis in rat s, but no histology was performed. 14


### Roncal et al. ( 2007 ) found that Pb accelerated arteriolopathy and tubulointerstitial injury 15 

### in non
- Pb

- related CKD. Sprague

- Dawley rats were administered Pb

- acetate at 150 ppm 16 

### for 4 weeks, then subjected to remnant kid ney surgery (left kidney mass reduced by 2/3 17 

### and right kidney removed), and subsequently exposed to Pb for an additional 12 weeks. 18 

### Pb

- treated rats had higher systolic BP, lower creatinine clearance, and higher proteinuria 19 

### than did controls. Most striking w as development of worse arteriolar disease, peritubular 20 

### capillary loss, tubulointerstitial damage, and macrophage infiltration. Pb treatment was 21 

### associated with significant worsening of pre

- glomerular vascular disease, as characterized 22 

### by an increase in th e media

- to

- lumen ratio. There was also a higher percentage of 23February 2012 5

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### segmental sclerosis within glomeruli and a tendency for a higher number of sclerotic 1 

### glomeruli. Additionally, a loss of peritubular capillaries, as reflected by a reduction in 2 

### thrombomodulin sta ining, was observed. This was associated with worse tubular injury 3 

### (osteopontin staining) due to more interstitial fibrosis (type III collagen staining) and a 4 

### greater macrophage infiltration in the interstitium. The increase in macrophages was 5 

### associated w ith higher renal MCP

- 1 mRNA. As a whole, these findings indicate that Pb 6 

### exposure concomitant with existing renal insufficiency due to surgical kidney resection 7 

### accelerated vascular disease and glomerular pathology. These findings are consistent with 8 

### th e p revious work of Bagchi and Preuss ( 2005 ) also showing that Pb

- exposed animals 9 

### with non
- Pb

- related CKD (remnant surgery) had kidney dysfunction including 10 

### impairment of the renin

- angiotensin system (Losartan challenge), elevated systolic BP, 11



### an d alterations in renal excretion of Pb, K + 

### , and Na + 

### . Thus, this model shows that low Pb 12 

### exposure may exacerbate pre

- existing underlying kidney disease. 13



### Histological Changes Historical studies discussed in previous Pb AQCDs have identified Pb

- related renal 14 

### damage by the presence of dense intranuclear inclusion bodies, which are capable of 15 

### sequestering Pb ( Goyer et al., 1970a )

. Chelators such as CaNa 2 

### EDTA removed these 16 

### inclusion bodies from affected nuclei ( Goyer et al., 1978 )

. Multiple endpoints indicate 17 

### dysfunction in the PT after Pb exposure. Pb

- induced formation of intranuclear inclusion 18 

### bodies in the PT is pro tective; Pb is sequestered such that it is not in its bioavailable, free, 19 

### toxicologically active form. Intranuclear inclusion bodies are found in the kidney with 20 

### acute ( i.e., < 4 weeks) Pb exposures but present to a lesser degree with chronic exposures 21 

### (Se e Section 5.2.3 for further discussion). Other PT ultrastructural changes in Pb

- induced 22 

### nephropathy include changes to the PT epithelium, endoplasm ic reticulum dilation, 23 

### nuclear membrane blebbing, and autophagosome enlargement ( Fowler et al., 1980 ; Goyer 24 

### et al., 1970b )

. Symptoms similar to the PT transport

- associated Fanconi syndrome appear 25 

### with Pb exposure, albeit often at high doses of Pb, i.e., Pb

- poisoning. These symptoms, 26 

### which include increased urinary electrolyte excretion (zi nc), decreased Na

- K

- ATPase 27 

### activity, mitochondrial aberrations, and aminoaciduria, also have been associated with 28 

### blood Pb levels in children. 29 

### New studies since the 2006 Pb AQCD are consistent with the historical findings and 30 

### build upon the literature ba se by including the role of antioxidants. Jabeen et al. ( 2010 ) 31 

### exposed pregnant albino BALB/c mice to a daily oral dose of Pb

- acetate (10 mg /kg body 32 

### weight, daily throughout pregnancy) until GD18, at which point the fetal kidneys were 33 

### processed for histological examination. Histology revealed Pb exposure induced 34 

### decreased kidney cortical thickness, decreased diameter of renal corpuscles, and 35 

### i ncreased renal tubular atrophy with desquamated epithelium and degenerated nuclei in 36February 2012 5

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– Do Not Cite or Quote 

### the distal and proximal tubules. Blood Pb levels were not reported in this study. 1 

### Nonetheless, these data show that in utero Pb exposure had significant histological effec ts 2 

### on the fetal kidney, which could contribute to altered renal function including clearance 3 

### of waste products, electrolyte balance, and vasoregulation. Massanyi et al. ( 2007 ) 4 

### reported on Pb

- induced alterations in male Wistar rat kidneys after single i.p. doses of 5 

### Pb

- acetate (50, 25, and 12.5 mg /kg); kidneys were removed and analyzed 48 hours after 6 

### Pb administration. Qualitative microscopic analysis detected dilat ed Bowman’s capsules 7 

### and dilated blood vessels in the interstitium with evident hemorrhagic alterations. 8 

### Quantitative histomorphometric analysis revealed increased relative volume of 9 

### interstitium and increased relative volume of tubules in the experimental groups. The 10 

### diameter of renal corpuscles and the diameter of glomeruli and Bowman’s capsule were 11 

### significantly increased. Measurement of tubular diameter showed dilatation of the tubule 12 

### with a significant decrease of the height of tubular epithelium compa tible with 13 

### degenerative renal alterations. These findings extend the observations of Fowler et al. 14 

### ( 1980 ) and Khalil

- Manesh et al. ( 1992a ; 1992b ) ; in particular, the enlarged glomerul i are 15 

### consistent with the early hyperfiltration caused by Pb. 16


### Abdel Moneim et al. ( 2011b ) reported histological evidence of inflammation after Pb 17 

### treatment in rats (i.p. 20 ppm , 5 days). This evidence included increased inflammatory 18 

### cellular infiltrations, cytoplasmic vacuolation, and dilatation of some kidney tubules. 19 

### Inflammation was accompanied by an increase in apoptotic cells and increased oxidative 20 

### stress. 21 

### A recent study has also reported inclusion body formation in the nuclei, cytoplasm , and 22 

### mitochondria of PT cells of Pb

- treated rats (50 mg Pb/kg bw i.p., every 48 hours for 14 23 

### days) ( Navarro

- Moreno et al., 2009 )

. These inclusion bodies were not observed in 24 

### chronically Pb

- exposed rats (500 ppm Pb in drinking water, 7 months). However, chronic 25 

### Pb exposure resulted in morphological alterations including loss of PT apical membrane 26 

### brush border, collapse and closure of the PT lumen, and formation of abnormal 27 

### intercellular junctions. 28 

### Vogetseder et al. ( 2008 ) examined the proliferative capacity of the renal PT (particularly 29 

### the S3 segment) following i.v. administration of Pb to juvenile and adult male Wistar 30 

### rats. P roliferation induction was examined by detection of Bromo

- 2’

- deoxyuridine 31 

### (BrdU), Ki

- 67 (labels S, G2, and M phase cells), and cyclin D1 (an essential cell cycle 32 

### progression protein). The cycling marker Ki

- 67 revealed a much higher proliferation rate 33 

### in th e S3 segment in control juvenile rats (4.8 ± 0.3%) compared with control adult rats 34 

### (0.4 ± 0.1%). Pb administration (3.8 mg /100 g bw) increased the proportion of Ki

- 67

- 35 

### positive cells to 26.1 ± 0.3% in juvenile rats and 31.9 ± 0.3% in adult rats. Thus, the 36 

### increased proliferation caused by Pb was age independent. The proliferation induction 37February 2012 5

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### caused by Pb administration may be a result of reduced cell cycle inhibition by p27 kip

- 1



### . 1 

### Acute Pb treatment increased the incidence of cyclin D1 labeling in the BrdU

- pos itive 2 

### cells suggesting Pb was able to accelerate re

- entry of cells into the cell cycle and cause 3 

### proliferation in the PT. Pb

- induced cellular proliferation has also been reported in the 4 

### retina with gestational and early postnatal rodent Pb exposure ( Giddabasappa et al., 5 

### 2011 )

. 6 

### Ademuyiwa et al

. ( 2009 ) examined Pb

- induced phospholipidosis and cholesterogenesis in 7 

### rat tissues. Sprague

- Dawley rats were exposed to 200, 300 and 400 ppm Pb

- acetate for 12 8 

### weeks. The Pb exposure resulted in induction of phospholipidosis in kidney tissue, 9 

### accompanied by depletion of renal cholesterol. The authors suggested that induction of 10 

### cholesterogenesis and phospholipidosis in kidney may be responsible for some o f the 11 

### subtle and insidious cellular effects found with Pb

- mediated nephrotoxicity. Drug

12 

### induced PT phospholipidosis is seen clinically with use of the potentially nephrotoxic 13 

### aminoglycoside drugs, including gentamicin ( Baronas et al., 2007 )

. 14


### Various antioxidants have been shown to attenuate Pb

- induced histopathological changes 15 

### to the kidney. Ozsoy et al. ( 2010 ) found L

- carnitine to be protective i n a model of 16 

### experimental Pb toxicity in female rats. Markers of histopathological change in the 17 

### kidney, including tubule dilatation, degeneration, necrosis, and interstitial inflammation 18 

### were rescued by L

- carnitine treatment in females. Male rats exposed to Pb (0.2% for 6 19 

### weeks) also displayed tubular damage, whereas concomitant treatment with Pb and an 20 

### extract of Achyranthes aspera ameliorated the observed damage ( Jayakumar et al., 2009 )

. 21 

### El

- Nekeety et al. ( 2009 ) found an extract of the folk medicine plant Aquilegia vulgaris to 22 

### be protective against Pb

- aceta te

- induced kidney injury in Sprague

- Dawley rats. Rats were 23 

### treated with Pb (20 ppm ; 2 weeks) and extract (administered before, during, or after Pb). 24 

### Pb treatment resulted in tubular dilatation, vacuolar and cloudy epithelial cell lining, 25 

### interstitial infla mmatory cell infiltration, hemorrhage, cellular debris, and glomerulus 26 

### hypercellularity. Concomitant exposure to Pb and extract produced histology 27 

### indiscernible from that in controls. Post treatment with extract partially rescued the Pb

28 

### induced histopathol ogy. El

- Neweshy and El

- Sayed ( 2011 ) studied the influence of 29 

### vitamin C supplementation (20 mg /kg pretreatment every other day) on histopathological 30 

### alterations in Pb

- exposed male rats (20 mg /kg by intragastric feeding once daily for 60 31 

### days). Control rats showed normal histology, while Pb

- treated rats exhibited karyomegaly 32 

### with eosinophilic intranuclear inclusion bodies in the epithelial cells of the proximal 33 

### tubules. Glomerular damage and tu bular necrosis with invading inflammatory cells were 34 

### also found. Rats treated with Pb

- acetate plus vitamin C exhibited relatively mild or no 35 

### karyomegaly with eosinophilic intranuclear inclusion bodies in the proximal tubules. 36 

### Normal glomeruli were noted in animals exposed to Pb and vitamin C. These findings are 37 

### presented in more detail in Section 5.5.5 but they consistently show that some 38February 2012 5

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### antioxidants are capable of preventing or rescuing Pb

- induced renal histopathological 1 

### changes. 2



### Alteration of Renal Vasculature and Reactivity

As discussed in Section 5.5.1 , changes in renal vasculature function or induction of 3 

### hypertension can contribute to further renal dysfunction. Pb can increase BP through the 4 

### promotion of oxidative stress and altered vascular reactivity. Also, Pb has been shown to 5 

### act on kn own vasomodulating systems in the kidney. In the kidney, two vascular tone 6 

### mediators, • 

### NO and ET

- 1, are found to be affected by Pb exposure. Antioxidants 7 

### attenuated Pb

- related oxidative/nitrosative stress in the kidney and abrogated the Pb

8 

### induced increase d BP ( Vaziri et al., 1999b )

. Administration of the vasoconstrictor 9 

### endothelin

- 1 (ET

- 1) affected mean arterial pressure (MAP) and decreased GFR ( Novak 10 

### and Banks, 1995 )

. Acute high

- dose Pb exposure (24 nmol/min for 15 or 30 minute s) 11 

### completely blocked this ET

- 1

- mediated GFR decrease but had no effect on MAP. 12 

### Depletion of the endogenous antioxidant glutathione using the drug buthionine 13 

### sulfoximine, a GSH synthase inhibitor, increased BP and increased kidney nitrotyrosine 14 

### formation without Pb exposure, demonstrating the importance of GSH in maintenance of 15 

### BP ( Vaziri et al., 2000 )

. Multiple studies have show n that Pb exposure depletes GSH 16 

### stores. Catecholamines are vascular moderators that are also affected by Pb exposure 17 

### ( Carmignani et al., 2000 )

. The effect on BP with Pb exposure is espe cially relevant to the 18 

### kidney because it is both a target of Pb deposition and a mitigator of BP. These historic 19 

### data detail the interaction of known modulators of vascular tone with Pb. 20 

### Recently, Vargas

- Robles et al. ( 2007 ) examined the effect of Pb exposure (100 ppm 21 

### Pb

- acetate for 12 weeks) on BP and angiotensin II vasoconstriction in isolated perfused 22 

### ki dney and interlobar arteries. Vascular reactivity was evaluated in the presence and 23 

### absence of the nitric oxide synthase inhibitor L

- NAME in both Pb

- treated and control 24 

### animals. Pb exposure significantly increased BP (134 ± 3 versus 100 ± 6 mmHg), eNOS 25 

### pro tein expression, oxidative stress, and vascular reactivity to angiotensin II. L

- NAME 26 

### potentiated the vascular response to angiotensin II in the control group, but had no effect 27 

### on the Pb

- treated group. Conversely, passive microvessel distensibility, measur ed after 28 

### deactivation of myogenic tone by papaverine, was significantly lower in the arteries of 29 

### Pb

- exposed rats. Nitrites released from the kidney under the influence of angiotensin II in 30 

### the Pb group were lower as compared to the control group whereas 3

- nitrotyrosine was 31 

### higher in the Pb group. The authors concluded that Pb exposure increases vascular tone 32 

### through nitric oxide

- dependent and

- independent mechanisms, increasing renal vascular 33 

### sensitivity to vasoconstrictors. 34February 2012 5

- 304 Draft

– Do Not Cite or Quote 

### Simões et al. ( 2011 ) reported that acute Pb treatment ( Pb

- acetate i.v. bolus dose of 1 

### 320 μ g/kg bw, blood Pb of 37 μ g/dL at 120 minute s after Pb administration) in adult 2 

### male Wistar rats increased systolic arterial pressure 60 minute s after treatment without 3 

### affecting diastolic arterial pressure or heart rate. With this single injection, serum 4 

### angiotensin converting enzyme (ACE) activity was significantly elevated. The Pb

5 

### induced altered s ystolic BP was under the control of the renin

- angiotensin system as 6 

### evidenced by attenuation of the effects of Pb in Losartan (Ang II receptor blocker) or 7 

### Enalapril (ACE inhibitor) co

- treated animals ( Simões et al., 2011 )

. These data agree with 8 

### earlier reports of Pb

- related increases in ACE activity in young rats exposed to Pb for 2

- 8 9 

### weeks ( Sharifi et al., 2004 ) and adult rats expos ed to Pb for 10 months ( Carmignani et al., 10 

### 1999 )

. 11



### Apoptosis and/or Ischemic Necrosis of Tubules and Glomeruli Apoptosis or programmed cell death in excess can cause cell atrophy while an 12 

### insufficiency can lead to uncontrolled cell proliferation, such as cancer. Pb exposure has 13 

### been shown to cause morphological changes to the kidney structure. Some of these Pb

14 

### induced changes are a result of cellular apoptosis or necrosis. Past studies have shown 15 

### Pb

- induced necrosis in proximal tubule cells ( Fowler et al., 1980 )

. Pb

- induced apoptosis 16 

### is known to act th rough the mitochondria ( Rana , 2008 )

. Pb

- induced calcium overload 17 

### may depolarize the mitochondria, resulting in cytochrome c release, caspase activation, 18 

### and apoptosis. The apoptosis is mediated by Bax translocation to the mitochondria and 19 

### can be blocked by overexpression of Bcl

- xl. Also, Pb

- induced ALA accumulation can 20 

### generate ROS, which may damage DNA leading to apoptosis. 21 

### Mitochondria are targets of Pb toxicity and often involved in apoptosis. Pb can induce 22 

### uncoupling of oxidative phosphorylation, decreased substrate utilization, and 23 

### modification of mitochondrial ion transport. ATP energetics are affected when ATP

- Pb 24 

### chelat es are formed and ATPase activity is decreased. ROS formation can contribute to 25 

### these mitochondrial changes and to other changes within the kidney. Antioxidant 26 

### supplementation after Pb exposure can remedy some changes. All of these outcomes, in 27 

### conjunction with Pb

- related depletion of antioxidants ( e.g., GSH) and elevation of lipid 28 

### peroxidation point to possible susceptibility of the kidney to apoptosis or necrosis. 29 

### Rodriguez

- Iturbe et al. ( 2005 ) reported that chronic exposure to low doses of Pb 30 

### (100 ppm in drinking water for 14 weeks) results in renal infiltration of immune cells, 31 

### apo ptosis, NF

- κB activation and overexpression of tubulointerstitial Ang ( II ) . Similarly, 32 

### higher level Pb treatment in rats (i.p. 20 mg /kg, 5 days) induced inflammatory cellular 33 

### infiltrations and an increase in apoptotic cells, accompanied by more pronounced B AX 34 

### staining in kidney tubule epithelial cells ( Abdel Moneim et al., 2011b )

. Pb treatment (0.5

- 35February 2012 5

- 305 Draft

– Do Not Cite or Quote 

### 1 μ M) of isolated rat proximal tubular cells increased cell death by apoptosis and necrosis 1 

### in a concentration

- and time

- dependent manner ( Wang et al., 2011b )

. This was 2 

### accompanie d by increased morphological changes typical of apoptosis such as 3 

### fragmented chromatin, condensed chromati n , and shrunken nuclei. These cells also 4 

### exhibited decreased mitochondrial membrane potential, decreased intracellular pH, 5 

### inhibition of Na+

- K+ ATPase and Ca2+

- ATPase activity, and increased intracellular 6 

### Ca2+ following Pb treatment. 7



### Navarro

- Moreno et al. ( 2009 ) examined the effect of 500 ppm Pb in drinking water over 8 

### 7 months on the structure (including intercellular junctions), function, and biochemical 9 

### properties of PT cells of Wistar rats. Pb effects in epithelial cells consisted of an early 10 

### loss of the apical microvill i , followed by a dec rement of the luminal space and the 11 

### respective apposition and proximity of apical membranes, resulting in the formation of 12 

### atypical intercellular contacts and adhesion structures. Inclusion bodies were found in 13 

### nuclei, cytoplasm, and mitochondria. Lipid pe roxidation (TBARS measurement) was 14 

### increased in the Pb

- treated animals as compared to controls. Calcium uptake was 15 

### diminished and neither proline nor serine incorporation that was present in controls was 16 

### noted in the PT of Pb

- exposed animals. The authors s peculated that Pb may compete with 17 

### calcium in the establishment and maintenance of intercellular junctions. 18


### Tubular necrosis was also observed in rats treated with Pb

- acetate (100 ppm s.c.) for 30 19 

### days ( El

- Sokkary et al., 2005 )

. Histological sections of kidneys from Pb

- treated rats 20 

### showed tubular degeneration with some necroti c cells. Similarly, El

- Neweshy and El

21 

### Sayed reported gl omerular damage and tubular necrosis with invading inflammatory 22 

### cells after Pb treatment (20 mg /kg by intragastric feeding once daily for 60 days) to male 23 

### rats. The incidence of necrosis was decreased in both of these studies by pretreatment 24 

### with either me latonin or vitamin C. Pretreatment with melatonin (10 mg /kg), an 25 

### efficacious free radical scavenger and indirect antioxidant, resulted in a near normal 26 

### tubular structure. The authors concluded that melatonin protected the liver and kidneys 27 

### from the damagin g effects of exposure to Pb through inhibition of lipid peroxidation and 28 

### stimulation of endogenous antioxidative defense systems ( El

- Sokkary et al., 2005 )

. 29 

### Vitamin C supplementation (20 mg /kg pretreatment every other day) protected the renal 30 

### architecture and histology ( El

- Newes hy and El

- Sayed, 2011 )

. 31 

### Wang et al. ( 2009c ) examined the effect of Pb

- acetate (0.25, 0.5 and 1 μ M) on cell death 32 

### in cultured rat primary PT cells. A progressive loss in cell viability, due to both apoptosis 33 

### and necrosis, was observed in cells exposed to Pb. Apoptosis predominated and could be 34 

### ameliorated with concomitant N

- acetylcysteine exposure, wh ereas necrosis was 35 

### unaffected. Elevation of ROS levels and intercellular calcium, depletion of mitochondrial 36 

### membrane potential, and intracellular glutathione levels was observed during Pb 37February 2012 5

- 306 Draft

– Do Not Cite or Quote 

### exposure. Pb

- induced apoptosis was demonstrated morphologically (Ho echst 33258 1 

### staining) with condensed/fragmented chromatin and apoptotic body formation. CAT and 2 

### SOD activities were significantly elevated, reflecting the response to accumulation of 3 

### ROS. 4


### Table 5

- 24 presents the acute and chronic renal effects of Pb exposure observed in recent 5 

### and past animal toxicology studies. 6



### Table 5
- 24 Effects of Pb on the kidney/renal system related to exposure duration

– evidence from animal toxicology studies
Effects with less than 3 months of exposure Effects with 6 or 12 months of exposure Mitochondrial dysfunction Renal cell apoptosis Nuclear Inclusion Body Formation Proximal Tubule Cytomegaly Glomerular Hypertrophy Increased GFR Mitochondrial dysfunction Renal cell apoptosis Oxidant redox imbalance Altered NO homeostasis ATPase dysfunction Aminoaciduria Increased electrolyte excretion Elevated blood pressure Decreased GFR



### 5.5.2.2 Epidemiology in Adults
A number of advances in research on the impact of Pb on the kidney in the 20 years 7 

### following the 1986 Pb AQCD ( U.S. EPA, 1986a ) were noted in the 2006 Pb AQCD ( U.S. 8 

### EPA, 2006b )

. T hese included research in general and CKD patient populations at much 9 

### lower blood Pb levels (5

- 10 μ g/dL) at the time of evaluation than were previously 10 

### studied. These advances contributed to the understanding of the effects of Pb exposure on 11 

### kidney dysfunc tion overall in the population. Pb, at much lower doses than those causing 12 

### chronic Pb nephropathy, may act as a cofactor with other more established kidney risks to 13 

### increase the risk for CKD and disease progression in susceptible patients. Maric and Hall 14 

### ( 2011 ) note that data from basic and clinical studies suggest that obesity, hypertension, 15 

### hyperglycemia, h yperlipedemia, and other elements of the metabolic syndrome are highly 16 

### interrelated and contribute to the development and progression of diabetic nephropathy 17 

### and thus represent populations potentially at increased risk for kidney dysfunction. 18


### In the 2006 P b AQCD ( U.S. EPA, 2006b ) , several key issues could not be completely 19 

### resolved based on the Pb

- kidney literature published to date. These included 20 

### characterizing the lowest Pb dose at which altered kidney function effects occur, t he 21February 2012 5

- 307 Draft

– Do Not Cite or Quote 

### impact of higher past exposures on associations with current Pb biomarker levels, the 1 

### impacts of Pb on the kidney in children, the use of paradoxical Pb

- kidney associations on 2 

### risk assessment in the occupational setting, and the impact of co

- exposure to other 3 

### environmental nephrotoxicants, such as cadmium. In the intervening five years, relevant 4 

### data addressing several of these challenges have been published. 5



### General Population Studies The 2006 Pb AQCD reported studies that examined associations between indicators of Pb 6 

### exposure and kidney function in general populations. This was a new approach to Pb

7 

### kidney research in the two decade time period covered by the 2006 Pb AQCD. As 8 

### illustrated in Figure 5

- 39 and Table 5

- 25 , studies consistently demonstrate associations 9 

### between higher blood Pb level and lower renal function in adults. The studies in this 10 

### category provided critical evidence that the effects of Pb on the kidney occur at much 11 

### lower doses than previously appreciated based on occupational exposure data. However, 12 

### because blood Pb leve l in nonoccupationally

- exposed adults reflects both recent and past 13 

### Pb exposures, the magnitude, timing, frequency, and duration of Pb exposure 14 

### contributing to the observed associations was uncertain. The evidence of Pb

- associated 15 

### renal effects in general population studies was substantiated by results that were adjusted 16 

### for multiple potential confounding factors including age, race, sex, education, household 17 

### income, smoking, alcohol use, cadmium exposure, and various health indicators such as 18 

### diabetes, SBP , BMI, and history of cardiovascular disease. 19 

### The landmark Cadmibel Study was the first large environmental study of this type that 20 

### adjusted for multiple kidney risk factors ( Staessen et al., 1992 )

. It included 965 men and 21 

### 1 , 016 women recruited from cadmium exposed and control areas in Belgium. Mean 22 

### concurrent blood Pb was 11.4 μg/dL (range 2.3

- 72.5) and 7.5 μg/dL (range 1.7

- 60.3) in 23 

### men and women, respectively. After adjustment, log transformed blood Pb was 24 

### negatively associated with measured creatinine clearance. A 10

- fold increase in blood Pb 25 

### was associated with a decrease in creatinine clearance of 10 and 13 mL/ min in men and 26 

### women, respectively. Blood Pb was also negatively associated with estimated creatinine 27 

### clearance. 28 

### Multiple analyses assessing the kidney impact of Pb exposure have been conducted in the 29 

### NAS population ( Tsaih et al., 2004 ; Wu et al., 2003a ; Kim et al., 1996 ; Payton et al., 30 

### 1994 )

. Participants in this study were originally recruited in the 1960s in the Greater 31 

### Boston area. Inclusion criteria included male sex, age 21 to 80 years, and absence of 32 

### chronic medical conditions. Longitudinal data contained in NAS publications remain 33



### essen tial, particularly in light of the dearth of prospective data on the kidney effects of 34 

### Pb. The first of these included 459 men whose blood Pb levels from periodic 35February 2012 5

- 308 Draft

– Do Not Cite or Quote 

### examinations, conducted every 3 to 5 years during 1979

- 1994, were estimated based on 1



### measurem ents in stored packed red blood cell samples adjusted for hematocrit level ( Kim 2 

### et al., 1996 )

. Participants were randomly selected to be representative of the entire NAS 3 

### population in terms of age and follow

- up. Kidney function was assessed with serum 4 

### creatinine. Data from four evaluations were available for the majority of participants. At 5 

### baseline, mean (SD) age, blood Pb l evel, and serum creatinine, at baseline, were 56.9 6 

### (8.3) years, 9.9 (6.1) μg/dL, and 1.2 (0.2) mg /dL, respectively. In the longitudinal 7 

### analysis, using random

- effects modeling with repeated measures, ln

- transformed blood 8 

### Pb was associated with an increase in serum creatinine from the previous to current 9 

### follow

- up period in the 428 participants whose highest blood Pb level was ≤ 25 μg/dL 10 

### (β= 0.027 mg /dL [95% CI: 0.0, 0.054] per unit increase in ln blood Pb); effect estimates 11 

### in the entire group and subsets w ith different peak blood Pb levels ( ≤ 10 or 40 μg/dL) 12 

### also were positive (and larger for blood Pb levels ≤ 10 μg/dL) but had p

- values between 13 

### 0.07 and 0.13. 14 

### This study made two other key contributions. In order to address the question of whether 15 

### nephrotox icity observed at current blood Pb levels is due to higher blood Pb levels from 16 

### past exposure, these authors performed a sensitivity analysis in participants whose peak 17 

### blood Pb levels, dating back to 1979, were ≤ 10 μg/dL. A statistically significant posi tive 18 

### association between blood Pb and concurrent serum creatinine remained in a cross

19 

### sectional analysis. These authors also addressed reverse causality, which attributes 20 

### increased blood Pb levels to lack of kidney excretion rather than as a causative fact or for 21 

### CKD, by showing in adjusted plots that the association between blood Pb and serum 22 

### creatinine occurred over the entire serum creatinine range (0.7

- 2.1 mg /dL), including the 23 

### normal range where reverse causality would not be expected. 24


### Cortical and trab ecular bone Pb measurements were obtained in addition to whole blood 25 

### Pb in evaluations performed in the NAS between 1991 and 1995. Associations between 26 

### baseline blood, tibia, and patella Pb and change in serum creatinine over an average of 6 27 

### years in 448 m en were reported in a subsequent NAS publication ( Tsaih et al., 2004 )

. At 28 

### baseline 6 and 26% of subjects had diabetes and hypertens ion, respectively. Mean blood 29 

### Pb levels and serum creatinine decreased significantly over the follow

- up period in the 30 

### group. Baseline blood Pb level was not associated with change in creatinine in all 31 

### participants. However, diabetes was observed to be an e ffect modifier of the relations of 32 

### blood and tibia Pb with change in serum creatinine. Per unit increase in ln blood Pb, the 33 

### increase in serum creatinine between follow

- up periods was substantially stronger in 34 

### diabetics (β = 0.076 mg /dL [95% CI: 0.031, 0.1 21]) compared to non
- diabetics (β = 35 

### 0.006 mg /dL [95% CI:

- 0.004, 0.016]). A similar relationship was observed for tibia Pb. 36 

### An interaction was also observed between tibia Pb and hypertension, although it is 37 

### possible that many of the 26 diabetics were also included in the hypertensive group and 38February 2012 5

- 309 Draft

– Do Not Cite or Quote 

### were influential there as well. Reverse causality was addressed in a sensitivity analysis of 1 

### participants whose serum creatinine was <1.5 mg /dL; the authors reported that 2 

### longitudinal associations did not materially c hange. 3February 2012 5

- 310 Draft

– Do Not Cite or Quote a Blood Pb data are presented as median and (IQR) in μg/dL for blood Pb. For uniform presentation, median and IQR were estimate d from the given distributional statistics by assuming normal distributions.
b The cross product of logged blood Pb and ranked urine Hg was included in the regression to model the interaction between these two variates. The significant hyperfilrative effect to these children could be due to a biphasic time course sometimes seen i n early exposure. Note: Results are presented first for kidney function tests where an increase is considered impaired function (black circles) then for tests where a decrease is considered impaired function ( blue circles, outlined in box). Within a category, results are presented first for longitudi nal analyses followed by cross

- sectional analyses. To compare results for linear and nonlinear modeling, effect estimates were standardized to a 1 μg/dL increase in blood Pb level within the 10th

- 90th percentile interval. Magnitudes of the effect should no t be compared among different kidney metrics. 

### Figure 5
- 39 Kidney metric slopes for blood Pb or bone Pb.February 2012 5

- 311 Draft

– Do Not Cite or Quote 

### The impact of Pb on the kidney has been examined in multiple NHANES datasets 1 

### obtained over the last few decades ( Figure 5

- 40 and Table 5

- 25 ). NHANES data analyses 2 

### benefit from a number of strengths including large sample size, ability to adjust for 3 

### numerous Pb risk factors, and the fact that the study population is representative of the 4 

### U.S. non
- institutionalized, civilian population. The results, cove ring different time 5 

### frames, have been consistent in providing support for Pb as a CKD risk factor, including 6 

### NHANES III, conducted from 1988

- 1994, in which adults with hypertension and diabetes 7 

### were observed to be susceptible populations ( Muntner et al., 2003 ) and NHANES 1999

- 8 

### 2002 ( Muntner et al., 2005 )

. However, because the various NHANES analyses were 9



### cross
- sectional in design, examining associations between concurrent measures of kidney 10 

### function and blood Pb levels, a common limitation is the uncertainty regardin g the 11 

### magnitude, timing, frequency, and duration of Pb exposure that contributed to the 12 

### observed associations. 13 

### A recent publication examined NHANES data collected from 1999 through 2006 ( Navas

14 

### Acien et al., 2009 )

. The geometric mean concurrent blood Pb level was 1.58 μg/dL in 15 

### 14,778 adults aged ≥ 20 years. After adjustment for survey year, sociodemographic 16 

### factors, CKD risk factors, an d blood cadmium, the odds ratios for albuminuria (≥ 17 

### 30 mg /g creatinine), reduced eGFR (<60 mL/min/1.73 m 2 

### ), and both albuminuria and 18 

### reduced eGFR were 1.19 (95% CI: 0.96, 1.47), 1.56 (95% CI: 1.17, 2.08), and 2.39 (95% 19 

### CI: 1.31, 4.37), respectively, compar ing the highest (> 2.4 μg/dL) to the lowest (≤ 1.1 20 

### μg/dL) blood Pb quartiles. Thus, in the subset of the population with the most severe 21 

### kidney disease (both reduced eGFR and albuminuria), the magnitude of association with 22 

### concurrent blood Pb was greater. When blood cadmium was included as a covariate, 23 

### blood Pb remained significantly associated. In fact, the most important contribution of 24 

### this recent NHANES analysis was the evaluation of joint Pb and cadmium exposure 25 

### (discussed in Section 5.5.4.1 ). 26 

### An important contribution of all NHANES publications is that they provide evidence that 27 

### blood Pb remains associated with reduced kidney function (<60 mL/mi n/1.73 m 2 

### as 28 

### estimated with the MDRD equation cross

- sectionally) despite steadily declining blood Pb 29 

### levels during the time periods covered. Additional studies in this category have also 30 

### reported worse kidney function related to blood Pb levels ( Lai et al., 2008a ; Hernandez

31 

### Serrato et al., 2006 ; Goswami et al., 2005 )

. 32February 2012 5

- 312 Draft

– Do Not Cite or Quote Note: These articles reported ORs of kidney function measures by grouping the population into quartiles of blood Pb and then comparing each group to the quartile with the lowest blood Pb (reference group). The blood Pb distribution of the examined gr oup is shaded black and the reference group is shaded gray. To express these odd s ratios in terms of blood Pb concentration, a log normal distribution was fit to the statistics presented and then the medians of each group were determined. The adjusted OR w as the exponentiated quantity (log(OR) divided by the difference in the medians of the groups compared). The resulting odds ratio is presented in terms of percent change=100*(OR

- 1). 

### Figure 5
- 40 Percent change for kidney outcomes associated with blood Pb.5102 10 -10 -40 50 Study Quartiles of Blood Pb Distribution Used

Muntner et al. ( 2003 )
Elevated Serum Creatinine Hypertensive

Normotensive

Navas

- Acien et al. ( 2009 ) Albuminuria >30 mg/g creatinine

eGFR <60 mL/min/1.73m 2

μ g/dL Blood Pb % Change per μ g/dL Blood PbFebruary 2012 5

- 313 Draft

– Do Not Cite or Quote 

### Table 5
- 25 Additional characteristics and quantitative data for associations of blood and bone Pb with kidney outcomes for results presented in Figure 5
- 39 and Figure 5
- 40

Reference Population Study Location; Time Period N Pb Biomarker Data Outcome Statistical Analysis Effect Estimate (95% CI)

FIGURE 5
- 39: Positive Effect Estimates Indicate Poorer Function Per 1 μg/dL increase in blood Pb within the 10th
- 90th percentile interval Longitudinal Results

Kim et al. ( 1996 ) Adult males Boston, MA; Multiple examination s 1979

- 1994 459 Median baseline blood = 8.6 μg/dL 10th

- 90th percentile: 4.0

- 17.5 Change in serum creatinine between visits x 10 (mg/dL) Random

- effects modeling adjusted for baseline age, time since initial visit, BMI, smoking status, alcohol ingestion, education level, hypertension, baseline serum creatinine, and time between visits Peak blood P b ≤ 40 μg/dL: 0.012 (

- 0.0001, 0.025) Peak blood Pb ≤ 25 μg/dL: 0.015 (0.0002, 0.03) Peak blood Pb ≤ 10 μg/dL: 0.021 (

- 0.005, 0.048)

Tsaih et al. ( 2004 ) Adult males Boston, MA; 8/1991

- 1995 with mean 6 year follow

- up 448 Mean (SD) Baseline Blood Pb = 6.5 (4.2) μg/dL 10th

- 90th percentile: 2.1

- 7.6 Tibia Pb = 21.5 (13.5) μg/g Patella Pb = 32.4 (20.5) μg/g Change in serum creatinine per year x 10 (mg/dL) Log linear regression adjusted for age, age squared, BMI, hypertension, diabetes, smoking status, alcohol consumption, analgesic use, baseline serum creatinine, serum creatinine squared With diabetes: 0.18 (0.07, 0.29) Without diabetes: 0.014 (

- 0.009, 0.037) With hypertension: 0.019 (

- 0.027, 0.065) Without hypertension: 0.021 (

- 0.007, 0.049)

Per unit increase in ln

transformed tibia Pb With diabetes: 0.082 (0.03, 0.14) Without diabetes: 0.005 (

- 0.01, 0.02) With hypertension: 0.023 (0.003, 0.04) Without hypertension: 0.0004 (

- 0.01, 0.01) Cross
- Sectional Results Kim et al. ( 1996 ) Adult males Boston, MA; Multiple examination s 1979

- 1994 459 Median baseline blood = 8.6 μg/dL 10th

- 90th percentile: 4.0

- 17.5 Serum creatinine (mg/dL) Random

- effects modeling adjusted for baseline age, time since initial visit, BMI, smoki ng status, alcohol ingestion, education level, hypertension. Peak blood Pb ≤ 40 μg/dL: 0.0017 (0.0005, 0.003) Peak blood Pb ≤ 25 μg/dL: 0.0021 (0.0007, 0.0035) Peak blood Pb ≤ 10 μg/dL: 0.0033 (0.0012, 0.0053)February 2012 5

- 314 Draft

– Do Not Cite or Quote Reference Population Study Location; Time Period N Pb Biomarker Data Outcome Statistical Analysis Effect Estimate (95% CI)

Tsaih et al. ( 2004 ) Adult males Boston, MA; 8/1991

- 1995 with mean 6 yr follow

- up 448 Mean (SD) baseline Blood Pb = 6.5 (4.2) μg/dL 10th

- 90th percentile: 2.6

- 11.5 Repeated measures 10th

- 90th percentile: 2.1

- 7.6 Tibia Pb = 21.5 (13.5) μg/g Patella Pb = 32.4 (20.5) μg/g Serum creatinine (mg/dL) Log linear regression adjusted for age, age squared, BMI, hypertension, diabetes, smoking status, alcohol consumption, analgesic use Baseline blood Pb With diabetes:

- 0.009 (

- 0.038, 0.020) Without diabetes:

- 0.004 (

- 0.010, 0.003) With hypertension: 0 (

- 0.013, 0.013) Without hypertension:

- 0.005 (

- 0.011, 0.002) Follow

- up blood Pb With diabetes: 0.053 (

- 0.032, 0.138) Witho ut diabetes: 0.034 (0.007, 0.061) With hypertension: 0.083 (0.038, 0.128) Without hypertension: 0.014 (

- 0.016, 0.044) De Burbure et al. ( 2006 ) Children, mean age = 10 years, age range = 8.5

- 12.3 years France, Czech Republic, and Poland; dates not provided 804 Concurrent Blood Pb Median (IQR) = 3.9 (2.6, 5.7) μg/dL 10th

- 90th percentile: 1.8

- 8.1 Log

- transformed serum creatinine, cystati n C, and β2

- microglobulin Log linear regression adjusted for cadmium, urinary creatinine, urinary mercury Log serum creatinine (mg/L):

- 0.062 (

- 0.106,

- 0.017) a Log Cystatin C:

- 1.3 (

- 2.4,

- 0.21) a Log β2

- microglobulin:

- 2.2 (

- 4.0,

- 0.54) a
FIGURE 5
- 39: Negative Effect Estimates Indicate Poorer Function Per 1 μg/dL increase in blood Pb within the 10th
- 90th percentile interval Longitudinal Results: Yu et al. ( 2004 ) Adult CKD patients Taipei, Taiwan; 48 month longitudinal study period 121 Mean (SD) Baseline blood = 4.2 (2.2) μg/dL 10th

- 90th percentile: 2.0

- 5.1 Change in MDRD eGFR over 4 yr/100 (mL/min/1.73 m2 body surface area) Generalized estimating equations adjusted for age, sex, BMI, hyperlipidemia, hypertension, smoking, use of ACE inhibitor, baseline serum creatinine, daily protein excretion, daily protein intake, underlying kidney disease - 0.040 (

- 0.072,

- 0.008) a

Cross
- Sectional Results: Akesson et al. ( 2005 ) WHILA, adult women Sweden; 6/1999

- 1/2000 820 M edian (5

- 95%) concurrent blood = 2.2 (1.1, 4.6) μg/dL 10th

- 90th percentile: 1.3

- 3.8 Creatinine clearance/100 (mL/min) Linear regression adjusted for age, BMI, diabetes, hypertension, regular use of nephrotoxic drug, smoking status - 0.018 (

- 0.03,

- 0.006)

Cystatin C

- based eGFR (Larsson et al. 2004)/100 (mL/min) - 0.02 (

- 0.03, 0.007)February 2012 5

- 315 Draft

– Do Not Cite or Quote Reference Population Study Location; Time Period N Pb Biomarker Data Outcome Statistical Analysis Effect Estimate (95% CI)

Staessen et al. ( 1992 ) Adults Belgium; 1985

- 1989 1,981 Concurrent Blood Pb Mean (SD) Males: 11.4 μg/dL Females: 7.5 μg/dL 10th

- 90th percentile: 3.7

- 15.1 Creatinine clearance/100 (mL/min) Log linear regression adjusted for age, age squared, sex, BMI, BP, ferritin level, smoking status, alcohol ingestion, rural/urban residence, analgesic and diuretic use, blood and urinary cadmium, diabetes, occupational exposure to heavy metals, and gamma glutamyl transpeptidase Females:

- 0.067 (

- 0.108,

- 0.027) a Males:

- 0.051 (

- 0.097,

- 0.047) a

Payton et al. ( 1994 ) Adult males Boston, MA; 1988

- 1991 744 Mean (SD) concurrent blood = 8.1 (3.9) μg/dL 10th

- 90th percentile: 4.1

- 12.9 Log

- transformed creatinine clearance (mL/min) Log linear regression adjusted for age, BMI, analgesic and diuretic use, alcohol consumption, smoking status, SBP, DBP - 0.040 (

- 0.079,

- 0.0015)

Fadrowski et al. ( 2010 ) NHANES, adolescents U.S.; 1988 1994 769 Median concurrent blood = 1.5 μg/dL 10th

- 90th percentile: 0.4

- 5.4 Q1: <1.0 Q2: 1.0 to 1.5 Q3: 1.6 to 2.9 Q4: >2.9 Cystatin C

- based eGFR/100 (mL/min/1.73 m 2 ; calculated using the Filler and Lepage equation) Log linear regression adjusted for age, sex, race/ethnicity, urban/rural residence, smoking, obesity, household income, education level of family reference person, BP, lipid levels, glucose levels - 0.022 (

- 0.038,

- 0.0054) Q1: Referent Q2:

- 1.4 (

- 7.4, 4.5) Q3:

- 2.6 (

- 7.3, 2.2) Q4:

- 6.6 (

- 12.6,

- 0.07)

FIGURE 5
- 40: Analysis of Blood Pb Quartiles: %change in kidney outcome
Muntner et al. ( 2003 ) NHANES III, adults U.S.; 1988

- 1994 4813 Mean (SD) concurrent blood Pb With Hypertension: 4.2 ( 0.14) μg/dL Q1: 0.7 to 2.4 Q2: 2.5 to 3.8 Q3: 3.9 to 5.9 Q4: 6.0 to 56.0

Without Hypertension: 3.3 (0.10) μg/dL Q1: 0.7 to 1.6 Q2: 1.7 to 2.8 Q3: 2.9 to 4.6 Q4: 4.7 to 52.9 Elevated Serum Creatinine (99th percentile of each race

- sex specific distribution for healthy young adults)

CKD Logistic regression adjusted for age, race, sex, diabetes, SBP, smoking, history of CVD, BMI, alcohol consumption, household income, education level, marital status, health insurance Q1: Referent With hypertension Q2: 47% (3, 110) Q3: 80% (34, 142) Q4: 141% (46, 297) Without hypertension Q2: 11% (

- 44, 121) Q3: 19% (

- 38, 125) Q4: 9% (

- 47, 122)
With hypertension Q2: 44% (0, 109 ) Q3: 85% (32, 159) Q4: 160% (52, 345) Without hypertension Q2:

- 10% (

- 63, 116) Q3: 0% (

- 55, 122) Q4: 9% (

- 59, 189) Navas

- Acien et al. ( 20 09 ) NHANES III, adults U.S.; 1999

- 2006 14,778 Geometric concurrent blood mean = 1.58 μg/dL Q1: ≤ 1.1 Q2: 1.2 to 1.6 Q3: 1.7 to 2.4 Q4: >2.4 eGFR <60 mL/minute/1.73 m 2 Logistic regression adjusted for survey year, age, sex, race/ethnicity, BMI, education, smoking, cotinine, alcohol intake, hypertension, diabetes, menopausal status Q1: Referent Q2: 10% (

- 20, 51) Q3: 36% (

- 1, 85) Q4: 56% (17, 108) Albuminuria and eGFR <60 mL/minute/1.73 m 2 Q2: 53% (

- 15, 177) Q3: 57% (

- 17, 198) Q4: 139% (31, 337)

a 95% CI estimated from given p

- value.February 2012 5

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### Patient Population Studies CKD as defined by the National Kidney Foundation

- Kidney Disease Outcomes Quality 1 

### Initiative (NKF

- K/DOQI) workgroup ( National Kidney Foundation, 2002 ) is the presence 2 

### of markers of kidney damage or GFR <60 mL/min/1.73 m 2 

### for ≥ 3 months. The MDRD 3 

### equation is the most common one used in the eGFR determination for this definition. 4 

### Notably, decreased GFR is not required for the first criterion and markers of kidney 5 

### damage are not required for the second criterion. 6 

### Several key studies in CKD patients provide prospective data to evaluate the impact of 7 

### baseline blood Pb level on CKD progression (kidney function decline) in patient 8 

### populations ( Table 5

- 26 ). Yu et al. ( 2004 ) , discussed in the 2006 Pb AQCD, followed 9 

### 121 patients over a four year period. Eligibility required well

- controlled CKD with serum 10



### cr eatinine between 1.5 and 3.9 mg /dL. Importantly, EDTA

- chelatable Pb <600 μg/72 h, a 11 

### level below that traditionally thought to indicate risk for Pb

- related nephrotoxicity, was 12 

### required at baseline. Patients with potentially unstable kidney disease were excl uded 13 

### ( i.e., due to systemic diseases such as diabetes). Mean blood Pb and EDTA

- chelatable Pb 14 

### levels were 4.2 μg/dL and 99.1 μg/72 hours, respectively. In a Cox multivariate 15 

### regression analysis, chelatable Pb was significantly associated with overall risk f or the 16 

### primary endpoint (doubling of serum creatinine over the 4

- year study period or need for 17 

### hemodialysis). When the group was dichotomized by EDTA chelatable Pb level, Kaplan

18 

### Meier analysis demonstrated that significantly more patients (15/63) in the hi gh

- normal 19 

### group (EDTA chelatable Pb level ≥ 80 but <600 μ g/72 hours) reached the primary end 20 

### point than did those in the lower EDTA chelatable Pb levels (<80 μ g Pb/72 hours) group 21 

### (2/58). Associations between baseline chelatable or blood Pb level and chang e in serial 22 

### measurements of eGFR (estimated by the MDRD equation ( Levey et al., 1999 ) were 23 

### modeled separately using generalized estimating equations. Based on these models, a 10 24 

### μg higher chelatable Pb level or 1 μg/dL higher blood Pb level reduced the GFR by 1.3 25 

### and 4.0 mL /min/1.73 m 2 

### , respectively, during the 4

- year stud y period. Recent studies 26 

### expanded the CKD patient populations in which this effect was observed to those with 27 

### diabetic nephropathy ( Lin et al., 2006a ) and with the lowest blood Pb levels studied to 28 

### date ( Lin et al., 2006b )

. Results of these observational studies have been summarized 29 

### ( Weaver and Jaar, 2010 )

. 30February 2012 5

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### Table 5
- 26 Patient population studies: kidney function decline

Study n Baseline mean (SD) blood Pb ( μ g/dL) Baseline mean (SD) chelatable Pb ( μ g/72 hours) Baseline mean (SD) eGFR ( mL /min/1.73 m 2 ) Years of follow
- up Decline in eGFR per 1 SD higher

Pb dose at baseline per year Comments

Lin et al. ( 2003 ) 202 5.3 (2.9) 104.5 (106.3) 41.6 (14.4) 2 0.16 Largest study to date

Yu et al. ( 2004 ) 121 4.2 (2.2) 99.1 (83.4) 36.0 (9.8) 4 2.7 (chelatable) 2.2 (blood Pb) Longest follow

- up; 1 μ g/dL higher blood Pb, at baseline, associated with 4.0 mL/min/1.73 m 2 reduction in eGFR over 4 years Lin et al. ( 2006a ) 87 6.5 (3.4) 108.5 (53.8) 35.1 (9.0) 1 3.87 Type II diabetics with nephropathy Lin et al. ( 2006b ) 108 2.9 (1.4) a 40.2 (21.2) (all <80) 47.6 (9.8) 2 1.1 Lowest Pb exposed CKD patients

a Notably, mean blood Pb level in this study was below that observed in a recent large general population study of 50

- to 70

- year olds in Baltimore, MD ( Martin et al., 2006 )

. Source: Reprinted with permission of UpToDate.co m , Weaver and Jaar ( 2010 ) 

### A recent population

- based case

- control study examined occupational Pb exposure as a 1 

### risk factor for severe CKD ( Evans et al., 2010 )

. The study included 926 cases with first 2 

### time elevations of serum creatinine >3.4 mg /dL for men and >2.8 mg /dL for women and 3 

### 998 population

- based controls. Occupational Pb exposure was assessed using an expert 4 

### rating method based on job histories. Eighty

- one cases and 95 controls were judged to 5 

### have had past occupational Pb exposure. Of those, 23 cases and 32 controls were thought 6 

### to have been exposed to Pb levels ≥ 30 μg/m 3 

### (the current U.S. OS HA limit is 50 μg/m 3 

### ). 7 

### In multivariable logistic regression modeling, the OR for CKD (adjusted for age, sex, 8 

### smoking, alcohol consumption, diabetes, education, and BMI) was 0.97 (95% CI: 0.68, 9 

### 1.38) in Pb

- exposed compared to non
- exposed participants. In an alyses comparing low 10 

### (≥ 3 to <10% of occupational exposure limit), medium (≥ 10 to < 30% of limit), and high 11 

### (≥ 30% of limit) exposure groups, although ORs were elevated (statistically 12 

### nonsignificant) in the medium exposure group compared to the never expo sed group for 13 

### average and lifetime cumulative exposure metrics, a monotonic increase in OR across 14 

### exposure groups was not observed. In the low and high exposure groups, odds of CKD 15 

### tended to be lower compared with the never exposed group. In addition, the CKD patients 16 

### were followed prospectively for a mean of 2.5 years for the 70 Pb

- exposed patients and 17 

### 2.4 years for the 731 patients without past occupational Pb exposure. Mean eGFRs (using 18 

### the MDRD equation) were 16.0 and 16.6 mL/min/1.73 m 2 

### in exposed and non
- exposed 19 

### patients, respectively, indicating severe disease in both groups. Using mixed

- effects 20 

### multivariable models, eGFRs declined by 4.27 and 3.39 mL/min/1.73 m 2 

### /y in ever and 21 

### most Pb

- exposed CKD patients, respectively, compared with 4.55 mL/min/1.73 m 2 

### /y in 22 

### patients without occupational Pb exposure. Thus, the results overall did not provide 23 

### strong evidence that Pb exposure was associated with renal effects. 24February 2012 5

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### Strengths noted by the authors included virtually complete case ascertainment and 1 

### minimal loss to follow

- up. Exposure assessment was listed as both a strength and a 2 

### limitation. Expert rating methods are commonly used when biological monitoring is not 3 

### an option and in case

- control studies where many occupational exposures are considered. 4 

### In Pb

- kidney research, this approach is uncommon except in the case

- control setting. 5 

### However, given the challenges of interpreting blood Pb in dialysis patients (discussed 6 

### below), this approach may have advantages in this study of such severe CKD. Other case

7 

### control s tudies examining occupational risk factors for CKD found Pb exposure to be a 8 

### risk factor ( Nuyts et al., 1995 ; Steenland et al., 1990 )

. Nuyts et al. ( 1995 ) found adults 9 

### with history of occupational Pb exposure to have elevated odds of CKD (OR for ever

10



### versus never
- exposed: 2.11 [95% CI: 1.23, 4.36]). The a ssociation was weaker in 11 

### Steenland et al. ( 1990 ) (OR for ever

- versus never
- expose d: 1.73 [95% CI: 0.82, 3.65]). 12 

### Regular moonshine consumption, also a potential source of Pb exposure, was a stronger 13 

### risk factor for CKD (OR: 2.42 [95% CI: 1.10, 5.36]). 14 

### The prospective observational aspect of Evans et al. ( 2010 ) is similar in design to the 15 

### work of Lin and colleagues but differs in several important respects. In Evans et al. 16 

### ( 2010 ) , only occupational Pb exposure was considered whereas the work in Taiwan 17 

### excluded occupational exposure and used blood and chelatable Pb measures. In the past 18 

### in dev eloped countries, environmental exposures were substantial. For example, mean 19 

### tibia Pb levels were 21.5 and 16.7 μ g/g bone mineral, in environmentally

- exposed 50

- to 20 

### 70

- year
- old African

- Americans and whites, respectively, in Baltimore ( Martin et al., 21 

### 2006 )

. In Korean Pb workers, mean baseline tibia Pb level was only twofold higher (35.0 22 

### μ g/g) ( Weaver et al., 2003a ) which illustrates the substantial body burden in middle

- and 23 

### older

- aged Am ericans from lifetime Pb exposure. Declines in blood Pb levels in Sweden 24 

### have been reported and attributed to the leaded gasoline phase

- out ( Strömberg et al., 25 

### 1995 ; Elinder et al., 1986 ) , although blood Pb levels were lower than those noted during 26 

### the U.S. phase

- out. Finally, the severe degree of CKD among subjects in Evans et al. 27 

### ( 2010 ) creates a survivor bias at enrollment and limits the eGFR decline possible during 28 

### follow

- up, thus limiting the ability to identify factors that influence that decline. 29



### ESRD Pati ent Studies End stage renal disease (ESRD) is a well

- established public health concern, and is 30 

### characterized by the use of dialysis to perform the normal functions of the kidney. 31 

### Incidence and prevalence in the U.S. continue to increase resulting in rates that are the 32 

### third highest among nations reporting such data ( U.S. Renal Data System, 2009 )

. Studies 33 

### in patients with CKD requiring chronic hemodialysis (ESRD) have also been published in 34 

### the past five years. A study of 271 adult patients on regular thrice weekly dialysis 35February 2012 5

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### reported much higher blood Pb levels than had been appreciated by the treating clinicians 1 

### ( Davenport et al., 2009 )

. Blood Pb levels ranged from 3 to 36.9 μg/dL; 25.5% had levels 2 

### >20 μg/dL, 59% had values of 10

- 20 μg/dL, and 15.5% were < 10 μg/dL. Few details on 3 

### the statistical analysis were provided which complicates interpretation of the findings. 4 

### However, blood Pb was positively correlated with hemodialysis vintage (months on 5 

### dialysis; Spearman r = 0.38, p <0.001); negatively correlated with urine output (r =

- 0.44, 6 

### p <0.001) and higher in patients using single carbon filter and reverse osmosis water 7 

### purification devices. Another recent publication reported higher Pb in dialysate than in 8 

### the tap water used in its preparation ( Chen et al., 2009a )

. A systematic review of a wide 9 

### range of trace elements in hemodia lysis patients reported higher Pb levels in patients 10 

### compared to controls although the difference was not large ( Tonelli et al., 2009 )

. These 11 

### data suggest that blood Pb monitoring in dialysis patients may be useful. 12



### Int erpretation of blood and bone Pb in patients on dialysis is challenging for several 13 

### reasons. First, renal osteodystrophy, the bone disease related to kidney disease, may 14 

### result in increased release of Pb from bone stores. Thus, interpretation of blood and even 15 

### bone Pb levels may require adjustment with one or more of a range of osteoporosis 16 

### variables. Secondly, as observed above ( Davenport et al., 2009 ) , residual kidney function 17 

### may have a substantial impact on blood Pb levels in populations with such minimal 18 

### excretion. Third, as illustrated in the studies cited above ( Chen et al., 2009a ; Davenport et 19 

### al., 2009 ) , water and concentrates used in dialysis may be variable sources of Pb. A 20 

### recent study reported decreased blood Pb in post

- dialysis compared to pre

- dialysis 21 

### samples ( Kazi et al., 2008 )

. Thus, substantial fluctuations in blood Pb are possible while 22 

### on dialysis. Finally, anemia is common in CKD and Pb is stored in red blood cells. Thus, 23 

### measurement of blood Pb in anemia may require adjustment for hemoglobin; no 24 

### standardized approach to thi s currently exists. 25 

### Given these caveats, a pilot study observed higher median blood Pb levels in 55 African

26 

### American dialysis patients compared to 53 age

- and sex

- matched controls (6 and 3 μg/dL 27 

### respectively; p <0.001) ( Muntner et al., 2007 )

. This study was unique in that tibia Pb 28 

### levels were assessed. Median tibia Pb was higher in ESRD patients although the 29 

### difference did not reach statistical significance (17 and 13 μg/g bone mineral, 30



### re spectively [p = 0.13]). In order to determine the potential impact of renal 31 

### osteodystrophy, median blood and tibia Pb levels in the dialysis patients were compared 32 

### by levels of serum parathyroid hormone, calcium, phosphorus, and albumin and were not 33 

### found to be significantly different ( Ghosh

- Narang et al., 2007 )

. A study of 211 diabetic 34 

### patients on hemodialys is ( Lin et al., 2008 ) found parathyroid hormone and serum 35



### cr eatinine to be associated with blood Pb level in crude but not adjusted associations. In 36 

### contrast, a study of 315 patients on chronic peritoneal dialysis observed parathyroid 37 

### hormone to be positively correlated and residual renal function to be negatively 38February 2012 5

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– Do Not Cite or Quote 

### correlated with logarithmic

- transformed blood Pb levels after adjustment ( Lin et al., 1 

### 2010 )

. In the prospective portion of this study, blood Pb levels at baseline were 2 

### categorized by tertile (range of 0.1 to 29.9 μg/dL with cut points of 5.62 and 8.66 μg/dL). 3 

### Cox multivariate analysis, after adjustment for parathyroid hormone level, resi dual renal 4 

### function, and 20 other variables, showed increased all

- cause mortality in the middle 5 

### (5.62

- 8.66 μg/dL) and highest (> 8.66 μg/dL) compared to the lowest (< 5.62 μg/dL) 6 

### tertiles after 18 months of follow

- up (hazard ratio= 2.1 [95% CI: 2.0, 2.2] a nd 3.3 [95% 7 

### CI: 1.3, 13.5], respectively). A recent publication of an 18

- month follow

- up of 927 8 

### patients on maintenance hemodialysis also reported increased hazard ratios for all

- cause 9 

### (4.7 [95% CI: 1.9, 11.5]), cardiovascular

- cause (9.7 [95% CI: 2.1, 23.3 ]), and infection

10 

### cause (5.4 [95% CI: 1.4, 20.8]) 18

- month mortality in the highest (> 12.64 μg/dL) 11 

### compared to the lowest tertile (< 8.51 μg/dL) of baseline blood Pb level, after adjustment 12 

### for sex, urban residence, hemodialysis vintage, hemoglobin, serum albumin, and ferritin 13 

### ( Lin et al., 2011 )

. Given other recent publications in hemodialysis patients by this group, 14 

### it would b e valuable to examine these risks after adjustment for hemoglobin A1C ( Lin

15 

### Tan et al., 2007a ) , and blood cadmium ( Yen et al., 2011 ; Hsu et al., 2009a )

. 16



### Clinical Trials in Chronic Kidney Disease Patients Randomized chelation trials in CKD patients, uncommon in nephrotoxicant research, 17 

### provide unique i nformation on the kidney impact of Pb. These studies have been 18 

### performed by Lin and colleagues in Taiwan and involve similar study designs. Initially, 19 

### patients are observed in order to compare CKD progression prior to chelation. Then, 20 

### CKD patients whose di agnostic EDTA chelatable Pb levels are within certain ranges 21 

### (generally 60

- 600 μ g/72 hours and thus below the level commonly considered for 22 

### chelation) are randomized. The treated group receives weekly chelation with 1 g EDTA 23 

### intravenously for up to 3 month s. The control group receives placebo infusions. In the 24 

### follow

- up period, chelation is repeated for defined indications such as increased serum 25 

### creatinine or chelatable Pb levels above specified cut

- offs. Placebo infusions are repeated 26 

### in the controls as w ell. The results of the most recent of these trials are summarized in 27 

### Table 5

- 27 below. 28February 2012 5

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### Table 5
- 27 Clinical randomized chelation trials in chronic kidney disease patients

Reference Group n Baseline mean (SD) blood Pb ( μ g/dL) Baseline mean (SD) chelatable Pb ( μ g/72 hr) Baseline mean (SD) eGFR ( mL /min/1.73 m 2 ) Months of treatment/ follow
- up Change in eGFR per yr ( mL /min/1.73

m 2 ) Comments

Lin et al. ( 2003 ) Chelated 32 6.1 (2.5) 150.9 (62.4) 32.0 (12.1) 27 + 1.07

Control 32 5.9 (3.0) 144.5 (87.9) 31.5 (9.0)

- 2.7 Lin et al. ( 2006a ) Chelated 15 7.5 (4.6) 148.0 (88.6) 22.4 (4.4) 15

- 3.5 Subjects with Type II diabetes and nephropathy Control 15 5.9 (2.2) 131.4 (77.4) 26.3 (6.2)

- 10.6 Lin et al. ( 2006b ) Chelated 16 2.6 (1.0) a 43.1 (13.7) 41.2 (11.2) 27 +3.0 Lowest Pb exposed and treated range Body Lead Burden (72 h urinary Pb excretion) ≥ 20

- <80 μ g Control 16 3.0 (1.1) 47.1 (15.8) 42.6 (9.7)

- 2.0 Lin

- Tan et al. ( 2007b ) Chelated 58 5.0 (2.2) 164.1 (111.1) 36.8 (12.7) 51

- 0.3 Subjects without diabetes Control 58 5.1 (2.6) 151.5 (92.6) 36.0 (11.2)

- 2.9

a Notably, mean blood Pb level in this study was below that observed in a recent large general population study of 50

- to 70

- year olds in Baltimore, MD ( Martin et al., 2006 )

.



### This study design requires replication in larger populations at multiple clinical centers. If 1 

### confirmed, the effect may be due to removal of Pb. However, chelation may also have a 2 

### direct beneficial effect on kidney function, regardless of Pb exposure. Antioxida nt effects 3 

### of CaNa 2 

### EDTA which may improve kidney function directly via improved blood flow to 4 

### the kidneys have been reported ( Saxena and Flora, 2004 ; Jacobsen et al., 2001 )

. EDTA 5 

### benefits in a Pb rodent model appeared to occur via reduced oxidation ( Saxena and Flora, 6 

### 2004 )

. EDTA administration reduced kidney damage in a rat model of acute renal failure 7 

### induced by ischemia ( Foglieni et al., 2006 )

. Similarly DMSA has be en reported to 8 

### prevent renal damage when co

- administered during induction of nephrosclerosis in a 9 

### nonPb

- exposed rat model ( Gonick et al., 1996 )

. Benefits from chelation reported in 10 

### rodent models of Pb

- related nephrotoxicity ( Sanchez

- Fructuos o et al., 2002a ; Sanchez

11 

### Fructuoso et al., 2002b ; Khalil

- Manesh et al., 1992b ) did not appear to occur via reversal 12 

### of structural damage ( Khalil

- Manesh et al., 1992b ) ; aga in suggesting that improved 13 

### hemodynamics from reduction of reactive oxidant species, which could be due to reduced 14 

### Pb and/or directly to the chelating agent, may be a mechanism ( Gon ick et al., 1996 )

. 15 

### However, the most parsimonious explanation for the combination of the observational 16February 2012 5

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### and experimental chelation work of Lin and colleagues is that reduced Pb is the 1 

### underlying reason. 2 

### The unique body of work in patient populations by Lin and co

- workers, both 3 

### observational and experimental, has numerous strengths including prospective study 4 

### design, randomization, Pb assessment that includes estimates of the bioavailable dose, 5 

### longitudinal statistical analysis, and control for multiple k idney risk factors. However, the 6 

### generalizability of the results to broader populations is unknown. In addition, the 7 

### association observed between Pb dose and decline in GFR has been variable; the annual 8 

### decline in eGFR per standard deviation (SD) higher Pb dose at baseline was much lower 9 

### in the 2003 study than in subsequent publications ( Table 5

- 27 above). Small sample sizes 10 

### and differences in renal dia gnoses between groups may be factors in this variability. 11 

### Additional research in large populations at multiple centers with assessment of 12 

### neuropsychological as well as kidney outcomes is needed. 13



### Occupational Studies The vast majority of studies in the lit erature on the impact of Pb on the kidney have been 14 

### conducted in the occupational setting. In general, study size and extent of statistical 15 

### analysis are much more limited than those in general population studies. Publications in 16 

### few populations have report ed adjusted results in occupationally exposed workers in the 17 

### five years since the 2006 Pb AQCD. In a two

- year prospective cohort study, generalized 18 

### estimating equations were used to model change in kidney function between each 19 

### evaluation in relation to tib ia Pb and concurrent change in blood Pb in 537 current and 20 

### former Pb workers ( Weaver et al., 2009 )

. Tibia Pb was evaluated at the beginning of each 21 

### follow

- up period (yearly on average) and Pb biomarker levels were adjusted for baseline 22 

### levels and other covariates. In males, serum creatinine decreased and calculated 23 

### creatinine clearance increased over th e course of the study; these changes were largest in 24 

### participants whose blood Pb declined concurrently or whose tibia Pb was lower at the 25 

### beginning of the follow

- up interval. In females, decreasing serum creatinine was 26 

### associated with declining blood Pb (a s in males); however, increasing blood Pb was 27 

### associated with a concurrent increase in serum creatinine. Women (25.9% of the study 28 

### population) were older and more likely to be former Pb workers than were men which 29 

### may have been important factors in the eff ect modification observed by sex. 30 

### Chia and colleagues observed a significant, positive association between concurrent 31 

### blood Pb and urine NAG in linear regression models after adjustment for age, sex, race, 32 

### exposure duration, ALAD G177C polymorphism and th e interaction between ALAD 33 

### genotype and blood Pb ( Chia et al., 2006 )

. Similar positive associations were observed 34 

### between blood Pb and a wider range of EBE markers in models that adjusted for age, sex, 35February 2012 5

- 323 Draft

– Do Not Cite or Quote 

### race, exposure duration, and the HpyCH4 ALAD polymorphism ( Chia et al., 2005 )

. 1 

### Other studies published in the last 5 years also focused on ALAD polymorphisms but did 2 

### not find e ffect modification to be in a consistent direction ( Gao et al., 2010a ; Wang et al., 3 

### 2009a ; Weaver et al., 2006 ; Weaver et al., 2005b )

. In adults with the ALAD2 genotype, 4 

### Pb has been associated with better and poorer renal function in separate cohorts of Pb 5 

### workers. 6 

### A study of 155 male workers reported significant, positive correlations between blood 7 

### and urine Pb and urine NAG and albumin after controlling for age and job duration ( Sun 8 

### et al., 2008a )

. An important additional study that analyzed occupational Pb exposure is 9 

### discussed below under patient population studies ( Evans et al., 2010 )

. 10 

### A few studies of occupationally

- exposed adults have performed benchmark dose 11 

### calculations for the effect of Pb on the kidney. Both used only EBE markers and found 12 

### NAG to be the most sensitive outcome; reported lower confidence limits on the 13 

### benchmark doses were 10.1 μg/dL ( Sun et al., 2008a ) , and 25.3 μg/dL ( Lin and Tai

- yi, 14 

### 2007 )

. 15 

### A number of other publications in the five years since the 2006 Pb AQCD have reported 16 

### significantly worse kidney outcomes in unadjusted analyses in occupationally

- exposed 17 

### adults compared to unexposed controls ( Onuegbu et al., 2011 ; Patil et al., 2007 ) and/or 18 

### significant correlations between higher levels of Pb biomarkers and worse kidney 19 

### function ( Alasia et al., 2010 ; Khan et al., 2008 ; Sun et al., 2008a ; Garcon et al., 2007 ; Lin 20 

### and Tai

- yi, 2007 ; Alinovi et al., 20 05 )

. One small st udy found no significant differences 21 

### ( Orisakwe et al., 2007 )

. In a study of 108 Pb workers with mean blood Pb level of 22 

### 36.2 μ g/dL, no significant correlations were obse rved between blood Pb concentration 23 

### and GFR, creatinine clearance, uric acid clearance or uric acid excretion fraction 24 

### ( Karimooy et al., 2010 )

. How ever, interpretation of this study is limited by the fact that 25 

### "only 30 subjects had a correct 24 hours urine volume" and no methods are described for 26 

### kidney outcome measurement or analysis. 27 

### Overall, the occupational literature published in the last five y ears on the kidney impact 28 

### of Pb exposure has been more consistent in reporting statistically significant associations 29 

### than were data reviewed for the 2006 Pb AQCD. This may reflect increased reliance on 30 

### EBE markers as more sensitive outcome measures, publi cation bias, or multiple 31 

### comparisons due to a greater number of outcomes assessed. 32 

### A small number of publications that include concentration

- response information provides 33 

### evidence of Pb

- related nephrotoxicity in the occupational setting across the blood P b 34 

### ranges analyzed ( Weaver et al., 2003a ; Ehrlich et al., 1998 )

. Data in 267 Korean Pb 35 

### workers in the oldest age tertile (mean age = 52 years) did not provide evidence of a 36February 2012 5

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– Do Not Cite or Quote 

### thre shold for a Pb effect on serum creatinine levels (added variable plot shown in Figure 1 

### 5

- 41 ) ( Weaver et al., 2003a )

. It is important to note the uncertainty regarding whether the 2 

### concentration

- response i nformation provided in these studies applies to lower blood Pb 3 

### levels or to populations with lower current environmental Pb exposures. 4

Source: Reprinted with permission of the BMJ Publishing Group, Weaver et al. ( 2003a ) Note: Both the adjusted regression line (straight line) and the line estimated by the smoothing method of the S

- PLUS statistical software function lowess (line with curves) are displayed. Both have been adjusted for covariates. For ease of interpretation , axes have been scaled, so that the plotted residuals are centered on the means, ra ther than zero. 

### Figure 5
- 41 Added variable plot of association between serum creatinine and blood Pb in 267 Korean Pb workers in the oldest age tertile.
A major challenge in interpretation of the occupational literature is the potential for Pb

5 

### related hyperfiltration. Hyperfiltration involves an initial increase in glomerular 6 

### hypertension which results in increased GFR. If persistent, the risk for subsequent CKD 7 

### increases. This pattern has been observed in dia betes, hypertension, and obesity ( Nenov 8 

### et al., 2000 )

. As discussed in the 2006 Pb AQCD, findings consistent with hyperfiltration 9 

### have been observed in occupational populations ( Weaver et al., 2003a ; Hsiao et al., 2001 ; 10 

### Roels et al., 1994 ) , a study of adults who were Pb poisoned as children ( Hu, 1991 ) , and a 11 

### study in European children ( De Burbure et al., 2006 )

. Longitudinal data in Pb

- exposed 12 

### rodents provide evidence of a hyperfiltration pattern of increased, followed by decrea sed 13 

### GFR, associated with Pb exposure and are critical in interpretation of the human Pb

14 

### kidney literature ( Khalil

- Manesh et al., 1992a )

. Pb could induce glomerular hypertension 15 

### resulting in hyperfiltratio n by several mechanisms including increased ROS, changes in 16 

### Adjusted blood Pb level ( μ g/dL)Adjusted serum creatinine (mg/dL)February 2012 5

- 325 Draft

– Do Not Cite or Quote 

### eicosanoid levels, and/or an impact on the renin

- angiotensin system ( Vaziri, 2008b ; Roels 1 

### et al., 1994 )

. Whether hyperfiltration contributes to pathology in humans is unclear; 2 

### longitudinal studies are needed. 3 

### Regardless, significant findings could be obscured if opposite direction associations are 4 

### present in different segments of the study population and in teraction models are not 5 

### performed to address this. In the Korean Pb workers ( Weaver et al., 2003a ; Weaver et al., 6 

### 2003b ) , significant associations in opposite directions were observed only when relevant 7 

### effect modifiers such as age or genetic variants in ALAD, VDR, and NOS were inc luded 8 

### in the model. This is a valid concern for risk assessment, since the factors involved in 9 

### these inverse associations in Pb

- exposed workers are not well defined at present. 10



### 5.5.2.3 Epidemiology in Children

Lead Nephrotoxicity in Children
Both the 2006 and 1986 Pb AQCDs noted that the degree of kidney pathology observed 11 

### in adult survivors of untreated childhood Pb poisoning in the Queensland, Australia 12 

### epidemic ( Inglis et al., 1978 ) has not been observed in other studies of childhood Pb 13 

### poisoning. Recent publications remain consistent with that conclusion; a recent study 14 

### ob served an impact of childhood Pb poisoning on IQ but not kidney outcomes ( Coria et 15 

### al., 2009 )

. Chelation was raised as a potential explanation for this discrepancy in the 2006 16 

### Pb AQCD. 17 

### With declining Pb exposure levels, recent work has focused on studies in children with 18 

### much lower blood Pb levels. However, insensitivity of the clinical kidney outcome 19 

### ( i.e., GFR) measures for early kidney damage is a particular problem in children who do 20 

### not have many of the other kidney risk factors that adults do, such as hypertension and 21 

### diabetes. As a result, su ch studies have utilized EBE markers. However, data to 22 

### determine the predictive value of such biomarkers for subsequent kidney function decline 23 

### in Pb exposed populations are extremely limited ( Coratelli et al., 1988 ) and may pose 24 

### particular challenges in children due to puberty

- related biomarker changes ( Sarasua et al., 25 

### 2003 )

. The few studies included the 2006 Pb AQCD that analyzed clinical kidney 26 

### outcome s in children found associations with indicators of Pb exposure that were 27 

### inconsistent in direction. Fels et al. ( 1998 ) found no difference in mean serum creatinine 28 

### between 62 children living near Pb

- producing factories and 50 control children living in 29 

### communities without Pb emission sources. In a study of 200 Belgian adolescents a ged 17 30 

### years, higher concurrent blood Pb level was associated with higher serum cystatin

- C in 31 

### 200 ( De Burbure et al., 2006 ) ; howev er, among 300

- 600 European children (n varied by 32February 2012 5

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### outcome), higher concurrent blood Pb level was associated with lower serum creatinine 1 

### and cystatin C ( Staessen et al., 2001 )

. 2


### Recent studies of children with elevated Pb exposure did not consistently indicate that Pb 3 

### exposure was associated with reduced kidney function. A study in 123 children of 4 

### workers in Pakistani Pb smelters and battery recycling plants and 123 control children, 5 

### ages 1

- 6 years, reported elevated blood Pb levels, serum creatinine and urea in children of 6 

### Pb

- exposed workers compared to controls (medians: 8.1 versus 6.7 μ g/dL; 56 versus 7 

### 52 μ M; and 4 , 500 versus 4 , 300 μ M, respectively (p ≤ 0.01 for all) in unadjusted analyses 8 

### ( Khan et al., 2010a )

. Blood Pb levels were correlated with serum creatinine (Spearman r 9 

### = 0.13; p = 0.05). However, a study of 77 participants, ages 10

- 25 years, who were 10 

### previously Pb poisoned through contaminated flour and chelat ed, reported no difference 11 

### in renal effects between children with blood Pb levels > 48 μ g/dL and < 43 μ g/dL 12 

### although lower IQ was observed in the subset who were exposed before the age of six 13 

### years ( Coria et al., 2009 )

. 14 

### One of the key gaps identified in the 2006 Pb AQCD was limited data in children and 15 

### adolescents particularly with respect to GFR measures and in populations without the 16 

### elevated Pb exposure associated with Pb poisoning, living near a Pb source, or having 17 

### parents with occupational Pb exposures. A recently published NHANES analysis in 18 

### adolescents begins to fill this gap ( Fadrowski et al., 2010 )

. Associations between 19 

### concurrent blood Pb and kid ney function were investigated in 769 adolescents aged 12

- 20 20 

### years in the U.S. NHANES III, conducted 1988

- 1994. Kidney function was assessed with 21 

### two eGFR equations. One utilized serum cystatin C and the other used the more 22 

### traditional marker, serum creati nine. Median concurrent blood Pb and cystatin C

- based 23 

### eGFR levels were 1.5 μg/dL and 112.9 mL/min/1.73 m 2 

### , respectively. Cystatin C

- based 24 

### eGFR was lower (

- 6.6 mL/min/1.73 m 2 

### [95% CI:

- 0.7,

- 12.6]) in participants with blood 25 

### Pb levels in the highest quartil e (≥ 3.0 μg/dL) compared with those in the lowest (<1 26 

### μg/dL). A doubling of blood Pb level was associated with a

- 2.9 mL/min/1.73 m 2 

### (95% 27 

### CI:

- 0.7,

- 5.0) lower eGFR. In contrast, the association between blood Pb and creatinine

28 

### based eGFR, although in the s ame direction, was not statistically significant. As these 29 

### children were born between 1968 and 1982, some likely had higher Pb exposures in 30 

### earlier childhood, although notably, not as high or as long in duration as did older adults 31 

### examined in aforemention ed studies. Nonetheless, in this study of NHANES adolescents, 32 

### there also is uncertainty regarding the magnitude, timing, frequency, and duration of Pb 33 

### exposure that contributed to the observed associations. Additional research in children is 34 

### warranted, in particular studies with longitudinal follow

- up, multiple outcome assessment 35 

### methods, and examination of children born after Pb was banned from gasoline. 36February 2012 5

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### 5.5.2.4 Associations between Lead Dose and New Kidney Outcome Measures
As noted above, in an effort to more ac curately estimate kidney outcomes, new equations 1 

### to estimate GFR based on serum creatinine have been developed, and the utility of other 2 

### biomarkers, such as cystatin C, as well as equations based on them, are being studied. 3 

### However, few publications have u tilized these state

- of

- the

- art techniques when 4 

### evaluating associations between Pb or cadmium dose and renal function. In addition to 5 

### the study in NHANES adolescents discussed above ( Fadrowski et al., 2010 ) , a cross
6 

### sectional study of Swedish women reported that higher concurrent blood Pb (median: 7 

### 2.2 μ g/dL) and cadmium (median: 0.38 μ g/L) levels were associated wi th lower eGFR 8 

### based on serum cystatin C alone (without age, sex, and race) after adjustment for socio

9 

### demographic and CKD risk factors ( Akesson et al., 2005 )

. Associations were comparable 10 

### to those using creatinine clearance as the kidney outcome for Pb; however associations of 11 

### cadmium dose measures were stronger for the cystatin C based outcome. Staessen et al. 12 

### ( 2001 ) found a statistically significan t association between concurrent blood Pb level and 13 

### serum cystatin C in a cross

- sectional study of adolescents; creatinine

- based measures 14 

### were not reported. However, in a cross

- sectional study of European children, higher 15 

### concurrent blood Pb levels were as sociated with lower serum cystatin C and creatinine; 16 

### these inverse associations were attributed to hyperfiltration ( De Burbure et al. , 2006 )

. A 17 

### very recent publication compared associations of blood Pb and eGFR using the traditional 18 

### MDRD equation to those with four new equations: CKD

- EPI, and cystatin C single 19 

### variable, multivariable, and combined creatinine/cystatin C, in 3 , 941 adul ts who 20 

### participated in the 1999

- 2002 NHANES cystatin C subsample ( Spector et al., 2 011 )

. 21 

### Similar to the NHANES adolescent analysis, associations with the cystatin C outcomes 22 

### were stronger. After multivariable adjustment, differences in mean eGFR for a doubling 23 

### blood Pb were

- 1.9 (95% CI:

- 3.2,

- 0.7),

- 1.7 (95% CI:

- 3.0,

- 0.5), and

- 1. 4 (95% CI:

- 2.3, 24



### - 0.5) mL/min/1.73 m 2 

### , using the cystatin C single variable, multivariable and combined 25 

### creatinine/cystatin C equations, respectively, reflecting lower eGFR with increased blood 26 

### Pb. The corresponding differences were

- 0.9 (95% CI:

- 1.9, 0.0 2) and

- 0.9 (95% CI:

- 1.8, 27 

### 0.01) using the creatinine

- based CKD

- EPI and MDRD equations, respectively. 28



### 5.5.2.5 Reverse Causality
As discussed briefly above, reverse causality has been considered as an alternative 29 

### hypothesis to explain associations observed between indicators of Pb exposure and renal 30 

### dysfunction in adults. The reverse causality hypothesis attributes increased blood and 31 

### bone Pb levels to reduced Pb excretion from nonPb

- related causes rather then implicating 32 

### Pb

- related renal dysfunction as a contributi ng factor to CKD. The 2006 Pb AQCD 33February 2012 5

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### concluded that "reverse causality is not likely to be a major explanatory factor accounting 1 

### for observed associations between Pb and renal dysfunction" ( U.S. EPA, 2006b )

. The 2 

### strongest evidence against reverse causality was provided by longitudinal studies in 3 

### which associations between baseline measurements of Pb biomarkers and subsequent 4 

### changes in renal function were demonstrated. In the NAS, baseline blood Pb levels were 5 

### associated with subseq uent declines in renal function over follow

- up periods ranging 6 

### from 3 to 6 years after adjustment for baseline renal function ( Tsaih et al., 2004 ; Kim et 7 

### al., 1996 )

. Similar re sults were reported in studies of patients with renal disease ( Yu et 8 

### al., 2004 ; Lin e t al., 2003 )

. The longitudi nal analyses in NAS studies also indicated that 9 

### associations between blood Pb level and serum creatinine persisted in the lower range of 10 

### serum creatinine levels, including those in the normal range ( Tsaih et al., 2004 ; Kim et 11 

### al., 1996 )

. Thus, the association was not limited to the segment of the population with 12 

### potentially clinically significant renal dysfunction in whom reduced Pb excretion would 13 

### be more likely. 14 

### Additional support against reverse causality was provided by findings in Swedish women 15 

### that both higher blood and urinary Pb were associated with lower creatinine clearance 16 

### ( Akesson et al., 2006 ; Akesson et al., 2005 )

. If reverse causality were the more likely 17 

### hypothesis for these associations, lower creatinine clearance would be associated with 18 

### lower urinary Pb, which it is not. Among adults with chronic kidney disease, renal failure 19 

### was not associated wi th increases in blood or bone Pb levels or chelatable Pb levels ( Van 20 

### De Vyver et al., 1988 )

. Batuman et al. ( 1983 ) found that chelatable Pb levels were similar 21 

### in adults with renal disease of unknown and known nonPb

- related causes. Study had bone 22 

### Pb levels (group means: 18 and 19 μ g/g) in the range of those measured in recent 23 

### epidemiologic studies. In summary, evidence that higher blood Pb levels are associated 24 

### with subsequent declines in renal function from baseline levels, that associations persist 25 

### among adults with normal renal function, and that renal failure does not incre ase Pb 26 

### biomarker levels collectively do not support reverse causality and thus demonstrate that 27 

### reverse causality does not provide a more suitable alternative hypothesis to explain the 28 

### associations consistently observed between Pb biomarker levels and rena l dysfunction. 29



### 5.5.3 Modes of Action for Lead
- Induced Nephrotoxicity

5.5.3.1 Altered Uric Acid
Higher occupational Pb exposure or blood Pb levels have been linked to increased risk 30 

### for both gout and kidney disease ( Shadick et al., 2000 ; Batuman, 1993 )

. Pb is thought to 31 

### increase serum uric acid by decreasi ng its kidney excretion ( Emmerson and Rave nscroft, 32February 2012 5

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### 1975 ; Ball and Sorensen, 1969 ; Emmerson, 1965 )

. Research during the past decade 1 

### indicates that uric acid is nephrotoxic at lower levels than previously recognized ( Johnson 2 

### et al., 2003 )

. Therefore, the 2006 Pb AQCD reviewed literature implicating increased uric 3 

### acid as a mechanism for Pb

- related nephrotoxicity ( Weaver et al., 2005a ; Shadick et al., 4 

### 2000 )

. However, this does not appear to be the only mechanism, since associations 5 

### between blood Pb and serum creatinine have remained significant even after adjustment 6 

### for uric acid ( Weaver et al., 2005a )

. These mechanistic relations have more than just 7 

### theoretical importance. Clinically relevant therapies may be possible since EDTA 8 

### chelation has been reported to improve both kidney function and urate clearanc e in 9 

### patients with kidney insufficiency and gout, even when EDTA

- chelatable Pb body 10 

### burdens were low ( Lin et al., 2001 )

. 11 

### Alterations in serum uric acid have been studied in animal models exposed to Pb. 12 

### Conterato et al. ( 2007 ) followed various parameters of kidney function after single or 13 

### multiple Pb injections in rats. The single dosing regimen consisted of a single i.p. 14 

### injection of 25 or 50 mg /kg Pb

- acetate , while the multiple injections involved once daily 15 

### i.p. injection of either vehicle or Pb

- acetate (5 or 25 mg /kg ) for 30 days. Single and 16 

### multiple injections at both dose levels increased plasma uric acid levels. Similarly, Abdel 17 

### Moneim et al. ( 2011b ) reported increased serum uric acid and urea levels after 5 days of 18 

### Pb

- acetate treatment (i. p. 20 mg /kg). In male rats exposed to Pb in drinking water from 19 

### lactation to puberty (40 days) or post

- puberty (65 days), Berrahal et al. ( 2011 ) found that 20 

### plasma urea levels increased after 40 days of exposure (puberty blood Pb level of 12.7 21 

### μg/dL) but decreased after 65 days of Pb exposure (post

- puberty blood Pb level of 7.5 22 

### μg/dL) ( Table 5

- 23 ). 23



### 5.5.3.2 Oxidative Damage
A role for ROS in the pathogenesis of experimental Pb

- induced hypertension and renal 24 

### disease has been well characterized ( Vaziri, 2008a , b ; Vaziri and Khan, 2007 )

. The 25 

### production of oxidative stress following Pb exposure is detailed in respect to modes of 26 

### action of Pb ( Section 5.2.4 ). Past studies have shown that Pb treatment (single or three 27 

### da ily i.p. injections) can elevate kidney GST levels, affecting glutathione metabolism 28 

### ( Daggett et al., 1998 ; Moser et al., 1995 ; Oberley et al., 1995 )

. 29 

### Animal studies continue to provide evidence for increased oxidative stress playing a role 30 

### in the pathogenesis of Pb

- induced renal toxicity. Increased ROS, serum NO, and renal 31 

### NO were observed after Pb injections in rats (i.p. 20 mg /kg, 5 days) ( Abdel Moneim et 32 

### al., 2011b )

. Pb exposure to rat proximal tubular cells (0.5

- 1 μ M) also increased ROS 33 

### production, in a concentration

- dependent manner ( Wang et al., 2011b )

. Oxidat ive stress 34February 2012 5

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### was also demonstrated by increased lipid peroxidation ( i.e., MDA) in serum and renal 1 

### tissue after Pb exposure ( Abdel Moneim et al., 2011b ; Lodi et al., 2011 ; Wang et al., 2 

### 2011b )

. Berrahal et al. ( 2011 ) reported increased MDA in Pb

- exposed (50 ppm 3 

### Pb

- acetate pre
- and post

- natally) rat kidney relative to controls at both 40 (puberty; blood 4 

### Pb 12.7 μg/dL) and 65 (post

- puberty; blood Pb 7.5 μg/dL) days of age. In addition, total 5 

### sulfhydryl groups were signif icantly decreased at 65 days. These increases in oxidative 6 

### stress were accompanied by age

- dependent Pb nephrotoxicity in male rats ( Table 5

- 24 ). 7


### Alte rations in endogenous antioxidants and antioxidant enzymes that may lead to 8 

### oxidative stress have also been reported after Pb exposure. Pb treatment decreased the 9 

### activity of the renal antioxidant enzymes, CAT, SOD, GST, GPx, and GR ( Abdel 10 

### M oneim et al., 2011b ) and protein levels of CAT and GSH ( Lodi et al., 2011 )

. 11 

### Additionally, proteomic analysis of high

- level Pb treated (1 , 500 ppm , 5 weeks; resulting 12 

### in blood Pb level of 53.4 μ g/dL) rat kidney identified decreased abundance of a rate

13 

### limiting enzyme in the synthesis of GSH (glutamate cysteine ligase) ( Chen et al., 2011 )

. 14


### Conterato et al. ( 2007 ) examined the effect of Pb

- acetate on the cytosolic thioredoxin 15 

### reductase activity and oxidative stress parameters in rat kidneys. A single injection of 16 

### Pb

- acetate consisted of a single i.p. injection of 25 or 50 mg /kg Pb

- acetate , while 17 

### repeated injections consisted of one daily i.p. injection of Pb

- acetate (5 or 25 mg /kg) for 18 

### 30 days. Measured were thioredoxin reductase

- 1, a selenoprotein involved in many 19 

### cellular redox processes, SOD, δ

- ALAD, GST, GPx, non protein thiol groups (NPSH), 20 

### CAT, as w ell as plasma creatinine, uric acid, and inorganic phosphate levels. The single 21 

### injection at the 25 mg Pb dose level resulted in increased SOD and thioredoxin reductase

22 

### 1 activity, while the 50 mg dose level increased CAT activity and inhibited δ

- ALAD 23 

### acti vity in the kidney. Repeated injections at the 5 mg dose level of Pb inhibited δ

- ALAD 24 

### and increased GST, NPSH, CAT, and thioredoxin reductase

- 1. Repeated injections at the 25 

### 25

- mg dose level reduced δ

- ALAD but increased GST, NPSH, and plasma uric acid 26 

### levels . No changes were observed in TBARS, GPx, creatinine or inorganic phosphate 27 

### levels after either single or repeated injection dosing. As both dosing regimens increased 28 

### thioredoxin reductase

- 1 activity, the authors suggest that this enzyme may be a sensitive 29 

### indicator of renal changes with low dose Pb treatment. 30


### Jurczuk et al. ( 2006 ) published a study of the involvement of some low molecular weight 31 

### thiols in the peroxidative mechanisms of action of Pb in the rat kidney. Wistar rats were 32 

### fed a diet containing 500 ppm Pb

- acetate for a period of 12 weeks and were compared t o 33 

### a control group receiving distilled water for the same time period. GSH, metallothionein 34 

### (MT), total and nonprotein SH groups (TSH and NPSH) were measured, as were the 35 

### blood activity and urinary concentration of δ

- ALA. The concentrations of GSH and 36 

### NPSH were decreased by Pb administration, while MT concentration was unchanged. δ

- 37February 2012 5

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### ALAD in blood was decreased, whereas urinary δ

- ALA was increased by Pb 1 

### administration. Negative correlations were found between the kidney GSH concentrations 2 

### and previously report ed concentrations of Pb and MDA in kidneys of these rats. It is 3 

### apparent from graphical presentation of the data that GSH was reduced by more than 4 

### 50% following Pb administration, while TSH was reduced by approximately 15%. No 5 

### values for either blood or ki dney Pb levels or kidney MDA were reported in this article. 6 

### In 2007, the same authors ( Jurczuk et al., 2007 ) reported on the renal concentrations of 7 

### the anti oxidants, vitamins C and E, in the kidneys of the same Pb

- treated and control rats. 8 

### Exposure to Pb significantly decreased vitamin E concentration by 13% and vitamin C 9 

### concentration by 26%. The kidney concentration of vitamin C negatively correlated with 10 

### M DA concentration. The authors concluded that vitamins E and C were involved in the 11 

### mechanism of peroxidative action of Pb in the kidney, and their protective effect may be 12 

### related to scavenging of free radicals. 13 

### Studies have used antioxidant compounds to i nvestigate the role of oxidative stress in Pb

14 

### induced nephrotoxicity. Abdel Moneim et al. ( 2011b ) reported that flaxseed oil treatment 15 

### protected rats from Pb

- induced (i.p. 20 mg /kg, 5 days) oxidative stress, inflammation, 16 

### and apoptosis. Howeve r, the flaxseed oil also decreased the accumulation of Pb in renal 17 

### tissue making it difficult to ascertain whether the protection was due to decreased 18 

### oxidative stress or to altered Pb uptake kinetics. 19


### El

- Neweshy and El

- Sayed ( 2011 ) studied the influence of vitamin C supplementation on 20 

### Pb

- induced histopathological alterations in male rats. Rats were given Pb

- acetate , 21 

### 20 mg /kg by intragastric feeding once daily for 60 days. Control rats were given 15 mg of 22 

### sod ium acetate per kg once daily, and an additional group was given Pb

- acetate plus 23 

### vitamin C (20 mg /kg every other day) 30 minute s before Pb feeding. Control rats showed 24 

### normal histology, while Pb

- treated rats exhibited karyomegaly with eosinophilic 25 

### intranuc lear inclusion bodies in the epithelial cells of the proximal tubules. Glomerular 26 

### damage and tubular necrosis with invading inflammatory cells were also seen in Pb

27 

### treated animals. Among rats treated with Pb

- acetate plus vitamin C, five exhibited 28 

### relativel y mild karyomegaly and eosinophilic intranuclear inclusion bodies of proximal 29 

### tubules and an additional five rats were normal. Normal glomeruli were noted in all. 30 

### Thus, vitamin C was shown to ameliorate the renal histopathological effects of Pb 31 

### intoxicatio n, however no measures of Pb accumulation were provided to clarify the 32 

### mechanism of action of vitamin C. 33



### Masso

- Gonzalez and Antonio

- Garcia ( 2009 ) studied the protectiv e effect of natural 34 

### antioxidants (zinc, vitamin A, vitamin C, vitamin E, and vitamin B6) against Pb

- induced 35 

### damage during pregnancy and lactation in rat pups. At weaning, pups were sacrificed and 36 

### kidneys were analyzed. Pb

- exposed pups had decreased body we ights. Blood Pb levels 37February 2012 5

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### were 1.43 μg/dL in the control group, 22.8 μg/dL in the Pb group, 21.2 μg/dL in the Pb 1 

### plus zinc plus vitamins group, and 0.98 μg/dL in the zinc plus vitamin group. The kidney 2 

### TBARS were significantly elevated in Pb exposed pups, whi le treatment with vitamins 3 

### and zinc returned TBARS to control levels. Kidney CAT activity was significantly 4 

### increased above control with Pb treatment; however supplementation with zinc and 5 

### vitamins reduced CAT activity toward normal. Pb exposure inhibited kidney Mn

6 

### dependent SOD but not Cu

- Zn

- dependent SOD activity. Thus, supplementation with zinc 7 

### and vitamins during gestation and lactation was effective in attenuating the redox 8 

### imbalance induced by developmental, chronic low

- level Pb exposure, likely throu gh the 9 

### alteration of Pb accumulation. 10


### Bravo et al. ( 2007 ) reported further that mycophenolate mofetil (an immunosuppressive 11 

### agent used in renal transplantation which inhibits T and B cell proliferation) 12 

### administration reduces renal inflammation, oxidative stress and hypertension in Pb

13 

### expos ed rats. Thus, an inflammatory immune and oxidative stress component can be seen 14 

### as contributing to Pb

- induced renal effects and hypertension. 15 

### Although the majority of studies of the effects of Pb exposure have been conducted in 16 

### male rats, a couple of stud ies have compared the response of male rats with female rats 17 

### ( Sobekova et al., 2009 ; Alghazal et al., 2008a )

. Sobekova et al. ( 2009 ) contrasted the 18 

### activity response to Pb on the antioxidant enzymes, GPx and GR, and on TBARS in both 19 

### male and female Wistar rats of equal age. Males weighing 412 ± 47 g and females 20 

### weighing 290 ± 19 g were fed diets containing eith er 100 ppm or 1 , 000 ppm Pb

- acetate 21 

### for 18 weeks. In the male rats, kidney Pb content increased by 492% on the 100 ppm Pb 22 

### diet and by 7 , 000% on the 1 , 000 ppm Pb diet. In the female rats, kidney Pb content 23 

### increased by 410% on the 100 ppm Pb diet and by 23,0 00% on the 1 , 000 ppm Pb diet. 24 

### There was virtually no change in GPx in the kidney of male rats given the 100 ppm Pb 25 

### diet but there was a significant reduction in GPx in the female rats on both the 100 ppm 26 

### diet and 1 , 000 ppm diet. In male rats, GR was increa sed from 182 units/gram of protein 27 

### in control kidneys to 220 units on the 100 ppm Pb diet and 350 units on the 1 , 000 ppm 28 

### diet. In female rats, kidney GR decreased from 242 units in control animals to 164 units 29 

### in animals on the 100 ppm Pb diet and 190 unit s in animals on the 1 , 000 ppm diet. In 30 

### male rats, kidney TBARS content increased from 7.5 units/gram protein to 10.0 units 31 

### (1 , 000 ppm Pb diet group). In female rats, there was a reduction in TBARS from 14.4 32 

### units per gram protein to 10.0 units in rats on t he 100 ppm Pb diet and to 11 units in rats 33 

### on the 1 , 000 ppm Pb diet. 34 

### Alghazal et al. ( 2008a ) compared the activity responses of the antioxidant enzyme, SOD 35 

### and the detoxifying enzyme, GST, of the same rats exposed to 100 ppm or 1 , 000 ppm 36 

### Pb

- acetate for 18 weeks. Similar to the previous study, kidney TBARS were increased 37February 2012 5

- 333 Draft

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### only in male rats given the higher dose of Pb. Kidney SOD activity, on the other hand, 1 

### was increased in both males and females at the higher dose of Pb, while GST activity was 2 

### increased in kidney of males at the higher dose of Pb and decreased at the lower dose, but 3 

### was decreased at both doses of Pb in females. Thus there were significant differences in 4 

### the responses of male and female rats to Pb exposure. Differences may be ac counted for 5 

### in part due to the greater deposition of Pb in female rat kidneys. Another explanation, 6 

### offered by the authors, is that male rats are known to metabolize some foreign 7 

### compounds faster than do females, so the biological half

- life of xenobiotics in the 8 

### females may be longer. 9



### 5.5.3.3 Renal Gangliosides
Gangliosides are constituents of the plasma membrane that are important for control of 10 

### renal GFR because they can act as receptors for various molecules and have been shown 11 

### to take part in cell

- cell interact ions, cell adhesion, recognition and signal transduction. 12 

### Aguilar et al. ( 2008 ) studied changes in renal gangliosides following Pb exposure 13 

### (600 ppm Pb

- acetate in drinking water for 4 months) in adult male Wistar rats. Pb 14 

### exposure caused an increase in blood Pb from 2.1 to 35.9 μ g/dL. There was no change in 15 

### serum creatinine or in hemoglobin, but there was an increase in urinary δ

- ALA. The 16 

### following renal gangliosides were measured by immunohistochemistry and by thin layer 17 

### chromatography: GM1, GM2, GM4, and 9

- O

- acetylated modified form of the GD3 18 

### gangliosid e (9

- O

- Ac

- GD3). The ganglioside pattern was mainly characterized by a 19 

### decrease in the GM1 ganglioside as well as by a mild increase in GM4 and GM2 20 

### gangliosides, while the strongest alteration was observed in the 9

- O

- Ac

- GD3, which was 21 

### overexpressed. The lat ter was observed only in the glomerular zone. This was associated 22 

### with a decrease in apoptotic glomerular cells, as assessed by the TUNEL assay. The 23 

### authors hypothesized that the increase in GD3

- O

- acetylation could represent a strategy to 24 

### attenuate the nor mal renal apoptotic process and therefore contribute to cell survival 25 

### during Pb exposure. 26



### 5.5.3.4 Role of Metallothionein

Yu et al. ( 2009 ) described dichotomous effects of Pb

- acetate on the expression of MT in 27 

### the liver and kidney of mice. Male mice were i.p. injected with Pb

- acetate in doses of 28 

### 100, 200, and 300 μ mol/kg and sacrificed 4, 8, and 24 hours after Pb treatment. 29 

### Administration of Pb increased the levels of MT

- 1 mRNA in the liver and kidneys but 30 

### increased MT protein only in the liver. Treatment of mouse PT cells in vitro with Pb also 31 

### resulted in an increase in MT mRNA but litt le increase in MT protein. Thus, Pb appears 32February 2012 5

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### to exert a dual effect on MT expression in the kidney: enhancement of MT gene 1 

### transcription but suppression of MT mRNA translation. 2


### Zuo et al. ( 2009 ) examined the potential role of α

- Synuclein (Scna) and MT in Pb

3 

### induced inclusion body formation. They used MT

- I/II double knockout (MT

- null) and 4 

### parental wild type (WT) cell lines to explore the formation process of Pb

- induced 5 

### inclusion bodies. Unlike WT cells, MT

- null cells did not form inclusion bodies after Pb 6 

### exposure. Western blot of the cytosol showed that soluble MT protein in WT cells was 7 

### lost during Pb exposure as inclusion bodies formed. However, transfection of MT

- 1 into 8 

### MT

- null cells allowed inclusion body f ormation after Pb exposure. As Scna is a protein 9 

### with a natural tendency to aggregate into oligomers, Scna was measured in WT cells and 10 

### MT

- null cells after Pb exposure. Scna protein showed poor basal expression in MT

- null 11 

### cells, and Pb exposure increased S cna expression only in WT cells. MT transfection 12 

### increased Scna transcript to WT levels. In both of these cell lines Pb

- induced Scna 13 

### expression rapidly increased and then decreased over 48 hours as Pb

- induced inclusion 14 

### bodies were formed. A direct interact ion between Scna and MT was confirmed ex vivo 15 

### by an antibody pull down assay, where the proteins co

- precipitated with an antibody to 16 

### MT. Pb exposure caused increased colocalization of MT and Scna proteins. In archival 17 

### kidney samples of renal cortex from WT mice chronically treated with Pb, MT was 18 

### localized to the surface of inclusion bodies. Thus, Scna may be a component of Pb

19 

### induced inclusion bodies and, with MT, may play a role in inclusion body formation. 20



### 5.5.4 Effects of Exposure to Lead Mixtures

The effect of Pb on other cations, specifically calcium, is well established in the kidney 21 

### literature. Calcium

- mediated processes involving receptors, transport proteins, and 22 

### second messenger signaling among other endpoints have been shown to be significantly 23 

### affecte d by Pb exposure. The disposition of Pb in the soft tissues (kidney and spleen) can 24 

### change with exposure to Pb and other compounds. Pb plus Cd exposure changed Pb 25 

### disposition with increased blood Pb (versus Pb alone group) and decreased metal 26 

### concentration in the kidney and liver (versus Pb alone). An iron deficient diet 27 

### significantly increased Pb deposition in adult animals ( Hashmi et al., 1989 ) , pregnant 28 

### dams, and maternally

- exposed fetuses ( Singh et al., 1991 )

. Di etary thiamine plus zinc 29 

### slightly reduced blood and kidney Pb in exposed animals ( Flora et al., 1989 )

. Selenium, a 30 

### cofactor for GPx, attenuated P b

- induced lipid peroxidation and abrogated the Pb

- induced 31 

### attenuation of GR and SOD. Concomitant exposure to the cations aluminum and Pb 32 

### protected animals from ensuing nephropathy ( Shakoor et al., 2000 )

. In summary, Pb has 33 

### been shown to affect processes mediated by endogenous divalent cations. In addition, 34February 2012 5

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### exposure to other metals or divalent cations c an modulate Pb disposition and its effects in 1 

### the body. 2



### 5.5.4.1 Lead and Cadmium
Cd shares many similarities with Pb; it has been shown to be a ubiquitous PT 3 

### nephrotoxicant and accumulates in the body. Despite the similarities, few studies have 4 

### evaluated associat ions between Cd exposure and CKD or the impact of joint exposure of 5 

### Pb and Cd or other metals on CKD. As discussed in the 2006 Pb AQCD, environmental 6 

### exposure to Cd, at levels common in the U.S. and other developed countries, has been 7 

### shown to impact subst antially associations between indicators of Pb exposure and the 8 

### kidney EBE marker, NAG, even in the presence of occupational level Pb exposure. In an 9 

### occupational study, mean NAG, although higher in the Pb

- exposed worker group 10 

### compared to controls, was cor related with urine Cd but not blood or tibia Pb ( Roels et al ., 11 

### 1994 )

. In another occupational population where both metals were significantly 12 

### associated with NAG, a 0.5 μg/g creatinine increase in Cd had the same effect on NAG as 13 

### did a 66.9 μg/g bone mineral increase in tibia Pb ( Weaver et al., 2003a )

. 14 

### The 2006 Pb AQCD noted that data examining the concentration

- response relation 15 

### between environmental Cd and the kidney were too scarce to determine the impact of Cd 16 

### exposure on relations between Pb exposure and other kidney outcomes. A recent 17 

### publication i n NHANES data collected from 1999 through 2006 addresses this need; 18 

### (results pertaining solely to Pb were discussed in Section 5.5.2.2 ) ( Navas

- Acien et al., 19 

### 2009 )

. Geometric mean concurrent blood Cd level was 0.41 μg/L in 14,778 adults aged ≥ 20 

### 20 years. After adjustment for survey year, sociodemographic factors, CKD risk factors, 21 

### and blood Pb, the ORs for albuminuria (≥ 30 mg /g creatinine), reduced eGFR (<60 22 

### mL/min/1.73 m 2 

### ), and both albuminuria and reduced eGFR were 1.92 (95% CI: 1.53, 23 

### 2.43), 1.32 (95% CI: 1.04, 1.68), and 2.91 (9 5% CI: 1.76, 4.81), respectively, comparing 24 

### the highest with the lowest blood Cd quartiles. Both Pb and Cd remained significantly 25 

### associated after adjustment for the other. Effect modification was not observed; however, 26 

### ORs were higher for adults in the hi ghest quartiles of both metals compared with the ORs 27 

### for the highest quartiles of concurrent blood Cd or Pb alone ( Table 5

- 25 ). Compared with 28 

### adults wi th blood Cd levels ≤ 0 .2 μg/L and blood Pb levels ≤ 1 .1 μg/dL, adults with blood 29 

### Cd levels > 0.6 μg/L and blood Pb levels > 2.4 μg/dL had ORs (95% CIs) of 2.34 (95% 30 

### CI: 1.72, 3.18) for albuminuria, 1.98 (95% CI: 1.27, 3.10) for reduced eGFR, and 4.10 31 

### (95% CI: 1.58, 10.65) for albuminuria and reduced eGFR together. These findings are 32 

### consistent with other recent publications ( Akesson et al., 2005 ; Hellstrom et al., 2001 ) , 33 

### support consideration of both metals as independent CKD risk factors in the general 34February 2012 5

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### population, and provide novel evidence of increased risk in those with higher 1 

### environmental exposure to both metals. 2 

### However, a very recent study suggests that interpretation of Cd associations with GFR 3 

### measures may be much more complex. Conducted in Pb workers to address the fact that 4 

### few studies have examined the impact of environmental Cd exposure in workers who are 5 

### occupationally exposed to other nephrotoxica nts such as Pb, the study assessed Cd dose 6 

### with urine Cd, which is widely considered the optimal dose metric of cumulative Cd 7 

### exposure. In 712 Pb workers, mean (SD) blood and tibia Pb, urine Cd, and eGFR using 8 

### the MDRD equation were 23.1 (14.1) μ g/ dL , 26.6 (28.9) μ g/g, 1.15 (0.66) μ g/g 9 

### creatinine, and 97.4 (19.2) mL /min/1.73m 2 

### , respectively ( Weaver et al., 2011 )

. After 10 

### adjustment for age, sex, BMI, urine creatinine, smoking, alcohol use, education, annual 11 

### income, diastolic BP, current or former Pb worker job status, new or returning study 12 

### participant, and blood and tibia Pb, higher urine Cd was associated with higher calculated 13 

### creatinine clearance, eGFR ( β = 8.7 mL /min/1.73 m 2 

### [95% CI: 5.4, 12.1] per unit 14 

### increase in ln

- transformed urine Cd) and ln

- NAG, but lower serum creatinine. These 15 

### unexpected para doxical associations have been reported in a few other publications 16 

### ( De Burbure et al., 2006 ; Hotz et al., 1999 ) and have been observed in other populations. 17 

### Potential explanations for these paradoxical results included a normal physio logic 18 

### response in which urine Cd levels reflect renal filtration; the impact of adjustment for 19 

### urine dilution with creatinine in models of kidney outcomes; and Cd

- related 20 

### hyperfiltration. 21 

### Wang et al. ( 2009c ) studied the effects of Pb and/or Cd on oxidative damage to rat kidney 22 

### cortex mitochondria. In this study young female Sprague Dawley rats were fed for 8 23 

### weeks with either Pb

- acetate (300 ppm ), Cd chloride (50 ppm ), or Pb and Cd together in 24 

### the same dos age. Lipid peroxidation was assessed as MDA content. Renal cortex pieces 25 

### were also processed for ultrastructural analysis and for quantitative rtPCR to identify the 26 

### mitochondrial damage and to quantify the relative expression levels of cytochrome 27 

### oxidase s ubunits (COX

- I/II/III). Cytochrome oxidase is the marker enzyme of 28 

### mitochondrial function, and COX

- I, II, and III are the three largest mitochondrially

29 

### encoded subunits which constitute the catalytic functional core of the COX holoenzyme. 30 

### Mitochondria were altered by either Pb or Cd administration, but more strikingly by Pb 31 

### plus Cd administration, as indicated by disruption and loss of mitochondrion cristae. 32 

### Kidney cortex MDA levels were increased significantly by either Pb or Cd, given 33 

### individually, but mo re so by Pb plus Cd. COX

- I/II/III were all reduced by either Pb or Cd 34 

### administration, but more prominently by Pb plus Cd administration. This study adds to 35 

### knowledge of the synergistic effects of Pb and Cd on kidney mitochondria. 36February 2012 5

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### 5.5.4.2 Lead, Cadmium, and Arsenic
Wang and Fowler ( 2008 ) present a general review of the roles of biomarkers in 1 

### evaluating interactions among mixtures of Pb, Cd, and arsenic. Past studies have found 2 

### that addition of Cd to treatment of rats with Pb or Pb and As sig nificantly reduced the 3 

### histological signs of renal toxicity from each element alone; on the other hand, animals 4 

### exposed to Cd in addition to Pb or Pb and As showed an additive increase in the urinary 5 

### excretion of porphyrins, indicating that, although measu red tissue burdens of Pb were 6 

### reduced, the biologically available fraction of Pb is actually increased ( Mahaffey et al., 7 

### 1981 ; Mahaffey and Fowler, 1977 )

. 8 

### Stress proteins were examined after exposure to mixtures of Pb and other metals. 9 

### Induction of MT was strongest in groups with Cd treatment. However, co

- exposure to Pb 10 

### and As induced higher levels of MT protein than did either Pb or As exposure alone in 11 

### kidney tubule cells. Heat shock proteins (Hsps) are commonly altered with exposure to 12 

### metal mixtures. A study found in vitro (low dose) and in vivo that Pb induced Hsps in a 13 

### metal/metalloid

- , concentration

- and time

- specific manner ( Wang et al., 2005 )

. Additive 14 

### or more than additive interactions occurred among Pb, Cd and As under combined 15 

### exposure conditions. 16



### 5.5.4.3 Lead and Zinc
Zinc has been investigated as a protective compound against the ef fects of Pb. Pb 17 

### treatment (35 mg /kg i.p. for 3 days) caused a significant fall in hemoglobin content, 18 

### significant increases in lipid peroxidation and decreased level of reduced glutathione in 19 

### liver, together with diminished total protein content in liver a nd kidney. Co

- treatment of 20 

### Pb with zinc (10 mg /kg i.p.) or ascorbic acid (10, 20 and 30 mg /kg i.p.) showed a 21 

### moderate therapeutic effect when administered individually, but more pronounced 22 

### protective effects after combined therapy ( Upadhyay et al., 2009 )

. 23


### Jamieson et al. ( 2008 ) studied the effect of dietary zinc content on renal Pb deposition. 24 

### Weanling Sprague Dawley rats were assigned to marginal zinc (MZ, 8 mg Zn/kg diet), 25 

### zinc adequate control (CT, 30 mg Zn/kg), zinc

- adequate diet

- restricted (30 mg Zn/kg), or 26 

### supplemental zinc (SZn, 300 mg Zn/kg) groups, with or without Pb

- acetate (200 ppm for 27 

### 3 weeks). Pb exposure did not result in nephromegaly or histological alterations. The MZ 28 

### rats had higher renal Pb (35%) and lower renal zinc (16%) concentrations than did CT 29 

### rats. On the other hand, SZn was more protective than the CT diet was against renal Pb 30 

### accumulation (33% lower). Standard procedures for indirect immunoperoxidase staining 31 

### were used to determine MT localization in the kidney. Pb had no effect on MT staining 32February 2012 5

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### intensity, distribution, or relative protein amounts. Western blot analysis confirmed that 1 

### MT levels were responsive to dietary zinc but not to Pb exposure. 2



### 5.5.4.4 Lead and Mercury

Stacchiotti et al. ( 2009 ) studied stress proteins and oxidative damage in a renal

- derived 3 

### cell line exposed to inorganic mercury and Pb. The time course of the expression of 4 

### several Hsps, glucose

- regulating proteins and MTs in a rat proximal tubular cell line 5 

### (NRK

- 52E) exposed to subcytotoxic doses of inorganic mercury (HgCl 2 

### , 1

- 40 μM) and 6 

### Pb (PbCl 2 

### , 2

- 500 μM) were analyzed. ROS and reactive nitrogen species (RNS) were 7 

### detected by flow cytometric analysis. Endogenous total GSH content and the enzymatic 8 

### activity of GST were determined in cell homogenates. Western blot analysis an d 9 

### immunohistochemistry were used for quantification of hsps and MTs. Reverse 10 

### transcription PCR was used for quantification of metallothionein. The higher doses of 11 

### mercury (20 μM and 40 μM) were shown to markedly inhibit growth of the cell line 12 

### while the hi gher doses of Pb (60 μM to 500 μM) inhibited cell growth to a lesser degree. 13 

### After 24 hours of exposure at 20 μ M mercury, the cells presented abnormal size and 14 

### pyknotic nuclei, swollen mitochondria and both apoptosis and overt necrosis. In the 15 

### presence of 60 or 300 μ M Pb, the cells lost cell

- cell and cell

- matrix contacts, showed a 16 

### round size, irregular nuclear contour and often mitotic arrest, but no apoptosis or overt 17 

### necrosis at 24 hours. Mercury induced a significant increase in both ROS and RNS, 18 

### maximal RNS at 24 hours, and maximal ROS at 48 hours. Pb (60 or 300 μ M) did not 19 

### cause an increase in ROS or RNS beyond the levels measured in control cells. Total GSH 20 

### significantly increased in cells grown in the presence of Pb; the effect was concentration

21 

### depen dent and GSH reached its maximal value at a dose of 300 μ M Pb. The effect of 22 

### mercury was biphasic: 10 μM significantly enhanced GSH by 600%, while the amount of 23 

### GSH detected after 20 μ M mercury only increased by 50% compared to control levels. 24 

### GST activity was enhanced by both Pb and mercury. Hsp25 and Hsp72 were up

- regulated 25 

### by mercury but there was no effect on Grp78 as compared to control. On the contrary, Pb 26 

### treatment only upregulated Grp78. Mercury induced a time

- dependent effect on MT 27 

### mRNA expression, which reached its maximal value 3 hours after beginning treatment 28 

### and reverted to control values at 24 hours. With Pb, on the other hand, mRNA 29 

### transcription was concentration

- and time

- dependent. The transcripts remained 30 

### overexpressed compared to controls up to 72 hours. The results of this study with regard 31 

### to the Pb effect on MT synthesis clearly differ from those of Jamieson et al. ( 2008 ) , 32 

### which found no increase in MT following Pb exposure. This discrepancy remains to be 33 

### clarified. 34February 2012 5

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### 5.5.5 Impact of Treatment with Antioxidants on Renal Lead Accumulation and Pat hology

5.5.5.1 Treatment with Antioxidants
Wang et al. ( 2010d ) assessed the protective effect of N

- acetylcysteine (NAC) on 1 

### experimental chronic Pb nephrotoxicity in immature female rats. NAC is a potent oxygen 2 

### free radical scavenger, a metal chelator, and the precursor to the antioxidant glutathione. 3 

### Sprague

- Dawley rats received Pb

- acetate (300 ppm in drinking water) and/or NAC 4 

### (100 mg /kg/day, by i.p. injection) for 8 weeks to investigate the protective effect of NAC 5 

### on Pb

- induced renal damage and oxidative stress. Serum and renal cortical Pb levels were 6 

### markedly increased in the Pb

- treated animals, but reduced in the Pb plus NAC treated 7 

### animals. There were time

- related increases in urinary alkaline phosphatase, urinary GGT, 8 

### urinary NAG, urinar y total protein, urinary β

- 2 microglobulin, and urinary microalbumin, 9 

### which were all decreased by NAC. Serum urea nitrogen was significantly increased by Pb 10 

### administration and reduced toward normal by Pb plus NAC. Alterations in proximal 11 

### tubular structures were observed in most kidney samples from Pb

- treated rats, but 12 

### animals treated with combination Pb plus NAC showed well

- preserved cell structures and 13 

### organelles. Indices of oxidative stress (MDA, SOD, GSH, GPx, and CAT) were altered 14 

### by Pb treatment and re stored to or toward normal by Pb plus NAC treatment (MDA 15 

### increased and the remainder decreased). Thus NAC was shown to have both an anti

16 

### oxidative and a chelator effect on Pb intoxication. 17


### Saxena et al. ( 2005 ) investigated the beneficial role of monoesters of meso

- 2, 3

- 18 

### dimercaptosucci nic acid in the mobilization of Pb and recovery of tissue oxidative injury 19 

### in rats. Dimercaptosuccinic acid (DMSA) is known as a Pb chelator and as an antioxidant 20 

### by virtue of its possession of thiol groups. In this study, DMSA, and two of its analogues, 21



### m onomethyl dimercaptosuccinic acid (MmDMSA) and mono

- cyclohexyl 22 

### dimercaptosuccinic acid (MchDMSA) were assessed for their capability to reduce Pb 23 

### concentration in blood and soft tissues and to recover Pb

- induced oxidative stress. Male 24 

### Wistar rats were expos ed to Pb

- acetate (0.1% in drinking water) for 20 weeks. Rats were 25 

### then treated orally once daily for five days with DMSA or its two analogues at doses up 26 

### to 100 mg /kg. Exposure to Pb caused a rise in blood Pb levels to approximately 25 μg/dL. 27 

### Exposure to P b also caused a significant decrease in blood ALAD activity and GSH 28 

### levels, accompanied by inhibition of kidney ALAD and an increase in δ

- aminolevulinic 29 

### acid syn the tase (ALAS) activity in liver and kidneys. Pb exposure also resulted in 30 

### increased blood and soft tissue (brain, liver, and kidney) Pb and TBARS levels and 31 

### decreased GSH levels. These were restored by treatment with DMSA and its analogues, 32 

### particularly MchDMSA. 33February 2012 5

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### Abdallah et al. ( 2010 ) examined the effect of Pb on coenzyme Q levels in rat tissues. 1 

### Coenzyme Q acts as an electron and proton carrier in mitochondria and functions as an 2 

### antioxidant in its reduced form (ubiquinol). Both coenzyme Q9 and coenzyme Q10 were 3 

### measured in rat tissues as coenzyme Q9 is the predominant form found in the rat. Male 4 

### albino r ats were injected i.p. with Pb

- acetate at a dose of 5 mg /kg daily for 6 weeks. No 5 

### blood Pb levels were reported. TBARS were elevated above levels in controls in serum, 6 

### liver, kidney and brain while non
- protein sulfhydryl groups (indicative of GSH) were 7 

### dec reased in serum and kidney. Both oxidized and reduced coenzyme Q9 levels were 8 

### significantly reduced in kidneys from Pb

- treated rats as contrasted to controls (mean 9 

### [SE]: respectively, 48.6 [5.6] versus 95.5 [10.1] nmol/g tissue for oxidized form and 35.4 10 

### [ 3.0] versus 61.4 [5.1] nmol/g tissue for reduced form). On the other hand, levels of 11 

### oxidized and reduced coenzyme Q10 were unchanged. Thus, the reduced levels of 12 

### coenzyme Q attributable to Pb exposure may be a factor in the diminished antioxidant 13 

### defense mechanism. 14


### El

- Sokkary et al. ( 2005 ) evaluated the effect of melatonin agai nst Pb

- induced hepatic and 15 

### renal toxicity in male rats. Melatonin is known to be efficacious as a free radical 16 

### scavenger and indirect antioxidant. Three groups of animals were used: control, 17 

### Pb

- acetate

- treated (100 ppm ) and Pb

- acetate and melatonin (10 mg / kg) given 18 

### subcutaneously for 30 days. Lipid peroxidation was measured as the sum of MDA and 4

- 19 

### hydroxyalkenals (4

- HAD). Pb increased kidney lipid peroxidation products, but these 20 

### were reduced toward normal with melatonin co

- treatment. Both SOD and GSH level s 21 

### were reduced by Pb and were increased by melatonin. Histological section of kidneys of 22 

### Pb

- treated rats showed tubular degeneration with some apparently necrotic cells, while 23 

### melatonin

- treated rats demonstrated a near normal structure. The authors conclud ed that 24 

### melatonin protected the liver and kidneys from the damaging effects of exposure to Pb 25 

### through inhibition of lipid peroxidation and stimulation of endogenous antioxidative 26 

### defense systems. 27 

### Ozsoy et al. ( 2010 ) studied the protective effects of L

- carnitine on experimental Pb 28 

### toxicity in rats. Female two month

- old rats were fed 0.5 mg /kg Pb

- acetate alone or with 29 

### daily injections of 0.5 mg /kg L

- carnitine for 60 days. Control animals were injected with 30 

### physiological saline. Pb caused an increase in serum creatinine and histopathological 31 

### changes in the kidney, consisting of tubule dilatation, degeneration and necrosis and 32 

### interstitial inflammation. In the Pb plus L

- carnitine group, serum cre atinine was reduced 33 

### to control values and the histopathological changes were reversed. Immunological 34 

### staining indicated Cu/Zn

- SOD stimulation by Pb feeding alone and reduction by L

35 

### carnitine co

- treatment. The authors attributed the beneficial effects of L

- carnitine to its 36 

### antioxidant effect. 37February 2012 5

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### Reddy et al. ( 2010 ) treated Sprague

- Dawley rats with 10 mg /kg/day of Pb

- acetate and/or 1 

### thiamine (25 mg /kg/day) for 7 weeks. Thiamine treatment normalized the Pb

- induced 2 

### alterations in blood ALAD activity and urinary NAG activity. 3 

### The effectiveness of various plant or bacterial extracts as antioxidants in the kidney was 4 

### examined in a few studies. Kharoubi et al. ( 2008a ) described the prophylactic effects of 5 

### Wormwood ( Artemisia absinthium L. ) plant extracts on kidney function in Pb

- exposed 6 

### animals. Male Wistar rats were exposed to Pb

- acetate (750 ppm in drinking water ) for 11 7 

### weeks, and then received Wormwood extract (200 mg /kg) for 4 weeks. Significant 8 

### differences in blood and urinary Pb concentration were observed between the Pb group 9 

### and the Wormwood group ( e.g., 55.6 μg/dL blood Pb versus 22.3 μg/dL, respectively). 10 

### Pb induced lipid peroxidation (TBARS and protein carbonyls in the kidney), but these 11 

### levels were reduced by Wormwood extract. Wormwood extract also attenuated the 12 

### effects of Pb on renal function. These results indicated that Wormwood extract had 13 

### significa nt antioxidant activity and protected the kidney from Pb

- induced toxicity. 14 

### Jayakumar et al. ( 2009 ) evaluated the effect of a methanolic extract of the Indian herb, 15 

### Achyranthes aspera , in preventing Pb

- induced nephrotoxicity in rats. Male albino Wistar 16 

### rats, received Pb

- acetate (0.2% for 6 weeks) or Pb

- acetate plus A. aspera (200 mg /kg for 17 

### 6 weeks) simultaneously. A. aspera partially prevented the increases in kidney weight, 18 

### BUN, serum uric acid, and serum creatinine caused by Pb administration. The levels of 19 

### urinary marker enzymes, GGT, β

- glucuronidase, NAG, Cathepsin D, and LDH, which 20 

### were reduced by Pb administration, wer e increased to or toward normal by A. aspera

. 21 

### Kidney histology revealed that Pb

- treated animals showed tubular damage, whereas the 22 

### Pb plus A. aspera

- treated animals showed a reduction in tubular damage. 23


### El

- Nekeety et al. ( 2009 ) evaluated the protective effect of an extract of the folk medicine 24 

### plant Aquilegia vulgaris against Pb

- acetate

- induced oxidative stress in Sprague

- Dawley 25 

### rats. The experimental group was treated with 200 ppm Pb

- acetate and/or 100 ppm of an 26 

### extract of A. vulgaris for 2 weeks prior to Pb

- acetate

. Pb

- acetate increased serum urea 27 

### and decreased serum total protein and albumin. These changes were reversed by 28 

### treatment with the extract. Hist ological examination of kidneys of rats treated with Pb 29 

### showed tubular dilatation, interstitial inflammatory cells, hemorrhage, cellular debris, and 30 

### hypercellularity in the glomerulus, with apoptotic nuclei in renal tubular epithelial cells. 31 

### The rats treat ed simultaneously with Pb and the extract showed essentially normal renal 32 

### tubules and glomeruli while rats treated with Pb and then the extract showed 33 

### improvement in tubular structure, but interstitial fibrosis was still present. This 34 

### experiment indicated that exposure to Pb generates free radicals, and that an extract of A. 35 

### vulgaris resulted in restoration of the different parameters tested. The second experiment 36 

### in this group was by Ponce

- Canchihuaman et al. ( 2010 ) who evaluated the antioxidant 37February 2012 5

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### activity of the cyanobacterium Spirulina maxima against Pb

- acetate

- induced 1 

### hyperlipidemia and oxidative damage in the liver and kidney of male rats. Male Wista r 2 

### rats were treated with Pb

- acetate by i.p. injection (25 mg /rat on a weekly basis for 3 3 

### weeks), and a 5% supplement of Spirulina was given in food. The findings in the kidney 4 

### were similar to those in the liver (see Section 5.9.1 ). Thus, Pb

- induced oxidative stress 5 

### and renal damage can be attenuated by treatment with Spirulina extract. 6 

### Finally, there is a need to examine whether the chelator, CaNa 2 

### EDTA, acts also as an 7 

### antioxidant and promotes increased vasodilatation and subsequent increased renal blood 8 

### flow by enhancing the delivery of NO. This question arose because of the observations of 9 

### Lin et al. ( 200 6b ) that repeated injections of CaNa 2 

### EDTA lead to improvement in kidney 10 

### function in patients with chronic renal failure, even in individuals with very low body Pb 11 

### stores as indicated by EDTA mobilization tests ( i.e., < 80 μg 72 hour urinary Pb 12 

### excretion ). Jacobsen et al. ( 2001 ) examined the anti

- oxidative effects of Gallic acid, 13 

### EDTA, and an emulsifier in mayonnaise enriched with 16% fish oil. EDTA was shown to 14 

### be an efficient antioxidant in the fish oil enriched mayonnaise as it stro ngly inhibited the 15 

### formation of free radicals and volatile oxidation compounds. The authors suggested that 16 

### the antioxidative effect appears to be due to its ability to chelate free iron in egg yolk at 17 

### the oil

- water interface. 18



### 5.5.5.2 Treatment with Antioxidants p lus Chelators

Santos et al. ( 2006a ) assessed the potentiating effects of chelators (2,3

- 19 

### dimercaptopropanol [BAL], 2,3

- dimercaptopropane

- l

- sulfonic acid [DMPS], and meso

20 

### 2,3

- di mercaptosuccinic acid [DMSA]) given simultaneously with Pb

- acetate on δ

- ALAD 21 

### activity, both in vivo and ex vivo. Ex vivo, human blood was pre

- incubated with BAL or 22 

### DMSA (10 μM) or DMPS (1 μM) then Pb

- acetate added to the reaction mixture. In vivo, 23 

### mice wer e given daily injections of 50 mg /kg Pb

- acetate for 15 days and then injected 24 

### with 1/3 of LD50 of the chelating agents. In human blood, the inhibitory effect of 25 

### Pb

- acetate (1 and 100 μM) on δ

- ALAD activity was markedly increased in the presence 26 

### of BAL and DMPS, whereas DMSA ameliorated the enzyme inhibition caused by 1 μM 27 

### Pb

- acetate . In vivo, Pb

- acetate inhibited δ

- ALAD activity by 42%. Parallel to the ex vivo 28 

### results, BAL and DMPS, but not DMSA, increased the inhibitory potency of Pb in blood. 29 

### In the kidney, BAL and DMSA but not DMPS increased inhibitory activity. The authors 30 

### conjectured that the chelators may deplete the cells of zinc, an essential element for δ

31 

### ALAD activ ity. These observed effects with chelators were supported by Bradberry and 32 

### Vale ( 2009 ) , Hamidinia et al. ( 2006 ) , and Aslani et al. ( 2010 ) who found decreased 33 

### kidney Pb content post

- chelation. 34February 2012 5

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### 5.5.6 Sum mary and Causal Determination
The 2006 Pb AQCD concluded that “in the general population, both circulating and 1 

### cumulative Pb was found to be associated with a longitudinal decline in renal function,” 2 

### evidenced by increased serum creatinine and decreased cr eatinine clearance or eGFR 3 

### over follow

- up of 4 to 15 years in association with higher baseline blood and bone Pb 4 

### levels ( U.S. EPA, 2006b )

. Data in general and patient populations of adults provided 5 

### consistent evidence of Pb

- assoc iated lower renal function in populations with mean 6 

### concurrent or baseline blood Pb levels of 2

- 10 μg/dL ( Akesson et al., 2005 ; Tsaih et al., 7 

### 2004 ; Yu et al., 2004 ; Kim et al., 1996 ) ; associations with lower eGFR were observed in 8 

### adults with hypertension with a mean concurrent blood Pb level of 4.2 μg/dL ( Muntner et 9 

### al., 2003 )

. The conclusion from the 2006 Pb AQCD was substantiated by the coherence 10 

### of effects observed across epidemiologic and toxicological studies. Both human and 11 

### animal studies observed Pb

- associated hyper filtration. In animals during the first 3 12 

### months after Pb exposure, effects were characterized by increased GFR and increased 13 

### kidney weight due to glomerular hypertrophy. However, exposure for 6 or 12 months 14 

### resulted in decreased GFR, interstitial fibrosis , and kidney dysfunction. Additionally, 15 

### toxicological studies found that early effects of Pb on tubular cells were generally 16 

### reversible, but continued exposure resulted in chronic irreversible damage. Toxicological 17 

### studies provided mechanistic evidence to support the biological plausibility of Pb

18 

### induced renal effects, including oxidative stress leading to • 

### NO inactivation. Despite the 19 

### strong body of evidence presented in the 2006 Pb AQCD, uncertainty remained on the 20 

### contribution of past Pb exposures to ass ociations observed in adults, the impact in 21 

### children, and the implication of hyperfiltration. 22 

### Recent epidemiologic studies in adult general and patient populations continue to support 23 

### Pb

- related nephrotoxicity with consistently observed associations of bl ood and bone Pb 24 

### levels with worse kidney function. These studies benefit from a number of strengths that 25 

### vary by study but include comprehensive assessment of Pb dose with measurements of 26 

### blood Pb, bioavailable Pb, and bone Pb as a biomarker of cumulative exposure; 27 

### prospective study design; and statistical approaches that utilize a range of exposure and 28 

### outcome measures, while adjusting for numerous potential confounding factors including 29 

### age, race, sex, education, household income, smoking, alcohol use, ca dmium exposure, 30 

### and various health indicators such as diabetes, SBP, BMI, and history of cardiovascular 31 

### disease. Large sample sizes provide strength to the general population studies. Re

32 

### examination of a study from the 2006 Pb AQCD provided data to conclud e that a 10

- fold 33 

### increase in concurrent blood Pb ( e.g., from 1 to 10 μg/dL) was associated with an 18 34 

### mL/min decrease in estimated creatinine clearance or a 25% decrease from the mean, and 35 

### that an increase in blood Pb from the 5th to the 95th percentile (3 .5 μg/dL) had the same 36 

### negative impact on eGFR as did an increase of 4.7 years in age or 7 kg/m 2 

### in body mass 37February 2012 5

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### index ( Akesson et al., 2005 )

. At current blood Pb levels in the U.S. adult population, a 1 

### downward shift in kidney function of the entire population due to Pb may not result in 2 

### CKD in identifiable individuals; however , that segment of the population with the lowest 3 

### kidney reserve may be at increased risk for CKD when Pb is combined with other kidney 4 

### risk factors. For example, in adults with mean (concurrent or baseline measured 4

- 6 years 5 

### before kidney function tests) b lood Pb levels that are comparable to that of the general 6 

### U.S. population (1.6 to 4.2 μg/dL), higher blood Pb level was found to be associated with 7 

### clinically

- relevant effects ( e.g., eGFR < 60 mL/min/1.73 m 2 

### , doubling of serum 8 

### creatinine) ( Fadrowski et al., 2010 ; Yu et al., 2004 ) and larger magnitudes of effect in 9 

### potentially at

- risk populations with cormorbidities for CKD such as diabetes mellitus 10 

### ( Tsaih et al., 2004 ) and hypertension ( Tsaih et al., 2004 ; Muntner et al., 2003 ) or higher 11



### co
- exposure to other environmental nephrotoxicants such as cadmium ( Navas

- Acien et 12 

### al., 200 9 )

. 13 

### Recent NHANES analyses added to the evidence for Pb

- associated lower renal function 14 

### in populations with low concurrent mean blood Pb levels (< 2 μg/dL) ( Fadrowski et al., 15 

### 2010 ; Navas

- Acien et al., 2009 )

. However, because of uncertainties concerning the 16 

### magnitude, timing, frequency, and duration of Pb exposure that contributed to the 17 

### observed associations, it is difficult to assess whether a threshold exists for Pb

- related 18 

### renal effects. 19


### Research in the occupational setting has traditionally been far less consistent than that in 20 

### environmentally exposed populations ( Section 5.5.2.2 ). A number of explanatory factors 21 

### for this inconsistency, all due to limitations of the occupational literature, were discussed 22 

### in the 2006 Pb AQCD. The observation of paradoxical or inverse associations (higher Pb 23 

### dose with lower serum creatinine, and/or higher eGFR o r calculated or measured 24 

### creatinine clearance) in several of these studies reflects limitations inherent in the study 25 

### design. Irrespective of the mechanism, these associations have risk assessment 26 

### implications. If associations are in opposite directions in different subgroups of the 27 

### population and the relevant effect modifier is not considered, null associations will be 28 

### observed. For these reasons, nonsignificant associations or paradoxical associations in the 29 

### occupational setting cannot be used as a ration ale for discounting Pb

- related 30 

### nephrotoxicity at lower environmental levels. 31 

### Important data on the effects of Pb on the kidney in children were reported in a recent 32 

### NHANES analysis in adolescents, ages 12

- 20 years, which observed an association 33 

### between hig her concurrent blood Pb (mean: 1.5 μg/dL) and lower cystatin C

- based eGFR 34 

### ( Fadrowski et al., 2010 )

. These find ings are consistent with results from a rodent model 35 

### study in which a low dose of Pb (50 ppm ) administered from birth resulted in renal 36 

### impairment (elevated serum creatinine as compared to control rats), but these 37February 2012 5

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### observations require confirmation by measu rement of GFR and renal pathology ( Berrahal 1 

### et al., 2011 )

. This recent epidemiologic study along with several previous studies that 2 

### included children with higher Pb exposures (due to residence near sources, Pb poisoning, 3 

### or parental occupational exposure) provide evidence that renal function in children may 4 

### be affected by Pb exposure; however, additional research is warranted. It is important to 5 

### recognize that the NHANES adolescents from Fadrowski et al. ( 2010 ) likely had higher 6 

### Pb exposures earlier in childhood, thus, the magnitude, timing, frequency, and duration of 7 

### Pb exposure contributing to the observed association is uncertain. 8 

### CKD results in substantial morbidity and mortality, and, even at earlier stages than those 9 

### requiring kidney dialysis or transplantation, is an important risk factor for cardiac disease. 10 

### As kidney dysfunction can increase BP and increased BP can lead to further damage to 11 

### the kidneys, Pb

- induced damage to either or both renal or cardiovascular systems may 12 

### result in a cycle of further increased severity of disease. Pb exposure has b een causally 13 

### linked to both increased BP and other cardiovascular effects ( Section 5.4 ). Interestingly, 14 

### animal studies have shown Pb

- induced vascula r injury in the kidney was associated with 15 

### increased glomerular sclerosis, tubulointerstitial injury, increased collagen staining, and 16 

### an increase in macrophages associated with higher levels of MCP

- 1 mRNA. It is possible 17 

### that the cardiovascular and renal effects of Pb observed are mechanistically linked and 18 

### are contributing to the progression of the diseases. 19 

### Recently available animal toxicological studies strengthen the evidence regarding the 20 

### modes of action for Pb exposure leading to renal alterations, including the influence of 21 

### Pb

- induced oxidative stress. The mode of action of Pb in the kidneys has been extended 22 

### to the field of immunology, evidenced by observations that Pb exposure resulted in 23 

### infiltration of lymphocytes and macrophages associated with increased expression of NF

24 

### κB in proximal tubules and infiltrating cells ( Roncal et al., 2007 )

. Additionally, recent 25 

### findings expand on the evidence of acute effects of Pb, including mitochondrial 26 

### dysfunction, renal cell apoptosis, and glomerular hyper trophy. These mechanisms are 27 

### useful in understanding the occurrence of acute hyperfiltration followed by chronic 28 

### kidney dysfunction. As indicated in Figure 5

- 38 and Table 5

- 22 , studies found 29 

### dysfunction in various kidney function measures, including urinary flow, ALP, 30 

### microalbumin, and NAG in animals with blood Pb levels > 20 μ g/dL ( Wang et al., 31 

### 2010d )

. Lower concentration Pb exposures and lower blood Pb levels in animals have not 32 

### been examined widely. 33 

### In summary, new epidemiologic and toxicological studies evaluated in the current review 34 

### support or expand upon the strong body of evidence presented in the 2006 Pb AQCD 35 

### indicating that Pb exposure is associated with renal effects. The weight of epidemiolog ic 36 

### evidence demonstrates consistently a relationship between higher blood Pb level and 37February 2012 5

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### kidney dysfunction ( e.g., lower creatinine clearance, higher serum creatinine, and lower 1 

### GFR) in nonoccupationally

- exposed adults with mean concurrent or baseline blood Pb 2 

### levels of 2

- 10 μ g/dL. A few analyses find higher blood Pb levels to be associated with a 3 

### greater longitudinal decrease in kidney function over time (4

- 15 years), suggesting that 4 

### past Pb exposures may contribute to ongoing renal effects. The epidemiologi c evidence is 5 

### strength en ed by associations between Pb biomarker levels and r enal function that were 6 

### observed after adjustment for multiple potential confounding factors such as age, sex, 7 

### comorbid cardiovascular conditions, BMI, smoking, and alcohol use. Si milar findings 8 

### among adults with CKD indicate that in populations with pre

- existing renal disease, Pb 9 

### exposure may be associated with greater progression of disease. Because blood Pb level 10 

### in nonoccupationally

- exposed adults reflects both recent and past P b exposures, the 11 

### magnitude, timing, frequency, and duration of Pb exposure contributing to the observed 12 

### associations is uncertain. Coherence for epidemiologic findings is provided by 13 

### observations in animal models that Pb exposure for greater than 6 months decreases GFR 14 

### and increases serum creatinine. The weight of evidence in animal studies indicates Pb

15 

### induced histopathological changes, including tubular atrophy and sclerosis. Overall, 16 

### reduced renal function and increased kidney damage in animals are obser ved with 17 

### chronic Pb (> 4 weeks) exposure that result in blood Pb levels > 20 μ g/dL. By 18 

### demonstrating Pb

- induced renal oxidative stress, inflammation, mitochondrial 19 

### dysfunction, apoptosis, and glomerular hypertrophy, toxicological studies provide 20 

### biological plausibility for the associations observed in epidemiologic studies between 21 

### blood Pb levels and kidney dysfunction. Collectively, the evidence integrated across 22 

### epidemiologic and toxicological studies as well as across the spectrum of renal outcomes 23 

### is su fficient to conclude that there is a causal relationship between Pb exposures and renal 24 

### health effects. 25



## 5.6 Immune System Effects

5.6.1 Introduction
With respect to studies conducted in laboratory animal and in vitro models, the immune 26 

### effects of Pb exposure have be en extensively examined over several decades. Animal 27 

### studies of the effects of Pb exposure on host resistance date back to the 1960s while those 28 

### focusing on Pb

- induced immune functional alterations, including developmental 29 

### immunotoxicity, were first conduc ted during the 1970s. Despite this long history of 30 

### research, Pb

- associated immune effects in animals with blood Pb levels in the range of 31 

### current U.S. population levels ( i.e., <10 μ g/dL), particularly early in life, have been 32 

### observed only relatively recen tly within the last 10

- 15 years ( Dietert and McCabe, 2007 )

. 33February 2012 5

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### Over the last 10

- 15 years, advances in the understanding of Pb

- associated changes in 1 

### immunological parameters in humans have substantiated the immune effects of Pb. 2 

### The 2006 Pb AQCD presented consistent evidence fo r immune system effects associated 3 

### with Pb exposure ( U.S. EPA, 2006b )

. Rather than producing overt cytotoxicity or 4 

### pathology, Pb exposure was found to be associated with alterations in several subclinical 5 

### parameters related to ce llular and humoral immunity ( Figure 5

- 42 ). These conclusions 6 

### were based most heavily on toxicological evidence in animals and in vitro models for the 7 

### direct effects of Pb exposure in inducing changes in a wide spectrum of immune 8 

### outcomes, but principally, a shift away from T

- derived lymphocyte helper (Th)1 9 

### cytokines toward Th2 cytokines, suppression of Th1

- dependent delayed type 10 

### hypersensitivity (DTH), elevation in Th2

- driven immunoglobulin E (IgE), and 11 

### modulation of macrophages into a hyperinflammatory phenotype. Relatively fewer 12 

### studies were available in humans and for fewer immune

- related endpoints. A majority of 13 

### studies in humans were conducted in Pb

- exposed male workers, and while a larger 14 

### number of immune outcomes were examined, the most consistent evidence comprised 15 

### effects on neutrophil functionality. In a smaller body of studies of humans without 16 

### occupational exposures, the weight of evidence ind icated associations of higher blood Pb 17 

### levels with lower abundance of T lymphocyte cells and, in concordance with 18 

### toxicological studies, higher IgE levels in children. Due to limited examination, the 19 

### immune effects of Pb exposure in adults without occupati onal exposures were not well 20 

### characterized. 21February 2012 5

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### Figure 5
- 42 Immunological pathways by which Pb exposure may increase risk of immune
- related diseases.
The pathways by which Pb exposure may alter immune cell func tion and consequently 1 

### increase the risk of immune

- related diseases are presented in Figure 5

- 42 . Both 2 

### toxicological and epidemiologic studies of childr en indicated Pb exposure effects on T 3 

### cells. A large body of toxicological evidence demonstrated Pb

- induced effects on 4 

### macrophages. Neutrophil functionality was found to be reduced with higher Pb exposure, 5 

### based on studies of Pb

- exposed workers with mean b lood Pb levels > 40 μ g/dL. 6 

### Alterations in immune cells can lead to changes in cell

- to

- cell interactions, multiple 7 

### signaling pathways, and inflammation that affect both innate and acquired immunity, that 8 

### in turn, influence the risk of developing infectious, allergic and autoimmune diseases as 9 

### well as exacerbating inflammatory responses in other organ systems. Studies conducted 10 

### in animal and in vitro models provided consistent evidence for Pb exposure inducing 11 

### effects on the range of immune effects presented in this continuum. Among the hallmarks 12 

### reported for Pb

- induced changes in functional pathways were: (1) a suppression of T

13 

### derived lymphocyte helper (Th)1

- driven cell

- mediated immunity (as measured by a DTH 14 

### response); (2) an increase in Th2

- driven IgE anti body and Th2 cytokine production; and 15 Increased risk of atopy and allergic disease 

# Pb

Macrophages and Other Innate Immune Cells

Reduced: Phagocytosis Nitric oxide production Peroxynitrite production Lysosomal activity

Reduced host resistance to bacterial infection Elevated TNF

- α O verproduction of ROS Depleted antioxidant defenses

Damaged epithelia and mucosal barriers Tissue damage and de novo antigen appearance Removal of normal myelomonocytic suppression

Inappropriate T cell proliferation activation

Increased risk of autoimmunity Increased tissue inflammation (e.g. lung, gut, skin) Dendritic Cells

Elevated IL

- 10 Suppressed IL

- 12 T Cells

Elevated IL

- 4 and IL

- 5 Suppressed INF

- γ

Skewed Th2

- biased responses

Increased IgE production

Increased risk of later

- life cancer Suppressed Th1

- mediated anti

- tumor host defense Increased lipid and DNA oxidation in tissues

Increased tumor cell formation B Cells

Increased risk of tissue inflammatory diseasesFebruary 2012 5

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### (3) a pro

- inflammatory shift in macrophage function. The latter was characterized by 1 

### increased production of reactive oxygen species (ROS), prostaglandin E 2 

### (PGE 2 

### ), and 2 

### inflammatory cytokines such as tumor necrosis fac tor

- α lpha (TNF

- α) and interleukin 3 

### (IL)

- 6 and decreased production of IL

- 12 and nitric oxide (NO). While epidemiologic 4 

### studies did not provide evidence for this full spectrum of immune effects, a shift to a Th2 5 

### phenotype was indicated by associations observ ed between higher blood Pb level and 6 

### higher serum IgE levels in children. 7 

### Reflecting an inhibition of Th1 activity, toxicological evidence presented in the 2006 Pb 8 

### AQCD linked Pb exposure of animals to impaired host resistance to bacteria ( U.S. EPA, 9 

### 2006b )

. Indicating a hyperinflammatory state and local tissue damage, a few available 10 

### toxicological studies found Pb exposure

- induced generation of autoantibodies, suggesting 11 

### an elevated risk of autoimmune reactions. Additionally, the shift toward a Th2 response 12 

### suggested that Pb could elevate the risk of atopy and allergic responses. While 13 

### toxicological evidence for Pb

- induced Th2 activity, elevated IgE, and inflammation 14 

### supported the biological plausibility of such effects, epidemiologic evidence was too 15 

### sparse to draw conclusions regarding associations between blood Pb levels and these 16 

### broader indicators of immune dysfunction in humans. 17 

### Changes in the spectrum of immune endpoints were associated with a wide range of 18 

### blood Pb levels. Several toxicological studies found blood Pb levels in the range of 7

- 19 

### 100 μ g/dL to be associated with juvenile and/or adult immune effects ( e.g., suppressed 20 

### DTH, elevated IgE, changes in cytokine levels). Most epidemiologic studies examined 21 

### and found lower T cell abundance and higher serum IgE levels in association with 22 

### population mean (or quantiles) concurrent blood Pb levels > 10 μ g/dL. 23 

### With respect to critical lifestages of Pb exposure, animal studies provided strong 24 

### evidence for immune effects induced by prenatal Pb exposures and b y postnatal 25 

### exposures in adult animals. There was uncertainty regarding critical lifestages of Pb 26 

### exposure in humans as epidemiologic studies of children primarily were cross

- sectional 27 

### in design and examined concurrent blood Pb levels. Several other limita tions of 28 

### epidemiologic studies were noted, including small sample sizes; inconsistent adjustment 29 

### for potential confounders such as age, sex, smoking, and comorbid conditions; and 30 

### reliance on comparisons of immune endpoints among groups with different blood Pb 31 

### levels, which provided limited information on the concentration

- response function. 32 

### Collectively, the small numbers of toxicological and epidemiologic studies published 33 

### since the 2006 Pb AQCD supported the previous findings of Pb

- associated immune 34 

### effec ts. Epidemiologic studies supported previous findings in children and provided new 35 

### evidence for effects in nonoccupationally

- exposed adults. Recent studies also expanded 36 

### on the array of immunological parameters affected by Pb exposure as presented in Figure 37February 2012 5

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– Do Not Cite or Quote 

### 5

- 42 . For example, a new toxicological study indicated that Pb may modulate the 1 

### function of dendritic cells. Results from new toxicological and epid emiologic studies 2 

### strengthened the link between Pb

- associated effects on immune cells and immune

- and 3 

### inflammatory

- based diseases by providing evidence for changes in intermediary signaling 4 

### and inflammatory pathways ( Figure 5

- 42 ). Several new epidemiologic studies examined 5 

### signaling molecules such as pro

- inflammatory cytokines and NO to produce findings 6 

### parallel with toxicological studies. New toxicolog ical studies expanded knowledge of the 7 

### broader role of Pb

- associated immune modulation in mediating Pb effects in 8 

### nonlymphoid tissues ( e.g., in the nervous, reproductive, and respiratory systems). 9 

### Although primarily cross

- sectional in design, recent epidem iologic studies improved on 10 

### the design of earlier studies through greater examination of children and adults with 11 

### blood Pb levels more comparable to contemporary levels in the U.S. population and 12 

### greater consideration of confounding by age, sex, smoking, c omorbid conditions, and 13 

### SES

- related variables. 14



### 5.6.2 Cell
- Mediated Immunity

5.6.2.1 T Cells
A majority of the evidence indicating effects of Pb exposure on T cells was provided by 15 

### older toxicological and epidemiologic studies reviewed in the 2006 Pb AQCD ( U.S. 16 

### EPA, 2006b )

. Toxicological studies demonstrated Pb exposure

- induced shifts in the 17 

### partitioning of CD4+ (T helper) cell populations to favor Th2 cells (10

- 100 μ M in vitro) 18 

### and the production of Th2 cytokines and to suppress production of T h1 cytokines (wide 19 

### range of Pb exposures) ( Section 5.6.5.4 ). Previous epidemiologic findings were limited 20 

### largely to associations between higher concurrent blood Pb level and lower T cell 21 

### abundance in children. 22 

### Consistent with previous epidemiologic findings, in a recent study of 7 week

- old Wistar 23 

### rats administered 200 ppm Pb

- acetate , the percentages of CD4+ and CD8+ cytotoxic T 24 

### cells were decrease d (with CD4

- CD8

- cells elevated) in the submaxillary lymph nodes, 25 

### but only with i.p. Pb dosing (p <0.05) and not oral exposure (200 ppm Pb

- acetate both 26 

### routes of administration) ( Teijon et al., 2010 )

. Limited recent toxicological investigation 27 

### added mechanistic information by indicating that Pb may induce a shift to Th2 responses 28 

### via T cell

- dependent and

- independent pathways. Previously, in cultures of human CD4+ 29 

### T cells, Pb (1 μ M, 30 minute s) was shown to activate transcription factor NF

- κB 30 

### (regulates T cell activation) ( Pyatt et al., 1996 ) and to increase, in a concentration

31 

### dependent manner (10 and 50 μ M PbCl 2 

### , 24 hours), the expression of MHC class II 32February 2012 5

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### surface antigens ( e.g., HLA

- DR), which mediate the CD4+ response to exogeno us 1 

### antigens ( Guo et al., 1996b )

. Recent studies examined higher Pb exposure concentrations 2 

### than did previous studies. Heo et al. ( 2007 ) recently showed that Pb (25 μ M) blocked 3 

### production of the Th1 cytokine interferon

- γ (I FN

- γ) in cultures of stimulated mouse T 4 

### cells not by affecting gene expression but by suppressing translation of the protein. This 5 

### blockage was rescued with the addition of IL

- 12, which promotes Th1 activity. These 6 

### results demonstrated a T cell

- dependent p athway to skewing toward Th2 responses. 7 

### However, Pb was able to decrease the IFN

- γ to IL

- 4 ratio (indicating a shift to Th2) in the 8 

### absence of STAT6, the preferential signaling pathway for T cells. Thus, Pb also was 9 

### found to skew toward Th2 responses via a T cell

- independent pathway. Similar 10 

### observations were made in vivo by Kasten

- Jolly et al. ( 2010 )

. In this study, 11 

### developmental Pb exposure of mice (100 μ M Pb

- acetate in drinking water of dams from 12 

### GD8 to PND21, resulting in pup blood Pb levels of 10

- 30 μ g/dL) induced gene 13 

### expression of IL

- 4 and suppressed production of IFN

- γ in splenic cells. These changes 14 

### occurred in the absence of STAT4 or STAT6 and occurred with concomitant increases in 15 

### adenylate cyclase 8 and phosphatidy linositol 3

- kinase, adding to the evidence that Pb 16 

### may promote Th2 activity via T cell

- independent pathways. 17 

### While a few available recent epidemiologic studies found associations of blood Pb levels 18 

### with lower levels of Th1 cytokines and higher levels of Th 2 cytokines in humans 19 

### ( Section 5.6.5.4 ), the extant evidence for effects on T cells in humans is derived largely 20 

### from older studies describing diffe rences in the abundance of several T cell subtypes that 21 

### mediate acquired immunity responses to antigens. In most studies of children, higher 22 

### blood Pb levels were associated with lower T cell abundance, primarily CD3+ cells. Such 23 

### associations were observed in studies that adjusted for potential confounding factors (as 24 

### described below) and studies that compared mean cell abundances among groups with 25 

### different blood Pb levels. Blood Pb level was less consistently associated with lower 26 

### abundance of other T cell subtypes such as CD4+ and CD8+. 27 

### The weight of evidence supported lower T cell abundance in association with concurrent 28 

### blood Pb level and in groups of children with levels > 10 μ g/dL ( Zhao et al., 2004 ; 29 

### Sarasua et al., 2000 ; Lutz et al., 1999 )

. Associations were less consistent in comparisons 30 

### of children with lower blood Pb levels. In analyses of 331 children in Germany, Karmaus 31 

### et al. ( 2005 ) found that children (ages 7

- 10 years) with concurrent blood Pb levels 2.2

- 32 

### 2.8 μ g/dL (2nd quartile) had a 9 to 11% lower abundance of several T cell subtypes (p < 33 

### 0.05, t

- test) compared with children with blood Pb levels < 2.2 μ g/dL (lowest quartile). 34 

### Unlike other studies in children, Karmaus et al. ( 2005 ) considered potential c onfounding 35 

### by adjusting for sex, age, number of infections in the past 12 months, passive smoke 36 

### exposure, serum lipids, and serum organochlorine levels. Other studies that examined 37February 2012 5

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### blood Pb levels < 10 μ g/dL (population mean < 2 μ g/dL or quantile 5

- 9 μ g/dL ) did not 1 

### find associations with T cell abundance ( Hegazy et al., 2011 ; Belles

- Isles et al., 2002 )

. 2 

### Another study of U.S. (multiple unspecified locations) children that considered 3 

### confounding also found an association between higher concurrent blood Pb level and 4 

### lower T cell abundance; however, it was limited to the youngest subjects ( Sarasua et al., 5 

### 2000 )

. Among 241 children 6

- 35 months in age, a 1 μ g/dL higher blood Pb level was 6 

### associated with a 0.18% (95% CI:

- 0.34,

- 0.02) lower CD3+ cell count, a 0.10% (95% CI: 7



### - 0.24, 0.04) lower CD4+ cell count, and a 0.04% (95% CI:

- 0.15, 0.07) lower CD8+ cell 8 

### count, adjusting for city of residence, age, and sex. In older age groups (36

- 71 months, 6

- 9 

### 15 years), many effect estimates were positive. Analysis of blood Pb level categories 10 

### indicated that associations were driven by lower T cell abundance (3

- 6%) among children 11 

### 6

- 35 months in age with blood Pb levels > 15 μ g/dL. It is important to note that 76% of 12 

### subjects lived near a Pb smelting operation. These subjects living near Pb sourc es likely 13 

### had higher blood Pb levels and may have driven the observed associations. Neither 14 

### Karmaus et al. ( 2005 ) nor Sarasua et al. ( 2000 ) found a monotonic decrease in T cell 15 

### abundance across blood Pb level groups. Neither of these studies adjusted for SES, which 16 

### has been associated with blood Pb levels and immune

- related conditions such as asthma, 17 

### allergy, and viral infections. However, it is difficult to assess the potential for 18 

### confounding by SES as neither study reported the SES characteristics of the study 19 

### population. 20 

### In the limited investigation of nonoccupationally

- exposed adults, higher concurrent blood 21 

### Pb levels were associated with higher T cell abundance ( Boscolo et al., 2000 ; Sarasua et 22 

### al., 2000 ; Boscolo et al., 1999 ) ; how ever, o ccupational studies conducted in the U.S. and 23 

### Asia did not find Pb

- exposed workers consistently to have lower or higher abundance of 24 

### T cells ( Mishra et al., 2010 ; Pinkerton et al., 1998 ; Yucesoy et al., 1997b ; Undeger et al., 25 

### 1996 ; Fischbein et al., 1993 )

. Some studies found that compared with unexposed 26 

### controls, Pb

- exposed workers had a lower ratio of CD4+/CD8+ cells ( Mishra et al., 2010 ; 27 

### Fischbein et al., 1993 )

. In particular, Fischbein et al. ( 1993 ) found this lower ratio in New 28 

### York area firearms instructors with a relatively lower mean blood Pb level of 14.6 μ g/dL 29 

### and after adjusting for age and smoking. Changes in the CD4+/CD8+ ratio have not be en 30 

### examined in populations with lower blood Pb levels. 31 

### Although several epidemiologic studies have found lower T cell abundance in association 32 

### with higher blood Pb levels, after adjusting for a range of potential confounding factors, it 33 

### is not clear what e ffects these small magnitudes of change may have on the cell

- to

- cell 34 

### interactions that mediate downstream acquired immune responses. In children, groups 35 

### with higher blood Pb level had lower CD3+ cell abundance that ranged between 1 and 36 

### 9%. Larger decreases (20

- 35%) were observed in studies of occupationally

- exposed 37February 2012 5

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### males with higher blood Pb levels than those found in the general U.S. population (means 1 

### 15 and 75 μ g/dL) ( Undeger et al., 1996 ; Fischbein et al., 1993 )

. 2 

### In summary, a majority of the evide nce indicating effects of Pb ex osure on T cells was 3 

### provided by previous toxicological and epidemiologic studies reviewed in the 2006 Pb 4 

### AQC D. Previous toxicological studies demonstrated Pb

- induced expansion of Th2 cells 5 

### and increased Th2 cytokine production. A small number of new toxicological studies 6 

### expanded the evidence by describing that Pb

- induced Th2 skewing may occur via T cell

7 

### depende nt pathways ( Heo et al., 2007 ) and

- independent pathways ( Kasten

- Jolly et al., 8 

### 2010 ; Heo et al., 2007 )

. Previous studies did not provide consistent evidence that higher 9 

### blood Pb levels or Pb exposure was associated with lower T cell abundance in 10 

### nonoccupational ly

- exposed or occupationally

- exposed adults, respectively. A new study 11 

### of children did not find children with higher blood Pb level to have lower T cell 12 

### abundance ( Hegazy et al., 2011 )

. Epidemiologic findings were limited largely to 13 

### associations between higher concurrent blood Pb level (> 10 μ g/dL) a nd lower T cell 14 

### abundance observed in previous studies of children ( Karmaus et al., 2005 ; Zhao et al., 15 

### 2004 ; Sarasua et al., 2000 ; Lutz et al., 1999 )

. 16



### 5.6.2.2 Lymphocyte Activation
A majority of the evidence indicating that Pb exposure stimulates lymphocyte activation 17 

### is provided by previous toxicological studies in which exposures to high concentrations 18 

### of Pb (10

- 100 μ M) induced an expansion of alloreactive B and T lymphocytes ( U.S. 19 

### EPA, 2006b )

. Lymphocyte act ivation occurs as a result of reversing the normal 20 

### suppression that is mediated by a macrophage

- like subpopulation. In the limited recent 21 

### investigation of Pb

- induced lymphocyte activation, toxicological studies characterized 22 

### potential mechanisms underlying this effect. Gao et al. ( 2007 ) described a potential role 23 

### for dendritic cells. Dendritic cells that matured in the presence of 25 μ M PbCl 2 

### promoted 24 

### enhanced alloreactive T cell prolifer ation compared to control dendritic cells. In addition, 25 

### using the local lymph node assay (LLNA), Carey et al. ( 2006 ) found that PbCl 2 

### (injection 26 

### doses 25

- 50 μ g) was able to provide a costimulatory signal to antigens that could activate 27 

### T cells in adult female mice. The exact mechanistic basi s for this is not known. As 28 

### discussed in Section 5.6.5.2 , changes in NO production appear to be involved in Pb

29 

### induced lymphocyte activation ( Farrer et al., 2008 )

. 30 

### The available epidemiologic evidence for Pb

- associat ed lymphocyte activation was 31 

### provided by a small number of previous studies in children and nonoccupationally

32 

### exposed adults. Instead of directly measuring lymphocyte proliferation, these studies 33 

### provided indirect evidence by measuring the abundance of cel ls that expressed HLA

- DR, 34February 2012 5

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– Do Not Cite or Quote 

### an indicator of activated cells. It is important to note that HLA

- DR+ cells also may 1 

### indicate the presence of activated monocytes. Further, these studies did not adjust 2 

### extensively for potential confounding. In the only study of c hildren (ages 9 months

- 6 3 

### years, Missouri), the mean percentage of HLA

- DR+ cells was approximately 2

- fold 4 

### higher (p > 0.05, Kruskal

- Wallis) in the 19 children with concurrent blood Pb levels 15

- 5 

### 19 μ g/dL than in children with blood Pb levels 10

- 14 μ g/dL (n = 61) or < 10 μg/dL 6 

### (n = 178) after adjusting for age ( Lutz et al., 1999 )

. However, activated cells were not 7 

### elevated in children with blood Pb levels 20

- 44 μ g/dL. Studies of nonoccupationally

8 

### exposed adults in Italy found that concurrent bl ood Pb level was correlated positively 9 

### with the percentage of HLA

- DR expressing cells in men with and without allergies 10 

### (Spearman r = 0.51, p < 0.002, n = 17 each, median blood Pb level both groups 11 

### combined: 11 μ g/dL) ( Boscolo et al., 1999 ) but only in women without allergies 12 

### (Spearman r = 0.44, p < 0.05, n = 25, median blood Pb level: 5.5 μ g/dL) ( Boscolo et al., 13 

### 2000 )

. 14 

### Comparisons of Pb

- exposed workers and unexposed controls indicated similar levels of 15 

### lymphocyte proliferation (≤ 1% differ ence) between groups ( Queiroz et al., 1994b ; 16 

### Cohen et al., 1989 ) or lower lymphocyte proliferation among Pb

- exposed workers (8

- 17 

### 25%) ( Mishra et al., 2003 ; Fischbein et al., 1993 ; Alomran and Shleamoon, 1988 ; Kimber 18 

### et al., 1986 ) Toxicological studies have demonstrated the selective expansion of Th2 cells 19 

### and suppression of Th1 cells ( U.S. EPA, 2006b )

. Therefore, the differential activation of 20 

### specific subtypes may not be discernable in epidemiologic studies that measure overall 21 

### lymphocyte proliferation. Additionally, because occupational studies did not provide 22 

### concentration

- response information, it is difficult to infer whether findings apply to 23 

### populations with lower blood Pb levels. Toxicological studies also did not provide 24 

### information on Pb

- induced lymphocyte activation at exposure concentrations relevant to 25 

### humans without occupational Pb exposures. 26



### 5.6.2.3 Delayed
- type Hypersensitivity
Although recent investigation was limited, a large body of previous toxicological studies 27 

### reviewed in the 2006 Pb AQCD ( U.S. EPA, 2006b ) and recent reviews ( Mishra, 2009 ; 28 

### Dietert and McCabe, 2007 ) identified a suppressed DTH response as one of the most 29 

### consistently observed and well

- documented immunomodulatory effects of Pb exposure in 30 

### animal models. A new study indicated th at this effect may be mediated by dendritic cells. 31 

### The DTH assay is commonly used to assess the T cell

- mediated adaptive immune 32 

### response, i.e., induration and erythema resulting from the activation of T cells and 33 

### recruitment of monocytes to the site of ant igen deposition. The DTH response is largely 34 

### Th1

- dependent in that Th1 cytokines drive the production of antigen

- specific T cells 35February 2012 5

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### directed against the antigen (sensitizing phase) and the recruitment of antigen

- specific T 1 

### cells and monocytes to the site of antigen deposition (elicitation phase). 2 

### Previous toxicological studies demonstrated suppressed DTH responses in animals after 3 

### gestational ( Chen et al., 2004 ; Bunn et al., 2001a ; Bunn et al., 2001b , c ; Lee et al., 2001b ; 4 

### Chen et al., 1999 ; Miller et al., 1998 ; Faith et al., 1979 ) and postnatal ( McCabe et al., 5 

### 1999 ; Laschi

- Loquerie et al., 1984 ; Muller et al., 1977 ) Pb exposures. Blood Pb level 6 

### data were not available in all studies; however, DTH was suppressed in animals with a 7 

### wide range of blood Pb levels (11 to > 100 μ g/dL), with the associations of DTH with 8 

### lower blood Pb levels occurring as a result of gestational exposure. 9 

### In some studies that examined Pb exposures at multiple stages of gestation, exposures 10 

### later in gestation suppressed DTH in animals ( Bunn et al., 2001c ; Lee et al ., 2001b )

. 11 

### These latter findings may reflect the status of thymus and T cell development. A recent 12 

### study contributed to the robust evidence by indicating a role for dendritic cells in the Pb

13 

### induced suppression of the DTH response. Gao et al. ( 2007 ) exposed bone marrow

14 

### derived dendritic cells in vitro to PbCl 2 

### (25 μ M, 10 days) then the antigen ovalbumin 15 

### (OVA) and injected the cells into naïve adult mice. Mice treated with Pb

- ex posed 16 

### dendritic cells had a diminished OVA

- specific DTH footpad response compared with 17 

### mice treated with non Pb

- exposed dendritic cells. 18 

### The capability of Pb to suppress the DTH response is strongly supported by mechanistic 19 

### studies in which Pb suppresses T h1 cytokine production ( Section 5.6.5.4 ). In some 20 

### animal studies, the suppressed DTH response was accompanied by a decreased 21 

### production of IFN

- γ ( Lee et al., 2001b ; Chen et al., 1999 ) , which i s the primary cytokine 22 

### that stimulates recruitment of macrophages, a key component of the DTH response. 23 

### Observations of a concomitant decrease in IFN

- γ further link Pb

- induced inhibition of 24 

### Th1 functional activities with suppression of the DTH response. Fu rther, coherence is 25 

### provided by associations observed between Pb exposure or blood Pb levels and other 26 

### responses in animals related to the inhibition of Th1

- driven adaptive immune responses, 27 

### including decreased host resistance ( Section 5.6.4.1 ). 28



### 5.6.2.4 Macrophages and Monocytes
As reported in the 2006 Pb AQCD, based on a large body of toxicological evidence, Pb

29 

### induced promotion of a hyperinflammatory phen otype in macrophages was considered to 30 

### be a hallmark of Pb

- associated immune effects ( U.S. EPA, 2006b )

. Pb

- induced 31 

### hyperinflammation was indicated by the enhanced production of ROS, suppressed 32 

### production of NO, enhanced productio n of TNF

- α, excessive metabolism of arachidonic 33 

### acid into immunosuppressive metabolites ( e.g., PGE 2 

### ), impaired growth and 34February 2012 5

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– Do Not Cite or Quote 

### differentiation of cells, and potentially altered receptor expression [ e.g., toll

- like 1 

### receptors]). Several of these findings are described in detail in Sections 5.6.5.2 and 2 

### 5.6.5.4 . Because macrophages are major resident populations in most tissues and organs 3 

### and also are highly mobile in response to microbial signals and tissue alterations, their 4 

### functional impairment in response to Pb exposure may serve as a link betwe en Pb

5 

### induced immune effects and impaired host defense, tissue integrity, and organ 6 

### homeostasis in numerous physiological systems ( Section 5.6.4.5 ). A small number of 7 

### available recent studies continue to support previous findings. A study in Pb

- treated 8 

### (40 ppm by oral gavage, once daily for 40 days) adult mice reported decreased 9 

### macrophage yield and viability, and phagocytic index in the kidney ( Lodi et al., 2011 )

. In 10 

### addition, several studies reinforced the effect of Pb exposure to alter a broad spectrum of 11 

### functional alterations in human monocytes (the blood form of macrophages) or mouse 12 

### macrophages in vitro ( Khan et al., 2011 ; Bussolaro et al., 2008 ; Mishra et al., 2006a )

. In 13 

### particular, Bussolaro et al. ( 2008 ) found reduced phagocytic activity with a relatively low 14 

### concentration of Pb exposure (0.2 μ M Pb nitrate, 72 hours). Khan et al. ( 2011 ) found that 15 

### exposure of human monocytes to 25

- 100 μ M Pb

- acetate for 3

- 6 hours induced increases 16 

### in the pro

- inflammatory cytokine TNF

- α . Mishra et al. ( 2006a ) found that 100 ppm 17 

### Pb

- acetate suppressed lipopolysaccharide (LPS)

- induced NO production. 18 

### Epidemiologic studies have not widely examined the effects of Pb exposure on altered 19 

### macrophage functional activity in humans, and the indices of macrophage function have 20 

### varied among the f ew available studies. Pineda

- Zavaleta et al. ( 2004 ) was unique in 21 

### examining the hyperinflammatory state specifically in macrophages, albeit in relation to 22 

### blood Pb levels higher than those in the current U.S. population (range of blood Pb 23 

### levels: 3.5

- 47.5 μ g/dL). This study included children in Lagunera, Mexico, attending 24 

### schools at varying distances from an active Pb smelter. Consistent with the large body of 25 

### toxicological evidence, higher concurrent blood Pb level was associated with lower NO 26 

### production and higher superoxide anion production in macrophages isolated from child 27 

### sera ( Section 5.6.5.2 ). Model covaria tes included sex, age, and presence of allergies. 28 

### Other studies in humans examined occupationally

- exposed adults and did not find a clear 29 

### association between concurrent measurements of blood Pb level and macrophage 30 

### abundance. Adjusting for age, race, smoki ng, and workshift, Pinkerton et al. ( 1998 ) found 31 

### a lower abundance of monocytes among 145 U.S. Pb smelter workers with a mean blood 32 

### Pb level of 39 μ g/dL (7.8%) than among 84 unexposed controls with a mean blood Pb 33 

### level of <2 μ g/dL (8.5%) (p = 0.03). Conterato et al. ( In Press ) examined a group of male 34 

### Pb

- exposed painters with blood Pb levels ranging between 1.4 and 14 .0 μ g/dL (mean: 35 

### 5.4 μ g/dL), lower than those in other occupational studies. The mean level and 36 

### percentage of monocytes in the 50 painters were similar to those in the 36 controls (mean 37 

### blood Pb level: 1.5 μ g/dL) and in the 23 battery workers with much high er blood Pb 38February 2012 5

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### levels (mean: 50 μ g/dL). Fischbein et al. ( 1993 ) found lower (p < 0.001) abundance of 1 

### HLA

- DR+ cells in two groups of New York metropolitan area firearms instructors with 2 

### mean blood Pb levels of 14.6 μ g/dL (n = 36, 8.8%) and 31.4 μ g/dL (n = 15, 8.7%) than 3 

### among the 36 unexposed controls (15. 2%). HLA

- DR+ is an indicator of activated 4 

### functional state of antigen presenting cells (APCs, e.g., macrophages, dendritic cells, T 5 

### cells) and is upregulated in response to cell signaling. While this study did not provide 6 

### direct evidence for Pb effects on macrophages, the investigators cited previous 7 

### observations that 70% of monocytes express HLA

- DR antigen compared with 15% of 8 

### lymphocytes to suggest that the reduced expression of HLA

- DR antigen in Pb

- exposed 9 

### workers was due primarily to a reduction in acti vated monocytes. 10 

### In summary, a small body of new in vitro studies ( Khan et al., 2011 ; Bussolaro et al., 11 

### 2008 ; Mishra et al., 2006a ) adds to the extensive base toxicological evidence indicating 12 

### that Pb exposure decreases functionality of macrophages and promotes a 13 

### hyperinflammatory phenotype. The sparse epidemiologic data are provided primarily by 14 

### older studies and are not conclusive. Whereas an association between blood Pb level and 15 

### a hyperinflammatory state of macrophages was observed in a previous study of children 16 

### ( Pineda

- Zavaleta et al., 2004 ) , studies of occupationally

- exposed adults did not clearly 17 

### indicate Pb exposure effects on macrophages. A new study did not find large differences 18 

### in m onocyte abundance between Pb

- exposed workers and unexposed controls ( Conterato 19 

### et al., In Press )

. 20



### 5.6.2.5 Neutrophils
In the 2006 Pb AQCD, Pb exposure was not judged to have strong effects on neutrophils 21 

### ( U.S. EPA, 2006b )

. This conclusion was based on the relatively limited available 22 

### toxicological evidence as compared with that for effects on other immune cells. Ho wever, 23 

### the modulation of neutrophil activity may have important consequences on the 24 

### dysregulation of inflammation and ability of organisms to respond to infectious agents. 25 

### Studies of cultured human polymorphonuclear cells (PMNs) ( Governa et al., 1987 ) and 26 

### occupationally

- exposed adults ( Queiroz et al., 1994a ; Queiroz et al., 1993 ; Valentino et 27 

### al., 1991 ; Bergeret et al., 1990 ) found Pb

- associated reductions in PMN functional ity, as 28 

### indicated by reduced chemotactic response, phagocytic activity, respiratory oxidative 29 

### burst activity, or reduced ability to kill ingested antigen. Important limitations to applying 30 

### epidemiologic findings broadly include male

- only study populations, the relatively high 31 

### blood Pb levels of workers (range of mean levels: 33.1

- 71 μ g/dL), lack of concentration

32 

### response information for associations between blood Pb level and neutrophil function, 33 

### and lack of consideration of potential confounding factors. 34February 2012 5

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### Instead of examining neutrophil functional activities, a few available recent studies of 1 

### animals and occupationally

- exposed adults examined the effects of Pb exposure on 2 

### neutrophil counts, an increase in which has been interpreted by some investigators to be a 3 

### compensatory response to Pb

- induced impairment in neutrophil chemotactic activity and 4 

### a hyperinflammatory response. In male rats with Pb spheres implanted in their brains, 5 

### neutrophils were a major responding cell ( Kibayashi et al., 2010 )

. Compared with rats 6 

### implanted with glass spheres, rats with Pb spheres had greater neutrophil infiltration with 7 

### inflammatory

- related damage that included apoptosis and indications of 8 

### neurodegeneration. It is important to acknowledge the uncertain relevance of these 9 

### findings to those expected fro m typical routes of Pb exposure in humans. 10 

### In a group of 68 ceramic, Pb recycling, or bullet manufacturing workers and 50 controls 11 

### selected among food plant workers, DiLorenzo et al. ( 2006 ) provided information on the 12 

### concentration

- response r elationship and adjusted for potential confounding variables. 13 

### Among all subjects, a 1 μ g/dL higher concurrent blood Pb level was associated with a 14 

### 21.8 cells/μL (95% CI: 11.2, 32.4 cells/μL) higher absolute neutrophil count (ANC) 15 

### adjusted for age, BMI, and smoking status. The geometric mean (range) of concurrent 16 

### blood Pb levels was 20.5 μ g/dL ( range: 3.2 to 120 μ g/dL ) among workers and 3.5 μ g/dL 17 

### ( range: 1 to 11 μ g/dL ) among controls. Eight workers described to have medium to high 18 

### Pb exposures (exact blood P b levels not reported) , but no controls had neutrophilia 19 

### (n >7,500 cells/mm 3 

### ), suggesting that chronic, higher

- level Pb exposures can lead to a 20 

### biologically meaningful excess of circulating neutrophils. Additionally, in analyses 21 

### comparing three blood Pb le vel groups, controls, workers with blood Pb levels ≤ 22 

### 30 μ g/dL, and workers with blood Pb levels >30 μ g/dL, ANC was observed to increase 23 

### monotonically across increasing blood Pb groups, supporting a blood Pb concentration

24 

### dependent relationship. When the th ree blood Pb groups were further stratified by current 25 

### smoking, two

- way ANOVA indicated an interaction between concurrent blood Pb level 26 

### and current smoking. Higher blood Pb level was associated with higher ANC only in 27 

### current smokers. Among nonsmokers, AN Cs were similar across blood Pb groups. In 28 

### contrast, Conterato et al. ( In Press ) found lower neutrophil concentrations among two 29 

### gro ups of Pb

- exposed male workers with mean concurrent blood Pb levels of 50.0 and 30 

### 5.4 μ g/dL than among controls with a mean blood Pb level of 1.5 μ g/dL. Pb

- exposed 31 

### workers did not consistently have higher levels of other immune cells such as 32 

### eosinophils, bas ophils, monocytes, or total lymphocytes either. 33 

### Additional evidence for the effects of Pb exposure on neutrophils is provided by findings 34 

### that blood Pb level is associated with mediators of neutrophil proliferation, survival, 35 

### maturation, and functional act ivation. These mediators include cytokines such as TNF

- α 36 

### ( Section 5.6.5.4 ) and complement. The complement system is a component of the innate 37 

### immune system that controls various cell

- mediated immune responses such as 38February 2012 5

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### chemotaxis of macrophages and neutrophils and ph agocytosis of antigens. The effects of 1 

### Pb exposure on complement have not been widely examined; however the limited data 2 

### suggest Pb may suppress complement activity. Both Ewers et al. ( 1982 ) and Undeger et 3 

### al. ( 1996 ) measured lower serum complement C3 protein among Pb

- exposed workers 4 

### compared with unexposed controls, with Ewers et al. ( 1982 ) additionally observing an 5 

### association between higher concurrent blood Pb level and lower C3 in a regression 6 

### analysis combining Pb

- exposed workers and controls. However, the broad applicability 7 

### of these findings may be limited due to the high blood Pb levels in these occupationally

8 

### exposed groups (range of blood Pb levels: 18.6

- 85.2 μ g/dL and 38

- 100 μ g/dL, 9 

### respectively) a nd by the lack of adjustment for potential confounding variables. 10 

### In summary, a majority of evidence indicating the effects of Pb exposure on neutrophils 11 

### was provided by previous studies that found that compared with unexposed controls, Pb

12 

### exposed workers had lower neutrophil functionality ( Queiroz et al., 1994a ; Queiroz et al., 13 

### 1993 ; Valentino et al., 1991 ; Bergeret et al., 1990 ) and lower complement, which is a 14 

### mediator of phagocyte functionality ( Undeger et al., 1996 ; Ewers et al., 1982 )

. In the 15 

### limited new epidemiologic investigation that examined neutrophil abundance, studies did 16 

### not conclusively find Pb

- exposed workers to have higher or lower neutrophil abundance. 17 

### Overall, the epidemiologic evidence is limited by the high blo od Pb levels (range of me an 18 

### levels: 33.1 to 71 μ g/dL) with which neutrophil functionality was observed to be 19 

### decreased and the lack of consideration of potential confounding. 20



### 5.6.2.6 Dendritic Cells
Since the publication of the 2006 Pb AQCD, new results ( from both an ex vivo and in 21 

### vitro model s) suggest that the effects of Pb exposure on suppressing Th1 activity and 22 

### promoting Th2 activity may be a consequence of the direct action of Pb on the function 23 

### of dendritic cells (a major APC). Prior research on the effects of Pb in favoring Th2 over 24 

### Th1 activity emphasized the direct measurement of Th1 versus Th2 T

- cell populations 25 

### and cytokine profiles. But new research techniques have been developed ( Gao and 26 

### Lawrence, 2010 ) that provide an opportunity to look upstream at how dendritic cells may 27 

### be involved in mediating the effects of Pb on acquired immunity. Gao et al. ( 2007 ) used 28 

### bone marrow cultures exposed to Pb to examine the impact of Pb on dendritic cell 29 

### maturation and function. PbCl 2 

### (25 μ M, 10 days) was found to alter the course of 30 

### dendritic ce ll maturation by changing the ratio of cell surface markers, such as the 31 

### CD86/CD80 ratio, that promote Th2 cell development. Additionally, upon activation with 32 

### LPS, Pb

- matured dendritic cells produced less IL

- 6, TNF

- α, and IL

- 12 (stimulates growth 33 

### and diff erentiation of T cells) than did control cells but the same amount of IL

- 10 34 

### (inhibits production of Th1 cytokines). The effect of Pb in altering the cytokine 35February 2012 5

- 360 Draft

– Do Not Cite or Quote 

### expression profile of dendritic cells, in particular, the lower IL

- 12/IL

- 10 ratio, may serve 1



### as an important signal to shift naïve T cell populations toward a Th2 phenotype. 2 

### Strengthening the role of dendritic cells in mediating Pb immune effects were ex vivo 3 

### results from the same study which showed that Pb

- naïve adult BALB/c mice implanted 4 

### with Pb

- exp osed dendritic cells were skewed toward Th2 activity as indicated by 5 

### inhibited DTH ( Section 5.6.2.3 ) and IgG2a antibody ( Section 5.6.3 ) responses ( Gao et al., 6 

### 2007 )

. 7



### 5.6.2.7 Natural Killer Cells
Based mostly on epidemiologic studies, pr evious and recent evidence does not clearly 8 

### indicate that the innate immune natural killer (NK) cells are affected to a large extent by 9 

### Pb exposure. In previous studies of nonoccupationally

- exposed adults in Italy, concurrent 10 

### blood Pb levels were correlate d with NK cell abundance among men with and without 11 

### allergy (Spearman r = 0.49, p ~ 0.05, n = 17 each, median blood Pb level both groups 12 

### combined: 11 μ g/dL) ( Boscolo et al., 1999 ) and women without allergies (Spearman r = 13 

### 0.44, p < 0.05) ( Boscolo et al., 2000 ) In a study of children from St. Lawrence River 14 

### communities in Quebec, Canada, cord blood Pb level was not significantly associated 15 

### with cord blood NK cell abundance (quantitative results not reported ( Belles

- Isles et al., 16 

### 2002 )

. Similar means of NK cell abundance or functional activity were observed in Pb

17 

### exposed workers and unexposed controls ( García

- Lestón et al., 2011 ; Mishra et al., 2003 ; 18 

### Pinke rton et al., 1998 ; Yucesoy et al., 1997b ; Undeger et al., 1996 ; Fischbein et al., 1993 ; 19 

### Kimber et al., 1986 )

. The lack of evidence for a strong effect on NK cells is underscored 20 

### by epidemiologic observations within individual studies that blood Pb levels are 21 

### associated with T cell abundance but are not associated with NK cell abundance or their 22 

### level of functiona l activity ( Karmaus et al., 2005 ; Sarasua et al., 2000 ; Pinkerton et al., 23 

### 1998 )

. Consistent with the epidemiologic evidence, toxicological evidence is not 24 

### conclusive. In a recent in vitro study comparing the toxicities of metals for different 25 

### populations of immune cells, Fortier et al. ( 2008 ) found that PbCl 2 

### (7.5

- 20.7 μ g/dL) did 26 

### not affect NK cytotoxicity compared with the DMSO vehicle. However, PbCl 2 

### was not 27 

### found to affect o ther immune parameters ( e.g., monocyte phagocytic activity or 28 

### lymphocyte proliferation) either. A recent study did show a decrease in NK cell activity 29 

### in mice albeit with high

- level Pb exposure (1 , 300 ppm Pb

- acetate in drinking water, 10 30 

### days, blood Pb level ~100 μ g/dL) ( Queiroz et al., 2011 )

. 31February 2012 5

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### 5.6.3 Humoral Immunity
The 2006 Pb AQCD described another hallmark effect of Pb on the immune system to be 1 

### an enhanced humoral immune response as characterized by increased prod uction of IgE 2 

### antibodies ( U.S. EPA, 2006b )

. Several previous toxicological and epidemiologic studies 3 

### ( Table 5

- 28 ) demonstrat ed Pb

- associated increases in IgE production, which is strongly 4 

### implicated in mediating allergic responses and inflammation in allergic asthma. In studies 5 

### of juvenile rodents, Pb exposures ( Pb

- acetate ) through drinking water from gestation 6 

### through lactatio n induced concomitant increases in IgE and IL

- 4 production (a Th2 7 

### cytokine) by T cells ( Snyder et al., 2000 ; Chen et al., 1999 ) , consistent with the 8 

### hypothesis that Th2

- mediated mechanisms can induce class switching of B cells to 9 

### produce IgE. Evidence from both earlier toxicological and epidemiologic studies ( Table 10 

### 5

- 28 ) did not consistently indicate that Pb exposure w as associated with changes in other 11 

### classes of Igs including IgG, IgM, and IgA. Previous epidemiologic studies of children 12 

### ( Table 5

- 28 ) and adults [ Table 5

- 28 with group comparisons and ( Boscolo et al., 2000 ; 13 

### Boscolo et al., 1999 ) with correlation analyses] did not find a consistent association 14 

### between blood Pb level and the abundance of B cells, which produce IgE and other 15 

### antibodies. 16 

### Although previous evidence for effects on Ig classes other than IgE was inconsistent, a 17 

### small number of available new toxicological studies found Pb

- induced increases in IgG 18 

### and pointed to a role for T cell

- mediated mechanisms in Pb

- induced activation of B cells 19 

### and production of Ig antibodies. Important limitations of this e vidence include high Pb 20 

### exposures tested and use of injection routes of exposure that may have little relevance to 21 

### human routes of exposure. Fernandez

- Cabezudo et al. ( 2007 ) reported evidence for a 22 

### subtle shi ft toward a Th2 immune response following Salmonella infection in mice 23 

### exposed to high concentrations of Pb

- acetate (5 , 000

- 10,000 μ M, 16 weeks, resulting in 24 

### blood Pb levels of 20.5

- 106 μ g/dL). Serum levels of Salmonella

- specific IgG1 antibodies 25 

### were increa sed in the Pb

- exposed mice compared to controls, whereas IgG2a levels were 26 

### increased in control but not Pb

- exposed mice following infection. The impaired Th1 and 27 

### enhanced Th2 response also was evident by the decreased secretion of IL

- 12 and 28 

### increased produ ction of IL

- 4 by spleen cells taken from Pb

- exposed (blood Pb levels 29


### > 20.5 μ g/dL) mice ( Fernandez

- Cabezudo et al., 2007 )

. 30 

### In a highly

- specialized strain of knockout adult mice lacking the ability to produce IFN

- γ, 31 

### i.p. injection with 50 μ g PbCl 2 

### increased the IgG2a/IgG1 ratio ( Gao et al., 2006 )

. This 32 

### result was surprising given evidence that IFN

- γ usually directs secretion of IgG2a; 33 

### however, the authors suggested that in these knockout mice, Pb may initiate a Th1 34 

### response via an IFN

- γ independent pathway to enhance IgG2a production. Other animal 35 

### studies that administered Pb via the i.p., found Pb

- induced humoral responses to 36February 2012 5

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– Do Not Cite or Quote 

### mediated preferentially via Th2 mechanisms. Carey et al. ( 2006 ) treated BALB/c mice 1 

### with subsensitizing doses of a T cell

- independent (Trinitrophenyl

- Ficoll [TNP

- Ficoll]) o r 2 

### T cell

- dependent (TNP

- ovalbumin [TNP

- OVA]) hapten

- protein conjugate with or 3 

### without co

- exposure to PbCl 2 

### . Seven days later, investigators examined the effects of Pb 4 

### on the LLNA response to TNP

- Ficoll or TNP

- OVA. A bolus injection of PbCl 2 5 

### (25

- 50 μ g) incr eased the numbers of T and B cells in the lymph node against both TNP

6 

### Ficoll and TNP

- OVA. Further, in a concentration

- dependent manner, Pb induced 7 

### statistically significant elevations of IgM

- , IgG2a

- , and IgG1

- producing cells in the 8 

### lymph node. While the i ncrease in IgM

- producing cells against TNP

- Ficoll indicated a T

9 

### cell independent mechanism, the increases in IgG2a

- and IgG1

- producing cells against 10 

### both antigens indicated a Th1

- and Th2

- mediated mechanism, respectively. Despite 11 

### finding increases in both IgG1

- and IgG2a

- producing cells, the authors concluded that Pb 12 

### skewed the response toward Th2 and had considerable potential for promoting allergic 13 

### sensitization against T

- dependent antigens. 14 

### In a recent microarray study in BALB/c mice, Kasten

- Jolly et al. ( 2010 ) found that early

15 

### life Pb exposure (100 μ M Pb

- acetate in drinking water of dams from GD8 to PND21, 16 

### resulting in pup blood Pb levels 10

- 30 μ g/dL) produced statistically significant increases 17 

### in the expression of genes encoding Ig antibodies or those involved in B lymphocyte 18 

### function and activation. These genes included those for the heavy chain of IgM, IL

- 4, IL

19 

### 7 and IL

- 7 receptor, IL

- 21, RAG

- 2, CD antigen 27, B

- cell leukemia/lymphoma 6, RNA 20 

### binding motif protein 24, Hi stocompatibility class II antigen A (beta 1), Notch gene 21 

### homolog 2, and histone deacetylase 7A. 22 

### In epidemiologic studies, several of which are new, associations between biomarkers of 23 

### Pb exposure and serum IgE level were demonstrated primarily in children, although a 24 

### monotonic dose

- dependent increase was not consistently observed ( Hegazy et al., 2011 ; 25 

### Hon et al., 2010 ; Hon et al., 2009 ; Karmaus et al., 2005 ; Annesi

- Maesano et al., 2003 ; 26 

### Sun et al., 2003 ; Lutz et al., 1999 ) ( Table 5

- 28 ). Karmaus et al. ( 2005 ) had the most 27 

### extensive adjustment for potential confounding factors and examined differences with 28 

### lower blood Pb levels. Compared with ch ildren with concurrent blood Pb level 29 

### <2.2 μ g/dL (quartile 1), children with blood Pb levels 2.84

- 3.41 μ g/dL (quartile 3) and 30 

### >3.4 μ g/dL (quartile 4) had 28% higher serum IgE levels (p = 0.03, F

- test). These 31 

### differences were observed after the adjustment f or potential confounding by age, 32 

### biomarkers of various organochlorine exposures, number of infections in the previous 12 33 

### months, serum lipids, and passive smoke exposure. Similar differences in IgE count on 34 

### basophils were not observed among the blood Pb qu artiles; however, it is important to 35 

### note that although serum IgE and basophil

- bound IgE have been correlated in adults 36 

### ( Malveaux et al., 1978 ; Conroy et al., 1977 ) , little data are available in children ( Dehlink 37 

### et al., 2010 )

. A recent study in children found that serum IgE levels were not correlated 38February 2012 5

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### with basophil

- bound IgE but correlated (Spearman r =

- 0.003) with other IgE receptor

1 

### expressing cells such as dendritic cells and monocytes (Spearman r = 0.43 to 0.65, p < 2 

### 0.05) ( Dehlink et al., 2010 )

. The number of IgE

- bound basophils also has been found to 3 

### be highly variable across individuals , particularly children ( Hausmann et al., 2011 ; 4 

### Dehlink et al., 2010 )

. Thus, it is not unexpected that higher blood Pb level was associated 5 

### with higher serum IgE but not basophil

- bound IgE counts in Karmaus et al. ( 2005 )

. In 6 

### this study, blood Pb level was not associat ed with serum levels of IgG, IgA, and IgM or B 7 

### cell abundance. Lutz et al. ( 1999 ) found higher serum IgE in children after adjusting for 8 

### age, albeit with concurrent blood Pb levels > 10 μ g/dL. 9 

### Recent studies in children also reported ass ociations between concurrent blood Pb level 10 

### and elevated serum IgE but did not adjust for potential confounding variables ( Hegazy et 11 

### al., 2011 ; Hon et al., 2010 ; Hon et al., 2009 )

. The studies by Hon and colleagues ( 2010 ; 12 

### 2009 ) demonstrated associations in children with low blood Pb levels (range: 1.4

- 13 

### 6.0 μ g/dL) and found that blood Pb level was correlated with both serum IgE and atopic 14 

### dermatitis, a condition commonly characterized by elevated IgE levels. 15February 2012 5

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### Table 5
- 28 Comparison of serum immunoglobulin levels and B cell abundance among various blood Pb groups

Study Population/Study Details Blood Pb Level Mean or Range (μg/dL) IgE a IgG b IgM b IgA b B cells c
Children Karmaus et al. ( 2005 ) 331 children, ages 7

- 10 yr, Hesse, Germany Results were adjusted for age, sex, passive smoke exposure, number of infections in the previous 12 months, serum lipid concentration, an d organochlorine exposures. <2.2 2.21

- 2.83 2.84

- 3.41 >3.41 46 (1.0) 30 (0.65) 59 (1.28) 59 (1.28) d 1210 1214 1241 1201 150 143 153 148 123 121 133 136 418 e (1.0) 353 (0.84) 389 (0.93) 393 (0.94) Hegazy et al. ( 2011 ) 318 children, ages 6 mo

- 7 yr, Egypt < 5 5

- 9 10

- 14 15

- 19 2 0

- 44 45

- 69 13.0 (1.0) 12.0 (0.92) 20.8 (1.60) 14.9 (1.15) 20.4 (1.57) 10.2 (0.78) d Sarasua et al. ( 2000 ) 382 children, ages 6

- 30 mo, Multiple U.S. locations Results were adjusted for age, sex, and study location. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 609 666 d 680 d 630 103 108 105 124 d 50.1 55.0 58.2 61.4 d 19.1 (1.0) 20 (1.05) 20.4 (1.07) 22.2 (1.16) Sarasua et al. ( 2000 ) 562 children, ages 36

- 71 mo, Multiple U.S. locations Results were adjuste d for age, sex, and study location. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 817 813 856 835 120 116 125 121 88.6 90.9 96.3 94.1 18.4 (1.0) 17.6 (0.96) 19.2 (1.04) 18.6 (1.01) Sarasua et al. ( 2000 ) 675 children ages 5

- 16 yr, Multiple U.S. locations Results were adjusted for age, sex, and study location. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 1 , 031 1 , 094 d 1 , 048 1 , 221 128 131 136 106 140 143 140 108 16.1 (1.0) 15.8 (0.98) 15.3 (0.95) 20.1 (1.25) Lutz et al. ( 1999 ) 279 children, ages 9 mo

- 6 yr, Springfield, MO Results were adjusted for age. <10 10

- 14 15

- 19 20

- 44 51.8 (1.0) 74.0 (1.43) 210.7 (4.07) 63.7 (1.23) d 13.4 (1.0) 12.6 (0.94) 16.9 (1.26) 11.1 (0.83) Zhao et al. ( 2004 ) 75 children, ages 3

- 6 yr, Zhejiang Province, China <10 ≥ 10 16.58 (1.0) 16.82 (1.01) Adults without Occupational Pb Exposures Sarasua et al. ( 2000 ) 433 ch ildren and adults, ages 16

- 75 yr, Multiple U.S. locations Results were adjusted for age, sex, study location, and smoking. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 1 , 099 1 , 085 1 , 231 1 , 169 175 175 262 d 139 252 242 283 193 13.9 (1.0) 13.0 (0.94) 12.4 (0.89) 14.8 (1.06) Adults with Occupational Pb Exposures Fischbein et al. ( 1993 ) 36 unexposed controls, mean age 47 yr 36 firearms instructors, mean age 49 yr 15 firearms instructors, mean age 48 yr New York metropolitan area NR 14.6 31.4 8.6 (1.0) 10.5 (1.22) 11.2 (1.30) d

Kimber et al. ( 1986 ) 21 unexposed male controls, ages 20

- 60 yr 39 male tetraethyl Pb plant workers, ages 25

- 61 yr U.K. 11.8 38.4 1062 1018 1294 1040 2235 2425February 2012 5

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– Do Not Cite or Quote Study Population/Study Details Blood Pb Level Mean or Range (μg/dL) IgE a IgG b IgM b IgA b B cells c
Pinkerton et al. ( 1998 ) 84 unexposed controls, mean age 30 yr 145 Pb smelter workers, mean age 33 yr U.S., exact location NR Results were adjusted for age, race, current smoking status, and workshift . <2 39 1 , 090 1 , 110 94.5 106.2 180 202 14.6 (1.0) 13.2 (0.90)

Heo et al. ( 2004 ) 606 Pb battery plant workers Korea <10 10

- 29 ≥ 30 112.5 (1.0) 223.3 (1.99) 535.8 (4.76) d Anetor and Adeniyi ( 1998 ) 50 male controls, ages 22

- 58 yr 80 male Pb

- exposed workers, ages 21

- 66 yr, Nigeria 30.4 56.3 1 , 997 1 , 187 d 215 191 188 144 d Ewers et al. ( 1982 ) 53 male unexposed controls, ages 21

- 54 yr 72 male Pb battery/smelter workers, ages 16

- 58 yr, Germany 11.7 59.0 193 f 171 161 f 127 140 f 128
Undeger et al. ( 1996 ) 25 unexposed male controls, ages 22

- 56 yr 25 male Pb battery plant workers, ages 22

- 55 yr, Turkey 16.7 74.8 1 , 202.1 854.6 d 140.4 93.3 d 210.3 168.1 545.5 e (1.0) 635.9 (1.17)

Alomran and Shleamoon ( 1988 ) 18 unexposed age

- matched controls 39 Pb battery workers, mean age 35 yr, Iraq NR NR 1713 1610 183 170 545.5 e (1.0) 635.9 (1.17)

a IgE data are presented as IU/mL. ( In parentheses are the ratio of IgE in the higher blood Pb group to IgE in the lowest blood Pb group. ) b Other Ig data are presented as mg/dL unless otherwise specified.

c B cell data are presented as the percentage of B cells among all lymphocytes unless otherwise specified. ( In parentheses are the ratio of B cells in the higher blood Pb group to B cells in the lowest blood Pb group. ) d p <0 .05 for group differences.

e Data represent the number of cells/μL serum. f Data are presented as IU/mL.



### Sarasua et al. ( 2000 ) found associations of higher concurrent blood Pb level with higher 1 

### IgA, IgG, and IgM in U.S. children. Blood Pb level was associated with higher levels of 2 

### all three Igs (0.8 [95% CI: 0.2, 1.4], 4.8 [95% CI: 1.2, 8.4], and 1.0 [95% CI: 0.1, 3 

### 1.9] mg /dL higher IgA, IgG, and IgM, respectively, per 1 μ g/dL higher blood Pb level, 4 

### adjusted for age, sex, and location) in the youngest age group (6

- 35 months) but not in 5 

### older age groups (36

- 71 months, 6

- 15 years, 16

- 75 years), suggesting elevated risk in 6 

### very young children. Among infants aged 6

- 35 months with concurrent blood Pb levels ≥ 7 

### 15 μ g/dL, serum levels of all three examined Igs were elevated over levels measur ed in 8 

### infants with blood Pb levels < 5 μ g/dL. In this study, IgE was not examined. 9 

### While most epidemiologic studies examined concurrent blood Pb levels, some studies 10 

### indicated that prenatal Pb exposure may impact Ig levels in newborns ( Annesi

- Maesano 11 

### et al., 2003 ; Belles

- Isles et al., 2002 )

. These studies also pointed to an increased risk in 12 

### infants; however, important limitations of these studies are the lack of extensive 13 

### consideration of potential confounding variables. Belles

- Isles ( 2002 ) examined newborns 14 

### in St. Lawrence River communities in Quebec, Canada and found an association between 15February 2012 5

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### higher cord blood Pb level and higher cord serum IgG in a regression analysis adjusted 1 

### for prenatal smoke exposure. Annesi

- Maesano et al. ( 2003 ) found that infant hair Pb level 2 

### but not cord or placental Pb level was associated with cord serum IgE in newborns in 3 

### Paris. From these findings, the authors inferred a stronger effect of Pb exposure 4 

### integrated over the entire gestational period compared to exposures closer to birth. 5 

### Cotinine was not associated significantly with Pb biomarker levels or with IgE. The 6 

### magnitude o f association was larger in the subgroup of infants with mothers without 7 

### allergies (Spearman r = 0.21, p < 0.01 in infants with mothers without allergies versus r = 8 

### 0.12 in infants with mothers with allergies, pointing to the possible masking of a blood 9 

### Pb association with IgE by the stronger association of family history of allergy. 10 

### Associations between blood Pb level and IgE also were reported in studies of adults, 11 

### without ( Pizent et al., 2008 ) and with occupational Pb exposure ( Heo et al., 2004 )

. In a 12 

### study of urban adults aged 19 to 67 years and of similar SES ( i.e., white

- collar office 13 

### workers) in Zagreb, Croatia, a statistically significant association between higher 14 

### concurrent blood Pb level and higher IgE was found in women but not men ( Pizent et al., 15 

### 2008 )

. Several covariates were considered in a stepwise multiple regression, including 16 

### age, smoking intensity, and alcohol consumption. Among women not on hor mone 17 

### replacement therapy or oral contraceptives, a 1 μ g/dL higher blood Pb level was 18 

### associated with a 0.60 higher log of IgE (95% CI: 0.58, 1.18). Concurrent blood Pb levels 19 

### were low in these women who were aged 19

- 67 years (mean: 2.16 μ g/dL, range 0.56

- 20 

### 7 .35 μ g/dL); however, due to the cross

- sectional nature of this study, it is difficult to 21 

### characterize the timing, level, frequency, and duration of Pb exposure that contributed to 22 

### the observed association. Because of likely higher past Pb exposures of adul ts and the 23 

### mobilization of Pb from bone to blood, the associations may reflect effects of higher past 24 

### Pb exposures. Investigators did not report an effect estimate in men because it did not 25 

### attain statistical significance. Without quantitative results, it was difficult to ascertain 26 

### whether there was suggestion of association in men but insufficient power to indicate 27 

### statistical significance due to the smaller number of men examined (50 men versus 166 28 

### females). Another study of 34 men in Italy also did not r eport quantitative results but 29 

### indicated a lack of statistically significant correlation between blood Pb level and IgE in 30



### men ( Boscolo et al., 1999 )

. 31 

### A majority of the epidemiologic evidence for the effects of Pb on IgA, IgG, and IgM 32 

### levels is provided by previous studies of Pb

- exposed workers ( Anetor and Adeniyi, 1998 ; 33 

### Pinkerton et al., 1998 ; Undeger et al., 1996 ; Queiroz et al., 1994b ; Alomran and 34 

### Shleamoon, 1988 ; Kimber et al., 1986 ; Ewers et al., 1982 )

. Consistent with the collective 35 

### body of toxicological findings for these other Ig classes, epidemiologic evidence is 36 

### mixed, with studies reporting higher, lower, and similar Ig levels in Pb

- exposed workers 37 

### compared with une xposed controls. Some studies reporting lower Ig levels in Pb

- exposed 38February 2012 5

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### workers included workers with the highest mean blood Pb levels (> 50 μ g/dL) ( Anetor 1 

### and Adeniyi, 1998 ; Undeger et al., 1996 )

. The lack of rigor ous statistical analyses in 2 

### occupational studies precludes characterization of the factors that may contribute to 3 

### inconsistent associations. 4 

### In summary, a majority of the evidence for Pb exposure affecting humoral immunity is 5 

### provided by previous toxicolo gical and epidemiologic studies that found Pb

- associated 6 

### increases in IgE. Previous toxicological studies provided evidence for the independent 7 

### effects of Pb exposure increasing IgE levels. A few available recent epidemiologic 8 

### studies add to the large body of extant evidence supporting associations between higher 9 

### concurrent blood Pb level and higher IgE levels in children; however, they did not adjust 10 

### for potential confounding variables ( Hegazy et al., 2011 ; Hon et al., 2010 ; Hon et al., 11 

### 2009 )

. Collectively, epidemiologic evidence indicates higher IgE in children with 12 

### concurrent blood Pb levels > 10 μ g/dL. The epidemiologic evidence in children equally 13 

### comprises studies that did not and did adjust for potential confounding. Studies that 14 

### adjusted for potential confounding varied in t he number and the specific variables 15 

### included as model covariates ( Table 5

- 28 ) but were consistent in finding an association 16 

### between blood Pb and IgE. None of the studies in children adjusted for SES or allergen 17 

### exposure. Lower SES, which may indicate poorer housing conditions, is associated with 18 

### higher exposures to Pb as well as cockroach, rodent, and other allergens. Allergen 19 

### exposure and lower SES are associated with higher IgE and IgE

- related conditions such 20 

### as allergies and asthma ( Bryant

- Stephe ns, 2009 ; Dowd and Aiello, 2009 ; Aligne et al., 21 

### 2000 )

. Studies generally did not provide detailed demographic or residential information 22 

### to assess whether SES and/or allergen exposure potentially confounded the observed 23 

### associations between blood Pb level and IgE. Thus, uncertainty remains as to the exten t 24 

### to which blood Pb

- IgE associations in children are confounded by unmeasured SES 25 

### and/or allergen exposure. Evidence in adults is limited and thus, inconclusive. A new 26 

### study of environmentally

- exposed adults found an association between concurrent blood 27 

### Pb level and IgE in women ( Pizent et al., 2008 )

. Evidence integrated across toxicological 28 

### and epidemiologic studies does not consistently indicate that Pb exposure affects other 29 

### classes of Igs; however, a few new toxicological studies found Pb

- induced increases in 30 

### IgG ( Fernandez

- Cabezudo et al., 2007 ; Gao et al., 2006 ) or increases in the expression of 31 

### genes encoding Ig antibodies ( Kasten

- Jolly et al., 2010 )

. 32February 2012 5

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### 5.6.4 Immune
- based Diseases

5.6.4.1 Host Resistance
The capability of Pb to reduce host resistance of animals to bacteria has been known for 1 

### almost 40 years and was supported by several animal studies described in the 2006 Pb 2 

### AQCD ( U.S. EPA, 2006b )

. A recent animal study provided supporting evidence for such 3 

### an effect. Biolog ical plausibility for these findings has been provided by observations that 4 

### Pb affects mechanisms underlying diminished host r esistance, e.g., suppressed 5 

### Th1

- driven acquired immune responses and increased inflammatory responses in target 6 

### tissue resulting i n further damage to host protective barriers. Host resistance to bacteria 7 

### such as Listeria requires effective Th1

- driven responses including the production of IL

8 

### 12 and IFN

- γ ( Lara

- Tejero and Pamer, 2004 ) and these have been found to be inhibited 9 

### by Pb exposure. The lack of IFN

- γ can inhibit appropriate and time ly macrophage 10 

### activation. Nitric oxide is produced by macrophages during cellular activation and plays a 11 

### role in host defense against bacterial infection ( U.S. EPA, 2006b ) , and NO production 12 

### also has been found to be suppressed b y Pb exposure. A recent animal study 13 

### characterized a potential mechanism by which Pb may impact both innate immune cells 14 

### and natural host defense barriers. Kasten

- Jolly et al. ( 2010 ) showed that developmental 15 

### exposure of mic e to Pb (100 μ M Pb

- acetate in drinking water of dams from GD8 to 16 

### PND21, resulting in pup blood Pb levels of 10

- 30 μ g/dL) resulted in an upregulation of 17 

### splenic RNA of caspase

- 12, a cysteine protease that inhibits the clearance of bacteria both 18 

### systemically and in the gut mucosa ( Saleh et al., 2006 )

. 19 

### In the few available epidemiologic studies, a range of indicators of Pb exposure ( i.e., cord 20 

### blood Pb, concurrent blood Pb, Pb content in total deposition samples, Pb con tent in 21 

### lichen) were associated with viral and bacterial infections in children. Collectively, study 22 

### limitations, including lack of consideration for potential confounding variables ( Karmaus 23 

### et al., 2005 ; Rabinowitz et al., 1990 ) , lack of statistical analysis ( Karmaus et al., 2005 ) , 24 

### and ecological design ( Carreras et al., 2009 ) , limit the ability to draw inferences 25 

### regarding the effects of Pb exposure on viral or bacterial infections in children. Similar to 26 

### these studies in children, an occupational study finding higher frequency of self

- reported 27 

### colds or influenza among Pb battery or smelter plant workers with higher blood Pb levels 28 

### than among unexposed controls is limited by a lack of statistical analysis ( Ewers et al., 29 

### 1982 )

. Studies that address the aforementione d limitations are needed to characterize the 30 

### relationship between Pb exposure and resistance to bacterial and viral infection in 31 

### humans. 32February 2012 5

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### With limited investigation, the effect of Pb on host resistance to parasitic agents is 1 

### unclear. The 2006 Pb ACQD descri bed a study in which high

- level Pb

- exposed (≥ 2 

### 10 μ M) mouse macrophages had diminished ability to kill Leishmania enrietti parasites 3 

### ( Mauel et al., 1989 )

. However, given the well

- characterized effect of Pb in promoting 4 

### Th2 activity, it is plausible for Pb to enhance host resistance to parasites that require 5 

### robust Th2 responses ( e.g., h elminths) ( U.S. EPA, 2006b )

. In a recent study, high

- level 6 

### Pb exposure (≥ 10 μ M) enhanced host resistance to malaria ( Koka et al., 2007 )

. However, 7 

### this was attributed to the capability of Pb to induce eryptosis and the rapid removal of 8 

### malaria

- infected erythrocytes and not to Pb

- induced alte rations in immune function. 9 

### Nriagu et al. ( 2008 ) also reported that higher blood Pb level was associated with lower 10 

### malaria prevalence among ch ildren (ages 2

- 9 years) from three Nigerian cities. A 11 

### majority of children (75%) had blood Pb levels below 10 μ g/dL. The association 12 

### persisted after adjusting for age, sex, number of siblings, and other comorbidities such as 13 

### headaches, depressed mood, and irritability. 14



### 5.6.4.2 Asthma and Allergy
Toxicological evidence and to a relatively limited extent, epidemiologic evidence, have 15 

### supported the effects of Pb exposure on multiple immune parameters, including elevated 16 

### production of Th2 cytokines such as IL

- 4, increased IgE antibody production ( Table 17 

### 5

- 24 ), and increased inflammation. These are well

- recognized pathways in the 18 

### development and exacerbation of allergy and allergic disease, including asthma. This 19 

### mechanistic evidence is coherent w ith a small body of epidemiologic studies that found 20 

### associations of blood Pb levels with asthma or allergy in children ( Figure 5

- 43 and Table 21 

### 5

- 29 ). Children examined in these studies encompassed a wide range of ages ( i.e., less 22 

### than 1 year to 12 years) and across studies, blood Pb was measured duri ng different 23 

### lifestages. This body of evidence in children included large studies with multivariate 24 

### analyses and studies with prospective follow

- up of subjects in which disease occurrence 25 

### was ascertained after the measurement of blood Pb levels. 26February 2012 5

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– Do Not Cite or Quote Note: Re sults are organized by endpoint and for asthma in order of increasing blood Pb level. For analyses with blood Pb level as a continuous variable, odds ratios are standardized to a 1 μg/dL increase in blood Pb level. SPT = skin prick test, BR = bronch ial res ponsiveness. Black diamond represents associations with concurrent blood Pb levels, green triangles represent associations wi th prenatal (cord) blood Pb levels, and blue circles represent associations with blood Pb levels measured in childhood up to 12 mon ths prior to outcome assessment. 

### Figure 5
- 43 Associations of blood Pb levels with asthma
- and allergy
- related conditions in children. Study

Rabinowitz et al. (1990)

Jedrychowski et al. (2011)

Rabinowitz et al. (1990)

Pugh Smith et al. (2011) Joseph et al. (2005) Population

Children

Children

Children

Children Children, Caucasian Children, African American Children, African American Outcome

Eczema

Positive SPT

Asthma

Asthma Incident Asthma Requiring Medical Care Blood Pb Level Mean or Group (μg/dL)

≥10 vs. <10

prenatal: 1.16 concurrent: 2.02

≥10 vs. <10

≥10 vs. <10 ≥5 vs. <5 ≥5 vs. <5 ≥10 vs. <5

►

0 1 2 3 4 5 6 7 8 Odds ratio (95% CI)February 2012 5

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### Table 5
- 29 Additional characteristics and quantitative results for studies presented in Figure 5
- 43

Study Population/Location Blood Pb Level Data (μg/dL) Statistical Analysis Outcome Odds Ratio (95% CI) a
Rabinowitz et al. ( 1990 ) 1 , 768 children foll owed from birth to unspecified age Boston area, MA Prenatal (cord) ≥ 10 vs. < 10 a Logistic regression with no additional covariates included in model Eczema 1.0 (0.6, 1.6)

Jedrychowski et al. ( 2011 ) 224 children followed prenatally to age 5 yr Krakow, Poland Prenatal (cord): GM: 1.16 (95% CI: 1.12, 1.22) Concurrent: GM: 2.02 (95% CI: 1.95, 2.12) Logistic regression adjusted for sex, parity, maternal age, matern al education, maternal atopy, maternal smoking Positive SPT 2.3 (1.1, 4.6) b 1.1 (0.7, 1.6) b

Joseph et al. ( 2005 ) 4 , 634 children, ages 1

- 3 yr followed prospectively for 12 months Southeastern MI Measured up to 12 mos before outcome Caucasian ≥ 5 vs. Caucasian <5 c African American ≥ 5 vs. African American <5 c African American ≥ 10 vs. African American <5 c Logistic regres sion adjusted for sex, birth weight, and annual income Incident asthma requiring medical care 2.7 (0.9, 8.1) 1.1 (0.8, 1.7) 1.3 (0.6, 2.6) Rabinowitz et al. ( 1990 ) 1 , 768 children fol lowed from birth to unspecified age Boston area, MA Prenatal (cord blood) ≥ 10 vs. <10 a Logistic regression with no additional covariates included in model Prevalent asthma 1.3 (0.8, 2.0)

Pugh Smith and Nriagu ( 2011 ) 356 children, ages 0

- 12 yr Saginaw, MI Levels ascertained from statewide database, timing unreported but varied among subjects ≥ 10 vs. <10 a Logistic regression adjusted for age, sex, number of stories in unit, cat in home, dog in home, cockroach problem, number of persons in home, household smoking, clutter, candles/incense, type of cooking stove, main heating source, months of residency, housing tenure, type of air conditioning, peeling paint, ceiling/wall damage, age of housi ng, water dampness/mold/mildew Prevalent asthma 7.5 (1.3, 42.9)

GM = geometric mean, SPT = skin prick test.

a Odds ratio in children with blood Pb level ≥ 10 μg/dL with children with blood Pb level <10 μg/dL serving as the reference gr oup b Odds ratio pres ented per 1 μg/dL increase in blood Pb level.

c Relative risk in each specified subgroup with children with blood Pb level <5 μg/dL serving as the reference group.


### In a study of 4 , 634 children in southeastern Michigan, blood Pb levels were measured at 1 

### ages 1 to 3 years, up to 12 months prior to asthma assessment. In analyses that controlled 2 

### for annual income, birth weight, and sex, an elevated risk of incident asthma requiring a 3 

### doctor visit or medication (indicator of severe asthma) was reported in ass ociation with 4 

### blood Pb levels ≥ 5 μ g/dL among Caucasian children (relative risk [RR]: 2.7 [95% CI: 5 

### 0.9, 8.1] compared with Caucasian children with blood Pb levels < 5 μ g/dL) ( Joseph et 6 

### al., 2005 ) ( Figure 5

- 43 and Table 5

- 29 ). In analyses restricted to African Americans, 7 

### children with blood Pb l evels ≥ 10 μ g/dL had an elevated risk of asthma requiring 8 

### medical care (RR: 1.3 [95% CI: 0.6, 2.6] compared with children with blood Pb level < 9 

### 5 μ g/dL) ( Figure 5

- 43 and Table 5

- 29 ). In analyses that used Caucasian children with 10 

### blood Pb level <5 μ g/dL as the reference group, blood Pb level was associ ated with 11 

### increased risk of asthma requiring medical care among African American children in all 12 

### blood Pb level categories. Collectively, the results indicated a stronger association with 13 

### race/ethnicity than with blood Pb level. It is important to recogniz e the small numbers of 14February 2012 5

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### children with asthma requiring medical care in the high blood Pb level categories, which 1 

### likely accounted for the wide 95% CIs (5 Caucasian children with blood Pb ≥ 5 μ g/dL 2 

### and 9 African American children with blood Pb level ≥ 10 μ g/ dL). 3 

### Consistent with Joseph et al. ( 2005 ) , a recent cross

- sectional study conducted in Saginaw, 4 

### Michigan found a higher prevalence of asthma in children (ages ≤ 12 years) with blood 5 

### Pb levels ≥ 10 μ g/dL ( Pugh Smith and Nriagu, 2011 )

. Of the 356 children randomly 6 

### selected from a statewide surveillance database of blood Pb measurements, 7 8% were 7 

### African American. This study had greater power than did Joseph et al. ( 2005 ) due to the 8 

### larger percentage of children wit h blood Pb levels ≥ 10 μ g/dL (18.6%). Compared with 9 

### children with initial blood Pb levels < 10 μ g/dL, children with initial blood Pb levels ≥ 10 

### 10 μ g/dL had a higher odds of having a doctor diagnosis of asthma within the past 12 11 

### months (OR: 7.5 [95% CI: 1.3, 42.9]). A strength of this study was the adjustment for a 12 

### large number of potential confounding variables such as age, sex, pets in the home, 13 

### housing characteristics, and household smoking. However, because children were 14 

### identified from a statewide databa se of initial blood Pb measurements collected at 15 

### unspecified ages, the timing of blood Pb varied among children. In a study that did not 16 

### consider potential confounding variables, Rabinowitz et al. ( 1990 ) reported an increased 17 

### risk of asthma in children (age of assessment not reported) in association with c ord blood 18 

### Pb levels > 10 μ g/dL relative to cord blood Pb levels ≤ 10 μ g/dL ( Figure 5

- 43 and Table 19 

### 5

- 29 ). 20 

### While the aforementioned studies examined blood Pb levels measured at a single point in 21 

### time, a recent prospective birth cohort study compared associations of prenatal (cord and 22 

### maternal) a nd concurrent blood Pb levels with risk of allergic sensitization at age 5 years 23 

### ( Je drychowski et al., 2011 )

. Cord and prenatal maternal blood Pb level were associated 24 

### with greater risk of positive skin prick test (SPT) to dust mite, dog, or cat allergen than 25 

### was child concurrent blood Pb level ( Figure 5

- 43 and Table 5

- 29 ). For prenatal Pb 26 

### biomarkers, similar effect estimates were obtained before and after adjusting for sex, 27 

### parity, maternal age, maternal education, maternal atopy, and environmental tobacco 28 

### smoke exposure. Cord and concurrent blood Pb levels were weakly correlated (r = 0.29), 29 

### providing support for an independent as sociation for prenatal Pb biomarkers. The 30 

### independent effects of Pb also were substantiated by observations that indicators of other 31 

### exposures, including blood levels of mercury, polycyclic aromatic hydrocarbon DNA 32 

### adducts, and residential levels of dust m ite or pet allergen were associated with lower 33 

### risks of SPT than was blood Pb level. While associations were observed with relatively 34 

### low cord blood Pb levels (geometric mean: 1.16 μ g/dL [95% CI: 0.12, 1.22]), it is 35 

### uncertain the extent to which higher pas t Pb exposures of the mothers may have 36 

### influenced their pregnancy blood Pb levels and newborn cord blood Pb levels. Among 37 

### children approximately age 10 years in Hong Kong, Hon et al. ( 2010 ; 2009 ) found 38February 2012 5

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### correlations between low concurrent blood Pb levels (population means < 2 μ g/dL) and 1 

### severity of atopic dermatitis (Spearman r = 0.46, p <0.001 and r = 0.33, p <0.005), 2 

### another inflammator y condition commonly related to elevated IgE levels. Neither 3 

### analysis considered potential confounding variables. 4 

### Among the studies in children that found associations of blood Pb level with asthma and 5 

### allergy

- related outcomes, several adjusted for potenti al confounding by SES

- related 6 

### variables. Joseph et al. ( 2005 ) adjusted for annual income. Pugh Smith et al. ( 2011 ) , who 7 

### examined a primarily low SES population of children in Michigan, adjusted for vario us 8 

### factors associated with SES, including multiple indices of housing condition, and 9 

### presence of pets and cockroaches in the home, which are indicators of allergens in the 10 

### home. Jedrychowski et al. ( 2011 ) adjusted for maternal educa tion , and found similar 11 

### magnitudes of association between cord blood Pb level ( and positive SPT ) as those in the 12 

### unadjusted analysis. Further, residential levels of dust mite or pet allergen were 13 

### associated with lower risks of SPT than was blood Pb level. Thus, while SES and/or 14 

### allergen exposure has been associated with both Pb exposure and asthma and allergy in 15 

### children ( Bryant

- Stephens, 2009 ; Dowd and Aiello, 2009 ; Aligne et al., 2000 ) , these 16 

### collective findings in different populations do not demonstrate that confounding by SES 17 

### and/or allergen exposure fully accounts for the associations ob served between blood Pb 18 

### levels and asthma and allergy. As allergic sensitization, asthma, and elevated IgE have 19 

### been correlated in children, the evidence of association of blood Pb level with asthma and 20 

### allergy after adjusting for SES, housing conditions, and allergen exposures, may provide 21 

### support for the associations observed between higher blood Pb and higher IgE in children 22 

### ( Section 5.6.3 ). 23


### Studi es in nonoccupationally

- exposed adults did not find associations of biomarkers of 24 

### Pb exposure with asthma or allergy ( Pizent et al., 2008 ; Mendy et al., In Press )

. The 25 

### largest of these studies was a U.S. NHANES 2007

- 2008 analysis of adults ages 20 years 26 

### and older, in which urinary Pb level was not associated with an increase in asthma (OR: 27 

### 0.72 [95% CI: 0.46, 1.12] per 1 μ g/g increase in creatinine

- adjusted urine) ( Mendy et al., 28 

### In Press )

. The results did not provide strong evidence that urinary Pb level was associated 29 

### with other respiratory conditions such as emphysema or chronic bronchiti s either. In a 30 

### study of 216 adults without occupational Pb exposures, Pizent et al. ( 2008 ) found th at 31 

### among women, the association between concurrent blood Pb level and serum IgE was 32 

### statistically significant, whereas the association with positive SPT to common inhaled 33 

### allergens was not. Among men, higher concurrent blood Pb level was associated with 34 

### lo wer odds of positive SPT (OR: 0.92 [95% CI: 0.86, 0.98] but was not statistically 35 

### significantly associated with IgE. These findings appeared to be discordant because an 36 

### increase in IgE commonly mediates the acute inflammatory response to allergens. 37 

### However , the interpretation of the findings is difficult because only statistically 38February 2012 5

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### significant effect estimates were reported; thus it is not known whether odds ratios were 1 

### in the same direction for SPT and IgE. Bener et al. ( 2001b ) found hig her prevalence of 2 

### asthma and allergy

- related conditions such as rhinitis and dermatitis among Pb industrial 3 

### workers than among control subjects; however, the blood Pb levels in both the Pb

4 

### exposed group and the control group (geometric means: 77.5 and 19.8 μ g/dL, 5 

### respectively) were higher than those in the current U.S. general adult population. 6 

### In summary, an available recent epidemiologic study added evidence for associations 7 

### between blood Pb level and asthma in children ( Pugh Smith and Nriagu, 2011 ) , and a few 8 

### recent studies provided new eviden ce for associations with allergic conditions in children 9 

### ( Jedry chowski et al., 2011 ; 2010 ; Hon et al., 2009 )

. This epidemiologic evidence was 10 

### strengthened by several observations that associations were robust to the adjustment of 11 

### multiple potential confounding factors, in particular, SES an d allergen exposures. 12 

### Because of the heterogeneity in the relatively small body of evidence, it was difficult to 13 

### identify whether the strength of association with asthma and allergy differed by age of 14 

### children, lifestage of blood Pb measurement (prenatal, sometime in childhood prior to 15 

### outcome assessment, concurrent), or level of blood Pb. The epidemiologic evidence for 16 

### associations of blood Pb level with asthma and allergy is well supported by toxicological 17 

### and epidemiologic evidence indicating Pb effects on increasing IgE ( Section 5.6.3 ), Th2 18 

### cytokines ( Section 5.6.5.4 ), and inflammation ( Section 5.6.5.1 ). In the few recent studies 19 

### that investigated nonoccupationally

- exposed adults, Pb bi omarker levels were not 20 

### associated significantly with greater asthma or allergy ( Pizent et al., 2008 ; Mendy et al., 21 

### In Press )

. 22



### 5.6.4.3 Other Respiratory Effects
The respiratory effects of Pb have been examined primarily in a small number of recent 23 

### epidemiologic studies of nonoccupationally

- exposed adults, and as with evidence for 24 

### asthma, evidence for Pb

- associated respiratory effects in adults is weak. Increased 25 

### bronchial responsiveness (BR) is a characteristic feature of asthma and other respirator y 26 

### diseases and can result from the activation of innate immune responses and increased 27 

### airway inflammation. In a study of 525 middle

- aged adults in Seoul, Korea, Min et al. 28 

### ( 2008a ) found an association between concurrent blood Pb level and BR. A 1 μ g/dL 29 

### higher concurrent blood Pb level was associated with a higher BR index (log [% decline 30 

### in forced expiratory volume in 1 second (FEV 1 

### )/log of final methacholine concentration 31 

### in mg/dL]) of 0.018 (95% CI: 0.004, 0. 03), adjusting for age, sex, height, smoking, lung 32 

### function, and asthma diagnosis ( Min et al., 2008a )

. The concurrent blood Pb levels in 33 

### these adults were low (mean [SD]: 2.90 [1.59] μ g/dL); however, it is uncertai n what 34 

### timing, level, frequency, and duration of Pb exposures contributed to the observed 35February 2012 5

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### association. In contrast to Min et al. ( 2008a ) , Pizent et al. ( 2008 ) found that higher 1 

### concurrent blood Pb level was associated with lower BR in men (2.4% decrease [95% CI: 2



### - 4.2,

- 0.52%] in percent change FEV 1 

### post

- hist amine challenge per 1 μ g/dL increase in 3 

### blood Pb level). Although this finding was not discussed in details by investigators, it was 4 

### consistent with the observation that higher blood Pb level was associated with lower odds 5 

### of positive SPT among men in this study. 6 

### In studies of male Pb workers, Pb

- associated respiratory effects were not clearly 7 

### indicated. None of the studies directly examined associations between blood Pb levels 8 

### and lung function. In bus drivers in Hong Kong, China, ( Jones et al., 2008 ; Jones et al., 9 

### 2006 ) , drivers of non
- air conditioned buses had lower exposures to PM 10 

### , lower blood Pb 10 

### levels (mean 3.7 μ g/dL versus 5.0 μ g/dL in air conditioned buses) but lower indices of 11 

### lung function than did drivers of air conditioned buses ( Jones et al., 2006 )

. In this study, 12 

### the authors attributed the slightly higher blood Pb levels of air conditioned bus drivers to 13 

### the poor efficiency in the filters and higher PM 10 

### levels measured on those buses versus 14 

### the non
- air conditioned buses. In a comparison of roadside vendors and adjacent 15 

### shopkeepers, blood Pb levels and various lung function parameters were similar between 16 

### groups ( Jones et al., 2008 )

. Pb industrial workers in the United Arab Emirates had higher 17 

### prevalence of respiratory symptoms such as cough, phlegm, shortness of breath, and 18 

### wheeze than did unexposed controls ( Bener et al., 2001b )

. Blood Pb levels in both the Pb

19 

### exposed group and the control group (geometric means: 77.5 and 19.8 μ g/dL, 20 

### respectively) were higher than those in the current U.S. adult gen eral population, and the 21 

### analysis did not consider potential confounding variables. 22 

### Toxicological evidence for Pb

- associated respiratory effects is provided by observations 23 

### that exposure to Pb

- containing PM induces a range of inflammatory

- related effects in the 24 

### airways of animals and in cultured airway cells ( Section 5.6.6 ). Specific effects on the 25 

### lung also were demonstrated in a recent study of ra ts injected with Pb

- acetate (25 mg /kg, 26 

### 3 consecutive days, resulting in 2.1 μ g/dL blood Pb) ( Kaczynska et al., 2011 )

. Pb

- treated 27 

### rats exhibited ultrastructural changes in lung tissue, including substantial pulmona ry 28 

### fibrosis containing numerous lipofibroblasts, collagens, and elastin filaments in the 29 

### interstitium. Mast cells also were present in the interstitium following Pb exposure. 30 

### Pulmonary inflammation was observed, evidenced by the increased recruitment of 31



### mo nocytes and thrombocytes inside the capillary vessels and increased macrophage 32 

### accumulation in the alveolar space. Evidence of damaged surfactant lining and 33 

### destruction of the laminae inside lamellar bodies of epithelial type II cells also was 34 

### observed. Wh ile the observed pulmonary histological changes have been linked with 35 

### functional pulmonary decrements in other studies (unrelated to Pb exposure), they were 36 

### observed with injected Pb. It is not clear whether routes of Pb exposure more relevant to 37 

### those in humans would result in similar effects. 38February 2012 5

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### 5.6.4.4 Autoimmunity
Evidence for the effects of Pb on increasing the risk of autoimmunity is provided 1 

### primarily by a small number of older toxicological studies reviewed in the 2006 Pb 2 

### AQCD in which Pb exposure of animals ( pre
- and postnatal) was associated with the 3 

### generation of autoantibodies ( Hudson et al., 2003 ; Bunn et al., 2000 ; El

- Fawal et al., 4 

### 1999 ; Waterman et al., 1994 )

. Whereas some evidence linked this risk of autoimmunity 5 

### to a sh ift toward Th2 responses, other evidence pointed to a shift toward Th1 responses. 6 

### While recent studies did not examine Pb

- induced production of autoantibodies, some 7 

### provided indirect evidence by indicating that the changes induced by Pb had broader 8 

### implica tions for increasing risk of autoimmunity. For example, Kasten

- Jolly et al. ( 2010 ) 9 

### examined the impact of developmental (100 μ M Pb

- acetate in drinking water of dams 10 

### from GD8 to PND21, resulting in pup blood Pb levels 10

- 30 μ g/dL) Pb exposure of mice 11 

### on changes in gene expression in the spleen. Investigators found that Pb upregulated 12 

### digestive and catabolizing enzymes that could lead to the generation of self

- peptides, 13 

### which in conjunction with other Pb

- induced immunomodulator y effects, had the potential 14 

### to induce the generation of autoantibodies. In Carey et al. ( 2006 ) , the activation of 15 

### neoantigen

- specific T cells in PbCl 2 

### - treated adult mice (25

- 50 μ g i.p.) also indicated the 16 

### potential for autoantibody generation. Evidence of Pb

- associated autoimmune responses 17 

### in humans is limited to an older study of male Pb battery workers with blood Pb levels 18 

### ranging from 10 to 40 μ g/dL ( El

- Fawal et al., 1999 )

. In this study, the Pb

- exposed 19 

### workers had higher levels of IgM and IgG autoantibodies to neural proteins compared 20 

### with unexposed controls (blood Pb levels not reported) ( El

- Fawal et al., 1999 )

. Pb also 21 

### was found to modify neural proteins in rats ( Waterman et al., 1994 )

. The generation of 22 

### self

- peptides and modificatio n of proteins can result in formation of neoantigens, thereby 23 

### increasing the risk of autoimmune reactions. 24



### 5.6.4.5 Specialized Cells in Other Tissues

As discussed in Section 5.6.2.4 , Pb exposure consistently has been found to alter the 25 

### function of macrophages. Nonlymphoid tissues also contain specialized macrophages 26 

### whose altered function can contribute to organ/tissue dysfunction, cell death, tissue 27 

### patholo gy and tissue

- specific autoimmune reactions. Among the specialized macrophages 28 

### are microglia and astrocytes in the brain, Kupffer cells in the liver, alveolar macrophages 29 

### in the lung, keratinocytes and Langerhans cells in the skin, osteoclasts in the bone, and 30 

### preadipocytes in adipose tissue. The evidence demonstrating the effects of Pb on 31 

### specialized macrophages, which was provided by recent toxicological studies, is 32 

### important as it demonstrates the contribution of immune dysfunction to the effects of Pb 33



### o n dysfunction in nonlymphoid tissues ( Figure 5

- 44 ). Because these specialized cells are 34February 2012 5

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### not always recognized as macrophages, the resulting diseases an d conditions are not 1 

### always recognized as being linked with Pb

- induced immune dysfunction. It is important 2 

### to note that in this small body of available studies, several studies treated animals by Pb 3 

### injection, and it is unclear whether the effects would be observed with routes of exposure 4 

### that are more relevant for humans. 5



### Figure 5
- 44 Specialized macrophages in nonlymphoid tissue may serve as a link between Pb exposure and disease in multiple organ systems.

Fan et al. ( 2009b ) reported that Kupffer cells undergo significant changes in phenotypic 6 

### expression ( e.g., CD68 and ferritin light chain), organization, and functional activity 7 

### connected to Pb

- induced apoptosis in the liver. Dosing of juvenile Wistar rats with Pb 8 

### injections (15 mg /kg of Pb

- acetate daily for 2 weeks, resulting in a mean blood Pb level 9 

### of 30 μ g/dL) during early postnatal maturation was observed to produce chronic glial 10 

### activ ation, increase pro

- inflammatory cytokines, and increase neurodegeneration, as 11 

### indicated by an increase in GFAP, increase in IL

- 1β, TNF

- α and IL

- 6 cytokines, and a 12 

### decrease in synaptophysin (component of presynaptic vesicles), respectively, in brain 13 

### tissue ( Struzynska et al., 2 007 )

. In bone, osteoclasts regulate osteoblast function ( Chang 14 

### et al., 2008a ) , and osteoblasts have been shown to be affected by Pb exposure. Effects on 15 

### these cell types can contribute to later life diseases such as arthritis [reviewed in Zoeger 16 

### et al. ( 2006 ) ] . Pb

- induced elevation of TGF

- β production was found to be involved in 17 

# Pb

Osteoblasts and Osteoclasts

Periodontitis and Chondrogenesis Kupffer cells

Altered liver metabolism Oxidative damage Glutathione status Microglia and Astrocytes

Neurobehavioral and neurodegenerative conditions Alveolar macrophages

Bronchial hyper

- reactivity Respiratory allergy Asthma Susceptibility to infections Resident bone macrophagesFebruary 2012 5

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### chondrogenesis in bone ( Zuscik et al., 2007 )

. Kaczynska et al. ( 2011 ) reported effects on 1 

### alveolar macrophages after Pb treatment (i.p. 25 mg /kg, 3 days, resulting in blood Pb 2 

### levels of 2.1 μ g/dL) in rats. Macrophage recruitment increased, and this macrophage 3 

### infiltration limited air space available to gas exchange and contained part s of 4 

### phagocytized surfactant and alveolar lining. 5 

### Resident immune cells in reproductive organs have been shown to be affected by Pb 6 

### exposure. Pace et al. ( 2005 ) reported that Pb exposure in mice contributed to poor 7 

### reproductive performance that was concomitant with altere d homeostasis of the testicular 8 

### macrophage population in that organ. The authors proposed that increased oxidative 9 

### damage and apoptosis among these macrophages and reduced potential to maintain organ 10 

### homeostasis contributed to the observed pattern of male sterility. 11



### 5.6.4.6 Tumors
While toxicological evidence indicates that high concentration Pb exposures directly 12 

### promote tumor formation or induce mutagenesis and genotoxicity ( Section 5.10 ), 13 

### evidence for involvement of the immune system is limited. Kerkvliet and Baecher

14 

### Steppan ( 1982 ) observed that male C57Bl/6 mice exposed to 130 and 1 , 300 ppm of 15 

### Pb

- acetate in drinking water had enhanced moloney sarcoma virus

- induced tumor growth 16 

### compared with control animals. The findings indicated that Pb

- induced 17 

### immunomodulation affecting tumors likely resulted from a combination of suppressed 18 

### Th1 responses and increased inflammation leading t o excessive release of ROS into 19 

### tissues. The promotion of cancer is a relatively common outcome in chemical

- induced 20 

### immunotoxicology, particularly when early life exposures are involved ( Dietert, 2011 )

. 21



### 5.6.5 Modes of Action for Lead Immune Effects

5.6.5.1 Inflammation
The 2006 Pb AQCD indicated that misregulated inflammation represents one of the 22 

### major immune

- related effects of Pb and a major mode of action for Pb effects in multiple 23 

### organ systems such as the liver, kidney, and vasculature ( Section 5.2.5 ). In the 2006 Pb 24 

### AQCD, several lines of toxicological evidence demonstrated Pb

- induced inflammation: 25 

### Pb

- induced increases in PGE 2 

### and ROS ( Section 5.6.5.2 ) and increases in pro

26 

### inflammatory cytokines ( Section 5.6.5.4 ), both of which promote a hyperinflammatory 27 

### phenotype in immune cells. As described in sections that follow, these findings are 28February 2012 5

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### corroborated by a small number of available recent toxicologica l studies. A few available 1 

### recent epidemiologic studies provided new evidence of Pb

- associated inflammation, as 2 

### indicated by associations of blood Pb levels with pro

- inflammatory cytokines and other 3 

### nonspecific indicators of inflammation. 4


### Enhanced inflamma tion and tissue damage occurs through the modulation of 5 

### inflammatory cell function and production of pro

- inflammatory cytokines and 6 

### metabolites. It is important to note that while inflammation can mediate immune

- related 7 

### conditions such as asthma, respirato ry infections, and BR, inflammation can exacerbate 8 

### disease and damage in almost any organ given the distribution of immune cells as both 9 

### permanent residents and infiltrating cell populations. Among the problems presented by 10 

### this immunomodulation are the ov erproduction of ROS and an apparent depletion of 11 

### antioxidant protective enzymes and factors ( e.g., selenium). For example, toxicological 12 

### studies have found that Pb

- induced inflammatory damage involves the depletion of 13 

### antioxidants such as glutathione and c atalase ( Lodi et al., 2011 ; Chetty et al., 2005 )

. 14 

### While several processes have been proposed to explain the mechanisms of Pb

- induced 15 

### oxidative damage, the exact combination of processes involved remains to be determined 16 

### ( Section 5.2.4 ). 17 

### In epidemiologic studies, Pb

- associated changes in pro

- inflammatory cell function 18 

### ( Section 5.6.2 ) and cytokine production ( Section 5.6.5.4 ) have been found in the 19 

### cumulative body of evidence. A few recent epidemiologic studies added to this evidence 20 

### with observations of associations between blood Pb level and other nonspecific i ndicators 21 

### of inflammation that may be related to multisystemic effects as have been demonstrated 22 

### in animal and in vitro studies. Using 1999

- 2004 NHANES data, Songdej et al. ( 2010 ) 23 

### examined the relationship between concurrent blood Pb levels and the inflammation 24 

### markers, C

- reactive protein (CRP), fibrinogen, and white blood cell (WBC) count in 25 

### adults 40 years of age or older. Adjusting for age, sex, race/ethnicity, education, income, 26 

### BMI, physical activity, smoking status, diabetes status, inflammator y disease status, and 27 

### cardiovascular disease status, investigators found larger magnitudes of association 28 

### between blood Pb and inflammation in men compared with women. Among women, 29 

### most ORs for associations between quintiles of blood Pb level and tertiles of CRP, 30 

### fibrinogen, and WBC count were less than 1.0 whereas corresponding ORs in men tended 31 

### to be greater than 1.0 but were not always statistically significant. For example, compared 32 

### with men with concurrent blood Pb levels less than 1.16 μ g/dL, men with blood Pb levels 33 

### of 1.16

- <1.63 μ g/dL, 1.63

- <2.17, 2.17

- <3.09 μ g/dL, and ≥ 3.09 μ g/dL had elevated odds 34 

### of elevated CRP (OR [95% CI]: 2.22 [1.14, 4.32], 1.67 [0.85, 3.28], 2.12 [1.07, 4.21], and 35 

### 2.85 [1.49, 5.45], respectively). For all inflammation markers , although the OR was 36 

### highest in the highest quintile of blood Pb level (≥ 3.09 μ g/dL), monotonic concentration

37 

### dependent increases were not observed. 38February 2012 5

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### Consistent with these findings, among men in Incheon, Korea without occupational Pb 1 

### exposures, Kim et al. ( 2007 ) reported associations of higher concurrent blood Pb level 2 

### with higher levels of WBCs and IL

- 6. Larger effects were estimated for men in the upper 3 

### two q uartiles of blood Pb levels, 2.51

- 10.47 μ g/dL than for the full range of blood Pb 4 

### levels. The findings are consistent with Pb effects on promoting a Th2 phenotype. Th2 5 

### cells produce IL

- 6 which is the primary stimulus for expression of CRP and fibrinogen 6 

### ( Hage and Szalai, 2007 ; Fuller and Zhang, 2001 )

. Despite the low concurrent blood Pb 7 

### level of adul ts in these aforementioned studies, it is important to acknowledge that the 8 

### relative contributions of recent versus past Pb exposures to the associations observed with 9 

### concurrent blood Pb levels are not delineated. 10 

### In a genome

- wide association study that included 37 children with autism and 15 children 11 

### without autism (ages 2

- 5 years; blood Pb level range: 0.37 to 5.2 μ g/dL) in California, in 12 

### models that included age, sex, and autism diagnosis, concurrent blood Pb level was 13 

### associated with the expression of several genes related to immune function and 14 

### inflammation, including human leukocyte antigen genes (HLA

- DRB) and MHC Class II

15 

### associated invariant chain CD74 (involved in antigen presentation) ( Tian et al., 2011 )

. 16 

### Although blood Pb levels were similar between children with and withou t autism and 17 

### correlations were observed in both groups, they were in opposite directions (positive 18 

### among children with autism and negative among children without autism). Pb has been 19 

### shown to increase MHC molecule surface expression in mouse and human HLA antigen 20 

### presenting cells ( Guo et al., 1996a ; McCabe and Lawrence, 1991 ) ; however, additional 21 

### larger studies with a priori hypotheses regarding specific indices of inflammation are 22 

### warranted to characterize Pb

- associated changes in inflammation in children. 23



### 5.6.5.2 Increased Prostaglandi n E 2 

### and Decreased Nitric Oxide
Consistent with the large body of evidence presented in the 2006 Pb AQCD ( U.S. EPA, 24 

### 2006b ) , a small number of available recent studies continued to indicate that Pb exposure 25 

### alters the levels of signaling molecules such as PGE 2 

### and NO. Collectively, the weight of 26 

### evidence was provided by toxicological studies. These signaling molecules are invol ved 27 

### in mediating inflammation and host resistance ( Figure 5

- 42 ). A recent in vitro study with 28 

### human neuroblastoma cells found increases in PGE 2 

### with l ower Pb concentrations (0.01

- 29 

### 1 μ M) than those previously reported ( Chetty et al., 2005 )

. A l arge body of evidence 30 

### reviewed in the 2006 Pb AQCD demonstrated a Pb

- associated decreased production of 31 

### NO by macrophages [see 2006 Annex Table AX5.9.6 ( U.S. EPA, 2006h ) ]

. 32 

### In adult animal models, decreases in NO were observed with short

- term exposures (hours 33 

### to days) to a wide range of Pb concentrations. Decreases in NO can impact not only 34February 2012 5

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### innate host defenses, but also, acquired immunity. In a recent study, Farrer et al. ( 2008 ) 1 

### found that high

- level Pb glutamate exposure (5 μ M) of lymphocyte cult ures led to 2 

### decreased NO production, decreased inducible NO synthase function in myeloid cells, 3 

### but no change in its gene expression. Additionally, Pb abrogated the myeloid cell 4 

### (CD11b+)

- mediated suppression of CD4+ T cell proliferation. Together, these fi ndings 5 

### indicated that Pb may indirectly enhance T cell proliferation through its effect on 6 

### decreasing NO production. Combined with the observation that Pb can alter antigen 7 

### processing ( Farrer et al., 2005 ) and, hence, the quality and magnitude of the acquired 8 

### immune response signal against pathogenic challenge, evidence indicated that multiple 9 

### arms of the host defense against infectious challenge can be c ompromised. The loss of 10 

### NO production in innate immune cells such as macrophages would be expected to affect 11 

### other physiological systems ( e.g., neurological, cardiovascular, endocrine) that require 12 

### NO signaling cascades. 13


### Relative to studies in animal and in vitro models, fewer epidemiologic studies have 14 

### examined the effects of Pb on signaling molecules; however the limited data supported 15 

### associations of blood Pb level with suppressed NO production ( Barbosa et al., 2006a ; 16 

### Pineda

- Zavale ta et al., 2004 ) and increased ROS production ( Pineda

- Zavaleta et al., 17 

### 2004 ) in populations living near Pb sources. In a previous s tudy of children in Mexico, 18 

### with increasing residential proximity to the Pb smelter, mean concurrent blood Pb levels 19 

### increased (7.02 to 20.6 to 30.38 μ g/dL) as did superoxide anion release from 20 

### macrophages (directly activated by IFN

- γ/LPS) isolated from ch ildren ( Pineda

- Zavaleta 21 

### et al., 2004 )

. NO release from macrophages (indirectly activated by phytohemagglutinin, 22 

### PHA) was lower with higher blood Pb levels. After adjusting for age and sex, a 1 μ g/dL 23 

### higher blood Pb level was associated with a higher level of superoxide anion of 0.00389 24 

### (95% CI: 0.00031, 0.00748) μ mol/mg protein and a lower level of NO of 0.00089 (95% 25 

### CI:

- 0.0017,

- 0.00005) nmol/μg protein. Because PHA activates macrophages indir ectly 26 

### through the activation of lymphocytes and IFN

- γ directly activates macrophages, these 27 

### results indicated that Pb suppressed T cell

- mediated macrophage activation and 28 

### stimulated cytokine

- induced macrophage activation. Group

- level comparisons indicated 29 

### that associations likely were driven by changes observed in the group of children living in 30 

### closest proximity to the smelter who had blood Pb levels 10.31

- 47.49 μ g/dL. Results also 31 

### demonstrated a larger magnitude of association between blood Pb levels and superoxide 32 

### anion release in males. Although not described in detail, higher blood Pb level was not 33 

### associated with lower NO in girls. Barbosa et al. ( 2006a ) also observed an association 34 

### between higher blood Pb level and lower plasma NO in a group of adults in Sao Paolo, 35 

### Brazil residing near a closed battery plant, particularly among adults with the TC or CC 36 

### eNOS genotype (r = 0.23, p = 0.048) which is associated with reduced promoter activity 37 

### and potentially reduced gene expression. Q uantitative results were not reported for 38 

### analysis of all adults combined or adults with the TT genotype, but p

- values were greater 39February 2012 5

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### than 0.05. Because NO was measured in plasma, it was not possible to identify immune 1 

### cells as the specific sources of NO. 2


### S tudies of occupationally

- exposed adults provided less clear indication of associations of 3 

### blood Pb level with NO and ROS. Despite large differences in blood Pb levels between 4 

### 30 male Pb recycling plant workers (mean: 106 μ g/dL) and 27 unexposed controls (m ean: 5 

### 4.5 μ g/dL), levels of ROS released from neutrophils (indicators of respiratory burst) were 6 

### similar between groups ( Mishra et al., 2006a )

. In a study of male foundry workers (mean 7 

### blood Pb level: 21.7 μ g/dL), pottery workers (mean blood Pb level: 9.7 μ g/dL), and 8 

### unexposed workers (mean blood Pb level: 3.9 μ g/dL), Valentino et al. ( 2007 ) found 9 

### similar plasma NO levels in controls compared with Pb

- exposed workers. Also, although 10 

### quantitative results were not reported, blood Pb level was reported not to be correlated 11 

### with NO. 12



### 5.6.5.3 Cellular Death (Apoptosi s, Necrosis)
The 2006 Pb AQCD reviewed a small number of toxicological studies in which Pb 13 

### exposure had contrasting effects on the apoptosis of macrophages. Since then, a few 14 

### available recent toxicological studies found that Pb exposure induces apoptosis o r 15 

### mediators of apoptosis in immune cells. In a study in mice, Bishayi and Sengupta ( 2006 ) 16 

### found that Pb treatment of adult mice (10 mg /kg, i.p.) elevated DNA fragmentation in 17 

### splenic macrophages. Using mouse resident peritoneal macrophages, Gargioni et al. 18 

### ( 2006 ) found that 20 and 40 μ M Pb nitrate induced both necrosis and apoptosis in vitro. 19 

### While the exact pathways involved were not determined, the authors concluded that 20 

### activation of the Bax pro

- apoptotic protein was not the key eff ect of Pb on inducing 21 

### macrophage apoptosis. In an in vivo study in 3 week

- old mice, Xu et al. ( 2008 ) found 22 

### that a 4

- week administration of Pb

- acetate (50

- 100 mg /kg, oral) significantly elevated 23 

### both ROS a nd malondialdehyde (an indicator of ROS

- induced peroxidation) levels in 24 

### peripheral blood lymphocytes. Pb also induced DNA damage (determined by the comet 25 

### assay), which was accompanied by elevations in p53 and Bax expression with no change 26 

### in Bcl

- 2 expressi on (creating a Bax/Bcl

- 2 imbalance). The authors proposed that 27 

### oxidative stress was a likely route to Pb

- induced apoptosis and tumorigenesis. Because of 28 

### the high Pb concentrations administered and systemic administration of Pb employed, 29 

### other studies are w arranted to examine whether apoptosis of macrophages represent a 30 

### mode of action relevant to human Pb exposures. 31February 2012 5

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### 5.6.5.4 Cytokine Production
The 2006 Pb AQCD presented a large body of toxicological evidence that clearly 1 

### demonstrated Pb

- induced changes in cytokine p roduction in vivo and in vitro ( U.S. EPA, 2 

### 2006b )

. Previous toxicological studies indicated that Pb affects immune cytokine 3 

### production via action on T cells and macrophages. The combination of cytokine changes 4 

### induced by Pb in mul tiple cell types can create a hyperinflammatory state among innate 5 

### immune cells to skew acquired immunity responses away from Th1 responses and toward 6 

### Th2 responses. As illustrated in Figure 5

- 42 , downstream effects include altered IgE 7 

### production, ROS production, and inflammation. In support of a Pb

- induced skew toward 8 

### Th2 responses, several studies in rats, mice, and chickens found t hat pre

- and postnatal 9 

### Pb exposure suppressed the production of Th1 cytokine IFN

- γ and/or increased 10 

### production of Th2 cytokines such as IL

- 4 [ Table 5

- 7 of the 2006 Pb AQCD ( U.S. EPA, 11 

### 2006g ) ] . Recent toxicological studies continued to find such Pb

- induced changes in 12 

### cytokine production, and a study provided new evidence that Th2 skewing may be 13 

### mediated via effects on dendritic cells. 14


### Che ng et al. ( 2006 ) found that Pb exposure affected TNF

- α production in vitro in A/J 15 

### mice macrophages by affecting the mitogen

- activated protein kinase (MAPK) signaling 16 

### pathway. Pb

- acetate (10 μ M) co

- administered with LPS stimulated the phosphorylation 17 

### of p42/44 MAPK and TNF

- α expression ( Cheng et al., 2006 )

. Blocking protein kinase C 18 

### or MAPK reduced TNF

- α production of macrophages in vitro, which in turn, protected 19 

### against Pb + LPS

- induced liver injury in vivo. Thus, Pb exposure may induce local tissue 20 

### damage through the modulation of immune responses. These findings were consistent 21 

### with those from recent studies. Gao et al. ( 2007 ) showed that treatment of mouse 22 

### dendritic cells with 25 μ M PbCl 2 

### prod uced an increased phosphorylation of the 23 

### Erk/MAPK signaling molecule, and Khan et al. ( 2011 ) showed that mon ocytes treated 24 

### with 25

- 50 μ M Pb

- acetate increased TNF

- α through ERK1/2 and p38 signaling. 25 

### In an in vivo study conducted across a lifetime (developmental through adulthood) in 26 

### Swiss mice (females and males) using a broad range of dietary Pb concentrations, Iavicoli 27 

### et al. ( 2006a ) found a nonlinear hierarchical cytokine response. At the lowest dietary Pb 28 

### concentration (0.11 ppm Pb

- acetate , resulting in blood Pb level: 1.6 μ g/dL), IL

- 2 and 29 

### IFN

- γ were decreased compared to those in the controls (0.02 ppm Pb

- acetate , resulting 30 

### in blood Pb level: 0.8 μ g/dL), indicating a suppressed Th1 response. As dietary Pb 31 

### exposure increased (resulting in blo od Pb levels 12

- 61 μ g/dL), a Th2 phenotype was 32 

### observed with suppressed IFN

- γ and IL

- 2 and elevated IL

- 4 production. These findings 33 

### support the notion that the immune system is differentially modulated by low

- level versus 34 

### high

- level Pb exposures. Other stu dies found variable Pb

- induced changes in IL

- 2, with 35 

### no change or elevated production, depending upon the protocol used. Recently, Gao et al. 36February 2012 5

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### ( 2007 ) found that Pb

- treated dendritic cells (25 μ M) promoted a slight but statistically 1 

### significant increase in IL

- 2 production (quantitative results not reported) among 2 

### lymphocytes. 3 

### In vitro studies also reported a Pb

- induced shift to production of Th2 cytokines. In 4 

### conjunction with other indicato rs of Th2 skewing des cribed in Sections 5.6.2.3 , 5.6.2.6 , 5 

### and 5.6.3 , Gao et al. ( 2007 ) observed that 25 μ M Pb elevated the production of Th2 6 

### cytokines such as IL

- 4, IL

- 5 and IL

- 6 in dendritic cells. In cultures of human PMNs 7 

### activated with Salmonella enteritidis or with monoclonal antibodies of CD3, CD28, and 8 

### CD40, Pb

- acetate concentrations of 0.15 μ g/dL and higher suppressed expression of Th1 9 

### cytokines, IFN

- γ, IL

- 1β, and TNF

- α, and increased secretion of Th2 cytokines, IL

- 5, IL

10 

### 6, and IL

- 10 ( Hemdan et al., 2005 )

. 11 

### Consistent with toxicological studies, a few available epidemiologic studies also found 12 

### higher concurrent blood Pb levels in children and occupationally

- exposed adults to be 13 

### associated with a shift toward producti on of Th2 cytokines relative to Th1 cytokines. The 14 

### evidence in children was based on comparisons of serum cytokine levels among groups 15 

### with different blood Pb levels without consideration of potential confounding factors. 16 

### Among children ages 9 months to 6 years in Missouri, Lutz et al. ( 1999 ) found that 17 

### children with concurrent blood Pb levels 15

- 19 μ g/dL had higher serum levels of IL

- 4 (p 18 

### = 0.08, Kruskal Wallis) and IgE ( Section 5.6.3 ) than did children with lower blood Pb 19 

### levels. However, IL

- 4 levels in children with blood Pb levels 20

- 44 μ g/dL were lower 20 

### than those in children with blood Pb levels < 15 μ g/dL. The elevated IL

- 4 and IgE in 21 

### children with blood Pb levels 15

- 19 μ g/dL were consistent with the mode of action for 22 

### IL

- 4 to activate B cells to induce B cell class switching to IgE. In another study of 23 

### children in grades 5 and 6 in Taiwan, investigators did not group children b y blood Pb 24 

### levels but by potential for Pb exposures due to age of home and location of residence 25 

### ( Hsiao et al. )

. Concurrent blood Pb levels did not differ by residence in old versus new 26 

### homes or by urban versus rural residence (means: 3.2

- 3.8 μ g/dL) but were higher among 27 

### children living near an oil refinery, in particular, among children with known respiratory 28 

### allergies (mean: 8.8 μ g/dL). This latter group of children also had the lowest serum levels 29 

### of IFN

- γ (45

- fold) and highest levels of IL

- 4 (6

- fo ld) (lower p < 0.05 for comparisons 30 

### with any subgroup). There was no direct comparison of cytokine levels between blood Pb 31 

### level groups in the population overall; however, cytokine levels were similar between 32 

### healthy and allergy groups in the other Pb sour ce groups that had similar blood Pb levels 33 

### (p > 0.05 for comparisons with any subgroup). Thus, the differences in cytokine levels 34 

### between healthy and allergic children living near the oil refinery may have been 35 

### influenced by differences in their blood Pb l evels. 36February 2012 5

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### Evidence of association between blood Pb levels and cytokine levels in 1 

### nonoccupationally

- exposed adults was equivocal. Among adult university students in 2 

### Incheon, Korea, Kim et al. ( 2007 ) found associations of concurrent blood Pb level with 3 

### serum levels of TNF

- α and IL

- 6 that were larger among males in the upper two quartiles 4 

### of blood Pb levels, 2.51

- 10.47 μ g/dL. Notably, the relativ e contributions of recent versus 5 

### past Pb exposures to these associations are not known. In models that adjusted for age, 6 

### BMI, and smoking status, a 1 μ g/dL higher blood Pb level was associated with a 0.75 7 

### (95% CI: 0.14, 1.36) pg/ mL higher TNF

- α and a 0.18 (95% CI:

- 0.02, 0.38) pg/ mL higher 8 

### IL

- 6. The association between levels of blood Pb and plasma TNF

- α was greater among 9 

### men who were GSTM1 null (1.14 [95% CI: 0.20, 2.10] pg/ mL higher per 1 μ g/dL higher 10 

### blood Pb level) than men who were GSTM1 sufficient (0. 12 [95% CI:

- 0.43, 0.67] 11 

### pg/ mL ). Blood Pb also was associated with a larger increase in TNF

- α among men who 12 

### had the TNF

- α GG genotype (0.80 [95% CI: 0.15, 1.45] pg/ mL higher per 1 μ g/dL higher 13 

### blood Pb level) than men who had the GA or AA genotype (

- 0.21 [ 95% CI:

- 1.1, 0.71] 14 

### pg/ mL ). For the association between blood Pb level and plasma IL

- 6, the effect estimate 15 

### was slightly elevated in TNF

- α GG genotype but not elevated in the GSTM1 positive 16 

### group. The effects of Pb on several physiological systems have bee n hypothesized to be 17 

### mediated by the generation of ROS ( Daggett et al., 1998 )

. Thus, it is biologically 18 

### plausible that the null variant of GSTM1, which is associated with reduced elimination of 19 

### ROS, may increase the risk of Pb

- associated immune eff ects. The results for the TNF

- α 20 

### polymorphism were difficult to interpret. The GG genotype is associated with lower 21 

### expression of TNF

- α, and the literature is mixed with respect to which variant increases 22 

### risk of inflammation

- related conditions. Among adult s in Italy, concurrent blood Pb 23 

### levels were not statistically significantly correlated with either Th2 or Th1 cytokine levels 24 

### in men ( Boscolo et al., 1999 ) or women ( Boscolo et al., 2000 ) (quantitative results not 25 

### reported). 26


### Results from studies of occupationally

- exposed adults also suggested that Pb exposure 27 

### may be associated with decreases in Th1 cytokines and increases in Th2 cytokines; 28 

### however, analysis were mostly limited to comparisons of mean cytokine levels among 29 

### different blood Pb groups or Pb exposure groups ( Di Lorenzo et al., 2007 ; Valentino et 30 

### al., 2007 ; Yucesoy et al., 1997a ) that did not consider potential confounding variables. 31 

### The exception was a study of male foundry workers, pottery workers, and unexposed 32 

### workers ( Valentino et al., 2007 )

. Multiple regression analyses were performed with age, 33 

### BMI, smoking, and alcohol consumption in cluded as covariates; however, regression 34 

### coefficients describing the concentration

- response functions were not reported. Pb

35 

### exposed workers had higher IL

- 10 and TNF

- α (ANOVA, p < 0.05). Levels of IL

- 2, IL

- 6, 36 

### and IL

- 10 also increased from the lowest to hig hest blood Pb group (ANOVA, p > 0.05). 37 

### In contrast with most other studies, both exposed worker groups had lower IL

- 4 levels 38 

### compared with controls (ANOVA, p > 0.05). In a similar analysis, DiLorenzo et al. 39February 2012 5

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### ( 2007 ) separated exposed workers into intermediate (9.1

- 29.4 μ g/dL) and high (29.4

- 1 

### 81.1 μ g/dL) blood Pb level groups, with unexposed workers comprising the low exposure 2 

### group (blood Pb levels 1

- 11 μ g/dL). Mean TNF

- α levels show ed a monotonic increase 3 

### from the low to high blood Pb level group. Results also indicated a potential interaction 4 

### between blood Pb level and smoking. Among current smokers, a 12

- to 16

- fold difference 5 

### in TNF

- α levels was observed among blood Pb groups. Amo ng nonsmokers, the 6 

### differences were less than two fold. In Yucesoy et al. ( 1997a ) , levels of the Th1 7 

### cytokines, IL

- 1β and IFN

- γ, were lower in workers than in controls. 8 

### In summary, a large body of previous toxicological evidence clearly demonstrate d Pb

9 

### induced increases in Th2 cytokines and decreases in Th1 cytokines. Several recent 10 

### toxicological studies added to this evidence by showing Pb

- induced increases in TNF

- α 11 

### that were mediated by MAPK signaling pathways ( Khan et al., 2011 ; Gao et al., 2007 ; 12 

### Cheng et al., 2006 ; Iavicoli et al., 2006a ; Hemdan et al., 2005 ) , a nd demonstrating Pb

13 

### induced increases in cytokine production in dendritic cells ( Gao et al., 2007 )

. The 14 

### collective epidemiologic evidence is sparse; however, in the few available new studies , 15 

### higher concurrent blood Pb level was associated with higher levels of various pro

16 

### inflammatory cytokines in children, nonoccupationally

- exposed adults, and 17 

### occupationally

- exposed adults. The recent epidemiologic study of children in Taiwan 18 

### found an assoc iation between higher IL

- 4 levels in children with higher blood Pb levels 19 

### ( Hsiao et al. ) , similar to a previous study of children in Missouri ( Lutz et al., 1999 )

. 20 

### Neither study of children considered potential confounding variabl es. In contrast to 21 

### previous studies, a recent study in nonoccupationally

- exposed adults found associations 22 

### between higher concurrent blood Pb level and higher TNF

- α and IL

- 6 cytokine levels in 23 

### men in Korea ( Kim et al., 2007 )

. Recent studies of occupationally

- exposed adults added 24 

### to the evidence for higher levels of several cytokines among Pb

- exposed workers 25 

### compared to unexposed controls ( Di Lorenzo et al., 2007 ; Valentin o et al., 2007 )

. Due to 26 

### the limited investigation, it is difficult to draw conclusions about the effects of Pb 27 

### exposure on cytokine levels in any particular group in the human population. 28



### 5.6.6 Air
- Lead Studies

Al though comprising a smaller body of evidence than do immune studies of Pb 29 

### biomarkers, several recent studies used Pb measured in PM 10 

### and PM 2.5 

### air samples to 30 

### represent Pb exposures. Some studies analyzed the Pb component individually, whereas 31 

### others analy zed Pb as part of a group of correlated components using source 32 

### apportionment techniques or principal component analysis. In concordance with blood Pb 33 

### studies, recent time

- series epidemiologic studies that examined ambient air Pb

- PM 34 

### concentrations found as sociations with respiratory morbidity in children ( Gent et al., 35February 2012 5

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### 2009 ; Hong et al., 2007b )

. Adjusting for season, day of week, and date, Gent et al. ( 2009 ) 1 

### found that an increase in lag 0

- 2 average Pb

- PM 2.5 

### was associated with elevated odds of 2 

### wheeze (OR: 1.03, p = 0 .13 per 5 ng/m 3 

### increase in Pb PM 2.5 

### ), shortness of breath (OR = 3 

### 1.12, p = 0.01), and fast

- acting inhaler use (OR: 1.04, p = 0.10) among children with 4 

### asthma (ages 4

- 12 years), followed for 1 year during 2000

- 2003. Among children in 5 

### Korea (grades 3

- 6), a 1 log increase in lag 1 Pb PM 10 

### was associated with a

- 6.83 L/min 6 

### decrease (p < 0.01) in morning peak expiratory flow and a − 6.37 L/min decrease 7 

### (p < 0.01) in daily average peak expiratory flow adjusting for age, sex, height, weight, 8 

### smoking exposure, and m eteorological factors ( Hong et al., 2007b )

. Older and recent 9 

### toxicological studies also found Pb

- containing CAPs to induce pulmonary inflammation. 10 

### Uzu et al. ( 2011 ) specifically tested particles emitted at a Pb recycling plant and found 11 

### that these Pb

- rich particles increased the release of the cytokine granuloc yte

- macrophage 12 

### colony

- stimulating factor from human epithelial cells. Other toxicological studies found 13 

### pulmonary inflammation induced by CAPs in which Pb was one of numerous 14 

### components ( Wei et al., 2011 ; Duvall et al., 2008 ; Godleski et al., 2002 ; Saldiva et al., 15 

### 2002 )

. As with blood Pb, epidemiologic studies did not consistently find associations of 16 

### ambie nt air Pb

- PM concentrations with respiratory

- related hospitalizations or mortality in 17 

### older adults. Both studies adjusted for meteorological factors and for temporal trends. 18 

### Among adults ages 65 years and older in 6 California counties, a 4 ng/m 3 

### increase in lag 19 

### 3 Pb

- 

### PM 2.5 

### was associated with an increased relative risk of respiratory mortality all year 20 

### of 1.01 (95% CI: 0.99, 1.03) and during summer months (quantitative results not 21 

### reported) ( Ostro et al., 2007 )

. However, among adults ages 65 years and older in 106 U.S 22 

### counties, Bell et al. ( 2009 ) found that an increase in lag 0 Pb

- 

### PM 2.5 

### was associated with a 23 

### decrease in respiratory hospital admissions. The 95% CI was wide, indicating lack of 24 

### precision in effect estimate. 25 

### Although limited available recent findings suggest a relationship between respiratory 26 

### effects in children and short

- term (over several days) changes in ambient air Pb

- PM 27 

### concentrations, it is important to note uncertainties that limit the ability to draw 28 

### conclusio ns regarding airborne Pb exposure. Size distribution data for Pb

- PM are 29 

### relatively limited, so it is difficult to assess the representativeness of these concentrations 30 

### to population exposure (Section 3.5.3). Moreover, data on the relationship between blood 31 

### Pb and air Pb are relatively limited (see Section 4.5.1) and do not characterize 32 

### relationships of varying Pb

- PM size distribution with blood Pb level. I n several air

- Pb 33 

### studies, other PM components such as elemental carbon (EC) , copper (Cu) , and zinc (Zn) 34 

### also were associated with respiratory effects. In the absence of detailed data on 35 

### correlations among all PM components, measurements on other co

- occurring ambient 36 

### pollutants, or results adjusted for copollutants, it is difficult to exclude confounding by 37 

### ambient air exposures to other PM components or ambient pollutants. In several studies 38 

### that analyzed PM component mixtures, of which Pb particles comprised one component, 39February 2012 5

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### it is not possible to attribute the observed associations or lack of associations spe cifically 1 

### to Pb ( Sarnat et al., 2008 ; Andersen et al., 2007 ; Veranth et al., 2006 ; Maciejczyk and 2 

### Chen, 2005 )

. 3



### 5.6.7 Immune Effects of Lead within Mixtures
Several toxicological studies published since the 2006 Pb AQCD examined immune 4 

### effects resulting from exposures to metal mixtures. Some studies indicated that immune 5 

### effects may be observed with lower levels of Pb exposure when they occur in conjunction 6 

### wi th other metals. In a study of mice treated with Pb

- acetate (10 mg /kg i.p. injection by 7 

### weight, daily for 15 days), As (0.5 mg /kg i.p. injection by weight, daily for 15 days), or 8 

### both, Bishayi and Sengupta ( 2006 ) reported a greater than additive ef fect of co

9 

### administered Pb and As on macrophages in decreasing bacterial resistance, 10 

### myeloperoxidase (MPO) release, and NO production. Investigators assessed the Pb

- As 11 

### interaction on MPO release using the multivariate ANOVA and constructing an 12 

### isobologram by running an ordinary least squares regression between effects (% MPO 13 

### release) and dose levels of metals (single and multimetal) in log

- linear form. 14 

### Epidemiologic studies have not widely examined interactions between Pb and other 15 

### metals. However, consiste nt with Bishayi and Sengupta ( 2006 ) , Pineda

- Zavaleta et al. 16 

### ( 2004 ) ( Section 5.6.5.2 ) found interactions between Pb and As among children in Mexico 17 

### aged 6

- 11 years. Contamination of drinking water by both Pb and As was a concern in 18 

### the study area; however, urinary As levels were higher in children who had lower blood 19 

### Pb levels. In multiple regression analyse s, urinary As was associated with lower NO 20 

### release from macrophages (similar to blood Pb). An interaction was observed between Pb 21 

### and As, which indicated that high internal doses of both metals were associated with a 22 

### larger decrease in NO than was either m etal alone (p for interaction = 0.037). Urinary As 23 

### was associated with lower superoxide anion release (opposite direction of Pb). The 24 

### interaction between Pb and As indicated that higher internal doses of both Pb and As 25 

### were associated with a larger increas e in superoxide anion than was blood Pb level alone 26 

### (p for interaction = 0.042). Due to the high blood Pb in these children (means in three 27 

### groups at varying distances from a Pb smelter: 7, 20.6, 30.4 μ g/dL), it is not clear 28 

### whether these relationships wou ld apply to children with lower blood Pb levels. 29


### Results from Institoris et al. ( 2006 ) indicated that metal co

- exposures potentiated the 30 

### effects of Pb. Lymph node weight decreased with exposure of 4 week

- old rats to 31 

### 20 mg /kg Pb

- acetate by drinking water plus a second metal (Cd or Hg) but not with 32 

### 20 mg /kg of Pb alone. In contrast with t he aforementioned studies, Fortier et al. ( 2008 ) 33 

### did not find Hg co

- exposure to increase the effects of Pb. PbCl 2 

### - exposed (7.5

- 20.7 μ g/dL) 34February 2012 5

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### human leukocytes did not have alterations in lymphocyte prol iferation, monocytic 1 

### phagocytic activity, or NK cell activity. The combination of 20.7 μ g/dL PbCl 2 

### plus 2 

### 12.0 μ g/dL methylmercuric chloride (MeHgCl) decreased lymphocyte proliferation; 3 

### however, these effects were attributed to MeHgCl, which had a stronger s uppressive 4 

### effect independently. Other toxicological studies found immune effects of multiple metal 5 

### mixtures that included Pb ( e.g., decreased antibody titers, increased neutrophil counts) 6 

### ( Jadhav et al., 2007 ; Massadeh et al., 2007 ) but did not test each metal individually. Thus, 7 

### it cannot be ascertained whether findings are due to interactions between Pb and other 8 

### components within the mixture. Overall, results indicated that exposure s to Pb

- containing 9 

### metal mixtures are associated with immune effects. Moreover, several studies found an 10 

### interaction between Pb and metals such as As, Cd, and Hg, suggesting that a threshold for 11 

### producing Pb

- induced immune effects may be lower if additiona l metals are present. 12



### 5.6.8 Summary and Causal Determination
The collective body of evidence demonstrates that Pb exposure is associated with 13 

### immune effects. The evidence indicates that rather than resulting in overt cytotoxicity to 14 

### lymphoid tissues, Pb exposure is associated predominantly with subtle changes in a 15 

### spectrum of cellular and humoral immune responses, most notably, altered function of T 16 

### lymphocytes and macrophages, suppression of the DTH response and resistance to 17 

### bacterial infection, increased IgE p roduction, changes in cytokine production, and 18 

### inflammation. The strength of evidence for Pb

- associated immune effects is derived not 19 

### only from the consistency of findings within particular endpoints but also from the 20 

### coherence of findings among the spectr um of immune changes operating within the same 21 

### pathway. In particular, the evidence integrated across the examined spectrum of immune 22 

### outcomes clearly indicates that the prominent immune effect of Pb exposure is to shift 23 

### responses from a Th1 phenotype towa rd a Th2 phenotype. 24 

### The weight of evidence is provided largely by an extensive body of animal studies 25 

### characterizing effects on the broad range of immune endpoints as described above. 26 

### Comprising a smaller body of evidence, epidemiologic studies in children are consistent 27 

### with toxicological studies in reporting associations of blood Pb levels with indicators of 28 

### increased Th2 activity, principally, higher levels of IgE, asthma, and allergy in children. 29 

### Across outcomes, because most studies examined concurrent blood Pb levels, there is 30 

### uncertainty regarding the critical timing, frequency, duration, and level of Pb exposures 31 

### in children that contributed to observed associations. In toxicological studies, the shift to 32 

### a Th2 phenotype is well characterized by obse rvations that Pb exposures suppress the 33 

### production of Th1 cytokines ( e.g., IFN

- γ) and increase production of Th2 cytokines 34 

### ( e.g., IL

- 4) ( Section 5.6.5.4 ). In animal studies, the shift to Th2 cytokine production was 35February 2012 5

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### observed in juvenile animals with prenatal Pb exposures and in adult animals with long

1 

### term (> 4 weeks) Pb exposures [ Table 5

- 7 in the 2006 Pb AQCD ( U.S. EPA, 2006g ) ]

. 2 

### Blood Pb levels of animals were reported infrequently, and a wide range of Pb exposures 3 

### was found to induce changes in cytokine levels. In a recent study, lifetime Pb exposure 4 

### that resulted in blood Pb levels between 2

- 3 μ g/dL suppressed Th1 cytokine production 5 

### in adult mice ( Iavicoli et al., 2006a )

. A few available epidemiologic studies in children 6 

### ( Hsiao et al., 2001 ; Lu tz et al., 1999 ) found higher lev els of IL

- 4, a Th2 cytokine, in 7 

### groups of children with higher concurrent blood Pb levels ( e.g., mean 8.8 μ g/dL or range 8 

### 15

- 19 μ g/dL, respectively); however, these studies are limited by their lack of 9 

### consideration of potential confounding variables and la ck of information on the 10 

### concentration

- response function. 11


### Coherence for a Pb

- associated skewed ratio of cytokine production is provided by 12 

### evidence demonstrating Pb

- induced activation of T cells in animals, with recent studies 13 

### describing mechanisms underly ing T cell activation (Sections 5.6.2.1 and 5.6.2.2 ). A 14 

### recent toxicological study expanded the extant evidence by showing in vivo and in vitro 15 

### that Pb may promote Th2 responses by directly increasing production of Th2 cytokines in 16 

### dendritic c ells, the major effector in antigen response ( Gao et al., 2007 )

. Pb

- induced T 17 

### cell activation also is indicated by a relatively small body of evidence for prenatal and 18 

### postnatal Pb exposures of animals resulting in the generation of autoantibodies in 19 

### response to new antigens. These findings suggest that Pb exposure may increase the risk 20 

### of developing autoimmune conditions ( Section 5.6. 4.4 ). In epidemiologic studies, 21 

### evidence for the effects of Pb on T cells comprised associations of concurrent blood Pb 22 

### levels > 10 μ g/dL in children with lower T cell abundance, in particular CD3+ cells. 23 

### However, the functional relevance of these changes is unclear. 24 

### In addition to T cell responses, a prominent effect of Pb exposure, as demonstrated in an 25 

### extensive historical toxicological evidence base, was the induction of macrophages into a 26 

### hyperinflamm atory state as characterized by enhanced production of ROS, suppressed 27 

### production of NO, enhanced production of TNF

- α, and excessive metabolism of 28 

### arachidonic acid into immunosuppressive metabolites ( e.g., PGE 2 

### ). Consistent with these 29 

### observations, a previ ous epidemiologic study examined and found greater release of ROS 30 

### and lower release of NO from macrophages, primarily in children with concurrent blood 31 

### Pb levels 10.31

- 47.49 μ g/dL ( Pineda

- Zavaleta et al., 2004 )

. Misregulated inflammation 32 

### represents one of the major modes of action for Pb

- induced immune effects. 33 

### Toxicological studies provide evidence for the modulation of inflammatory cell function, 34 

### production of pro

- inflammatory cytokines and metabolites, enhance d inflammatory 35 

### chemical messengers, and pro

- inflammatory signaling cascades. In addition to the 36 

### associations reported with IL

- 4, epidemiologic evidence for Pb effects on inflammation is 37 

### limited to a few recent studies in nonoccupationally

- exposed adults in which concurrent 38February 2012 5

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### blood Pb level was associated with other indicators of inflammation such as CRP 1 

### ( Songdej et al., 2010 ) and IL

- 6 ( Kim et al., 2007 )

. The studies commonly adjusted for 2 

### potential confounding by age, sex, BMI, and smoking status. However, because only 3 

### concurrent blood Pb levels were examined, there is uncertainty regarding the magnitude, 4 

### timing, frequency, and duration of Pb exposures tha t contributed to the observed 5 

### associations. 6 

### The toxicological evidence for Pb

- induced production of Th2 cytokines provides 7 

### biological plausibility for the evidence linking Pb exposure with elevated IgE levels. An 8 

### increase in IL

- 4 from activated Th2 cells induces differentiation of B cells into antibody

9 

### producing cells, thereby amplifying B cell expansion to secrete IgE, IgA, and IgG. 10 

### Animal studies describe Pb

- induced (resulting in blood Pb levels 10

- 30 μ g/dL) increases 11 

### in IgE ( Snyder et al., 2000 ; Miller et al., 1998 ; Heo et al., 1996 )

. Additionally, 12 

### epidemiologic studies in children consistently demonstrated associations between higher 13 

### concurrent blood Pb levels and higher in serum IgE ( Section 5.6.3 ). While most studies 14 

### found elevated IgE in groups of children with blood Pb levels > 10 μ g/dL, Karmaus et al. 15 

### ( 2005 ) found higher serum in IgE in children with blood Pb levels 2.8

- 3.4 μ g/dL 16



### c ompared with children with lower blood Pb levels. A few studies considered potential 17 

### confounding by factors such as age ( Karmaus et al., 2005 ; Lutz et al., 1999 ) , smoking 18 

### exposure, serum lipids, organochlorine b iomarkers, and number of previous infections 19 

### ( Karmaus et al., 2005 ) but not SES or allergen exposure. It is important to acknowledge 20 

### the extensive evidence in animals for Pb

- induced increases in IgE that is not subject to 21 

### confounding by SES. Additional sup port for effects on IgE is provided by toxicological 22 

### evidence for Pb

- induced increases in Th2 cytokines. Epidemiologic studies did not find 23 

### blood Pb level to be associated consistently with B cell abundance. 24 

### The toxicological evidence for Pb

- induced suppre ssion of Th1 cytokine production is 25 

### coherent with historical observations in animals that Pb exposure decreases responses to 26 

### antigens and bacterial infection ( U.S. EPA, 2006b )

. Toxicological studies provide clear, 27 

### consistent evidence that prenatal and postnatal Pb exposure of animals (resulting in blood 28 

### Pb levels 11

- >100 μ g/dL) suppresses the DTH response to antigens ( Section 5.6.2.3 ), and 29 

### a recent in vitro study indicates such effects may be mediated by dendritic cells ( Gao et 30 

### al., 2007 )

. Recent toxicologica l studies provided additional support for Pb

- induced 31 

### decreased host resistance by demonstrating that Pb exposure impairs phagocytic and 32 

### chemotactic activity of macrophages ( Lodi et al., 2011 ; Bussolaro et al., 2008 )

. These 33 

### observations reflect suppressed Th1 activity given the role of Th1

- dependent IFN

- γ in 34 

### enhan cing the killing capacity of macrophages. Epidemiologic evidence for Pb

- associated 35 

### diminished response to bacterial infection is limited to previous observations of reduced 36 

### neutrophil functionality in Pb

- exposed workers with mean blood Pb levels > 30 μ g/dL

. 37 

### While a few previous epidemiologic studies in children found higher prevalence of 38February 2012 5

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### respiratory infections in children with higher blood Pb levels ( Karmaus et al., 2005 ; 1 

### Rabinowitz et al., 1990 ) , th e findings are limited by the weak statistical methods of 2 

### studies and the lack of consideration of potential confounding variables. 3 

### The effects of Pb exposure on macrophages also suggest a role for the immune system in 4 

### mediating Pb

- associated effects in mu ltiple other physiological systems. A small body of 5 

### new toxicological studies indicated Pb

- induced changes in specialized macrophages in 6 

### nonlymphoid tissue such as alveolar macrophages, testicular macrophages, and brain 7 

### microglia ( Section 5.6.4.5 ); however, these studies primarily used the i.p. route to 8 

### administer Pb. Thus, the relevancy of observations to those expected from typical routes 9 

### of human e xposure is not clear. 10


### Th2

- dependent increases in IgE mediate type 1 hypersensitivity resulting in various 11 

### allergic conditions and asthma. Observations of Pb

- associated increases in Th2 cytokines 12 

### and circulating IgE levels provide biological plausibility fo r the small body of available 13 

### epidemiologic evidence indicating associations of blood Pb levels with asthma and 14 

### allergic conditions in children ( Jedrychowski et al., 2011 ; Pugh Smith and Nriagu, 2011 ; 15 

### Joseph et al., 2005 )

. Several of these studies considered a larger set of potential 16 

### confounding variables than did studies of IgE in children. While the set of particular 17 

### factors varied among studies, studies frequently considered SES indicators and/or 18 

### residential allergen measurement. Low SES has been associated with higher blood Pb 19 

### lev els, poorer housing conditions, higher exposures to mouse and cockroach allergen, and 20 

### with conditions such as asthma and allergy. Jedrychowski et al. ( 2011 ) found similar 21 

### magnitudes of association between cord blood Pb level and cord blood IgE in models that 22 

### did and did not adjust for potential confounding variables, including maternal edu cation. 23 

### Studies examining cognitive effects in children also found that associati ons with blood Pb 24 

### levels with and without adjustment for SES

- related variables ( Section 5.3 ). Pugh Smith et 25 

### al. ( 2011 ) and Jedrychowski et al

. ( 2011 ) also indicated lack of confounding by allergen 26 

### exposures and other indicators of housing condition by their inclusion as model 27 

### covariates or analysis of their independent associations with outcomes. The robust 28 

### association between blood Pb level and allergic sensitization (which indicates elevated 29 

### allergen

- specific IgE) observed in Jedrychowski et al. ( 2011 ) , provides support for the 30 

### associations observed between blo od Pb and IgE, in children, in which confounding by 31 

### SES and allergen was not considered. Collectively, these findings do not indicate that 32 

### confounding by SES or allergen exposure alone accounts for associations observed 33 

### between blood Pb levels and immune e ffects in children. This evidence for associations 34 

### of blood Pb level with clinical conditions such as asthma, allergic sensitization, and 35 

### allergic diseases in children expanded by results from recent studies also supports the 36 

### public health significance for Pb

- associated immune effects. 37February 2012 5

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### With respect to lifestages of Pb exposure, animal studies found that gestational Pb 1 

### exposures, encompassing a wide range of concentrations, affected endpoints such as IgE, 2 

### cytokine levels, and DTH but also found postnatal sho rt

- term (multiple days) long

- term 3 

### (multiple weeks) Pb exposures to affect cytokine levels in adult animals. The blood Pb 4 

### levels and Pb exposure lifestage, magnitude, frequency, and duration associated with 5 

### immune effects are not well characterized in human s. Several epidemiologic studies 6 

### examined associations (of IgE, asthma, and allergy) with concurrent blood Pb levels of 7 

### children > 10 μ g/dL. A few studies found cord blood or newborn hair Pb levels to be 8 

### associated with endpoints such as Ig levels ( Annesi

- Maesano et al., 2003 ; Belles

- Isles et 9 

### al., 2002 ) and allergic sensitization ( Jedrychowski et al., 2011 )

. The small body of 10 

### epidemiologic studies of nonoccupationally

- exposed adults examined different e ndpoints 11 

### and found associations with concurrent blood Pb levels, which are influenced by current 12 

### Pb exposures as well as cumulative Pb stores in bone. 13 

### In summary, recent toxicological and epidemiologic studies support the strong body of 14 

### evidence presented in the 2006 Pb AQCD that Pb exposure is associated with a broad 15 

### spectrum of changes in both cell

- mediated and humoral immunity that cumulatively 16 

### promote a Th2 phenotype and hyperinflammatory state. The principal findings are Pb

17 

### induced increased production of Th2 cytokines, suppressed production of Th1 cytokines, 18 

### increased inflammation, and elevated IgE, with the weight of evidence provided by 19 

### toxicological studies. Collectively, these findings are coherent with the observed effects 20 

### of Pb exposure on decrea sing responses to antigens ( e.g., DTH, bacterial resistance) in 21 

### animals. Both toxicological and epidemiologic studies in children provide evidence for 22 

### Pb

- associated increases in IgE. The toxicological and epidemiologic findings for Th2 23 

### cytokines, IgE, and inflammation provide biological plausibility for associations 24 

### observed for blood Pb levels with asthma and allergic conditions in children. 25 

### Associations with asthma and allergy were observed after considering potential 26 

### confounding by several factors, inclu ding, SES and allergen exposure. Animal studies 27 

### found a range of immune effects with prenatal exposure in juvenile animals and long

28 

### term postnatal (> 4 weeks) Pb exposures in adult animals. The blood Pb levels and Pb 29 

### exposure lifestage, magnitude, frequenc y, and duration associated with immune effects 30 

### are not well characterized in children or adults. Epidemiologic studies of children and 31 

### adults primarily examined concurrent blood Pb levels. Little information was provided on 32 

### concentration

- response functions . In epidemiologic studies, higher IgE and higher asthma 33 

### prevalence were examined and found in children with blood Pb levels > 10 μ g/dL. In the 34 

### large body of studies of adults (mostly males) with occupational Pb exposures, the most 35 

### consistent findings were decreased neutrophil functionality in workers with mean blood 36 

### Pb levels 21

- 71 μ g/dL. Recent epidemiologic studies provided new evidence in adults 37 

### without occupational Pb exposures; however, each examined a different immune 38 

### endpoint, for example, IgE, eNO, IL

- 6. These endpoints were associated with concurrent 39February 2012 5

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### blood Pb levels in populations of adults with mean blood Pb levels of 1.9

- 7 μ g/dL; 1 

### however, there is uncertainty regarding the contributions of current Pb exposures and 2 

### cumulative Pb stores in bone. Th e consistency and coherence of findings across the 3 

### continuum of related immune parameters that demonstrate a stimulation of Th2 responses 4 

### in toxicological studies combined with the supporting epidemiologic evidence in children 5 

### are sufficient to conclude th at there is a causal relationship between Pb exposures and 6 

### immune system effects. 7



## 5.7 Effects on Heme Synthesis and Red Blood Cell Function

5.7.1 Summary of F indings from 2006 Pb AQCD
The 2006 Pb AQCD reported that Pb affects developing red blood cells (RBCs) in 8 

### chi ldren and occupationally exposed adults as noted by anemia observed with blood Pb > 9 

### 40 μ g/dL. Pb

- induced anemia is thought to occur due to decreased RBC life span and 10 

### effects on hemoglobin (Hb) synthesis. The exact mechanism for these effects was not 11 

### known , although Pb

- induced changes on iron uptake or inhibition of enzymes in the heme 12 

### synthetic pathway may be responsible. 13 

### The 2006 Pb AQCD indicated that Pb crosses RBC membranes through passive 14 

### ( i.e., energy
- independent) carrier

- mediated mechanisms includi ng a vanadate

- sensitive 15 

### Ca 2+ 

### pump. Once Pb enters the cells, it is predominantly found in protein

- bound form, 16 

### with Hb and aminolevulinic acid dehydratase (ALAD) both identified as targets. Pb 17 

### poisoning (blood Pb levels > 100 μ g/dL) was found to decrease RB C survival in 18 

### laboratory animals, as well as alter RBC mobility and morphology, although the precise 19 

### mechanisms by which it does so are not known. Pb exposure has been found to 20 

### significantly decrease several hematological parameters including Hb, hematocri t (Hct), 21 

### mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean 22 

### corpuscular hemoglobin concentration (MCHC). Pb has also been observed to exert 23 

### multiple effects on RBC membranes, including altered microviscosity and fluidity, 24 

### decreased sialic acid content, decreased lamellar organization, decreased lipid resistance 25 

### to oxidation (possibly mediated by perturbations in RBC membrane lipid profiles), and 26 

### increased permeability. These alterations to RBC membranes potentially lead to RBC 27 

### fragil ity, abnormal cellular function, RBC destruction, and ultimately anemic conditions. 28 

### Pb exposure also has been shown to result in increased activation of RBC scramblase, an 29 

### enzyme responsible for the expression of phosphotidylserine (PS) on RBC membranes. 30 

### T his expression of PS decreases the life span of RBCs via phagocytosis by macrophages. 31 

### Pb exposure has been observed to alter the phosphorylation profiles of membrane 32February 2012 5

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### proteins, which may influence the activity of membrane enzymes and the functioning of 1



### rece ptors and channels located on the membrane. 2 

### The 2006 Pb AQCD reported that Pb affects heme synthesis through the inhibition of 3 

### multiple key enzymes, most notably ALAD, the enzyme that catalyzes the second, rate

4 

### limiting step in heme biosynthesis ( Figure 5

- 45 presents a schematic representation of the 5 

### heme biosynthetic pathway). The 2006 Pb AQCD further reported that decreased RBC 6 

### ALAD activity is the most sensitive measure of human Pb exposure, i n that measurement 7 

### of ALAD activity is correlated with blood Pb levels. Concentration

- response changes in 8 

### the ratio of activated/nonactivated ALAD activity in avian RBCs was observed to be not 9 

### dependent on the method of Pb administration. The inhibition of the ALAD enzyme was 10 

### observed in RBCs from multiple species, including birds, Cynomolgous monkeys, and 11 

### humans. Pb was also observed to inhibit other enzymes responsible for heme 12 

### biosynthesis, including ferrochelatase, porphobilinogen (PBG) deaminase, and 13



### c oproporphyrinogen oxidase. Pb also potentially alters heme biosynthesis through 14 

### inhibition of transferrin (TF) endocytosis and iron transport. 15 

### Pb has been found to alter RBC energy metabolism through inhibition of enzymes 16 

### involved in anaerobic glycolysis and the pentose phosphate pathway. Pb was also found 17 

### to inhibit pyrimidine 5'

- nucleotidase (P5N) activity and the 2006 Pb AQCD indicated that 18 

### this might be another possible biomarker of Pb exposure. Inhibition of P5N results in an 19 

### intracellular increase in pyrimidine nucleotides leading to hemolysis and potentially 20 

### ultimately resulting in anemic conditions. The 2006 Pb AQCD indicated that 21 

### perturbations in RBC energy metabolism may be related to significant decreases in levels 22 

### of nucleotide pools, including nicotinamide adenine nucleotide (NAD), possibly due to 23 

### decreased NAD synthase activity, and nicotinamide adenine nucleotide phosphate 24 

### (NADP) accompanying significant increases in purine degradation products. 25 

### Pb was found to alter the activity of membrane

- bound ion pumps. Potassium (K + 

### ) 26 

### permeability was found to be increased by Pb due to altered sensitivity of the membrane 27 

### calcium (Ca 2+ 

### )

- binding site that caused selective efflux of K + 

### ions from the RBC 28 

### membrane. Inhibition of RBC sodium (Na + 

### )

- K + 

### adenosine t riphosphate synthase 29 

### (ATPase), acetylcholinesterase (ACh), and NADH dehydrogenase was also observed. In 30 

### human RBCs, Na +



### - K + 

### ATPase activity was more sensitive to Pb exposure than were Ca 2+ 31 

### or magnesium (Mg 2+ 

### ) ATPases. 32 

### The 2006 Pb AQCD identified oxidative s tress as an important potential mechanism of 33 

### action by which Pb exposure induced effects on RBCs. Increased lipid peroxidation and 34 

### inhibition of antioxidant enzymes ( e.g., superoxide dismutase [SOD], catalase [CAT]) 35 

### were observed following exposure to Pb. 36February 2012 5

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### 5.7.2 Red Blood Cell Functions
As stated in the 2006 Pb AQCD, Pb poisoning has been associated with anemia resulting 1 

### from shortened RBC life span and Pb effects on Hb synthesis. As of 2006, the 2 

### mechanism for this was not clear, but it was determined not to be d ue to iron deficiency, 3 

### which can be found to occur independently of Pb exposure. However, Zimmerman et al. 4 

### ( 2006 ) found that blood Pb levels were statistically significantly lower in non
- or mildly 5 

### anemic, iron

- deficient 5

- to 9

- year old children in India fed an iron

- fortified diet for 3 0 6 

### weeks compared to 14 weeks (mean [range]: 8.1 [3.1

- 219] μ g/dL versus 12.1 [3.7

- 7 

### 26.8] μ g/dL; p < 0.02); blood Pb levels were not lower in children receiving the no iron 8 

### diet for 30 weeks compared to 14 weeks (mean [range]: 10.2 [4.4

- 25.3] μ g/dL versus 9 

### 12. 0 [3.8

- 25.5] μ g/dL). Although a number of epidemiologic studies find decreases in 10 

### RBCs and/or Hct levels associated with blood Pb, it is not known whether this is due to 11 

### reduced cell survival or a decrease in RBC cell production. However, decreased RBC 12



### sur vival and hematopoiesis can be expected to occur simultaneously, and any effect on 13 

### RBC numbers is likely a combination of the two modes of action. 14



### 5.7.2.1 Pb Uptake, Binding, and Transport into Red Blood Cells
The 2006 Pb AQCD reported that Pb uptake into human RBCs occurs via passive anion 15 

### transport mechanisms. Although Pb can passively cross the membrane in both directions, 16 

### little of the Pb is found to leave the cell after entry. Simons ( 1993b ) found that in vitro 17 

### uptake of 203 

### Pb (1

- 10 μ M) occurred via an anion exchanger while the effl ux occurred via 18 

### a vanadate

- sensitive pathway. After entry into the RBC, radioactive Pb was found to 19 

### partition with Hb at a ratio estimated to be about 6000:1 bound to unbound ( Simons, 20 

### 1986 )

. However, Bergdahl et al. ( 1997a ) suggested that ALAD was the primary Pb 21 

### binding protein and not Hb. The 2006 Pb AQCD also reported that the majority 22 

### (approximatel y 98%) of Pb accumulates in RBC cytoplasm bound to protein and only 23 

### about 2% is found in the membrane. This is related to the high ratio of Pb in RBCs 24 

### compared to plasma Pb. Further information on Pb binding and transport in blood can be 25 

### found in the kinet ics section of 0 ( Section 4.2 ). 26 

### Although no studies were indentified that examined transport of Pb into RBCs, Lind et al. 27 

### ( 2009 ) recently observed that several zinc (Zn) io nophores (8

- hydroxyquinoline 28 

### derivatives and Zn and Na pyrithione) were able to effectively transport Pb out of RBCs 29 

### into the extracellular space. 30February 2012 5

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### 5.7.2.2 Red Blood Cell Survival, Mobility, and Membrane Integrity
Although Pb exposure has been consistently shown t o shorten the RBC life span and alter 1 

### RBC mobility, as of the 2006 Pb AQCD the mechanism of this was not well understood. 2 

### While the mechanism is still not fully understood, there has been some indication for a 3 

### role of free Ca 2+ 

### . There are also newer studie s that examine the relationship between Pb 4 

### and RBC survival, mobility, and membrane integrity. Occupational studies investigating 5 

### severely Pb

- intoxicated worker populations (mean blood Pb > 28 μ g/dL) observed 6 

### increased intracellular RBC free calcium levels ([Ca 2+ 

### ] i 

### ) and decreased RBC membrane 7 

### Ca 2+



### - Mg 2+



### - ATPase activity in workers compared with unexposed controls ( Abam et al., 8 

### 2008 ; Quintanar

- Escorza et al., 2007 )

. [Ca 2+ 

### ] i 

### levels were highly correlated with blood Pb 9 

### levels even among unexposed control populations with blood Pb levels < 10 μ g/dL (mean 10 

### 9.9 ± 2 μ g/dL) ( Quintanar

- Escorza et al., 2007 )

. Changes in [Ca 2+ 

### ] i 

### were associated with 11 

### increased fragility of the RBCs and dr amatic morphological alterations, including the 12 

### increased presence of ec h inocytes (cells without normal biconcave shape) and crenocytes 13 

### (speculated cells) in Pb

- exposed workers. 14 

### Similar to the associations observed in Quintanar

- Escorza et al. ( 2007 ) , concentration

15 

### dependent effects were observed when RBCs from healthy human volunteers were 16 

### incubated in vitro with Pb nitrate at physiologically

- relevant concentrations. [Ca 2+ 

### ] i 17 

### increased in a concentration

- dependent manner when RBCs were exposed to 0.2 or 18 

### 0.4 μ M Pb nitrate for 24 or 120 hours (0.4 μ M Pb nitrat e roughly approximates 10 μ g/dL 19 

### Pb, although concentrations in exposure media are not directly comparable to blood Pb 20 

### levels) ( Quintanar

- Escorza et al., 2010 )

. [C a 2+ 

### ] i 

### levels were further increased at higher, 21 

### less physiologically

- relevant concentrations ( i.e., 2

- 6 μ M Pb nitrate). The increase in 22 

### [Ca 2+ 

### ] i 

### levels was observed to be related to increased Ca 2+ 

### influx: RBCs exposed to 23 

### 0.4 μ M Pb nitrate for 24 hours incorporated twice as much Ca 2+ 

### as controls did, whereas 24 

### exposure for 120 hours resulted in a fourfold increase. Concomitant to increased influx of 25 

### Ca 2+ 

### , efflux of Ca 2+ 

### was observed due to reduced Ca 2+



### - Mg 2+ 

### ATPase activity; exposure to 26 

### 0.4 μ M Pb nitrate fo r 24 or 120 hours reduced activity by 33% and 83%, respectively, 27 

### compared to controls. As was observed among severely Pb

- intoxicated workers, changes 28 

### in [Ca 2+ 

### ] i 

### were associated with increased fragility of the RBCs (measured as increased 29 

### hemolysis) and dram atic morphological alterations, including the increased presence of 30



### ec h inocytes (cells without normal biconcave shape) and crenocytes (speculated cells) 31 

### following exposure to 0.4 μ M Pb nitrate. Similarly, Ciubar et al. ( 2007 ) found that RBC 32 

### morphology was disrupted, with ≥ 50% RBCs having lost the typical discocytic 33 

### morphology and displaying moderate to severe echinocytosis following exposure to Pb 34 

### nitrate concentrations of 0.5 μ M or hig her for 24 hours at 37 °C. Exposure of RBCs to 35 

### higher concentrations (concentrations not stated) of Pb nitrate resulted in cell shrinkage. 36 

### Ademuyiwa et al. ( 2009 ) observed that the cholesterol content of RBC plasma 37February 2012 5

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### membranes, but not the phospholi pid content, was statistically significantly higher in rats 1 

### exposed to 200 ppm Pb

- acetate (resulting in mean [SD] blood Pb: 40.63 [9.21] μ g/dL) for 2 

### three months through drinking water compared to controls. Further, the 3 

### cholesterol/phospholipid ratio was increased in the Pb

- exposed rats with increased 4 

### cholesterol, indicating that RBC membrane fluidity was decreased. 5


### A number of studies have investigated the effect of occupational exposure to Pb on 6 

### various inter

- connected and related hematological parameters. Most occupationally

7 

### exposed cohorts represent populations highly exposed to Pb, with mean blood Pb levels 8 

### ranging from 26

- 74 μ g/dL. Alth ough effects observed within these cohorts may not be 9 

### generalizable to the population as a whole, they are useful in demonstrating consistent 10 

### effects on a number of hematological parameters, including Hb, MCV, MCH, MCHC, 11 

### total RBCs, and packed cell volume (PCV) ( Khan et al., 2008 ; Patil et al., 2006a ; Patil et 12 

### al., 2006b ; Karita et al., 2005 )

. Additionally, these studies were cross

- sectional in design; 13 

### thus, there is uncertainty regarding the magnitude, timing, frequency, and duration of Pb 14 

### exposure that contributed to the observed observations. Workers o ccupationally exposed 15 

### to Pb may also have been co

- exposed to other contaminants than can affect the 16 

### hematological system although the potential for co

- exposure was not assessed in most 17 

### studies. A few occupational studies did investigate the effect of moder ate occupational Pb 18 

### exposure on hematological parameters. Ukaejiofo et al. ( 2009 ) studied the hematological 19 

### effec ts of Pb in 81 male subjects moderately exposed to Pb at three different 20 

### manufacturing companies in Nigeria for durations between six months and 20 years. Two 21 

### control groups were used for comparison (30 individuals from the same industries not 22 

### involved in handling Pb and 20 individuals from the same locality but not involved in Pb 23 

### handling). The exposed individuals had a mean blood Pb level of 7.00 μ g/dL compared to 24 

### 3 μ g/dL in controls drawn from industries not involved in Pb handling (control group I) 25 

### and 2 μ g/dL in controls drawn from the general population (control group II) (p < 0.05). 26 

### Pb

- exposed workers had significantly reduced Hb and PCV levels and increased 27 

### percentage of reticulocytes. Although the differences were statistically significant 28 

### between t he exposed and control subjects, the study authors state that the levels in the 29 

### exposed subjects were at the lower range of normal for Nigerians. The percent cell lysis 30 

### did not differ between controls and exposed workers; however, when workers and 31 

### controls were stratified by age, there was a significant increase in cell lysis in workers 32 

### under age 30 compared to similarly aged controls in group II (p < 0.01). Conterato et al. 33 

### ( In Press ) investigated hematological parameters in automotive painters exposed to Pb in 34 

### Brazil. Exposed painters had a mean [SEM] blood Pb concentration of 5.4 [0.4] μ g/dL 35 

### compared to 1.5 [0.1] μ g/dL in controls. The mean [SEM] duration of exposure to Pb in 36 

### painters was 133.9 [14.5] months, whereas the controls were not occupationally exposed 37 

### to Pb. Although hematocrit, hemoglobin concentration, and the number of RBCs were 38 

### statistically significan tly decreased in painters compared to controls, they were not 39February 2012 5

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### correlated with blood Pb levels. These parameters were correlated with blood Cd levels 1 

### which were also statistically significantly elevated in painters compared to controls 2 

### (mean [SEM]: 1.606 [0 .074] versus 0.003 [0.001] μ g/dL). MCHC and RBC distribution 3 

### width (a measure of the variability of RBC widths) were correlated (r = 0.23 and 0.44, 4 

### respectively; p < 0.05) with blood Pb although these measures were not statistically 5 

### different in painters c ompared to controls. In an investigation of petrol workers in 6 

### Sarajevo, Yugoslavia, examinations on hematological parameters were performed on the 7 

### same population five years apart, in 2003 and 2008. Workers (mean [SD] duration of 8 

### exposure: 12.1 [9.1] years ) had increased blood Pb (mean: 5.96 μ g/dL) in 2008 compared 9 

### to 2003 (mean: 4.07 μ g/dL; mean [SD] duration of exposure: 10.4 [5.5]) ( Cabaravdic et 10 

### al., 2010 )

. In 2008, levels of MCH and MCHC were significantly decreased from lev els 11 

### in 2003, whereas RBC numbers, Hb, Hct, and MCV were elevated. Positive correlations 12 

### were observed in all subjects between blood Pb and RBC count, Hb, and MCH (r = 13 

### 0.241, 0.201, and 0.213, respectively; p < 0.05). Taken together, the above occupational 14 

### studies provide consistent evidence that very high (blood Pb > 26 μ g/dL) and moderate 15 

### (blood Pb ~ 5.5

- 7.0 μ g/dL) occupational exposure to Pb reduces the number of RBCs in 16 

### circulation. Although this decrease in RBCs observed in highly exposed worker cohorts 17 

### may be explained by both decreased cell survival and/or disruption of hematopoiesis, the 18 

### observation of increased reticulocytes in Ukaejiofo et al. ( 2009 ) seems to represent 19 

### compensation for decreased RBC survival due to Pb exposure. In a non
- occupational 20 

### study, the associations between blood Pb levels, calcium, iron, and hemoglobin were 21 

### investigated in 55 pregnant Brazilian women (21.9% 14

- 19 years old, 74.5% 20

- 34 years 22 

### olds, and 3.6% ≥ 35 years old) ( Zentner et al., 2008 )

. The majority of women (across all 23 

### age groups) had concurrent blood Pb levels below 5 μ g/dL (58.2%), although the mean 24 

### blood Pb level was not reported; only 5.4% of women had blood Pb levels above 25 

### 10 μ g/dL. The vast majority of women (78.2%) were also observed to have adequate 26 

### levels of hemoglobin (≥ 11 g/dL). In a multiple linear regression model, blood Pb was 27 

### observed to be negatively correlated with h emoglobin (β =

- 0.359 g/dL). 28 

### Studies in children were generally supportive of effects on hematological parameters 29 

### ( e.g., Hb, MCV, MCH) observed in occupational adult populations. In two cross

30 

### sectional studies of children measuring blood hemoglobin as the independent variable, 31 

### blood Pb levels were observed to decrease with increasing blood Hb. Riddell et al. ( 2007 ) 32 

### found that 21% of children 6 months to 5 years of age living in rural Philippines had 33 

### concurrent blood Pb levels greater than 10 μ g/dL (total populatio n mean: 6.9 μ g/dL). Hb 34 

### levels were inversely related to blood Pb, with a decrease of 3% blood Pb associated with 35 

### every 1 g/dL increase in Hb. Similarly, in children aged 6

- 36 months (n = 222) living in 36 

### Montevideo, Uruguay, 32.9% of children had blood Pb gr eater than 10 μ g/dL (population 37 

### mean [SD]: 9.0 [6.0] μ g/dL) ( Que irolo et al., 2010 )

. The mean [SD] Hb concentration 38 

### was 10.5 [1.5] g/dL, and 44.1% of children were diagnosed as anemic (Hb < 10.5 g/dL). 39February 2012 5

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### Blood Pb levels were higher in anemic children compared to non
- anemic (mean [SD]: 1 

### 10.4 [6.8] versus 7.9 [5.1] μ g/dL ), and in bivariate regression models, blood Pb decreased 2 

### 0.65 μ g/dL for every 1 g/dL increase in Hb. In children younger than 18 months of age, 3 

### blood Pb was, on average, 3.5 ± 1.1 μ g/dL higher in anemic children. In children 4 

### aged 5

- 9 years (n = 189) livin g in Cartegena, Columbia, a smaller percentage (4.7%) of 5 

### children had blood Pb > 10 μ g/dL (mean [SD]: 5.49 [0.23] μ g/dL). The only 6 

### hematological parameters that fell outside of their refe re nce values were MCV and MCH, 7 

### which were negatively correlated with blood Pb (r =

- 0.159 [p = 0.029] and

- 0.171 [p = 8 

### 0.019], respectively) ( Olivero

- Verbel et al., 2007 )

. RBC count, whic h was not observed 9 

### to differ from reference values, was positively correlated with blood Pb level (r = 0.208, p 10 

### = 0.004). Ahamed et al. ( 2006 ) studied 39 male urban adolescent s in India who were 11 

### separated into groups according to their blood Pb level (group 1: <10 μ g/dL [mean 12 

### 7.4 μ g/dL], group 2: >10 μ g/dL [mean 13.27 μ g/dL]). Although the groups were similar 13 

### in age (mean [SD]: 16.59 [0.91] versus 16.76 [0.90] years, respective ly), height, weight, 14 

### and body mass index, group 2 had a significantly lower PCV compared to group 1. In a 15 

### related study, Ahamed et al. ( 2007 ) investigated the relationship between blood Pb, 16 

### anemia, and other hematological parameters in urban children in India (n = 75). Children 17 

### were split into two groups as above: group 1 had blood Pb <10 μ g/dL (mean [SD]: 6.89 18 

### [2.44] μ g/dL, n = 19), whereas group 2 had blood Pb >10 μ g/dL (mean [SD]: 21.86 19 

### [7.58] μ g/dL, n = 56 ). As with the earlier study, ages were similar between the two 20 

### groups: mean [SD]: 4.68 [1.49] and 4.11 [1.77] years, respectively. Hb and Hct were 21 

### significantly decreased in group 2, compared to group 1, and children in group 2 had an 22 

### increased odds of an emia (OR: 2.87 [95% CI: 1.60, 2.87]) compared to group 1 after 23 

### adjustment for age, sex, and area of residence. Similarly, in a study of 340 children (aged 24 

### 1

- 5 years) from Karachi, Pakistan, mildly and sever e ly anemic children (mean [SD] Hb 25 

### levels: 8.9 [0.9 ] and 7.4 [0.5] g/dL, respectively) had higher blood Pb levels compared to 26



### non
- anemic children (mean [SD] Hb: 12.1 [1.3] g/dL). Mean [SD] blood Pb levels in the 27 

### mildly anemic, severely anemic, and nonanemic children were 14.9 [0.81], 21.4 [2.7], 28 

### and 7.9 [1 .7] μ g/dL, respectively (p < 0.01) ( Shah et al., 2010 )

. A dditionally, Hct, RBC 29 

### count, and MCV were all decreased in anemic children versus non
- anemic children. 30 

### Although statistical analyses were not reported, the levels of Hb, Hct, RBC count, and 31 

### MCV in anemic children all fell outside of the reported normal ran ge for these 32 

### parameters, whereas the reported values in non
- anemic children did not. Blood Pb was 33 

### negatively correlated with Hb level in all groups, with the magnitude of negative 34 

### correlation increasing with increasing severity of anemia: r =

- 0.315 ( non
- a nemic 35 

### children),

- 0.514 (mild anemia), and

- 0.685 (severe anemia). In iron

- deficient anemic 36 

### children (n = 23) from Denizli, Turkey, mean (SD) serum Pb levels were statistically 37 

### increased compared to healthy children (n = 179): 0.013 (0.004) versus 0.008 38 

### (0 .001) μ g/dL, respectively ( Turgut et al., 2007 )

. The iron

- deficient children were 39February 2012 5

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### observed to have decreased Hb, MCV, and ferritin compared to controls, but increased 1 

### RBC and RDW. In 140 children from southern Brazil aged 2

- 11 years, living within 25 2 

### km of a Pb smelter, blood Pb levels were not observed to differ between anemic and 3



### non
- anemic children (mean [SD]: 10.36 [ 6.8] versus 9.73 [5.8] μ g/dL, p = 0.98) ( Rondo 4 

### et al., 2006 )

. However, blood Pb levels were significantly negatively correlated with Hb 5 

### in anemic children (r =

- 0.41, p = 0.01); this relationship was not observed in non
- anemic 6 

### childr en (r = 0.018, p = 0.84). 7 

### Contrary to the observations in the above studies, Huo et al. ( 2007 ) found that children 8 

### (less than 6 years of age) living near an area where electronic waste was recycled in 9 

### China had significantly higher mean blood Pb levels than did children in the neighboring 10 

### town with no waste recycling (15.3 versus 9.94 μ g/dL). No difference was detected in the 11 

### mean Hb levels of the children in the two towns (127.55 g/L in children from the waste 12 

### recycling town versus 123.46 g/L in children from the town with no recycling). 13 

### Equivocal findings in studies investigating associations of blood Pb levels with 14 

### hematological effects in children may be due to the comparatively shorter time period 15 

### and magnitude of exposure versus those seen in occupational studies. 16 

### A number of animal toxicology studies support associations of b lood Pb levels with 17 

### hematological parameters observed in epidemiologic studies. Baranowska

- Bosiacka et al. 18 

### ( 2009 ) examined the effects of Pb on RBC hemolysis both in vitro measuring lysate in 19 

### human RBCs incubated with Pb at concentrations ranging from 0.1

– 100 μ M for 5

– 20 

### 30 minute s, and in vivo using a rat RBC lysate from rats exposed to Pb

- acetate (0.1 %) in 21 

### d rinking water for 9 months. Rats exposed to Pb in the in vivo portion of the study 22 

### achieved a blood Pb concentration of 7.1 μ g/dL. The concentration of Hb in the plasma 23 

### of chronically

- exposed rats, a marker of RBC hemolysis, was statistically significantly (p 24 

### = 0.01) increased compared to that in control rats. The in vitro studies demonstrated a 25 

### similar concentration

- dependent increase in the amount of hemolysis, with a significant 26 

### (fourfold) increase even at the lowest concentration tested ( i.e., 0.1 μ M). Pb

- induced 27 

### hemolysis in these experiments may be due to inhibition of RBC 28 

### phosphoribosyltransferases ( Section 5.7.5.1 ). Lee et al. ( 2005 ) observed that rats orally 29 

### administered Pb (25 mg /kg) via gavage once a week for 4 weeks had an average plasma 30 

### Pb level of 6.5 μ g/dL (9.6

- fold higher than that in controls, p < 0.05), and had significant 31 

### decreases in Hct, Hb, and RBCs (p < 0.05). Male mice orally administered 50 mg /kg Pb 32 

### nitrate in distilled wat er via gavage for 40 days had mean [SD] final blood Pb levels of 33 

### 1.72 [0.02] μ g/dL versus 0.09 [0.011] μ g/dL in control mice. Pb

- exposed mice had 34 

### significantly reduced total RBC counts, total leukocyte counts, Hb, lymphocytes, and 35 

### monocytes compared to con trols (p < 0.001) ( Sharma et al., 2010b )

. Rats exposed to 2 36 

### g/L Pb

- acetate in drinking water for 30 days (blood Pb not reported) had significantly 37 

### decreased RBCs, Hb, PCV, MCH, and MCHC compared to controls (p < 0.05) ( Simsek 38February 2012 5

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### et al., 2009 ) , and not a disruption of hematopoiesis. Mice exposed to 1 g/L Pb

- acetate in 1 

### drinking water (blood Pb not reported) for 90 days, but not those exposed for 15 or 45 2 

### days`, had significantly decreased RBC counts and Hct compared to controls (p< 0.05) 3 

### ( Marques et al., 2006 )

. Spleen weights were observed to be increased relative to body 4 

### weight in animals exposed to Pb for 45 days. Mice injected daily with 50 mg /kg 5 

### Pb

- acetate sub cutaneously (blood Pb not reported) had significantly reduced Hb, MCV, 6 

### MCH, and MCHC compared to controls injected with 5% dextrose ( Wang et al. )

. In 7 

### weanling rats (age = 25 days, n = 10) whose dams were exposed to 2.84 mg /mL 8 

### Pb

- acetate (approximating mean [SD] daily exposures of 342.57 [28.11] and 744.47 9 

### [29.27] mg /kg during gestation and lactation, respectively), blood Pb was significantly 10 

### elevated compared to controls (mean [SE]: 6 98.1 [78.2] versus 5.4 [0.8] ng/g). The only 11 

### hematological parameter affected by Pb exposure was Hct, which was decreased in 12 

### exposed rats (mean [SE]: 27.3 [0.5] versus 33.4 [0.3] percent) ( Molina et al., 2011 )

. 13 

### Some toxicological studies found no evidence of hematological effects in animals 14 

### following exposure to Pb. Male rats administered Pb

- acetate in the drinking water for 4 15 

### weeks at concentrations ranging from 100

– 1 , 000 ppm had a concentration

- dependent 16 

### increase in blood Pb (range: 6.57

– 22.39 μ g/dL) compared to controls (0.36 μ g/dL), but 17 

### there were no significant changes in any of the hematologica l parameters (complete blood 18 

### cell count performed) measured at the end of treatment ( Lee et al., 2006b )

. Slight, 19 

### statistically n onsignificant increases in PS expression on RBC membranes were also 20 

### observed. Similarly, exposure of male rats to 0.5% Pb nitrate in drinking water (blood Pb 21 

### not reported) for three weeks had no affect on any measured hematological parameter 22 

### ( Gautam and Flora, 2010 )

. In vitro experiments with rat and human blood did not 23 

### de monstrate a significant increase in hemolysis after 4 hours of treatment with Pb

- acetate 24 

### at concentrations up to 10 μ M. 25


### Khaĭrullina et al. ( 2008 ) observed that the surface profiles of RBC membrane shadows 26 

### incubated with 0.5

- 10 μ M Pb

- acetate for three hours were much smoother than were 27 

### untreated RBC membranes when examined by atomic force mic roscopy. The authors 28 

### postulate that the observed smoothing in Pb

- treated RBC membranes may be due to 29 

### clusterization of band 3 protein. Band 3 (anion exchanger 1 [AE1]), is a 30 

### chloride/bicarbonate (Cl - 

### /HCO 3 - 

### ) exchanger and is the most abundant protein in RBC 31 

### membranes. AE1 is integral in carbon dioxide (CO 2 

### ) transport and linkage of the cellular 32 

### membrane to the underlying cytoskeleton ( Akel et al., 2007 ; Su et al., 2007 )

. The 33 

### observed smoothing of the RBC membrane may due to Pb interfering with how the 34 

### membrane attaches to the cytoskeletal structure of the RBC through p erturbation of the 35 

### normal activity of AE1. 36February 2012 5

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### Eryptosis Eryptosis is the suicidal death of RBCs. It is characterized by cell shrinkage, membrane 1 

### blebbing, and cell membrane phospholipid scrambling associated with PS exposure on 2 

### the cell membrane that leads t o cell destruction via macrophages ( Föller et al., 2008 ; 3 

### Lang et al., 2008 )

. As previously reported in the 2006 Pb AQCD, Kempe et al. ( 2005 ) 4 

### found that exposing human RBCs to Pb at concentrations ranging from 0.3

– 3 μ M caused 5 

### increased activation of K + 

### channels that led to cell shrinkage and scramblase activation. 6 

### The activation of scramblase increased the exposure to PS on the cell mem brane, which 7 

### causes an increase in the destruction of the RBCs by macrophages. 8


### Shin et al. ( 2007 ) found that in vitro ex posure of human RBCs to 1

– 5 μ M Pb

- acetate 9 

### increased PS expression in a time

- and concentration

- dependent manner. The maximum 10 

### mean [SE] increase in expression of PS was 26.8 ± 3.15% (compared to deionized water), 11 

### observed after incubation with 5 μ M Pb for f our hours. The expression of PS in RBCs is 12 

### considered to be regulated through a Ca 2+ 

### dependent mechanism and, correspondingly, 13 

### [Ca 2+ 

### ] i 

### was observed to increase with exposure to Pb (mean [SE]: 0.24 [0.21] μ M in 14 

### controls to 6.88 [1.13] μ M in RBCs treated wit h 5 μ M Pb for one hour). Consistent with 15 

### this finding, Shin et al. ( 2007 ) also observed that scramblase activity, which i s important 16 

### for induction of PS exposure and is activated by [Ca 2+ 

### ] i 

### , was increased in Pb

- exposed 17 

### RBCs. Flippase, which translates PS exposure to inner membranes, is inhibited by high 18 

### levels of [Ca 2+ 

### ] i 

### and was observed to exhibit reduced activity following Pb exposure. The 19 

### inhibition of flippase is additionally influenced by the depletion of cellular adenosine 20 

### triphosphate (ATP). ATP levels were decreased in a concentration

- dependent manner 21 

### following exposure to Pb. To corroborate these findings in vivo, Sh in et al. ( 2007 ) treated 22 

### male rats i.p. to 25, 50, or 100 mg /kg Pb

- acetate (blood Pb not reported). Expression of 23 

### PS was observed to increase in a concentration

- dependent manner at concentrations ≥ 24 

### 50 mg /kg, confirming the in vitro results. No hemolysis or microvesicle formation was 25 

### observed in the in vitro and in vivo experiments. In a follow

- up study, the same lab 26 

### observed that exposure of human RBCs to low concentrations of Pb

- acetate (0.1

- 0.5 μ M) 27 

### induced PS expression. Most notably, exposure to 0.1 μ M Pb for 24 hours increased PS 28 

### expression on RBC membranes by approximately 20% ( Jang et al., 2011 )

. 29 

### Accompanying the expression of PS were abnormal, echinocytic RBCs following 30 

### incubation with Pb. Unlike the above study, incubation with low concentrations of Pb 31 

### (0.1 μ M) induced the generation of microvesicles, which also expressed PS on their 32 

### membranes. Flippase was inhibited by 0.1 μ M Pb following incubation for one hour, but 33 

### scramblase activity was not changed at any Pb exposure concentration. The intracellular 34 

### concentration of ATP was decreased at Pb concentrations 0.25 μ M and greater, but 35 

### [Ca 2+ 

### ] i 

### did not increase following exposure. This decrease in ATP levels, but lack of 36 

### affect on [Ca 2+ 

### ] i 

### may explain why flippase activity, but not scramblase, was altered 37February 2012 5

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### following Pb incubat ion. At 0.5 μ M, Pb

- exposed RBCs with externalized PS were 1 

### observed to be targeted and engulfed by differentiated macrophages. Similar in vitro 2 

### effects were observed in rat erythrocytes, although higher concentrations were generally 3 

### required. PS expression on rat erythrocytes was observed ex vivo in rats exposed to 10 or 4 

### 50 mg /kg Pb. To corroborate these in vitro and ex vivo findings, rats were exposed to 0, 5 

### 50, 250, or 1,000 ppm Pb

- acetate in drinking water for 4 weeks. At 100 ppm , Hb and Hct 6



### were significantly decreased relative to control, and liver and spleen weights were 7 

### increased. At doses greater than 50 ppm , iron accumulation was observed in the spleen, a 8 

### clear sign of increased RBC clearance via phagocytosis. 9


### Ciubar et al. ( 2007 ) also found that exposure to Pb nitrate (0.5

– 2 μ M) resulted in an 10 

### increase in PS exposure to RBCs and cell shrinkage, which authors stated were indicators 11 

### of cell apoptosis. As reported above, Khaĭ rullina et al. ( 2008 ) observed Pb

- induced RBC 12 

### membrane smoothing that may be due to alte rations in AE1 activity. Disruptions in AE1 13 

### activity may also result in enhanced PS exposure and premature cell death. Akel et al. 14 

### ( 2007 ) observed that in AE1 - /

- 

### knockout mice, Pb

- induced PS exposure was much greater 15 

### than that in wild type mice. Decreased RBCs and increased reticulocytes were also 16 

### observed, an indication of high cell turnover. 17



### 5.7.2.3 Red Blood Cell Hematopoiesis
Erythropoietin is a glycoprotein hormon e excreted by the kidney to promote the 18 

### development of RBCs in the bone marrow. Sakata et al. ( 2007 ) examined the relationship 19 

### between blood Pb level and serum erythropoietin levels in Pb

- exposed nonanemic 20 

### tricycle taxi drivers (n=27) working in Kathmandu, Nepal (mean [SD] age: 5.6 [2.6] 21 

### years). The average blood Pb l evel in the taxi drivers was 6.4 μ g/dL compared to 22 

### 2.4 μ g/dL in nondrivers. Drivers had a significantly lower mean level of serum 23 

### erythropoietin (12.7 versus 18.8 mU/mL) compared to the nondrivers and there was an 24 

### inverse relationship between the level of serum erythropoietin and blood Pb (r =

- 0.68, p 25 

### < 0.001). Blood Pb level was not associated with any other hematological effects. The 26 

### Sakata et al. ( 2007 ) study demonstrated that serum erythropoietin levels are affected by 27 

### Pb even at levels low enough not to cause anemia. While decreased erythropoietin is 28 

### generally co nsidered a measure of kidney toxicity, it can also indicate that Pb could 29 

### possibly affect the level of RBCs through decreasing levels of serum erythropoietin. 30


### Celik et al. ( 2005 ) observed that exposure of female rats to 140, 250, or 500 mg /kg 31 

### Pb

- acetate via gavage once per week for 10 weeks (blo od Pb not reported) resulted in 32 

### decreased numbers of polychromatic RBCs (PCE) and increased numbers of 33 

### micronucleated PCEs, compared to controls (p < 0.001). Alghazal et al. ( 2008b ) exposed 34February 2012 5

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### male and female rats to 100 mg /L Pb

- acetate daily in drinking water for 125 days (blood 1 

### Pb not reported) and observed increases in micronucleated PCEs in female rats (p = 0.02) 2 

### but no significant reduction in the ratio of PCEs to normochromic RBCs (NCE). In male 3 

### rats, an increase in micronucleated PCEs was observed (p < 0.001) along with a decrease 4 

### in the PCE/NCE ratio (p = 0.02). While the results from Alghazal et al. ( 2008b ) indicate 5 

### that Pb is cytotoxic in male rats only, but is genotoxic in both sexes, results from Celik et 6 

### al. ( 2005 ) indicate that Pb is cytotoxic in female rats as well. Mice exposed to 1 g/L 7 

### Pb

- acetate in drinking water for 90 days (blood Pb no t reported) had statistically 8 

### significant increases in micronucleated PCEs; a small, but statistically nonsignificant 9 

### decrease in the PCE/NCE ratio was also observed ( Marques et al., 2006 )

. Cyto

- and 10 

### geno
- toxicity in RBC precursor cells are strong indications of altered hematopoiesis in 11 

### bone marrow. 12



### 5.7.2.4 Membrane Proteins
There have been few studies examining the effects of Pb on membrane proteins since the 13 

### 2006 Pb AQCD. According to the 2006 Pb AQCD, Pb has been found to affect RBC 14 

### membrane polypeptides in exposed workers ( Apostoli et al., 1988 ; Fukumoto et al., 15 

### 1983 )

. In Pb

- exposed workers, Fukumoto et al. ( 1983 ) found decreased levels of 16 

### polypeptides in band 3, which Apostoli et al. ( 1988 ) suggested may represent an anion 17 

### channel protein, and increases in the level of polypeptid es in bands 2, 4, 6, and 7. 18 

### Fukumoto et al. ( 1983 ) suggested that the changes in the RBC membrane polypeptides 19 

### may cause changes in membrane permeability. Apostoli et al. ( 1988 ) found that the 20 

### changes in membrane polypeptides in association with blood Pb levels greater than 21 

### 50 μ g/dL. Exposure to Pb

- acetate at concentrations above 0.1 μ M for 60 minute s has also 22 

### been found to increase the phosphory lation of proteins in human RBC membranes in 23 

### vitro ( Belloni

- Olivi et al., 1996 )

. Phosphorylation did not occur in cells depleted of 24 

### protein kinase C (PKC), indicating a PKC

- dependent mechanism. 25


### Huel et al. ( 2008 ) found that newborn hair and cord blood Pb levels (mean [SD]: 1.22 26 

### [1.41] μ g/g and 3.54 [1.72] μ g/dL) were negatively associated with Ca

- ATPase act ivity 27 

### in plasma membranes of RBCs isolated from cord blood; newborn hair Pb levels were 28 

### more strongly associated with cord Ca pump activity than were cord blood Pb (p < 29 

### 0.0001 versus p < 0.05). Maternal blood Pb levels were not correlated with Ca pump 30 

### acti vity in maternal or cord blood. Pb

- induced disruptions in Ca homeostasis in RBCs can 31 

### lead to cytotoxicity and necrosis, and these effects may be representative of cellular 32 

### dysfunction in other organ systems. 33February 2012 5

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### 5.7.3 Red Blood Cell Heme Metabolism

Pb has been foun d to inhibit several enzymes involved in heme synthesis, namely ALAD 1 

### (cytoplasmic enzyme catalyzing the second, rate

- limiting, step of the heme biosynthesis 2 

### pathway), coporphyrinogen oxidase (catalyses the sixth step in heme biosynthesis 3 

### converting coporph yrinogen III into protoporphyrinogen IX), and ferrochelatase 4 

### (catalyses the terminal step in heme synthesis converting protoporphyrin IX into heme) 5 

### ( Figure 5

- 45 ). The observations of decreased Hb (measured as total Hb, MCH, or 6 

### MCHC) in occupationally

- exposed adults ( Ukaejiofo et al., 2009 ; Khan et al., 2008 ; Patil 7 

### et al., 2006b ; Karita et al., 2005 ) and Pb

- exposed experimental animal models ( Sharma et 8 

### al., 2010b ; Simsek et al., 2009 ; Marques et al., 2006 ; Lee et al., 2005 ) and associations 9 

### with blood Pb levels in children ( Riddell et al., 2007 ) , is a direct indicator of decreased 10 

### heme synthesis due to Pb exposur e. 11

Note: Steps in the pathway potentially affected by Pb are indicated with curved arrows pointing to the affected enzyme, and e ffects are represented by ↑ and ↓ arrows. 

### Figure 5
- 45 Schematic representation of the enzymatic steps involved in the heme synthetic pathway.February 2012 5

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### 5.7.3.1 Red Blood Cell 5
- Aminolevulinic Acid Dehydratase

Decreases in RBC 5

- aminolevulinic acid dehydratase (ALAD) levels are strongly 1 

### associated with Pb exposure in humans to such an extent that RBC ALAD activity has 2 

### been used to assess Pb toxicity. Several epidemiologic studies published since the 2006 3 

### Pb AQCD evaluated the relationship between Pb exposure, blood Pb levels and ALAD 4 

### activity in adults and children (see below). These studies were cros s
- sectional in nature. 5 

### This limits their utility in assessing the magnitude, timing, frequency, and duration of Pb 6 

### exposure necessary to contribute to the observed effects. 7 

### As was seen with epidemiologic studies investigating Pb

- induced deficits in 8 

### hematol ogical parameters, most occupationally

- exposed cohorts investigated ALAD 9 

### levels in association with high blood Pb levels, i.e., > 27 μ g/dL (range: 27.0

- 74.4 μ g/dL). 10 

### Although effects observed within these cohorts regarding ALAD levels may not be 11 

### generalizable to the population as a whole, they are useful in demonstrating consistent 12 

### and negative effects of Pb on the activity of this enzyme ( Quintanar

- Escorza et al., 2007 ; 13 

### Patil et al., 2006a ; Patil et al., 2006b ; Ademuyiwa et al., 2005b )

. Occupationally

- exposed 14 

### adults had levels of inhibition of ALAD that were as great as 90% relative to con trol 15 

### ( Quintanar

- Escorza et al., 2007 )

. The re were few studies that investigated Pb

- associated 16 

### decrements in ALAD levels among moderately

- exposed workers. Painters in India with a 17 

### mean blood Pb level of 21.92 μ g/dL (mean [SD] duration of exposure: 126.08 [49.53] 18 

### months) had lower ALAD levels (p < 0 .01) compared to controls whose mean blood Pb 19 

### level was 3.06 μ g/dL ( Mohammad et al., 2008 )

. Stoleski et al. ( 2008 ) observed that 20 

### workers in a Pb smelter in Macedonia (mean [SD]: 16.4 [8.5] μ g/dL blood Pb; 18.8 [7.5] 21 

### years employment) had lower ALAD activity (p < 0.001) and higher ALA levels (p < 22 

### 0.0005) compared to workers with no exposure to Pb (mean [SD] blood Pb: 7.0 23 

### [5.4] μ g/dL). In automotive painters exposed to Pb in Brazil (mean [SD]: 5.4 [0.4] μ g/dL 24 

### blood Pb; 133.9 [14.5] months duration of exposure), the ALAD reactivation inde x was 25 

### increased over that in controls; ALAD activity did not differ between groups ( Conterato 26 

### et al., In Press )

. However, ALAD act ivity was negatively correlated with blood Pb (r =

- 27 

### 0.59, p < 0.05) but not blood Cd, whereas ALAD reactivation index was positively 28 

### correlated with blood levels of both metals (Pb: r = 0.84, p < 0.05; Cd: r = 0.27, p < 29 

### 0.05). In a benchmark dose (BMD)

- bas ed analysis, Murata et al. ( 2009 ) calculated the 30 

### BMD and 95% lower confidence limit of the BMD (BMDL) for decreased ALAD 31 

### activity in RBCs of exposed Pb workers. The calculated BMD and BMDL values of 2.7 32 

### and 2.3 μ g/dL, res pectively, were substantially lower than the BMDs (28.7

- 44.2 μ g/dL) 33 

### and BMDLs (19.4

- 29.6 μ g/dL) for decreased Hb, Hct, and RBC count in similarly 34 

### exposed workers, indicating decreases in ALAD activity can occur at blood Pb levels that 35 

### do not decrease RBC s urvival. 36February 2012 5

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### Wang et al. ( 2010f ) found that there was also a concentration

- dependent decrease in 1 

### ALAD activity in both children (4

- 13 years old) and adults ( 16

- 77 years old) (mean blood 2 

### Pb levels: 7.1 and 6.4 μ g/dL, respectively) in rural southwest China. Further, Wang et al. 3 

### ( 2010f ) observed that the relatio nship between blood Pb and ALAD activity was 4 

### nonlinear and exponential, with larger decreases in ALAD activity occurring with blood 5 

### Pb levels greater than 10 μ g/dL. No correlation was observed between urinary ALA 6 

### levels and blood Pb. Ahamed et al. ( 2006 ) studied male urban adolescents in India. The 7 

### 39 adolescents were separated into groups according to their blood Pb levels (group 1: 8 

### <10 μ g/dL [mean 7.4 μ g/dL], group 2: >10 μ g/dL [mean 13.27 μ g/dL]). Although group 9 

### 1 and 2 were similar in age (mean [SD]: 16.59 [0.91] versus 16.76 [0.90] years, 10 

### respectively), height, weight, and body mass index, group 2 had lower ALAD activity 11 

### than did group 1 (p < 0.001). When all 39 adolescen ts were examined together, an inverse 12 

### relationship was found between blood Pb and ALAD activity. Similar decreases in 13 

### ALAD activity were observed in other populations of Indian children (aged 4

- 12 and 1

- 7 14 

### years) with elevated blood Pb (mean [SD]: 11.39 [1. 39] and 21.86 [7.58] μ g/dL) 15 

### compared to children with lower blood Pb levels (mean [SD]: 3.93 [0.61] and 6.89 16 

### [2.44] μ g/dL) ( Ahamed et al., 2007 ; Ahamed et al., 2005 )

. Decreases were also observed 17 

### in children 3

– 6 years of age with > 10 μ g/dL, compared to children < 10 μ g/dL (mean 18 

### blood Pb concentration for groups not reported) in northeastern China ( Jin et al., 2006 )

. 19 

### Decreased ALAD activity in response to Pb exposure is also observed in toxicological 20 

### studies. Rats administered 500 ppm Pb

- acetate in drinking water for 15 or 30 days had 21 

### decreased blood ALAD activity that was related to duration of exposure and blood Pb 22 

### ( Rendón

- Ramirez et al., 2007 )

. Administration of Pb (25 mg /kg) to rats once a week for 4 23 

### weeks achieved a blood Pb level of 6.5 μ g/dL, which was associated with statistically 24 

### significant decreases (approximately 50% lower than control levels) in RBC ALAD 25 

### activity ( Lee et al., 2005 )

. Exposure of male Wistar rats to 0.5% Pb

- acetate via drinking 26 

### water for three weeks statistically significantly decreased ALAD activity 72% compared 27 

### to controls (mean [SD]: 7.35 [0.35] versus 26.14 [ 2.19] nM/min/mL RBCs) ( Gautam and 28 

### Flora, 2010 )

. 29



### 5.7.4 Other Heme Metaboli sm Enzymes
The 2006 Pb AQCD indicated that Pb affects RBC PBG synthase ( Simons, 1995 ; Farant 30 

### and Wigfield, 1990 , 1987 ) , PBG deaminase ( Tomokuni and Ichiba, 1990 ) , and TF 31 

### endocytosis and iron transport across membranes ( Qian and Morgan, 1990 ) , all of which 32 

### are directly or indirectly involved in heme synthesis. Although there a re no new studies 33 

### that examine the effect Pb has on the activities of other heme metabolism enzymes, a 34February 2012 5

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### number of studies investigated associations of blood Pb with concentrations of various 1 

### intermediate products in the heme biosynthetic pathway. 2


### Pb intoxi cation is known to inhibit the function of ferrochelatase, the enzyme that 3 

### catalyzes the last step in the heme biosynthetic pathway. Under normal conditions, 4 

### ferrochelatase incorporates ferrous iron (Fe 2+ 

### ) into protoporphyrin IX, converting it into a 5 

### heme molecule ( Figure 5

- 45 ). However, Pb has been shown to inhibit this insertion of 6 

### Fe 2+ 

### into the protoporphyrin ring and instead, Zn is inserted into the ring creating ZPP. A 7 

### number of recent studies have shown that blood Pb is statistically significantly associated 8 

### with increased RBC ZPP levels in adults occupationally exposed to high levels of Pb 9 

### (blood Pb level > 27

- 54 μ g/dL) ( Patil et al. , 2006b ; Ademuyiwa et al., 2005b ) , workers 10 

### exposed to moderate levels of Pb (blood Pb level = 21.92 μ g/dL) ( Mohammad et al., 11 

### 2008 ) , children aged 1

- 21 years (blood Pb 1 8

- 23 μ g/dL) ( Counter et al., 2009 , 2008 ; 12 

### Counter et al., 2007 ) , and animals exposed to 500 ppm Pb via drinking water for 15 or 30 13 

### days ( Rendón

- Ramirez et al., 2007 )

. Interestingly, Wang et al. ( 2010f ) found that in 14 

### children and adults living in a rural area of Southwest China, ZPP levels were negatively 15 

### correlated wi th blood Pb at blood Pb levels < 10 μ g/dL and were only positively 16 

### correlated with blood Pb at higher blood Pb concentrations ( i.e., > 10 μ g/dL). The authors 17 

### suggest that this may be representative of ALAD activities at low blood Pb levels, which 18 

### contribut es to lower ZPP levels. Scinicariello et al. ( 2007 ) performed a meta

- analysis and 19 

### observed that Pb

- exposed individ uals who carried the ALAD2 allele had slightly lower 20 

### concentrations of blood ZPP levels compared to carriers of the ALAD1 allele (overall 21 

### pooled standardized mean estimate:

- 0.09 [units not specified]; 95% CI:

- 0.22, 0.03, p = 22 

### 0.13). 23



### 5.7.5 Other Hematological Pa rameters

5.7.5.1 Energy Metabolism
RBCs use high energy purine nucleotides ( i.e., ATP and guanine triphosphate [GTP]) to 24 

### support basic metabolic functions. In mature RBCs, these nucleotides are synthesized via 25 

### salvage reactions via either an adenine pathway, which requires adenine 26 

### phosphoribosyltransferase (APRT), or an adenosine pathway, which requires adenosine 27 

### kinase. The 2006 Pb AQCD reported that Pb significantly reduces the nucleotide pool 28 

### including NAD and NADP, as well as increases purine degradation produc ts resulting in 29 

### altered RBC energetics. Since the 2006 Pb AQCD, there have been few studies 30 

### examining Pb effects on energy metabolism. Baranowska

- Bosiacka et al. ( 2009 ) 31 

### examined the effects of Pb on RBC APRT and hypoxanthine

- guanine 32February 2012 5

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### phosphoribosyltransferase (HPRT) due to in vitro and in vivo exposures. For the in vitro 1 

### exposure, APRT and HPRT were measured in lysate of human RBCs after exposure to 2 

### Pb at a concentration range from 0.1 to 100 μ M for 5

– 30 minute s. In vivo tests measured 3 

### APRT and HPRT in rat RBC lysate from rats exposed to Pb

- acetate (0.1 %) in drinking 4 

### water for 9 months. Both the in vivo and v itro studies found a significant decrease in both 5 

### HPRT and APRT levels. The levels were significantly decreased in vitro after only 6 

### 5 minute s of exposure to the 0.1 μ M concentration, but the decrease was also 7 

### concentration

- dependent. However, the study aut hors considered the inhibition moderate 8 

### (30

– 35%) even with the highest levels used in vitro. Shin et al. ( 2007 ) found a 9 

### concentration

- dependent decrease in intracellular ATP in human RBCs in vitro with 10 

### significant decreases found even with the lowest concentration ( i.e., 1 μ M). 11



### 5.7.5.2 Other Enzymes
The 2006 Pb AQCD reported that K + 

### permeability was increased by Pb due to altered 12



### s ensitivity of the membrane Ca 2+



### - binding site that caused selective efflux of K + 

### ions from 13 

### the RBC membrane. However, inhibition of the RBC Na +



### - K + 

### ATPase is more sensitive to 14 

### Pb exposure than is the inhibition of Ca 2+



### - Mg 2+ 

### ATPase. Few new studies were found 15 

### that examined the effects of Pb exposure on other enzymes. Ekinci et al. ( 2007 ) tested the 16 

### effects of Pb on two carbonic anhyd rase isozymes (I and II) isolated from human RBCs. 17 

### Carbonic anhydrases are metalloproteins that use Zn to catalyze the equilibrium between 18 

### carbon dioxide and bicarbonate in the cells of higher invertebrates. Although 19 

### investigators found that Pb nitrate inh ibited both carbonic anhydrase isozymes in a 20 

### concentration

- dependent manner, the concentrations used ( i.e., 200

- 1 , 000 μ 

### M) were above 21 

### those that would be physiologically relevant. Inhibition of isozyme I was noncompetitive, 22 

### while the inhibition for isozyme II was uncompetitive. Bitto et al. ( 2006 ) examined the 23 

### mechanisms of action of Pb

- induced inhibition of P5N, an enzyme important in the 24 

### pyrimidine salv age pathway that requires manganese for normal activity. Pb was 25 

### observed to bind directly to the active site of the enzyme in a different position than the 26 

### manganese, thus possibly resulting in improper protein folding and inhibition of activity. 27



### 5.7.6 Red Bloo d Cell Oxidative Stress
It has been suggested that the Pb

- associated decreases in ALAD activity result in 28 

### increased oxidative stress, owing to the buildup of ALA. ALA can act as an electron 29 

### donor in the formation of reactive oxygen species (ROS) ( Nemsadze et al., 2009 ; 30 

### Ahamed and Siddiqui, 2007 )

. Many epidemiologic and toxicological studies have found 31February 2012 5

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### an association between the level of blood Pb and lipid peroxidation, antioxidant levels, or 1 

### indicators of ROS production. 2



### 5.7.6.1 Oxidative Stress, Lipid Peroxid ation, and Antioxidant Enzymes
Malondialdehyde (MDA) is an end product of lipid peroxidation and is commonly used 3 

### as an indicator of lipid peroxidation. Numerous occupational studies demonstrated 4 

### increased lipid peroxidation in highly

- exposed worker popula tions (blood Pb levels 5 

### ranging from 29 to 74.4 μ g/dL) ( Kasperczyk et al., 2009 ; Khan et al., 2008 ; Quintanar

6 

### Escorza et al., 2007 ; Patil et al., 2006a ; Patil et al., 2006b )

. There was a correlation 7 

### between MDA levels and blood Pb even in the unexposed workers who had lower 8 

### ( i.e., <12 μ g/dL) blood Pb levels, although the magnitude of correlation in exposed 9 

### workers was greater. ( Quintanar

- Escorza et al., 2007 )

. Increases in C

- reactive protein and 10 

### decreases in RBC SOD, catalase, and plasma ceruloplasm were also observed in these 11 

### workers, further indicating increased RBC oxidative stress due to higher Pb exposure. 12 

### Evidence of lipid peroxidation was also observed in occupational cohorts mo derately 13 

### exposed to Pb. These studies were cross

- sectional in design; thus, there is uncertainty 14 

### regarding the magnitude, timing, frequency, and duration of Pb exposure that contributed 15 

### to the observed associations. In auto repair apprentices in Turkey (me an [SD]: 16.8 [1.2] 16 

### years of age, 3.8 [1.8] years duration of exposure) with blood Pb levels as low as 17 

### 7.9 μ g/dL ( Ergurhan

- Ilhan et al., 2008 ) , i ncreases in glutathione peroxidase (GPx) and 18 

### MDA, as well as decreases in α

- tocopherol and β

- carotene were observed compared with 19 

### controls (compared to 2.6 μ g/dL in controls; mean [SD] age: 16.3 [1] years). Decreases 20 

### were observed in SOD and CAT, but the r esults did not attain statistical significance. In 21 

### painters in India (mean [SD] duration of exposure: 126.08 [49.53 months]) with a mean 22 

### blood Pb level of 21.92 μ g/dL (compared to 3.06 μ g/dL in controls), there was a 23 

### significant decrease in SOD, glutathion e (GSH), and CAT accompanied by a significant 24 

### increase in oxidized GSH ( i.e., GSSG) and thiobarbituric acid reactive species (TBARS, 25 

### expressed in terms of MDA) measured in plasma and RBC lysate ( Mohammad et al., 26 

### 2008 )

. In automotive painters in Brazil (mean [SE] blood Pb: 5.4 [0.4] μ g/dL), 27 

### glutathione

- S

- transferase, GPx, and SOD were positively correlated with blood Pb (r = 28 

### 0.34, 0.38, and 0.32, respecti vely; p < 0.05) ( Conterato et al., In Press )

. Similar effects on 29 

### indices of oxidative stress were observed in in vitro studies: increased MDA and 30 

### decreased SOD and catalase in RBCs exposed to 2 μ M Pb ( Ciubar et al., 2007 ) , 31 

### decrease d glutathione reductase (GR) activity in human RBCs incubated with 5

- 18 μ M 32 

### Pb ( Coban et al., 2007 ) , and decreased GSH and increased GSSG and lipid peroxidation 33February 2012 5

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### in RBCs from healthy volunteers with no Pb exposure incu bated with 0.4 μ M Pb for 24

- 1 

### 120 hours ( Quintanar

- Escorza et al., 2010 )

. 2 

### Evidence of lipid peroxidation was also observed in children moderately exposed to Pb; 3



### as with the occupational studies, their cross

- sectional design limits their utility in 4 

### assessing the magnitude, timing, frequency, and duration of Pb exposure necessary to 5 

### elicit the observed effects. Ahamed et al. ( 2005 ) investigated the relationship between 6 

### blood Pb levels and antioxidant enzyme levels and lipid peroxidation in children aged 4

- 7 

### 12 years in Lucknow, India. A total of 62 children, with a mean [SD] blood Pb level of 8 

### 7.47 [3.06] μ g/dL, we re included in the study. Children were separated into three groups 9 

### based on their blood Pb levels. The means (SDs) were: 3.93 (0.61) μ g/dL group I; 7.11 10 

### (1.25) μ g/dL group II; and 11.39 (1.39) μ g/dL group III. Lipid peroxidation, measured as 11 

### blood MDA, wa s statistically significantly greater in group III, compared to group II and 12 

### I, whereas GSH was lower in group III relative to groups II and I. Catalase activity was 13 

### the only measure of oxidative stress that was statistically significantly elevated in grou p 14 

### II compared to group I. Additionally, blood Pb levels were found to be statistically 15 

### significantly positively correlated with MDA and CAT and negatively correlated with 16 

### GSH. Ahamed et al. ( 2006 ) additionally studied male urban adolescents in India. The 39 17 

### adolescents were separated into groups according to their blood Pb level (group 1: 18 

### <10 μ g/dL [mean 7.4 μ g/dL], group 2: >10 μ g/dL [mean 13.27 μ g/dL]). Although the 19 

### groups were similar in age (mean [SD]: 16.59 [0.91] versus 16.76 [0.90] years, 20 

### respectively), height, weight, and body mass index, group 2 had significantly higher 21 

### levels of CAT and MDA compared to group 1. There were no significant differences in 22 

### blood GSH level s. Examining all the study subjects together, investigators found a 23 

### correlation between blood Pb level and blood MDA and RBC CAT levels, as well as an 24 

### inverse relationship between ALAD activity and MDA and CAT levels. In a similar 25 

### study, Ahamed et al. ( 2008 ) examined oxidative stress in Indian childre n (aged 3

- 12 26 

### years) with neurological disorders. There was a significantly higher mean blood Pb level 27 

### in the study population compared to the control healthy population (18.60 versus 28 

### 10.37 μ g/dL). In addition, the following indicators of oxidative stress w ere observed in 29 

### the study population: increased blood MDA, RBC SOD, and CAT levels and decreased 30 

### blood GSH levels. GPx levels were similar between the two groups. Typical indicators of 31 

### Pb exposure (active/nonactive ALAD ratio) were found to be correlated w ith lipid 32 

### peroxidation and oxidative stress. Children aged 3

– 6 years old living near a steel refinery 33 

### in China with blood Pb levels ≥ 10 μ g/dL also had a significant increase in plasma MDA 34 

### compared to the children with blood Pb levels < 10 μ g/dL. However, levels of RBC 35 

### SOD, GSH, and GPx were not different from those in controls ( Jin et al., 2006 )

. 36 

### Administration of Pb (25 mg /kg) to rats once a week for 4 weeks, which was related to a 37 

### blood Pb level of about 6.5 μ g/dL, caused a significant increase in RBC MDA levels ( Lee 38February 2012 5

- 413 Draft

– Do Not Cite or Quote 

### et al., 2005 )

. Other indications of Pb

- induced oxidative stre ss included significant 1 

### increases in RBC SOD and CAT levels accompanied by significant decreases in GSH 2 

### and GPx. Exposure of rats to 750 mg /kg Pb

- acetate in drinking water for 11 weeks 3 

### resulted in decreased concentrations of plasma vitamin C, vitamin E, no nprotein thiol, 4 

### and RBC

- reduced glutathione, with simultaneous increased activity of SOD and GPx 5 

### ( Kharoubi et al., 2008b )

. CAT activity was also slightly elevated in Pb

- exposed rats, but 6 

### the increase failed to reach statistical significance. Exposure of male rats to 0.5% Pb 7 

### nitrate in drinking water (blood Pb not reported) for three weeks decreased GSH levels 8 

### compared to that in controls (mean [SE]: 1.91 [0.02] versus 2.44 [0.09] mg /mL, 9 

### respectively) ( Gautam and Flora, 2010 )

. SOD activity was significantly decreased in rats 10 

### injected with 15 mg /kg Pb i.p. for seven days, but not rats treated with 5 mg /kg Pb 11 

### ( Berrahal et al., 2007 )

. GPx activity and MDA concentrations were slightly elevated in 12 

### the exposed groups, but differences with the control group failed to reach statistical 13 

### significance. 14



### 5.7.6.2 Antioxidant Defense

In addition to the studies listed above t hat examine lipid peroxidation and oxidative 15 

### stress, there have been studies that indicate that the use of antioxidants and free radical 16 

### reactions is protective against Pb

- induced RBC oxidative stress. Rats treated with 17 

### 500 ppm Pb

- acetate in drinking water for 15 or 30 days had an increase in free RBC 18 

### protoporphyrin and TBARS that was related to length of exposure and blood Pb 19 

### ( Rendón

- Ramirez et al., 2007 )

. Vitamin E administration after exposure to Pb 20 

### significantly reduced the TBARS levels and increased ALAD activity, compared to 21 

### exposure to Pb alone. Co

- exposure to vitamin E and Pb simultaneously and exposure to 22 

### vitamin E before Pb exposu re also prevented Pb

- induced oxidative stress. In vitro studies 23 

### by Casado et al. ( 2007 ) , found that Pb

- induced hemolysis and RBC membr ane damage 24 

### was mediated via oxidative stress. The in vitro studies demonstrated a concentration

- and 25 

### time

- dependent formation in lipid peroxide that was inhibited with a number of 26 

### antioxidants, including desferrioxamine (iron chelator), trolox (chain break ing 27 

### antioxidant), and mannitol and Na formate ( • 

### OH scavengers). Results suggested the role 28 

### of singlet oxygen in Pb

- mediated membrane damage and hemolysis of exposed RBCs. In 29 

### rats exposed to 2 , 000 ppm Pb in drinking water for 5 weeks, MDA levels were 30 

### significantly increased, whereas vitamin E concentrations were significantly decreased 31 

### ( Caylak et al ., 2008 )

. In the case of MDA, co

- exposure to Pb and a number of sulfur

32 

### containing antioxidants ( e.g., L

- methionine, N

- acetylcysteine, and L

- homocysteine) 33 

### reduced concentrations to a level not statistically significantly different from that in 34February 2012 5

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– Do Not Cite or Quote 

### controls, but statistically smaller than concentrations observed with Pb alone. Exposure to 1 

### L

- methionine and N

- acetylcysteine also reduced Pb

- induced depletion of vitamin E. 2



### 5.7.7 Summary and Causal Determination
There is consistent toxicological and epidemiologic eviden ce indicating that exposure to 3 

### Pb affects hematological endpoints, including decreased RBC survival and function, 4 

### altered heme synthesis, and increased RBC oxidative stress. Pb has been shown to 5 

### preferentially partition into RBCs following exposure, with R BC concentrations 6 

### approximately 100

- fold greater than those observed in the plasma ( Jin et al., 2008 ; 7 

### Timchalk et al., 2006 )

. 8 

### Elaborating on the body of evidence presented in the 2006 Pb AQCD , recent 9 

### epidemiologic and toxicological st udies continue to demonstrate that Pb exposure is 10 

### associated with decreased RBC survival and function, with the largest body of evidence 11 

### consisting of populations of adults and children in which Pb is associated with effects on 12 

### several inter

- connected and related hematological parameters such as Hb, PCV, MCV, 13 

### MCH, and MCHC. For adult populations, the largest body of evidence consists of 14 

### occupationally

- exposed workers in which measures of RBC survival are altered when 15 

### compared with unexposed control populati ons ( Cabaravdic et al., 2010 ; Ukaejiofo et al., 16 

### 2009 ; Khan et al., 2008 ; Patil et al., 2006a ; Patil et al., 2006b ; Karita et al., 2005 ; 17 

### Conterato et al., In Press )

. Although the mean blood Pb level in most occupationally 18 

### exposed populations was in excess of 20 μ g/dL, decreases in Hb and PCV were observed 19 

### in adults with blood Pb levels of 7 μ g/dL (compared to controls with blood Pb levels of 20 

### 3 μ g/dL), and significant correlatio ns were observed between RBC distribution width and 21 

### MCHC and blood Pb levels in adult populations with mean blood Pb levels of 5.4 μ g/dL. 22 

### Only one non
- occupational study was found investigating the association of Pb with 23 

### hematological parameters; in pregna nt women, blood Pb levels were found to be 24 

### negatively correlated with Hb concentrations. Studies in children measuring concurrent 25 

### blood Pb levels are generally in agreement with those investigating occupationally

26 

### exposed adults regarding effects on hematol ogical parameters ( i.e., Hb, MCV, ( Queirolo 27 

### et al., 2010 ; Shah et al., 2010 ; Ahamed et al., 2007 ; Huo et al., 2007 ; Olivero

- Verbel et 28 

### al., 2007 ; Riddell et al., 2007 ; Turgut et al., 2007 ; Ahamed et al., 2006 ; Rondo et al., 29 

### 2006 )

. Any differences in the effects on specific hematological parameters observed 30 

### between study findings for adults and those for children may be due to the comparatively 31 

### shorter duration and lower magnitude of Pb exposure experienced by chi ldren compared 32 

### to adults, although there is uncertainty regarding the timing and duration of exposure 33 

### needed to induce effects in adults. In addition, Pb was shown to reduce Ca 2+



### - and Ca 2+



### - 34 

### Mg 2+



### - ATPase activity in RBC membranes, which leads to an increase i n RBC [Ca 2+ 

### ] i 

### , 35February 2012 5

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– Do Not Cite or Quote 

### increased membrane fragility, and abnormal morphological changes in studies of 1 

### occupationa l ly exposed adults ( Quintanar

- Escorza et al., 2007 ) and in in vitro studies 2 

### ( Quintanar

- Escorza et al., 2010 ; Ciubar et al., 2007 )

. Heul et al. ( 2008 ) observed a 3 

### reduction of RBC Ca 2+



### - Mg 2+



### - ATPase activity in children in association with a concurrent 4 

### group mean cord blood Pb level of 3.54 μ g/dL. Toxicological studies ha ve also observed 5 

### decreases in Hct and Hb and increases in hemolysis and reticulocyte density in rats and 6 

### mice with blood Pb levels as low as 6.6

- 7.1 μ g/dL ( Sharma et al., 2010b ; Baranowska

7 

### Bosiacka et al., 2009 ; Simsek et al., 2009 ; Lee et al., 2005 )

. Pb exposure has also been 8 

### observed to increase PS expression on RBC membrane s, leading to cell shrinkage, 9 

### erythro po ie sis, and destruction of the RBCs by macrophages ( Jang et al., 2011 ; Ciuba r et 10 

### al., 2007 ; Shin et al., 2007 )

. Suggestive eviden ce of disrupted hematopoiesis evidenced 11 

### by decreased serum erythropoietin was observed in occupationally

- exposed adults with a 12 

### mean blood Pb level of 6.4 μ g/dL ( Sakata et al., 2007 ) ; toxicological studies in rats also 13 

### indicate that Pb is cytotoxic to RBC progenitor cells ( Alghazal et al., 2008b ; Celik et al., 14 

### 2005 )

. Taken together, these studies p rovide consistent evidence that exposure to Pb 15 

### affects RBC function and survival, and leads to the reduction of RBCs in circulation. 16 

### Although this decrease in RBCs may be explained by both decreased cell survival and/or 17 

### disruption of hematopoiesis, the obs ervation of increased reticulocytes seems to represent 18 

### compensation for decreased RBC survival due to Pb exposure. 19 

### Confirming effects observed in the 2006 Pb AQCD , a large body of evidence consisting 20 

### of cross

- sectional epidemiologic studies measuring conc urrent blood Pb in adults and 21 

### children have found that decreases in RBC ALAD levels and activity are strongly 22 

### associated with higher blood Pb levels ( Wang et al., 2010f ; Mohammad et al., 2008 ; 23 

### Ahamed et al., 2007 ; Quintanar

- Escorza et al., 2007 ; Ahamed et al., 2006 ; Pa til et al., 24 

### 2006a ; Patil et al., 2006b ; Ademuyiwa et al., 2005b ; Ahamed et al., 2005 ; Conterato et 25 

### al., In Press )

. Although the body of evidence is smaller than for humans, decreases in 26 

### blood ALAD activity were also seen in rats with increased blood Pb levels, compared to 27 

### controls ( Lee et al., 2005 )

. In addition to ALAD, recent studies have shown that Pb 28 

### exposure inhibits the activity of ferrochelatase, leading to increased RBC ZPP in children 29 

### and occupationally

- exposed adults ( Counter et al., 2009 , 2008 ; Mohammad et al., 2008 ; 30 

### Counter et al., 2007 ; Patil et al., 2006b ; Ademuyiwa et al., 2005b ) and animals ( Rendón

31 

### Ramirez et al., 2007 )

. Pb has also been shown to inhibit the in vitro activities of other 32 

### enzymes in RBCs, including those involved in nucleotide scavenging, energy 33



### m etabolism, and acid

- base homeostasis ( Baranowska

- Bosiacka et al., 2009 ; Ekinci et al., 34 

### 2007 )

. 35 

### Lastly, Pb exposure induces lipid peroxidation and oxidative stress in RBCs. 36 

### Epidemiologic st udies have observed increases in MDA in occupationally

- exposed adult 37 

### populations ( Ergurhan

- Ilhan et al., 2008 ; Khan et al., 2008 ; Mohammad et al., 2008 ; 38February 2012 5

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– Do Not Cite or Quote 

### Quintanar

- Escorza et al., 2007 ; Patil et al., 2006a ; Patil et al., 2006b )

. Other changes in 1 

### oxidative stress parameters observed included lowered activities of SOD, GR, and CAT, 2 

### and increased CRP. Altered indices of RBC oxidative stress were also seen in adolescents 3 

### and children in association with blood Pb levels ( Ahamed et al., 2008 ; Ahamed et al., 4 

### 2006 ; Jin et al., 2006 )

. In vitro and vivo studies have also demonstrated that prior, 5 

### concurrent, or subsequent treatment with various antioxidants has been shown to 6 

### ameliorate at least partially Pb

- induced oxidative stress in RBCs ( Caylak et al., 2008 ; 7 

### Casadoa et al., 2007 ; Rendón

- Ramirez et al., 2007 )

. 8 

### Similar to the epidemiologic and toxicological studies that demonstrate an association 9 

### between Pb exposure and hematological effects in humans and laboratory animals, the 10 

### ecological literature has consistently reported on hematological responses in aquatic and 11 

### terrestrial invertebrates and vertebrates ( Section s 7.4.1.2 and 7.4.2.2 ). The most 12 

### consistently observed effect in metal impacted environments is decreased RBC ALAD 13 

### activity. This effect has been observed across a wide range of taxa, including bivalves, 14 

### fish, amphibians, birds, and mammals. More limited evidence exists regarding deleterious 15 

### effects of Pb on serum enzyme levels and white blood cell counts in birds and mammals. 16 

### In summary, new epidemiologic and toxicological studies included in the current review 17 

### provide strong evidence that exposure to Pb is associated with numerous deleterious 18 

### effects on the hematological system, including effects on RBC survival and function, 19 

### altered heme synthesis, and increased oxidative stress, and continue to confirm previous 20 

### conclusions from the 2006 Pb AQCD . The principal finding regarding RBC survival and 21 

### function are consistent Pb

- induced alterations in several inter

- connected and relat ed 22 

### hematological parameters such as Hb, Hct, and MCV across multiple studies, with the 23 

### weight of evidence provided by epidemiological studies in occupationally

- exposed adult 24 

### populations and children. In occupationally

- exposed adults, these findings are mos t 25 

### substantiated in populations with current blood Pb levels > 20 μ g/dL, although effects on 26 

### hematological parameters were observed in some occupationally

- exposed populations at 27 

### concurrent blood Pb levels in the range of 5

- 7 μ g/dL. In Pb

- exposed children, e ffects on 28 

### hematologi cal parameters were most substa ntiated in populations with blood Pb levels 29 

### less than 15 μ g/dL. The weight of evidence in adult rodents exposed long

- term to Pb (i.e, 30 

### ≥ 4 weeks), although less than the weight of evidence in humans, is coh erent with 31 

### epidemiologic studies regarding decrements in hematological parameters at blood Pb 32 

### levels as low as 6.6

- 7.1 μ g/dL. Regarding alterations in heme synthesis, the largest body 33 

### of evidence again is provided by decre a sed ALAD activity observed in epidemiologic 34 

### studies in occupationally

- exposed adult populations and children. In the occupationally

35 

### exposed adult populations, the observation of decreased ALAD activity was most often 36 

### observed in populations with concurrent blood Pb levels >15 μ g/dL. In children, 37 

### decreases in ALAD activity were observed in populations with concurrent blood Pb 38February 2012 5

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– Do Not Cite or Quote 

### levels in the range of 7

- 22 μ g/dL. Animal toxicological studies also provide to the weight 1 

### of evidence regarding altered ALAD activity, with effects seen in adult a nimals exposed 2 

### for 3

- 4 weeks with blood Pb levels as low as 6.5 μ g/dL. As for the effects listed above, 3 

### the weight of evidence for oxidative stress ( i.e., increased lipid peroxidation or alterations 4 

### in antioxidant enzyme levels) primarily comes from epidem iological studies in 5 

### occupationally

- exposed adults and children. The majority of evidence for increased 6 

### oxidative stress in Pb

- exposed adults comes from occupational cohorts with concurrent 7 

### blood Pb levels >15 μ g/dL. In children, concurrent blood Pb levels of 7

- 22 μ g/dL were 8 

### associated with measures of oxidative stress. Due to the cross

- sectional nature of the 9 

### above epidemiologic studies in adults and children, and the measurement of concurrent 10 

### blood Pb, the timing and duration of exposure necessary to alte r RBC survival and 11 

### function, heme synthesis, or the state of oxidative stress in RBCs is unclear. This 12 

### uncertainty is greatest in adults as concurrent blood Pb levels reflecting recent exposures 13 

### is likely to be less than blood Pb levels resulting from past exposures. The consistency of 14 

### findings in epidemiologic studies investigating effects in adults and children, and the 15 

### coherence of findings in the toxicological literature and coherence across the disciplines 16 

### is sufficient to conclude that a causal relati onship exists between Pb exposures and 17 

### effects on heme synthesis and red blood cell function. 18



## 5.8 Reproductive and Developmental Effects
The effect of Pb on reproductive outcomes has been of interest for years, starting in 19 

### cohorts of occupationally

- exposed i ndividuals. More recently, researchers have begun to 20 

### focus on reproductive effects in populations without occupational exposures, with 21 

### environmentally

- relevant levels of Pb exposure. In the toxicological and epidemiologic 22 

### literature, research on reproducti ve effects of Pb include female and male reproductive 23 

### function (hormone levels, fertility, puberty, and effects on reproductive organs and 24 

### estrus), birth defects, spontaneous abortions, infant mortality, preterm birth, low birth 25 

### weight/fetal growth, and ot her developmental effects. In epidemiologic studies, various 26 

### biological measures of Pb are used including Pb measured in blood and bone; 27 

### toxicological studies only report exposure using blood Pb. Bone Pb is indicative of 28 

### cumulative Pb exposure. Blood Pb ca n represent more recent exposure, although it can 29 

### also represent remobilized Pb occurring during times of bone remodeling and pregnancy 30 

### or lactation. More detailed discussion of these measures and Pb transfer via umbilical 31 

### cord blood Pb across the placenta , and via lactation is given in Section 4.3.5.2 on Pb 32 

### Toxicokinetics. A few studies of pregnancy

- induced hypertension and eclampsia have 33 

### been condu cted and are reported on in the section on hypertension ( Section 5.4.2.1 ). 34February 2012 5

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– Do Not Cite or Quote 

### Briefly, the relatively small number of studies found consistently positi ve associations 1 

### between blood Pb levels and pregnancy

- induced hypertension. 2 

### Overall, the recent reproductive literature continues to support associations reported in 3 

### earlier Pb AQCDs between Pb exposure and effects on various parameters of sperm 4 

### (function , motility, count, integrity, histology). The toxicological and epidemiologic 5 

### literature also indicates that Pb exposure is associated with delayed onset of puberty in 6 

### both males and females. The new information from epidemiologic and toxicological 7 

### studies is integrated with conclusions from previous Pb AQCDs below. 8



### 5.8.1 Effects on Female Reproductive Function
The epidemiologic studies on Pb and female reproductive function presented in the 2006 9 

### Pb AQCD ( U.S. EPA, 2006b ) provided litt le evidence for an association between Pb 10 

### biomarkers and effects on female reproduction and fertility. However, the 1986 and 2006 11 

### Pb AQCDs ( U.S. EPA, 2006b , 1986a ) reported toxicological findings that Pb exposure 12 

### was associated with effects on female reproductive function that can be classified as 13 

### alterations in female sexual maturation, effects on fertility and menstrual cycle, endocrine 14 

### disruption, and changes in m orphology or histology of female reproductive organs 15 

### including the placenta. Since the 2006 Pb AQCD, many epidemiologic studies have been 16 

### published regarding Pb biomarker levels in women and reproductive effects. For some 17 

### effects, there are inconsistent fi ndings, but for others, such as delayed puberty, there are 18 

### clear associations with blood Pb levels. In addition, recent toxicological studies add 19 

### further knowledge of Pb

- related effects on the female reproductive system. 20



### 5.8.1.1 Effects on Female Sex Endocrine Sy stem and Estrus Cycle
Multiple epidemiologic studies have examined the association between blood Pb levels 21 

### and hormone levels and the estrus cycle. Epidemiologic studies (characterized in Table 22 

### 5

- 30 ; all studies included in the table used measures of Pb and hormones that were either 23 

### concurrent or close in time) support the toxicological findings, which are the major body 24 

### of evidence on endocrine effects of Pb. 25February 2012 5

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### Table 5
- 30 Summary of recent epidemiologic studies of associations between Pb levels and hormones for females

Reference a Study, Location, and Years Outcome Study population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates Krieg et al. ( 2007 ) U.S. 1988

- 1994 FSH, LH Women aged 35

- 60 from the NHANES III study Blood Pb 2.8 Linear regression slope (95% CI) for log

- transformed Pb FSH: Post

- menopausal 22.2 (13. 5, 30.8) Pregnant 0.1 (

- 0.1, 0.3) Men struating at time of exam 2.1 (

- 2.1, 6.3) B oth ovaries removed 32.6 (10.1, 55.1) Birth control pills being used

- 6.3 (

- 10.0,

- 2.5) Pre

- menopausal 8.3 (3.8, 12.7)

LH: Post

- menopausal 6.2 (3.0, 9.5) Pregnant

- 0.8 (

- 1.9, 0.4) Mens truating at time of exam

- 0.3 (

- 1.8, 1.3) Both ovaries removed 10.0 (1.1, 18.9) Birth c ontrol pills being used:

- 0.6 (

- 2.9, 1.6) Pre

- menopausal 1.7 (

- 0.6, 4.1) Chang et al. ( 2006 ) Kaohsiung City, Taiwan 1999, 2000

- 2001 Estradiol Women receiving care at a infertility clinic in 2000

- 2001 or delivering a normal infant at a nearby medical center in 1999 Blood Pb 3.12 (0.19) Linear regression β (SE) for Pb 1.18 (0.60) p - value: 0.049

Poll ack et al. ( 2011 ) Buffalo, NY 2005

- 2007 FSH, estradiol, LH, progesterone, and cycle length Healthy, premenopausal women aged 18

- 44 with menstrual cycle length of 21

- 35 days, BMI of 18

- 35 kg/m 2 , not recently using birth control, not planning to become pregnant, and not breast feeding Blood Pb 0.93 IQR: 0.68, 1.20 Me an % Estradiol 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : 8.2 (

- 1.2, 18.6) 1.11

- 6.20 μg/ dL : 4.7 (

- 4.7, 15.2)
Amplitude Estradiol 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : - 0.01 (

- 0.06, 0.04) 1.11

- 6.20 μg/ dL : - 0.02 (

- 0.7, 0.03)

Phase Shift Estradiol 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : - 0.09 (

- 0.24, 0.05) 1.11

- 6.20 μg/ dL : 0.14 (

- 0.01, 0.29)

Mean % FSH 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : 8.0 (

- 0.9, 17.7) 1.11

- 6.20 μg/ dL : 3.6 (

- 5.3, 13.3)
Amplitude FSH 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : - 0.01 (

- 0.03, 0.02) 1.11

- 6.20 μg/ dL : - 0.02 (

- 0.04, 0.01)

Phase Shift FSHFebruary 2012 5

- 420 Draft

– Do Not Cite or Quote Reference a Study, Location, and Years Outcome Study population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : - 0.06 (

- 0.25, 0.12) 1.11

- 6.20 μg/ dL : - 0.02 (

- 0.21, 0.18)

Mean % LH 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : 5.1 (

- 5.1, 16.4) 1.11

- 6.20 μg/ dL : - 0.5 (

- 10.5, 10.7)
Amplitude LH 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : - 0.01 (

- 0.03, 0.02) 1.11

- 6.20 μg/ dL : - 0.02 (

- 0.04, 0.01)

Phase Shift LH 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : - 0.16 (

- 0.36, 0.03) 1.11

- 6.20 μg/ dL : - 0.11 (

- 0.32, 0.10)
Mean % Progesteron e 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : 7.5 (0.1, 15.4) 1.11

- 6.20 μg/ dL : 6.8 (

- 0.8, 14.9)
Amplitude Progesterone 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : 0.07 (0.01, 0.15) 1.11

- 6.20 μg/ dL : - 0.06 (

- 0.13, 0.01)
Phase Shift Progesterone 0.30

- 0.72 μg/ dL : Ref 0.73

- 1.10 μg/ dL : 0.04 (

- 0.06, 0.15) 1.11

- 6.20 μg/ dL : 0.15 (0.05, 0.26)
Linear models β (95% CI) Estradiol 0.03 (

- 0.05, 0.11)

FSH

- 0.01 (

- 0.07, 0.06)

LH 0.02 (

- 0.06, 0.10)
Progesterone 0.06 (

- 0.04, 0.17)

OR (95% CI) for anovulation per I μg/dL 1.20 (0.62, 2.34)February 2012 5

- 421 Draft

– Do Not Cite or Quote Reference a Study, Location, and Years Outcome Study population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates Jackson et al. ( 2011 ) Buffalo, NY 2005

- 2007 FSH, estradiol, LH, progesterone, and cycle length Healthy, pre

menopausal women aged 18

- 44 with menstrual cycle length of 21

- 35 days, BMI of 18

- 35 kg/m 2 , not recently using birth control, not planning to become pregnant, and not breast feeding Blood Pb Median: 0.87 IQR : 0.68, 1.20 Adjusted percent change (95% CI) in serum hormone level for change in blood Pb FSH:

- 2.5 (

- 11.2, 7.0) Estradiol: 4.9 (

- 5.0, 15.9) LH : 2.5 (

- 12.3, 19.9) Progesterone: 4.6 (

- 12.2, 24.6) Cycle length: 0.2 (

- 2.8, 3.3)
OR (95% CI) per unit Pb <25 d vs

. 25

- 35 d cycle length: 0.9 (0.4, 2.3) >35 d vs

. 25

- 35 d cycle length: 0.5 (0.1, 1.9)

a Studies are presented in order of first appearance in the text of this section.


### An epidemiologic study using the NHANES III data and including women aged 1 

### 35

- 60 years old examined the relationship between blood Pb levels (mean 2.8 μg/dL) and 2 

### serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) ( Krieg, 2007 )

. 3 

### Deviation from normal FSH and LH levels may indicate endocrine disruption related to 4 

### ovary functioning. Researchers found that higher blood Pb levels were associated with 5 

### higher levels of serum FSH and LH among both postmenopausal women and wome n 6 

### with both ovaries removed. There was also a trend of increasing serum FSH with blood 7 

### Pb levels for pre

- menopausal women who were not menstruating at the time of the exam 8 

### or pregnant, although the association was not statistically significant for LH. A li mitation 9 

### of this portion of the study is that FSH and LH were measured without attention to day of 10 

### a woman’s menstrual cycle and LH and FSH are known to vary throughout the cycle of 11



### non
- menopausal, cycling women who are not taking birth control pills. High er blood Pb 12 

### levels were associated with lower levels of serum FSH among women taking birth 13 

### control pills. The inverse association was also present for LH, but it was not statistically 14 

### significant. No associations between blood Pb and FSH or LH were apparen t for women 15 

### who were menstruating at the time of the exam or were pregnant. Further analysis 16 

### indicated that the lowest level of blood Pb for which a statistically significant association 17 

### between blood Pb and FSH could be observed was 1.7 μg/dL among women with their 18 

### ovaries removed. For LH, the lowest level of blood Pb for which a statistically significant 19 

### association between blood Pb and LH could be observed was 2.8 μg/dL among 20 

### postmenopausal women. Another epidemiologic study was performed in Kaohsiung Ci ty, 21 

### Taiwan among two groups of women aged 23

- 44 years: those who were seeking help at a 22 

### fertility clinic after one year of trying to conceive, and those who had previously 23 

### delivered an infant and were identified from medical records of a postpartum care un it 24 

### ( Chang et al., 2006 )

. The mean (SD) blood Pb in this study was 3.12 (0.19) μg/dL. The 25 

### study reported a positive association between blood Pb levels and serum estradiol 26February 2012 5

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– Do Not Cite or Quote 

### concentrations during the early follicular phase, which reflects ovary activity. 1 

### Associations between hormones and blood Pb level were also investigated using the 2 

### BioCycle study cohort ( Jackson et al., 2011 ; Pollack et al., 2011 )

. These women were 3 

### premenopausal with normal cycles and not on birth control. Neither study detected an 4 

### association between unit change in blood Pb and hormone levels. However, when 5 

### examining tertiles of Pb, women in the highest tertile blood Pb (1.11

- 6.20 μg/dL) had 6 

### higher mean progesterone and longer length of a phase shift compared to women in the 7 

### lowest tertile (0.30

- 0.72 μg/dL) ( Pollack et al., 2011 )

. Other associations were observed 8 

### but were not statistically significant ( Pollack et al., 2011 )

. No associations were detected 9 

### for anovulation ( Pollack et al., 2011 ) or for cycle length ( Jackson et al., 2011 )

. 10 

### The effect of Pb exposure on the female endocrine system was demo nstrated in 11 

### toxicological studies reviewed in the 1986 and 2006 Pb AQCD ( U.S. EPA, 2006b , 12 

### 1986a )

. However, the mechanism by which Pb affects the endocrine system has not been 13 

### fully elucidated. Several recent articles continue to demonstrate that Pb alters the 14 

### concentration of circu lating hormones in female experimental animals. As mentioned in 15 

### the previous AQCD, Pine et al. ( 2006 ) observed that maternal Pb exposure (during 16 

### gestation and lactation) caused a decrease in basal LH levels in pre

- pubertal female 17 

### Fisher 344 rat pups as compared to control, non
- Pb exposed pups. Dumitrescu et al. 18 

### ( 2008a ) observed alteration of hormone levels in female Wistar rats after ingesting 19 

### Pb

- acetate (50, 100, 150 ppb ) in drinking water for 6 months; measurements were made 20 

### during the pro

- estrous stage of the estrous cy cle to allow for consistent timing for 21 

### comparison of cyclic hormonal variation. The authors reported decreases in FSH, 22 

### estradiol, and progesterone levels with increases in LH and testosterone levels. 23 

### Nampoothiri and Gupta ( 2008 ) administered Pb

- acetate at a concentration that did not 24 

### affect reproductive performance, implantation or pregnancy outcome (0.05 mg /kg body 25 

### weight) to Charles Foster female rats 5 days before mating and during the gestational 26 

### p eriod. They observed a decrease in steroidogenic enzymes, 3β

- hydroxysteroid 27 

### dehydrogenase (HSD) and 17β

- HSD, activity in reproductive organs, as well as a 28 

### decrease in steroid hormones (progesterone and estradiol), suggesting that chronic 29 

### exposure to low l evels of Pb may affect reproductive function of mothers and their 30 

### offspring. Similarly, Pillai et al. ( 2010 ) reported impaired ovarian steroidogenesis in 31 

### Charles Foster adult female rats (PND56) from dams exposed gestationally and 32 

### lactationally to Pb

- acetate (subcutaneous daily injections of 0.05 μg/kg BW). Pillai 33 

### observed a decrease in steroido genic enzymes, 3β

- HSD and 17β

- HSD, but saw no 34 

### changes in ovarian steroidogenic acute regulatory protein (StAR) or CYP11 mRNA 35 

### levels indicating Pb

- induced inhibition of ovarian steroidogenesis. 36


### Kolesarova et al. ( 2010 ) conducted an in vitr o study to examine the secretory activity of 37 

### porcine ovarian granulose cells after Pb administration. The results of the study showed 38February 2012 5

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– Do Not Cite or Quote 

### that Pb

- acetate concentrations of 0.046 mg /mL and 0.063 mg /mL statistically 1 

### significantly inhibited insulin

- like growth factor

- 1 (IGF

- 1) release, but concentrations of 2 

### 0.25 mg /mL and 0.5 mg /mL did not influence IGF

- 1 release. Progesterone release was 3 

### not affected by Pb treatment; however, Pb caused a reduction in LH and FSH binding in 4 

### granulose cells and increased apoptosis as evidenced by increased expression of 5 

### caspase
- 3 and cyclin B1, suggesting a Pb

- induced alteration in the pathways of 6 

### proliferation and apoptosis of porcine ovarian granulose cells. Decreased gonadotropin 7 

### binding was also observed in rats after Pb exposu re ( Nampoothiri a nd Gupta, 2006 )

. 8 

### No recent toxicological studies were found that examined Pb

- induced effects on the 9 

### estrus cycle. 10 

### Overall, toxicological studies report alterations in hormone levels related to blood Pb 11 

### concentration. Similarly, epidemiologic studies rep orted associations between blood Pb 12 

### levels and hormone levels in female adults. Although Pb

- associated changes in hormone 13 

### levels are observed, there are discrepancies about the direction of the hormone changes 14 

### related to Pb. One explanation is that the dir ection of change could vary based on current 15 

### hormonal and reproductive status. 16



### 5.8.1.2 Effects on Fertility
Previous studies indicated that Pb exposure does not produce total sterility, but it can 17 

### disrupt female fertility ( U.S. EPA, 2006b )

. Recent epidemiologic studies and studies in 18 

### experimental animals support this finding. The epidemiologic studies are summarized in 19 

### Table 5

- 31 . Most of these studies examined biological measures of Pb collected at or 20 

### during the period of possible fertilization, although Bloom et al. ( 2011a ) measured blood 21 

### Pb at baseline and followed women for at least 12 men strual cycles (or until pregnancy). 22February 2012 5

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### Table 5
- 31 Summary of recent epidemiologic studies of associations between Pb levels and fertility for females

Reference a Study, Location, and Years Outcome Study population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates Chang et al. ( 2006 ) Kaohsiung City, Taiwan 1999, 2000

- 2001 Infertility Women receiving care at a infertility clinic in 2000

- 2001 or delivering a normal infant at a nearby medical center in 1999 Blood Pb 3.12 (0.19) OR (95% CI) Infertility ≤ 2 .5 μg/ dL : 1.00 (Ref) >2.5 μg/ dL : 2.94 (1.18, 7.34)

Al

- Saleh et al. ( 2008a ) Riyadh, Saudi Arabia 2002

- 2003 Achieving pregnancy and/or fertilization Women aged 19

- 50 undergoing IVF Blood Pb Follicular fluid Pb Blood Pb 3.34 (2.24) Blood Pb level s >10 μg/ dL : 1.7%

Follicular fluid 0.68 (1.82) OR (95% CI) (unit not given, assume results are per 1 μg/dL) Pregnancy Blood Pb 0.55 (0.23, 1.31) Follicular fluid Pb 1.36 (0.91, 2.02)

Fertilization Blood Pb 0.30 (0.08, 1.03) Follicular fluid Pb 1.45 (0.69, 3.02)
Note: In a reduced adjusted model for fertilization, the OR for blood Pb was 0.38 (0.14, 0.99) Silberstein et al. ( 2006 ) Providence, RI NS Achieving pregnancy Women undergoing IVF at the study hospital Follicular fluid Pb Not given quantitatively
From a figure in the paper: Median Pb in follicular fluid of pregnant women: ~1.3 Median Pb in follicular fluid of non

- pregnant women: ~2.2 P

- value for difference in medians by Mann

- Whitney U test: 0.0059
*note, study only included 9 women

Bloom et al. ( 2010 ) California 2007

- 2008 Oocyte maturity, oocyte fertilization Women who were part of the Study of Metals and Assisted Reproductive Technologies (SMART): women referred to the Cen ter for Reproductive Health of UCSF for infertility treatment and their first IVF procedure Blood Pb 0.82 (0.32) RR per 1 μg/dL Oocyte maturity (determined by Metaphase II arrest): 0.54 (0.31, 0.93) 0.25 (0.03, 2.50)*
Oocyte fertilization: 0.97 (0.66, 1.43) 1.09 (0.72, 1.65)*
*Controlling for CdFebruary 2012 5

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– Do Not Cite or Quote Reference a Study, Location, and Years Outcome Study population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates Bloom et al. ( 2011b ) California 2007

- 2008 Embryo cell number, embryo fragmentation score Women who were part of the Study of Metals and Assisted Reproductive Technologies (SMART) (women referred to the Center for Reproductive Health of UCSF for infertility treatment and their first IVF procedure) and who generated embryos Blood Pb 0.83 (0.30) OR per 1 μg/dL* Embryo cell number: 0.25 (0.07, 0.86)
Embryo fragmentation score: 1.71 (0.45, 6.56)
*Adjusted for Hg and Cd

Bloom et al. ( 2011a ) New York 1996

- 1997 Achieving pregnancy Women who were aged 18

- 34 years, were previously part of a study five years prior about fish consumption, and were not currently pregnant Blood Pb No positive pregnancy test: 1.55 (0.16) Positive pregnancy test: 1.54 (0.12) β (95% CI)

- 0.031 (

- 1.066, 1.004) per 0.6 μg/dL

a Studies are present ed in order of first appearance in the text of this section.


### Epidemiologic studies examined women having difficulty conceiving by performing 1 

### studies among patients of fertility clinics or undergoing in vitro fertilization (IVF). 2 

### Among women aged 23

- 44 years, a difference in blood Pb was reported between women 3 

### who were seeking help at a fertility clinic after one year of trying to conceive and women 4 

### who had previously delivered an infant and were identified from medical records of a 5 

### postpartum care unit at a medical center ( Chang et al., 2006 )

. Higher odds of infertility 6 

### were observed when comparing women with blood Pb levels >2.5 μg/dL to those with 7 

### blood Pb levels ≤ 2.5 μg/dL. Another study examining fertility reported on women in 8 

### Saudi Arabia aged 19

- 50 years who were undergoing IVF treatment ( Al

- Sale h et al., 9 

### 2008a )

. Women were categorized as having achieved a pregnancy versus not having 10 

### achieved a pregnancy and achieved fertilization versus not achieving fertilization. The 11 

### majority of women had follicular Pb levels that were below the limit of det ection, 12 

### whereas less than 2% of women had blood Pb levels below the limit of detection. In 13 

### addition, less than 2% of women had blood Pb levels that were above 10 μg/dL. 14 

### Follicular Pb levels were not correlated with the blood Pb. No association was observed 15 

### between blood or follicular Pb and pregnancy outcomes in either crude or adjusted 16 

### models. An association was not detected between follicular Pb and fertilization, but 17 

### higher blood Pb was associated with lower rates of fertilization. Finally, a study that 18 

### included nine women undergoing IVF treatment in Rhode Island ( Silberstein et al., 2006 ) 19 

### found that median follicular Pb levels in women who achieved pregnancy were lower 20 

### than the follicular Pb levels among nonpregnant women. One limitation present in these 21 

### studies is that the participants, especially in the later two studies, are women who ar e 22 

### seeking help for fertility problems. The participants are not samples of the general 23 

### population and therefore cannot be generalized to all women of childbearing age. The 24 

### Study of Metals and Assisted Reproductive Technologies (SMART) enrolled women 25February 2012 5

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### underg oing their first round of IVF and investigated multiple steps before pregnancy as 1 

### the outcomes ( Bloom et al., 2011b ; Bloom et al., 2010 )

. Higher blood Pb levels were 2 

### associated with lower oocyte matu rity although the lack of power made interpretation of 3 

### models controlling for Cd difficult. No association was observed between blood Pb and 4 

### oocyte fertilization ( Bloom et al., 2010 )

. When examining markers of IVF success, 5 

### inconsistent results were observed. Embryo cell number was lower in association with 6 

### higher blood Pb levels but no association was observed for embryo fragmentation score 7 

### ( Bloom et al., 2011b )

. The study was limited by the small number of participants. 8 

### Overall, these epidemiologic studies examine a variety of fertility

- related endpoints and 9 

### although some studies demonstrate an association between higher Pb levels and 10 

### fertility/pregnancy, as a whole the results are inconsistent across studies. 11 

### A prospective coho rt study enrolled women who previously participated in a study of fish 12 

### consumption for a length of <1 to >6 menstrual cycles and investigated the relationship 13 

### between blood Pb levels and having a positive pregnancy test ( Bloom et al., 2011a )

. No 14 

### association was observed between blood Pb and achieving pregnancy. 15 

### Animal toxicology studies following female fertility looked at various outcomes. Several 16 

### studies observed a decrease in litter size when females were expose d to Pb before mating 17 

### or during pregnancy ( Dumitrescu et al., 2008b ; Iavicoli et al., 2006b ; Teijon et al., 2006 )

. 18 

### Pups in a study by Teijon et al. ( 2006 ) receiving 400 ppm Pb

- acetate in drinking water 19 

### had blood Pb of 97 μg Pb/dL blood at 1 week post

- weaning and 18.2 μg Pb/dL blood at 20 

### 2 week post

- weaning. Dumitrescu et al. observed a modification in sex ratio of pups born 21 

### to dams exposed to Pb before mating and during pregnancy. As the dose of Pb increased, 22 

### the number of females per litter al so increased ( i.e., 1 male to 0.8 female in non
- Pb 23 

### exposed group; 1 male to 0.66 female in 50 ppb Pb

- acetate group; 1 male to 2.25 females 24 

### in 100 ppb group; and 1 male to 2.5 females in 150 ppb group). These results are not 25 

### consistent with earlier results of Ronis et al. ( 1998b ) , who did not observe differences in 26 

### sex ratio dams and offspring were exposed only during pregnancy. Thus, Pb exposure in 27 

### animal studies during or before pregnancy have shown effects on litter size and mixed 28 

### effects on sex ratio. 29


### Nandi et al. ( 2010 ) de monstrated a concentration

- dependent decline in viability rate, 30 

### maturation, fertilization, and cleavage rates of buffalo oocytes cultured in medium 31 

### containing 1

- 10 μg/mL Pb

- acetate . Karaca and Şimşek ( 2007 ) observed an increase in the 32 

### number of mast cells in ovary tissue after Pb exposure (2 , 000 μg/mL in drinking water) 33 

### suggesting that Pb may stimulate an in flammatory response in the ovaries which may 34 

### contribute to Pb

- induced female infertility. 35 

### In contrast, Nampoothiri and Gupta ( 2008 ) did not observe any statistically significant 36 

### change in fertility ra te or litter size in female rats subcutaneously administered Pb 37February 2012 5

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### (0.05 mg /kg body weight daily before mating and during pregnancy) with a resulting 1 

### blood Pb of 2.49 μg/mL. Although reproductive performance was not affected in this 2 

### study, the authors did rep ort an alteration in implantation enzymes. Cathepsin

- D activity 3 

### decreased and alkaline phosphatase activity increased after Pb exposure. 4 

### Recent epidemiologic and toxicological studies on the effect of Pb on fertility outcomes 5 

### have generated inconsistent re sults. However, the bulk of the evidence including the 6 

### current and historical Pb literature ( U.S. EPA, 2006b ) indicate that increased Pb exposure 7 

### may decrease fertility. 8



### 5.8.1.3 Effects on Puberty
Recent toxicological studies of rodents have examined the effects of Pb on pubertal and 9 

### reproductive organ development and on biomarkers of pubertal development. There have 10 

### also been recent epidemiologic studies examining associations between blood Pb levels 11 

### and onset of puberty, which are summa rized in Table 5

- 32 and in the text below. All of 12 

### the epidemiologic studies examined concurrently measured blood Pb and puberty and are 13 

### reported below. Additionally, Naicker et al. ( 2010 ) followed girls to determine their age 14 

### of menarche; however, blood Pb levels were measured once at 13 years of age. 15February 2012 5

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### Table 5
- 32 Summary of recent epidemiologic studies of associations between Pb levels and puberty for females

Reference a Study Location and Years Outcome Study Population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect E stimates Tomoum et al. ( 2010 ) Cairo, Egypt 2007 Hormones and pubertal development Healthy children aged 10

- 13 yr; seeking treatment for minor health problems and living in one of two designated areas (one with high

- risk for Pb contamination and one with no Pb source) Blood Pb NS for girls only (combined with boys in the study the mean was 9.46 [3.08] ) Breast Development <10 μg/ dL : Stage 2: 36.4% Stage 3: 63.6% ≥ 10 μg/ dL : Stage 2: 100% Stage 3: 0% Chi

- square p - value<0.01
Pubic Hair Development <10 μg/ dL : Stage 2: 36.4% Stage 3: 63.6% ≥ 10 μg/ dL : Stage 2: 77.8% Stage 3: 22.2% Chi

- square p - value>0.05

*Quantitative results for hormones not provided Denham et al. ( 2005 ) Akwesasne Mohawk Nation (boundaries of New York, Ontario, and Quebec NS Age at menarche 10

- to 16.9

- yr - old girls in the Akwesasne community Blood Pb 0.49 (0.905 )

Median: 1.2 Coefficients for binary logistic regression predicting menarche with Pb centered at the mean: log blood Pb

- 1.29 (p

- value 0.01) log blood Pb

- squared:

- 1.01 (p

- value 0.08) Non

- linear relationship observed and Pb below the mean did not appear to affect the odds of menarche. Increasing blood Pb from 0.49 to 0.98 μg/dL decreased the odds of menarche attainment by 72% Naicker et al. ( 2010 ) Johannesburg/Soweto, South Africa Born in 1990 Self

- reported Tanner staging at age 13 and age at menarche Girls of black or mixed ancestry who were enrolled in the Birth to Twenty (Bt20) cohort (born in 199 0) that lived in Johannesburg/Soweto for at least 6 mo after birth Blood Pb at 13 yr of age 4.9 (1.9) blood Pb levels ≥ 10 μg/dL: 1% OR (95% CI) Delay in breast development at age 13 <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 2.34 (1.45, 3.79) Delay in pubic hair d evelopment at age 13 <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 1.81 (1.15, 2.84) Delay in attainment of menarche at age 13 <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 2.01 (1.38, 2.94)February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Outcome Study Population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect E stimates Den Hond et al. ( 2011 ) Flanders 2003

- 2004 Tanner staging, age at menarche, regular menses Girls ages 14 and 15, in their 3rd year of secondary education and living in the same study areas for at least 5 years Blood Pb Median: 1.81 10th percentile: 0.88 90th percentile: 3.81 OR (95% CI) for pubic hair development with doubling of exposure 0.65 (0.45, 0.93) *Association was no longer statistically significant when PCB marker included in the model

No association between Pb and breast development (results not given) Wolff et al. ( 2008 ) ; Wolf et al. ( 2007 ) New York City, NY 1996

- 1997 Pubertal stages defined using standard drawings 9

- yr old girls from the study hospital and nearby pediatric offices Blood Pb Median: 2.4 PR (95% CI) (unit not given, assume results are per 1 μg/dL) Breast stage: 1.01 (0.79, 1.30) Pubic hair stage: 1.25 (0.83, 1.88 ) Wu et al. ( 2003b ) U.S.A. 1988

- 1994 Tanner staging and age at menarche Girls ages 8

- 16 from the NHANES III study Blood Pb 2.5 (2.2)
Weighted proportion of the sample wi th blood Pb 5.0

- 21.7: 5.9% OR (95% CI) Breast development 0.7

- 2.0 μg/ dL : 1.00 (Ref) 2.1

- 4.9 μg/ dL : 1.51 (0.90, 2.53) 5.0

- 21.7 μg/ dL : 1.20 (0.51, 2.85)
Pubic hair development 0.7

- 2.0 μg/ dL : 1.00 (Ref) 2.1

- 4.9 μg/ dL : 0.48 (0.25, 0.92) 5.0

- 21.7 μg/ dL : 0.27 (0.08, 0.93)

Menarche 0.7

- 2.0 μg/ dL : 1.00 (Ref) 2.1

- 4.9 μg/ dL : 0.42 (0.18, 0.97) 5.0

- 21.7 μg/ dL : 0.19 (0.08, 0.43)February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Outcome Study Population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect E stimates Selevan et al. ( 2003 ) U.S.A. 1988

- 1994 Tanner staging and age at menarche Girls ages 8

- 18 from the NHANES III study Blood Pb Geometric mean NHWhites: 1.4 NHBlacks: 2.1 Mexican

- Americans: 1.7

Blood Pb levels>5μg/dL: NHWhites: 2.7% NHBlacks: 11.6% Mexican

- Americans: 12.8%

Blood Pb levels >10 μg/dL: NHWhites: 0.3% NHBlacks: 1.6% Mexican

- Americans: 2.3% OR (95% CI) Breast development NH Whites: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.82 (0.47, 1.42) NH Blacks: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.64 (0.42, 0.97) Mexican Americans: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.76 (0.63, 0.91)

Pubic hair development NH Whites: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.75 (0.37, 1.51) NH Blacks: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.62 (0.41, 0.96) Mexican Americans: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.70 (0.54, 0.91)
HR (95% CI) *included only girls 8

- 16 Age at menarche NH Whites: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.74 (0.55, 1.002) NH Blacks: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.78 (0.63, 0.98) Mexican Americans: 1 μg/ dL : 1.00 (Ref) 3 μg/ dL : 0.90 (0.73, 1.11) Gollenberg et al. ( 2010 ) U.S.A. 1988

- 1994 Luteinizing hormone (LH) and inhibin B Girls ages 6

- 11 from the NHANES III study Blood Pb Median 2.5 (range 0.07, 29.4) blood Pb >10 μg/dL: 5% OR (95% CI) for exceeding pu bertal inhibin B cutoff (>35pg/mL) <1 μg/ dL : 1.00 (Ref) 1

- 4.9 μg/ dL : 0.38 (0.12, 1.15) ≥ 5 μg/ dL : 0.26 (0.11, 0.60)
OR (95% CI) for exceeding pubertal LH cutoff (>0.4 mIU/mL) <1 μg/ dL : 1.00 (Ref) 1

- 4.9 μg/ dL : 0.98 (0.48, 1.99) ≥ 5 μg/ dL : 0.83 (0.37, 1.87)
*a sensitivity analysis including only those with blood Pb <10 μg/ dL had similar results but ORs were slightly attenuated

a Studies are presented in order of first appearance in the text of this section.


### Several epidemiologic studies investigated the association between blood Pb and 1 

### indicators of puberty onset. A study among girls aged 10

- 13 years (median: 12 years) 2February 2012 5

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### reported lower levels of FSH and LH levels in the group with blood Pb of at least 1 

### 10 μ g/dL compared to the group with blood Pb less than 10 μ g/dL ( Tomoum et al., 2 

### 2010 )

. In addition, there were some indications of lower Tanner stages of breast 3 

### development associated with P b levels of at least 10 μ g/dL, but this relationship was not 4 

### present for stages of pubic hair development. A study of girls aged 10

- 16.9 years of age 5 

### in the Akwesasne Mohawk Nation reported a nonlinear positive association between 6 

### blood Pb and age at menar che ( Denham et al., 2005 )

. No association was observed below 7 

### blood Pb of 0.49 μ g/dL in a nonlinear model of the Pb

- menarche relationship. A study 8 

### conducted in South Africa reported a positive association between blood Pb levels and 9 

### age at first menarche and pubertal development ( Naicker et al., 2010 )

. Another study 10 

### reporting on girls with low blood Pb concentrations observed an association between 11 

### blood Pb and pubic hair but not breast development ( Den Hond et al., 2011 )

. The 12 

### association was no longer statistically significant when a marker for polychlorinated 13 

### biphenyl exposure was included in the model. A study perfo rmed in NYC among 9 year 14 

### old girls reported no association between Pb levels and pubertal development ( Wolff et 15 

### al., 2008 ) , bu t this age group may be too young to study when investigating delayed 16 

### puberty as the outcome. 17 

### Multiple studies have been performed examining blood Pb levels and puberty using 18 

### NHANES III data ( Gollenberg et al., 2010 ; Selevan et al., 2003 ; Wu et al., 2003b )

. A 19 

### study that included girls aged 8

- 16 years and re ported an association for delayed 20 

### attainment of menarche and pubic hair development, but not for breast development ( Wu 21 

### et al., 2003b )

. The associations were observed even at blood Pb levels of 2.1

- 4.9 μ g/dL 22 

### comp ared to girls with blood Pb levels <2.1 μ g/dL. Another NHANES III study included 23 

### girls 8

- 18 years of age and reported the results stratified by race ( Selevan et al., 2003 )

. 24 

### Higher blood Pb levels we re associated with lower Tanner stage of breast and pubic hair 25 

### development and later age at menarche among African Americans and with lower stage 26 

### of breast and pubic hair development among Mexican Americans. For whites, the 27 

### associations were in the same di rections, but none reached statistical significance. In a 28 

### study of girls ages 6

- 11 years old from NHANES III data, higher blood Pb levels were 29 

### associated with lower inhibin B, a protein that inhibits FSH production, but no 30 

### association was observed for LH. ( Gollenberg et al., 2010 )

. The inverse association 31 

### between blood Pb and inhibin B was greater among girls with iron deficiency compared 32 

### to t hose with high Pb but sufficient iron levels. Inhibin B and LH were chosen for this 33 

### study because, as the authors indicated, these hormones are, “believed to be relevant for 34 

### younger girls... near the onset of puberty and...serve as markers for hypothalamic

35 

### pit uitary

- gonadal functioning.” 36

37February 2012 5

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### Figure 5
- 46 Toxicological exposure
- response array for reproductive effects of Pb.February 2012 5

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### Table 5
- 33 Toxicological concentration
- response array summary for reproductive effects of Pb presented in Figure 5
- 46

Reference Blood Pb level with Effect (μg/ dL ) Altered Outcome Iavicoli I. et al. ( 2006b ) 8 & 13 Delayed onset female puberty Leasure et al. ( 2008 ) 10 & 42 10, 24 & 42 10 & 42 Neurotransmitter, Dopamine homeostasis Physical Development, Adult obesity (males) Aberrant response to amphetamine Fox et al. ( 2008 ) 12 Retinal aberrations Nava

- Hernandez et al. ( 2009 ) 19.5 Sperm affected via redox imbalance Moorman et al. ( 1998 ) 25

- 130 Semen quality affected Teijon et al. ( 2006 ) 40 & 100 40 & 100 40 & 100 100 Hematology Histology

- Offspring renal & hepatic Biomarker

- Offspring renal function Physica l development: birth weight Fox et al. ( 2008 ) 12 Retinal aberrations Nava

- Hernandez et al. ( 2009 ) 19.5 Sperm affected via redox imbalance Moorman et al. ( 1998 ) 25

- 130 Semen quality affected Teijon et al. ( 2006 ) 40 & 100 40 & 100 40 & 100 100 Hematology Histology

- O ffspring renal & hepatic Biomarker

- Offspring renal function Physical development: birth weight Fox et al. ( 2008 ) 12 Retinal aberrations



### Earlier studies showed that prenatal and lactational exposures to Pb can cause a delay in 1 

### the onset of female puberty in rodents. Recent studies corroborate these findings and 2 

### show that puberty onset is one of the more sensitive markers of effects of Pb exposure as 3 

### is demonstrated in the exposure response array ( Figure 5

- 46 and Table 5

- 33 Figure 5

- 45 ; 4 

### incl uding outcomes described in sections that follow). Dumitrescu et al. ( 2008b ) exposed 5 

### adult Wistar female rats to varying doses of Pb

- acetate (50

- 150 ppb ) in drinking water for 6 

### 3 months before m ating and during pregnancy. Vaginal opening, an indicator of sexual 7 

### maturation, was statistically significantly delayed in pups from all Pb treated groups 8 

### when compared to pups from non
- treated dams. The age at vaginal opening in female 9 

### pups from the Pb tr eated groups increased, in a concentration

- dependent manner, from 39 10 

### days to 43

- 47 days. The authors also observed a correlation between body weight and age 11 

### at vaginal opening meaning that as body weight decreased the age at vaginal opening 12 

### increased. This effect also exhibited a concentration

- dependent relationship. 13 

### In another recent study, Iavicoli et al. ( 2006b ) reported a statistically signific ant delay in 14 

### several indicators of sexual maturity in offspring (Swiss mice, F 1 

### generation) born to 15 

### dams that ingested 3.5

- 40 ppm Pb in their daily diet; offspring had continuous dietary 16 

### exposure until the termination of the experiment. Maternal ingestion of Pb at the various 17February 2012 5

- 434 Draft

– Do Not Cite or Quote 

### doses resulted in female pup blood Pb levels of 3.5

- 13 μ g/dL. For all diet groups in this 1 

### range (3.5

- 13 μ g/dL), there was a delay in age at vaginal opening, age of first estrus, age 2 

### of vaginal plug formation, and age of first parturiti on when compared to the group at 3 

### background Pb concentration (2 μ g/dL). A novel finding in the Iavicoli study was that 4 

### very low dose Pb (blood Pb of 0.7 μ g/dL, food concentration of 0.02 ppm continuous 5 

### through gestation, lactation and until the termination of the experiment) induced 6 

### statistically significant acceleration of markers of sexual maturation in female offspring 7 

### versus background Pb level animals (blood Pb of 2 μ g/dL). There were statistically 8 

### significant increases in time of vaginal opening (30% earlier), first estrous, first vaginal 9 

### plug formation, and first parturition at the very low Pb exposure versus 2 μ g/dL animals. 10 

### Thus, the timing of puberty is delayed in a concentration

- dependent fashion with very 11 

### low dose Pb having a statistically signif icant earlier onset of puberty than the background 12 

### Pb animals (2 μ g/dL). Also, the animals exposed to the higher dose of Pb (blood Pb up to 13 

### 13 μ g/dL) had statistically significant delays in onset of puberty when compared to the 14 

### other dose groups. 15


### In addit ion, Pb

- induced shifts in sexual maturity were observed in the subsequent 16 

### generation (F 2 

### generation) across that dose range. These F 2 

### animals continued to be 17 

### exposed to same concentrations of Pb over multiple generations through the diet. Results 18 

### in the F 2 

### generation closely resembled those of the F 1 

### generation, as both generations 19 

### received Pb exposure. The authors concluded that a modest elevation in blood Pb level 20 

### (13 μ g/dL) over background (2

- 3 μ g/dL) can result in a profound delay in the onset of 21 

### puberty (15

- 20%). In the F 2 

### generation, reduction in blood Pb (0.7 μ g/dL) below 22 

### background (2

- 3 μ g/dL) was associated with an earlier onset of sexual maturity (30% 23 

### increase) above background. 24 

### In the 2006 Pb AQCD ( U.S. EPA, 2006b ) , it was reported that a statistically significant 25 

### reduction in the circulating levels of insulin

- like growth factor 1 (IGF

- 1), LH, and 26 

### estradiol (E2) was associated with Pb

- induced delayed puberty in Fisher 344 pups. 27 

### Subsequently, Pine et al. ( 2006 ) evaluated whether IGF

- 1 replacement could reverse the 28 

### effects of Pb on delayed female puberty onset. The authors reported that offspring from 29 

### dams exposed to Pb during gestation and lactation ( daily oral gavage of dam with 1.0 mL 30 

### solution of Pb

- acetate 12 mg /mL; mean maternal blood Pb level 40 μ g/dL) exhibited a 31 

### marked increase in LH and luteinizing hormone releasing hormone (LHRH) secretion 32 

### after IGF

- 1 administration (200 ng 3 

### /μL i.p. injection twice daily from PND23 until the 33 

### appearance of vaginal opening which appears in control animals at ~ PND 40) resulting 34 

### in restored timing of vaginal to that of control animals. It should be noted that, IGF

- 1 35 

### replacement in Pb

- exposed animals did not cause advanced puberty over non
- Pb

- exposed 36 

### controls. The results of this study provide support to the theory that Pb

- induced delayed 37 

### onset of puberty may be due to disruption of pulsatile release of sex hormones ( U.S. 38February 2012 5

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– Do Not Cite or Quote 

### EPA, 2006b ) and not necessarily due to a direct toxic effect on the hypothalamic

1 

### pituitary

- gonadal axis ( Salawu et al., 2009 ) , and IGF

- 1 may play a prominent role in the 2 

### process. 3 

### In sum, epidemiologic studies consistently show an association between higher 4 

### concurrent blood Pb and delayed pubertal development in girls. This association is 5 

### apparen t even at low blood Pb levels. New evidence from the toxicology literature 6 

### continues to support Pb

- induced delays in the onset of puberty. Further, the biological 7 

### plausibility of delayed puberty is expanded with the toxicological literature that shows 8 

### this pathway is mediated by IGF

- 1. 9



### 5.8.1.4 Effects on Lactation
Experiments in laboratory animals have shown that dietary manipulation of maternal 10 

### fatty acid (FA) levels in diet can worsen Pb

- related behavioral effects of offspring after 11 

### lactational Pb exposure ( Lim et al., 2005 )

. To determine if compon ents of dam milk 12 

### contributed to this change, dam dietary fatty acids were altered via diet. Diets deficient in 13



### n
- 3 fatty acids can lead to a deficiency of DHA, which is essential for proper nervous 14 

### system development. Lim et al. ( 2005 ) found that dam Pb exposure (Long

- Evans rats, 15 

### 0.2% Pb

- acetate trihydrate/BW) during lactation (PND 0

- 21) led to a decrement in 16



### non
- essential fatty acids in the maternal organs at PND25 (mean [SD] blood Pb levels in 17 

### dams: 308 [56] μ g/dL). In animals with a diet deficient in n

- 3 FAs, there was a Pb

- diet 18 

### interac tion on the 20

- carbon n

- 6 PUFAs. In general, Pb exposure caused a decrement in 19 

### shorter chain monounsaturated and saturated FAs in maternal organs. 20 

### Dietary supplementation with calcium can be a n especially important contribu tor to Pb 21 

### mobilization during pe riods of high calcium demand including pregnancy/lactation. For 22 

### example, mothers with elevated blood Pb levels given calcium phosphate and as c orbic 23 

### acid supplementation during lactation had a 90% decrease in placental Pb content and a 24 

### 15% decrease in the c oncentration of Pb in breast milk ( Altmann et al., 1981 ) versus the 25 

### control group that did not receive dietary treatment. Another study ( Gulson et al., 2004a ) 26 

### has shown that calcium supplementation during the lactation is less beneficial in 27 

### modulating maternal blood Pb levels (mean blood Pb at first sampling was 2.4 μ g/dL); 28 

### the Gulson cohort was limited by power (n=10 women). In a cohort of women from 29 

### Mexico City, daily calcium supplementation during lactation reduced maternal blood lead 30 

### by 15

– 20% and lead in breast milk by 5

– 10% ( Ettinger e t al., 2004a )

. Another study by 31 

### the same investigators showed that using calcium supplements daily during pregnancy 32 

### also reduced blood lead levels during pregnancy ( Ettinger et al., 2009 ) with the effect 33 

### strongest in women with higher biomarkers of Pb exposure (elevated baseline bone Pb or 34February 2012 5

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– Do Not Cite or Quote 

### >5 μ g/dL blood Pb) or in women with higher Pb exposure (self

- reported use of lead

1 

### glazed ceramics). Thus, dietary modulation with calcium supplementation during 2 

### pregnancy and lactation may decrease the amount of Pb to which the developing fetus of 3 

### i nfant is exposed. The evidence for this seems especially strong for protection during 4 

### pregnancy and more mixed for protective effects of calcium during lactation. 5



### 5.8.1.5 Summary of Effects on Female Reproductive Function
In summary, Pb exposure was found to affec t female reproductive function as 6 

### demonstrated by both epidemiologic and toxicological studies. At low concurrent blood 7 

### Pb levels, associations are observed with delayed puberty. These associations are noted 8 

### among girls with mean blood Pb levels of 10 μ g/d L and lower. Some evidence is also 9 

### available regarding blood Pb levels and altered hormone levels in adults, although the 10 

### direction of the change varied among studies. Although studies reported inconsistent 11 

### findings for the association between Pb and ferti lity, there is some evidence of a potential 12 

### relationship. Most of the epidemiologic studies are cross

- sectional, thus there is 13 

### uncertainty regarding the lifestages of Pb exposure associated with the greatest risk. 14 

### Toxicological studies are often dealing wi th prenatal or early postnatal exposures, except 15 

### for puberty studies which use concurrent exposure. Although epidemiologic and 16 

### toxicological studies provide information on different exposure periods, both types of 17 

### studies support the conclusion that Pb aff ects at least some aspects of female reproductive 18 

### function. 19



### 5.8.2 Effects on Male Reproductive Function
The 2006 Pb AQCD ( U.S. EPA, 2006b ) reported on male Pb exposure or biomarker 20 

### levels and reproductive functions as measured by sper m count/motility/morphology, time 21 

### to pregnancy, reproductive history, and chromosomal aberrations. Despite limitations, 22 

### most of the studies found slight associations between high blood Pb levels ( i.e., ≥ 23 

### 45 μ g/dL) and reduced male fecundity or fertility ( U.S. EPA, 2006b )

. Evidence reviewed 24 

### in the 1986 Pb AQCD ( U.S. EPA, 1986a ) also demonstrated that Pb exposure affects 25 

### male reproductive function in humans and experimental animals. Recently published 26



### resea rch has continued to support an association between Pb and reproductive function in 27 

### males. These studies are described in the sections below. 28February 2012 5

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### 5.8.2.1 Effects on Sperm/Semen Production, Quality, and Function
Multiple epidemiologic and toxicological studies have ex amined the relationship between 1 

### Pb and sperm and semen production, quality, and function. These studies are summarized 2 

### in the text below. In addition, recent epidemiologic studies are included in Table 5

- 34

. 3 

### The biological samples in these studies are from concurrent measures. The biological 4 

### samples measuring Pb levels and outcomes were collected concurrently in the 5 

### epidemiologic studies. 6



### Table 5
- 34 Summary of recent epidemiologic studies of associations between Pb levels and effects on sperm and semen

Reference a Study Location and Years Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Naha and Manna ( 2007 ) Bangalore, India NS Non

- occupationally exposed controls and occupationally exposed workers Categorized by work history as controls, low exposure (7

- 10 yr of exposure for 8 h/day) and high exposure (>10 yr of exposure for 8 h/day) Blood Pb measurement Controls 10.25 (2.26) Lo w exposure 50.29 (3.45) High exposure 68.26 (2.49)

Semen Pb measurement Controls 2.99 (0.76) Low exposure 15.85 (1.95) High exposure 25.30 (2.28) p - values for difference across the three groups for mean values of semen profiles were <0.01 for: liquefactio n time, seminal volume, sperm count, sperm DNA hyploidy, sperm morphological abnormality, sperm motility, sperm ATPase activity, seminal plasma fructose, seminal plasma total protein, seminal plasma free amino acid, seminal plasma cholesterol

Naha and Chowdhury ( 2006 ) Kolkata, India NS Men aged 31

- 45 that were non

occupationa lly exposed controls and occupationally exposed workers) Categorized by work history as controls, low exposure (7

- 10 yr of exposure for 8 h/day) and high exposure (>10 yr of exposure for 8 h/day) Blood Pb measurement Controls 13.62 (2.45) Low exposure 48 .29 (4.91) High exposure 77.22 (1.25)

Semen Pb measurement Controls 3.99 (1.36) Low exposure 10.85 (0.75) High exposure 18.30 (2.08) p - values for difference across the three groups for mean values of semen profiles were <0.01 for: sperm count, sperm prote in, sperm DNA hyploidy, sperm DNA, sperm RNA, sperm viability, sperm membrane lipid peroxidation, seminal plasma total ascorbate, seminal plasma DHAA, sperm ATPase activity, sperm motility, sperm velocity, seminal plasma fructoseFebruary 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Hsu et al. ( 2009b ) Taiwan NS Men working at a battery plant Blood Pb
Categorized into 3 groups: <25 μg/ dL , 25

- 45 μg/ dL , >45 μg/ dL 40.2 p - values for difference across the three gro ups were <0.05 for: sperm head abnormalities, sperm neck abnormalities, sperm chromatin structure assay (αT, COMPαT)

p - values for difference across the three groups were >0.05 for: semen volume, sperm count, motility, sperm tail abnormalities, sperm immat urity, computer

- assisted semen analysis, % sperm with ROS production
Coefficients for regression analysis with blood Pb: Morphologic abnormality 0.271 (p

- value <0.0001) Head abnormality 0.237 (p

- value 0.0002) αT 1.468 (p

- value 0.011) COMPαT 0.233 (p

- value 0.21) Kasperczyk et al. ( 2008 ) Poland NS Healthy, non

smoking, fertile men that worked at the Zn and Pb Metalworks Blood Pb; seminal fluid Pb
Categorized as high exposure workers (blood Pb 40

- 81 μg/ dL ), low exposed workers (blood Pb 25

- 40 μg/ dL ), and controls (office workers with no history of occupational Pb exposure) Blood Pb High exposure workers: 53.1 (2.05) Low exposure workers: 34.7 (0.83) Controls: 8.47 (0.54)
Seminal plasma Pb High exposure workers: 2.02 (0.23) Low exposure workers: 2.06 (0.40) Controls: 1.73 (0.16) Mean (SE) Sperm volume (mL) Control s: 2.94 (0.32) Low exposure: 2.89 (0.22) High exposure: 2.98 (0.22) (p

- value for ANOVA: 0.993)
Sperm cell count (mln/mL) Controls: 43.1 (7.0) Low exposure: 44.6 (10.1) High exposure: 42.2 (5.86) (p

- value for ANOVA: 0.400)
Normal morphology (%) Contro ls: 63.3 (2.7) Low exposure: 57.3 (2.5) High exposure: 58.4 (2.1) (p

- value for ANOVA: 0.266)
Progressively motile sperm after 1 h (%) Controls: 16.4 (3.2) Low exposure: 14.8 (2.6) High exposure: 10.5 (1.9) (p

- value for ANOVA: 0.217)
Motile sperm after 24 h (%) Controls: 4.4 (1.8) Low exposure: 7.3 (1.7) High exposure: 3.1 (0.8) (p

- value for ANOVA: 0.188)

p - value for correlation between blood Pb and sperm cell motility after 1 h: 0.011February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Telisman et al. ( 2007 ) Croatia 2002

- 2005 Men aged 19

- 55, never occupationally exposed to metals and going to a clinic for infertility examination or for semen donation to be used for artificial insemination Blood Pb Median: 4.92 (range 1.13

- 14.91) Standardized regression coefficients for log bloo d Pb (units not given) Immature sperm: 0.13 (p

- value <0.07) Pathologic sperm: 0.31 (p

- value <0.0002) Wide sperm: 0.32 (p

- value <0.0001) Round sperm: 0.16 (p

- value <0.03)
Coefficients and p

- values not given if not statistically significant: semen volume, s perm concentration, slow sperm, short sperm, thin sperm, amorph sperm Meeker et al. ( 2008 ) Michigan NS Men aged 18

- 55 going to infertility clinics (distinction not made between clinic visits for male or female fertility issues) Blood Pb Median: 1.50 (IQR 1.10, 2.00) OR (95% CI) for having below reference

- level semen parameters Concentration 1st quartile: 1.00 (ref) 2nd quartile: 0.88 (0.32, 2.44) 3rd quartile: 2.58 (0.86, 7.73) 4th quartile: 1.16 (0.37, 3.60)
Motility 1st quartile: 1.00 (ref) 2nd quartile: 1.04 (0.43, 2.53) 3rd quartile: 1.95 (0.70, 5.46) 4th quartile: 1.66 (0.64, 4.29)
Morphology 1st quartile: 1.00 (ref) 2nd quartile: 0.83 (0.37, 1.87) 3rd quartile: 1.41 (0.54, 3.67) 4th quartile: 1.18 (0.50, 2.79)
Models with adjustment for multiple metals Concentration 1st quartile: 1.00 ( ref) 2nd quartile: 0.89 (1.57, 2.89) 3rd quartile: 3.94 (1.15, 13.6) 4th quartile: 2.48 (0.59, 10.4) Slivkova et al. ( 2009 ) NS Men aged 22

- 48 undergoing semen an alysis at an infertility clinic Semen Pb 1.49 mg/kg (0.40 mg/kg) Correlation between Pb and flagellum ball :

- 0.39 (p

- value not given)
*correlations not given for any other sperm pathological changes (therefore assume not statistically significant): broke n flagellum, separated flagellum, separated flagellum, small heads, retention of cytoplasmic drop, other pathological spermatozoa, large heads, acrosomal changes, and knob twisted flagellumFebruary 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Mendiola et al. ( 2011 ) Spain 2005

- 2007 Men attending infertility clinics and classified as either no rmal sperm (controls) or oligo

- astheno

teratozoospermia (cases) based on WHO semen quality criteria Seminal plasma Pb Blood Pb Seminal plasma: 2.90 (IQR 2.70, 3.20) Whole blood: 9.50 (IQR 7.50, 11.90) Blood plasma: 2.90 (IQR 2.70, 3.10)

Cases: Seminal pla sma: 3.0 ( 0.3) Whole blood: 9.8 (2.3) Blood plasma: 2.9 (0.2)

Controls: Seminal plasma: 2.9 ( 0.3) Whole blood: 9.7 (2.3) Blood plasma: 2.9 (0.3) β (95% CI) Sperm concentration Seminal plasma:

- 1.0 (

- 3.1, 2.3) Whole blood:

- 0.2 (

- 1.7, 1.6) Blood plasma: 0.08 (

- 4.1, 5.2)
% Immotile sperm Seminal plasma: 1.5 (0.37, 1.9) Whole blood: 0.05 (

- 0.32, 0.43) Blood plasma:

- 0.49 (

- 1.8, 6.2)
% Morphologically normal sperm Seminal plasma:

- 0.54 (

- 3.1, 2.0) Whole blood:

- 0.31 (

- 1.5, 0.89) Blood plasma:

- 0.08 (

- 3.5 , 3.4)
*Units not given (assume 1 μg/dL)
Note: No correlation in Pb levels among bloods or seminal plasma. There was correlation between Pb and other metals (Cd and Hg) within each body fluid. Other metals were not controlled for in models

a Studies are presented in order of first appearance in the text of this section.


### International epidemiologic studies of men occupationally exposed to Pb have reported 1 

### on associations between Pb exposure or biomarker levels and sperm count and quality 2 

### and semen qualit y. In most of these occupational studies, blood Pb levels over 40 μ g/dL 3 

### have been reported for individuals occupationally exposed to Pb. For example, studies 4 

### performed in India ( Naha and Manna, 2007 ; Naha and Chowdhury, 2006 ) reported that 5 

### men in the highest exposure group (men working in battery or paint manufacturing plants 6 

### for 10

- 15 years for 8 hours/day) had mean blood Pb levels of 77.22 μ g/dL ( Naha and 7 

### Chowdhury, 2006 ) and 68.26 μ g/dL ( Naha and Manna, 2007 )

. Control groups in these 8 

### studies (those without occupational Pb exposure) had mean blood Pb levels below 9 

### 15 μ g/dL. Increases in levels of Pb in semen were also noted across exposure group s. 10 

### Both studies report decreases in sperm count and in sperm velocity and motility with 11 

### increasing Pb exposure. Higher Pb exposure was also associated with greater hyploidy of 12 

### sperm DNA and morphologic abnormalities ( Naha and Manna, 2007 ; Naha and 13 

### Chowdh ury, 2006 )

. Decreased viability and increased lipid peroxidation were detected 14 

### ( Naha and Chowdhury, 2006 )

. A study performed in Taiwan among men with high levels 15 

### of blood Pb reported that men with higher blood Pb levels had increased sperm head 16 

### abnormalities, increased sperm DNA denaturation, and increased sensitivity to 17 

### denaturation compared to men with lower blood Pb levels ( Hsu et al., 2009b )

. No 18 

### difference was detected between three Pb exposure groups and semen volume, sperm 19 

### count, motility, velocity, and reactive oxygen species production. A similar study in 20February 2012 5

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### Poland included employees exposed to Pb and compared them with a group of male 1 

### office workers ( Kasperczyk et al., 2008 )

. Pb levels measured in seminal fluid were 2 

### slightly higher among those in the exposed groups although they were not statistically 3 

### different from the levels in the contr ol group. No difference was observed for semen 4 

### volume, sperm count, or sperm morphology among the groups. Sperm motility was lower 5 

### in the highest exposure group compared to both the control and moderate exposure 6 

### groups. Lipid peroxidation, which can induce tissue damage in sperm via reactive oxygen 7 

### species, was greater in the highest exposure group compared to the controls. 8 

### A study performed in Croatia recruited men who had never been occupationally exposed 9 

### to metals and therefore had lower blood Pb levels than the occupational studies ( Telisman 10 

### et al., 2007 )

. Increased blood Pb was associated with increased percentages of pathologic 11 

### sperm, wide sperm, and round sperm. There was also a slight increase in immature sperm 12 

### although it was not statistically sign ificant. Similar results were seen when other 13 

### biomarkers for Pb (erythrocyte protoporphyrin and δ

- aminolevulinic acid dehydratase 14 

### [ALAD]) were used instead. 15 

### A few studies examined blood or seminal plasma Pb levels and semen quality of men at 16 

### infertility cl inics ( Mendiola et al., 2011 ; Slivkova et al., 2009 ; Meeker et al., 2008 )

. In 17 

### general, these men had lower levels of Pb biomarkers than men that were occupationally 18 

### exposed. Meeker et al. ( 2008 ) detected no associations between higher blood Pb and 19 

### semen concentration, morphology, or motility (although a slight positive trend was 20 

### observed between higher Pb levels and motility in unadjusted models). In models that 21 

### include multiple metals, blood Pb was associated with being below the WHO limit of 22 

### sperm concentration levels (less than 20 million sperm/mL), although the 95% CI was 23 

### wide for the 4th quartile of Pb levels and included the null. Slivkova et al. ( 2009 ) 24 

### reported a negative correlation between semen Pb and pathological changes in sperm 25 

### (specifically, flagellum ball), but no correlations were observed for other alterations in 26 

### the sperm. Another study reported a positive association between seminal plasma Pb 27 

### concentration and percentage of immotile sperm, but this analysis did not adjust for 28 

### exposure to other metals reported to be correlated with Pb concentration in the seminal 29 

### plasma ( Mendiola et al., 2011 )

. No association was observed for seminal plasma Pb 30 

### concentration and sperm concentration or percentage of morph ologically normal sperm. 31 

### Additionally, neither Pb levels in whole blood or plasma were associated with sperm 32 

### concentration, percentage of immotile sperm, or percentage of morphologically normal 33 

### sperm. 34 

### An abundance of evidence in the toxicological literatu re demonstrates that Pb exposure is 35 

### detrimental to the quality and overall health of testicular germ cells. Earlier studies 36 

### showed that chronic Pb exposure (15 weeks) in adult male rabbits, resulting in blood Pb 37February 2012 5

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### of 16

- 24 μ g/dL, induced statistically signif icant decrements in semen quality and greater 1 

### testicular pathology ( Moorman et al., 1998 )

. Recent studies corroborate earlier findings 2 

### that Pb alters sperm parameters such as sperm count, viability, motility, and morphology. 3 

### Anjum et al. ( 2010 ) exposed 50 day old male albino Wistar/NIN rats to Pb

- acetate (273 4 

### or 819 mg /L in drinking water, 0.05% or 0.15%, respectively) for 45 days. Affected 5 

### endpoints included reduced epididymal sperm count, motile sperm, and viable sperm, 6 

### indi cating decreased sperm production and quality. Anjum did not report blood Pb 7 

### values. Wistar/NIN rats (0.15% Pb

- acetate in drinking water for 70 days) supplemented 8 

### with the herb Centella asiatica had significant attenuation of the Pb

- induced changes 9 

### observe d by Anjum et al. ( 2010 )

. Pillai et al. ( In Press ) found gestational and lactational 10 

### treatment with Pb

- acetat e in Charles Foster rats (subcutaneous injection of 0.05 mg /kg 11 

### BW/day) induced effects on sperm in adults (PND65) including significant decreases in 12 

### testicular sperm count, e pi didymal sperm count, and sperm motility. Oliveira et al. ( 2009 ) 13 

### observed a negative correlation between Pb dose and intact acrosomes. Rubio et al. 14 

### ( 2006 ) , Bis was and Ghosh ( 2006 ) , and Salawu et al. ( 2009 ) observed a decrease in 15 

### absolute testicular weight after Pb exposure. Rubio et al. ( 2006 ) and Biswas and Ghosh 16 

### ( 2006 ) also observed a Pb

- induced decrease in seminal vesicle and ventral prostate 17 

### weights and Rubio et al. ( 2006 ) reported that Pb

- acetate , in a exposure concentration

18 

### dependent manner (8

- 24 mg /kg body weight), reduced the length of certain st ages of the 19 

### spermatogenic cycle of rat seminiferous tubules and thus affected spermatogenesis. Oral 20 

### Pb

- acetate exposure (25 mg /kg bw in drinking water for 3 months, resulting in blood Pb 21 

### level of 5.3 μ g/dL) to adult male albino rats produced significant hi stological 22 

### seminiferous tubule damage (epithelium, spermatocytes, acrosomes) that was attenuated 23 

### with ascorbic acid treatment (Pb exposure + 100 mg /kg bw/day ascorbic acid, resulting in 24 

### blood Pb level of 4.7 μ g/dL) ( El Shafai et al., 2011 )

. Reshma Anjum et al. ( 2010 ) 25 

### reported decreased testicular and epididymal weights, sperm count, and viable sperm of 26 

### male rats exposed to Pb

- acetate (273 mg /L or 819 mg /L in drinking water) which were 27 

### significantly attenuated with Pb co

- exposure to the herb Centella asiatica ( Sa inath et al., 28 

### 2011 )

. Pb induced morphological abnormalities in sperm in a concentration

- dependent 29



### manner ( Allouche et al., 2009 ; Oliveira et al., 2009 ; Salawu et al., 2009 ; Shan et al., 30 

### 2009 ; Tapisso et al., 2009 ; Massanyi et al., 2007 ; Wang et al., 2006a )

. Sperm 31 

### abnormalities reported after Pb exposures were amorphous sperm head, abnormal tail, 32 

### and abnormal neck. Dong et al. ( 2009 ) reported decreased epididymis and body weights 33 

### in mice after exposure to 0 .6% Pb

- acetate in drinking water. However, the majority of 34 

### studies did not observe a statistically significant difference in body weight or 35 

### reproductive organ weights after Pb exposure at the doses used in the studies. Not all of 36 

### the aforementioned studies observed changes in every parameter. This may be due to the 37 

### use of different strains or species, chemical form of the Pb compound administered, 38February 2012 5

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### dosage schedule, duration of exposure, and age of animals at the time of the study 1 

### ( Oliveira et al., 2009 )

. 2 

### Data from recent studies suggest that a component of Pb

- induced toxicity is the 3 

### generation of reactive oxygen species (ROS) which can then affect antioxidant defense 4 

### systems of cells ( Pandya et al., 2010 )

. Salawu et al. ( 2009 ) observed a statistically 5 

### significant increase in malondialdehyde (MDA, oxidative stress marker) and a significant 6 

### decrease in the activity of antioxidant enzymes superoxide dismutase (SOD) and catal a se 7 

### (CAT) in plasma and testes of adult male Sprague Dawley rats after administration of 1% 8 

### Pb

- acetate in drinking water for 8 weeks. Supplementation with tomato paste (used as a 9 

### source of antioxidants) reduced Pb

- induced ROS production and prevented the Pb

10 

### induced increase in MDA formation and decrease in SOD and CAT activity. 11 

### Furthermore, co

- treatment of Pb with substances that are known to have antioxidant 12 

### properties [ i.e., tomato paste, Maca ( Lepidium meyenii ) , and ascorbic acid ] prevented the 13 

### Pb

- induced reduction in sperm count, sperm motility, and sperm viability ( Salawu et al., 14 

### 2009 ; Shan et al ., 2009 ; Madhavi et al., 2007 ; Rubio et al., 2006 ; Wang et al., 2006a )

. 15 

### Recent studies also demonstrate that Pb may be directly toxic to mature spermatozoa 16 

### ( Tapisso et al., 2009 ; Hernandez

- Ochoa et al., 2006 ) as well as primary spermatocytes 17 

### ( Nava

- Hernandez et al., 2009 ; Rafique et al., 2009 )

. Nava

- Hernandez et al. ( 2009 ) 18 

### exposed two groups of rodent to Pb via drinking water (L1 and L2). In their study, all 19 

### Pb

- treated animals had blood Pb levels statistically significantly higher than controls 20 

### (L1:19.54 μ g/dL and L2:21.90 μ g/dL); no statistically significant difference in blood Pb 21 

### levels existed between the two Pb exposure groups likely because the L2 group drank less 22 

### water than did the L1 group. Piao et al. ( 2007 ) reported that Pb exposure caused DNA 23 

### damage to sperm; the Pb exposed group had a blood Pb of 67 μ g/l. Piao et al. ( 2007 ) also 24 

### examined the effect of Zn supplementation on Pb

- induced sperm aberrations and found 25 

### that the proportion of abnormal sperm was statistically significantly higher in the Pb 26 

### group and the Pb+Zn group than in controls. However, the proportion of abnormal sperm 27 

### in Pb+Zn group was statistically significantly lower than in Pb alone group. 28 

### Hernandez

- Ochoa et al. ( 2006 ) reported that Pb reaches the sperm n ucleus in the 29 

### epididymis of mice chronically exposed (16 weeks in adult animals) to Pb (resulting in 30 

### mean blood Pb of 75.6 μ g/dL) by binding to nuclear sulfhydryl groups from the 31 

### DNA

- protamine complex, increasing sperm chromatin condensation, and thereby 32 

### i nterfering with the sperm maturation process without altering sperm quality parameters. 33 

### Tapisso et al. ( 2009 ) observed a statistically significant increase in the number of 34 

### micro nuclei and frequency of sister chromatid exchange with increasing treatment 35 

### duration in adult male mice administered 21.5 mg /kg body weight Pb

- acetate by i.p. 36 

### injection. Nava

- Hernandez ( 2009 ) reported a concentration

- dependent increase in DNA 37 

### damage in rat primary spermatocytes after a 13

- week exposure period to Pb

- acetate in 38February 2012 5

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### drinking water (resulting in mean blood Pb levels between 19.5 and 21.9 μ g/dL). Rafique 1 

### et al. ( 2009 ) reporte d degenerative changes from pyknosis to apoptosis in primary 2 

### spermatocytes. Hepatic expression of spermatogenic genes was transiently down

3 

### regulated in 8 week old male Wistar

- Kyoto (WKY) rats in response to Pb nitrate 4 

### (100 μ mol single i.v. injection) 3 hou rs after injection and recovered to baseline by 12 5 

### hours ( Nemoto et al., 2011 ) ; this effect was not seen in the stroke

- prone spontaneously 6 

### hypertensive rats, which are from a WKY background, or in Sprague

- Dawley rats, 7 

### demonstrating strain specificity. 8


### Pb

- induced apoptosis in germ cells within the seminiferous tubules is another sugge sted 9 

### mechanism by which Pb exerts its toxic effects on sperm production and function ( Wang 10 

### et al., 2006a )

. Dong et al. ( 2009 ) reported a exposure concentration

- related increase in 11 

### apoptosis in spermatogonia and spermatocytes of Kunming mice a fter exposure to 12 

### 0.15

- 0.6% Pb

- acetate in drinking water. Pb

- induced testicular germ cell apoptosis was 13 

### associated with up

- regulation of genes involved in the signal pathway of MAPK and 14 

### death receptor signaling pathway of FAS. For instance, up

- regulation of K

- ras and Fas 15 

### expressions was concomitant with activation of c

- fos and active caspase

- 3 proteins. 16 

### Wang et al. ( 2006a ) observed a exposure concentration

- dependent increase in the 17 

### expression of apop totic markers TGFβ1 and caspase

- 3 in spermatogenic cells, Sertoli 18 

### cells, and Leydig cells. Shan et al. ( 2009 ) also reported a statistically significant increase 19 

### in mRNA expression and protein levels of Fas, Fas

- L and caspase

- 3 after Pb ex posure. 20 

### Supplementation with ascorbic acid inhibited or reduced the Pb

- induced apoptosis in 21 

### germ cells and protected testicular structure and function ( El Shafai et al., 2011 ; Shan et 22 

### al., 2009 ; Wang et al., 2006a ) suggesting ROS generation is a major contributing factor in 23 

### decreased male fertility observed after chronic Pb exposure. 24 

### Similar to the results summarized in previous Pb AQCDs, recent epidemiologic and 25 

### toxicological studies indicate that high levels of Pb exposure have effects on sperm and 26 

### semen. In studies of men exposed to Pb in occupational settings, associations were 27 

### observed between blood Pb levels as low as 20

- 45 μ g/dL and sperm count and quality. 28 

### Multiple epidemiologic studies of occupational cohorts included control populations with 29 

### high blood Pb levels (close to or greater than 10 μ g/dL), which m akes identification of 30 

### effects at lower levels difficult. Future studies are warranted to determine whether this 31 

### association is observed at lower Pb levels. 32



### 5.8.2.2 Effects on Hormone Levels
The 2006 Pb AQCD ( U.S. EPA, 2006b ) provided ev idence that Pb acts as an endocrine 33 

### disruptor in males at various points along the hypothalamic

- pituitary

- gonadal axis. The 34February 2012 5

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– Do Not Cite or Quote 

### 2006 Pb AQCD also reported inconsistencies in the effects of Pb exposure on circulating 1 

### testosterone levels. Recent epidemiologic an d toxicological studies are reported below. 2 

### Epidemiologic studies are summarized in Table 5

- 35 (biological samples used for the 3 

### measurement of Pb in t hese studies were measured concurrently with hormone levels). 4



### Table 5
- 35 Summary of recent epidemiologic studies of associations between Pb levels and hormones for males

Reference a Study Location and Years Outcome Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Telisman et al. ( 2007 ) Croatia 2002

- 2005 FSH, LH, testosterone, estradiol, prolactin Men aged 19

- 55, never occupationally exposed to metals and going to a clinic for infertility examination or for semen donation to be used for artificial insemination Blood Pb Median: 4.92 (range 1.13

- 14.91) Standardized regression coefficients for log blood Pb (units not given) Testosterone: 0.21 (p

- value <0.0 03) Estradiol: 0.22 (p

- value <0.0008) Prolactin:

- 0.18 (p

- value <0.007)
Coefficients and p

- values not given if not statistically significant (LH, FSH) Naha and Manna ( 2007 ) Bangalore, India NS FSH, LH, testosterone Non

- occupationally exposed controls and occupationally exposed workers Categorized by work history as controls, low exposure (7

- 10 yr of exposure for 8 h/day) and high exposure (>10 yr of exposure for 8 h/day) Blood Pb measurement Controls 10.25 (2.26) Low exposure 50.29 (3.45) High exposure 68.26 (2.49)

Semen Pb measurement Controls 2.99 (0.76) Low exposure 15.85 (1.95) High exposure 25.30 (2.28) Mean FSH (SD) Control: 2.69 (1.22) Low exposure: 2.58 (1.94) High exposure: 2.16 (0.99) p - values for difference >0.05
Mean LH (SD) Control: 5.14 (2.35) Low exposure: 4.27 (2.52) High exposure: 3.9 (1.69) p - values for difference >0.05
Mean testosterone (SD) Control: 5.24 (2.40) Low exposure: 4.83 (1.21) High exposure: 4.59 (1.27) p - values for difference >0.05February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Outcome Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Hsieh et al. ( 2009a ) Taiwan 1 991

- NS FSH, LH, testosterone, inhibin B Workers at a Pb

- acid battery factory with annual blood Pb measures Current blood Pb, cumulative blood Pb, time

weighted cumulative blood Pb Current blood Pb: <10 μg/dL: 11.6% >40 μg/dL: 17.1% β from linear regression Inhibin B Current blood Pb: 0.40 (p

value 0.40) Cumulative blood Pb: 0.05 (p

value 0.02) Time

- weighted cumulative blood Pb: 1.33 (p

- value 0.007)

Pearson's correlations detected no correlations between current blood Pb levels and FSH, LH, or testosterone. Cumulative blood Pb levels were correlated with FSH and LH, but not testosterone. Time

weighted cumulative blood Pb levels were correlated with LH, but not FSH or testosterone.February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Outcome Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Meeker et al. ( 2010 ) Michigan NS FSH, LH, inhibin B, testosterone, SHBG, FAI, testosterone/LH Men aged 18

- 55 going to i nfertility clinics (distinction not made between clinic visits for male or female fertility issues) Blood Pb Median: 1.50 (IQR 1.10, 2.00) Regression coefficients (95% CI) FSH 1st quartile: 1.00 (ref) 2nd quartile: 0.13 (

- 0.10, 0.37) 3rd quartile: 0.10 (

- 0.15, 0.35) 4th quartile: 0.07 (

- 0.18, 0.31)

LH 1st quartile: 1.00 (ref) 2nd quartile:0.004 (

- 0.20, 0.21) 3rd quartile: 0.13 (

- 0.09, 0.35) 4th quartile: 0.88 (

- 0.14, 0.29)

Inhibin B 1st quartile: 1.00 (ref) 2nd quartile:

- 6.45 (

- 27.2, 14.3) 3rd quartil e: - 4.62 (

- 26.6, 17.4) 4th quartile:

- 7.79 (

- 29.0, 13.4)

Testosterone 1st quartile: 1.00 (ref) 2nd quartile: 28.6 (

- 6.82, 64.1) 3rd quartile: 15.8 (

- 21.8, 53.3) 4th quartile: 39.9 (3.32, 76.4)

SHBG 1st quartile: 1.00 (ref) 2nd quartile:

- 0.01 (

- 0.16, 0.15) 3rd quartile: 0.04 (

- 0.12, 0.21) 4th quartile: 0.07 (

- 0.10, 0.23)

FAI 1st quartile: 1.00 (ref) 2nd quartile: 0.8 (

- 0.04, 0.20) 3rd quartile: 0.03 (

- 0.10, 0.17) 4th quartile: 0.08 (

- 0.05, 0.21)

Testosterone/LH 1st quartile: 1.00 (ref) 2nd quartile: 0.07 (

- 0.16, 0.30) 3rd quartile:

- 0.05 (

- 0.29, 0.19) 4th quartile: 0.07 (

- 0.17, 0.31)February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Outcome Study Population Pb Biomarker or Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Mendiola et al. ( 2011 ) Spain 2005

- 2007 FSH, LH, testosterone Men attending infertility clinics and classified as either normal sperm (controls) or oligo

- astheno

teratozoospermia (cases) ba sed on WHO semen quality criteria Seminal plasma Pb Blood Pb Seminal plasma: 2.90 (IQR 2.70, 3.20) Whole blood: 9.50 (IQR 7.50, 11.90) Blood plasma: 2.90 (IQR 2.70, 3.10)

Cases: Seminal plasma: 3.0 ( 0.3) Whole blood: 9.8 (2.3) Blood plasma: 2.9 (0.2)

Controls: Seminal plasma: 2.9 ( 0.3) Whole blood: 9.7 (2.3) Blood plasma: 2.9 (0.3) Linear regression β (95% CI) FSH Seminal plasma: 0.05 (

- 0.24, 0.39) Whole blood: 0.04 (

- 0.03, 0.04) Blood plasma:

- 0.20 (

- 0.64, 0.25)

LH Seminal plasma: 0.14 (

- 0.13, 0.41) Whole blood: 0.05 (

- 0.05, 0.07) Blood plasma:

- 0.07 (

- 0.49, 0.31)

Testosterone Seminal plasma: 0.11 (

- 0.10, 0.31) Whole blood: 0.01 (

- 0.05, 0.02) Blood plasma:

- 0.12 (

- 0.40, 0.14)
*Units not given (assume 1 μg/dL)

a Studies are presented in order of first appearance in the text of this section.


### Hormone levels were measured in a few recent epidemiologic studies. In a study of men 1



### non
- occupationally exposed to Pb in Croatia, increased blood Pb level was associated 2 

### with increasing serum testosterone and estradiol but decreasing serum prolactin 3 

### ( Telisman et al., 2007 )

. In addition, the analysis of an interaction term for blood Pb and 4 

### blood cadmium levels demonstrated a synergistic effect on increasing serum testosterone 5 

### levels. No association was observed between blood Pb and FSH or LH. Another study of 6 

### men with high blood Pb levels reported no difference in serum FSH, LH, and testosterone 7 

### among the three groups (controls: mean blood Pb 10.25 μ g/dL, low exposure: mean 8 

### blood Pb 50.29 μ g/dL, high exposure: m ean blood Pb 68.26 μ g/dL) ( Naha and Manna, 9 

### 2007 )

. A study of occupationally

- exposed men in Taiwan reported an association 10 

### between measures of cumulative bl ood Pb levels and inhibin B levels, but no association 11 

### was detected when using current blood Pb levels ( Hsieh et al., 2009a )

. A correlation 12 

### between cumulative blood Pb measures and LH levels was detected but correlations were 13 

### not present when examining FSH or testosterone levels. No correlations were apparent 14 

### between FSH, LH, or testosterone and current blood Pb levels. 15 

### Among m en recruited from infertility clinics in Michigan, median blood Pb levels were 16 

### much lower than those observed in the other studies of Pb and hormone levels among 17February 2012 5

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– Do Not Cite or Quote 

### men ( Meeker et al., 2010 )

. No association was detected between blood Pb and levels of 1 

### FSH, LH, inhibin B, sex hormone

- binding globulin (SHBG), free androgen index (FAI) 2 

### or a measure of Leydig cell function (T/LH). A positive association between the highest 3 

### quartile of blood Pb and testosterone was present, but this association did not persist 4 

### when other metals were included in the model. Similarly, another study of men recruited 5 

### from infertility clinics observed no association between Pb concentrations from seminal 6 

### plasma, whole blo od, or blood plasma and FSH, LH, or testosterone ( Mendiola et al., 7 

### 2011 )

. 8 

### In a recent toxicological study, Rubio et al. ( 2006 ) observed a decrease in testosteron e 9 

### levels in Pb

- acetate

- treated rats in a exposure concentration

- related fashion (8

- 24 mg /kg 10 

### body weight), and this decrease correlated with reduced lengths of spermatogenic cycle 11 

### stages VII

- VIII (spermiation) and IX

- XI (onset of spermatogenesis). Anjum et al. ( 2010 ) , 12 

### who dosed 50 day old male rats with 273 or 819 mg /L Pb

- acetate in drinking water 13 

### (0.05% or 0.15%, respectively; blood Pb not re ported), found significant decreases in 14 

### serum testosterone and testicular 3β

- HSD and 17β

- HSD levels in Pb

- exposed animals 15 

### versus controls. Pandya et al. ( 2010 ) reported altered hepat ic stero i dogenic enzyme 16 

### activity. Pillai et al. ( In Press ) found gestational and lactational exposure to Pb

- acetate in 17 

### Charles Foster rats (subcutaneous injection of 0.05 mg /kg BW/day, blood Pb not 18 

### reported) induced significant decreases in testicular 17β

- HSD and serum testosterone. 19 

### Biswas and Ghosh ( 2006 ) reported a Pb

- induced decrease in serum testosterone and 20 

### gonado tropins (FSH, LH) with inhibition of spermatogenesis, however, there was a 21 

### statistically significant increase in adrenal steroidogenic enzyme, Δ5

- 3β

- HSD activity 22 

### and serum corticosterone levels indicating disruption of the adrenocortical process. 23 

### Exposure concentration

- dependent decreases in serum testosterone were reported in Pb

24 

### exposed male rats ( Reshma Anjum et al., 2010 )

. In contrast , Salawu et al. ( 2009 ) did not 25 

### observe a decrease in serum testosterone be tween control animals and animals 26 

### administered 1% Pb

- acetate in drinking water for 8 weeks. Allouche et al. ( 2009 ) not 27 

### only did not observe any statistically significant changes in serum FSH or LH, but 28 

### reported an increase in serum testosterone levels after 0.05

- 0.3% Pb

- acetate treatment in 29 

### drinking water (only statistically significant in animals administered 0.05% Pb

- acetate ). 30 

### The results of these recent studies further support the theory th at compensatory 31 

### mechanisms in the hypothalamic

- pituitary

- gonadal axis may allow for the adaptation of 32 

### exposed animals to the toxic endocrine effects of Pb ( Rubio et al., 2006 ; U.S. EPA, 33 

### 2006b )

. 34 

### Overall, recent epidemiologic and toxicological studies report mixed findings regarding 35 

### hormone aberrations in males associated with Pb exposure or Pb biomarker levels. These 36 

### results are similar to those from the 2006 Pb AQCD on the effects of Pb exposure on 37 

### circulating testosterone levels. 38February 2012 5

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### 5.8.2.3 Fertility
Epidemiologic studies have been performed comparing Pb and infertility in men. A study 1 

### conducted in Turkey reported that blood and seminal plasma Pb levels were different in 2 

### fertile (mean [SD] blood Pb: 23.16 [5.59] μ g/dL) and infertile men (mean [SD] blood Pb: 3 

### 36.82 [12.30] μ g/dL)(p <0.001, ANOVA) ( Kiziler et al., 2007 )

. The SMART study 4 

### examined the success of IVF treatment for women and their partners starting their first 5 

### round of treatment ( Bloom et al., 2011b ; Bloom et al., 2010 )

. A small number of the male 6 

### partners participated. Their mean (SD) blood Pb level was 1.50 (0.80) μ g/dL. Higher 7 

### blood Pb concentration was associated with greater oocyte fertilization (OR 1.08 [95% 8 

### CI: 0.97, 1.21] per 1 μ g/dL increase in blood Pb when adjusted for Cd), which is not the 9 

### expected direction ( Bloom et al., 2010 )

. However, higher blood Pb was associated with 10 

### lower embryo cell number (a predictor of IVF success) and associated with higher 11 

### embryo fragmentation score (an inverse predictor of IVF success) (OR for embryo cell 12 

### number: 0. 58 [95% CI: 0.37, 0.91]; OR for embryo fragmentation score: 1.47 [95% CI 13 

### 1.11, 1.94] per 1 μ g/dL, controlled for Cd and Hg) ( Bloom et al., 2011b )

. Another study 14 

### examined occupational Pb exposure (determined by self

- report of occupational exposure) 15 

### and detected no difference in reported exposure for infertile versus fertile men (OR 0.95 16 

### [95% CI: 0.6, 1.6]) ( Gracia et al., 2005 )

. Blood Pb was not measured but approximately 17 

### 5.0% of infertile men and 5.3% fertile men reported occupational exposure to Pb. As with 18 

### the fertility studies among women, a limitation present in these studies is that the cases 19 

### included are men who are seeking help at fertility clinics; the study populations are not a 20 

### sample of the general population regarding fertility. 21 

### A couple of recent animal toxicology studies assessed paternal

- mediated reproductive 22 

### fitness by examining the reproductive success of Pb

- exposed males with non
- exposed 23 

### control females. Anjum et al. ( 2010 ) found that adult male rats who were exposed to 273 24 

### or 819 mg /L Pb

- acetate in drinkin g water (0.05%or 0.15%, respectively; blood Pb not 25 

### reported) spent a significantly longer time copulating than did their control littermates. 26 

### The Pb

- exposed males were less successful copulators with only 73% of the 0.05% 27 

### Pb

- acetate exposed males, and 53% of the 0.15% exposed males generating copulatory 28 

### plugs in the unexposed female mates. While the number of pregnant females did not 29 

### significantly differ from controls, Pb exposed males contributed to the formation of 30 

### significantly fewer implantations/dam, a nd significantly fewer fetuses/dam. Pb

- exposed 31 

### males were able to sire offspring, but produced fewer offspring per litter. In a group of 32 

### males rats with co

- exposure to Pb and the herb Centella asiatica, these reproductive 33 

### decrements were attenuated relativ e to rats exposed to Pb alone ( Sainath et al., 2011 )

. 34February 2012 5

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### 5.8.2.4 Puberty
Research has also been published examining the association between blood Pb and onset 1 

### of puberty in males. These epidemiological studies are summarized in Table 5

- 36 . The 2 

### majority of studies used concurrent measures of blood Pb and puberty ( Den Hond et al., 3 

### 2011 ; Tomoum et al., 2010 ; Hauser et al., 2008 ) , b ut Williams et al. ( 2010 ) performed a 4 

### longitudinal analysis of blood Pb levels measured at ages 8

- 9 years and pubertal onset. 5February 2012 5

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### Table 5
- 36 Summary of recent epidemiologic studies of associations between Pb levels and puberty for males

Reference a Study Location Outcome Study Population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates Hauser et al. ( 2008 ) Chapaevsk, Russia 2003

- 2005 Pubertal stages defined using standard drawings Healthy boys aged 8

- 9 Blood Pb Median: 3 (I QR 2

- 5) blood Pb >10 μg/ dL : 3% OR (95% CI) Pubertal onset based on testicular volume <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 0.83 (0.43, 1.59) *after adjustment for macronutrients, the OR (95% CI) became 0.66 (0.44, 1.00)
Genital development <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 0.57 (0.34, 0.95) *after adjustment for macronutrients, the OR (95% CI) became 0.52 (0.31, 0.88)
Pubic hair development <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 0.74 (0.34, 1.60) Williams et al. ( 2 010 ) Chapaevsk, Russia 2003

- 2008 Pubertal stages defined using standard drawings Healthy boys aged 8

- 9 at enrollment who had annual follow

up evaluations Blood Pb at ages 8

- 9 Median: 3 (IQR 2

- 5) Blood Pb level >10 μg/ dL : 3% HR (95% CI) Pubertal onset based on testicular volume <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 0.73 (0.55, 0.97)
Genital development <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 0.76 (0.59, 0.98)
Pubic hair development <5 μg/ dL : 1.00 (Ref) ≥ 5 μg/ dL : 0.69 (0.44, 1.07)February 2012 5

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– Do Not Cite or Quote Reference a Study Location Outcome Study Population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates Tomoum et al. ( 2010 ) Cairo, Egypt 2007 Hormones and pubertal development Healthy children aged 10

- 13 seeking treatment for minor health problems and living in one of two designated areas (one with high

- risk fo r Pb contamination and one with no Pb source) Blood Pb NS for boys only (combined with girls in the stu dy the mean was 9.46 [3.08] ) Testicular size <10 μg/ dL : Stage 1: 0% Stage 2: 44.4% Stage 3: 55.6% ≥ 10 μg/ dL : Stage 1: 33.3% Stage 2: 66.7% Stage 3: 0% Chi

- square p - value<0.01
Pubic Hair Development <10 μg/ dL : Stage 1: 0% Stage 2: 55.6% Stage 3: 44.4% ≥ 10 μg/ dL : Stage 1: 33.3% Stage 2: 66.7% Stage 3: 0% Chi

- square p - value<0.05
Penile staging <10 μg/ dL : Stage 1: 11.1% Stage 2: 44.4% Stage 3: 44.4% ≥ 1 0 μg/ dL : Stage 1: 58.3% Stage 2: 41.7% Stage 3: 0% Chi

- square p - value<0.05

Mean testosterone level <10 μg/ dL : 4.72 (SD 1.52) ≥ 10 μg/ dL : 1.84 (SD 1.04)

*Quantitative results for LH and FSH not provided Den Hond et al. ( 2011 ) Flanders 2003

- 2004 Tanner staging and gynecomastia Boys ages 14 and 15, in their 3rd year of secondary education and living in the same study areas for at least 5 years Blood Pb Median: 2.50 10th percentile: 1.20 90th percentile: 5.12 OR (95% CI) for gynecomastia with doubling of exposure 1.84 (1.11, 3.05)
No association between Pb and pubic hair or genital development (results not given)

a Studies are presented in order of first appearance in the text of this section.


### Studies were performed among a cohort of Russian boys enrolled between ages 8

- 9 years 1 

### ( Williams et al., 2010 ; Hauser et al., 2008 )

. The area where these studies were performed 2 

### had various environmental contaminants such as dioxin, polychlorinated biphenyls, and 3 

### other metals, present. Both the cross

- sectional study ( Hauser et al., 2008 ) and the 4February 2012 5

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### prospective study with annual follow

- ups ( Williams et al., 2010 ) demonstrated an 1 

### association; higher blood Pb levels at 8

- 9 years of age was associated with later onset of 2 

### puberty. In a study of boys in Egypt, boys with higher blood Pb had delayed pubertal 3 

### development compared to those with lower levels (median age in the high blood Pb group 4 

### was 12.5 years compared to 13.0 years in the low blood Pb group) ( Tomoum et al., 5 

### 2010 )

. In addition, compared to the low blo od Pb group, those boys with higher blood Pb 6 

### had lower testosterone, FSH, and LH levels. A study in Flanders reported no associations 7 

### between blood Pb concentration and pubertal development among 14 and 15 year old 8 

### boys ( Den Hond et al., 2011 )

. However, higher blood Pb levels were associated with an 9 

### increased odds of gynecomastia. 10


### No recent toxic ological studies address Pb

- induced male sexual maturation and 11 

### development, but older studies do provide support to findings in epidemiologic cohorts. 12 

### Pb exposure resulted in delayed sexual maturity as measured by prostate weight in male 13 

### Sprague

- Dawley pup s at PND 35. These pups were exposed chronically to 0.15 or 0.45% 14 

### Pb

- acetate in dam or their own drinking water from GD5 until PND85 and had blood Pb 15 

### ranges from low to high of 88

- 196 and 120

- 379 μg/dL, respectively ( Ronis et al., 1998b )

. 16 

### Cynomolgus monkeys exposed to Pb over a lifetime (10 years, blood Pb levels ranging 17 

### from 30

– 60 μg/dL) had altered pituitary and Sertoli cell function along with decreases in 18 

### inhibin/FSH ratio and reduced gonadotropin

- releasing hormone (GnRH) stimulation o f 19 

### LH release in adulthood ( Foster et al., 1993 ; Foster, 1992 ) , all indicators that are 20 

### important in proper sexual maturation. Further me chanistic understanding of the effect of 21 

### Pb can be gleaned from studies in adult male Wistar rats exposed to Pb for 1 month 22 

### (starting at PND56, 0.1 or 0.3% Pb

- a cetate in drinking water, respective blood Pb levels 23 

### of 34 or 60 μg/dL) that showed significant decreases in FSH, ventral prostate weight and 24 

### serum testosterone but no change in serum LH ( Sokol et al., 1985 )

. These Pb

- exposed 25 

### adult male rats (0.3% Pb

- acetate in drinking water starting at PND56 for 30 days) 26 

### demonstrated an impaired pituitary release of LH in response to challenge of the 27 

### hypothalamic

– pituitary

– adrenal (HP A) axis with the opiate antagonist naloxone, an 28 

### enhanced release of LH from the pituitary in response to direct stimulation of the 29 

### pituitary with luteinizing hormone

- releasing hormone (LHRH), an enhanced response to 30 

### human chorionic gonadotropin (hCG) by th e testes, increased pituitary LH stores, and 31 

### increased GnRH mRNA levels in the hypothalamus ( Klein et al., 1994 ; Sokol, 1987 )

. 32 

### Thus, Pb likely interferes with the male HPA axis, contributing to its reproductive 33 

### toxicity. 34



### In summa ry, recent epidemiologic studies have demonstrated an inverse effect of Pb on 35 

### pubertal development among boys at low concurrent blood Pb levels. No recent 36 

### toxicological studies were found that addressed the effect of Pb on male sexual 37 

### development and matur ation; however, the 2006 Pb AQCD ( U.S. EPA, 2006b ) supported 38February 2012 5

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### earlier findings that Pb exposure may result in delayed onset of male puberty and altered 1 

### reproductive function later in life in experimental animals. 2



### 5.8.2.5 Effects on Morpho logy and Histology of Male Sex Organs
Recent toxicological studies further support historical findings that showed an association 3 

### between Pb exposure and changes in the sex organs as well as germ cells. Histological 4 

### changes of testes in Pb nitrate

- treated animals (a single i.p. dose of 12.5, 25, or 50 mg /kg 5 

### of BW and were sacrificed 48 hours later) included seminiferous tubule atrophy, Sertoli 6 

### cell and Leydig cell shrinkage with pyknotic nuclei ( Shan et al., 2009 ; Wang et al., 7 

### 2006a ) , dilatation of blood capillaries in the interstitium, undulation of basal membrane, 8 

### and occurrence of empty spaces in seminiferous epithelium ( Massanyi et al., 2007 )

. Pillai 9 

### et al. ( In Press ) found gestational and lactational exposure to Pb

- acetate in Charles Foster 10 

### rats (subcutaneous injection of 0.05 mg /kg BW/day) induced significant decreases in 11 

### absolute organ weight (testes and epididymis) and significant decreases in relative 12 

### epididymal weight. Anjum et al. ( 2010 ) , who exposed 50 day old male albino Wistar/NIN 13 

### rats to Pb

- acetate (273 or 819 mg /L in drinking water, 0.05% or 0.15%, respectively, 14 

### blood Pb levels not reported) for 45 days, reported significant decreases in relative 15 

### reprodu ctive organ weight (epididymis, testis, vas deferens, and seminal vesicle) in Pb

16 

### exposed animals. . 17



### 5.8.2.6 Summary of Effects on Male Reproductive Function
Evidence of associations between Pb exposure and male reproductive function vary by 18 

### outcome. The strongest evidence of an association is the relationship observed between 19 

### Pb and negative effects on sperm and semen in both recent epidemiologic and 20 

### toxicological studies and studies reviewed in previous Pb AQCDs. Many of the 21 

### epidemiologic studies included occupati onal cohorts, which had high blood Pb levels. 22 

### Recent toxicological studies also reported an association between Pb exposure and 23 

### decreases in reproductive organ weight, organ histological changes in the testes and germ 24 

### cells. Male rats exposed to Pb also sh owed subfecundity in that they produced smaller 25 

### litters when mated with unexposed females. In addition, recent epidemiologic studies 26 

### found blood Pb levels to be associated with delayed pubertal development in boys with 27 

### low concurrent blood Pb levels (mean/ median blood Pb levels <10 μ g/dL). This is 28 

### supported by earlier toxicological studies. Similar to the 2006 Pb AQCD ( U.S. EPA, 29 

### 2006b ) , recent epidemiologic and toxicological studies reported inconsistent results 30 

### regarding hormone aberrations associated with Pb exposure. Mixed findings were also 31 

### apparent among epidemiologic studies of fertility among men. 32February 2012 5

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### 5.8.3 Effects on Ovaries, Embryo Develo pment, Placental function, and Spontaneous Abortions

The 2006 Pb AQCD ( U.S. EPA, 2006b ) included studies of Pb exposure among men and 1 

### women and their associations with spontaneous abortions. The 2006 Pb AQCD 2 

### concluded that overal l there was little evidence to support an association between Pb 3 

### exposure among women and spontaneous abortion ( U.S. EPA, 2006b )

. Most of the 4 

### studies examined in the 2006 Pb AQCD assigned exposure based on living near a smelter 5



### o r working in occupations that often result in Pb exposure and the results of these studies 6 

### were inconsistent. Little evidence was available in the 2006 Pb AQCD to suggest an 7 

### association with paternal Pb levels ( U.S. EPA, 2006b ) , and no recent studies have been 8 

### performed to examine paternal Pb levels and spontaneous abortion. Since the 2006 Pb 9 

### AQCD , multiple epidemiologic studies have been published that examine Pb levels in 10 

### women and their possible association with spontaneous abo rtion. Additionally, 11 

### toxicological studies have studied the effects of Pb on fetal loss and the contribution of 12 

### the ovaries and placenta to fetal loss. 13February 2012 5

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### Table 5
- 37 Summary of recent epidemiologic studies of ass ociations between Pb levels and spontaneous abortions

Reference a Study Location Outcome Study population Pb Biomarker Mean Pb (SD) in μg/dL Adjusted Effect Estimates Yin et al. ( 2008 ) Shanxi P rovince, China 2004

- 2006 Anembryonic pregnancy Women age 25

- 35 yr old and at 8

- 12 weeks of gestation at study entry; cases were anembryonic pregnancies and controls were normal pregnancies that ended in a live birth between 37

- 42 weeks Maternal blood Pb a fter miscarriage for cases and at study enrollment for controls Cases: 5.3 (95% CI: 5.2, 5.9) Controls: 4.5 (95% CI: 3.7, 5.0) Comparisons between log

transformed blood Pb levels of cases and controls performed via Student’s t

- test had a p

- value of 0.03

Vigeh et al. ( 2010 ) Tehran, Iran 2006

- 2008 Pregnancy ended before 20 weeks of gestation Women who were non - smokers, non

obese, had no chronic health conditions, had their last menstrual period less than 12 weeks prior, and were pregnant with a singl eton infant Maternal blood Pb during weeks 8

- 12 of pregnancy 3.8 (2.0) Spontaneous abortion: 3.51 (1.42) Non

- spontaneous abortion: 3.83 (1.99) T

- test for difference in mean values: 0.41
OR: 0.331 (95% CI: 0.011, 10.096) for an increase in log

transformed blood Pb (units not given, assume 1 μg/dL)

Lamadrid

- Figueroa et al. ( 2007 ) Mexico City, Mexico 1997

- 1999, 2001

- 2004 Previous miscarriage Women who had a previous pregnancy and were currently pregnant with gestational age of ≤ 1 4 wks Maternal and umbilical cord blood Pb, maternal bone Pb Overall: Blood Pb: 6.2 (4.5) Plasma Pb: 0.014 (0.013) Cases: Blood Pb: 5.8 (3.4) Plasma Pb: 0.014 (0.013) Controls: Blood Pb: 6.5 (4.9) Plasma Pb: 0.013 (0.013) Categorized Plasma Blood Pb ratio: 1st tertile: 1.00 (Ref) 2nd tertile: 1.16 (p

- value 0.61) 3rd tertile: 1.90 (p

- value 0.015)
IRR (95%CI) Per 1 SD increase: Plasma Pb 1.12 (p

- value 0.22) Blood Pb 0.93 (p

- value 0.56) Plasma/Blood Pb ratio 1.18 (p

value 0.02) Patella Pb 1.15 (p

- value 0.39) Tibia Pb 1.07 (p

- value 0 .56) Gundacker et al. ( 2010 ) Vienna, Austria 2005 Previous miscarriage Women recruited during the second trimester of pregnancy Whole placentas shortly after birth Median (IQR): 25.8 (21.0, 36.8) Median Placenta Pb: Women who had not previously miscarried: 27 μg/kg Women who had previously miscarried: 39 μg/kg (p

- value for difference: 0.039)

a Studies are presented in order of first appearance in the text of this section.



### Table 5

- 37 , above, provides a summary of the recent epidemiologic studies examining 1 

### the association between Pb biomarker levels and past and current spontaneous abortion. 2 

### Yin et al. ( 2008 ) performed a study in the Shanxi Province of China to examine if plasma 3 

### Pb levels were associated with anembryonic pregnancies (spontaneous abortions during 4 

### the first trimester, which account for 15% of al l spontaneous abortions). Women were 5 

### enrolled at 8

- 12 weeks of gestation. Women who delivered a term pregnancy had mean 6 

### plasma Pb levels that were lower than those of women who had an anembryonic 7 

### pregnancy. Of note, among cases plasma Pb level was inversel y correlated with folate 8 

### and vitamin B12, but this correlation was not observed among those who delivered at 9 

### term; no models examining plasma Pb levels adjusted for nutrient status. Another study 10February 2012 5

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### examining spontaneous abortions occurring early in the pregn ancy was conducted in Iran 1 

### ( Vigeh et al., 2010 )

. Mean blood Pb concentrations were similar in women who did and 2 

### did not have spontaneous abortions. Higher blood Pb levels were not associated with 3 

### greater odds of spontaneous abortions before 20 weeks of pregnancy. A study in Turkey 4 

### reported on groups of women who either had a spontaneous abortion before the 5 

### 20th week of gestation or who had a viable pregnancy ( Faikoglu et al., 2006 )

. No 6 

### difference was detected between the blood Pb levels of the two groups (Pb levels not 7 

### reported here due to calcula tion errors discovered in the paper; errors do not appear to 8 

### affect conclusions). A study in Mexico City examined a group of pregnant women 9 

### (maximum gestational period at enrollment was 14 weeks) who had previously been 10 

### pregnant and either given birth or h ad a spontaneous abortion ( Lamadrid

- Figueroa et al., 11 

### 2007 )

. Women in the highest tertile of plasma/blood Pb ratio had higher rates of previous 12 

### spontaneous abortions than did women in the lowest tertile. The authors state that the 13 

### plasma/whole blood ra tio represents the bioavailability of Pb , which is capable of 14 

### crossing the placental barrier for a given blood concentration. No association was 15 

### observed when examining the relationship between Pb and spontaneous abortions using 16 

### whole blood, plasma, or bon e Pb alone. Similarly, a study of placental Pb levels among 17 

### pregnant women in Austria observed higher placenta Pb levels among women who had 18 

### miscarried a previous pregnancy compared to women who had not miscarried a previous 19 

### pregnancy ( Gundacke r et al., 2010 )

. It is important to note that the number of women 20 

### included in the study was small (only 8 women reported previously having a miscarriage) 21 

### In toxicological studies, isolated embryo cultures are often used to understand the 22 

### mechanisms res ponsible for aberrant embryo development as it may contribute to 23 

### teratogenesis, fetal loss or negative postnatal pup outcomes. Nandi et al. ( 2010 ) 24 

### demonstrated an exposure concentration

- dependent decline in embryo development of 25 

### fertilized buffalo oocy tes cultured in medium containing 0.05

- 10 μ g/mL Pb

- acetate as 26 

### evidenced by reduced morula/blastocyst yield and increased four

- to eight

- cell arrest, 27 

### embryo degeneration, and asynchronous division. This study provides evidence of the 28 

### negative effect of Pb on embryo development and contributes mechanistic understand to 29 

### Pb

- dependent pregnancy loss. 30 

### A possible explanation for reduced fertility and impaired female reproductive success as 31 

### a result of Pb exposure is changes in morphology or histology in female sex organs and 32 

### the placenta ( Dumitrescu et al., 2007 ; U.S. EPA, 2006b )

. Wang et al. ( 2009e ) observed 33 

### that elevated maternal blood Pb (0.6

- 1.74 μ M, ~12.4

- 36.0 μ g/dL) compared to control 34 

### (0.04 μ M, ~0.83 μ g/dL) were associated with decreased fetal body weight, pup body 35 

### length, and placental weight in Wistar rats. The aut hors reported that placentae from Pb

36 

### exposed groups showed concentration

- dependent increasing pathology of 37 

### cytoarchitecture and cytoplasmic organelles. The authors also reported a positive 38February 2012 5

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### expression of NF

- κB, a transcription factor that controls the expre ssion of genes involved 1 

### in immune responses, apoptosis, and cell cycle, in the cytotrophoblasts, decidual cells, 2 

### and small vascular endothelial cells in rat placenta under a low

- level Pb exposure 3 

### condition which correlated with low blood Pb levels. 4


### Pb

- exp osed (273 mg /L or 819 mg /L in drinking water, 0.05 or 0.15% Pb Acetate, 5 

### respectively) male rats from Reshma Anjum et al. ( 2010 ) that had an exposure 6 

### concentration

- dependent decreases in serum testosterone, decreased male reproductive 7 

### organ weight and decreased sperm were mated to untreated females. These untreated 8 

### dams had male related exposure concentration

- dependent decreased implantation r ate and 9 

### higher pre

- and post

- implantation loss, indicating paternally mediated fetal loss. The 10 

### magnitude of these effects in dams was dependent on the concentration of Pb exposure in 11 

### their male mating partners. 12 

### As observed in sperm cells, Pb stimulates changes in antioxidant enzyme activity in rat 13 

### ovaries indicating that oxidative stress may be a contributing factor in Pb

- induced 14 

### ovarian. Nampoothiri et al. ( 2007 ) observed a reduction in SOD activity and an increase 15 

### in CAT activity along with a decrease in glutathione c ontent and an increase in lipid 16 

### peroxidation in rat granulosa cells after 15 days of Pb treatment (0.05 mg /kg body 17 

### weight). 18 

### Previous studies demonstrated that Pb accumulates in the ovaries and causes histological 19 

### changes, thus contributing to Pb

- induced ef fects on female fertility ( U.S. EPA, 2006b )

. In 20 

### support of historical studies, recent studies demonstrate Pb

- induced histological changes 21 

### in ovarian cells of pigs ( Kolesarova et al., 2010 ) and rats ( Nampoothiri et al., 2007 ; 22 

### Nampoothiri and Gupta, 2006 )

. Kolesarova et al. ( 2010 ) observed a reduction of the 23 

### monolayer of granulosa cells after Pb addition (0.5 mg /mL). Nampoothiri and Gupta 24 

### ( 2006 ) reported that Pb exposure caused a decrease in cholesterol and tot al phospholipid 25 

### content in the membranes of granulosa cells which resulted in increased membrane 26 

### fluidity. A possible explanation for reduced fertility and impaired female reproductive 27 

### success as a result of Pb exposure is changes in morphology or histolog y in female sex 28 

### organs and the placenta ( Dumitrescu et al., 2007 ; U.S. EPA, 2006b )

. 29 

### Overall, the recent studies support the conclusions of the 2006 Pb AQCD that there is 30 

### insufficient evidence among epidemiologic studies to suggest an association between Pb 31 

### and spontaneous abort ions. It is important to note that studies of spontaneous abortions 32 

### are difficult to conduct. The majority of spontaneous abortions are during the first 33 

### trimester, which makes them difficult to capture. Women may miscarry before being 34 

### enrolled in a study a nd many women may not have known they were pregnant when they 35 

### miscarried. This limits the ability to detect subtle effects, especially if higher Pb levels do 36 

### lead to increased risk of early spontaneous abortions. Toxicological data provide 37February 2012 5

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### mechanistic unde rstanding of the contribution of Pb exposure to spontaneous abortions. 1 

### These laboratory data show that Pb exposure impaired placental function, induced 2 

### oxidative stress and histological changes in the ovaries, and affected embryo 3 

### development. The toxicolog ical and epidemiologic data provide mixed evidence on the 4 

### role of Pb in spontaneous abortions. 5



### 5.8.4 Infant Mortality and Embryogenesis
The 2006 Pb AQCD ( U.S. EPA, 2006b ) concluded that Pb exposure can increase fetal 6 

### mortality and produce sublethal effects (disrupt growth and development) in offspring of 7 

### Pb exposed dams at concentrations that do not result in clinical toxicity to the dams by 8 

### disrupting implantation and preg nancy, particularly at the blastocyst stage of 9 

### development. In rodent studies gestational exposure to Pb (blood Pb 32 to >70 μg/dL) 10 

### resulted in smaller litters and fewer implantation sites and in non
- human primates pre

11 

### and perinatal mortality was reported in squirrel monkeys exposed to Pb (mean dam blood 12 

### Pb of 54 μg/dL) in the last two

- thirds of gestation ( U.S. EPA, 2006b )

. There is substantial 13 

### evidence to show that there is no apparent maternal

- fetal barrier to Pb and it can eas ily 14 

### cross the placenta and accumulate in fetal tissue during gestation ( Pillai et al., 2009 ; 15 

### Wang et al., 2009e ; Uzbekov et al., 2007 )

. No recent studies have reported on the 16 

### relationship between Pb levels and infant mortality. 17



### 5.8.5 Birth Def ects
The 2006 Pb AQCD ( U.S. EPA, 2006b ) reported the possibility of small associations 18 

### between high Pb exposure and birth defects, but many of the epidemiologic studies used 19 

### occupational histories instead of actual measures of bl ood Pb levels. Among the studies 20 

### included in the 2006 Pb AQCD, a couple studies reported possible associations between 21 

### parental exposure to Pb and neural tube defects ( Irgens et al., 1998 ; Bound et al., 1997 )

. 22 

### Recent studies also examined indicators of Pb exposure and neural tube defects ( Table 23 

### 5

- 38 ). No other recent epidemiologic studies of Pb exposure and birth defects were 24 

### identified in the literature. No recent toxicological studies were found that investigated 25 

### Pb

- induced changes in morphology, teratology effects, or skeletal malformations of 26 

### developing fetuses as a result of maternal Pb exposure; however, in t he 2006 Pb AQCD 27 

### toxicological studies demonstrated associations between exposure to high doses of Pb 28 

### and increased incidences of teratogenic effect in experimental animals. 29February 2012 5

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### Table 5
- 38 Summary of recent epidem iologic studies of associations between Pb levels and neural tube defects

Reference a Study Location Study Population Pb Biomarkers and Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Zeyrek et al. ( 2009 ) Turkey NS Infants with gestational age of at least 20 wks Maternal and umbilical cord blood Pb taken 0.5h after birth Cases: Maternal: 15.5 (15.0) Umbilical cord: 18.2 (17.8) Controls: Maternal: 12.5 (12.7) Umbilical cord: 16.5 (16.1) P

- values for differences of Student’s t

- test or Mann

- Whitney U test (dependent on distribution) were 0.35 for maternal blood Pb and 0.63 for umbilical cord blood Pb Brender et al. ( 2006 ) Texas 1995

- 2000 Infants of Mexican

American women Maternal blood Pb taken 5

- 6 wks post

- par tum Cases: 2.4 (1.9) Controls: 2.5 (1.6) Blood Pb<6.0 μg/dL: 1.0 (Ref) Blood Pb≥ 6.0 μg/dL: 1.5 (95% CI: 0.6, 4.3) Huang et al. ( 2011b ) China 2002

- 2004 Live and still births of women living in the study area (villages in the Lvliang region of Shanxi province) 2 soil samples from each village 56.14 μg/g (11.43 μg/g) N/A

a Studies are presented in order of first appearance in the text of this section.


### Among the recent epidemiologic studies (described in Table 5

- 38 ), a study of women in 1 

### Turkey detected no difference between the blood Pb of mothers or the umbilical cord 2 

### blood Pb of the newborns for healthy infants compared with infants with neural tube 3 

### defects (cases of spina bifida occulta were excluded, but other forms of spina bifida were 4 

### included) ( Zeyrek et al., 2009 )

. Brender et al. ( 2006 ) performed a study of Mexican

5 

### American women living in Texas. Measurements were taken 5

- 6 weeks postpartum, 6 

### which is a limitation of this study because the blood Pb levels may be different from 7 

### those during the developmental period of gestation. The OR comparing women with at 8 

### least 6 μ g/dL blood Pb to those with less than 6 μ g/dL blood Pb was 1.5 (95% CI: 0.6, 9 

### 4.3). This increased after adjusting for breast feeding, although this variable was not a 10 

### confounder because it cannot be associated with neural tube defects. For these women, 11 

### neither occupational exposure to Pb nor proximity of residence to a facility with Pb a ir 12 

### emissions at the time of conception was associated with increased odds of neural tube 13 

### defects. A study with an ecologic design was performed in China and did not use 14 

### individual

- level biomarkers to determine Pb levels ( Huang et al., 2011b )

. A positive 15 

### association between Pb levels in soil samples and neural tube defects was reported 16 

### Exposure to multiple ot her trace elements also demonstrated a positive association but no 17 

### control for co

- exposures was included in the models for Pb. 18 

### Previous studies included in the 2006 Pb AQCD observed associations between Pb and 19 

### neural tube defects but were limited due to th e lack of biologi cally measured Pb [ Pb was 20 

### measured in drinking water ( Bound et al., 1997 ) and estimated from occupational reports 21 

### ( Irgens et al., 1998 ) ] . A recent ecologic study reported an association between Pb in the 22 

### soil and neural tube defects but was also limited by its lack of biological samples, as well 23 

### as a lack of individual

- level data and the prevalence of several other metals ( Huang et al ., 24February 2012 5

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### 2011b )

. Other recent epidemiologic studies of maternal blood Pb levels and neural tube 1 

### defects observed no associations ( Zeyrek et al., 2009 ; Brender et al., 2006 )

. 2



### 5.8.6 Preterm Birth
Epidemiologic studies on preterm birth inc luded in the 2006 Pb AQCD ( U.S. EPA, 3 

### 2006b ) reported inconsistent findings regarding the relationship between Pb and 4 

### gestational age. Recent studies have examined this potential association and again mixed 5 

### results were reported ( Table 5

- 39 ). Of these studies, the ones that categorized births as 6 

### preterm or term all defined preterm birth as less than 37 weeks of gestation. One 7 

### limitation to note for these studies is that if Pb af fects spontaneous abortion and length of 8 

### gestation via a similar pathway, then the studies that only collect data at delivery and not 9 

### at earlier stages of p regnancy would be biased toward the null. 10February 2012 5

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### Table 5
- 39 Summary of recent epidemiologic studies of associations between Pb levels and preterm birth

Reference a Study Location Outcome Study Population Pb Biomarkers or Exposure Measurement Mean Pb (SD) in μ g/dL Adjusted Effect estimates

Berkowitz et al. ( 2006 ) Idaho 1970

- 1981 Preterm birth (<37wk) Singleton births with 28

- 45 wk gest ation Three time periods of two locations (unexposed and exposed/near smelter): pre

- fire, “high

- exposure period” (when a fire happened at the smelter and resulted in damages leading to high air Pb concentrations for 6 mo), and “post

- fire” NS OR (90% CI) (u nexposed location is referent group): Pre

- fire 0.93 (0.67, 1.28) High exposure 0.68 (0.34, 1.35) Post

- fire 1.17 (0.95, 1.45)

Patel and Prabhu ( 2009 ) Nagpur, India NS Gestational age Consecutive births at the study hospital Umbilical cord blood Pb Umbilical cord blood Pb: 4.7 (12.1) >5 μg/dL: mean gestational age 38 wks ≤ 5 μg/dL: mean gestational age 39 wks
Linear regression: gestational age decreased 1 wk with every 1 μg/dL increase in umbilical cord blood Pb (exact values and 95% CI not given) Jones et al. ( 2010 ) Tennessee 2006 Gestational Age: preterm (<37wk) , term (37

- 40 wk), post

- term (>40 wk) Singleton births ≥ 27 wk gestation from mothers aged 16

- 45 living in the Shelby County area for at least 5 mo during pregnancy Umbilical cord blood Pb 2.4 (4.3) Geometric mean: 1.3 Geometric Mean: Preterm birth: 1.4 Term birth: 1.2 Post

- term birth: 1.3 p - value for difference: >0.10
Wells et al. ( 2011a ) Baltimore, MD 2004

- 2005 Gestational age Singleton births from the Baltimore Tracking Health Related to Environmental Exposures (THREE) study Umbilical cord Pb 0.84 (95%: CI 0.72, 0.96)

≥ 5 μg/ dL : 0.7% Ratio for Pb concentration per 10 days of gestation: 0.99 (0.93, 1.06)

Jelliffe

- Pawlowski et al. ( 2006 ) California 1995

- 2002 Preterm birth (<37 completed wk) Singleton births to non - smoking mothers with blood Pb measures during pregnancy from either the California Childhood Lead Poisoning Prevention Branch or the California Occupational Lead Poisoning Prevention Program Maximum maternal blood Pb during pregnan cy ≥ 10 μg/ dL : 30.9% Odd Ratios: ≤ 5 μg/ dL : 1.00 (Ref) 6

- 9 μg/ dL : 0.8 (0.1, 6.4) 10

- 19 μg/ dL : 1.1 (0.2, 5.2) 20

- 39 μg/ dL : 4.5 (1.8, 10.9) ≥ 40 μg/ dL : 4.7 (1.1, 19.9)
<10 μg/ dL : 1.00 (Ref) ≥ 10 μg/ dL : 3.2 (1.2, 7.4)

Vigeh et al. ( 2011 ) Tehran, Iran 2006 Preterm birth (20

- 37 wk) Singleton births from non - smoking, non

obese mothers aged 16

- 35 and referred for prenatal care during the 8th

- 12th week of gestation Maternal blood Pb 3.8 (2.0) Mean blood Pb (SD): Preterm birth: 4.52 (1.63) Term birth: 3.72 (2.03) p - value for differenc e: <0.05
OR (95% CI) 1.41 (1.08, 1.84) (unit not given, assume per 1 μg/dL)February 2012 5

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– Do Not Cite or Quote Reference a Study Location Outcome Study Population Pb Biomarkers or Exposure Measurement Mean Pb (SD) in μ g/dL Adjusted Effect estimates
Cantonwine et al. ( 2010a ) Mexico City 1997

- 1999 Preterm birth (<37wk), Gestational age Birth s to mothers with at least 1 blood Pb measurement during pregnancy and no chronic diseases requiring medication Maternal blood Pb during pregnancy Blood Pb Visit at <20wks pregnant 7.2 (5.2) Visit at 20

- 28 weeks pregnant 6.3 (4.3) Visit at >28 weeks pregnant 6.8 (4.5)

Plasma Pb Visit at <20wks pregnant 0.17 (0.16) Visit at 20

- 28 weeks pregnant 0.13 (0.10) Visit at >28 weeks pregnant 0.16 (0.26) Linear regression β (95% CI) Blood Pb Visit at <20wks

- 2.76 (

- 5.21,

- 0.31) Visit at 20

- 28 weeks

- 1.77 (

- 3.3 9,

- 0.15) Visit at >28 weeks

- 0.47 (

- 1.78, 0.84) Average

- 1.49 (

- 3.63, 0.64)

Plasma Pb Visit at <20wks

- 2.38 (

- 4.97, 0.21) Visit at 20

- 28 weeks

- 1.34 (

- 2.98, 0.29) Visit at >28 weeks

- 1.28 (

- 2.63, 0.06) Average

- 0.28 (

- 2.81, 2.25)

Plasma

- to

- blood Pb rati o Visit at <20wks

- 3.23 (

- 6.01,

- 0.44) Visit at 20

- 28 weeks

- 1.41 (

- 3.10, 0.29) Visit at >28 weeks

- 1.30 (

- 2.67, 0.07) Average

- 1.27 (

- 3.89, 1.35)

Cord blood Pb

- 0.68 (

- 2.37, 1.00) Zhu et al. ( 2010 ) New York 2003

- 2005 Preterm birth (<37 completed wk) Singleton births to mothers aged 15

- 49 with blood Pb measures before or on the date of delivery and blood Pb measuring <10 μg/ dL Maternal blood Pb 2.1 Odd Ratios: ≤ 1 .0 μg/ dL : 1.00 (Ref) 1.1

- 2.0 μg/ dL : 1.03 (0.93, 1.13) 2. 1

- 3.0 μg/ dL : 1.01 (0.92, 1.10) 3.1

- 9.9 μg/ dL : 1.04 (0.89, 1.22)

Chen et al. ( 2006a ) Taiwan 1993

- 1997 Preterm birth (<37 wk) Infants born to at least one parent who was part of the Program to Reduce Exposure by Surveillance System

– Blood Lead Levels co hort that monitored workers occupationally exposed to Pb Maternal blood Pb during pregnancy (or if that wasn’t available, the 1 year prior to fertilization) and/or paternal blood Pb during spermatogenesis (the 64 days before fertilization, or if that wasn’ t available, the 1 year prior to spermatogenesis) Maternal blood Pb 10.1 (10.4)

Paternal blood Pb 12.9 (13.8) Risk Ratios

Maternal blood Pb <10 μg/ dL : 1.00 10

- 19 μg/ dL : 1.97 (0.92, 3.86) ≥ 20 μg/ dL : 1.86 (0.68, 4.28)

Paternal blood Pb <10 μg/ dL : 1.00 10

- 19 μg/ dL : 1.17 (0.53, 2.32) ≥ 20 μg/ dL : 0.55 (0.19, 1.28) Orun et al. ( 2011 ) Turkey NS Preterm birth (<37 wk) Births to mothers not occupationally exposed to toxic metals and living in a suburban but non

- industrial area Breast milk 2 months post

partum Median: 20.6 μg/L
>WHO limit (5 μg/L): 87% Median Pb (IQR) >37 wk:20.6 (11.2, 29.2) μg/L ≤ 3 7 wk: 20.4 (14.4, 27.9) μg/L

p - value for Mann

- Whitney U test: ≥ 0.05

a Studies are presented in order of first appearance in the text of this section.


### A study of preterm birth included women living in two different residential areas over 1 

### three different time periods ( Berkowitz et al., 2006 )

. One residential area had consistently 2 

### lower exposures but the other had a period of high Pb emissions due to damage at a local 3February 2012 5

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### factory. Preterm birth rates were examined during three time periods: before, during, and 1 

### after the time of higher Pb exposure. No association was observed between women living 2 

### in the high exposure area compared t o those in the low exposure area during any of the 3 

### exposure time periods, but the number of preterm infants born during the period of higher 4 

### exposure was small. 5 

### In another study, measurements of umbilical cord blood were taken after birth at a 6 

### hospital in Nagpur, India ( Pa tel and Prabhu, 2009 )

. A sample of women had their blood 7 

### Pb measured and among this sample, maternal blood Pb was correlated with the umbilical 8 

### cord Pb levels. Mean gestational age differed between infants with >5 μg/dL cord blood 9 

### Pb and infants with ≤ 5 μ g/dL cord blood Pb. In a linear regression model, gestational 10 

### age was found to decrease with increasing umbilical cord Pb levels. A study of women in 11 

### Tennessee consisted primarily of African American women living in an urban setting 12 

### ( Jones et al., 2010 )

. The mean level of umbilical cord blood Pb was slightly higher 13 

### among infants born preterm but the difference was not statistically significant. Using 14 

### umbilical cord blood Pb measures, a stud y reported no association between cord blood Pb 15 

### levels and gestational age. The concentrations of cord blood Pb among study participants 16 

### were overall low (99.3% had umbilical cord blood Pb ≤ 5 μ g/dL) ( Wells et al., 2011a )

. 17 

### In a study taking place in California, women with information on blood Pb levels during 18 

### pregnancy based on their participation in a surveillance program (reason for participation 19 

### in the surveillance program was unknown but the authors speculate it was likely because 20 

### of potential Pb exposure) were matched with the birth certificates of their infants 21 

### ( Jelliffe

- Pawlowski et al., 2006 )

. Almost 70% of women had maximum blood Pb 22 

### measurements <10 μ g/dL with the majority being <5 μ g/dL. Preterm birth was associated 23 

### with higher blood Pb when comparing women with maximum blood Pb le vels 24 

### ≥ 10 μ g/dL to women with blood Pb levels <10 μ g/dL in adjusted analyses. In analyses 25 

### of maximum Pb levels further refined into additional categories, the odds of preterm birth 26 

### were elevated among women with maximum blood Pb measurement ≥ 20 μ g/dL 27 

### comp ared with women with maximum blood Pb levels ≤ 5 μ g/dL. A study in Iran also 28 

### reported higher maternal blood Pb for preterm births than for term births ( Vigeh et al., 29 

### 2011 )

. The women in this study had lower blood Pb levels than did those observed in the 30 

### Jelliffe

- Pawlowski et al. study ( Jelliffe

- Pawlowski et al., 2006 )

. Higher maternal blood 31 

### Pb level was associated with higher odds of preterm birth. Another study examining 32 

### blood Pb and gestational age among women with lower blood Pb levels reported an 33 

### inverse association bet ween maternal blood Pb concentration and gestational age, 34 

### especially for blood Pb levels early in pregnancy ( Cantonwine et al., 2010a )

. However, a 35 

### study conducted in New York among women with lower blood Pb levels (inclusion 36 

### criteria mandated that blood Pb concentration be less than 10 μ g/dL), no association was 37 

### observed between blood Pb levels and preterm birth ( Zhu et al., 2010 )

. Similarly, a study 38February 2012 5

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– Do Not Cite or Quote 

### of maternal and paternal blood Pb concentrations reported no association between 1 

### maternal or paternal blood Pb levels and preterm birth ( Chen et al., 2006a )

. 2 

### A study of breast milk in the second month post partum reported no difference in breast 3 

### milk Pb levels for those infants born preterm or term; however, a limitation of this study 4 

### is that Pb levels were not measured until two months after the birth ( Örün et al., 2011 )

. 5 

### In sum, as in the 2006 Pb AQCD, recent epidemiologic studies report inconsistent 6 

### findings for a relationship between indicators of Pb exposure and preterm birth. No 7 

### patterns we re apparent within type of exposure measurement or Pb level. 8



### 5.8.7 Low Birth Weight/Fetal Growth
The 2006 Pb AQCD reported inconsistent epidemiologic study results for the 9 

### associations between Pb and birth weight/fetal growth and concluded that there could be 10



### a small effect of Pb exposure on birth weight and fetal growth ( U.S. EPA, 2006b )

. Since 11 

### then, multiple epidemiologic studies on the relationship between Pb exposure and birth 12 

### weight and fetal growth have been published using vario us measures of exposure, such as 13 

### air levels, umbilical cord blood, and maternal blood and bone. These studies are 14 

### summarized in Table 5

- 40 below. Additionally, there have been a few recent 15 

### toxicological studies evaluating the effect of Pb exposure during gestation on birth 16 

### weight. 17February 2012 5

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### Table 5
- 40 Summary of recent epidemiologic studies of association s between Pb levels and low birth weight and fetal growth

Reference a Study Location Outcome Study population Pb Biomarkers and Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Lamb et al. ( 2008 ) Mitrovica and Pristina , Yugoslavia 1985

- 1986 Height and BMI at birth Participants of the Yugoslavia Study of Environmental Lead Exposure, Pregnancy Outcomes, and Childhood Development Mid

- pregnancy blood Pb Mitrovica: 20.56 (7.38) Pristina: 5.60 (1.99) Regression Coefficients (95% CI) for 1 μg/dL increase in Pb: BMI Mitrovica:

- 0.18 (

- 0.69, 0.33) Pristina:

- 0.14 (

- 0.69, 0.42) Height Mitrovica: 0.43 (

- 0.83, 1.69) Pristina: 0.35 (

- 0.64, 1.34) Jelliffe

- Pawlowski et al. ( 20 06 ) California 1995

- 2002 Low birth weight (<2,500g) Small for gestational age (birth weight for gestational age <10th percentile of race

- and gender

- specific norms Singleton births to non - smoking mothers with blood Pb measures during pregnancy from ei ther the California Childhood Lead Poisoning Prevention Branch or the California Occupational Lead Poisoning Prevention Program and matched to birth records Maximum maternal blood Pb during pregnancy ≥ 10 μg/ dL : 30.9% Odd Ratios: Low birth weight ≤ 5 μg/ dL : 1.00 (Ref) 6

- 9 μg/ dL : -- 10

- 19 μg/ dL : 2.7 (0.5, 14.8) 20

- 39 μg/ dL : 1.5 (0.3, 7.7) ≥ 40 μg/ dL : -- <10 μg/ dL : 1.00 (Ref) ≥ 10 μg/ dL : 3.6 (0.3, 40.0)
Small for gestational age ≤ 5 μg/ dL : 1.00 (Ref) 6

- 9 μg/ dL : -- 10

- 19 μg/ dL : 2.3 (0.6, 9.2) 20

- 39 μg/ dL : 2. 1 (0.7, 6.7) ≥ 40 μg/ dL : -- <10 μg/ dL : 1.00 (Ref) ≥ 10 μg/ dL : 4.2 (1.3, 13.9) Iranpour et al. ( 2007 ) Isfahan, Iran 2005 Low birth weight (≤ 2,500g, >37wk) Full

- term infants born at a hospital affiliated with Isfahan University Umbilical cord and maternal blood Pb within 12 h of delivery Maternal blood Pb: Cases: 12.5 (2.0) Con trols: 13.5 (2.7) Umbilical cord blood Pb: Cases: 10.7 (1.7) Controls: 11.3 (1.9) P

- values for t

- tests: Maternal blood Pb 0.07 Umbilical cord blood Pb: 0.20

P

- values for correlations: Maternal blood Pb and Birth weight: Low birth weight: 0.17 Normal birth weight: 0.3 P

- values for correlations: Umbilical cord blood Pb and birth weight: Low birth weight: 0.84 normal birth weight: 0.26February 2012 5

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– Do Not Cite or Quote Reference a Study Location Outcome Study population Pb Biomarkers and Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Gundacker et al. ( 2010 ) Vienna, Austria 2005 Birth length, birth weight, head circumference Infants of women recruited during their second trimester Maternal blood Pb between wk 34

- 38 of gestation, whole placentas and umbilical cord Pb shortly after birth, meconium samples in first five days after birth Median (IQR): Maternal blood Pb: 2.5 (1.8, 3.5) Umbili cal cord blood Pb: 1.3 (0.8, 2.4) Placenta Pb: 25.8 μg/kg (21.0, 36.8 μg/kg) Meconium Pb: 15.5 μg/kg (9.8, 27.9 μg/kg) Regression coefficients (units not given, assume results are per 10 μg/dL or 1 μg/kg) Birth length: Placenta Pb: 0.599 (SE 0.154, p

- va lue <0.001) Meconium Pb:

- 0.385 (SE 0.157, p

- value 0.012) Birth weight: Placenta Pb: 0.658 (SE 0.136, p

- value <0.001) Maternal blood Pb:

- 0.262 (SE 0.131, p

- value 0.058) Zhu et al. ( 2010 ) New York 2003

- 2005 Birth weight, sm all for gestational age (birth weight for gestational age <10th percentile based on national birth weight by gestational week from weeks 25

- 42 Singleton births to mothers aged 15

- 49 with blood Pb measures before or on the date of delivery and blood Pb mea suring <10 μg/ dL Maternal blood Pb 2.1 Difference in birthweight in grams: 0 μg/ dL : Ref 1 μg/ dL : - 27.4 (

- 37.8,

- 17.1) 2 μg/ dL : - 38.8 (

- 53.4,

- 24.1) 3 μg/ dL : - 47.5 (

- 65.4,

- 29.6) 4 μg/ dL : - 54.8 (

- 75.5,

- 34.2) 5 μg/ dL : - 61.3 (

- 84.4,

- 38.2) 6 μg/ dL : - 67.2 (

- 92.5,

- 41.8) 7 μg/ dL : - 72.5 (

- 99.9,

- 45.2) 8 μg/ dL : - 77.6 (

- 106.8,

- 48.3) 9 μg/ dL : - 82.3 (

- 113.3,

- 51.2) 10 μg/ dL : - 86.7 (

- 119.4,

- 54.0)

After exclusion of blood Pb <1 μg/ dL , a 1 μg/ dL increase in blood Pb was associated with a 7.0 g decrease in birthwe ight

Odd Ratios for small for gestational age: ≤ 1 .0 μg/ dL : 1.00 (Ref) 1.1

- 2.0 μg/ dL : 1.07 (0.98, 1.17) 2.1

- 3.0 μg/ dL : 1.06 (0.98, 1.16) 3.1

- 9.9 μg/ dL : 1.07 (0.93, 1.23)February 2012 5

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– Do Not Cite or Quote Reference a Study Location Outcome Study population Pb Biomarkers and Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Chen et al. ( 2006a ) Taiwan 1993

- 1997 Low birth weight (<2,500 g), small for gestational a ge (birth weight ≤ 1 0th percentile of sex

- and gestational wk weights for singletons in 1993

- 1996) Infants born to at least one parent who was part of the Program to Reduce Exposure by Surveillance System

– Blood Lead Levels cohort that monitored workers o ccupationally exposed to Pb Maternal blood Pb during pregnancy (or if that wasn’t available, the 1 year prior to fertilization) and/or paternal blood Pb during spermatogenesis (the 64 days before fertilization, or if that wasn’t available, the 1 year prior to spermatogenesis) Maternal blood Pb 10.1 (10.4)

Paternal blood Pb 12.9 (13.8) Risk Ratios
Low birth weight Maternal blood Pb <10 μg/ dL : 1.00 (Ref) 10

- 19 μg/ dL : 2.22 (1.06, 4.26) ≥ 20 μg/ dL : 1.83 (0.67, 4.20)

Paternal blood Pb <10 μg/ dL : 1.00 (Ref) 10

- 19 μg/ dL : 0.83 (0.34, 1.75) ≥ 20μg/ dL : 0.42 (0.12, 1.06)

SGA Maternal blood Pb <10 μg/ dL : 1.00 (Ref) 10

- 19 μg/ dL : 1.62 (0.91, 2.75) ≥ 20μg/ dL : 2.15 (1.15, 3.83)

Paternal blood Pb <10 μg/ dL : 1.00 (Ref) 10

- 19 μg/ dL : 0.94 (0.49, 1.66) ≥ 20 μg/ dL : 0.94 (0.51, 1.62) Kordas et al. ( 2009 ) Mexico City, Mexico 1994

- 1995 Head circumference, birth weight, birth length Infants of mothers receiving antenatal care at hospitals serving low

- to

middle income populations (cross

- sectional study of baseline info from Ca supplementation trial) Umbilical cord a nd maternal blood Pb within 12 h of delivery; maternal tibia Pb Maternal tibia Pb: 9.9 μg/g (9.8 μg/g)

Maternal blood Pb ≥ 10μg/ dL : 27% Umbilical cord blood Pb ≥ 10μg/ dL : 13.7% Regression coefficients (SE) (adjusted for maternal BMI, maternal height, inf ant gestational age, and other variables) for each 1 μg/g increase in tibia Pb: Birth weight:

- 4.9 (1.8) Birth length:

- 0.02 (0.01) Head circumference:

- 0.01 (0.01; p

- value<0.05)
Women with 4th quartile tibia Pb (15.6

- 76.5 μg/g) delivered infants 140 g less than women with tibia Pb in the lowest quartile Afeiche et al. ( 2011 ) Mexico City 1994

- 2005 Birth weight Term, singleton births, at least 2,500 grams enrolled in one of three birth cohorts recruited for other longitudinal studies Maternal patella and tibia Pb measured at 1 mo nth postpartum Patella Pb 10.4 (11.8) μg/g
Tibia Pb 8.7 (9.7) μg/g β (95% CI) for 1 SD increase in maternal patella Pb

Girls

- 45.7 (

- 131.7, 40.2)
Boys 72.3 (

- 9.8, 154.4)

No association for birth weight and tibia Pb among girls. A positive association was observed for tibia Pb and birth weight among boys. (results not given)February 2012 5

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– Do Not Cite or Quote Reference a Study Location Outcome Study population Pb Biomarkers and Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Cantonwine et al. ( 2010b ) Mexico City 1994

- 1995 Birthweight Infants who were part of a clinical trial to assess maternal calcium supplementation on bone lead mobilization during lactation Umbilical cord blood Pb Maternal tibia and patella Pb one month after delivery Umbilical cord blood Pb varied by genotype from 6.3 to 6.9

Umbilical cord blood Pb ≥ 10 μg/ dL : 12.6% Regression models β (95% CI)

Umbilical cord blood Pb

- 31.1 (

- 105.4, 43.3)

Maternal tibia Pb Overall

- 4.4 (

- 7.9,

- 0.9) <1

- 4.1 μg/g: Ref 4.1

- 9.2 μg/g: 17.2 (

- 75.6, 110.1) 9.2

- 15.4 μg/g:

- 19.1 (

- 112.1, 73.9) 15.4

- 43.2 μg/g:

- 95.4 (

- 189.9,

- 0.8) Llanos and Ronco ( 2009 ) Santiago, Chile NS Fetal growth restriction (1,000

- 2,500g) *note normal b irth weights were >3,000g Term births (37

- 40 wks) from non

smoking mothers Placenta Pb Fetal growth restricted: 0.21 μg/g (0.04 μg/g) Controls: 0.04 μg/g (0.009 μg/g) P

- value for Mann

- Whitney U

- test <0.01

Zentner et al. ( 2006 ) Santo Amaro, Brazil 2002 Birth weight and length Singleton births with maternal residence within 5 km of Pb smelter Umbilical cord blood Pb from delivery Umbilical cord blood Pb: 3.9 (3.6) Linear regression coefficient with umbilical cord blood Pb as the dependent variable in model with only length and weight (unit not given, assume per 1 μg/dL): Length

- 0.46 (p

- value 0.003 ) and Weight

- 0.275 (0.048) ( i.e., in this study, Pb is assessed as the outcome) Atabek et al. ( 2007 ) Turkey NS Birth weight, birth length, head circumference, mid

- arm circumference Term, singleton infants born to healthy mothers living in urban areas and assumed to have high Pb concentrations Umbilical cord blood Pb 14.4 (8.9)

Umbilical cord blood Pb ≥ 10 μg/ dL : 53.7%

Umbilical cord blood Pb ≥ 25 μg/ dL : 9.2% Regression models β (p

- value)
Birth weight

- 0 .81 (0.01)
Birth length 0.41 (0.05)

Mid

- arm circumference 0.30 (0.05) Al

- Saleh et al. ( 2008b ) Saudi Arabia 2004 Head circumference Infants with a gestational age of at least 34 weeks born to healthy mothers aged 17

- 46 years and non

- occupationally exposed to Pb Umbilical cord blood Pb 2.210 (1.691)

Umbilical c ord blood Pb >10 μg/ dL : 1.23% Regression models for those above the 75th percentile of cord blood Pb levels β (SE) per unit of log

transformed Pb

- 0.158 (0.718), p

- value: 0.036
Janjua et al. ( 2009 ) Karac hi, Pakistan 2005 Low birth weight (≤ 2,500g) Infants of randomly selected women who planned to deliver between 37

- 42 wk Umbilical cord blood Pb Umbilical cord blood Pb: 10.8 (0.2) Prevalence ratio: <10 μg/ dL : 1.00 (Ref) ≥ 10 μg/ dL : 0.82 (0.57, 1.17)

Jones et al. ( 2010 ) Tennessee 2006 Low birth weight (<2.500g) Singleton births ≥ 27 wks gestation from mothers aged 16

- 45 living in the Shelby County area for at least 5 mo during preg nancy Umbilical cord blood Pb 2.4 (4.3) Geometric mean: 1.3 Geometric Mean: Low birth weight: 1.2 Normal birth weight: 1.3 p - value for difference: >0.10February 2012 5

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– Do Not Cite or Quote Reference a Study Location Outcome Study population Pb Biomarkers and Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates Wells et al. ( 2011a ) Baltimore, MD 2004

- 2005 Birth weight Singleton births from the Baltimore Tracking Health Related to Environmental Exposures (THREE) study Umbilical cord Pb 0.84 (95%: CI 0.72, 0 .96)

≥ 5 μg/ dL : 0.7% Ratio for Pb concentration per 100g birth weight: 1.01 (0.99, 1.02)

Örün et al. ( 2011 ) Turkey NS Birth weight and head circumferen ce Births to mothers not occupationally exposed to toxic metals and living in a suburban but non - industrial area Breast milk 2 months post

- partum Median: 20.6 μg/L
>WHO limit (5 μg/L): 87%
Median (IQR) <2500g: 20.4 (8.5, 27.1) μg/L ≥ 2500g: 20.6 (11.8, 29.5) μg/L Correlations for breast milk Pb and z

- scores of head circumference Girls 0.087 Boys 0.029

Correlations for breast milk Pb and z

- scores of birth weight Girls 0.097 Boys 0.045
*All p

- values for correlations>0.05 Williams et al. ( 2007 ) Tennessee 2002 Birth weight Infants from singleton births or the firstborn infant in a set of multiples Air Pb levels during first trimester of pregnancy 0.12 μg/m3 (0.04 μg/m3) p - value for multilevel regression of Pb with birth weight: 0.002 Increase of Pb from 0 to 0.04 relates to a 38g decrease in birth weight In crease of Pb from 0 to 0.13 (maximum) relates to a 124g decrease in birth weight Berkowitz et al. ( 2006 ) Idaho 1970

- 1981 Low birth weight (<2,500 g and ≥ 37 wk) Small for gestational age (birth weight ≤ 5 th percentile of sex

- and gestational wk weights for singletons in Idaho) Singleton infants with 28

- 45 wk gestation Three time p eriods of two locations (unexposed and exposed/near smelter): pre

fire, “high

- exposure period” (when a fire happened at the smelter and resulted in damages leading to high air Pb concentrations for 6 mo), and “post

- fire” Not specified Term Low birth weight : OR (90% CI) (unexposed location is referent group): Pre

- fire 0.81 (0.55, 1.20) High exposure 2.39 (1.57, 3.64) Post

- fire 1.28 (0.95, 1.74) Small for gestational age: OR (90% CI) (unexposed location is referent group): Pre

- fire 0.98 (0.73, 1.32) High exposure 1.92 (1.33, 2.76) Post

- fire 1.32 (1.05, 1.67)

a Studies are presented in order of first appearance in the text of this section.


### Women residing in two different towns in Yugoslavia (one with a Pb smelter and one 1 

### without a Pb smelter) were recruited during their first prenatal visit ( Lamb et al., 2008 ) 2 

### (study based on previous work by Factor

- Litvak et al. ( 1991 )

. The mid

- pregnancy blood 3 

### Pb levels were greater in women from the town with a Pb smelter. No association was 4 

### reported between maternal blood Pb and height or BMI at birth for the infants of these 5 

### women despite the differences in maternal blood Pb betw een the two towns. Multiple 6 

### studies were conducted that examined the association between maternal blood Pb and 7 

### birth weight/fetal growth. In California, blood Pb measurements of women during 8 

### pregnancy were matched with the corresponding birth certificates ( Jelliffe

- Pawlowski et 9February 2012 5

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– Do Not Cite or Quote 

### al., 2006 )

. The adjusted OR for low birth weight that compared women with blood Pb 1 

### levels ≥ 10 μ g/dL to women with levels <10 μ g/dL was elevated. However, it was 2 

### diffic ult to draw conclusions about the relationship between blood Pb and birth weight 3 

### due to small numbers (n = 9 for low birth weight) and the subsequently wide 95% CI. An 4 

### association was detected for high blood Pb and having an infant who was small of his/her 5 

### gestational age (SGA). A study of term births in Iran reported no difference in blood Pb 6 

### of women giving birth to a normal weight infant and women giving birth to an infant with 7 

### low birth weight ( Iranpour et al., 2007 )

. A study in Vienna, Austria reported an inverse 8 

### association between maternal blood Pb levels and birth weight but no as sociations for 9 

### birth length or head circumference ( Gundacker et al., 2010 )

. Similarly, increased 10 

### maternal blood Pb was associated with decreased birth weight, with the strongest 11 

### associations observed at the lowest blood Pb level ( Zhu et al., 2010 )

. No association was 12 

### observed between maternal blood Pb and SGA. A study in Taiwan examined both 13 

### maternal and paternal blood Pb levels among those occupationally exposed to Pb and 14 

### their associations with birth weight and SGA ( Chen et al., 2006a )

. Paternal blood Pb 15 

### levels were not associated with increased risk of low birth weight or SGA. Higher 16 

### maternal blood Pb concentration was associated with higher risk of low birth weight and 17 

### SGA, altho ugh not all of the associations were statistically significant. There were small 18 

### numbers of infants with low birth weight or SGA, especially at the highest blood Pb 19 

### levels (≥ 20 μ g/dL). 20 

### A study examining the association between Pb biomarker levels and bir th weight used 21 

### tibia bone measurements from mothers living in Mexico City ( Kordas et al., 2009 )

. Tibia 22 

### Pb levels were inversely associated with birth weight but not with birth length. This 23 

### association between Pb and birth weight was not modified by maternal folate 24 

### consumption or maternal or infant MTHFR genotyp e, although the association between 25 

### tibia Pb levels and birth weight was greater in magnitude among women with certain 26 

### genotypes (statistical tests not reported). Another study in Mexico City reported no 27 

### association between maternal tibia Pb levels and bir th weight among girls but reported a 28 

### positive association for boys ( Afeiche et al., 2011 )

. No associations were observed with 29 

### maternal patella Pb concentration, although among boys, the relationship was positive but 30 

### not statistically significant. One of the c ohorts used by Afeiche et al. ( 2011 ) was also 31 

### evaluated in another study ( Cantonwine et al., 2010b )

. An inverse association was 32 

### observed between tibia Pb and birth weight, especially at higher levels. This association 33 

### was stronger among those mothers with variants of the hemochromatosis iron gene 34 

### (HFE). 35 

### Multiple studies examined the relationship between Pb level and birth weigh t using Pb 36 

### measured from the placenta or umbilical cord. Researchers in Chile collected the 37 

### placentas from term births and compared the Pb levels for those born with normal birth 38February 2012 5

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– Do Not Cite or Quote 

### weights to those with low birth weights ( Llanos and Ronco, 2009 )

. Pb levels were g reater 1 

### in the placentas of infants with low birth weights. In addition, the authors note that 3 low 2 

### birth weight infants had extremely high Pb levels in the placentas (>1.5 μ g/g) and were 3 

### excluded from these analyses. A study in Brazil examined Pb levels i n umbilical cord 4 

### blood from term births of women residing within 5 km of a Pb smelter ( Zentner et al., 5 

### 2006 )

. The cord blood Pb level was found to be inversely correlated with length and 6 

### weight of the infants. A study with high Pb concentrations in umbilical cord blood 7 

### repor ted an inverse association between Pb levels and birth weight ( Atabek et al., 2007 )

. 8 

### However, no correlation was detected in an analysis restricted to umbilical cord Pb less 9 

### than 10 μ g/dL. No association with other measures of growth, such as birth length and 10 

### mid

- arm circumference, w ere detected. In Saudi Arabia, a study was conducted among 11



### non
- occupationally exposed women ( Al

- Saleh et al., 2008b )

. Umbilical cord blood Pb 12 

### concentrations were low and an association was observed between umbilical cord Pb and 13 

### head circumference. Another study recruited women in Pakistan ( Janjua et al., 2009 )

. 14 

### Umbilical cord blood Pb levels were not associated with low birth weight. The study by 15 

### Iranpour et al. ( 2007 ) discussed above investigated the association with umbilical cord 16 

### blood Pb levels in addition to their examination of maternal whole blood Pb. They again 17 

### report no difference in levels between term infants of normal and low birth weight. A 18 

### study comparing geometric mean umbilical cord blood Pb levels reported no difference in 19 

### the levels for normal and low birth weight infants born to women living primarily in 20 

### urba n areas of Memphis, TN ( Jones et al., 2010 )

. A study performed in Baltimore, MD 21 

### also reported no association between umbilical cord blood Pb concentration and birth 22 

### weight ( Wells et al., 2011a )

. This study had low blood Pb levels, with only 0.7% of 23 

### participants having umbilical cord blood Pb measuring > 5 μ g/dL. A study previously 24 

### mentioned that observed an inverse association between maternal tibia Pb and birth 25 

### weight in Mexico City reported no association between umbilical cord blood Pb 26 

### concentration and birth weight ( Cantonwine et al., 2010b )

. Finally, a study in Vienna 27 

### measured Pb in the placenta ( Gundacker et al., 2010 )

. A positive correlation was 28 

### observed between placenta Pb and birth length and weight; however, in the same study, 29 

### maternal blood Pb was inversely related to birth weight. 30 

### A study performed in Turkey examined the relationship between Pb levels in brea st milk 31 

### two months postpartum and size at birth ( Örün et al., 2011 )

. No association was observed 32 

### between breast milk Pb concentration and bir th weight or head circumference. 33 

### A few studies examined air exposures and reported inverse associations between air Pb 34 

### concentrations and birth weight. Williams et al. ( 2007 ) examined Pb concentrations in the 35 

### air during the first trimester. The purpose of their study was to demonstrate the use of 36 

### hierarchical linear models and they used the example of air pollution and birth weight in 37 

### Tennessee. The model results showed an association between ambient Pb concentration 38February 2012 5

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### and birth weight, with an estimated decrease in birth weight of 38 grams for every 1 

### 0.04 μ g/m 3 

### ( i.e., one standard deviation) increase in Pb concentration. Another study of 2 

### air Pb levels was conducted in Idaho and included two areas over three time periods. One 3 

### study area was affected by damage to a local factory that lead to high Pb emissions 4 

### durin g one of the time periods under study ( Berkowitz et al., 2006 )

. No levels of Pb are 5 

### provided. Mean birth weight for term births was decreased among infants born to women 6 

### living in the high exposure area during the period of high exposure compared to those 7 

### living in the lower exposure area. The difference in birth weig ht of term births remained, 8 

### but was reduced, between the two areas during the time period after the exposure ended. 9 

### During the period of higher exposure, the odds of low birth weight among term births 10 

### was increased among those living in the higher exposed area compared to those in the 11 

### lower exposed area, but the odds were not different between the two study areas during 12 

### the time periods before or after the high level of exposure. An increase in SGA infants 13 

### (defined as infants with weights less than or equal to the lowest 5th percentile of birth 14 

### weight for their sex and age) was also associated with living in the higher exposed area 15 

### during the time period of higher exposure. The odds of SGA infants decreased during the 16 

### time period after the exposure but the o dds were still elevated compared to those residing 17 

### in the lower exposed area. 18 

### Evidence from previous toxicological studies has shown an association between 19 

### gestational Pb exposure and reduced birth weight and impaired postnatal growth ( U.S. 20 

### EPA, 2006b )

. More recent studies have reported conflicting results. Wang et al. ( 2009e ) 21 

### demonstrated a statistically significant decrease in fetal body weight and body length of 22 

### Wistar rats after maternal exposure to 0.025 % Pb

- acetate during gestation days 1

- 10, 11

- 23 

### 20, or 1

- 20. The greatest decrease in fetal body weight and length was observed in the 24 

### group exposed to Pb during gestation days 1

- 20 followed by the group exposed to Pb 25 

### during gestation days 11

- 20. Teijón et al. ( 2006 ) observed that when pregnant dams were 26 

### administered 200 ppm or 400 ppm Pb

- acetate in drinking water, litter weight was 27 

### significantly decreased (400 ppm Pb only) versus controls due to signi ficant decrements 28 

### in female pup birth weight; male birth weight was unaffected. This effect did not persist 29 

### in the postnatal growth of the rats. The results of these studies indicate that as Pb 30 

### exposure increases, the body weight of exposed offspring decre ases. Massó

- González and 31 

### Antonia

- García ( 2009 ) also observed an 8

- 20% decrease in body weight of pups from rat 32 

### dams given 300 mg /L Pb

- acetate in drinking water (exposur e during gestation and 33 

### lactation resulting in mean blood Pb level of 22.8 μ g/dL), but no changes in body length 34 

### were reported. In contrast, Leasure et al. ( 2008 ) reported a statistically significant inverse 35 

### relationship between Pb exposure and body weight for male mice exposed to low 36 

### (27 ppm )

- an d high (55 ppm )

- levels of Pb during gestation. Male mice exposed to the low 37 

### and high Pb concentrations during gestation were 26% and 13% heavier than controls at 38 

### 1 year of age, respectively. In this study, dams were administered 27 ppm (low), 55 ppm 39February 2012 5

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### (moder ate), and 109 ppm (high) Pb in drinking water beginning 2 weeks before mating 1 

### and continuing until PND10. Resulting blood Pb levels ranged from 10 μ g/dL or less in 2 

### the low

- exposure offspring to 42 μ g/dL in the high

- exposure offspring at PND10. The 3 

### authors also reported that when dams received low or moderate levels of Pb in drinking 4 

### water from birth to weaning neither male nor female offspring exposed to Pb postnatally 5 

### exhibited a difference in body weight when compared to control offspring. 6 

### In summary, ass ociations were observed between Pb and low birth weight in a study of 7 

### maternal bone Pb and studies of Pb air exposures and birth weight. However, the 8 

### associations were less consistent when using maternal blood Pb or umbilical cord and 9 

### placenta Pb as the ex posure measurement. Previous toxicological studies observed an 10 

### association between gestational Pb exposure and reduced birth weight with moderate to 11 

### high dose Pb. More recent findings using low dose Pb exposure reported increased 12 

### offspring body weight afte r developmental Pb exposure. 13



### 5.8.8 Effects on Postnatal Stature and Body Weight
Findings from previous toxicological studies of rodents and primates have demonstrated 14 

### Pb induced impairment of postnatal growth ( U.S. EPA, 2006b )

. Severa l recent 15 

### epidemiologic studies examining the association of various biomarkers of Pb exposure 16 

### with stature and body weight have been conducted and the evidence reported is mixed. 17February 2012 5

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### Table 5
- 41 Summary of recent epidemiologic studies of associations between Pb levels and postnatal growth

Reference a Study Location and Years Study Population Pb Biomarker Mean Pb (SD) Effect Estimates
Afieche et al. ( 2011 ) Mexico City, Mexico Children born between 1994 and 2005 n=522 boys n=477 girls Mat ernal bone Pb Patella: 10.4 (11.8) μg/g Change in weight at 5 years of age (g) per 1 SD increase in maternal bone Pb Girls: 130.9 (95% CI:

- 227.4 to

- 34.4) Boys: 13.0 (95%CI

- 73.7, 99.9)
Adjusted for cohort, maternal age, calf circumference, height, educ ation, number of pregnancies, breast feeding for 6 mo nth s, calcium treatment, child’s gestational age at birth, height. Sanna et al. ( 2011 ) Sardinia, Italy Data collected in 1998, 2002 and 2007 n=825 children 11

- 14 yrs old Pb in hair 1998: 5.84 (6.56) μg/g 2002: 1.49 (1 .72) μg/g 2007: 0.78 (0.93) μg/g Height 1998: β log Pb=

- 0.121 (p=0.0021) 2002: β log Pb=

- 0.115 (p=0.0349) 2007: β log Pb=

- 0.011 (p=0.8665) Sitting Height 1998: β log Pb=

- 0.117 (p=0.0017) 2002: β log Pb=

- 0.036 (p=0.5149) 2007: β log Pb=0.028 (p=0.6633) ELL 1998: β log Pb=

- 0.103 (p=0.0209) 2002: β log Pb=

- 0.164 (p=0.0057) 2007: β log Pb=

- 0.008 (p=0.9058)
Adjusted for age and sex Ignasiak et al. ( 2006 ) South

- western Poland 1995
(Industrial area with copper smelters and refineries) school children 7

- 15 years n=463 boys n= 436 girls Concurrent blood Pb 7.7 (3.5) μg/dL Estimated decrement per 10 μg/dL increase in blood Pb Weight: Boys: 2.8 kg Girls: 3.5 kg Height: Boys: 3.2 cm Girls: 4.0 cm Trunk length: Boys: 1.2 cm Girls: 1.1 cm Leg length: Boys: 2.1 cm Girls: 2.9 cm Arm length: Boys: 1.8 cm Girls: 1.9 cm
Adjusted for age, age2, and education level of motherFebruary 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Study Population Pb Biomarker Mean Pb (SD) Effect Estimates
Hauser et al. ( 2008 ) Chapaevsk, Russia May 2003

– May 2005 n=489 boys 8

- 9 yrs old Concurrent blood Pb 3 (2

- 5) μg/dL Median (25

- 75 percentile) Regression coefficient (95 % CI) Height (cm):

- 1.439 (

- 2,25,

- 0.63) Weight (kg):

- 0.761 (

- 1.54, 0.02) BMI:

- 0.107 (

- 0.44, 0.23) Adjusted for birth weight, gestational age, and age at exam

Little et al. ( 2009 ) Dallas, Texas 1980

- 1989 and 2002 n=191 (1980s) n=169 (2002) 2

- 12 yrs old Concurrent blood Pb 1980s: 23.6 (1.3 SE) μg/dL 2002: 1.6 (0.2 SE) μg/dL Blood Pb effect per 10μg/dL Pb increase

– Mean(95%CI) Height (cm):

- 2.1 (

- 1.9,

- 2.3) Weight (kg):

- 1.9 (

- 1.7,

- 2.1) BMI (kg/m2):

- 0.5 (

- 0.4,

- 0.7)
Adjusted for age, age2, sex and cohort effect Min et al. ( 2008b ) Seoul, South Korea Date(s) not specifi ed n=62 boys n= 46 girls 5

- 13 yrs Concurrent blood Pb 2.4 (0.7) μg/dL Linear model estimate (SE; p) Height:

- 1.449 (0.639; p=0.026) Total arm length:

- 1.804 (0.702; p=0.012) Body weight:

- 0.646 (0.718; p=0.370) BMI:

- 0.006 (0.272; p=0.982)
Adjusted for age, sex, and father's education Schell et al. ( 2009 ) Albany, New York 1986

- 1992, 1992

- 1998 n=244 Maternal blood Pb during second trimester, third trimester, and delivery; Infant blood Pb at delivery, 6 months, and 12 months Maternal blood Pb during second trimester 2.8 (2.6) μg/dL, maternal blood Pb during third trimester: 2.6 (2.2) μg/dL, maternal blood Pb at delivery: 2.8 (2.4) μg/dL Infant blood Pb at delivery: 2.3 (2.7) μg/dL, infant blood Pb at 6 months: 3.2 (3.3) μg/dL, and infant blood Pb at 12 months: 6.3 (4.8) μg/dL Linear model for maternal second trimester Pb:

- 0.242 (p

- value 0.01) for 6

- month head circumference.
When examining second t rimester maternal Pb ≥ 3 μg/dL, associations were observed for 6 mo weight for age, 6 mo weight for length, 6 and 12 mo head circumference, and 12 mo upper arm circumference for age
Adjusted for infant sex, infant birth weight, infant nutrition, maternal age, marital status, employment, race, height, parity, second trimester smoking, and education.February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Study Population Pb Biomarker Mean Pb (SD) Effect Estimates
Lamb et al. ( 2008 ) Kosovo, Yugoslavia 1985

- 1986 n=309 mother child pairs Maternal blood Pb Pristina: 5.60 (1.99) μg/dL Mitrovica: 20.56 (7.38) μg/dL Regression coefficients relating maternal blood Pb to height (95% CI): Pristina Birth: 0.35 (

- 0.64, 1.34) 1 yr:

- 0.61 (

- 2.24, 1.03) 4 yr: 0.79 (

- 1.71, 3.29) 6.5 yr: 0.15 (

- 2.43, 2.74) 10 yr:

- 0.09 (

- 3.69, 3.52)

Mitrovica Birth: 0.43 (

- 0.83, 1 .69) 1 yr:

- 0.30 (

- 2.55, 1.96) 4 yr:

- 0.72 (

- 3.26, 1.82) 6.5 yr:

- 1.87 (

- 4.38, 0.64) 10 yr:

- 2.87 (

- 6.21, 0.47)
To BMI (95% CI): Pristina Birth:

- 0.14 (

- 0.69, 0.42) 1 yr: 0.61 (

- 0.28, 1.50) 4 yr: 0.17 (

- 0.67, 1.00) 6.5 yr: 0.61 (

- 0.09, 1.30) 10 yr:

- 0.49 (

- 1.45, 0.46)

Mitrovica Birth:

- 0.18 (

- 0.69, 0.33) 1 yr: 0.23 (

- 0.84, 1.30) 4 yr: 0.16 (

- 0.66, 0.98) 6.5 yr:

- 0.12 (

- 0.90, 0.66) 10 yr: 1.31 (

- 0.95, 3.57)
Adjusted for sex, ethnicity, parity, maternal height or maternal BMI, maternal education, gestational age at delivery, gestational age at blood sample, and HOMES score Zalina et al. ( 2008 ) Kuala Lumpur, Malaysia n=269 children 6.5

- 8.5 yrs old n=169 urban n=100 industrial Concurrent blood Pb Industrial: 3.75 μg/dL Urban: 3.56 μg/dL Correlation with blood lead: Height for age: Urban:

- 0.095 (p=0.219) Industrial:

- 0.037 (p=0.716) Weight for age: Urban: 0.019 (p=0.806) Industrial:

- 0.063 (p=0.535) Weight for height: Urban: 0.136 (p=0.079) Industrial:

- 0.069 (p=0.493) Left arm circumference: Urban: 0.041 (p=0.595) Industrial:

- 0.055 (p=0.587)February 2012 5

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– Do Not Cite or Quote Reference a Study Location and Years Study Population Pb Biomarker Mean Pb (SD) Effect Estimates
Tomoum et al. ( 2 010 ) Cairo, Egypt Jan

- Jun 2007 n=45 boys and girls 10

- 13 yrs old Concurrent blood Pb 9.46 (3.08) μg/dL Percentage of the median (SD): Pb<10 μg/dL Weight: Boys: 127.56 (16.26)% Girls: 114.8 (10.8)% Height: Boys: 98.06 (3.19)% Girls: 96.75 (2.91)%

Pb≥ 10 μg/dL Weight: Boys: 122.0 (16.71)% Girls: 123.11 (12.52)% Height: Boys: 99.5 (5.04)% Girls: 100.33 (4.53)%
Adjusted for age and sex Olivero

- Verbel et al. ( 2007 ) Cartegena, Columbia Jun

- Aug 2004 n=189 children 5

- 9 yrs old Concurrent blood Pb 5.49 (0.23) μg/dL Spearman correlation coefficient (p

value) between blood Pb and body size:

- 0.224 (0.002)
*no significance in partial correlation between blood Pb and size when controlled for age:

- 0.096 (0.189) Liu et al. ( 2011a ) Guiyu, China Chendian, China Jan

- Feb 2008 n=303 3

- 7 yr s old Concurrent blood Pb Guiyu: 13.2 (4.0

- 48.5) μg/dL Chendian: 8.2 (0

- 21.3) μg/dL Median (range) Mean chest circumference in girls was lower among those with higher blood Pb levels (>10μg/dL)

Mean chest and head circumference in children > 6 years old g reater among those with higher blood Pb levels (>10 μg/dL)
No multivariate adjustment, stratification by age/sex Mahram et al. ( 2007 ) Zanjan province,, Iran Date(s) not specified n=42 boys n= 39 girls 7

- 11 yrs Concurrent blood Pb Area with lead smelters: 37.0 (24.7) μg/dL

Area without lead smelters: 15.6 (13.4) μg/dL Comparison of control and study groups Height, standardized for age: p - value 0.52 Weight, standardized for age: p

value 0.8

*Estimated Lower Limb Length

a Studies are presented in order of first appearance in the text of this section.


### Results from recent epidemiologic studies of postnatal growth are summarized in Table 1 

### 5

- 41 . Afeiche et al. ( 2011 ) conducted a longitudinal study of children in Mexico City, 2 

### born between 1994 and 2005. Maternal bone Pb during pregnancy was associated with a 3 

### significant decrease in BW at age 5 years in girls but not in boys. The findings were 4 

### robust to additional adjustment for child’s blood Pb level. In a study of children in 5 

### Sardinia Italy, Sanna et al. ( 2011 ) measured Pb in hair at three points in time (1997, 2002, 6 

### and 2007) and reported cross

- sectional results from regress ion analyses for each of these 7 

### time periods. Pb in hair decreased over time and significant associations of Pb in hair 8February 2012 5

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### with height were observed only in earlier time periods when hair Pb levels were relatively 1 

### high. 2 

### Ignasiak et al. ( 2006 ) studied school children aged 7

- 15 years living close to copper 3 

### smelters and refineries in Poland to assess the impact of Pb exposure on their growth 4 

### status. There was a statistically significant li near relationship between concurrent blood 5 

### Pb and reduced weight, height, trunk, leg and arm lengths. This decrease in height was 6 

### more influenced by decreases in leg length than trunk length. These results also indicated 7 

### that there was attenuation in osteo blast activity associated with higher blood Pb levels, 8 

### consistent with animal toxicological studies ( Long et al., 1990 )

. Hauser et al. ( 2008 ) 9 

### investigated the relationship between blood Pb and height in boys living in Chapaevsk, 10 

### Russia. In a mult ivariate adjusted regression analysis, height significantly decreased with 11 

### increasing blood Pb. Statistically nonsignificant decreases in weight and BMI were also 12 

### observed. The association of blood Pb with height, weight and BMI was examined 13 

### among two coho rts of children living near Pb smelters in Texas ( Little et al., 2009 )

. The 14 

### first cohort included children 2

- 12 years old in 1980 and the second cohort included 15 

### children of the same age in 2002 when blood Pb levels were substantially lower. 16 

### Decreases in height, weight and BMI with increasing blood Pb levels were observed 17 

### among children in both cohorts and increases in height and weight were observed 18 

### comparing children from the 2002 cohort to those from the 1980 cohort. In a study with 19 

### Korean children, Min et al. ( 2008b ) observed that height and total arm length decreased 20 

### significantly with inc reasing blood Pb in multivariate adjusted regression models. A 21 

### statistically nonsignificant decrease in body weight was observed with increasing blood 22 

### Pb while no effect on BMI reported. A study in New York reported an association 23 

### between maternal blood Pb during the second trimester of pregnancy and various 24 

### measures of growth, especially among those mothers with blood Pb levels of at least 25 

### 3 μg/dL ( Schell et al., 2009 )

. These associations did not persist for those with maternal 26 

### blood Pb levels less than 3 μ g/dL. Among infants, 6 month blood Pb levels were not 27 

### associated with 12 month measures of growth. In comparisons of changes in blood Pb 28 

### levels over time, high maternal blood Pb combined with low 12 month blood Pb among 29 

### infants (indicating a decrease in blood Pb over time) resulted in the greatest growth, even 30 

### compared to those with both low maternal and infant blood Pb measures. 31 

### Contrary to the results summ arized above, several studies do not observe associations 32 

### between blood Pb levels and impaired growth. In a prospective study of 309 mother

- child 33 

### pairs from Yugoslavia, the relationship between maternal blood Pb and attained height in 34 

### children was investig ated in those living in an highly exposed town with a smelter and 35 

### battery plant and those living in a relatively lower exposed town ( Lamb et al., 2008 )

. In 36 

### multivariate adjusted regression models, neither attained height (at birth, 1, 4, 6.6, or 37 

### 10 years age) nor rate of height change per month (at birth

- 1 year, 1

- 4 years, 4

- 6.5 years, 38February 2012 5

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### 6.5

- 10 years age) was associated with maternal pregnancy blood Pb levels in either the 1 

### industrial or less exposed town. Weight was also not associated with maternal blood Pb 2 

### in this study. In a study with a similar design, Zailina et al. ( 2008 ) studied the relationship 3 

### of blood Pb and height in 7 year
- old Malaysian school children comparing those 4 

### attending two schools in an urban setting to those attending a school near an industrial 5 

### area. After adjustment for age no statistically significant associations between concurrent 6 

### blood Pb and physical development were observed. Tomoum et al. ( 2010 ) investigated 7 

### the association between blood Pb and height in pubertal children in Cairo, Egypt. Neither 8 

### boys nor girls with concurrent blood Pb levels > 10 μ g/dL differed significantly in height 9 

### or weight when compared to those with blood Pb < 10 μ g/dL. In a simple correlation 10 

### analysis of children aged 5

- 9 years in Colombia, Olivero

- Verbel et al. ( 2007 ) reported 11 

### that concurrent blood Pb levels were negatively associated with body size (r =

- 0.224, p < 12 

### 0.002). However, when a partial correlation analysis was performed controlling for age, 13 

### the association between blood Pb and body size was no longer statistically significant. In 14 

### a study of school children in China chest and head circumference were found to differ 15 

### between high (>10μg/dL) and low co ncurrent blood Pb level groups; however, the 16 

### direction of the difference was not consistent ( Liu et al., 2011a )

. Among girls, comparing 17 

### those with high and low blood Pb levels, a reduction in head circumference was 18 

### observed. Among children greater than 6 years of age, those with higher blood Pb levels 19 

### were reported to have greater head and chest circumferences. In a study of children aged 20 

### 7

- 11 years and living in an area of Iran with or without Pb smelters, age

- standardized 21 

### weight and height did not vary by study area ( Mahram et al., 2007 )

. 22 

### Recent toxicological studies report significant ch anges in postnatal or adult body weight 23 

### after Pb exposure during different developmental windows. Masso

- Gonzalez and 24 

### Antonio

- Garcia ( 2009 ) found Pb

- induced decreased bo dy weights at weaning (PND21) in 25 

### rat pups from dams exposed to Pb during pregnancy and lactation. Blood Pb level in the 26 

### control group was 1.43 μg/dL, in the Pb group it was 22.8 μg/dL. Dong et al. ( 2009 ) 27 

### reported decreased body weight in adult Kunming mice after exposure to 0.6% Pb

- acetate 28 

### in drin king water for 8 weeks. In contrast, Leasure et al. ( 2008 ) reported a statistically 29 

### significant inverse relationship between Pb exposure and body weight for male mice 30 

### exposed to lower (27 ppm ), moderate (55 ppm ) and higher levels (109 ppm ) levels of Pb 31 

### during gestation and lactation (2 weeks befo re mating, through gestation and to PND10) 32 

### with those exposed to the lowest dose having the highest adult body weight among the 33 

### overweight Pb

- exposed animals. Male mice exposed to the lower and higher Pb 34 

### concentrations during gestation were 26% and 13% hea vier than were controls at 1 year 35 

### of age, respectively. In this study, dams were administered 27 ppm (low), 55 ppm 36 

### (moderate), and 109 ppm (high) Pb in drinking water beginning which resulted in 37 

### respective blood Pb levels from 10 μg/dL or less in the low

- e xposure offspring to 42 38 

### μg/dL in the high

- exposure offspring at PND10. Leasure et al. ( 2008 ) also exposed a 39February 2012 5

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### separate group of mice to Pb only during the postnatal period (PND0

- PND21, lactation 1 

### only exposure) and mice exposed to the same aforementioned low or high dose of Pb did 2 

### not exhibit a difference in body weight when compared to control offspring. Wang et al. 3 

### ( 2009e ) observed a statistically significant decrease in fetal body weight and body length 4 

### of Wistar rats at GD20 after maternal exposure to 0.025% Pb

- acetate du ring gestation 5 

### days 1

- 10, 11

- 20, or 1

- 20. Also, associations were reported between elevated maternal 6 

### blood Pb levels (0.6, 1.3, or 1.74 μ M, respectively or ~12.4, 26.9, or 36.0 μ g/dL, 7 

### respectively) compared to control (0.04 μ M or ~ 0.83 μ g/dL) and decrease d pup body 8 

### length, and placental weight in Wistar rats at GD20. The greatest decrease in fetal body 9 

### weight and length was observed in the group exposed to Pb during gestation days 1

- 20 10 

### followed by the group exposed to Pb during gestation days 11

- 20. Teijón et al. ( 2006 ) 11 

### observed reductions in birthweight of litters administered 200 ppm or 400 ppm Pb

- acetate 12 

### in drinking water, but found that this effect did not persist in the postnatal growth of the 13 

### rats. 14 

### Notably, previous toxicological studies observed reductions in postnatal weight as well as 15 

### birth weight after exposure to Pb, albeit often at higher concentrations of Pb exposure. 16 

### Ronis et al. ( 2001 ; 1998a ; 1998b ; 1996 ) have published a series of papers exposing rats to 17 

### Pb over different developmental windows, showing associations between Pb exposure 18



### a nd deficits in gro wth. Sprague

- D awley rats with lifetime Pb exposure to 0.6% Pb

- acetate 19 

### in drinking water (gestational

- termination of experiment Pb exposure, maximum blood 20 

### Pb of 316 μ g/dL in males and 264 μ g/dL in females) had sex

- independent pre

- pubertal 21 

### growth suppression , male

- specific suppression of pubertal growth and loss of growth 22 

### effects postnatally but still maintained an overall decreased body size out to PND60 due 23 

### to earlier deficits. In a follow up study using the same exposure duration with a dose of 24 

### 0.45% Pb

- ac etate (resulting in blood Pb of 263 μ g/dL at PND85) yielded the same results 25 

### ( Ronis et al., 1996 ) with mechanistic insight showing decre ments in insulin

- like growth 26 

### factor 1 (IGF1) accompanying the decreases in growth rates. 27 

### The body of toxicological literature on postnatal growth with Pb exposure indicates that 28 

### Pb exposure can induce decrements in both height/body length and BW that may be 29 

### persistent and differ by sex. However, findings from epidemiologic studies of postnatal 30 

### growth are not consistent. Animal toxicology studies give insight to mechanistic changes 31 

### that may contribute to this Pb

- induced decrement and to the windows of expos ure that 32 

### may contribute greatest to these decrements. 33February 2012 5

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### 5.8.9 Toxicological Studies of Developmental Effects

5.8.9.1 Developmental Effects on Blood and Liver
The 1986 and 2006 Pb AQCDs reported studies that suggest Pb may alter hematopoietic 1 

### and hepatic function during d evelopment. Some recent studies provide evidence that 2 

### support these findings; however recent results are not consistent among the studies. 3


### Massó et al. ( 2007 ) reported a decreas e in liver weights of pups born to dams that 4 

### consumed 300 mg /L Pb in drinking water during gestation and lactation. They also 5 

### reported an increase in the number of erythrocytes; however the erythrocyte size was 6 

### diminished by 62%. Pb produced microc y tic ane mia as evidenced by decreased 7 

### hemoglobin content and hematocrit values without changes in mean corpuscular 8 

### hemoglobin (MCH) concentration. Alkaline phosphatase (ALP) activity, CAT activity, or 9 

### thiobarbituric acid reactive substances (TBARS) production did not change in pups at 10 

### postnatal 0, but increased statistically significantly by PND21 indicating reactive oxygen 11 

### generation. No change in acid phosphatase (ACP) activity was observed in the livers of 12 

### pups at PND0 or 21. 13


### Massó

- González and Antonia

- García ( 2009 ) reported normochromic and microcytic 14 

### anemia and a significant decrease in hematocrit values and blood δ

- aminolevulinic acid 15 

### dehydratase (ALAD) activity (90% reduction) in pups from dams administered 300 mg /L 16 

### Pb

- acetate in drinking water during gestation. The authors also reported that erythrocyte 17 

### osmotic fragility was four times greater in Pb

- exposed pups than in control pups. Massó

18 

### González and Antonia

- García ( 2009 ) reported increases in TBARS and CAT activity in 19 

### the liver after Pb exposure. Intoxication with Pb also resulted in decreased liver protein 20 

### concentrations and ma nganese
- dependent SOD activity. Abnormalities in liver function 21 

### were further exemplified by increases in liver concentrations of ALP and ACP. 22 

### Teijón et al. ( 2006 ) observed that gestational exposur e to Pb caused a decrease in 23 

### erythrocytes, hemoglobin, and MCH at weaning; however, by 1 and 3 months 24 

### postweaning, these parameters had returned to normal values. The authors observed a 25 

### slight increase in serum ALP, alanine aminotransferase (ALT), and aspa rtate 26 

### aminotransferase (AST) levels after Pb exposure in the absence of liver histological 27 

### changes. 28


### Pb

- induced effects on SOD activity in the liver of fetuses after Pb intoxication was 29 

### supported by a study by Uzbekov et al. ( 2007 )

. The authors reported an initial increase in 30 

### SOD activity in livers of pups exposed to 0.3 mg /L and 3.0 mg /L Pb nitrate during 31 

### gestation for 1 month (mean daily consumption 27 μ g /kg). In contrast, long

- term 32February 2012 5

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### exposure (5 months) to the same concentrations of Pb nitrate concentration during 1 

### gestation resulted in decreased hepatic SOD activity. 2 

### Effects on hepatic Phase I and Phase II enzymes after early developmental exposure of 3 

### offsp ring to Pb during gestation and lactation was evaluated by Pillai et al. ( 2009 )

. In the 4 

### study, pregnant Charles Foster rats were administered 0.05 mg /kg body weight Pb 5 

### subcutaneously throughout gestation until PND21. Pups were evaluated on PND56. 6 

### Results of the study show that Phase I xenobiotic

- metabolizing enzymes (NAD PH

- and 7 

### NADH cytochrome c reductase) and Phase II xenobiotic

- and steroid

- metabolizing 8 

### enzymes (δ

- glutamyl transpeptidase, UDPGT, glutathione

- s
- transferase, and 17β

9 

### hydroxysteroid oxidoreductase) were reduced in both male and female pups by PND56. 10 

### Only inh ibition in glutathione

- s
- transferase and 17β

- hydroxysteroid oxidoreductase 11 

### activities demonstrated a sex

- specific pattern (glutathione

- s
- transferase inhibition in 12 

### males; 17β

- hydroxysteroid oxidoreductase inhibition greater in females). Observed Pb

13 

### induced histological changes included massive fatty degeneration in hepatocytes, large 14 

### vacuoles in cytoplasm, appearance of pyk notic nuclei, and infiltration of lymphocytes in 15 

### the liver. Antioxidant enzymes (SOD, CAT, glutathione peroxidase, and glutathione 16 

### reduct ase) were also reduced after Pb intoxication. Alterations in biochemical parameters 17 

### included decreased DNA, RNA, and cholesterol content. 18



### 5.8.9.2 Developmental Effects on Skin
The 2006 Pb AQCD ( U.S. EPA, 2006b ) reported a study that demo nstrated Pb

- induced 19 

### abnormalities in skin development. No current studies were identified that addressed Pb

20 

### induced skin alterations. 21



### 5.8.9.3 Developmental Effects on the Retina

The 2006 Pb AQCD concluded that Pb exposure during early postnatal development 22 

### (result ing in blood Pb levels ~20 μ g/dL) impaired retinal development in female Long

23 

### Evans hooded rats. A more recent study ( Fox et al., 2008 ) exposed female Long

- Evans 24 

### hooded rats to low (27 ppm ), moderate (55 ppm ), and high (109 ppm ) levels of 25 

### Pb

- acetate in drinking water beginning 2 weeks before mating, throughout gestat ion, and 26 

### until PND10. Blood Pb levels measured in these pups on postnatal days 0

- 10 were 27 

### 10

- 12 μ g/dL (low), 21

- 24 μ g/dL (moderate), and 40

- 46 μ g/dL (high). Results of the study 28 

### demonstrated supernormal persistent rod photoreceptor

- mediated (scotopic) 29 

### elect roretinograms (ERGs) ( Figure 5

- 46 , Table 5

- 33 ) in adult rats similar to ERG 30 

### findings in male and female children after gestational exposure to low

- and moderate

- 31February 2012 5

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### levels of Pb. Low

- and moderate

- levels of Pb increased neurogenesis of rod 1 

### p hotoreceptors and rod bipolar cells without affecting Müller glial cells and statistically 2 

### significantly increased the number of rods in central and peripheral retina ( Figure 5

- 46 , 3 

### and Table 5

- 33 ). High

- level Pb exposure (109 ppm ) statistically significantly decreased 4 

### the number of rods in central an d peripheral retina. Pb

- exposure induced concentration

5 

### dependent decreases in adult rat retinal dopamine synthesis and utilization/release. 6



### 5.8.9.4 Developmental Effects on Teeth
Pb has been associated with multiple health effects including dental caries, however , 7 

### there is very limited information available on the temporal and spatial incorporation of Pb 8 

### in dental tissue ( Arora et al., 2005 )

. Arora et al. ( 2005 ) demonstrated that Wistar rat pups 9 

### exposed to Pb during gestation and lactation (40 mg /L of Pb nitrate in drinking water of 10 

### pregnant dams) had higher concentrations of Pb on the surface of enamel and in the 11 

### dentine immediately adjacent to the pulp. The authors concluded that additional research 12 

### is needed on the intracellular uptake of Pb during tooth development to fully understand 13 

### the spatial distribution of Pb in teet h. 14



### 5.8.10 Summary and Causal Determination
Many epidemiologic and toxicological studies of the effects of Pb on reproductive 15 

### outcomes have been conducted since the 2006 Pb AQCD. These studies covered 16 

### outcomes such as female and male reproductive function, birth defects, spontaneous 17 

### abortions, infant mortality, preterm birth, low birth weight, and developmental effects. 18 

### There is an abundance of evidence in the literature demonstrating that Pb induces 19 

### reproductive and developmental effects in laboratory animals exp osed to Pb during 20 

### gestation and/or lactation. Many of the Pb

- induced effects occur in a concentration

21 

### dependent manner and have been observed at maternal blood Pb levels that do not result 22 

### in clinical toxicity in the dams. Additionally, epidemiologic studi es have demonstrated 23 

### strong evidence of an association between Pb and delayed puberty as well as decrements 24 

### to sperm/semen quality and function. 25 

### Many of the animal toxicology studies included in the 2006 Pb AQCD examined the 26 

### effect of Pb on reproduction an d development at blood Pb levels greater than 40 μ g/dL, a 27 

### dose where maternal toxicity can develop during pregnancy. Data from the 2006 Pb 28 

### AQCD on male fertility showed Pb exposure or biomarkers of Pb

- exposure were 29 

### associated with decrements in semen quali ty. Recent studies have shown the effects of Pb 30 

### exposure during early development to include disruption of endocrine function; delay in 31February 2012 5

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### the onset of puberty and alteration in reproductive function later in life; and changes in 1 

### morphology or histology in se x organs and placenta. Additionally, recent epidemiologic 2 

### studies of reproductive factors among males and females investigated whether Pb 3 

### biomarker levels were associated with hormone levels, fertility, and onset of puberty. 4 

### Epidemiologic studies showed as sociations between blood Pb and hormone levels for 5 

### females. Studies of Pb and fertility are limited and inconsistent for females and males. 6 

### Strong and consistent associations were observed between Pb levels in males in 7 

### occupational settings with blood Pb l evels as low as 20

- 45 μ g/dL and sperm count and 8 

### quality. 9 

### Delayed puberty has been linked to decreased peak bone mass and increased risk of 10 

### osteoporotic fractures ( Gilsanz et al., 2011 ; Naves et al., 2005 )

. Multiple epidemiologic 11 

### studies of Pb and puberty have shown associations between concu rrent blood Pb levels 12 

### and delayed pubertal development for girls and boys (evidence is stronger among girls). 13 

### These associations are consistently observed in multiple epidemiologic studies in 14 

### populations with blood Pb levels <10μg/dL. Confounders considere d in the 15 

### epidemiologic studies that performed regression analyses varied. Most studies controlled 16 

### for age and BMI. Other variables, such as measures of diet and SES and race/ethnicity, 17 

### were included in some of the studies. Many of the studies demonstrating positive 18 

### associations adjusted for many of the potential confounders. No patterns were detected in 19 

### the associations between Pb and puberty based on inclusion of specific confounders. 20


### Pb

- mediated changes in levels or function of reproductive and growth ho rmones have 21 

### been demonstrated in past and more recent toxicological studies; however the findings 22 

### are inconsistent. More data are needed to determine whether Pb exerts its toxic effects on 23 

### the reproductive system by affecting the responsiveness of the hypo thalamic

- pituitary

24 

### gonad axis or by suppressing circulating hormone levels. More recent toxicological 25 

### studies suggest that oxidative stress is a major contributor to the toxic effects of Pb on 26 

### male and female reproductive systems. The effects of ROS may in volve interference with 27 

### cellular defense systems leading to increased lipid peroxidation and free radical attack on 28 

### lipids, proteins, and DNA. Several recent studies showed an association between 29 

### increased generation of ROS and germ cell injury as evidence d by destruction of germ 30 

### cell structure and function. Co

- administration of Pb with various antioxidant compounds 31 

### either eliminated Pb

- induced injury or greatly attenuated its effects. In addition, many 32 

### studies that observed increased oxidative stress also observed increased apoptosis which 33 

### is likely a critical underlying mechanism in Pb

- induced germ cell DNA damage and 34 

### dysfunction. 35 

### Overall, results of pregnancy outcomes were similar to those of the 2006 Pb AQCD ; 36 

### inconsistent evidence of a relationship with Pb was available for preterm birth and little 37February 2012 5

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### evidence was available to study the associations with spontaneous abortions. The 2006 1 

### Pb AQCD included a few studies that reported potential associations between Pb and 2 

### neural tube defects, but the recent epid emiologic studies found no association. Some 3 

### associations were observed between Pb and low birth weight when epidemiologic studies 4 

### used measures of maternal bone Pb or air exposures, but the associations were less 5 

### consistent when using maternal blood Pb or umbilical cord and placenta Pb (maternal 6 

### blood Pb or umbilical cord and placenta Pb were the biomarkers most commonly used in 7 

### studies of low birth weight). Effects of Pb exposure during early development on 8 

### toxicological studies included reduction in litt er size, implantation, birth weight and 9 

### postnatal growth. Findings from epidemiologic studies of postnatal growth are 10 

### inconsistent. 11 

### Toxicological studies demonstrated that the effects of Pb exposure during early 12 

### development include impairment of retinal de velopment and alterations in the developing 13 

### hematopoietic and hepatic systems. Negative developmental outcomes were also noted 14 

### including effects on the eyes and teeth. 15 

### Similar to toxicological and epidemiologic studies that observed Pb to be associated wit h 16 

### delayed puberty, delays of dynamic changes in the HPT axis are seen in the ecological 17 

### literature, i.e., delayed metamorphosis in Pb exposed frogs. Additionally, Pb exposure 18 

### has been shown to have detrimental effects on sperm, albeit often at higher blood Pb 19 

### levels in epidemiology studies but in lower doses in the toxicology literature. Again, 20 

### these findings agree with the ecological literature where Pb

- dependent sperm effects are 21 

### seen in rotifers, earthworms, and trout ( Sections 7.3.5.2 , 7.2.4.2 , and 7.3.5.3 ). 22 

### In conclusion, the recent toxicological and epidemiologic literature provides strong 23 

### evidence that Pb exposure is associated with effects on reproduction and development. 24 

### The weight of the evidence supports the association of Pb exposure with delayed onset of 25 

### puberty in both males and females and detrimental effe cts on sperm and semen quality in 26 

### occupationally

- exposed males and in laboratory animals. In cross

- sectional 27 

### epidemiologic studies of girls (ages 6

- 18 years) with mean and/or median concurrent 28 

### blood Pb levels less than 5 μ g/dL consistent associations with delayed pubertal 29 

### development (measured by age at menarche, pubic hair development, and breast 30 

### development) were observed. Toxicological studies indicate that prenatal and lactational 31 

### exposures to Pb can cause a delay in the onset of female puberty at blood Pb levels as low 32 

### as 8 μ g/dL ( Iavicoli et al., 2006b ; Iavicoli et al., 2004 )

. Recent studies show that pubertal 33 

### onset is one of the more sensitive markers of Pb exposure with effects observed after 34 

### maternal exposures leading to blood Pb levels in the pup of 3.5

- 13 μ g/dL ( Iavicoli and 35 

### Carelli, 2007 )

. In boys (ages 8

- 15 years), fewer studies were conducted but associations 36 

### were observed in most. Male animal toxicology studies have reported delayed sexual 37February 2012 5

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### maturity as measured with prostate weight, among other outcomes, seeing significant 1 

### decrements at b lood Pb levels of 34 μ g/dL ( Sokol et al., 1985 )

. Additionally, Pb expos ure 2 

### has been shown to have detrimental effects on sperm. These were observed in 3 

### epidemiologic studies at population mean blood Pb levels of 30 μg/dL and greater among 4 

### men occupationally exposed (mean blood Pb levels in study controls around 10 μg/dL) 5 

### and i n animal toxicological studies with rabbits exposed to subcutan e ous Pb 3 times 6 

### per week for 15 weeks with blood Pb levels of 20 μg/dL ( Moorman et al., 1998 )

. The 7 

### data on preterm birth, low birth weight, spontaneous abortions, birth defects, hormonal 8 

### influences, and fecundity are less consistent between the toxicological and epidemiologic 9 

### li terature. The collective body of evidence integrated across epidemiologic and 10 

### toxicological studies with a focus on the strong relationship observed with detrimental 11 

### effects on sperm and delayed pubertal onset is sufficient to conclude that there is a caus al 12 

### relationship between Pb exposures and reproductive and developmental effects. 13



## 5.9 Effects on Other Organ Systems

5.9.1 Effects on the Hepatic System
Hepatotoxic effects of Pb indicated in various animal models and human populations 14 

### include alterations in hepatic metabolism, hepatic cell proliferation, changes in 15 

### cholesterol metabolism, as well as oxidative stress

- related injury. Animal studies have 16 

### also shown that exposure to Pb causes a decrease in Phase I along with a simultaneous 17 

### increase in Phase II enzymes fo llowing exposure to Pb. Induction of oxidative stress by 18 

### Pb exposure is well supported by an increase in lipid peroxidation along with a decrease 19 

### in glutathione (GSH) levels and catalase (CAT), superoxide dismutase (SOD) and 20 

### glutathione peroxidase (GPx) ac tivities. 21



### 5.9.1.1 Summary of Key Findings of the Effects on the Hepatic System from the 2006 Lead AQCD
The 2006 Pb AQCD stated that the large experimental animal database indicated 22 

### hepato to xic effects, including liver hyperplasia, at very high dose Pb exposures. Other 23 

### effects noted in the liver following exposure to Pb included altered cholesterol synthesis, 24 

### DNA synthesis and glucose

- 6

- phosphotase dehydrogenase (G6DP) activity . The 2006 Pb 25 

### AQCD reported that cytochrome (CYP) P450 levels decreased following single doses of 26 

### Pb nitrate. Inhibition of induced and constitutive expression of microsomal CYP 1A1 and 27 

### 1A2 was observed among various P450 isozymes. Inhibition of Phase I enzymes was 28February 2012 5

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### accompanied by an increase in Phase II enzymes following exposure to Pb nitrat e and 1 

### other Pb compounds, suggesting that Pb is capable of causing a biochemical phenotype 2 

### similar to hepatic nodules. Studies relating to Pb

- induced hepatic hyperplasia suggested 3 

### alterations in the gluconeogenic mechanism, DNA hypomethylation along with c hanges 4 

### in proto

- oncogene expression as well as cholesterol synthesis. Cholesterol metabolism 5 

### changes following exposure to Pb were reportedly mediated as a result of induction of 6 

### several enzymes related to cholesterol metabolism as well as a decrease in th e cholesterol 7 

### catabolizing enzyme, 7 α

- hydroxylase. Tumor necrosis factor α lpha (TNF

- α ) was 8 

### reported to be one of the major mitogenic signals that mediated Pb nitrate

- induced 9 

### hepatic hyperplasia in studies using inhibitors to block TNF

- α activity. Other Pb

- related 10 

### effects presented in the 2006 Pb AQCD include liver cell apoptosis mediated b y Kupffer 11 

### cell derived signals and Pb

- induced oxidative stress in vitro cell cultures. The 2006 Pb 12 

### AQCD further suggested that alterations in liver heme metabolism may involve changes 13 

### in 5

- aminolevulinic acid dehydrogenase (ALAD) activity, porphyrin metabo lism, 14 

### transferrin (TF) gene expression and changes in iron metabolism. 15 

### With regard to human studies, the 2006 Pb AQCD stated that nonspecific liver injury 16 

### generally observed as increases in liver enzymes in the serum was reported in adults with 17 

### occupation al Pb exposure, although associations specifically with Pb exposures have not 18 

### been well established. In addition, similar to effects noted in animal studies, cytochrome 19 

### P450 activity was also suppressed in children and adults (drawn from the general 20 

### popula tion) following exposure to Pb. The 2006 Pb AQCD reported that hepatic effects 21 

### occurred only at high Pb exposure levels (blood Pb levels > 30 μ g/dL). 22



### 5.9.1.2 New Epidemiologic Studies
A few occupational epidemiologic studies examined liver biochemical parameters e ffects 23 

### on antioxidant status and oxidative stress resulting from exposure to Pb. However all of 24 

### these occupationally

- exposed cohorts represented populations highly exposed to Pb, with 25 

### blood Pb levels ranging from 29 to 53 μ g/dL. Although the hepatotoxicity observed 26 

### within these cohorts may not be generalizable to the general population as a whole, they 27 

### are useful in demonstrating consistent effects on a number of liver outcomes, including 28 

### altered liver function ( i.e., changes in the level of liver function enzymes), oxidative 29 

### stress, and antioxidant status ( Can et al., 2008 ; Khan et al., 2008 ; Patil et al., 2007 )

. 30 

### Additionally, these studies were cross

- sectional in design; thus, there is uncertainty 31 

### regarding the ma gnitude, timing, frequency, and duration of Pb exposure that contributed 32 

### to the observed associations. 33February 2012 5

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### In one of the occupational cohorts (spray painters in western Maharashtra, India, exposed 1 

### to Pb for > 6 hours/day for 2 to 20 years) examined by Patil et al. ( 2007 ) , mean (SD) 2 

### blood Pb levels in workers were 22.32 (8.87) μ g/dL. The blood Pb levels in workers were 3 

### statistically significantly higher compared to those in the concurrent control group (mean 4 

### [SD]: 12.52 [4.08] μ g/dL), who had no history of Pb exposure. Liver function enzymes, 5 

### including serum glutamic oxaloacetic transaminase (SGOT)/AST, and serum glutamic 6 

### pyruvic transaminase (SGPT)/ALT levels were statistically sign i ficantly increased in 7 

### spray painters compared to those in controls, whereas total serum protein levels were 8 

### decreased. In another occupational epidemiolgic study, Conterato et al

. ( In Press ) 9 

### investigated liver function parameters in automotive painters exposed to Pb in Brazil. 10 

### Exposed painters had a mean (SD) blood Pb concentration of 5.4 (0.4) μ g/dL compared 11 

### to 1.5 (0.1) μ g/dL in controls. The mean (SD) duration of exposure to Pb in painters was 12 

### 133.9 (14.5) months, whereas the controls were not occupationally exposed to Pb. In 13 

### exposed workers, the levels of aspartate aminotransferase (AST), but not γ
14 

### glutamyltranseferase, were increased approximately 2

- fold compared to levels in controls 15 

### (p < 0.05). The activity of AST was positively correlated with blood Pb levels (r = 0.26, p 16 

### < 0.05). The authors suggested that confounding exposures to toxic constituents of the 17 

### paints regularly used by painters, and not Pb, may be the etiological cause of decrements 18 

### in AST function as these effects were not also seen in battery workers with much higher 19 

### blood Pb levels (49.8 μ g/dL). Co

- exposure to other environmental contaminants may also 20 

### explain the effects seen in occupationall y
- exposed spray

- painters in Patil et al. ( 2007 )

. 21



### 5.9.1.3 New Toxicological Studies

Hepatic Metabolism As stated in the 2006 Pb AQCD, acute exposures of rodents to Pb nitrate and other Pb 22 

### compounds cause a decrease in Phase I enzymes accompanied by a simultaneo us increase 23 

### in Phase II enzymes. The conclusions presented in the 2006 Pb AQCD were also 24 

### reviewed by Mudipalli ( 2007 )

. 25 

### Changes in biochemical parameters, suggestive of liver damage, in undernourished male 26 

### Wistar rats (f ed low

- protein diet without mineral supplements) treated with 500 ppm 27 

### Pb

- acetate in drinking water over a 10 month period, included decreases in serum protein 28 

### and albumin levels as well as increases in aspartate aminotransferase (AST), alanine 29 

### aminotransferase (ALT), serum alkaline phosphatase (ALP), and gamma glutamyl 30 

### transpeptidase (GGT) levels ( DS et al., 2009 )

. In Pb

- treated animals, the blood Pb levels 31 

### steadily increased throughout the initial portion of the study period, reaching a maximum 32 

### of approximately 30 μ g/dL after 2 months. After this time, blood Pb levels rapidly 33February 2012 5

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### increased to approximately 110 μ g/dL (greatly in excess of blood levels in the general 1 

### human population) by six months time and remained at this level until the termination of 2 

### exposure at 10 months. The study authors reported that similar biochemical changes were 3 

### not observed in animals treated with Pb

- acetate maintained on protein

- adequate, mineral 4 

### rich diet and concluded that nutritional management is important in managing Pb

- related 5 

### poisoning. Mice gavaged with 50 mg /kg Pb nitrate for 4 0 days demonstrated similarly 6 

### increased activities of AST, ALT, ALP, and acid phosphatase (ACP) compared to 7 

### controls ( Sharma et al., 2010a )

. Upadhyay et al. ( 2009 ) reported that treatment of 8 

### Sprague

- Dawley rats with 35 mg /kg Pb via i.p. injection for 3 days (blood Pb not 9 

### reported) significantly increased the activities of ALT, AST, serum ALP, and acid 10 

### phosphatase over those in controls, whereas alkaline phosphatase activity was decreased 11 

### in Pb

- treated animals. Concomitant treatment with zinc and varying levels of ascorbic 12 

### acid were observed to ameliorate the toxic effects of Pb. The serum activities of glutamic 13 

### pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) were similarly 14 

### significantly increased over those in controls in mice subcutaneously injected with 15 

### 50 mg /kg Pb

- acetate daily for 15 days (blood Pb not reported) ( Wang et al. )

. Swarup et 16 

### al. ( 2007 ) investigated serum biochemical changes in cows living in Pb

- contaminated 17 

### environments. Serum levels of ALT, AST, alkaline phosphatase, total protein, albumin, 18 

### globulin, and A/G ratio were statistically significantly altered in cows living near Pb

- Zn 19



### s melters (mean [SD] blood Pb: 86 [6] μ g/dL, greatly in excess of blood levels in general 20 

### human populations) compared to control cows (mean [SD] blood Pb: 7 [1] μ g/dL). 21 

### Significant positive correlations were found between blood Pb and ALT and AST, 22 

### whereas a negative correlation was observed between blood Pb and total lipids, protein, 23 

### and albumin. 24


### Pillai et al. ( 2009 ) investigated effects on hepatic phase I and II enzymes in male and 25 

### female rats born to dams that were treated with 50 μ g/kg Pb

- acetate via subcutaneous 26 

### injection daily throughout gestation and continuing until PND21. Thus, the offspring of 27 

### treated dams were exposed to Pb via placental and lactational transfer. The female and 28 

### male pups were then allowed to reach sexual maturity (PND55

- 56) to assess continuing 29 

### exposure to bioaccumulated Pb. The activities of hepa tic phase I enzymes NADPH

- and 30 

### NADH

- cytochrome c reductase were statistically significantly reduced in Pb

- exposed 31 

### male and female rats on PND56 (blood Pb not reported), compared to controls. In rats 32 

### treated with 25 μ g/kg Pb and Cd, the effect on phase I en zymes was increased. Pb 33 

### treatment additionally decreased the activities of phase II enzymes uridine diphosphate

34 

### glucoronyl transferase and GST in males and females, but no effect was observed on 35 

### GGT or 17β

- hydroxysteroid oxidoreductase. Additionally, no ef fect was observed in Pb

36 

### treated rats on serum glutamate pyruvate dehydrogenase or ALP activities in males or 37 

### females. Histological observations in both male and female rats demonstrated fatty 38 

### degeneration, vacuolization, and pycnotic nuclei, indicating gen eral hepatotoxicity 39February 2012 5

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### following Pb treatment. In a similar study, Teijon et al. ( 2006 ) exposed Wistar rats to 200 1 

### or 400 ppm throughout gestation, lactation, and 3 months postweaning, or only 1 mont h 2 

### postweaning. In the animals exposed continuously throughout gestation and lactation, the 3 

### concentrations of Pb in the liver were elevated in the 200

- and 400

- ppm groups 1 and 3 4 

### months postweaning. Liver concentrations of Pb were greater in the 200 ppm ani mals 5 

### compared to the 400 ppm animals at one month postweaning (mean [SE]: 1.19 μ g Pb/g 6 

### tissue [0.30] versus 0.76 [0.06], respectively), but were similar between the 2 dosing 7 

### regimens at 3 months postweaning (mean [SE]: 0.054 [0.06] versus 0.55 [0.07], 8 

### resp ectively). ALP activity was increased at 2 weeks postweaning in animals 9 

### continuously exposed to Pb throughout gestation and lactation, whereas ALT activity was 10 

### decreased only at 2 and 3 months postweaning. In animals exposed only for 1 month 11 

### postweaning, s erum ALP activity was significantly increased, although not in a 12 

### concentration

- dependent manner. ALT and AST activities were not affected. 13 

### Cheng et al. ( 2006 ) studied the mechanism of Pb effects on bacterial lipopolysaccharide 14 

### (LPS)

- induced TNF

- α expression. A/J mice were injected via i.p with 100 μ mol/kg Pb, 15 

### with or without 5 mg /kg LPS. Pb alone did not affect liver function (measured as AST or 16 

### ALT activity) or the level of TNF

- α in the serum. In comparison, treatment of mice with 17 

### low doses of Pb and LPS together caused a statistically significant increase in TNF

- α 18 

### induction as well as enhanced liver injury, suggesting that Pb potentiated LPS

- in duced 19 

### inflammation. In an in vitro study, the authors reported that co

- exposure of Pb and LPS 20 

### stimulated the phosphorylation of p42/44 mitogen

- activated protein kinase (MAPK) and 21 

### increased TNF

- α expression in mouse whole blood cells, peritoneal macrophages , and 22 

### RAW264.7 cells (a macrophage cell line) and concluded that monocytes/macrophages 23 

### (rather than hepatocytes) were primarily responsible for Pb increasing LPS

- induced 24 

### TNF

- α levels via the protein kinase C (PKC)/MAPK pathway. Similarly, Pb chloride 25 

### pote ntiated bovine serum albumin (BSA)

- induced inflammation in the livers of mice 26 

### subcutaneously injected with Pb ( Sá et al., In Press )

. 27



### Lipid Metabolism In a lipid metabolism study, Ademuyiwa et al. ( 2009 ) reported that male albino Sprague 28 

### Dawley rats exposed to 200, 300 and 400 ppm Pb in drinking water had mean (SD) blood 29 

### Pb levels of 40.63 (9.21), 61.44 (4.63), and 39.00 (7.90) μ g/dL, respectively. Animals 30 

### exposed to 200 ppm Pb had mean (SD) liver Pb concentrations of 10.04 (1.14) μ g/g, 31 

### compared to 3.24 (1.19) and 2.41 (0.31) in animals exposed to 300 or 400 ppm Pb, 32 

### respectively. Animals exposed to Pb exhibited increased hepatic cholesterogenesis at all 33 

### doses tested compared to controls. Additionally, a decrease in triglyceride was observed 34 

### at 300 and 400 ppm Pb; a decrease in phospholipid levels was observed at 400 p pm Pb. 35 

### The authors also reported positive correlations between tissue cholesterol and 36February 2012 5

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### phospholipids and Pb accumulation in liver across all doses. In contrast, the association 1 

### between tissue triglyceride levels and Pb accumulation was negative. In related studies, 2 

### Khotimchenko and Kolenchenko ( 2007 ) reported that adult male albino rats treated with 3 

### 100 mg /kg Pb

- acetate for as little as 14 days (blood Pb not reported) exhibited disorders 4 

### in lipid metabolism that were supported by increased levels of total cholesterol and 5 

### triglyceride levels in the liver tissue, whereas Sharma et al. ( 2010a ) reported increased 6 

### liver cholesterol in mice gavaged with 50 mg /kg Pb nitrate for 40 days. Pill ai et al. ( 2009 ) 7 

### observed decreases in total liver cholesterol in PND56 male and female rats that had been 8 

### exposed to 50 μ g/kg Pb

- acetate continuously throughout gestation and lactation. These 9 

### results suggest that induction of cholesterogenesis and phospholipidosis in the liver by Pb 10 

### may cause subtle effects at the cellu lar level that may lead to hepatotoxicity. Kojima and 11 

### Degawa ( 2006 ) examined the sex

- related differences in the hepatic sterol regulatory 12 

### element binding protein

- 2 (SREBP

- 2) and 3

- hydroxy

- 3

- methylglutary l

- CoA reductase 13 

### (HMGR) gene expressions in male and female Sprague Dawley rats injected with 14 

### 100 μ mol/kg body weight of Pb nitrate intravenously (blood Pb not reported). The 15 

### SREBP

- 2 expression, which is a transcription factor for the HMGR gene, was 16 

### signifi cantly increased in males and females with the increase occurring earlier in male 17 

### rats (6

- 12 hours, compared to 24

- 36 hours in females). In contrast, expression of the 18 

### HMGR gene, a rate limiting enzyme in cholesterol biosynthesis, was significantly 19 

### increas ed in both Pb

- exposed males and females at earlier time frames (3

- 48 hours in 20 

### males; 12

- 48 hours in females) compared to the SREBP

- 2 gene expression. Significant 21 

### increases in total liver cholesterol were also observed in Pb

- exposed males and females at 22 

### 3

- 4 8 and 24

- 48 hours, respectively. These results suggest that the SREBP

- 2 and HMGR 23 

### gene expressions and increase in total cholesterol levels in the liver in response to Pb 24 

### occur earlier in males compared to females and also suggest that the HMGR gene 25 

### express ion and increase in total cholesterol levels in the liver occur before an increase in 26 

### the SREBP

- 2 gene expression in either sex. 27



### Hepatic Oxidative Stress A number of studies pertaining to hepatic oxidative stress as a result of exposure to 28 

### various Pb comp ounds were identified. Adegbesan and Adenuga ( 2007 ) reported that 29 

### protein undernourished male Wistar rats injected with 100 μ mol/kg Pb nitrate (blood Pb 30 

### not reported) exhibited increased lipid peroxidation, increased CAT activi ty, decreased 31 

### SOD activity, and increased GSH levels, compared to undernourished rats not exposed to 32 

### Pb. Increased lipid peroxidation and decreased CAT and SOD activity were also 33 

### observed when comparing undernourished Pb

- exposed rats to well

- nourished cont rol rats. 34 

### Study authors concluded that malnutrition exacerbated Pb exposure effects on liver lipid 35 

### peroxidation and the involvement of free radicals in Pb toxicity. Male Foster rats treated 36February 2012 5

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### with 0.025 mg /kg Pb via i.p. injection (blood Pb not reported) als o exhibited statistically 1 

### significant increases in lipid peroxidation levels and decreases in SOD, CAT, and 2 

### glucose

- 6

- phosphatase dehydrogenase (G6PD) levels in liver mitochondrial and 3 

### postmitochondrial fractions ( Pandya et al., 2010 )

. Statistically nonsignificant decreases 4 

### were also observed in GSH levels and GPx and GR activities in Pb

- treated animals. In 5 

### mice gavaged with 50 mg /kg Pb nitrate for 40 days, lipid peroxidation was increased and 6 

### SOD, CAT, and GSH were decreased compared to controls ( Sharma et al., 2010a )

. 7 

### Add i tionally, exposure to Pb nitrate resulted in histopathological changes in the structure 8 

### of the liver: hepato cytes were damaged and were marked by cytoplasmic vacuolization 9 

### and pycnotic nuclei. Yu et al. ( 2008 ) reported similar concentration

- dependent increases 10 

### in lipid peroxide levels and decreases in GSH levels and CAT, SOD and GPx activities in 11 

### castrated boars that received a supplemental diet with 0, 5, 10, or 20 mg /kg Pb. The level 12 

### of hepatic CuZnSOD mRNA was also reduced in Pb

- treated animals. The study authors 13 

### suggested that this decrease in SO D mRNA expression and activity of antioxidant 14 

### enzymes may lead to a reduction free radical scavenging capability, along with increased 15 

### lipid peroxidation, potentially causing serious damage to hepatic function and structure. 16 

### Khotimchenko and Kolinchenko ( 2007 ) also reported an increase in lipid peroxidation 17 

### and development of hepatitis in male albino rat liver parenchyma following treatment 18 

### with 100 mg /kg Pb

- acetate for as little as 14 d ays. Lipid peroxidation was demonstrated 19 

### by increases in malondialdehyde (MDA) levels along with decreases in GSH and thiol 20 

### groups indicating injury in the liver antioxidant system. In another experiment, Jurczuk et 21 

### al. ( 2007 ) reported that male Wistar rats treated with 500 mg /L Pb in drinking water 22 

### (blood Pb not reported) exhibited decreases in liver vitamin E and GSH levels along with 23 

### an increase in lipid pe roxidation. The study authors hypothesized that vitamin E is 24 

### involved in the mechanism of peroxidative action of Pb in the liver, and concluded that 25 

### the suggested protective role of vitamin E in the potential toxicity by Pb may be related to 26 

### scavenging of free radicals that are generated either directly or indirectly by Pb. In a 27 

### study examining the role of low molecular weight thiols on peroxidative mechanisms, 28 

### Jurczuk et al. ( 2006 ) stated that male Wistar rats treated with 500 mg /L Pb

- acetate in 29 

### drinking water exhibited a decrease in blood ALAD as well as decreases in GSH and 30 

### nonprotein sulfhydryl (NPSH) levels in the liver. Metallthionein levels were also reported 31 

### to be higher in the liver following exposure to Pb. Levels of hepatic lipid peroxidation 32 

### were observed to be significantly increased in rats treated with 35 mg /kg Pb via i.p. 33 

### injection (blood Pb not reported), whereas hepatic GSH was significantly decreased 34 

### ( Upadhyay et al., 2009 )

. In vitro exposure of human embryonic hep atocytes (WRL

- 68) to 35 

### 5 μ M Pb

- acetate for 30 days resulted in increase production of reactive oxygen species 36 

### (ROS) throughout the incubation period ( Hernández

- Franco et al., 2011 )

. Concurrent 37 

### with this increase in ROS generation, the activities of SOD and the levels of membrane 38February 2012 5

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### lipid peroxidative damage also increased throughout the first 24 days of exposure, but 1 

### returned to normal levels by the end of the incubation period. 2 

### In a study examining the effects of Pb e xposure to fetuses, Massó et al. ( 2007 ) exposed 3 

### pregnant Wistar rats with 300 mg /L Pb in drinking water starting at day 1 of pregnancy to 4 

### parturition or until weaning to determi ne the effects of Pb exposure in the fetal liver. 5 

### Blood Pb levels were higher at parturition (mean [SD]: 31.5 [0.80] μ g/dL) than at 6 

### weaning (mean [SD]: 22.8 [0.50] μ g/dL). Pups exhibited liver damage that was 7 

### accompanied by an increase in thiobarbituric ac id

- reactive species (TBARS) production 8 

### and increased CAT activity compared to controls. In addition, increased ALP and acid 9 

### phosphatase activity was also observed. Uzbekov et al. ( 2007 ) exposed female Wistar rats 10 

### to 0.3 and 3.0 mg /L Pb nitrate for 1 and 5 months prior to, and continuing during 11 

### pregnancy, and measured fetal hepatic SOD activity on GD20. Control rats had a mean 12 

### (SD) blood Pb level of 16.1 (0 .63) μ g/dL, whereas rats exposed to 0.3 and 3.0 mg /L Pb 13 

### had mean blood Pb levels that were 26.5% (20.4 μ g/dL) and 51.8% (24.4 μ g/dL) higher, 14 

### respectively. In the fetuses from dams exposed for 1 month prior to pregnancy, a 15 

### concentration

- dependent increase i n liver SOD activity was observed, whereas SOD 16 

### activity was decreased in the fetuses from dams exposed for 5 months prior to pregnancy. 17 

### The increase in SOD activity in the livers of fetuses from dams exposed to 0.3 or 18 

### 3.0 mg /L Pb nitrate for one month sugg ests that activation of SOD in response to 19 

### increased free radical production, while the decrease in SOD production in fetal livers 20 

### from dams exposed to the same concentrations for 5 months suggests that longer 21 

### durations of Pb exposure impairs the antioxida nt defense mechanism. No effects on GSH 22 

### or MDA levels were observed in PND56 male and female rats following continuous 23 

### exposure to 50 μ g/kg Pb

- acetate throughout gestation and lactation (blood Pb not 24 

### reported) ( Pillai et al., 2009 )

. 25 

### The studies presented above all support the possible oxidative stress impacts following 26



### ex posure to various doses of Pb administered in various forms and the potential for 27 

### hepatotoxicity as a result of oxidative stress. 28



### Hepatic Apoptosis

Fan et al. ( 2009b ) reported that a sing le i.v. injection (tail vein) of 200 μ mol/kg Pb nitrate 29 

### resulted in an increase in the expression of ferritin light

- chain (FLT) in rats (mean [SD]: 30 

### 3.5 [1.0]

- fold increase) over that in controls. Immunohistochemical analysis revealed that 31 

### hepatocytes aroun d the central vein were heavily stained by anti

- FTL antibodies, as were 32 

### nonparenchymal cells identified as Kupffer cells. The authors hypothesized that the 33 

### expression of FTL in Kupffer cells may be the result of phagocytosis of apoptotic cells: 34 

### in Pb

- treat ed rats, apoptotic hepatocytes represented a mean (SD) 2.5 (1.4)% of total cells 35February 2012 5

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### whereas only a mean (SD) 0.31 (0.31)% of hepatocytes were apopto t i c in control 1 

### animals. FTL expression in Kupffer cells was not increased in rats treated with clofibrate, 2 

### whic h induced hepatocellular proliferation, but not apoptosis. 3



### 5.9.2 Effects on the Gastrointestinal System
Gastrointestinal effects of Pb exposure resulting in blood Pb levels ranging from 30 up to 4 

### 80 μ g/dL in humans primarily include abdominal pain, constipation, and internal 5 

### paralysis. In animals, degeneration of the intestinal epithelial mucosa and a decrease in 6 

### duodenal motility has been reported following Pb exposure. 7



### 5.9.2.1 Summary of Key Findings on the Effects on the Gastrointestinal System from the 2006 Lead AQCD
The 2006 Pb AQCD stated that a number of factors influence the gastrointestinal 8 

### absorption of Pb including the chemical and physical form of Pb, the age at Pb intake, as 9 

### well as various nutritional factors. Potential malabsorption of Pb as a result of 10 

### deg eneration of the intestinal epithelial mucosa has been observed in rats exposed to Pb. 11 

### In suckling rat pups, casein micelles incidences were reported as a result of Pb present in 12 

### bovine and rat milk and in infant milk formula. Pb ingestion through water wa s more 13 

### toxic compared to Pb ingestion via milk. Pb ingested in milk was reported to be taken up 14 

### by the ileal tissue, whereas Pb administered intragastrically as a soluble salt was 15 

### primarily accumulated in the duodenum irrespective of vehicle used for admin istration. 16 

### Decreases in duodenal motility and the amplitude of contractility in the intestinal tract 17 

### were observed in rats following Pb exposure. Nutritional studies examining different 18 

### dietary levels of Pb in rats, calcium, and vitamin D indicated competi tion in absorption 19 

### between Pb and calcium. Dietary supplement with vitamin D led to an increase in 20 

### intestinal absorption of Pb and calcium. In instances where severe calcium deficiency 21 

### was noted, ingestion of Pb caused a clear decrease in 1,25

- dihydroxy vi tamin D (1,25

- 22 

### (OH)2D3) levels. Overall, the 2006 Pb AQCD stated that studies in rat intestine have 23 

### shown that the largest amount of Pb absorption occurs in the duodenum with the 24 

### mechanisms of absorption involving active transport and diffusion via the inte stinal 25 

### epithelial cells. Absorption has been reported to occur, through both saturable and 26 

### nonsaturable pathways based on results from various animal studies. The 2006 Pb AQCD 27 

### reported evidence that symptoms associated with gastrointestinal colic (abdomina l pain, 28 

### constipation, intestinal paralysis) were prevalent in occupationally exposed adults with 29 

### blood Pb levels ≥ 50 μ g/dL. 30February 2012 5

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### 5.9.2.2 New Epidemiologic Studies
The 2006 Pb AQCD reported that in humans, gastrointestinal effects generally include 1 

### abdominal pain, con stipation, and internal paralysis. Kuruvilla et al. ( 2006 ) reported 2 

### gastrointestinal effects including stomach pain and gastritis along with other Pb

- related 3 

### clinical manifestations in painters (mean [SD] blood Pb: 8.04 [5.04] μ g/dL) 4 

### occupationally exposed to Pb in India. Case reports involving children and individuals 5 

### occupationally exposed to Pb with very high blood Pb levels (> 15 μ g/dL) were 6 

### consistent with these observations of GI pain and gastritis ( Fonte et al., 2007 )

. 7



### 5.9.2.3 New Toxicological Studies
A few new studies pertaining to gastrointestinal effects of Pb exposure were identified. 8 

### Santos et al. ( 2006b ) examined the impact of Pb exposure on nonadr energic 9 

### noncholinergic (NANC) relaxations in rat gastric fundus. Male Wistar rats treated with 10 

### 0.008% Pb

- acetate (80 ppm ) via drinking water for 15, 30, and 120 days (blood Pb not 11 

### reported) exhibited a significant difference in NANC relaxations in the gast ric fundus 12 

### following electrical field stimulus (EFS). While frequency

- dependent relaxations were 13 

### observed in all groups, including the control group, the relaxations were significantly 14 

### inhibited in rats treated with Pb

- acetate for all three durations. When gastric fundus strips 15 

### from rats were incubated with L

- nitroarginine (L

- NOARG), a nitric oxide synthase 16 

### (NOS) inhibiter, no additional inhibition in relaxations was observed. In contrast, 17 

### incubation with sodium nitroprusside and 8

- Br

- GMPc (a Cyclic guanosi ne 18 

### monophosphate [cGMP] analog), resulted in a concentration

- dependent relaxation in 19 

### strips in the control group and group exposed to Pb

- acetate for 120 days. Study authors 20 

### concluded that chronic exposure to Pb causes inhibition in NANC relaxation probably 21 

### due to the modulated release of NO from the NANC nerves or due to interaction with the 22 

### intracellular transducer mechanism in the rat gastric fundus. 23 

### In another study examining Pb

- induced oxidative stress in the gastric mucosa, Olaleye et 24 

### al. ( 2007 ) treated Albino Wistar rats with 100 or 5,000 mg /L of Pb

- acetate for 15 weeks 25 

### (blood Pb not reported). Exposure to Pb

- acetate caused a significant increase in gastric 26 

### mucosal damage caused by pretreatment with acidified ethanol. Study authors reported 27 

### that though the basal gastric acid secretory rate was not altered, s tomach response to 28 

### histamine was significantly higher in animals treated with Pb

- acetate compared to that in 29 

### the controls. Additionally, there was a significant increase in gastric lipid peroxidation at 30 

### both the 100 and 5,000 mg /L dose levels. In contrast, CAT, and SOD activities and nitrite 31 

### levels were significantly decreased in the gastric mucosa. Study authors concluded that 32February 2012 5

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### exposure to Pb may increase the formation of gastric ulcers as a result of changes in the 1 

### oxidative metabolism in the stomach. 2



### 5.9.3 Effe cts on the Endocrine System
Endocrine processes that are most commonly found to be impacted by Pb exposure 3 

### include changes in the thyroid, such as changes in the thyroid stimulating hormone 4 

### (TSH), triiodothyronine (T3), and thyroxine (T4). 5



### 5.9.3.1 Summary of Key Findings of the Effects on the Endocrine System from the 2006 Lead AQCD

The 2006 Pb AQCD reported that endocrine processes impacted by occupational Pb 6 

### exposure include thyroid hormone levels, changes in male sex hormone levels, as well as 7 

### changes in the pr oduction of 1,25

- (OH)2D3 levels. However, these effects were reported 8 

### to be observed only with blood Pb levels exceeding 30

– 40 μ g/dL. In addition, alterations 9 

### in calcitropic hormones were affected in children with blood Pb levels ranging from 10

- 10 

### 120 μ g/dL. A summary of key findings pertaining to reproductive hormones in males and 11 

### females in the current document is presented in the section on reproductive and 12 

### developmental effects ( Sections 5.8.1 and 5.8.2 ). 13



### 5.9.3.2 New Epidemiologic Studies
Recent epidemiologic studies have reported associations betwee n indicators of exposure 14 

### to Pb and thyroid hormone levels. In workers highly exposed to Pb (blood Pb = 15 

### 71.1 μ g/dL) thyroid stimulating hormone (TSH) and free T4, but not free T3, were 16 

### increased over levels in controls (mean blood Pb level of 0.2 μ g/dL), al though these 17 

### results are most likely not generalizable to the general public due to the high blood Pb 18 

### levels of exposed workers ( Pekci ci et al., 2010 )

. Abdelouahab et al. ( 2008 ) performed a 19



### cross
- sectional study in a Can adian population characterized by high consumption of 20 

### freshwater fish. The median concurrent blood Pb level was 3.1 μ g/dL for men and 21 

### 1.7 μ g/dL for women. It is important to note that the median blood Pb level for women 22 

### was lower than the limit of detectio n for Pb in the blood (2.1 μ g/dL), effectively meaning 23 

### that greater than 50% of women in the study had nondetectable levels of Pb in their 24 

### blood. The study authors conducted a stratified analysis and concluded that TSH levels 25 

### were negatively correlated wit h blood Pb in women who consumed fish contaminated 26 

### with Pb and other environmental pollutants. No associations T3 and T4 levels were 27February 2012 5

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### reported in women. TSH, T3 and T4 levels were not observed to be correlated with blood 1 

### Pb in males. However, study authors stated that occupational exposure to Pb in men can 2 

### affect pituitary thyroid axis homeostasis and the relation between low

- level Pb exposure 3 

### thyroid hormone homeostasis in men and women needs to be investigated further. The 4 

### authors also concluded that envir onmental contaminants not investigated ( e.g., As) may 5 

### be influencing TSH levels. Dundar et al. ( 2006 ) examined associations of blood Pb with 6 

### thyroid func tion in 42 male adolescent auto repair workers exposed long term to Pb. A 7 

### control group comprising 55 healthy subjects was also used for comparison purposes. 8 

### Mean blood Pb levels were reported to be higher in the auto repair workers compared to 9 

### the control subjects (mean [SD]: 7.3 [2.92] versus 2.08 [1.24] μ g/dL). Free T4 (FT4) 10 

### levels were significantly lower in the study group compared to the control group, which 11 

### had no abnormal FT4 levels reported. In contrast, free T3 (FT3) and TSH levels were 12 

### comparable between the study and control group. Blood Pb level was reported to be 13 

### negatively correlated with FT4 levels. Based on the study outcome, the study authors 14 

### reported that long

- term Pb exposures that result in the studied blood Pb levels may lead 15 

### to lower F T4 levels without impact on T3 and TSH levels in adolescents. The study 16 

### authors stated that this effect is likely secondary to the toxic effects of Pb on the pituitary

17 

### thyroid axis and to the earlier findings of primary hypothyroidism as a result of impair ed 18 

### production of peripheral thyroid hormones. Similar findings were reported by Croes et al. 19 

### ( 2009 ) in a study conducted in Belgium. Croes et al. ( 2009 ) examined the hormone levels 20 

### in 1 , 679 adolescents residing in nine s tudy areas with varying exposures to multiple 21 

### industrial pollutants including Pb. The median concurrent blood Pb of the participants 22 

### from the nine different regions ranged from 1.6 to 2.8 μ g/dL. The study authors reported 23 

### that, after adjustment for potenti al confounding, significant interregional differences were 24 

### observed FT3 hormone levels. When individual neighborhoods were analyzed within the 25 

### larger study areas, altered levels of FT3 levels were also observed. Though varying levels 26 

### of FT3 levels were obs erved, the study authors reported that these changes were not 27 

### wholly due to exposure to various pollutants, including Pb that were measured in the 28 

### study and stated that other pollutants and environmental factors may also have 29 

### contributed to the effects not ed. In a prospective study of 309 mother

- child pairs from 30 

### Yugoslavia, the relationship between maternal TSH and T4 and blood Pb was 31 

### investigated in those living in a highly exposed town with a smelter and battery plant (n = 32 

### 156 mother

- child pairs) and thos e living in a relatively unexposed town (n = 153 mother

33 

### child pairs) ( Lamb et al., 2008 )

. The mid

- pregnancy blood Pb levels were highly elevated 34 

### in the industrial town compared to the unexposed town (mean [SD]: 20.5 6 [7.38] versus 35 

### 5.60 [1.99] μ g/dL). Mid

- pregnancy maternal free T4 levels were observed to be inversely 36 

### related with maternal blood Pb levels, but this association was not observed in the 37 

### unexposed town. In 24 newborns delivered in Tokyo, Japan, neither TS H nor free T4 38February 2012 5

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### (sampled 4

- 6 days postpartum) was associated with cord blood Pb sampled at delivery 1 

### (mean: 0.67 μ g/dL) ( Iijima et al., 2007 )

. 2 

### Gump et al. ( 2008 ) examined cortisol response to acute stress in children (aged 9.5 years) 3 

### whose prenatal and postnatal blood Pb levels had been determined prior to the study at 4 

### birth (from cord blood) and at a mean (SD) age of 2.62 (1.2) years, r espectively. For 5 

### prenatal blood Pb, the children were divided into the following quartiles: ≤ 1, 1.1

- 1.4, 6 

### 1.5

- 1.9, and 2.0

- 6.3 μ g/dL. For postnatal blood Pb, the quartiles were 1.5

- 2.8, 2.9

- 4.1, 7 

### 4.2

- 5.4, and 5.5

- 13.1 μ g/dL. The study authors reported that blood Pb was not associated 8 

### with initial salivary cortisol levels. However following an acute stressor, which 9 

### comprised submerging the dominant arm for a minute in a gallon of one part ice to one 10 

### part water, increasing prenatal and postnatal blood Pb level s were statistically 11 

### significantly associated with increases in salivary cortisol responses. Children in the 2nd, 12 

### 3rd and 4th prenatal blood Pb quartiles and in the 4 th postnatal quartile had increased 13 

### salivary cortisol responses compared to children in th e 1 st quartile. When blood Pb was 14 

### treated as a continuous variable, regression analysis showed that both prenatal and 15 

### postnatal blood Pb levels were significantly correlated to salivary cortisol reactivity. 16 

### Based on these results, the study authors reporte d that relatively low prenatal and 17 

### postnatal blood Pb levels, notably those well below 10 μ g/dL, can alter adrenocortical 18 

### responses of children following acute stress and the health impact and behavioral aspects 19 

### of this Pb

- induced HPA deregulation in child ren needs to be further examined. 20 

### In another study on the impact of Pb in children, Kemp et al. ( 2007 ) examined the blood 21 

### Pb levels in 142 young, U.S. urban African

- American and Hispanic children in winter 22 

### and summer to determine the seasonal increase in blood Pb and its association with 23 

### vitamin D (1,25

- (OH)2D3), age and race. There was a winter/summer (W/S) increase in 24 

### blood Pb levels in children aged b etween 1 and 3 years (mean [SE]: 4.94 [0.45] μ g/dL in 25 

### winter, 6.54 [0.82] μ g/dL in summer), with a smaller W/S increase observed in children 26 

### aged between 4 and 8 years (mean [SE]: 3.68 [0.31] μ g/dL in winter, 4.16 [0.36] μ g/dL 27 

### in summer). Additionally, the winter and summer blood Pb levels were highly correlated 28 

### with one another. The percentage of African

- American children with blood Pb levels ≥ 29 

### 10 μ g/dL increased from 12.2% in winter to 22.5% in summer. In children aged 4

- 8 30 

### years, the concentrations of 1,2 5

- (OH)2D3 were greater in the summer compared to the 31 

### winter (mean [SE]: 33.8 [1.1] μ g/L in summer versus 25.3 [1.2] μ g/L in winter). No 32 

### difference in seasonal 1,25

- (OH)2D3 was observed in children 1

- 3 years old. 33 

### Additionally, winter and summer concentratio ns of 1,25

- (OH)2D3 were highly correlated 34 

### (r = 0.635, p < 0.0001). There was a significant correlation between seasonal differences 35 

### in blood Pb and serum 1,25

- (OH)2D3 in all children and African

- American children 36 

### between 4 and 8 years. Based on these resul ts, the study authors concluded that higher 37 

### summertime increase in serum 1,25

- (OH)2D3 levels in children between 4 and 8 years is 38February 2012 5

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### most likely due to increased sunlight

- induced vitamin D synthesis and may be a 1 

### contributing factor to seasonal changes in bloo d Pb levels. 2



### 5.9.3.3 New Toxicological Studies
. In a study examining the effects of Pb and cadmium in adult cows reared in a polluted 3 

### environment in India, Swarup et al. ( 2007 ) stated that the mean plasma T3 and T4 levels 4 

### were significantly higher in cows near Pb and zinc smelters (mean [SD] blood Pb: 86 5 

### [6] μ g/dL) and near closed Pb and operational zinc smelters (mea n [SD] blood Pb: 51 6 

### [9] μ g/dL) when compared to cows in unpolluted areas (mean [SD] blood Pb: 7 7 

### [1] μ g/dL). Regression analyses from 269 cows examined in the study showed a 8 

### significant positive correlation between blood Pb and plasma T3 and T4 levels, wher eas 9 

### the correlation between blood Pb and plasma cortisol was nonsignificant. Mean plasma 10 

### estradiol level was significantly higher in cows near closed Pb and operational zinc 11 

### smelter industries compared to the control group. Based on these results, the stud y 12 

### authors concluded that endocrine profile in animals can be impacted following exposure 13 

### to Pb in polluted environments. 14


### Biswas and Ghosh ( 2006 ) investigated the effect of Pb treatment on adrenal and male 15 

### gonadal functions in Wistar rats treated with 8.0 mg /kg Pb

- acetate via i.p. injection for 21 16 

### days (blood Pb not reported). Pb treatment was observed to significantly increase adrenal 17 

### steroidogen ic enzyme activity and serum corticosterone levels. Accessory sex organ 18 

### (prostate and seminal vesicle) weights were decreased in Pb

- treated animals, whereas 19 

### adrenal weights were increased. Spermatogenesis was decreased and the percent of 20 

### spermatid degenera tion was increased in animals treated with Pb. Lastly, serum 21 

### concentrations of testosterone, FSH, and LH, were decreased in Pb

- treated animals. 22 

### Supplementation with testosterone during the last 14 days of Pb treatment was observed 23 

### to ameliorate these effec ts. 24



### 5.9.4 Effects on Bone and Teeth
Primary effects on bone as a result of Pb exposure have included an increase in 25 

### osteoporosis, increased frequencies of falls and fractures, changes in bone cell function as 26 

### a result of replacement of bone calcium with Pb and depression in early bone growth. 27 

### Similar to bone, calcium in the teeth can be easily substituted by Pb following Pb 28 

### exposure. Exposure of animals to high levels of Pb (30 mg /kg body weight) may result in 29 

### the formation of “Pb line” and Pb can also cause a d ecrease in cell proliferation, 30 

### procollagen type I production, intracellular protein, and osteocalcin in human dental pulp 31February 2012 5

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### cell cultures. Accumulation of Pb was also associated with tooth loss and higher 1 

### incidence of periodontitis. 2



### 5.9.4.1 Summary of Key Findings o f the Effects on Bone and Teeth from the 2006 Lead AQCD
The 2006 Pb AQCD reported many effects on bone and some in teeth in animals and 3 

### following exposure to Pb. Pb easily substituted for calcium in bone and was taken up by 4 

### the bone causing changes in bone cell function. Exposure of animals to Pb during 5 

### gestation and immediate postnatal period was reported to significantly depress early bone 6 

### growth with the effects showing a concentration

- dependent trends,. In mature animals, 7 

### long

- term exposure (up to one y ear) to Pb, along with poor nutrition (low calcium) 8 

### affected bone growth as well bone density. Systemic effects of Pb exposure included 9 

### disruption in bone mineralization during growth, alteration in bone cell differentiation 10 

### and function due to alterations in plasma levels of growth hormones and calcitropic 11 

### hormones such as 1,2

- [OH]2D3 and impact on calcium binding proteins and increases in 12 

### calcium and phosphorus concentrations in the bloodstream. Bone cell cultures exposed to 13 

### Pb had altered vitamin D

- stimu lated production of osteocalcin accompanied by inhibited 14 

### secretion of bone

- related proteins such as osteonectin and collagen. In addition, Pb 15 

### exposure caused suppression in bone cell proliferation most likely due to interference 16 

### from factors such as growth hormone (GH), epidermal growth factor (EGF), transforming 17 

### growth factor

- beta 1 (TGF

- β1), and parathyroid hormone

- related protein (PTHrP). 18 

### As in bone, Pb can easily substitute for calcium in the teeth and is taken and incorporated 19 

### into developing teeth in experimental animals. Since teeth do not undergo remodeling 20 

### like the bone does during growth, most of the Pb in the teeth remains in a state of 21 

### permanent storage. High dose exposure of Pb to animals (30 mg /kg body weight) has 22 

### lead to the formation of a “Pb line” that is visible in both the enamel and dentin and is 23 

### localized in areas of recently formed tooth structure. Areas of mineralization are easily 24 

### evident in the enamel and the dentin within these “Pb lines.” Pb has also been shown to 25 

### decrease cell prol iferation, procollagen type I production, intracellular protein, and 26 

### osteocalcin in human dental pulp cell cultures. Adult rats exposed to Pb have exhibited an 27 

### inhibition of the posteruptive enamel proteinases, delayed teeth eruption times, as well as 28 

### decr ease in microhardness of surface enamel. Pb was reported to be widely dispersed and 29 

### incorporated into developing apatite crystal during enamel formation process; however, 30 

### post formation, Pb was reported to be capable of entering and concentrating in enamel 31 

### areas that were calcium deficient. The 2006 Pb AQCD also reported that a number of 32 

### epidemiologic and animal studies have both separately suggested that Pb is a caries

33 

### promoting element. 34February 2012 5

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### 5.9.4.2 New Toxicological and Epidemiologic Studies
As reported in the 2006 Pb AQCD, Pb appears to be capable of causing effects in bones 1 

### of humans and animals following exposure. The association between blood Pb levels and 2 

### osteoporosis was examined in several epidemiologic studies. Most studies were cross

3 

### sectional in design; thu s, there is uncertainty regarding the magnitude, timing, frequency, 4 

### and duration of Pb exposure that contributed to the observed associations. Campbell and 5 

### Auigner ( 2007 ) examined subjects ≥ 50 years of age using NHANES III for association 6 

### between concurrent blood Pb level and osteoporosis. The study authors used the bone 7 

### mineral density in the hip as the primary outcome in groups comprising non
- Hispanic 8 

### white men (mean blood Pb: 4 .9, range: 0.7 to 48.1 μ g/dL), non
- Hispanic white women 9 

### (mean blood Pb: 3.6, range: 0.7 to 28.7 μ g/dL), African

- American men (mean blood Pb: 10 

### 7.7, range: 0.7 to 52.9 μ g/dL), and African

- American women (mean blood Pb: 4.5, range: 11 

### 0.7 to 23.3 μ g/dL). The resu lts indicated that the adjusted mean total hip bone mineral 12 

### density in the non
- Hispanic white males who had the lowest blood Pb levels (actual 13 

### concentration not reported) was statistically significantly higher than that in the males 14 

### with higher blood Pb le vels. Similar associations, although not statistically significant, 15 

### were reported among white females. Likely due to the small sample size, similar results 16 

### were not observed among African

- American men and women. No association was 17 

### observed between blood Pb and osteoporotic fractures in any sex/race group. Since the 18 

### NHANES study comprised a cross

- sectional design, no inferences could be made 19 

### regarding the temporal sequence of the observed association. The study authors 20 

### concluded that further inquiry was need ed to study the possible causal association 21 

### between Pb exposure and osteoporosis. In a similar study, Sun et al. ( 2008b ) examined 22 

### the association between concurrent blood Pb levels and osteoporosis in 155 males and 37 23 

### females in China occupationally

- exposed to Pb (mean blood Pb: 20.22 and 15.5 μ g/dL, 24 

### respectively). Bone mineral density was reported to be statistically si gnificantly lower in 25 

### exposed females compared to exposed males. When all participants (including 36 male 26 

### and 21 female unexposed controls) were divided into groups according to blood Pb and 27 

### urinary Pb levels, the study authors reported that there were sign ificant decreases in bone 28 

### mineral density in groups that had high urinary Pb levels (≥ 5 μ g/g creatinine) compared 29 

### to groups with low urinary Pb in both sexes. In contrast, a significant difference was 30 

### observed between blood Pb and bone mineral density onl y in males with blood Pb 31 

### >30 μ g/dL. Prevalence of osteoporosis was reported to increase significantly with 32 

### increasing blood Pb in a linear manner. Khalil et al. ( 2008 ) reported similar associations 33 

### between blood Pb level and osteoporosis in older women. The stu dy authors conducted a 34 

### prospective study using 533 women aged 65

- 87 years with a mean (SD) blood Pb of 5.3 35 

### (2.3) μ g/dL to determine the association between blood Pb and recurring fractures. 36 

### Analysis of bone mineral density was conducted in 1986

- 1990 and 19 93

- 1994, while 37February 2012 5

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### blood Pb was measured from 1990

- 1991. The bone mineral density was 7% lower in the 1 

### total hip (p <0.02) and 5% lower in the femoral neck (p <0.03) in the highest blood Pb 2 

### group (≥ 8 μ g/dL) compared to the lowest blood Pb group (≤ 3 μ g/dL). Th e trend across 3 

### all dose groups was also observed to be statistically significant for hip and femoral neck 4 

### bone mineral density. In addition, hip, femoral neck, and calcaneus bone loss was 5 

### observed to be greater in the medium (blood Pb: 4

- 7 μ g/dL) and high Pb groups 6 

### compared to the low Pb group, but the observed trend was only significant for calcaneus 7 

### bone loss. Multivariate analysis indicated that women with high blood Pb levels had an 8 

### increased risk of non
- spine fracture and women with medium or high bloo d Pb levels had 9 

### a higher risk of falls compared to the low blood Pb level group. Based on these results, 10 

### the study authors concluded that blood Pb is associated with an increased risk of falls and 11 

### fractures leading to osteoporosis

- related fractures. 12



### To ex amine the association between biomarkers of joint tissue metabolism and blood Pb 13 

### levels, Nelson et al. ( In Press ) performed a cross

- sectional analysis of 329 male and 342 14 

### female participants in the Johnson County Osteoarthritis Project Metals Exposure Sub

15 

### study. In women (mean age = 62 years), the median concurrent blood Pb level was 16 

### 1.9 μ g/dL (range: 0.5

- 25.4 μ g/dL). Blood Pb levels were higher in African

- American 17 

### compared to Caucasian women. Unadjusted cor relation analyses demonstrated significant 18 

### positive correlations between blood Pb and uNTX

- I (a marker of bone 19 

### resorption/turnover), uCTX

- II (a marker associated with the progression of radiographic 20 

### knee and hip osteoarthritis), and COMP (a cartilage bioma rker related to osteroarthritis); 21 

### however, significant associations only remained for uNTX

- I and uCTX

- II after adjusting 22 

### for age, BMI, race, and smoking status. In men (median age = 65), the median blood Pb 23 

### level was 2.2 μ g/dL (range: 0.5

- 25.1 μ g/dL). As w ith women, African

- American men 24 

### had higher blood Pb levels than did Caucasian men. In unadjusted correlation analyses, 25 

### blood Pb levels in men were positively associated with uCTX

- II, COMP, and C2C:CPII 26 

### ratio (an indication of the balance between cartilage collagen degradation and synthesis), 27 

### and negatively associated with CPII (a marker of collagen synthesis). After controlling 28 

### for age, BMI, race, and smoking, only the positive association between blood Pb and 29 

### COMP remained borderline statistically signific ant in men. The authors concluded that 30 

### blood Pb is associated with bone turnover and mineralized cartilage turnover in women 31 

### and non
- mineralized cartilage turnover in men; however, as this study was cross

- sectional 32 

### in nature, it is impossible to conclude w hether increased cartilage turnover is a product of 33 

### increased blood Pb, or whether cartilage turnover itself results in increased Pb. Similarly, 34 

### Machida et al. ( 2009 ) investigated bone matrix turnover rates in Japanese women related 35 

### to menopause status and blood Pb . Perimenopausal women (n = 319) were observed to 36 

### have significantly higher geometric mean blood Pb (2.0 μ g/dL), than did premenopausal 37 

### women (n = 261, blood Pb = 1.6 μ g/dL), younger postmenopausal women (n = 397, 38 

### blood Pb = 1.8 μ g/dL), or older postmenopa usal women (n = 248, blood Pb = 1.7 μ g/dL). 39February 2012 5

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### In all subjects and perimenopausal women, markers of bone matrix turnover (osteocalcin 1 

### [OC], bone

- specific alkaline phosphatase [BALP], and N

- telopeptide cross

- linked 2 

### collagen type I [NTx]) were significantly pos itively associated with blood Pb in 3 

### unadjusted correlation analyses. In multivariate regression models controlling for bone

4 

### mineral density, NTx, and age, OC was additionally statistically positively associated 5 

### with blood Pb in all subjects and perimenopau sal women. Bone mineral density and NTx 6 

### were also positively associated with blood Pb in these models for all subjects. As with 7 

### Nelson et al. ( In Press ) , the cross

- sectional nature of this study precludes a determination 8 

### whether higher blood Pb is cause of increased bone matrix turnover biomarkers, or a 9 

### consequence of increased bone turn over. 10 

### To understand the importance of bone as a target tissue of Pb toxicity, Jang et al. ( 2008 ) 11 

### studied the effect of Pb on calcium release activated calcium influx (CRACI) using 12 

### primary cultures of human osteoblast

- like cells (OLC). When cells were incubated with 13 

### 1 , 000 or 3 , 000 μ M Pb, a concentration

- dependent impact on the CRACI was observed, as 14



### was a conc entration

- dependent increase in the influx of Pb into human OLC. These 15 

### results suggest that Pb interferes with CRACI in human OLCs by initiating the CRACI 16 

### ( i.e., the measurable influx of calcium upon re

- addition of calcium is partially inhibited 17 

### by Pb) and the influx of Pb is enhanced after CRACI is induced. Since studies have found 18 

### associations between higher blood Pb level and reduced skeletal growth in children, 19 

### Zuscik et al. ( 2007 ) conducted a study using murine limb bud mesenchymal cells (MSCs) 20 

### to test the hypothesis that Pb alters chondrogenic commi tment of mesenchymal cells and 21 

### also to assess the effects of Pb on various signaling pathways. Exposure to 1 μ M Pb 22 

### caused increased basal and TGF

- β/BMP induction of chondrogenesis in MSCs which was 23 

### supported by nodule formation and upregulation of Sox

- 9, t ype 2 collagen, and aggrecan 24 

### which are all key markers of chondrogenesis. The study authors also observed enhanced 25 

### chondrogenesis during ectopic bone formation in mice that had been pre

- exposed to Pb in 26 

### drinking water (55 or 233 ppm , corresponding to 14 or 40 μ g/dL blood Pb). MSCs 27 

### exposed to Pb exhibited an increase in TGF

- β, but BMP

- 2 signaling was inhibited. Pb 28 

### was also reported to induce NF

- κB and inhibit AP

- 1 signaling. Based on these results, the 29 

### study authors concluded that chondrogenesis following ex posure to Pb most likely 30 

### involved modulation and integration of multiple signaling pathways including TGF

- β, 31 

### BMP, AP

- 1, and NF

- κB. 32 

### Effects of Pb exposure on teeth were examined in a few epidemiologic studies. Since 33 

### individuals may be impacted by the releas e of Pb stored in their skeletal compartments, 34 

### Arora et al. ( 2009 ) examined the association between bone Pb concentrations and loss of 35 

### natural teeth in 333 male participants of the NAS. Tooth loss in men was categorized as 36 

### 0, 1

- 8 or ≥ 9. Individuals with ≥ 9 teeth missing had significantly higher tibia and patella 37 

### Pb concentrations (measured within 3 years of dental assessment) compared to those with 38February 2012 5

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### no tooth loss; no significant difference in blood Pb levels (measured within 3 years of 1 

### dental assessment [ ( Hu et al., 1996a ) ] ) was observed between the categories of teeth loss. 2 

### Following adjustment for age, education, smoking status, pack

- years of smoking, and 3 

### diabetes, m en with the highest tibia Pb concentrations (>23 μ g/g) had higher odds of 4 

### tooth loss (OR: 3.03 [95% CI: 1.60, 5.75]) compared to men with tibia Pb ≤ 15 μ g/g. 5 

### Men with the highest patellar Pb (>36 μ g/g) also had higher odds of tooth loss (OR: 2.41 6 

### [95% CI: 1.30, 4.49]) compared to men with patellar Pb ≤ 22.0 μ g/g. Tooth loss was not 7 

### statistically associated with blood Pb levels. Based on these results, the study authors 8 

### concluded that long

- term cumulative exposure to Pb is associated with increased odds of 9 

### t ooth loss. In a study examining the effects of Pb exposure on periodontitis in the U.S, 10 

### Saraiva et al. ( 2007 ) analyzed data for 2,500 men and 2,399 women aged between 30 and 11 

### 5 6 years from NHANES III. The analysis took into account various covariates including 12 

### age, NHANESIII phase, cotinine levels, poverty ration, race/ethnicity, education, bone 13 

### mineral density, diabetes, calcium intake, dental visits, and menopause in women. Af ter 14 

### adjusting for these covariates and comparing individuals with a concurrent blood Pb level 15 

### of >7 μ g/dL to those with a blood Pb level of <3 μ g/dL, the prevalence ratios of 16 

### periodontitis was 1.70 (95% CI: 1.02, 2.85) for men and 3.80 (95% CI: 1.66, 8.73) for 17 

### women. Based on these results, the study authors concluded that there was a positive 18 

### association between periodontitis and blood Pb levels for both men and women. In a 19 

### similar study, Yetkin et al. ( 2007 ) recruited 60 male subjects (30 apprentices with Pb 20 

### exposure, 30 controls), to examine the impact of occupational exposure to Pb on 21 

### periodontal stat us and association between periodontitis and blood Pb or oxidative stress. 22 

### The results of their analysis indicated that blood Pb was significantly higher in 23 

### apprentices exposed to Pb compared to controls (mean [SD]: 7.38 [4.41] versus 2.27 24 

### [1.49] μ g/dL, re spectively). No clinical periodontal or oxidative stress parameters were 25 

### significantly different between apprentices and controls. While the correlation between 26 

### blood Pb and periodontal parameters was not reported, significant correlations between 27 

### plaque i ndex and CAT, probing depth and SOD, clinical attachment level and SOD, and 28 

### clinical attachment level and malondialdehyde in Pb

- exposed apprentices were observed. 29 

### These results demonstrate that there is significant association between clinical 30 

### periodontal parameters and oxidative stress/damage indices in Pb

- exposed apprentices. In 31 

### a multiple regression analysis, a statistically significant association between gingival 32 

### index and working status, family income and either probing depth or clinical attachment 33 

### le vel was noted. 34February 2012 5

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### 5.9.5 Effects on Ocular Health
Ocular effects most commonly indicated to be associated with exposure to Pb include 1 

### formation of cataract, impaired vision, edema and retinal stippling. 2



### 5.9.5.1 Summary of Key Findings of the Effects on Ocular Health from the 2006 Lead AQCD
The 2006 Pb AQCD stated that various changes in the visual system were observed with 3 

### Pb poisoning including retinal stippling and edema, cataract, ocular muscle paralysis and 4 

### impaired vision. The 2006 Pb AQCD reported that retinal respon ses were observed in 5 

### children of mothers with a blood Pb range of 10.5 to 32.5 μg/dL during pregnancy, while 6 

### cataracts were noted in middle

- aged male with tibia bone Pb levels of 31

- 126 μg/g. 7



### 5.9.5.2 New Toxicological and Epidemiologic Studies

A small number of hu man studies pertaining to ocular effects of Pb were identified. 8 

### Mosad et al. ( 2010 ) studied the association between subcapsular cataract and Pb, 9 

### cadmium, vitamin C, vitamin E, and beta carotene blood levels in middle

- aged male 10 

### smokers compared to nonsmokers. Blood Pb was statistically significantly elevated in 11 

### light (mean [SD]: 14.5 [0.41] μ g/dL), moderate (14.5 [0.41] μ g/dL), and heavy smokers 12 

### (18.7 [1.24] μ g/dL) compared to nonsmokers (12.2 [0.21] μ g /dL). Blood Pb 13 

### concentrations were also observed to be statistically higher in the cataracts of smokers 14 

### versus nonsmokers. Similar associations were also observed for cadmium blood and lens 15 

### levels, while vitamins C, E, and beta carotene levels were signifi cantly decreased in 16 

### smokers. Based on these results, the study authors concluded that the Pb and cadmium 17 

### present in high concentration in smokers were associated with cataracts due to oxidative 18 

### stress which was indicated by reduced levels of antioxidants s uch as vitamins C, and E 19 

### and beta carotene. Erie et al. ( 2009 ) investigated the association between age

- related 20 

### macular degeneration and Pb and Cd in retinal tissue of human eye donors. The authors 21 

### observed that Pb, but not Cd, was significantly elevated in the neural retina tissue of 22 

### donors with ag e
- related macular degeneration (n = 36 donors, 72 eyes; median [IQR]: 23 

### 12.0 [8

- 18] ng/g Pb) versus normal control donors (n = 25 donors, 50 eyes; median 24 

### [IQR]: 8.0 [0

- 11] ng/g Pb). Neither heavy metal was significantly elevated in the retinal 25 

### pigment epithe lium

- choroid complex in donors with age

- related macular degeneration 26 

### and normal controls. 27February 2012 5

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### New animal studies pertaining to the ocular effects of Pb have investigated endpoints 1 

### such as retinal progenitor cell proliferation and neurogenesis,( Section 5.3.4.3 ). An in 2 

### vitro study was found that investigated whether exposure of cultured lenses from rats (4

- 6 3 

### weeks age) to 1 μ M Pb nitrate increased opacity with or without secondary oxidative 4 

### challenge ( Neal et al., 2010b )

. Lenses incubated for 3 days in the presence of 1 μ M Pb 5 

### were transparent, with no difference in amino acid incorporation compared to control 6 

### lenses. The authors concl uded this indicated that short

- term Pb exposure does not induce 7 

### osmotic swelling or lens shrinkage. However, exposure to lenses to Pb for 5 days 8 

### dramatically decreased the percentage of transparent lenses (30%) compared to controls 9 

### (80%). In Pb

- exposed len ses, 30% displayed "definite cataracts" compared to only 2.5% 10 

### in control lenses. By culture day 8, all exposed lenses were described either as clearly 11 

### opaque or definite cataracts, while only 7% of control lenses displayed these 12 

### characteristics, indicating that prolonged exposure o f lenses to Pb induced an accele rated 13 

### formation of opacity/cataract compared to unexposed lenses. Pb

- exposed lenses cleared 14 

### the media of hydrogen peroxide more rapidly than did control lenses, potentially due to 15 

### increased CAT acti vity. Exposure to hydrogen peroxide resulted in total opacity in Pb

16 

### exposed lenses at culture day 7, compared to less than 20% in control cells. Exposure to 17 

### Pb additionally altered epithelial nutrient transport and lens histology relative to that in 18 

### contro ls. 19



### 5.9.6 Effects on the Respiratory System
The collective body of toxicological and epidemiologic studies demonstrates Pb

20 

### associated effects on multiple immunological pathways, including a shift from a Th1 to a 21 

### Th2 phenotype, increased IgE antibody production, and increased inflammatory 22 

### responses ( Sections 5.2.5.1. and 5.6). These are well recognized pathways that contribute 23 

### to increased susceptibility to infections and also to the development of respiratory 24 

### diseases such as asthma. Recent investigation of the respiratory effects of Pb exposure 25 

### has been limited; however, cross

- sectional studies have indicated an association of 26 

### increasing blood Pb level with increased prevalence of asthma in children 27 

### ( Section 5.6.4.2 ). As described in Section 5.2.4 , Pb has been shown to induce the 28 

### generation of ROS. R OS are implicated in mediating increases in bronchial 29 

### responsiveness and activating neural reflexes leading to decrements in lung function. 30 

### Studies investigating these airway responses also are limited in number and collectively 31 

### do not provide strong evide nce of an association with blood Pb ( Section 5.6.6 ). 32 

### Collectively, panel and time

- series epidemiologic studies demonstrate associations 33 

### between Pb m easured in PM 2.5 

### or PM 10 

### air samples and decreases in lung function and 34 

### increases in respiratory symptoms, and asthma hospitalizations in children but not adults 35February 2012 5

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### ( Section 5.6.4.3 ). Toxicological studies have found pulmonary inflammation induced by 1 

### CAPs in which Pb was one of numerous components ( Wei et al., 2011 ; Duvall et al., 2 

### 2008 ; Godleski et al., 2002 ; Saldiva et al., 2002 )

. Despite this evidence for respiratory 3 

### effects related to air

- Pb concentrations, it is impor tant to note the limitations of air

- Pb 4 

### studies, including the limited data on the size distribution of Pb

- PM (Section 3.5.3), the 5 

### uncertain relationships of Pb

- PM 10 

### and Pb

- PM 2.5 

### with blood Pb levels, and the lack of 6 

### adjustment for other correlated PM chemi cal components. 7



### 5.9.7 Summary
There is evidence from epidemiologic and toxicological studies that substantial exposure 8 

### to Pb can result in altered liver function and hepatic toxicity. Biochemical changes 9 

### indicative of liver injury, including decreases in serum p rotein and albumin levels and 10 

### increased AST, ALT, ALP, and GGT activities, have been observed in occupationally

11 

### exposed humans (blood Pb > 22 μ g/dL) ( Can et al., 2008 ; Khan et al., 2008 ; Patil et al., 12 

### 2007 ) mature animals exposed to high levels of Pb during adulthood ( Sharma et al., 13 

### 2010a ; Wang et al. ; DS et al., 2009 ; Cheng et al., 2006 ) , and animals exposed during 14 

### gestation and lactation ( Pillai et al., 2009 ; Teijon et al., 2006 )

. In humans with mean 15 

### blood Pb levels 5.4 μ g/dL, altered AST levels were observed, but increases may be 16 

### related to exposure to contaminants other than Pb ( Conterato et al., In Press )

. Increased 17 

### hepatic cholesterogenesis, altered triglyceride and phospholipid levels, and disorders in 18 

### lipid metabolism accompanied by increased levels of total cholesterol and tri glycerides 19 

### have been reported in the animal literature ( Ademuyiwa et al., 2009 ; K hotimchenko and 20 

### Kolenchenko, 2007 )

. These results suggest that induction of cholesterogenesis and 21 

### phospholipids in the liver may cause subtle effects at the cellular level, leading to hepatic 22 

### injury. Multiple studies in humans and animals have observed hepatic oxidative stress, 23 

### generally in dicated by an increase in lipid peroxidation along with a decrease in GSH 24 

### levels and CAT, SOD, and GPx activities following exposure to Pb ( Pandya et al., 2010 ; 25 

### Sharma et al., 2010a ; Khan et al ., 2008 ; Yu et al., 2008 ; Adegbesan and Adenuga, 2007 ; 26 

### Jurczuk et al., 2007 ; Khotimchenko and Kolenchenko, 2007 ; Jurczuk et al., 2006 )

. 27 

### Indices of increased oxidative stress were additionally observed in the livers of fetuses 28 

### exposed to Pb throughout gestation ( Massó et al., 2007 )

. 29 

### Relatively few human studies have been conducted on the gastrointestinal toxicity of Pb 30 

### since the completion of the 2006 Pb AQCD. A case study reporting on GI symptoms in a 31 

### child reported that elevated blood Pb wa s associated with nonlocalized abdominal pain, 32 

### vomiting, nausea, constipation, lack of appetite, fatigue, and headaches ( Cabb et al., 33 

### 2008 )

. Symptoms were reported to diminish following cessation of exposure. Similar GI 34 

### sym ptoms (stomach pain and gastritis) were observed in battery works and painters 35February 2012 5

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### exposed to Pb in India with blood Pb levels ranging from 0.4

- 116.6 μ g/dL ( Kuruvilla et 1 

### al., 2006 )

. Toxicological evidence for Pb

- induced GI effects in rats includes inhibition of 2 

### NANC relaxations in the gastric fundus and th e observation of oxidative stress (lipid 3 

### peroxidation, decreased SOD and CAT) in the gastric mucosa ( Olaleye et al., 2007 ; 4 

### Santos et al., 2006b )

. The observation of oxidative stress was accompanied gastric 5 

### mucosal damage. 6 

### The endocrine processes most impacted by exposure to Pb include changes in thyroid 7 

### function. FT4, but not FT3, was decreas ed in adolescent male auto repair workers 8 

### ( Dundar et al., 2006 ) and mid

- pregnancy T4 levels were negatively associated in women 9 

### living in a highly contami nated town in Yugoslavia, whereas this association was not 10 

### observed in a non
- contaminated town ( Lamb et al., 2008 )

. Toxicological evidence for 11 

### similar effects was found in adult cows reared in an environment contaminated with Pb. 12 

### A positive correlation was reported between blood Pb and plasma T3 and T4 levels 13 

### ( Swarup et al., 2007 )

. In children challenged with an acute stressor, increasing prenatal 14 

### maternal and age 2

- year blood Pb levels were associated with significant increases in 15 

### salivary cortisol responses at age 9 years, even with blood Pb levels less than 10 μ g/dL 16 

### ( Gump et al., 2008 )

. A summary of key findings pertaining to reproductive hormones in 17 

### males and females in the current document is presented in the section on reproductive and 18 

### developmental effects ( Sections 5.8.1 and 5.8.2 ). 19 

### Numerous epidemiologic studies investigated the association between Pb biomarkers and 20 

### osteoporosis in adults. Higher blood Pb was observed to be associated with decreased 21 

### bo ne mineral density in non
- Hispanic white males ( Campbell and Auinger, 2007 ) , 22 

### whereas urinary Pb, but not blood Pb, was associated with decreased bone mineral 23 

### density in Chin ese individuals occupationally exposed to Pb ( Sun et al., 2008b )

. In 24 

### elderly women, blood Pb levels were positively associated with risk of falls and 25 

### osteoporosis

- related fractures ( Khalil et al., 2008 )

. Linear skeletal growth was reduced in 26 

### children living near copper smelters and refiners (concurrent mean blood Pb level = 27 

### 7 .7 μ g/dL) ( Ignasiak et al., 2006 )

. In vitro studies indicate that Pb interferes with CARCI 28 

### in human OLCs and that Pb perturbs multiple signaling pathways during murine limb bud 29 

### gro wth, potentially resulting in altered skeletal development ( Jang et al., 2008 ; Zuscik et 30 

### al., 2007 )

. Blood Pb levels have also been shown to be related to biomarkers of joint 31 

### tissue metabolism in elderly populations, but the cross

- sectional nature of these analyses 32 

### prevents conclusions being drawn on whether Pb increases bone and joint turnover 33 

### biomarkers, or whether increased turnover releases Pb into the bloodstream ( Machida et 34 

### al., 2009 ; Nelson et al., In Press )

. Epidemiologic studies investigating Pb exposure and 35 

### tooth loss indicate that long

- term, cumulative exposure to Pb is associated with increased 36 

### odds of tooth loss, periodontitis in men and women, and that periodontitis is associated 37February 2012 5

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### with oxidative stress/damage in individuals exposed in an occupational setting ( Arora et 1 

### al., 2009 ; Saraiva et al., 2007 ; Yetkin

- Ay et al., 2007 )

. 2 

### New toxicology studies have reported ocular effects ( i.e., retinal progenitor cell 3 

### proliferation) due to Pb exposure ( Section 5.3.4.3 ). and studi es in humans report 4 

### associations between heavy smoking, increased blood Pb levels, and cataracts ( Mosad et 5 

### al., 2010 ) and retinal Pb concentrations and age

- related macular degeneration ( Erie et al., 6 

### 20 09 )

. Investigation of the respiratory effects of Pb exposure has been limited; however, 7



### cross
- sectional studies have indicated an association of higher blood Pb levels with 8 

### increased prevalence asthma in children ( Section 5.6.4.2 ). 9 

### In summary, recent toxicological and epidemiologic evidence regarding the effects of Pb 10 

### exposure on the liver, GI tract, endocrine system, bone and teeth, eyes, and res piratory 11 

### tract largely are supportive of those effects noted in the 2006 Pb AQCD. However, recent 12 

### evidence of these effects is relatively limited, and therefore no causal determinations are 13 

### made regarding Pb

- induced effects in these organ systems. 14



## 5.10 Cancer

T he previous epidemiologic studies included in the 2006 Pb AQCD ( U.S. EPA, 2006b ) 15 

### “provide[d] only very limited evidence suggestive of Pb exposure associations with 16 

### carcinogenic or genotoxic effects in humans” and the studies were summarized as 17 

### follows: 18
“The epidemiologic data ...suggest a relationship between Pb exposure and cancers of the lung and the stomach... Studies of genotoxicity consistently link Pb

- exposed populations with DNA damage and micronuclei formation, although less c onsistently with chromosomal aberrations.” 

### The International Agency for Research on Cancer (IARC) recently classified inorganic 19 

### Pb compounds as probable human carcinogens (Group 2A of IARC classifications) based 20 

### on stronger evidence in animal studies than human studies, and organic Pb compounds as 21 

### not classifiable (Group 3 of IARC classifications) ( IARC, 2006a ; Rousseau et al., 2005 )

. 22 

### Additionally, the National Toxicology Program has listed Pb and Pb compounds as 23 

### “reasonably anticipated to be human car cinogens” ( NTP, 2004 )

. The typical cancer 24 

### bioassays used by IARC or NTP as evidence of Pb

- induced carcinogenicity used rodents 25 

### that were continuously exposed to Pb

- acetate in chow or drinking water for 18 months to 26 

### two years in duration. These two year c ancer bioassays and the doses administered are 27 

### typical of cancer bioassays used with other chemicals. 28 

### In the following sections, recent epidemiologic and toxicological studies published since 29 

### the 2006 Pb AQCD regarding Pb and cancer mortality and incidence are examined. In 30February 2012 5

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### addition, recent studies of Pb and DNA and cellular damage, as well as epigenetics 1 

### studies, are summarized. When the information is available, the form of the Pb compound 2 

### under study ( e.g., inorganic, organic) is indicated. In epidemiolog ic studies, various 3 

### biological measures of Pb are used including Pb measured in blood and bone. Bone Pb is 4 

### indicative of cumulative Pb exposure. Blood Pb can represent more recent exposure, 5 

### although it can also represent remobilized Pb occurring during tim es of bone remodeling. 6 

### Toxicological studies only report exposure by blood Pb or exposure dose. More detailed 7 

### discussion of these measures is given in Section 4.3.5

. 8



### 5.10.1 Cancer Incidence and Mortality
Recent studies have included epidemiologic evaluations of the associations between Pb 9 

### and both specific cancers, such as lung cancer and brain cancer, and overall cancer. Table 10 

### 5

- 42 provides an overview of the study characteristics and results for the epidemiologic 11 

### studies that reported effect estimates. This section also evaluates toxicological evidence 12 

### on the potential carcinogenicity of Pb. 13February 2012 5

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### Table 5
- 42 Summary of recent epidemiologic studies a 

### of cancer incidence and mortality

Reference a Study Location Cancer Outcome Study Population Measure of Pb Exposure Mean Pb (SD) Adjusted Effect Estimates Cancer Mortality: Menke et al. ( 2006 ) Multiple U.S. locations Overall cancer mortality NHANES III cohort with Blood Pb measures in 1988

- 1994 A t least 12 years of follow

- up B lood Pb <10 μg/dL Blood Pb at baseline 2.58 μg/dL (geometric mean)
Tertile 1: <1.93 μg/dL Tertile 2: 1.94

- 3.62 μg/dL Tertile 3: ≥ 3.63 μg/dL Tertile 1: 1.00 Tertile 2: 0.72 (95% CI: 0.46, 1.12) Tertile 3: 1.10 (95% CI: 0.82, 1.47)

Schober et al. ( 2006 ) Multiple U.S. locations Overall cancer mortality NHANES III cohort A t least 40 years of age Blood Pb at baseline Blood Pb<5 μg/dL: 67.7% Blood Pb 5

- 9 μg/dL: 26.0% Blood Pb>=10 μg/dL: 6.3% Blood Pb<5 μg/dL: 1.00 Blood Pb 5

- 9 μg/dL: 1.44 (95% CI: 1.12, 1.86) Blood Pb≥ 10 μg/dL: 1.69 (95% CI: 1.14, 2.52) Note: Modification by age assessed and associations varied slightly Weisskopf et al. ( 2009 ) Boston, MA Overall cancer mortality NAS I ncluded men only M ean follow

- up period for this study: 8.9 yr Blood Pb at b aseline, P atella Pb at baseline Blood Pb: 5.6 μg/ dL (3.4)

Tertile 1 of Blood Pb: <4 μg/dL Tertile 2 of Blood Pb: 4

- 6 μg/dL Tertile 3 of Blood Pb: >6 μg/dL
Tertile 1 of patella Pb: <22μg/g Tertile 2 of patella Pb: 22

- 35μg/g Tertile 3 of patella Pb: >35 μg/g Blood Pb Tertile 1: 1.00 Blood Pb Tertile 2: 1.03 (95% CI: 0.42, 2.55) Blood Pb Tertile 3: 0.53 (95% CI: 0.20, 1.39)

Patella Pb Tertile 1: 1.00 Patella Pb Tertile 2: 0.82 (95% CI: 0.26, 2.59) Patella Pb Tertile 3: 0.32 (95% CI: 0.08, 1.35)

Khalil et al. ( 2009a ) Baltimore, MD, and Monongahela Valley, PA Overall cancer mortality Subgroup of the Study of Osteoporotic Fractures cohort I ncluded white women aged 65

- 87 ; 12 yr (+/

- 3 yr) follow

- up Blood Pb at baseline Blood Pb Level 5.3 (2.3) μg/dL Blood Pb<8 μg/dL: 1.00 Blood Pb≥ 8 μg/dL: 1.64 (95% CI: 0.73, 3.71)

Lung Cancer: Lundstrom et al. ( 2006 ) Sweden Lung cancer (incidence and mortality) Male Pb smelter workers first employed for ≥ 3 months between 1928 and 1979 Followed up for mortality from 1955

- 1987 Median peak blood Pb level

Median number of yr with at least one blood sample obtained

Median cumulative blood Pb index (sum of annual blood Pb Level) Median peak blood Pb Level: cas es 2.4 μmol/L, controls 2.7 μmol/L
Median number of yr with at least one blood sample obtained: cases 4.5 yr, controls 6.0 yr

Median cumulative blood Pb index: cases 9.0 μmol/Pb, controls 11.9 μmol/Pb Median peak blood Pb Level: 1.00 (95% CI: 0.71, 1.42)
Median number of yr with at least one blood sample obtained: 0.96 (95% CI: 0.91, 1.02) per μmol/L
Median cumulative Blood Pb index: 0.99 (95% CI: 0.96, 1.02) per μmol/L

Note: similar results were observed when restricted to smokers onlyFebruary 2012 5

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– Do Not Cite or Quote Reference a Study Location Cancer Outcome Study Population Measure of Pb Exposure Mean Pb (SD) Adjusted Effect Estimates Jones et al. ( 2007 ) Humberside, UK Lung cancer mortality Male tin smelter employees Personnel record cards and air sampling conducted from 1972

- 1991
Three exposure scenarios determined for working lifetime cumulative exposure
– all have similar medians of approximately 2 mg yr/m 3 NA RR for Pb exposure weighted age and time since exposure: 1.54 (90% CI: 1.14, 2.08)
Note: Similar results for other exposure determination scenarios.

Rousseau et al. ( 2007 ) Montreal, Canada Lung cancer and other cancer incidence Men aged 35

- 79 Interview of job history and exposure matrix Ever exposed to: Organic Pb 3.0% Inorganic Pb 17.0% Pb in gasoline emissions 38.6% Organic Pb exposure compared to no exposure: Lung 1.3 (95% CI: 0.5, 3.1)
Inorganic Pb exposure compared to no exposure: Lung 1.1 (95% CI: 0.7, 1.7)
Pb in gasoline emissions exposure compared to no exposure: Lung 0.8 (95% CI: 0.6, 1.1)
Note: results are for comparisons using population

- based controls; results for controls with other types of cancers were similar Brain Cancer:

van Wijngaarden and Dosemeci ( 2006 ) Multiple U.S locations Brain cancer mortalit y National Longitudinal Mortality Study

– included individuals with occupational information

- included follow

- up from 1970

- 1989 Interview about current or most recent job within the past 5 years and a job exposure matrix NA Any Pb exposure compared to no e xposure 1.56 (95% CI: 1.00, 2.43)
Note: HRs were greatest among those with high probabilities of exposure and medium/high exposure intensity

Rajaraman et al. ( 2006 ) Phoenix, AZ, Boston, MA, and Pittsburgh, PA Brain cancer incidence NCI Brain Tumor Study

– included individuals >=18 yr diagnosed with brain cancer less than 8 wk befo re hospitalization; frequency

- matched controls were individuals admitted to the same hospitals for non

neoplastic conditions Interviews of lifetime work history and exposure databases NA Meningioma: Ever exposure to Pb 0.8 (95% CI: 0.5, 1.3)

Glioma: Ever exposure to Pb 0.8 (95% CI: 0.6, 1.1)
Note: positive associations between Pb exposure and meningioma incidence was observed among individuals with ALAD2 genotypes, but not individuals with ALAD1 genotypes; these associations were not observed for glioma incidenceFebruary 2012 5

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– Do Not Cite or Quote Reference a Study Location Cancer Outcome Study Population Measure of Pb Exposure Mean Pb (SD) Adjusted Effect Estimates Bhatti et al. ( 2009 ) Phoenix, AZ, Boston, MA, and Pittsburgh, PA Brain cancer incidence NCI Brain Tumor Study

– included non

Hispanic whites ≥ 1 8 yr diagnosed with brain cancer less than 8 wk before hospitalization; frequency

- matched cont rols were individuals admitted to the same hospitals for non

neoplastic conditions Interviews of lifetime work history and exposure databases Glioma: 70.5 μg/m 3 y (193.8 μg/m 3 y)

Glioblastoma multiform: 97.5 μg/m3y (233.9 μg/m 3 y)
Meningioma: 101.1 μg/m 3 y (408.7 μg/m 3 y)

Controls: 69.7 μg/m 3 y (248.8 μg/m 3 y) Per 100 μg/m 3 y increase in cumulative Pb exposure Glioma: 1.0 (95% CI: 0.9, 1.1)
Glioblastoma multiform: 1.0 (95% CI 0.9, 1.1)
Meningioma: 1.1 (95% CI: 1.0, 1.2)
Note: modification by SNPs was conducted and associations varied by SNP

Breast Cancer: Pan et al. ( 2011 ) Canada Breast cancer incidence National Enhanced Cancer Surveillance System (NECSS)

– population

- based sample of cancer cases and controls with information collected from 1994

- 1997 Self

- reported previous addresses and their proximity to Pb smelters (determined using Environmental Quality Database [EQDB]) NA Residing >3.2 km from Pb smelter or no nearby smelter: 1.00
Residing 0.8

- 3.2 km from Pb smelter: 0.41 (95% CI: 0.11, 1.51)
Residing <0.8 km from Pb smelter: 0.61 (95% CI: 0.11, 3.42) Multiple and Other Cancers: Absalon and Slesak ( 2010 ) Silesia province, Poland Overall cancer incidence Children living in this province at least five years Pb

- related air pollution measures NA Reported correlations between changes in Pb and cancer incidence

– no/low correlations observed (correlation coefficients between

- 0.3 and 0.2) Obhodas et al. ( 2007 ) Island of Krk, Croatia Incidence rates for neoplasms Individuals living in the Island of Krk from 1997

- 2001 Soil and vegetation samples, household potable water samples, children’s hair samples NA No association observed between Pb in the samples and incidence of neoplasm (numerical results not provided)

Mendey et al. ( In Press ) Multiple U.S. locations Incidence of cancer or “malignancy of any kind” 2007

- 2008 NHANES cohort

– at least 20 yea rs of age Concurrently measured creatinine

adjusted urinary Pb Geometric mean for creatinine

- adjusted urinary Pb marker: 0.59 μ g/g (95% CI: 0.57, 0.61) Greater than log

- transformed mean creatinine

- adjusted urinary Pb level compared to less than log

- transfo rmed mean creatinine

adjusted urinary Pb level: 0.76 (95% CI: 0.44, 1.33)February 2012 5

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– Do Not Cite or Quote Reference a Study Location Cancer Outcome Study Population Measure of Pb Exposure Mean Pb (SD) Adjusted Effect Estimates Rousseau et al. ( 2007 ) Montreal, Canada Lung cancer and other cancer incidence Men aged 35

- 79 Interview of job history and exposure matrix Ever exposed to: Organic Pb 3.0% Inorganic Pb 17.0% Pb in gasoline emissions 38.6% Never exposed is referent group
Organic Pb: Esophageal 1.7 (95% CI: 0.5, 6.4) Stomach 3.0 (95% CI: 1.2, 7.3) Colon 1.5 (95 % CI: 0.7, 3.6) Rectum 3.0 (95% CI: 1.2, 7.5) Pancreas 0.9 (95% CI: 0.1, 5.2) Prostate 1.9 (95% CI: 0.8, 4.6) Bladder 1.7 (95% CI: 0.7, 4.2) Kidney 2.3 (95% CI: 0.8, 6.7) Non

- Hodgkin’s lymphoma 0.4 (95% CI: 0.1, 2.2)
Inorganic Pb: Esophageal 0.6 (95% CI: 0.3, 1.2) Stomach 0.9 (95% CI: 0.6, 1.5) Colon 0.8 (95% CI: 0.5, 1.1) Rectum 0.8 (95% CI: 0.5, 1.3) Pancreas 0.9 (95% CI: 0.4, 1.8) Prostate 1.1 (95% CI: 0.7, 1.6) Bladder 1.1 (95% CI: 0.7, 1.5) Kidney 1.0 (95% CI: 0.6, 1.7) Melanoma 0.4 (95% CI: 0.2, 1.0) Non

- Hodgkin’s lymphoma 0.7 (95% CI: 0.4, 1.2)

Pb in gasoline emissions: Esophageal 0.6 (95% CI: 0.4, 1.1) Stomach 1.0 (95% CI: 0.7, 1.4) Colon 0.8 (95% CI: 0.6, 1.1) Rectum 1.0 (95% CI: 0.7, 1.4) Pancreas 0.9 (95% CI: 0.5, 1.4) Prostate 0.9 (95% CI : 0.7, 1.2) Bladder 0.8 (95% CI: 0.6, 1.1) Kidney 1.0 (95% CI: 0.7, 1.5) Melanoma 0.8 (95% CI: 0.5, 1.4) Non

- Hodgkin’s lymphoma 0.7 (95% CI: 0.5, 1.0)

Note: results are for comparisons using population

- based controls; results for controls with other type s of cancers were similar except no association was present between organic Pb and rectal cancerFebruary 2012 5

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– Do Not Cite or Quote Reference a Study Location Cancer Outcome Study Population Measure of Pb Exposure Mean Pb (SD) Adjusted Effect Estimates Santibanez et al. ( 2008 ) Valencia and Alicante, Spain Esophageal cancer incidence PANESOES study included 30

- 80 yr old men hospitalized in any of the participating study hospitals Interviews to determine occupational history and a job expo sure matrix NA All esophageal cancers: Unexposed: 1.00 Low workplace Pb exposure ( ≤ 0 .237 μmol/L): 0.79 (95% CI: 0.43, 1.46) High workplace Pb exposure (>0.237 μmol/L): 1.69 (95% CI: 0.57, 5.03)
Esophageal squamous cell carcinoma: Unexposed: 1.00 Low workplace Pb exposure ( ≤ 0 .237 μmol/L): 0.70 (95% CI: 0.34, 1.43) High workplace Pb exposure (>0.237 μmol/L): 0.91 (95% CI: 0.22, 3.75)

Adenocarcinoma: Unexposed: 1.00 Low workplace Pb exposure ( ≤ 0 .237 μmol/L): 0.95 (95% CI: 0.32, 2.82) High workplace Pb exposure (>0.237 μmol/L): 5.30 (95% CI: 1.39, 20.22)
*associations not changed or slightly increased when restricted to occupational exposures ≥ 1 5yr

a Studies listed in order of appearance in the text.



### 5.10.1.1 Overall Cancer Mortality

Several recent epidemiologic studies examined the association between Pb levels and 1 

### cancer mortality, including multiple analyses of the NHANES III population. In one 2 

### NHANES III analysis, the cohort was followed for 12 years and individuals with blood 3 

### Pb levels greater t han 10 μ g/dL were excluded from the study (mean baseline blood Pb 4 

### level was 2.58 μ g/dL). No association was observed between blood Pb and cancer 5 

### mortality (HR of highest tertile [ ≥ 3 .63 μ g/dL]compared to lowest tertile [<1.93 μ g/dL]: 6 

### 1.10 [95% CI: 0.82, 1.47]) ( Menke et al., 2006 )

. Another analysis of the NHANES III 7 

### population, which was restricted to individuals 40 years and older at the time of blood Pb 8 

### collection and included individuals with all blood Pb levels (including those greater than 9 

### 10 μ g/dL), reported ass ociations between blood Pb and cancer mortality ( Schober et al., 10 

### 2006 )

. In this study, median follow

- up time was 8.6 years. The RRs were 1.69 (95% CI: 11 

### 1.14, 2.52) for individuals with blood Pb levels of at least 10 μ g/dL and 1.44 (95% CI: 12 

### 1.12, 1.86) for blood Pb levels of 5

- 9 μ g/dL compared to individuals with blood Pb levels 13 

### less than 5 μ g/dL. When stratified by age, point estima tes comparing blood Pb levels 14 

### of 5

- 9 versus less than 5 μ g/dL were similar across all age groups but only statistically 15 

### significant among 75

- 84 year olds. The odds of mortality associated with blood Pb levels 16 

### ≥ 1 0 μ g/dL in the groups aged 40

- 74 years and 8 5 years and older were elevated. A study 17 

### of men from the greater Boston area enrolled in the NAS found no association between 18February 2012 5

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– Do Not Cite or Quote 

### blood or bone Pb and cancer mortality in adjusted analyses. The mean (SD) blood Pb 1 

### level for this population was 5.6 (3.4) μ g/dL a nd blood Pb was poorly correlated with 2 

### measured bone Pb ( Weisskopf et al., 2009 )

. As part of the Study of Osteoporotic 3 

### Frac tures , white women aged 65

- 87 were included in a sub

- study of blood Pb level and 4 

### cancer mortality and were followed for approximately 12 years ( Khalil et al., 2009a )

. The 5 

### mean (SD) blood Pb levels were 5.3 (2.3) μ g/dL and no association was detected between 6 

### blood Pb and cancer mortality in the study population. 7 

### Overall, epidemiologic studies of blood Pb levels and cancer mortality reported 8 

### inconsistent results. One ep idemiologic study using NHANES III data demonstrated an 9 

### association between blood Pb and increased cancer mortality; however, other studies 10 

### reported weak or no associations. 11



### 5.10.1.2 Lung Cancer
Most of the evidence regarding lung cancer incidence is provided by s tudies of 12 

### occupationally

- exposed adults. In a study of smelter workers, no association was 13 

### observed between several metrics of Pb exposure (peak blood Pb values, number of years 14 

### Pb samples were obtained, and cumulative blood Pb index) and lung cancer incid ence 15 

### and mortality combined ( Lundstrom et al., 2006 )

. The median follow

- up in the study was 16 

### about 30 years and the median peak blood Pb values during employment were 49.7 μ g/dL 17 

### for lung cancer cases and 55.9 μ g/dL for controls. In a study of tin smelters workers, no 18 

### association was observed between Pb exposure and lung cancer mortal ity in unweighted 19 

### analyses, but when the analyses were weighted by age and time since exposure, positive 20 

### associations were apparent ( Jones et al., 2007 )

. In this study, Pb exposure was calculated 21 

### by combining historical air sampling data and personnel record cards, which specified 22 

### work histories. The median Pb exposure was estimated to be approximately 2 mg
23 

### year/m 3



### . It is important to note that the smelter workers were exposed to other metals as 24 

### well, such as arsenic and ant imony. A population

- based case

- control study 25 

### performed among men in Montreal, Canada assessed Pb exposure via interviews 26 

### regarding job histories and calculated the likely Pb exposures associated with the job 27 

### activities ( Rousseau et al., 2007 )

. No association was apparent between organic Pb, 28 

### inorganic Pb, or Pb from gasoline emissions and lung cancer. 29 

### Studies of Pb and lung cancer that compared the lung tissue of individuals with lung 30 

### cancer to those without lung cancer were also conducted. The controls for these studies 31 

### were individuals with metastases in the lung from other primary cancers ( De Palma et al., 32 

### 2008 ) and individuals with non
- cancerous lung diseases ( De Palma et al., 2008 ; Kuo et 33 

### al., 2006 )

. Findings are mixed among the studies. De Palma et al. ( De Palma et al., 2008 ) 34February 2012 5

- 51 9 Draft

– Do Not Cite or Quote 

### reported higher Pb concentr ations in the cancerous and non
- cancerous lung tissue of 1 

### individuals with non
- small cell lung cancer compared to control groups (although the 2 

### authors report these results may be confounded by smoking). Kuo et al. ( Kuo et al., 2006 ) 3 

### found no statistical difference in Pb levels for lung tissue of individuals with lung cancer 4 

### compared to controls . 5 

### Some studies in the 2006 Pb AQCD reported associations between Pb exposure and lung 6 

### cancer; however, more recent epidemiologic studies of l ung cancer reported no 7 

### associations. Overall, these recent epidemiologic studies included only men, limiting the 8 

### generalizability. The studies by Jones et al. ( 2007 ) and Rousseau et al. ( 2007 ) also have 9 

### the disadvantage of not obtaining actual measures of Pb levels. In addition, studies 10 

### limited to occupational exposures may be confounded by other workplace exposures. 11



### 5.10.1.3 Brain Cancer

A f ew studies of brain cancer examined the association between cancer and Pb using 12 

### exposures determined via exposure databases and patient interviews about past jobs and 13 

### known exposures. Interpretation of these results is limited due to the lack of biological Pb 14 

### measures and the potential confounding by other occupational exposures. The National 15 

### Longitudinal Mortality Study, a study that included a national sample of the U.S. 16 

### population, estimated Pb exposure based on current/most recent employment among 17 

### indiv iduals ( Van Wijngaarden and Dosemeci, 2006 )

. Although not all estimates are 18 

### statistically significant, a pattern of increased associations between Pb exposure and brain 19 

### cancer mortality was observed in the study pop ulation. In a case

- control study of brain 20 

### tumors, glioma was reported to have no association with any Pb exposure metric; 21 

### however, positive associations were observed between high cumulative Pb exposure and 22 

### meningioma among individuals with ALAD2 genotypes (OR 2.4 [95% CI 0.7, 8.8] 23 

### comparing individuals ever exposed to Pb with those not exposed to Pb; OR 12.8 [95% 24 

### CI 1.4, 120.8] comparing individuals with cumulative Pb exposure ≥ 1 00 μ g/m 3 

### y to those 25 

### not exposed to Pb) ( Rajaraman et al., 2006 )

. This association was not present among 26 

### individuals with the ALAD1 genotypes (OR 0.5 [95% CI 0.3, 1.0] comparing individuals 27 

### ever exposed to Pb with those not exposed to Pb; OR 0.7 [95% CI 0.2, 1.8] comparing 28 

### individuals with cumulative Pb exposure ≥ 1 00 μ g/m 3 

### y to those not exposed to Pb). 29 

### Another study of the associatio n between Pb exposure (measured using self

- reported 30 

### occupational exposure history) and brain tumors reported none or slight overall 31 

### associations with types of brain tumors; however, positive associations were observed 32 

### among individuals with certain genetic single nucleotide polymorphisms (SNPs) ( Bhatti 33 

### et al., 2009 )

. After control for multiple comparisons, individuals with GPX1 variants 34 

### (rs1050450) had positive associations between cumulative Pb exposure and glioblastoma 35February 2012 5

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### multiforme and meningioma. Indiv iduals without RAC2 variants (rs2239774) showed a 1 

### positive association between Pb and glioblastoma multiforme. Also, individuals without 2 

### XDH variants (rs7574920) displayed a positive association between Pb and meningioma. 3 

### Overall, associations between Pb exposure and brain cancer incidence and mortality were 4 

### found to vary according to several genetic variants. These studies were limited in their 5 

### methods because they do not have individual level biological measures and the potential 6 

### for confounding by other workplace exposures exist. Future research will be important in 7 

### confirming these associations and the modification by various genetic variants. 8



### 5.10.1.4 Breast Cancer
A population

- based case

- control study in Canada examined the proximity to a Pb smelter 9 

### based on r esidential addresses ( Pan et al., 2011 )

. No associa tion was reported between 10 

### proximity of a Pb smelter and breast cancer incidence, but the study was limited by the 11 

### small number of women who resided near a Pb smelter (n=13 lived ≤ 3 .2 km from Pb 12 

### smelter). 13


### A few studies examined Pb levels and breast tumors among individuals with and without 14 

### breast tumor and/or cancer present. A study of newly diagnosed breast cancer patients 15 

### and controls examined Pb levels in blood and hair samples and reported higher levels of 16 

### both for cancer cases, although the difference in the Pb content in hair samples was not 17 

### statistically significant ( Alatise and Schrauzer, 2010 )

. Siddiqui et al. ( 2006 ) observed 18 

### higher blood Pb levels in women with benign and malignant tumors compared to 19 

### controls. Additionally, although blood Pb levels were higher among those with malignant 20 

### breast tumors compared to those with benign tumors, both had similar levels of Pb 21 

### detected in breast tissues. Another study of Pb levels present in breast tissue also reported 22 

### no statistical difference in Pb levels ( Pasha et al., 2008b )

. However, one study of breast 23 

### tissue did observe a statistically significant difference between Pb levels in the breast 24 

### tissue of cancer cases and controls ( Ionescu et al., 2007 )

. Finally, a study of Pb levels in 25 

### urine reported a positive association between urine Pb and breast cancer, but this 26 

### association became null when wome n taking nonsteroidal aromatase inhibitors but not 27 

### taking bisphostphonates (a combination responsible for bone loss) were excluded from 28 

### the analysis ( McElroy et al., 2008 )

. 29 

### Overall, these studies demonstrate the possibility that women with breast cancer may 30 

### have higher Pb levels in blood measurement, whereas the results for actual breast tissue 31 

### are mixed. However, these studies are limited by their study design. The samples are 32 

### taken after cancer is already present in the cases, leading to issues of temporality for the 33February 2012 5

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### Pb levels. Additionally, the sample sizes are often small and the studies may be 1 

### underpowered. 2



### 5.10.1.5 Other Cancers
Studies of all cancers combined, multiple different cancers, or cancers not listed above 3 

### have also been performed. An ecologic analysis compared levels of Pb in the air from 4 

### 1990 to 2005 with incidence rates of cancer (cancer sites not specified) among children 5 

### during this time period ( Absalon and Slesak, 2010 )

. The highest Pb levels were measured 6 

### in 1990 when over 50% of the study area exceeded the limit of 1 μ g/m 2 

### ∙year. No 7



### cor relation was observed both overall and in sex

- specific analyses. A similar study 8 

### examined correlations between Pb concentrations in soil, water, vegetation, and hair 9 

### samples with incidence of neoplasms ( Obhodas, 2007 )

. The Pb concentrations were not 10 

### correlated with incidence of neoplasms. A recent study using the 2007

- 2008 NHANES 11 

### cohort reported no association between higher creatinine

- adjusted urine Pb levels and 12 

### having ever had cancer or a malignancy ( Mendy et al., In Press )

. The timing of cancer 13 

### diagnosis in relation to the urine sample collection was not identified. 14 

### A study performed among men evaluated multiple cancer outcomes and determi ned 15 

### exposures to organic Pb, inorganic Pb, and Pb from gasoline emissions via interviews 16 

### regarding job histories and then subsequent exposure approximations by chemists and 17 

### hyg i enists ( Rousseau et al., 2007 )

. Adults exposed to organic Pb exposure had greater 18 

### odds of stomach cancer compared to adults never exposed to organic Pb. A positive 19 

### association was also observed for rectal cancer when population

- based controls w ere 20 

### used but was null when the control population was limited to individuals with other types 21 

### of cancers. No association was detec ted for cancers of the esophag us, colon, pancreas, 22 

### prostate, bladder, kidney, melanoma, or non
- Hodgkin’s lymphoma. None of the cancers 23 

### were associated with exposure to inorganic Pb. When occupational exposure to Pb in 24 

### gasoline was categorized as unexposed, nonsubstantial level, and substantial level, a 25 

### positive association with stomach cancer was observed when cancer controls wer e used 26 

### as the comparison group; however the association was not present when population 27 

### controls were utilized as the control group). Another case

- control study using participant 28 

### interviews and a job exposure matrix, including only men, reported no associa tion 29 

### between Pb exposure and esophageal squamous cell carcinomas, but an association was 30 

### present between high Pb exposure and adenocarcinoma of the esophagus ( Santibanez et 31 

### al., 2008 )

. However, neither of these studies was able to quantify Pb levels using 32 

### biological measurements. 33February 2012 5

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### Several studies compared Pb levels in blood, tissue, and ur ine of individuals who have 1 

### cancer with individuals who are cancer

- free. Compared to control groups, higher Pb 2 

### levels were observed in the blood and bladder tissue of individuals with bladder cancer 3 

### ( Golabek et al., 2009 ) , the kidney tissue of individuals with renal cell carcinoma (with 4 

### highest levels among those with the highest stage tumors ) ( Calvo et al., 2009 ) , the tissue 5 

### (but not serum) of individuals with laryngeal cancer ( Olszewski et al., 2006 ) , the blood of 6 

### individuals with gastric cancer ( Khorasani et al., 2008 ) , the plasma and hair of 7 

### individuals with gastrointestinal cancer ( Pasha et al., 2010 ) , the blood and hair of 8 

### individuals with non
- specified types of cancer ( Pasha et al., 2008c ; Pasha et al., 2007 ) , 9 

### and the hair of individuals with benign tumors ( Pasha et al., 2008a )

. No statistical 10 

### difference in Pb levels was reported for colon tissue of individuals with colorectal polyps 11 

### ( Alimonti et al., 2008 ) or urine of individuals with bladder cancer ( Lin et al., 2009 ) 12 

### compared to control groups. A study examining Pb levels in kidney tissue reported the 13 

### highest levels of Pb in normal kidney tissue samples that were adjacent to neoplastic 14 

### tumors. The Pb levels reported in the kidney tissue of neoplastic tumors were elevated 15 

### compared to those detected in corpses without neoplastic tumors of the kidney ( Cerulli et 16 

### al., 2006 )

. All of these comparison studies are limited by the inability to examine 17 

### temporality as Pb biomarkers were measured after the cancer di agnosis; the level of Pb 18 

### may be due to changes that result from having cancer, not changes that result in cancer. 19 

### Many of these studies attempted to control for this by including only cases who have not 20 

### undergone certain treatments. Additionally, studies a re limited by their small sample size 21 

### and the selection of the control populations. Control populations are supposed to 22 

### represent the general population from which the cases are drawn; some of the control 23 

### subjects in these studies are individuals with dise ases/conditions warranting tissue 24 

### resections, which are not prevalent in the general population. 25 

### In sum, epidemiologic studies reported no associations between various measures of Pb 26 

### exposure and overall cancer incidence. Studies examining specific cancer s reported 27 

### varying associations. Associations were null for Pb and most cancer sites examined; 28 

### however a positive association was observed between Pb exposure and adenocarcinoma 29 

### of the esophagus as well as exposure to organic Pb and stomach cancer. Associa tions 30 

### between organic Pb exposure and rectal cancer and exposure to Pb in gasoline and 31 

### stomach cancer were inconsistent. These conclusions are limited by the small number of 32 

### studies and a lack of biological measurements of Pb. 33February 2012 5

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### 5.10.1.6 Toxicological Models of Carci nogenicity

Carcinogenicity in Animal Models Previous AQCDs have established that Pb has been shown to act as a carcinogen in 1 

### animal toxicology models, albeit at relatively high concentrations. The 2006 Pb AQCD 2 

### pointed out that because Pb is a "well

- established animal carcinogen...., focus has been 3 

### more on the mechanism of neoplasia and possible immunomodulatory effects of Pb in the 4 

### promotion of cancer." This focus continues to date. The kidneys are the most common 5 

### target of Pb

- dependent carcinoge nicity ( Kasprzak et al., 1985 ; Koller et al., 1985 ; Azar et 6 

### al., 1973 ; Van Esch and Kroes, 1969 ) but the testes, brain, adrenals, prostate, pituitary, 7 

### and mammary gland have also been affected ( IARC, 2006a )

. The typical cancer 8 

### bioassays used by IARC or NTP as evidence of Pb

- induced carcinogenicity were 9 

### designed using rodents, typically males but som etimes animals of both sexes, that were 10 

### continuously exposed to Pb

- acetate in chow ( i.e., 0.1 or 1% Pb

- acetate ) or drinking water 11 

### ( i.e., 26 or 2 , 600 ppm Pb

- acetate ) for 18 months to two years in duration ( Kasprzak et al., 12 

### 1985 ; Koller et al., 1985 ; Azar et al., 1973 ; Van Esch and Kroes, 1969 )

. These two year 13 

### cancer bioassays and the doses employed are typical of cancer bioassays employed by 14 

### other chemicals. Recognition of the importance of windows of exposure in Pb

- induced 15 

### cancer bioassays is a focu s of more recent studies. In one study, early life gestational and 16 

### lactational exposure of laboratory rodents to inorganic Pb induced carcinogenicity in 17 

### adult offspring ( Waalkes et al., 1995 )

. Another recent study considered Pb

- dependent 18 

### carcinogenesis in laboratory animals with early life Pb exposure. Tokar et al. ( 2010 ) 19 

### considered tumorigenesis in homozygous metallothionein I/II knockout mice and their 20 

### corresponding wild type controls (groups of ten mice each) that were exposed by 21 

### drinking water to 2,000 or 4,000 ppm Pb

- acetate and compared to untreated controls. 22 

### Study animals were exposed in utero, through birth and lactatio n, and then postnatally to 23 

### drinking water until 8 weeks old. The Pb

- exposed metallothionein I/II knockout mice had 24 

### increased testicular teratomas and renal and urinary bladder preneoplasia. Pb exposed 25 

### wild

- type mice were not statistically significantly dif ferent than controls. The data 26 

### suggest that metallothionein can protect against Pb

- induced tumorigenesis. Concerns with 27 

### the study are that the doses are at levels of Pb to which humans would not likely be 28 

### exposed and there is no metallothionein null condit ion in humans, though there is 29 

### variability in the expression of metallothionein. The data do not address whether this 30 

### variability would have any impact on Pb

- dependent carcinogenesis in humans. Thus, the 31 

### animal toxicology data demonstrate that Pb is a well

- established animal carcinogen in 32 

### studies employing high dose Pb exposure over a continuous extended duration of 33 

### exposure ( i.e., 2 years), which is typical of cancer bioassays. Newer studies are showing 34February 2012 5

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### early life maternal Pb exposure can contribute to car cinogenicity in offspring and have 1 

### shown that metallothionein is protective against cancer in this pathway. 2



### Neoplastic Transformation Studies, Human Cell Cultures Carcinogenesis can be measured in cell culture systems through neoplastic transformation 3 

### mode ls that monitor change by following morphological transformation of cells, 4 

### i.e., formation of a focus (or foci) of cell growth.. Xie et al. ( 2007 ) treated BEP2D cells 5 

### (human papilloma virus

- immortalized human bronchial cells) with 0, 1, 5, or 10 μ g/cm2 6 

### PbCrO 4 

### for 120 h. PbCrO 4 

### induced foci formation in a concentration

- dependent manner. 7 

### Xie et al. ( 2008 ) treated BJhTERT cells (hTERT

- immortalized human skin fibroblasts) 8 

### and ATLD

- 2 cells (hTERT

- immortalized human skin fibroblasts deficient in Mre11) with 9 

### 0, 0.1, 0.5, and 1 μ g/cm 2 

### PbCrO 4 

### for 120 h. PbCrO 4 

### induced foci formation in a 10 

### concentration

- dependent manner in the Mre11 deficient cells. Mre11 was required to 11 

### prevent PbCrO 4 

### - induced neoplastic transformation. 12



### Immune Modulation o f Tumori genesis by Pb

As described in the 2006 Pb AQCD ( U.S. EPA, 2006b ) , Pb

- induced immunotoxicity can 13 

### contribute to increased risk of cancer, primarily due to the intersection of suppressed Th1 14 

### responses and misregulated inflammation. First, Pb

- induced misregulati on of 15 

### inflammation involving innate immune cells has been shown to result in chronic insult to 16 

### tissues. These insults, excessive lipid and DNA oxidation production by overproduction 17 

### of ROS and weakened anti

- oxidant defenses, can increase the likelihood of mutagenesis, 18 

### cellular instability, and tumor cell formation. For example, Xu et al. ( 2008 ) found 19 

### toxicological evidence that supports the association with Pb exposure and DNA damage 20 

### and concluded that it is a possible route to increased Pb

- induced tumorigenesis. The 21 

### second component of increased risk of cancer involves Pb

- induced suppression of Th1

- 22 

### dependent anti

- tumor immunity as acquired immunity shifts statistically significantly 23 

### toward Th2 responses. W ith cytotoxic T lymphocytes and other cell

- mediated defenses 24 

### dramatically lessened, the capacity to resist cancer may be compromised. 25



### 5.10.2 Cancer Biomarkers
A study of men aged 21

- 40 years without occupational history of metals exposure 26 

### examined prostate specif ic antigen (PSA), a biomarker for prostate cancer. This study 27 

### reported a positive association between Pb levels and PSA levels (measured in the same 28 

### blood samples) in regression models adjusted for confounders, including other metals 29February 2012 5

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### (Cd, Zn, Se, and Cu) ( Pizent et al., 2009 )

. The median blood Pb level was 2.6 μ g/dL 1 

### (range 1.0

- 10.8 μ g/dL). The authors note that the study population was young and at 2 

### lowe r risk of prostate cancer than are older men. 3



### 5.10.3 DNA and Cellular Damage
Multiple studies have been performed examining the relationship between Pb and DNA 4 

### and cellular damage. Details of the recent epidemiologic and toxicological studies follow. 5



### 5.10.3.1 Epidemiologi c Evidence for DNA and Cellular Damage
Multiple studies examined the relationship between Pb and sister chromatid exchange 6 

### (SCE). SCEs are exchanges of homologous DNA material between chromatids on a 7 

### chromosome and is a test for mutagenicity or DNA damage. A study of male policemen 8 

### reported mean blood Pb levels for the study population of 43.5 μ g/dL ( Wiwanitkit et al., 9 

### 2008 )

. When dichotomized as having high or low blood Pb levels (cut

- off at 49.7 μ g/dL), 10 

### the higher blood Pb group was observed to have higher mean SCE. Another study of 11 

### adult males compared the SCE of storage battery manufacturing workers (mean blood Pb 12 

### levels of 40.14 μ g/dL) and off ice workers (mean blood Pb levels of 9.77 μ g/dL) ( Duydu 13 

### et al., 2005 )

. The exposed workers had higher SCE levels and also a greater number of 14 

### cells in which the SCEs per cell were higher than the 95th percentile of the population. 15 

### Finally, a study of children aged 5

- 14 years old (mean [SD] blood Pb levels of 7.69 16 

### [4.29] μ g/dL) reported no correlation between blood Pb levels and SCE ( Mielzyńska et 17 

### al., 2006 )

. However, the study did report a positive association between blood Pb and 18 

### micronuclei (MN) levels. 19 

### Other studies of DNA damage have reported mixed results. A study of children ages 6

- 11 20 

### years old and environmentally

- ex posed to Pb (children attending a school far from a Pb 21 

### smelter: median blood Pb levels 4.6 μ g/dL; children attending a school near a Pb smelter: 22 

### median blood Pb levels 28.6 μ g/dL for) reported no association between blood Pb and 23 

### baseline DNA damage or repa ir ability after a peroxide challenge ( Méndez

- Gómez et al., 24 

### 2008 )

. Another study included adult participants aged 50

- 65 years and reported an 25 

### association between blood Pb and carcinoembryonic antigen (CEA) but not with DNA

26 

### strand breaks, MN freq uency, or oxidative DNA damage (median blood Pb level of the 27 

### study population: 3.92 μ g/dL) ( De Coster et al., 2008 )

. A study conducted among 28 

### workers exposed to Pb (mean blood Pb level: 30.3 μ g/dL) and unexposed controls (mean 29 

### blood Pb level: 3.2 μ g/dL) reported greater cytogenetic damage (measured by MN 30 

### frequency), chromosomal aberr ations, and DNA damage in the Pb

- exposed group 31February 2012 5

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### (although this was not statistically significant in linear regression models controlling for 1 

### age) ( Grover et al., 2010 )

. A study of painters in India, where Pb concentrations in paint 2 

### are high, reported a mean (SD) blood Pb level of 21.56 (6.43) μ g/dL among painters who 3 

### reported painting houses for 8

- 9 hours/day for 5

- 10 years ( Khan et al., 2010b ) ; the mean 4 

### (SD) blood Pb level was 2.84 (0.96) μ g/dL for healthy workers who had not been 5 

### occupationally exposed to Pb. Cytogenetic damage was higher among the painters 6 

### compared to the healthy controls. Another study compared the blood Pb of metal workers 7 

### and office workers and reported higher blood Pb levels (both current and 2 year average) 8 

### among the metal workers (blood Pb level ≥ 2 0 μ g/dL) compared to the office workers 9 

### (blood Pb level < 10 μ g/dL for) ( Olewińska et al., 2010 )

. Overall, the workers had 10 

### increased DNA strand breaks versus the office workers (this held true at various blood Pb 11 

### levels). Finally, a study of Pb battery workers with symptoms of Pb toxicity and a group 12 

### of controls were examined ( Shaik and Jamil, 2009 )

. Higher chromosomal aberrations, 13 

### MN frequency, and DNA damage were reported for the battery workers as compared to 14 

### the controls. 15



### 5.10.3.2 Toxicological Evidence for DNA and Cellular Damage

Sister Chromatid Exchanges Tapisso et al. ( 2009 ) , considered sister chromatid exchanges (SCE) in Algerian mice 16 

### (groups of six mice each) that were exposed by i.p. injection to 5 or 10 doses of 17 

### 0.46 mg /kg Pb

- acetate . The SCE in bone marrow were elevated after Pb exposure alone, 18 

### which increased with time. Co

- exposure with cadmium or zinc further increased SCE 19 

### levels. 20 

### SCE was also followed in cultured human cells. Ustundag and Duydu ( 2007 ) , considered 21 

### the ability of N

- acetylcysteine and melatonin to reduce Pb nitrate

- induced SCE in a 22 

### single human donor. Cells were treated with 0, 1, 5, 10, or 50 μ M Pb nitrate. SCE 23 

### statistically significantly increased at every Pb concentration in a concentration 24 

### dependent manner. Both 1 and 2 mM N

- ac etylcysteine and melatonin were able to 25 

### statistically significantly reduce SCE levels in Pb

- exposed animals. In another study, 26 

### Turkez et al. ( In Press ) considered the ability of boron compounds to prevent Pb chloride

27 

### induced SCE in human lymphocytes. Cells were obtained from 4 non
- smoking donors. 28 

### Both 3 and 5 ppm Pb chloride induced a statistically significant increase in SCE levels 29 

### over controls. Boron w as able to statistically significantly diminish these levels. For both 30 

### studies, exposure times were not provided and the full interpretation of these data is 31 

### limited by the limited number of donors and the absence of an exposure time for the SCE 32 

### assay. 33February 2012 5

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### Mic ronuclei Formation The 2006 Pb AQCD stated "studies of genotoxicity consistently find associations of Pb 1 

### exposure with DNA damage and MN formation" and the current document continues to 2 

### report these associations. Alghazal et al. ( 2008b ) , considered the ability of Pb

- acetate 3 

### trihydrat e to induce MN in bone marrow of Wistar rats. Animals were given a daily dose 4 

### of 100 mg /l in their drinking water for 125 days. The mean number of MN in male and 5 

### female rats was statistically significantly higher in Pb

- exposed animals than in unexposed 6



### con trols. Tapisso et al., ( 2009 ) , considered Pb alone, Pb plus zinc and Pb plus cadmium

7 

### induced MN in rodents. Algerian mice were exposed by i.p. injection to 5 or 10 doses of 8 

### 0.46 mg /kg Pb

- acetate and compared to untreated controls. The MN in bone marrow 9 

### were elevated after Pb exposure and increased with time. Co

- exposure with cadmium or 10 

### zinc did not further increase MN levels. 11


### MN formation has also been followed in cultured human cells. Ustundag and Duydu 12 

### ( 2007 ) considered the ability of N

- acetylcysteine and melatonin to reduce Pb nitrate

13 

### induced MN in a single human donor. Cells were treated with 0, 1, 5, 10, or 50 μ M Pb 14 

### nitrate. MN formation statistically significantly increased at the two highest Pb 15 

### concentrations in a concentration dependent manner. Both 1 and 2 mM N

- acetylcysteine 16 

### and melatonin were not able to statistically significantly reduce MN levels. In another 17 

### study, Turkez et al. ( In Press ) considered the ability of boron compounds to prevent Pb 18 

### chloride

- induced MN in human lymphocytes. Cells were obtained from 4 non
- smoking 19 

### donors. Both 3 and 5 ppm Pb chloride induced a statistically significant increase in MN 20 

### levels over controls. Boron induced a statistically significant attenuation of these Pb

21 

### induced levels. For both studies, exposure times were not provided, and the full 22 

### interpretation of these data is limited by the limited number of donors and the abs ence of 23 

### an exposure time for the MN assay. Gastaldo et al. ( 2007 ) evaluated the ability of Pb to 24 

### induce MN. Human endothelial HMEC cell line was treated with 1

– 1 , 000 μ M Pb nitrate 25 

### for 24 hours. MN increased in a statistically significant, concentration

- dependent manner. 26



### Hypoxanthine
- guanine phosphoribosy l transferase Mutations The potential mutagenicity of Pb in human or animal cells was e valuated by monitoring 27 

### mutations at the hypoxanthine

- guanine phosphoribosytransferase (HPRT) locus. Li et al. 28 

### ( 2008a ) evaluated Pb

- acetate

- induced HPRT in the non
- small

- cell lung carcinoma tumor 29 

### cell line, CL3 and in normal human diploid fibroblasts (specific tissue source not 30 

### reported). All cells were exposed to 0, 100, 300 or 500 μ M Pb

- acetate for 24 hours in 31



### serum
- free medium ± a 1

- hour pretreatment with a MKK1/2 inhibitor or a PKC

- alpha 32 

### inhibitor. Pb alone did not induce HPRT mutations. Inhibiting the ERK pathway via 33 

### either inhibitor statistically significantly increased Pb

- induced mutagenesis. Wang et al. 34February 2012 5

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### ( 2008c ) , investigated Pb

- acetate

- induced HPRT mutations in CL3 cells. All cells were 1 

### exposed t o 0, 100, 300 or 500 μ M Pb

- acetate for 24 hours in serum

- free medium ± a 2 

### 1

- hour pretreatment with a PKC

- alpha inhibitor or siRNA for PKC

- alpha. Pb alone did 3 

### not induce HPRT mutations. Inhibiting PKC

- alpha via either inhibitor statistically 4 

### significantly in creased Pb

- induced mutagenesis. McNeill et al. ( 2007 ) considered 5 

### Pb

- acetate induced HPRT mutations in Chinese hamster ovary AA8 cells and AA8 cells 6 

### overexpressing human Ape1. Cells were treated with 5 μ M Pb

- acetate for 6 hours. No 7 

### increases in HPRT mutations were observed after Pb exposure in either cell line but with 8 

### specific pathway perturbations (PKC

- alpha or ERK) Pb was able to induce HPRT 9 

### mutations. 10



### Chromosomal Aberrations
Chromosomal aberrations, an indicator of cancer risk, were followed in P b

- exposed 11 

### rodents ( El

- Ashmawy et al., 2006 )

. Dietary exp osure to Pb

- acetate administered as a 12 

### single dose of 0.5% w/w to male Swiss albino mice caused statistically significant 13 

### increased levels of chromosomal aberrations in the Pb treatment alone group, particularly 14 

### with respect to fragments, deletions, ring ch romosomes, gaps, and end

- to

- end 15 

### associations. In addition, the authors found turmeric and myrrh powders were protective. 16 

### Concerns with the study include the use of only a single dose of Pb

- acetate along with the 17 

### high levels of unusual aberrations such as r ing chromosomes and end

- to

- end associations. 18 

### Typically, these aberrations are rare after metal exposure, but were the most commonly 19 

### observed aberration in this study raising questions about the quality of the metaphase 20 

### preparations. An additional concern w as that only 50 metaphases per dose were analyzed 21 

### instead of the more common 100 metaphases per dose. The authors did not explain why 22 

### their spectrum of aberrations was so different, why they only used one dose, or analyzed 23 

### fewer metaphases per dose. 24


### Multi ple studies considered the ability of Pb to induce chromosomal aberrations in 25 

### cultured human cells. The ability of Pb nitrate to induce chromosomal aberrations was 26 

### examined in primary human peripheral blood lymphocytes obtained from healthy, 27 

### nonsmoking don ors ( Pasha Shaik et al., 2006 )

. Cells were treated with 0, 1.2 or 2 mM 28 

### Pb

- nitrate for 2 hours. No increase in chromosomal aberrations was reporte d. Some 29 

### aneuploidy was observed. Concerns with the study are that only a 2 hour exposure was 30 

### used, which may not be long enough for DNA damage to be expressed as a chromosomal 31 

### aberration. It also appears from the data presentation that only three subjects were used; 32 

### one for a control, one for the low dose and one for the high dose. Experiments were not 33 

### repeated, thus given the small number of subjects, this study may not have had sufficient 34 

### power to detect any effects. Holmes et al. ( 2006a ) , treated WHTBF

- 6 cells (hTERT

35 

### immortalized human lung cells) with 0, 0.1, 0.5, or 1 μ g/cm 2 

### Pb chromate for 24

- 120 36February 2012 5

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### hours or with 0, 0.1, 0.5, 1, 5 or 10 μ g/cm 2 

### Pb oxide for 24 or 120 hou rs. Pb chromate 1 

### induced statistically significant, concentration

- dependent increases in centrosome 2 

### abnormalities and aneuploidy. Wise et al. ( 2006b ) treated BEP2D cells with 0, 0.5, 1, 5, 3 

### or 10 μ g/cm 2 

### Pb chromate for 24 hours. Pb chromate induced statistically significant 4



### concen tration

- dependent increases in chromosomal aberrations. Holmes et al. ( 2006b ) , 5 

### treated WHTBF

- 6 cells with 0, 0.1, 0.5, or 1 μ g/cm 2 

### Pb chromate for 24

- 72 hours. Pb 6 

### chromate induced statistically significant, concentration

- dependent increases in 7 

### chromosomal aberrations. The effects of the chromate anion cannot be ruled out as 8 

### causative in inducing these chromosomal aberrations. Wise et al. ( 2006a ) , treated 9 

### WHTBF

- 6 cells with 0, 0.1, 0.5, or 1 μ g/cm 2 

### Pb chromate for 24

- 120 hours. Pb chromate 10 

### induced statistically significant, concentration

- dependent increases in spindle assembly 11 

### checkpoint disruption, effects of mitosis and aneuploidy. By contrast, chromate

- free Pb 12 

### oxide did not induce centrosome amplification. The effects were likely attributable to the 13 

### chromate anion. Xie et al. ( 2007 ) treated BEP2D cells with 0, 1, 5, or 10 μ g/cm 2 

### Pb 14 

### chromate for 24 hours. Pb chromate induced statistically significant, concentration

15 

### dependent increases in chromosomal aberrations and aneuploidy. Wise et al. ( 2010 ) 16 

### treated WHTBF

- 6 cells with 0, 0.1, 0.5, or 1 μ g/cm 2 

### Pb chromate for 24 hours in a study 17 

### comparing 4 chromate compounds. Pb chromate induced statistically significant, 18 

### concentration

- dependent increases in chromosomal aberrations 19 

### Multiple investigators considered the ability of Pb chromate to induce chromosome 20 

### aberrations in rodent cell cultures. Grlickova Duzevik et al. ( 2006 ) treated Chinese 21 

### hamster ovary (CHO) cells with 0, 0.1, 0.5, or 1 μ g/cm 2 

### Pb chromate for 24 h. Specific 22 

### CHO lines used included AA8 (wildtype) EM9 (XRCC1

- deficient), and H9T3 (EM9 23 

### complemented with human XRCC1 gene). Pb chromate induced statistically significant, 24 

### concentration

- dependent increases in chromosomal aberrations that were statistically 25 

### significantly increased by XRCC1 deficiency. Nestmann and Zhang ( 2007 ) treated 26 

### Chinese hamster ovary cells (clone WB(L )) with 0, 0.1, 0.5, 1, 5, or 10 μ g/cm 2 

### Pb 27 

### chromate (as pigment yellow) for 18 h. No increases in chromosomal aberrations were 28 

### observed. Savery et al. ( 2007 ) treated CHO cells with 0, 0.1, 0.5, 1, or 5 μ g/cm 2 

### Pb 29 

### chromate for 24 h. Specific CHO lines used included AA8 (wildtype) , KO40 30 

### ( Fancg

- deficient), and 40BP6 ( Fancg

- complemented). Pb chromate induced statistically 31 

### significa nt, concentration

- dependent increases in chromosomal aberrations that were 32 

### increased by Fancg

- deficiency. The Fancg gene plays an important role in cellular 33 

### resistance to DNA interstrand crosslinks, protecting against genetic instability. Camrye et 34 

### al., ( 2007 ) treated CHO cells with 0, 0.1, 0.5, 1, 5, or 10 μ g/cm 2 

### Pb chromate for 24 35 

### hours. S pecific CHO lines used included CHO

- K1 (parental), xrs

- 6 (Ku80 deficient), and 36 

### 2E (xrs

- 6 complemented with Ku80 gene). Pb chromate induced statistically significant, 37 

### concentration

- dependent increases in chromosomal aberrations that were not affected by 38 

### Ku8 0 deficiency. Ku80 is a gene a gene involved in nonhomologous end

- joining repair 39February 2012 5

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### and its absence can contribute to genetic instability. Stackpole et al. ( 2007 ) treated CHO 1 

### and Chinese hamster lung (CHL) cells with 0, 0.1, 0.5, or 1 μ g/cm 2 

### Pb chromate for 24 2 

### hours. Specific CHO lines used included A A8 (wildtype), irs1SF (XRCC3

- deficient), and 3 

### 1SFwt8 (XRCC3 complemented). XRCC3 is DNA repair enzyme involved in 4 

### homologous recombination. CHL lines used included V79 (wildtype), irs3 (Rad51C 5 

### deficient) and irs3#6 (Rad51C complemented). Rad51C is a gene wh ich encodes strand

6 

### transfer proteins that are thought to be involved in recombinational repair of damaged 7 

### DNA and in meiotic recombination. Pb chromate induced statistically significant, 8 

### concentration

- dependent increases in chromosomal aberrations that wer e statistically 9 

### significantly increased by both XRCC3 and Rad51C deficiency. 10 

### Multiple studies considered the ability of Pb chromate to induce chromosome aberrations 11 

### in marine mammal cell cultures. Li Chen et al. ( 2009 ) treated primary North Atlantic 12 

### right whale lung and skin fibroblasts with 0, 0.5, 1.0, 2.0, and 4.0 μ g/cm 2 

### Pb chromate for 13 

### 24 hours. Wise et al. ( 2009 ) treated primary Steller sea lion lung fibroblasts with 0, 0.1, 14 

### 0.5, 1 and 5 μ g/cm 2 

### Pb chromate for 24 hours. Wise et al. ( 2011 ) treated primary sperm 15 

### whale skin fibroblasts with 0, 0.5, 1, 3, 5, and 10 μ g/cm 2 

### Pb chromate for 24 hours. In all 16 

### three studies, Pb chromate induced statistically significant, concentration

- dependent 17 

### increases in chromosomal aberrations. 18 

### In summary, exposure of various cell models and an in vivo model to Pb (acetate, 19 

### chromate, or nitrate) induced significant increases in chromosomal aberration that often 20 

### responded in a concentration dependent manner. The use of various cell lines deficient in 21 

### specific DNA repair enzymes helpe d to elucidate which pathways may be most sensitive 22 

### to Pb

- dependent chromosomal aberration. 23



### COMET Assay Multiple studies considered the ability of Pb to induce DNA single strand breaks in 24 

### laboratory animals using the comet assays. The COMET assay measures DNA damage 25 

### assessed by single cell electrophoresis of a lysed cell and measurement of the fragmented 26 

### DNA or tail length. Xu et al. ( 2008 ) considered the ability of Pb

- acetate to induce DNA 27 

### damage measured by the comet assay in lymphocytes of male ICR mice. Animals (5 per 28 

### group) were given Pb

- acetate by gavage at doses of 0, 10, 50, or 100 mg /kg body weight 29 

### every other day for 4 weeks. Pb exposure statistically significantly increased both tail 30 

### length and t ail moment in a dose

- dependent manner. Nava

- Hernandez et al. ( 2009 ) 31 

### considered the ability of Pb

- acetate to induce DNA damage in primary spermatocyte 32 

### DNA of male Wista r rats. Animals (3 per group) were treated for 13 weeks with 0, 250 or 33 

### 500 mg /L Pb in their drinking water. There was statistically significantly less DNA 34 

### damage in the controls compared to the two treatment groups. Narayana and Al

- Bader 35February 2012 5

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### ( 2011 ) considered the ability of Pb nitrate to induce DNA damage in liver tissue of adult 1 

### male Wistar rats. Animals (8 per group) were treated for 60 days with doses of 0, 0.5 or 2 

### 1% Pb(NO 3 

### )2 in their drinking water. There were no statistical differences between 3 

### t reated groups and controls. Drosophila melanogaster larvae (72 hours old) exposed to Pb 4 

### nitrate (2 , 000, 4 , 000, and 8 , 000 μ M in culture media for 24 hours) yielded haemocytes 5 

### that tested positive in the comet assay; Pb chloride (8 , 000 μ M) did not cause DNA 6 

### damage with the comet assay ( Carmona et al. )

. 7


### Other studies used the COMET assay in cultured human cells. Pasha Shaik et al. ( 2006 ) 8 

### treated primary human peripheral blood lymphocytes obtained from healthy, nonsmoking 9 

### donors with 0, 2.1, 2.4, 2.7, 3.0, 3.3 Pb nitrate for 2 hours and found dose

- dependent 10 

### increases in Comet tail length. Concerns with the study are that apparently no untreated 11 

### control was used. It also appears from the data presentation that only five subjects were 12 

### used; one for each dose. Experiments were not repeated. Thus, given the small number of 13 

### subjects and the absence of a negative control, this study may only be detecting 14 

### background levels. Xie et al. ( 2008 ) treated BJhTERT cells (hTERT

- immortalized human 15 

### sk in fibroblasts) and ATLD

- 2 cells (hTERT

- immortalized human skin fibroblasts 16 

### deficient in Mre11) with 0, 0.1, 0.5, and 1 μ g/cm 2 

### Pb chromate for 24 hours. Mre11 is a 17 

### component of the MRN complex and plays a role in telomere maintenance and double

18 

### strand brea k repair. Pb chromate induced a concentration

- dependent increase in DNA 19 

### double strand breaks measured by the comet assay. In another study, Pb nitrate exposure 20 

### (30 μ g /mL) induced statistically significant increased DNA damage in human liver 21 

### HepG2 cells that was statistically significantly attenuated with co

- exposure with the 22 

### antioxidant NAC (500 μ M) ( Yedjou et al., 2010 )

. 23 

### Other studies used the comet assay to examine Pb

- induced DNA single strand breaks in 24 

### rodent cell cultures. Xu et al. ( 2006 ) , treated PC12 cells with 0, 0.1, 1 or 10 μ M 25 

### Pb

- acetate. Both tail length and tail moment statistically significantly increased in a 26



### conce ntration

- dependent manner. Kermani et al. ( 2008 ) exposed mouse bone marrow

27 

### mesenchymal stem cells to 60 μ M Pb

- acetate for 48 hour s. There was an increase in 28 

### several comet assay measurements including tail length. 29



### Other Assays Other studies considered the ability of Pb to induce DNA double strand breaks by 30 

### measuring gamma

- H2A.X foci formation in cultured human cells. Xie et al. ( 2008 ) 31 

### treated BJhTERT cells (hTERT

- immortalized human skin fibroblasts) and ATLD

- 2 cells 32 

### (hTERT

- immortalized human skin fibroblasts deficient in Mre11) with 0, 0.1, 0.5, and 33 

### 1 μ g/cm 2 

### Pb chromate for 24 hours. Pb chromate induced a concentration

- dependent 34 

### increase in DNA double strand breaks measured by gamma

- H2A.X foci formation. 35February 2012 5

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### Gastaldo et al. ( 2007 ) e valuated the ability of Pb to induce DNA double strand breaks 1 

### with both gamma

- H2A.X foci formation and pulse

- field gel electrophoresis in cultured 2 

### human cells. The human endothelial HMEC cell line was treated with 1 to 1,000 μ M Pb 3 

### nitrate for 24 hours. DNA double strand breaks increased in a concentration

- dependent 4 

### manner. Wise et al. ( 2010 ) treated WHTBF

- 6 cells with 0, 0.1, 0 .5, or 1 μ g/cm 2 

### Pb 5 

### chromate for 24 hours in a study comparing four chromate compounds. Pb chromate 6 

### induced statistically significant, concentration

- dependent increases in DNA double strand 7 

### breaks measured by gamma

- H2A.X foci formation, at a similar level t o the other three 8 

### compounds. Two studies demonstrated the ability of Pb to destabilize DNA by forming 9 

### DNA

- histone cross links, which can lead to histone aggregation. Extracts of rat liver co

10 

### incubated with Pb nitrate (<300 μ M) were shown to react with chro matin components 11 

### and induce chromatin aggregation via histone

- DNA cross links ( Rabbani

- Chadegani et 12 

### al., 2011 ; Rabbani

- Chadegani et al., 2009 )

. 13 

### Genotoxicity testing of Drosophila melanogaster larvae (72 hours old) using the Wing 14 

### Spot test showed that neither Pb chloride nor Pb nitrate (at concentrations of 2 , 000, 4 , 000 15 

### and 8 , 000 μ M in culture media with exposure until pupation) was able to induce 16 

### significant increases in the frequency of wing spots ( Carmona et al. )

. Further, wing spot 17 

### assays employing Pb co

- exposure with gamma radiation showed no effect of Pb on 18 

### gamma radiation induced spotting frequency. The wing spo t test can detect mitotic 19 

### recombination and multiple mutational events such as point mutations, deletions, and 20 

### certain types of chromosome aberrations ( Graf and Würgler, 19 86 )

. 21 

### Multiple studies considered Pb and DNA repair. Most were conducted in cultured cells, 22 

### and one was done in an animal model. El

- Ghor et al. ( In Press ) followed microsatellite 23 

### instability (MSI) in Pb

- acetate trihydrate exposed adult male rats; MSI reflects impaired 24 

### DNA mismatch repair, and contributes to an increased risk of cancer. DNA from 25 

### leukocytes of male albino rats exposed to Pb

- acetate (acute: single oral dose of 26 

### 467 mg /kg BW or sub

- chronic: 47 mg /kg BW six d/wk for 4 wk) showed increased MSI 27 

### at three microsatellite loci (D6mit3, D9 mit2, and D15Mgh1). This study is limited by its 28 

### small sample size (n=2 to 3 rodents per treatment group). Li et al., ( 2008a ) , evaluated 29 

### Pb

- acetate

- induced effects on nucleotide excision repair efficiency in CL3 cells. All cells 30 

### were exposed to 0, 100, 300 or 500 μ M Pb

- acetate for 24 hours in serum

- free medium. 31 

### Pb increased nucleotide excision repair efficiency. Gastaldo et al. ( 2007 ) evaluated the 32 

### ability of Pb to affect DNA repair in cultured human cells. The human endothelial HMEC 33 

### cell line was treated with 100 μ M Pb nitrate for 24 hours. Pb inhibi ted non
- homologous 34 

### end joining (NHEJ) repair, over activated MRE11

- dependent repair and increased Rad51

- 35 

### related repair. Xie et al. ( 2008 ) treated BJhTERT cells (hTERT

- immortalized human skin 36 

### fibroblasts) and ATLD

- 2 cells (hTERT

- immortalized h uman skin fibroblasts deficient in 37 

### Mre11) with 0, 0.1, 0.5, and 1 μ g/cm 2 

### Pb chromate for 24 or 120 hours. Mre11 was 38February 2012 5

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### required to prevent Pb chromate

- induced DNA double strand breaks. McNeill et al, 1 

### ( 2007 ) considered Pb

- acetate effects on Ape1. Chine se hamster ovary cells (AA8) were 2 

### treated with 0, 0.5, 5, 50, or 500 μ M Pb

- acetate and then whole cell extracts were used to 3 

### determine AP site incision activity. The data show that Pb reduced AP endonuclease 4 

### function. Finally, studies considered Pb

- induced cellular proliferation in laboratory 5 

### animals. An earlier study in rats showed Pb nitrate

- induced increased proliferation of 6 

### liver cells after a partial hepatectomy, effects that were more prominent in males than 7 

### females (sexual dimorphism) ( Tessitore et al., 1995 )

. The newer studies showed similar 8 

### trends in males. Fortoul et al. ( 2005 ) exposed adult male CD1 mice (24 animals per 9 

### group) to 0.01 M Pb

- acetate , 0.006 M cadmium chloride or a mixture of the two 10 

### chemicals for 1 h twice a week for 4 weeks by inhalation. The lungs were then examined 11 

### by electron microscopy for changes. Pb induced cellular proliferation in the lungs. 12 

### Kermani et al. ( 2008 ) exposed mouse bone marrow

- mesenchymal stem cells to 0

- 100 μ M 13 

### Pb

- acetate for 48 hours. As measured by the MTT assay, Pb decreased cell proliferation 14 

### at all concentrations tested. 15



### 5.10.4 Effects of Lead within Mixtures
Several studies considered the impact of mixtures with Pb. All considered genotoxicity. 16 

### Mendez

- Gomez et al., ( 2008 ) , evaluated 65 children from Mexico exposed to both 17 

### arsenic and Pb. DNA damage and decreased DNA repair were seen using the comet assay 18 

### and other assays, but did not correlate with urinary arsenic or blood Pb levels. Tapisso et 19 

### al., ( 2009 ) , considered Pb alone, Pb plus zinc and Pb plus cadmium

- induced MN in 20 

### rodents. Algerian mice (groups of six mice each) were exposed (i.p.) to 5 or 10 doses of 21 

### 0.46 mg /kg Pb

- acetate and compared to untreated controls. The MN in bone marrow 22 

### were elevated after Pb treatment alone and increased with time. Co

- exposure with 23 

### cadmium or zinc did not further increase MN levels but did increase SCE levels. Glahn et 24 

### al., ( 2008 ) performed a gene array study in primary normal human bronchial epithelial 25 

### cells from four donors treated with 550 μ g/L Pb chloride, 15 μ g/L cadmium sulf ate, 26 

### 25 μ g/L cobalt chloride or all three combined for 72 hours. There was a clear interaction 27 

### of all three metals impacting RNA expression. 28 

### No recent studies of the protective role of calcium in Pb

- dependent carcinogen esis or 29 

### genotoxicity were found. There were some data suggesting that boron, melatonin, 30 

### N

- acetylcysteine, turmeric and myrrh protect cells against Pb

- induced genotoxicity 31 

### ( Sections 5.10.3.2 and 5.10.6 ). 32 

### A new study details Pb and selenium interactions in v irus

- dependent carcinogenesis in 33 

### laboratory animals. Schrauzer ( 2008 ) considered the impact of selenium on 34February 2012 5

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### carcinogenesis by studying 4 groups of weanling virgin female C3H/St mice infected 1 

### with murine mammary tumor virus (groups of 20

- 30 mice), w hich induces mammary 2 

### tumor formation. One set of two groups were fed a diet containing 0.15 ppm selenium 3 

### and then were exposed via drinking water to acetic acid (control group) or 0.5 ppm 4 

### Pb

- acetate (treated group). The second set of two groups were fed a diet containing 5 

### 0.65 ppm selenium and then similarly exposed to acetic acid or 0.5 ppm Pb

- acetate . The 6 

### study was primarily focused on the general effects of a low selenium diet. The data 7 

### suggest that selenium is anticarcinogenic as in the groups without Pb exposure, the 8 

### animals exposed to the higher selenium levels had fewer mammary tumors and these 9 

### tumors had a delayed onset of appearance. Pb exposure with low selenium caused the 10 

### same delayed onset as did the higher dose of selenium and also caused some re duction in 11 

### the tumor frequency. Pb exposure with higher selenium increased the tumor frequency 12 

### and the onset of the tumors. Pb also induced weight loss at 14 months in both exposed 13 

### groups. The data suggest that there may be interactions of Pb and selenium, but they 14 

### suggest that Pb mimics or antagonizes selenium. They do not suggest that selenium is 15 

### protective of Pb

- induced toxicity or carcinogenesis. 16



### 5.10.5 Modes of Action
The carcinogenic mechanism of action of Pb is poorly understood. It is unclear whether 17 

### the m echanism of action of Pb is best understood within the framework of multistage 18 

### carcinogenesis, genomic instability or epigenetic modification. For example, multistage 19 

### carcinogenesis involves a series of cellular and molecular changes that result from the 20 

### p rogressive accumulation of mutations that induce alterations in cancer

- related genes. Pb 21 

### does not appear to follow this paradigm and the literature suggests it is weakly 22 

### mutagenic. Pb does appear to have some ability to induce chromosomal mutation and 23 

### DNA damage, i.e., clastogenicity. However, the ability of Pb to alter gene expression 24 

### (epigenetic effects) and to interact with proteins may be a means by which Pb induces its 25 

### carcinogenicity. It is known that Pb can replace zinc in zinc

- binding (zinc

- finger) 26 

### proteins, which include hormone receptors, cell

- cycle regulatory proteins, the Ah 27 

### receptor, estrogen receptor, p53, DNA repair proteins, protamines, and histones. These 28 

### zinc

- finger proteins all bind to specific recognition elements in DNA. Thus, Pb may act at 29 

### a post

- translational stage to alter protein structure of Zn

- finger proteins, which can in turn 30 

### alter gene expression, DNA repair and other cellular functions. To recapitulate, cancer 31 

### develops from one or a combination of multiple mechanisms including m odification of 32 

### DNA via epigenetics or enzyme dysfunction and genetic instability or mutation(s). These 33 

### modifications then provide the cancer cells with a selective growth advantage. In this 34 

### schematic, Pb appears to contribute to epigenetic changes, and chr omosomal aberrations. 35February 2012 5

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### The genomic instability paradigm requires a cascade of genome

- wide changes caused by 1 

### interfering with DNA repair, kinetochore assembly, cellular checkpoints, centrosome 2 

### duplication, microtubule dynamics or a number of cell maintenance processes. These 3 

### processes have been rarely studied for Pb, thus there are few data that suggest Pb may 4 

### interfere with some of these processes. Furthermore, the bulk of the literature in this area 5 

### involves Pb chromate and it is unclear if the effects are due to Pb or chromate. Epigenetic 6 

### modifications can lead to cancer by altering cellular functions without altering the genetic 7 

### material. The most commonly studied epigenetic change is methylation alterations. A 8 

### small number of studies show that Pb can indu ce epigenetic changes, but studies are still 9 

### missing to clearly tie these effects to Pb

- induced carcinogenesis and genotoxicity. Thus, 10 

### the mechanism is difficult to define but, if Pb is a human carcinogen, the mechanism 11 

### likely involves either genomic insta bility or epigenetic modification paradigms or some 12 

### combination of the two. However, Pb

- dependent carcinogenicity is it not likely to occur 13 

### by a multistage paradigm. More work is needed to determine the mechanism. 14 

### Exposure to mixtures can also contribute to understanding of modes of action. No recent 15 

### studies of the protective role of calcium or zinc in Pb

- dependent carcinogenesis or 16 

### genotoxicity were found. There were some data suggesting that metallothionein protects 17 

### rodents from Pb

- induced cancers. There were some data suggesting that boron, 18 

### melatonin, N

- acetylcysteine, turmeric and myrrh protected cells against Pb

- induced 19 

### genotoxicity. There were some data suggesting that Pb mimics or antagonizes selenium 20 

### in rodents. These data are discussed in more deta il elsewhere in the cancer section and 21 

### point to the relevance of mixtures in assessing toxicity. 22



### 5.10.5.1 Epigenetics
Air pollution exposure is being linked increasingly with epigenetic changes ( Pavanello et 23 

### al., 2010 ; Baccarelli and Bollati, 20 09 ; Tarantini et al., 2009 ; Bollati et al., 2007 )

. 24 

### Epigenetic changes involve changes in DNA expression without actual changes in the 25 

### DNA sequence and these changes may be heritable. Epigenetic changes are mediated by 26 

### histone modification, DNA methylation, miRNA changes , or pathways that affect these 27 

### three mediators. Differential epigenetic modification has the possibility to contribute to 28 

### disease. Epigenetic studies have been conducted to examine the associations between Pb 29 

### biomarker levels and global DNA methylation ma rkers [Alu and long interspersed 30 

### nuclear element

- 1 (LINE

- 1)] in humans ( Wright et al., 2010 ; Pilsner et al., 2009 )

. Wright 31 

### et al. ( 2010 ) utilized a sample of participants from the NAS with mean (SD) Pb levels of 32 

### 20.5(14.8)g/g for tibia measur es, 27.4 (19.7)g/g for patella measures, and 4.1 (2.4) μ g/dL 33 

### for blood measures. In both crude and adjusted analyses, patella Pb levels were inversely 34 

### associated with LINE

- 1 methylation but not with Alu, both of which are indicators of 35February 2012 5

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### global methylation. When examining the relationship between patella Pb and LINE

- 1 1 

### more closely, a non
- linear trend was observed with a smaller magnitude of effect 2 

### estimated for higher patella Pb ( ≥ 4 0 μ g/g). No associations were observed for tibia or 3 

### blood Pb and either LINE

- 1 or Alu. Another study included maternal

- infant pairs from 4 

### the Early Life Exposures in Mexico to Environmental Toxicants study and measured 5 

### LINE

- 1 and Alu methylation in umbilical cord blood samples ( Pilsner et al., 2009 )

. In 6 

### unadjusted models, maternal pregnancy tibia Pb levels [mean (SD) 10.5 (8.4) μ g/g] were 7 

### inversely associated with Alu methylation; maternal patella Pb levels [mean (SD) 12.9 8 

### (14.3) μ g/g] were inversely associated with LINE

- 1 methylation. The associations 9 

### persisted in adjusted models although the association between patella Pb and LINE

- 1 was 10 

### only apparent when the adjusted models also included umbilical cord blood Pb levels. No 11 

### association was detected between umbilical cord Pb levels and the DNA methylati on 12 

### markers. Overall, the studies consistently demonstrate an association between patella Pb 13 

### levels and LINE

- 1 methylation. 14 

### Toxicological studies have been performed examining Pb

- dependent epigenetic changes 15 

### and gene expression, DNA repair, and mitogenesis . Glahn et al., ( 2008 ) performed a gene 16 

### array study in primary normal human bronchial epithelial cells from four donors after in 17 

### vitro treatment of the cells with 550 μ g/L Pb chloride, 15 μ g/L cadmium sulfate, 25 μ g/L 18 

### cobalt chloride or all three combined fo r 72 hours. The authors describe a pattern of RNA 19 

### expression changes indicating “ ...coordinated stress

- response and cell

- survival signaling, 20 

### deregulation of cell proliferation, increased steroid metabolism, and increased expression 21 

### of xenobiotic metaboliz ing enzymes.” These are all known targets of possible epigenetic 22 

### changes, but full interpretations of the data as epigenetic changes are complicated by the 23 

### absence of a measure to determine if these changes were a result of genotoxic effects. A 24 

### recent publ ication using HepG2 cells in tissue culture showed that cells exposed to a high 25 

### dose of Pb (100 μ M Pb

- acetate ) experienced ALAD gene promoter hypermethylation and 26 

### decreased ALAD transcription. This was in agreement with findings in battery plant 27 

### workers who showed ALAD hypermethylation (versus non
- occupationally exposed 28 

### controls) and an association of this hyperm ethylation with elevated risk of Pb poisoning 29 

### ( Li et al., 2011 )

. 30

31



### 5.10.6 Summa ry and Causal Determination
In summary, the toxicological literature on the genotoxic, mutagenic, and carcinogenic 32 

### potential of Pb provide strong evidence of effects in laboratory animals. In laboratory 33 

### studies, high

- dose Pb has been demonstrated to be an animal carcinogen. There are data 34February 2012 5

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### to suggest Pb is a human carcinogen among toxicological studies, but they are not 1 

### definitive. Multiple toxicological studies showed neoplastic transformation in cultured 2 

### cells, but all focused on Pb chromate and the positi ve response to the chromate ion and 3 

### not the Pb likely contributed to these findings. Multiple epidemiologic studies have been 4 

### performed examining the association between cancer incidence and mortality and Pb 5 

### exposures, estimated with biological measures an d exposure databases. Mixed results 6 

### have been reported fo r cancer mortality studies; a large NHANES epidemiologic study 7 

### demonstrated a positive association between blood Pb and cancer mortality, but the other 8 

### studies reported null results. Although the 200 6 Pb AQCD reported some studies that 9 

### found an association between Pb exposure indicators and lung cancer, current studies 10 

### mostly included occupationally

- exposed adults and observed no associations. Most 11 

### studies of Pb and brain cancer were null among the ov erall study population, but positive 12 

### associations were observed among individuals with certain genetic variants. However the 13 

### studies of Pb and brain cancer were all performed among occupational cohorts using 14 

### interviews instead of biological measurements to represent Pb exposure. A limited 15 

### amount of research has been performed on other types of cancer. The 2006 Pb AQCD 16 

### reported evidence that suggested an association between Pb exposure and stomach 17 

### cancer, but recent studies of stomach cancer are lacking. One study examining Pb and 18 

### stomach cancer has been performed since the last AQCD and the results of the study are 19 

### mixed 20 

### Among epidemiologic studies, high Pb levels were associated with SCEs among adults 21 

### but not children. Other epidemiologic studies of DNA dam age reported inconsistent 22 

### results. Consistent with previous toxicological findings, Pb does appear to have genotoxic 23 

### activity in animal and in vitro models, inducing SCE, MN and DNA strand breaks, but 24 

### continues to not produce chromosomal aberrations except for Pb chromate; this again is 25 

### likely due to the chromate. Pb does not appear to be very mutagenic as the HPRT assays 26 

### were typically negative unless a cell signaling pathway was disturbed. 27 

### Mechanistic understanding of the carcinogenicity of Pb is expandi ng with work on the 28 

### antioxidant selenium and metallothionein, a protein that binds Pb and reduces its 29 

### bioavailability. Metallothionein has been shown to be protective against the effect of Pb 30 

### on carcinogenicity. Pb is clastogenic and mutagenic in some but not all models. 31 

### Clastogenicity and mutagenicity may be possible mechanisms contributing to cancer but 32 

### are not absolutely associated with the induction of cancer. Because Pb has a higher 33 

### atomic weight than does zinc, Pb replaces zinc at many zinc binding si tes or zinc finger 34 

### proteins. This substitution has the potential to induce indirect effects that can contribute 35 

### to carcinogenicity via interactions at hormone receptors, at cell

- cycle regulatory proteins, 36 

### with tumor suppressor genes like p53, with DNA repa ir enzymes, with histones, etc. 37February 2012 5

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### These indirect effects may act at a post

- translational level to negatively alter protein 1 

### structure and DNA repair. 2 

### Epigenetic changes associated with Pb exposure, particularly with respect to methylation 3 

### and effects on DNA r epair, are beginning to appear in the literature. These modifications 4 

### may further alter DNA repair or change the expression of a tumor suppressor gene or 5 

### oncogene. A small number of epidemiologic studies examining Pb and global epigenetic 6 

### changes including LINE

- 1 and Alu demonstrated an inverse association between patella 7 

### Pb and LINE

- 1 methylation, an emerging area of research. Toxicological studies show 8 

### that Pb can activate or interfere with a number of signaling and repair pathways, though it 9 

### is unclear w hether these are due to epigenetic responses or genotoxicity. Thus, an 10 

### underlying mechanism is still uncertain, but likely involves either genomic instability, 11 

### epigenetic modifications, or both. 12 

### In conclusion, the toxicological literature provides the strongest evidence for Pb exposure 13 

### and cancer with supporting evidence provided by the epidemiologic literature. This is 14 

### substantiated by the findings of other agencies including IARC, which has classified 15 

### inorganic Pb compounds as a probable human carcino gen and the National Toxicology 16 

### Program, which has listed Pb and Pb compounds as “reasonably anticipated to be human 17 

### carcinogens.” Strong evidence from toxicological studies demonstrates an association 18 

### between Pb and cancer, genotoxicity/clastogenicity or epigenetic modification. 19 

### Carcinogenicity in historical animal toxicology studies with Pb exposure has been 20 

### reported in the kidneys, testes, brain, adrenals, prostate, pituitary, and mammary gland, 21 

### albeit at high doses of Pb. Epidemiologic studies of cancer incidence and mortality 22 

### reported inconsistent results; one strong epidemiologic study demonstrated an association 23 

### between blood Pb and increased cancer mortality, but the other studies reported weak or 24 

### no associations. In the 2006 Pb AQCD, Pb exposure was found to be associated with 25 

### stomach cancer, but there was only one recent study on stomach cancer and Pb exposure, 26 

### which reported mixed findings. Similarly, some studies in the 2006 Pb AQCD reported 27 

### associations between Pb exposure and lung cancer. More r ecent epidemiologic studies of 28 

### lung cancer focused on occupational exposures and reported no associations. The 29 

### majority of epidemiologic studies of brain cancer had null results overall, but positive 30 

### associations between Pb exposure and brain cancer were o bserved among individuals 31 

### with certain genotypes. In toxicological studies, chromosomal aberrations after Pb 32 

### exposure are most often reported with Pb chromate exposure, which is likely due to 33 

### toxicity of the chromate moiety. Mechanistic understanding of Pb and its effect on cancer 34 

### and genotoxicity is expanding through toxicological work focusing on antioxidants and 35 

### other proteins that sequester Pb or reduce its bioavailability. The collective body of 36 

### evidence integrated across toxicological and epidemiologi c studies is sufficient to 37 

### conclude that there is a likely causal relationship between Pb exposure and cancer

. 38February 2012 5

- 539 Draft

– Do Not Cite or Quote 1

2

3



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