May 2011 i DRAFT

– DO NOT CITE OR QUOTE 

# Chapter 5 Contents

Chapter 5. Integrated Health Effects of LeadExposure

________________________________ _____________________ 5

- 1
5.1. Introduction

________________________________ ________________________________ _______________ 5

- 1
5.2. Modes of Action

________________________________ ________________________________ ___________ 5

- 2
5.2.1. Introduction

________________________________ ________________________________ _______ 5

- 2
Figure 5

- 1. Schematic representation of the relationships between the various MOAs by which Pb exerts its toxic effects.

________________________________ _______ 5

- 2 5.2.2. Altered Ion Status

________________________________ ________________________________ ___ 5

- 3
5.2.2.1. Disruption of Ca 2+ Homeostasis

________________________________ ________________ 5

- 3
5.2.2.2. Disr uption of Ion Transport Mechanisms

________________________________ _________ 5

- 5
5.2.2.3. Displacement of Metal Ions and Perturbed Protein Function

__________________________ 5

- 8

Table 5

- 1. Enzymes and proteins potentially affected by exposure to Pb and the metal cation cofactors necessary for their proper physiological activity

____________ 5

- 12 5.2.2.4. Mitochondrial Abnormality

________________________________ __________________ 5

- 12
5.2.3. Protein Binding

________________________________ ________________________________ ___ 5

- 15
5.2.3.1. Intranuclear and Cytoplasmic Inclusion Bodies

________________________________ ___ 5

- 15
5.2.3.2. Cytosolic Lead Binding Proteins

________________________________ ______________ 5

- 16
5.2.3.3. Erythrocytic Lead Binding Proteins

________________________________ ____________ 5

- 17
5.2.3.4. Metallothionein

________________________________ ___________________________ 5

- 18
5.2.4. Oxidative Stress

________________________________ ________________________________ ___ 5

- 19
5.2.4.1. δ

- ALA Oxidation

________________________________ __________________________ 5

- 20
5.2.4.2. Membrane and Lipid Peroxidation

________________________________ _____________ 5

- 20
5.2.4.3. NAD( P)H Oxidase Activation

________________________________ ________________ 5

- 22
5.2.4.4. Antioxidant Enzyme Disruption

________________________________ _______________ 5

- 22
5.2.4.5. Nitric Oxide Signaling

________________________________ ______________________ 5

- 24
5.2.5. Inflammation

________________________________ ________________________________ _____ 5

- 25
5.2.5.1. Cytokine Production

________________________________ ________________________ 5

- 26
5.2.6. Endocrine Disruption

________________________________ _______________________________ 5

- 28
5.2.6.1. Hypothalamic

- Pituitary

- Gonadal Axis

________________________________ __________ 5

- 28
5.2.6.2. Hypothalamic

- Pituitary

- Thyroid Axis

________________________________ __________ 5

- 29
5.2.7. Ce ll Death and Genotoxicity

________________________________ _________________________ 5

- 30
5.2.7.1. DNA Damage

________________________________ _____________________________ 5

- 30
5.2.7.2. Mutagenicity

________________________________ ______________________________ 5

- 32
5.2.7.3. Clastogenicity

________________________________ _____________________________ 5

- 33
5.2.7.4. Epigenetic Effects

________________________________ __________________________ 5

- 38
5.2.7.5. Gene Expression

________________________________ ___________________________ 5

- 39
5.2.7.6. Apoptosis

________________________________ ________________________________ 5

- 40
5.2.8. Summary

________________________________ ________________________________ ________ 5

- 40

Table 5

- 2. Related health effects resul ting from the MOAs of Pb and the lowest level eliciting the MOA reported as blood Pb level and dose delivered

_____________ 5

- 41 5.3. Neurological Effects

________________________________ ________________________________ _______ 5

- 43
5.3.1. Introduction

________________________________ ________________________________ ______ 5

- 43
5.3.2. Neurocognitive Function and Learning

________________________________ _________________ 5

- 46
5.3.2.1. Epidemiologic Studies of Cognitive Function in Children

___________________________ 5

- 46
Figure 5

- 2. Associations of blood Pb levels with full

- scale IQ (FSIQ) a mong children.

_____ 5

- 48

Table 5

- 3. Additional characteristics and quantitative results for studies presented in Figure 5

- 2

________________________________ _______________________ 5

- 49 Figure 5

- 3. Effect modification of association between concurrent blood Pb level and IQ by blood Mn level.

________________________________ _________________ 5

- 51May 2011 ii DRAFT

– DO NOT CITE OR QUOTE Figure 5

- 4. Associations of blood Pb levels with standardized scores for specific indices of cognitive function in children.

________________________________ ______ 5

- 54

Table 5

- 4. Additional characteristics and quantitative results for studies presented in Figure 5

- 4

________________________________ _______________________ 5

- 54 Figure 5

- 5. Comparing model results for 4th

- grade EOG mathematics scores.

____________ 5

- 59 Figure 5

- 6. Reduction in EOG achievement test scores at each percentile of the test distribution.

________________________________ ______________________ 5

- 60 Figure 5

- 7. Associations of blood Pb measures at various lifestages with cognitive function in children.

________________________________ _______________ 5

- 63 Figure 5

- 8. Associations of Pb biomarkers at various lifestages with behavioral indices in children.

________________________________ ______________________ 5

- 64

Table 5

- 5. Additional characteristics and quantitative results for studies presented in Figures 5

- 7 and 5

- 8

________________________________ ________________ 5

- 65 Figure 5

- 9. Regress ion of fitted MDI score at 36 months on log

- transformed concentration of cord blood Pb level by sex.

_____________________________ 5

- 68 Figure 5

- 10. Associations of cognitive function in children with different degrees of changes in blood Pb levels over time.

________________________________ __ 5

- 70

Table 5

- 6. Additional chara cteristics and quantitative results for studies presented in Figure 5

- 10

________________________________ ______________________ 5

- 71 Figure 5

- 11. Estimated IQ in combined Cincinnati and Rochester cohorts for 3 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 level (white squares).

________________________________ _______________ 5

- 73 Figure 5

- 12. Neurological summary array of toxicological outcomes after Pb exposure. Dosimetric representation reported by blood Pb level. (ID corresponds to Table 5

- 7.)

________________________________ _______________________ 5

- 74

Table 5

- 7. Summary of findings from neurotoxicological exposure

- response array presented in Figure 5

- 12.

________________________________ ___________ 5

- 75 5.3.2.2. Toxicological Studies of Neurocognition, Memory and Learning

_____________________ 5

- 75
Figure 5

- 13. Mean basal corticosterone levels of female and male offspring exposed to lifetime Pb (0, 50, 150 ppm) and/or stress (PS (dam stress) or OS (offspring stress)).

________________________________ _________________________ 5

- 79 Figure 5

- 14. Changes in FI performance (FI overall performance, run rate, PRP) in female offspring with maternal Pb exposure with various s tressors in adulthood (restraint, cold, novelty).

________________________________ ___ 5

- 81 Figure 5

- 15. Changes in FI performance (FI overall performance, run rate, PRP ) in male offspring with maternal Pb exposure with various stressors in adulthood (restraint, cold, novelty).

________________________________ ____________ 5

- 81

Table 5

- 8. Summar y of effects of maternal and lifetime Pb exposure on FI performance

water a

.

________________________________ __________________________ 5

- 83 5.3.2.3. Toxicological Studies on the Effects of Ch elation

________________________________ _ 5

- 84
5.3.2.4. Epidemiologic Studies of Cognitive Function in Adults

_____________________________ 5

- 86
Figure 5

- 16. Associations of blood and bone Pb levels with cognitive function among adults without occupational exposures to Pb.

____________________________ 5

- 88

Table 5

- 9. Additional characteristics and quantitative results for studies presented in Figure 5

- 16

________________________________ ______________________ 5

- 89 Figure 5

- 17. Exploration of nonlinear association of tibia Pb concentration with annual rate of cognitive decline, by class of HFE genotype.

_______________________ 5

- 94 Figure 5

- 18. Nonlinear association between patella bone Pb concentration and the relative change in response latency over time on the pattern comparison test (reference = 0 at mean of patella P b concentration).

______________________ 5

- 95 5.3.3. Neurobehavioral Effects

________________________________ _____________________________ 5

- 98
5.3.3.1. Epidemiologic Studies of Behavioral Effects in Children

___________________________ 5

- 98
Figure 5

- 1 9. Associations of blood Pb levels with behavioral indices in children.

__________ 5

- 100

Table 5

- 10. Additional characteristics and quantitative results for studies presented in Figure 5

- 19.

________________________________ ____________________ 5

- 100 Figure 5

- 20. Estimates and 95% CI of direct and indirect effects of concurrent blood Pb concentrations at age 7 on BASC

- TRS.

________________________________ 5

- 103May 2011 iii DRAFT

– DO NOT CITE OR QUOTE Figure 5

- 21. Estimates and 95% CI of direct and indirect effects of concurrent blood Pb concentrations at age 7 on BASC

- PRS.

________________________________ 5

- 103 Figure 5

- 22. Adjusted odds ratio for ADHD among U.S. children (ages 4

- 15 years) from NHANES 1999

- 2002 by quintile of blood Pb level.

_______________________ 5

- 104 5.3.3.2. Epidemiologic Studies of Behavior, Mood, and Psychiatric Effects in Adults

___________ 5

- 107
5.3.3.3. Toxicological Studies of Neurobehavioral Outcomes

_____________________________ 5

- 108
5.3.3.4. Toxicological Studies of Mood Alterations

________________________________ _____ 5

- 110
Figure 5

- 23. Animal toxicology evidence of possible Pb

- dependent contri butors to the development of mood disorders.

________________________________ _____ 5

- 111 Figure 5

- 24. Schematic representation of the contribution of Pb exposure to the development of a phenotype consistent with schizophrenia.

________________ 5

- 112 Figure 5

- 25. Neurogenesis (production of new cells) in the rat dentate gyrus after postnatal Pb exposure.

________________________________ ____________ 5

- 113 5.3.4. Se nsory Acuity

________________________________ ________________________________ ___ 5

- 114
5.3.4.1. Epidemiologic Studies of Children

________________________________ ____________ 5

- 114
5.3.4.2. Epidemiologic Studies of Adults

________________________________ _____________ 5

- 114
5.3.4.3. Toxicological Studies of Sensory Organ Function

________________________________ 5

- 116

Table 5

- 11. Summary of toxicological studies of Pb on the retina.

_____________________ 5

- 117 Figure 5

- 26. Retinal a

- wave and b

- wave ERG amplitude in G LE adult males.

____________ 5

- 118 Figure 5

- 27. Retinal dopamine metabolism in adult control and GLE rats.

_______________ 5

- 118 5.3.5. Neurodegenerative Diseases

________________________________ _________________________ 5

- 119
5.3.5.1. Epidemiologic Studies of Adults

________________________________ _____________ 5

- 119
5.3.5.2. Toxicological Studies

________________________________ ______________________ 5

- 122
5.3.6. Studies of Mechanisms of the Neurological Effects of Lead

________________________________ 5

- 126
5.3.6.1. Effects on Brain Physiology and Activity

________________________________ ______ 5

- 126
5.3.6.2. Cholesterol and Lipid Homeos tasis

________________________________ ___________ 5

- 129
5.3.6.3. Oxidative Stress

________________________________ __________________________ 5

- 12 9
5.3.6.4. Nitrosative Signaling and Nitrosative Stress

________________________________ ____ 5

- 131
5.3.6.5. Synaptic Changes

________________________________ _________________________ 5

- 131
5.3.6.6. Blood Brain Barrier

________________________________ _______________________ 5

- 133
5.3.6.7. Cell Adhesion Molecules

________________________________ ___________________ 5

- 134
5.3.6.8. Glial Effects

________________________________ _____________________________ 5

- 134
5.3.6.9. Neurotransmitters

________________________________ _________________________ 5

- 135
5.3.6.10. Neurite Outgrowth

________________________________ ________________________ 5

- 138
5.3.6.11. Epigenetics

________________________________ ______________________________ 5

- 139
5.3.7. Examination of the Lead Concentration

- Response Relationship

_____________________________ 5

- 139
Figure 5

- 28. Comparison of associations between blood Pb and cognitive function among various blood Pb strata.

________________________________ ___________ 5

- 141

Table 5

- 12. Additional characteristics and quantitative results for studies presented
in Figure 5

- 28

________________________________ ___________________ 5

- 141 5.3.8. Summary and Causal Determination

________________________________ __________________ 5

- 146
Figure 5

- 29. Snapshot of evidence for the spectrum of effects to the nervous system associated with Pb exposure.

________________________________ _______ 5

- 151 5.4. Cardiovascular Effects

________________________________ ________________________________ _____ 5

- 152
5.4.1. Introduction

________________________________ ________________________________ _____ 5

- 152
5.4.2. Blood Pressure and Hypertension

________________________________ ____________________ 5

- 153
5.4.2.1. Epidemiology

________________________________ ____________________________ 5

- 153
Figure 5

- 30. Slope of BP (mmHg) per μg/dL blood Pb level at 1 μg/dL or per 10 μg/g bone Pb (95% CI) for associations of blood Pb (closed circles) and bone Pb (open circles) with systolic BP (SBP; blue), diastolic BP (DBP; red), and pulse pressure (PP; purple).

________________________________ ________ 5

- 154

Table 5

- 13. Additiona l characteristics and quantitative data for associations of blood and bone Pb with BP measures for results presented in Figure 5

- 30

_________ 5

- 155 Figure 5

- 31. Odds ratio (95% CI) for associations of blood and bone Pb with hypertension measures.

________________________________ ____________ 5

- 158May 2011 iv DRAFT

– DO NOT CITE OR QUOTE Table 5

- 14. Additional characteristics and quantitative data for associations of blood and bone Pb with hypertension measures for results presented in Figure 5

- 31

________________________________ ____________________________ 5

- 159 Figure 5

- 32. The relationship between tibia Pb and estimated SBP for those with high self

- reported stress versus those with low self

- reported stres s.

______________ 5

- 162
5.4.2.2. Toxicology

________________________________ ______________________________ 5

- 165
Figure 5

- 33. Rat blood Pb levels reported to be associated with changes in SBP from the current ISA and 2006 Pb AQCD.

________________________________ ____ 5

- 167 Figure 5

- 34. Changes in BP after Pb exposure (as blood Pb level) in unanesthetized adult rats across studies.

________________________________ _______________ 5

- 168
5.4.2.3. Hypertension Mechanisms

________________________________ __________________ 5

- 168
5.4.2.4. Summary

________________________________ _______________________________ 5

- 174
Figure 5

- 35. Change in SBP, association size in mmHg with 95% CI, associated with a doubling in the blood Pb concentration.

_______________________________ 5

- 176 Figure 5

- 36. Prospective and cross

- sectional increase in SBP and DBP and relative risk of hypertension per 10 μg/g increase in bone Pb levels.

___________________ 5

- 178 5.4.3. Vascular Effects and Cardiotoxicity

________________________________ ___________________ 5

- 179
5.4.3.1. Effects on Vascular Cell Types

________________________________ ______________ 5

- 179
5.4.3.2. Cholesterol

________________________________ ______________________________ 5

- 181
5.4.3.3. Heart Rate Variability

________________________________ ______________________ 5

- 181
5.4.3.4. Peripheral Artery Disease

________________________________ ___________________ 5

- 182
5.4.3.5. Ischemic Heart Disease

________________________________ ____________________ 5

- 183
5.4.3.6. Atherosclerosis

________________________________ ___________________________ 5

- 184

Table 5

- 15. Characteristics and quantitative data for associations of blood and bone Pb with other CVD measures.

________________________________ __________ 5

- 185 5.4.3.7. Summar y

________________________________ _______________________________ 5

- 186
5.4.4. Mortality Associated with Long

- Term Lead Exposure

________________________________ ____ 5

- 186
Figure 5

- 37. Multivariate adjusted relative hazards of all

- cause and cardiovascular mortality.

________________________________ _______________________ 5

- 188 Figure 5

- 38. Multivariate adjusted relative hazard (left axis) of mortality associated with blood Pb level between 1 μg/dL and 10 μg/dL.

__________________________ 5

- 189 Figure 5

- 39. Relative risk of all cause mortality for different blood Pb levels compared with referent level of 1.5 μg/dL (12.5th percentile).

______________________ 5

- 190 Figure 5

- 40. Nonlinear association between patella bone Pb concentration and the log of HR (logHR) for all

- cause, cardiovascular, and ischemic heart disease adjusted for age, education, smoking status, and pack

- years of smoking among participants without ischemic heart disease at baseline.

_____________ 5

- 191 Figure 5

- 41. Multivariate adjusted relative hazard (left axis) of mortality associated with blood Pb levels between 1 μg/dL and 15 μg/dL.

_________________________ 5

- 192 5.4.4.1. Summary

________________________________ _______________________________ 5

- 192
Figure 5

- 42. Hazard ratios between blood Pb (closed markers), bone Pb (open markers), all

- cause mortality (diamonds), and cardiovascular mortality (circles).

_______ 5

- 194

Table 5

- 16. Additional characteristics and quantitative data for associations of blood and bone Pb with CVD mortality for results presented in Figure 5

- 42.

_______ 5

- 195 5.4.5. Air Lead

- PM Studies

________________________________ ______________________________ 5

- 196
5.4.5.1. Hospital Admissions

________________________________ _______________________ 5

- 196
5.4.5.2. Mortality

________________________________ ________________________________ 5

- 197
5.4.6. Summary and Causal Determina tion

________________________________ __________________ 5

- 197
5.5. Renal Effects

________________________________ ________________________________ ____________ 5

- 200
5.5.1. Introduction

________________________________ ________________________________ _____ 5

- 200
5.5.1.1. Kidney Outcome Measures

________________________________ _________________ 5

- 201
5.5.2. Nephrotoxicity and Renal Pathology Related to Lead Effects

_______________________________ 5

- 202
5.5.2.1. Toxicology

________________________________ ______________________________ 5

- 202

Table 5

- 17. Indicators of renal damage in male rats exposed to 50 ppm Pb for 40 and 65 days, starting at parturition

________________________________ _________ 5

- 204May 2011 v DRAFT

– DO NOT CITE OR QUOTE Table 5

- 18. Acute and chronic effects of Pb on the kidney/renal system

– evidence from animal toxicology studies.

________________________________ __________ 5

- 209 5.5.2.2. Epidemiology in Adults

________________________________ ____________________ 5

- 209
Figure 5

- 43. Kidney metric slopes on blood Pb or bone Pb.

__________________________ 5

- 212 Figure 5

- 44. Percent change for kidney outcomes associated with blood Pb.

_____________ 5

- 214

Table 5

- 19. Additional characteristics and quantitative data for associations of blood and bone Pb with kidney outcomes for results presented in Figures 5

- 43 and
5

- 44

________________________________ ___________________________ 5

- 215 Figure 5

- 45. Added variable plot of association between serum creatinine and blood Pb in 267 Korean Pb workers in the oldest age tertile.

______________________ 5

- 218

Table 5

- 20. Patient population studies: kidney function decline

_______________________ 5

- 220

Table 5

- 21. Clinical randomized chelation trials in chronic kidney disease patients

_______ 5

- 223 5.5.2.3. Epidemiology in Children

________________________________ __________________ 5

- 224
5.5.2.4. Associatio ns between Lead Dose and New Kidney Outcome Measures

_______________ 5

- 225
5.5.3. Mechanisms of Lead Nephrotoxicity

________________________________ __________________ 5

- 226
5.5.3.1. Altered Uric Acid

________________________________ _________________________ 5

- 226
5.5.3.2. Oxidative Damage

________________________________ ________________________ 5

- 227
5.5.3.3. Lead Effect on Ren al Gangliosides

________________________________ ___________ 5

- 229
5.5.3.4. Role of Metallothionein

________________________________ ____________________ 5

- 230
Figure 5

- 46. Dose

- responsive representation of the effect of Pb on renal outcomes in animal toxicology studies.

________________________________ __________ 5

- 231

Table 5

- 22. Additional characteristics for results of toxicological studies presented in Figure 5

- 46

________________________________ _____________________ 5

- 232 5.5.4. Effects of Exposure to Lead Mixtures

________________________________ _________________ 5

- 232
5.5.4.1. Lead and Cadmium

________________________________ _______________________ 5

- 233
5.5.4.2. Lead, Cadmium, and Arsenic

________________________________ ________________ 5

- 234
5.5.4.3. Lead and Zinc

________________________________ ____________________________ 5

- 235
5.5.4.4. Lead and Mercury

________________________________ ________________________ 5

- 235
5.5.5. Impact of Treatment with Antioxidants on Renal Lead Accumulation and Pathology

_____________ 5

- 236
5.5.5.1. Treatment with Antioxidants

________________________________ ________________ 5

- 236
5.5.5.2. Treatment with Antioxidants plus Chelators

________________________________ ____ 5

- 239
5.5.6. Summary and Causal Determination

________________________________ __________________ 5

- 240
5.6. Immune System Effects

________________________________ ________________________________ ____ 5

- 242
5.6.1. Introduction

________________________________ ________________________________ _____ 5

- 242
Figure 5

- 47. Immunological pathways by which Pb exposure may increase risk of
immune

- related diseases.

________________________________ __________ 5

- 243 5.6.2. Cell

- Mediated Immunity

________________________________ ___________________________ 5

- 245
5.6.2.1. T Cells 5

- 245
Figure 5

- 48. Comparisons of the relative abundance of CD3+ T cells among groups with increasing blood Pb level (μg/dL).

________________________________ ___ 5

- 247

Table 5

- 23. Comparison of serum abundance of T

- cell subtypes a among various blood Pb groups.

________________________________ ______________________ 5

- 247 5.6.2.2. Lymphocyte Activation

________________________________ ____________________ 5

- 248
5.6.2.3. Delayed

- type H ypersensitivity

________________________________ _______________ 5

- 249
5.6.2.4. Macrophages and Monocytes

________________________________ ________________ 5

- 250
5.6.2.5. Neutrophils

________________________________ ______________________________ 5

- 251
5.6.2.6. Dendritic Cells

________________________________ ___________________________ 5

- 252
5.6.2.7. Natural Killer (NK) Cells

________________________________ ___________________ 5

- 253
5.6.3. Humoral Immunity

________________________________ ________________________________ 5

- 253
Figure 5

- 49. Comparison of IgE levels among groups with increasing blood Pb level ( μ g/dL).

________________________________ ________________________ 5

- 256 Figure 5

- 50. Comparison of the relative abundance of B cells among groups with increasing blood Pb level ( μ g/dL).

________________________________ ___ 5

- 256

Table 5

- 24. Additional characteristics and quantitative data a for results presented in Figures 5

- 49 and 5

- 50

________________________________ _____________ 5

- 257May 2011 vi DRAFT

– DO NOT CITE OR QUOTE 5.6.4. Immune

- based Diseases

________________________________ ____________________________ 5

- 258
5.6.4.1. Host Resistance

________________________________ __________________________ 5

- 258
5.6.4.2. Asthma and Allergy

________________________________ _______________________ 5

- 259
Figure 5

- 51. Associations of blood Pb levels with immune

- based conditions.

_____________ 5

- 260

Table 5

- 25. Additional characteristics and quantitative data for results presented in Figure 5

- 51.

________________________________ ____________________ 5

- 261
5.6.4.3. Other Respiratory Effects

________________________________ ___________________ 5

- 262

Table 5

- 26. Associations of air

- Pb with respira tory effects

__________________________ 5

- 264
5.6.4.4. Autoimmunity

________________________________ ___________________________ 5

- 264
5.6.4.5. Specialized Cells in Other Tissues

________________________________ ____________ 5

- 265
Figure 5

- 52. Specialized macrophages in nonlymphoid tissue may serve as a significant link between Pb and disease in multiple organ systems.

___________________ 5

- 266
5.6.4.6. Tumors

________________________________ ________________________________ _ 5

- 267
5.6.5. Mechanisms of Lead

- Induced Immunomodulation

________________________________ _______ 5

- 267
5.6.5.1. Inflammation

________________________________ ____________________________ 5

- 267
5. 6.5.2. Increased Prostaglandin E 2 and Decreased Nitric Oxide

___________________________ 5

- 268
5.6.5.3. Cellular Death (Apoptosis, Necrosis)

________________________________ __________ 5

- 270
5.6.5.4. Cytokine Production

________________________________ _______________________ 5

- 270
5.6.6. Immune Effects of Lead within Mixtures

________________________________ _______________ 5

- 272
5.6.7. Summary and Causal Determination

________________________________ __________________ 5

- 273
5.7. Effects on Heme Synthesis and Red Blood Cell Function

________________________________ __________ 5

- 276
5.7.1. Summary of Findings from 2006 Pb AQCD

________________________________ ____________ 5

- 276
5.7.2. Effects on Red Blood Cell Functions

________________________________ __________________ 5

- 278
5.7.2.1. Lead Uptake, Binding, and Transport into Red Blood Cells

________________________ 5

- 278
5.7.2.2. Red Blood Cell Survival, Mobility, and Membrane Integrity

________________________ 5

- 278
5.7.2.3. Red Blood Cell Hematopoiesis

________________________________ _______________ 5

- 283
5.7.2.4. Membra ne Proteins

________________________________ ________________________ 5

- 283
5.7.3. Effects on Red Blood Cell Heme Metabolism

________________________________ ___________ 5

- 284
Figure 5

- 53. Schematic representation of the enzymatic steps involved in the heme synthetic pathway.

________________________________ ________________ 5

- 285 5.7.3.1. Red Blood Cell 5

- Aminolevulinic Acid Dehydratase

______________________________ 5

- 285
5.7.4. Other Heme Metabolism Enzymes

________________________________ ____________________ 5

- 287
5.7.5. Effects on Other Hematological Parameters

________________________________ _____________ 5

- 287
5.7.5.1. Energy Metabolism

________________________________ _______________________ 5

- 287
5.7.5.2. Other Enzymes

________________________________ ___________________________ 5

- 288
5.7.6. Red Blood Cell Oxidative Stress

________________________________ _____________________ 5

- 288
5.7.6.1. Oxidative Stress, Lipid Peroxidation, and Antioxidant Enzymes

_____________________ 5

- 289
5.7.6.2. Antioxidant Defense

________________________________ _______________________ 5

- 291
5.7.7. Summary and Causal Determination

________________________________ __________________ 5

- 291
5.8. Reproduc tive Effects and Birth Outcomes

________________________________ ______________________ 5

- 293
5.8.1. Effects on Female Reproductive Function

________________________________ ______________ 5

- 294
5.8.1.1. Effects on Female Sex Endocrine System and Estrus Cycle

________________________ 5

- 294
5.8.1.2. Effects on Fertility

________________________________ ________________________ 5

- 296

Table 5

- 27. Summary of recent epidemiologic studies of ef fects on fertility for females

_____ 5

- 296
5.8.1.3. Effects on Puberty

________________________________ ________________________ 5

- 298

Table 5

- 28. Summary of recent epidemiologic studies of effects on puberty for females

____ 5

- 298 Figure 5

- 54. Toxicological Exposure

- Response Array for Reproductive Effects of Pb.

______ 5

- 301

Table 5

- 29

. Toxicological Exposure

- Response Array Summaries for Reproductive Effects of Pb presented in Figure 5

- 54

________________________________ 5

- 302 5.8.1.4. Summary of Effects on Female Reproductive Function

____________________________ 5

- 303
5.8.2. Effects on Male Reproductive Function

________________________________ ________________ 5

- 304
5.8.2.1. Effects on Sperm/Semen Production, Quality, and Function

________________________ 5

- 304

Table 5

- 30. Summary of recent epidemiologic studies of effects on sperm and semen

______ 5

- 304May 2011 vii DRAFT

– DO NOT CITE OR QUOTE 5.8.2.2. Hormone Levels

________________________________ __________________________ 5

- 309

Table 5

- 31. Summary of recent epidemiologic studies of effects on hormones for males

____ 5

- 310 5.8.2.3. Fertility

________________________________ ________________________________ _ 5

- 312
5.8.2.4. Puberty

________________________________ ________________________________ _ 5

- 312

Table 5

- 32. Summary of recent epidemiologic studies of effects on puberty for males.

_____ 5

- 313 5.8.2.5. Effects on Morphology and Histology of Male Sex Organs

_________________________ 5

- 314
5.8.2.6. Summary of Effects on Male Reproductive Function

______________________________ 5

- 314
5.8.3. Effects on Ovaries, Embryo Development, Placental function, and Spontaneous Abortions

________ 5

- 315

Table 5

- 33. Summary of recent epidemiologic studies of effects on spontaneous abortions.

________________________________ ______________________ 5

- 315 5.8.4. Infant Mortality and Embryogenesis

________________________________ __________________ 5

- 318
5.8.5. Birth Defects

________________________________ ________________________________ ____ 5

- 318

Table 5

- 34. Summary of recent epidemiologic studies of effect s on neural tube defects.

____ 5

- 318 5.8.6. Preterm Birth

________________________________ ________________________________ ____ 5

- 319

Table 5

- 35. Summary of recent epidemiologic studies of effects on preterm birth.

_________ 5

- 320 5.8.7. Low Birth Weight/Fetal Growth

________________________________ _____________________ 5

- 321

Table 5

- 36. Summary of recent epidemiologic studies of effects on low birth weight and fetal growth.

________________________________ ____________________ 5

- 322 5.8.8. Toxicological Studies of Developmental Effects

________________________________ _________ 5

- 326
5.8.8.1. Developmental Effects on Blood and Liver

________________________________ _____ 5

- 326
5.8.8.2. Developmental Effects on Skin

________________________________ ______________ 5

- 328
5.8.8.3. Developmental Effects on the Retina

________________________________ __________ 5

- 328
5.8.8.4. Developmental Effects on Teeth

________________________________ _____________ 5

- 328
5.8.9. Summary and Causal Determination

________________________________ __________________ 5

- 328
5.9. Effects on Other Organ Systems

________________________________ _____________________________ 5

- 330
5.9.1. Effects on the Hepatic System

________________________________ _______________________ 5

- 330
5.9.1.1. Summary of Key Findings of the Effects on the Hepatic System from the 2006 Lead AQCD 5

- 331
5.9.1.2. New Epidemiologic Studies

________________________________ _________________ 5

- 331
5.9.1.3. New Toxicological Studies

________________________________ __________________ 5

- 333
5.9.2. Effects on the Gastrointestinal System

________________________________ _________________ 5

- 337
5.9.2.1. Summary of Key Findings on the Effects on the Gastrointestinal System from the 2006 Lead AQCD

________________________________ _____________________________ 5

- 337
5.9.2.2. New Epidemiologic Studies

________________________________ _________________ 5

- 337
5.9.2.3. New Toxicological Studies

________________________________ __________________ 5

- 338
5.9.3. Effects on the Endocrine System

________________________________ _____________________ 5

- 339
5.9.3.1. Summary of Key Findings of the Effects on the Endocrine System from the 2006 Lead AQCD 5

- 339
5.9.3.2. New Epidemiologic Studies

________________________________ _________________ 5

- 339
5.9.3.3. New Toxicological Studies

________________________________ __________________ 5

- 341
5.9.4. Effects on Bone and Teeth

________________________________ __________________________ 5

- 342
5.9.4.1. Summary of Key Findings of the Effects on Bone and Teeth from the 2006 Lead AQCD 5

- 342
5.9.4.2. New Toxicological and Epidemiologic Studies

________________________________ __ 5

- 343
5.9.5. Effects on Ocular Health

________________________________ ___________________________ 5

- 346
5.9.5.1. Summary of Key Findings of the Effects on Ocular Health from the 2006 Lead AQCD

___ 5

- 346
5.9.5.2. New Toxicology and Epidemiology Studies

________________________________ ____ 5

- 346
5.9.6. Effects on the Respiratory System

________________________________ ____________________ 5

- 346
5.9.7. Summary

________________________________ ________________________________ _______ 5

- 347
5.10. Cancer

________________________________ ________________________________ _________________ 5

- 349
5.10.1. Cancer Incidence and Mortality

________________________________ ______________________ 5

- 349

Table 5

- 37. Sum mary of epidemiologic studies of cancer incidence and mortality

_________ 5

- 350 5.10.1.1. Overall Cancer Mortality

________________________________ ___________________ 5

- 353May 2011 viii DRAFT

– DO NOT CITE OR QUOTE 5.10.1.2. Lung Cancer

________________________________ _____________________________ 5

- 354
5. 10.1.3. Brain Cancer

________________________________ _____________________________ 5

- 354
5.10.1.4. Breast Cancer

________________________________ ____________________________ 5

- 355
5.10.1.5. Other Cancers

________________________________ ____________________________ 5

- 356
5.10.1.6. Toxicological Models of Carcinogenicity

________________________________ ______ 5

- 357
5.10.2. Cancer Biomarkers

________________________________ ________________________________ 5

- 358
5.10.3. DNA and Cellular Damage

________________________________ _________________________ 5

- 358
5.10.3.1. Epidemiologic Evidence for DNA and Cellular Damage

___________________________ 5

- 358
5.10.3.2. Toxicological Evidence for DNA and Cellular Damage

___________________________ 5

- 3 59
5.10.3.3. Mechanisms of Action

________________________________ _____________________ 5

- 365
5.10.3.4. Effects of Le ad within Mixtures

________________________________ ______________ 5

- 366
5.10.4. Epigenetics

________________________________ ________________________________ ______ 5

- 367
5.10.5. Summary and Causal Determination

________________________________ __________________ 5

- 368
5.11. Overall Summary

________________________________ ________________________________ _________ 5

- 369

Table 5

- 38. Summary of causal d eterminations for Pb.

_____________________________ 5

- 369 Chapter 5. References

________________________________ ________________________________ ___________ 5

- 370May 2011 5

- 1 DRAFT

– DO NOT CITE OR QUOTE 

# Chapter 5. Integrated Health Effects of LeadExposure

5.1. Introduction

This chapter reviews, summarizes, and integrates the evidence for the broad spectrum of health 

### 1
effects associated with exposure to Pb. The chapter begins (Section 5.2) with a discussion of the evidence 

### 2
for the modes of action that mediate the health effects of Pb, including those common to all health effects 

### 3
evaluated in the ISA and those specific to particular endpoints. Subsequent sections consist of 

### 4

asse ssments of the epidemiologic and toxicological evidence for the effects of Pb exposure on major 

### 5
health effect categories such as neurological effects (Section 5.3), cardiovascular effects (Section 5.4), 

### 6
renal effects (Section 5.5), immune effects (Section 5.6), effects on heme synthesis and red blood cell 

### 7
function (Section 5.7), and reproductive effects and birth outcomes (Section 5.8). Section 5.9 provides 

### 8

reviews of the evidence for Pb effects on health outcomes for which a fewer number of studies are 

### 9

ava ilable, including those related to the hepatic system (Section 5.9.1), gastrointestinal system (Section 

### 10
5.9.2), endocrine system (Section 5.9.3), bone and teeth (Section 5.9.4), ocular health (Section 5.9.5), and 

### 11
respiratory system (Section 5.9.6). Chapter 5 concludes with a discussion of the evidence for Pb effects 

### 12
on cancer (Section 5.10). 

### 13
Individual sections for major health effect categories (e.g., neurological, cardiovascular, renal) 

### 14
include a brief summary of conclusions from the 2006 Pb AQCD and an evaluation of recent evidence 

### 15
that is intended to build upon evidence from previous reviews. Within each of these sections, results are 

### 16
organized by endpoint (e.g., cognitive function, behavior, neurodegenerative diseases) then by specific 

### 17

scientific disci pline (i.e., epidemiology, toxicology). Each major section (e.g., neurological, 

### 18
cardiovascular, renal effects) concludes with an integrated summary of the findings and a conclusion 

### 19
regarding causality. Based upon the framework described in Chapter 1, a det ermination of causality is 

### 20
made for a broad health effect category, such as neurological effects, with coherence and biological 

### 21
plausibility being based on evidence available across disciplines and also across the suite of related health 

### 22
endpoints. 

### 23

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific literature used by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (I SA) and the Integrated Risk Information System (IRIS)

.May 2011 5

- 2 DRAFT

– DO NOT CITE OR QUOTE 

# 5.2. Modes o f Action

5.2.1. Introduction

The diverse health effects of Pb are dependent on multiple factors, including the concentration and 

### 1
duration of exposure, the particular Pb compounds constituting the exposure, and which tissues are 

### 2
targeted. A mode of action is the s equence of key events (i.e., empirically observable precursor steps) that 

### 3
cumulatively result in the formation of negative health outcomes. Although the toxic effects of Pb appear 

### 4
to be mediated through multiple modes of action, alteration of cellular ion status (including disruption of 

### 5
calcium homeostasis, altered ion transport mechanisms, and perturbed protein function through 

### 6
displacement of metal cofactors) seems to be the major unifying mode of action underlying all subsequent 

### 7
modes of action (Figure 5

- 1). The following section draws information from all of the health effects 

### 8

sectio n s in the current document and identifies the major modes of action operating at the molecular, 

### 9
cellular, and tissue/organ level . In turn, individual health effect sections b ridge the se effects to toxicities 

### 10
observed on the organismal level. Accordingly, this section differs in structure and content from other 

### 11

health effects sections as it does not primarily focus on the literature published since the 2006 Pb AQCD

, 

### 12

but rather incorporates that information with older studies that represent the current state of the science on 

### 13
the possible modes of action of Pb. 

### 14

Note: The 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 toxic effects.May 2011 5

- 3 DRAFT

– DO NOT CITE OR QUOTE 

## 5.2.2. Altered Ion Status

Physiologically

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

### 1
functions in biological systems, including roles as charge ca rriers, intermediates in enzymatically



### 2
catalyzed reactions, and as structural elements in the proper maintenance of tertiary protein conformations 

### 3
( Garza et al., 2006 ) . I t is through disruption of these biological functions that Pb effects its toxic action, 

### 4
ultimately adversely affecting such tightly regulated processes as cell signaling, intracellula r ion 

### 5
homeostasis, ion transport, energy metabolism, and enzymatic function. 

### 6

5.2.2.1. Disruption of Ca 2+ 

### Homeostasis

Calcium is one of the most important carriers of cell signals and regulates virtually all aspects of 

### 7
cell function, including energy metabolism, sig nal transduction, hormonal regulation, cellular motility, 

### 8
and apoptosis ( Carafoli, 2005 )

. Ca 2+ homeostasis is maintained through a tightly regulated balance of 

### 9
cellular transport and intracellular storage ( Pentyala et al., 2010 ) . Disruption of Ca 2+ homeostasis by Pb 

### 10
has been observed in a number of different cell types and cell

- free environments, indicating that this is a 

### 11
major mode of action for Pb

- induced toxicity on a cel lular level. 

### 12

Ca 2+ homeostasis is particularly important in bone cells, as the skeletal system serves as the major 

### 13
dynamic reservoir of Ca 2+ in the body ( Long et al., 1992 ; Wiemann et al., 1999 ) . Bone cells also are 

### 14
unique in that they exist in a microenvironment that is high in both Ca 2+ and Pb concentrations, 

### 15

pot entially increasing their relative susceptibility to Pb

- induced toxicity ( Long et al., 1992 ) . A series of 

### 16
studies from the laboratory of Long, Dowd, and Rosen have indicated that exposure of cultured 

### 17
osteoblastic bone cells to Pb disrupts intracellular Ca 2+ levels ( [Ca 2+ ] i ). Exposure of osteoblasts to 1, 5, or 

### 18
25 μM Pb for 40

- 300 minutes resulted in prolonged increases in [ Ca 2+ ] i of 36, 50 and 120% over baseline, 

### 19

res pectively ( Schanne et al., 1989 ; Schanne et al., 1997 ) . Long et al. ( 1992 ) observed that exposure of 

### 20
osteoblasts to either 400 ng parathyroid hormone (PTH)/ml culture for 1 hour or 25 μM Pb for 20 hours 

### 21

in creased [Ca 2+ ] i . Pretreatment of Pb

- exposed cells with PTH increased [Ca 2+ ] i above concentrations 

### 22
observed in either single exposure, indicating that Pb may disrupt bone cell’s ability to respond to normal 

### 23

hormonal control. A similar additive increase in [ Ca 2+ ] i was also observed when bone cells were co



### 24
treated with epidermal growth factor (EGF) and Pb versus Pb alone ( Long & Rosen, 1992 ) . Pb

- induced 

### 25

increases in [Ca 2+ ] i were blocked by a protein kinase C (PKC) inhibitor, indicating that PKC activation 

### 26
may serve as the mechanism by which Pb perturbs [Ca 2+ ] i ( Schanne et al., 1997 ) . Schirrmacher et al. 

### 27
( 1998 ) also observed alterations in Ca 2+ homeostasis in osteoblasts exposed to 5 μM Pb for 50 minutes 

### 28

du e to potential disruption of Ca 2+

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

### 29

to 5 or 12.5 μM Pb inhibited the Ca 2+ release activated calcium (CRAC) influx of Ca 2+ independent of 

### 30

any inhibitory effect on Ca 2+

- ATPases. 

### 31May 2011 5

- 4 DRAFT

– DO NOT CITE OR QUOTE Ca 2+ homeostasis has also been shown be to disturbed in erythrocytes due to Pb exposure 

### 1
( Quintanar

- Escorza et al., 2010 ; Quintanar

- Escorza et al., 2007 ; Shin et al., 2007 ) . In blood samples taken 

### 2

from Pb

- exposed workers (blood Pb level = 74.4 ± 21.9 μg/dL), the [Ca 2+ ] i was approximately 2.5

- fold 

### 3
higher than that seen in nonexposed workers (blood Pb level = 9.9 ± 2 μg/dL) ( Quintanar

- Escorza et al., 

### 4

2007 ) . The increase in [ Ca 2+ ] i was associated with higher osmotic fragility and modifications in 

### 5
erythrocyte shape. When erythrocytes from 10 healthy volunteers were exposed to Pb at concentrations o f 

### 6
0.2 to 6.0 μM for 24 or 120 hours, dose

- related increases in [Ca 2+ ] i were observed across all 

### 7
concentrations for both durations of exposure ( Quintanar

- Escorza et al., 2010 ) . Subsequent exposures of 

### 8
erythrocytes to either 0.4 or 4.0 μM Pb (corresponding to 10 or 80 μg/dL in exposed workers ( Quintanar



### 9
Escorza et al., 2007 ) ) for 12

- 120 hours demonstrated duration

- related increases at durations >12 hours. 

### 10
Osmotic fragility (measured as percent hemolysis) was increased in erythrocytes exposed to 0.4 μM Pb 

### 11

for 24 hours. Co

- incubation with a vitamin E analog mitigated these effe cts, indicating that the increase in 

### 12
[Ca 2+ ] i is dependent on the oxidative state of the erythrocytes. Shin et al. ( 2007 ) observed that incubation 

### 13
of human erythrocytes with 5 μM Pb for 1 hour resulted in a 30

- fold increase in [ Ca 2+ ] i in vitro, inducing 

### 14
the pro

- coagulant activity of exposed erythrocytes. Induction of pro

- coagulant activity in erythrocytes 

### 15
could lead to thrombus formation and negatively contribute to overall cardiovascular health, whereas 

### 16
increased osmotic fragility cou ld substantially reduce erythrocyte life span and ultimately lead to anemic 

### 17

conditions. 

### 18
Similar to effects seen in erythrocytes, Ca 2+ homeostasis has been observed in platelets and white 

### 19
blood cells. Dowd and Gupta ( 1991 ) observed statistically significant increases in [ Ca 2+ ] i in human 

### 20
platelets exposed to as little as 1 μM Pb for 3.5 hours. The observed increase in Ca 2+ levels was attributed 

### 21

to increased influx of external Ca 2+ , possibly through receptor

- operated Ca 2+ channels. In mouse splenic 

### 22
lymphocytes exposed to Pb, [ Ca 2+ ] i was increased at exposure levels as low as 1 μM when incubated for 

### 23
10 minutes or greater ( S. Li et al., 2008 ) . These increases in Ca 2+ appeared to be reversible as [Ca 2+ ] i 

### 24

returned to baseline after one hour. Pretreatment with a calmodulin antagonist slightly mitigated the 

### 25
effects of Pb exposure, indicating a role for calmodulin in disruption of Ca 2+ homeostasis in lymphocytes. 

### 26
In rat tail arteries exposed to 1.2 μM Pb acetate for 1 hour, intracellular stores o f Ca 2+ increased over 

### 27
controls, possible through increased transmembrane influx of Ca 2+ ( Piccinini et al., 1977 )

. 

### 28
Exposure of the microsomal fraction of rat brain cells to as little as 0.25 μM Pb for 2 minutes 

### 29

resulted in increased release of Ca 2+ into the media ( Pentyala et al., 2010 ) . Further, Pb exposure also 

### 30
decreased the activity of the microsomal Ca 2+

- ATPase, thus decreasing the sequestration of Ca 2+ into 

### 31

micro somes. The results of this study suggest that disruption of microsomal release and re

- uptake of Ca 2+ 

### 32
may alter Ca 2+ homeostasis, ultimately leading to altered signal transduction and neuronal dysfunction. 

### 33
However, Ferguson et al. ( 2000 ) observed that [Ca 2 + ] i was decreased in rat hippocampal neurons in 

### 34
response to exposure to 100 nM Pb for 1

- 48 hours, although the observed decreases were not time

- 

### 35May 2011 5

- 5 DRAFT

– DO NOT CITE OR QUOTE dependent. The decrease in [Ca 2+ ] i was shown to be due to increased efflux of Ca 2+ out of the neuron via a 

### 1

calmo dulin

- regulated mechanism, possibly through stimulated Ca 2+ efflux via Ca 2+

- ATPase. 

### 2

5.2.2.2. Disruption of Ion Transport Mechanisms

As described above, deregulation of Ca 2+ homeostasis results in negative effects in multiple organ 

### 3
systems. Under normal conditions , cytosolic concentrations of free Ca 2+ are maintained at low levels (0.1 

### 4
μM) by extrusion and internal compartmentalization processes ( Huel et al., 2008 ) . An important 

### 5
component of the maintenance of Ca 2+ homeostasis is transmembrane transport of Ca ions via Ca 2+

- 

### 6
ATPase and voltage

- sensitive gates ( Carafoli, 2005 ) . Pb has been shown to disrupt the normal move ment 

### 7

of Ca 2+ ions, as well as other physiologically important ions through interactions with these transport 

### 8

mechanisms. 

### 9
Multiple studies have reported the effects of Pb exposure on Na +

- K +

- ATPase, Ca 2+

- ATPase, and 

### 10
Mg 2+

- ATPases in animal models. Decreases i n the activity of all three ATPases were observed in the 

### 11
kidneys and livers of rats exposed to 750 ppm Pb in drinking water for 11 weeks (blood Pb = 55.6 ± 6.3 

### 12
μg/dL) ( Kharoubi, Slimani, Aoues, et al., 2008 ) and in erythrocytes of rats exposed to 0.2% Pb in 

### 13
drinking water for 5 weeks (blood Pb = 97.56 ± 11.8 μg/dL) ( Sivaprasad et al., 2003 ) . Increases in lipid 

### 14
peroxidation were seen in both studies and the decrements in ATPase activities may be explained by 

### 15
generation of free radicals in Pb

- expos ed animals. A decrease in the activity of Na +

- K +

- ATPase was 

### 16
observed in rabbit kidney membranes exposed to 0.01 to 10 μM Pb, possibly due to Pb inhibiting the 

### 17
hydrolytic cleavage of phosphorylated intermediates in the K

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

### 18

2009 ) . Similar decreases in Na +

- K +

- ATPase activity were observed in synaptosomes isolated from rats 

### 19
exposed to 200 mg/L Pb in drinking water for 3 months (blood Pb = 378 μg/dL ) ( Rafalowska et al., 

### 20

1996 ) or 15 mg/kg Pb injected intraperitoneally for 7 days (blood Pb = 112.5 μg/ dL) ( Struzynska, 

### 21

Dabrowska

- Bouta, et al., 1997 ) . The activity of Ca 2+

- ATPase in the sarcoplasmic reticulum of rabbits 

### 22
exposed to 0.01 μmol/L Pb was similarly decreased ( Hechtenberg & Beyersmann, 1991 ) . The inhibitory 

### 23
effect of Pb was diminished in the presence of high MgATP concentrations. The activity of generic 

### 24
ATPase was reported to b e altered in the testes of rats exposed to 300 mg/L Pb acetate gestationally, and 

### 25
in drinking water after weaning to the age of 6, 8, 10, or 12 weeks ( H. T. Liu et al., 2008 ) . In rats fed a Pb



### 26
depleted (20 ± 5 μg/kg) or control (1 mg/kg) diet during gestation and lactation, no difference was 

### 27
observed in the activity of Na +

- K +

- ATPase and Ca 2+

- Mg 2+

- ATPase in the P 0 generation ( Eder et al., 1990 )

. 

### 28
However, the F 1 generation of Pb

- depleted rats displayed decreased activities in both enzymes (meaning 

### 29
that animals with higher exposure to Pb, i.e., “control” animals, had higher enzymatic act ivities). A 

### 30
similar increase in the activity Na +

- K +

- ATPase was observed in rats exposed to 20 mg/kg Pb 

### 31
intraperitoneally for 14 consecutive days ( Jehan & Motlag, 1995 ) . Co

- exposure of Pb with zinc and 

### 32
copper greatly reduced the increase in ATPase activity observed. Although the precise m echanism was not 

### 33May 2011 5

- 6 DRAFT

– DO NOT CITE OR QUOTE investigated, Navarro

- Moreno et al. ( 2009 ) reported that Ca 2+ uptake was diminished in proximal renal 

### 1
tubule cells in rats chronically exposed to 500 ppm Pb in drinking water for 7 months (blood Pb = 43.0 ± 

### 2
7.6 μg/dL). 

### 3
In vitro studies of ATPase activities in human erythrocyte ghosts ha ve also shown that Pb affects 

### 4
the transport of metal ions across membranes. Calderon

- Salinas et al. ( 1999 ) observed that 10

- 50 mM Pb 

### 5

and Ca 2+ were capable of inhibiting the passive transport of each other in human erythrocyte ghosts 

### 6
incubated with both cations. Subsequent inhibition experiments indicated that both cations share the same 

### 7
electrogenic transport pathway ( Sakuma et al., 1984 ) . Further study by this group ( Ca lderon

- Salinas, 

### 8
Quintanar

- Escorza, et al., 1999 ) demonstrated that Pb can noncompetitively block the transport of Ca 2+ by 

### 9
inhibiting the activity of Ca 2+

- Mg 2+

- ATPase at concentrations of 1

- 5 mM. Mas

- Oliva ( 1989 ) demonstrated 

### 10
that the activity of Ca 2+

- Mg 2+

- ATPase in human erythrocyte gh osts was inhibited by incubation with 0.1

- 

### 11
100 μM Pb. The inhibitory action was most likely due to direct reaction with sulfhydryl groups on the 

### 12
ATPase at Pb concentrations greater than 1 μM, but due to the action of Pb on calmodulin at lower 

### 13

concentrations . Grabowska and Guminska ( 1996 ) observed that the activity of Na +

- K +

- ATPase was 

### 14
decreased in erythrocyte ghosts exposed to concentrations as low a s 10 μg/dL Pb; activity of Ca 2+

- Mg 2+

- 

### 15
ATPase was less sensitive to Pb exposure and Mg 2+

- ATPase activity was not affected. 

### 16
In a study investigating ATPase activities in occupationally

- exposed workers in Nigeria, Abam et 

### 17

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

- bound Ca 2+

- Mg 2+

- ATPase was decreased 

### 18
by roughly 50% in all occupational gr oups (range of mean ± SD blood Pb level across nine occupational 

### 19
groups = 28.75 ± 11.31

- 42.07 ± 12.01 μg/dL) compared to nonexposed controls (blood Pb = 12.34 ± 

### 20
2.44 (males) and 16.85 ± 6.01 μg/dL (females)). Increased membrane concentrations of Ca 2+ and 

### 21
magnesium were 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. Huel et al. ( 2008 ) found that 

### 23
newborn hair and cord blood Pb levels (1.22 ± 1.41 μg/g and 3.54 μg/dL) were negatively associated with 

### 24

Ca 2+

- ATPase activity in plasma membranes of erythrocyte s isolated from cord blood; newborn hair Pb 

### 25
levels were more strongly associated with cord Ca 2+ pump activity than cord blood Pb 

### 26
Pb has also been shown to disrupt cation transport mechanisms through direct action on voltage



### 27

sensitive cation channels. Audesirk and Audesirk ( 1991

, 1993 ) demonstrated that extracellular free Pb 

### 28
inhibits the action of multiple voltage

- sensitive Ca 2+ channels, with free Pb IC 50 (half maximal inhibitory 

### 29
concentration) values of 0.7 μM for L

- type chan nels and 1.3 μM for T

- type channels in neuroblastoma 

### 30
cells, and IC 50 values as low as 0.03 μM for L

- type channels in cultured hippocampal neurons. Sun and 

### 31

Suszkiw ( 1995 ) confirmed the inhibitory action of extracellular Pb on Ca 2+ channels, demonstrating an 

### 32

IC 50 value of 0.3 μM in adrenal chromaffin cells. The o bserved disruption of the Ca 2+ channels most 

### 33
likely reflects competition between Pb and Ca 2+ for the extracellular Ca 2+ binding domain of the channel. 

### 34
Research by other laboratories supported these findings: Pb inhibited the action of multiple Ca 2+ channel s 

### 35
in human embryonic kidney cells transfected with L

- , N

- , and R

- type channels (IC 50 values of 0.38 μM, 

### 36May 2011 5

- 7 DRAFT

– DO NOT CITE OR QUOTE 1.31 μM, and 0.10 μM, respectively) ( Peng et al., 2002 ) and P

- type channels in cultured hippocampal 

### 1
neurons at concentrations up to 3 μM ( Ujihara et al., 1995 ) . However, intracellular Pb was observed to 

### 2

enhance Ca 2+ currents through attenuation of the Ca 2+ depen dent deactivation of Ca 2+ channels at an EC 50 

### 3
value of 0.2 nM, possibly through blocking the intracellular Ca 2+ binding domain, or through Ca 2+ 

### 4
dependent dephosphorylation of the channel ( L. R. Sun & Suszkiw, 1995 )

. 

### 5
Pb also disrupts the action of Ca 2+

- dependent potassium channels. Alvarez et al. ( 1986 ) observed 

### 6
that Pb promoted the efflux of potassium from inside

- out erythrocyte vesicles in a dose

- dependent manner 

### 7

at concentrations of 1

- 300 μM, either through action on a Mg modulatory site or through direct 

### 8

interaction with the Ca 2+ binding site. Fehlau et al. ( 1989 ) also demonstrated Pb

- induc ed activation of the 

### 9
potassium channel in erythrocytes. However, Pb only activated the potassium channels at concentrations 

### 10
below 10 μM; higher concentrations of Pb completely inhibited the channel’s activity, indicating the 

### 11
modulation of potassium permeab ility is due to alterations in channel gating. Silken et al. ( 2001 ) observed 

### 12
that Pb activated potassium channels in erythrocytes from the marine teleost S corpaena porcus in a dose



### 13
dependent manner after a 20

- minute incubation; minor loss of potassium was seen at Pb concentrations of 

### 14

1

- 2 μM, whereas exposure to 20

- 50 μM Pb resulted in approximately 70% potassium loss. Competitive 

### 15
and inhibitory binding assays suggest that Pb directly activates potassium channel s in S. porcus

. 

### 16

Disruption of Neurotransmitter Release

Pb has been shown to inhibit the evoked release of neurotransmitters by inhibiting Ca 2+ transport 

### 17
through voltage

- sensitive channels in in vitro experiments ( Cooper & Manalis, 1984 ; J. Suszkiw et al., 

### 18

1984 ) . However, concentrations of Pb a s low as 5 μM were also observed to actually increase the 

### 19
spontaneous release of neurotransmitters in these same experiments. Subsequent research by other groups 

### 20

confirmed that Pb demonstrates Ca 2+

- mimetic properties in enhancing neurotransmitter release f rom cells 

### 21

in the absence of Ca 2+ and Ca 2+

- induced depolarization. Tomsig and Suszkiw ( 1993

, 1995 ) reported that 

### 22
Pb exposure induced the relea se of norepinephrine (NE) from bovine adrenal chromaffin cells, and was 

### 23
considerably more potent at doing so than Ca 2+ (K 0.5 of 4.6 nM for Pb versus 2.4 μM for Ca 2+ ). Activation 

### 24
of protein kinase C (PKC) was observed to enhance the Pb

- induced release of NE. Westrink and Vijverber 

### 25
( 2002 ) observed that Pb acted as a high affinity substitute for Ca 2+ , and triggered enhanced catecholamine 

### 26

release fr om PC12 cells at 10 μM in intact cells and 30 nM in permeabilized cells. The suppression of 

### 27

Ca 2+

- induced evoked release of neurotransmitters combined with the ability of Pb to enhance spontaneous 

### 28
releases could result in higher noise in the synaptic transm ission of nerve impulses in Pb

- exposed 

### 29
animals. In rats exposed to Pb at concentrations of 0.1

- 1.0% in drinking water beginning at GD15

- 16 and 

### 30
continuing to 120 days postnatal, decreases in total potassium

- stimulated hippocampal GABA release 

### 31

were seen at e xposure levels of 0.1

- 0.5% (blood Pb = 26.8 ± 1.3

- 61.8 ± 2.9 μg/dL) ( Lasley & Gilbert, 

### 32

2002 ) . Maximal effects were observed at 0.2% Pb in drinking water, but effects were less evident at 0.5%, 

### 33May 2011 5

- 8 DRAFT

– DO NOT CITE OR QUOTE and were absent at 1.0%. In the absence of Ca 2+ , po tassium

- induced release was increased in the two 

### 1
highest exposure concentrations, suggesting a Pb

- induced enhancement of evoked release of GABA. The 

### 2
authors suggest that this pattern of response indicates that Pb is a potent suppressor of evoked release at 

### 3
low concentrations, but a Ca 2+ mimic in regard to independently evoking exocytosis and release at higher 

### 4

concentrations ( Lasley & Gilbert, 2002 ) . Suszkiw ( 2004 ) reports that augmentation of spontaneous 

### 5
release of neurotransmitters may involve Pb

- induced activation of CaMKII

- dependent phosphorylation of 

### 6
synapsin I or direct activation of synaptotagmin I . Further, Suszkiw ( 2004 ) suggests that unlike the 

### 7
intracellularly mediated effects of Pb on spontaneous rele ase of neurotransmitters, Pb

- induced inhibition 

### 8
of evoked transmitter releases is largely due to extracellular blockage of the voltage

- sensitive Ca 2+ 

### 9

channels. 

### 10

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 specific 

### 11
cellular function of many proteins may be altered by conformational changes ( Kirberger & Yang, 2008 )

. 

### 12
Metal binding sites on proteins are generally ion

- specific and are influenced by multiple factors, including 

### 13
binding geometries, ligand preferences, ionic radius, and metal coordination numbers ( Garza et al., 2006 ; 

### 14
Kirberger & Yang, 2008 ) . The coordination chemistry that normally regulates metal

- protein binding 

### 15
makes many proteins particularly susceptible to perturbation from Pb, as it is able to function with 

### 16
flexible coordination numbers and can bind multiple ligands ( Garza et al., 2006 ; Kirberger & Yang, 

### 17

2008 ) . However, due to differences in its physical properties, Pb induces abnormal conformational 

### 18
changes when it binds to proteins ( Bitto et al., 2006 ; Garza et al., 2006 ; Kirberger & Yang, 2008 ; Magyar 

### 19
et al., 2005 ) , and these structural changes elic it altered protein function. It is known that [ Ca 2+ ] i is an 

### 20
important second messenger in cell signaling pathways, and operates by binding directly to and activating 

### 21
proteins such as calmodulin and protein kinase C (PKC) ( Goldstein, 1993 ) . Alterations in the functions of 

### 22
both of these proteins due to direct interaction with Pb have been well documented in the literature. 

### 23
PKC is a family of serine/threonine protein kinases critical for cell signaling and important for 

### 24
cellular processes, including growth and differentiation ( Goldstein, 1993 ) . PKC contains a C2 Ca 2+

- 

### 25
binding domain and requires the cation, as well as diacylglycerol and phospholipids, for proper cellular 

### 26
activity ( Garza et al., 2006 ) . Markovac and Goldstein ( 1988b ) observed that , in the absence of Ca 2+

, 

### 27
exposure to picomolar concentrations of Pb for 5 minutes directly activated PKC purified from rat brains. 

### 28
The activation of PKC by Pb was more potent than Ca 2+

- dependent activation by five orders of 

### 29
magnitude. Long et al. ( 1994 ) confirmed these findings, reporting that Pb had a K act 4800 times smaller 

### 30

than Ca 2+ (55 pM versus 25 μM, following a 3 minute exposure). However, Ca 2+ had a higher maximal 

### 31
activation of PKC than Pb. This possibly indicates the presence of multiple Ca 2+

- binding sites on the 

### 32
protein, and that Pb ma y bind the first site more efficiently than Ca 2+ , but not subsequent sites. Tomsig 

### 33May 2011 5

- 9 DRAFT

– DO NOT CITE OR QUOTE and Suszkiw ( 1995 ) further demonstrated the ability of Pb to activate PKC at picomolar concentrations in 

### 1

adrenal chrom affin cells incubated with Pb for 10 minutes, but also reported that activation of PKC by Pb 

### 2
was only partial (approximately 40% of the maximum activity induced by Ca 2+ ) and tended to decrease at 

### 3
concentrations greater than one nanomolar. 

### 4
Contrary to the above findings, Markovac and Goldstein ( 1988a ) observed that Pb and Ca 2+ 

### 5
activated PKC at equivalent concentrations and efficacies when broken cell preparations of rat brain 

### 6
microvessels were incubated with either cation for 45 minutes. However, when PKC activation was 

### 7
investigated in whole vessel preparations, no activation w as observed, but PKC did become redistributed 

### 8
from the cytosolic to the particulate fraction. This suggests that Pb redistributes PKC at micromolar 

### 9
concentrations, but does not activate the protein in brain microvessels. In human erythrocytes exposed to 

### 10

Pb acetate for 60 minutes, the amount of PKC found in erythrocyte membranes and total PKC activity 

### 11
was increased at concentrations greater than 100 nM ( Belloni

- Olivi et al., 1996 ) . The observation that 

### 12

neither Ca 2+ nor diacylgycerol was increased due to exposure indicates that Pb

- induced activation of PKC 

### 13
is due to direct interaction with the protein. Pb

- induced alterations in PKC have al so been observed in 

### 14
other tissues, including increased activity in rabbit mesenteric arteries at picomolar concentrations of Pb 

### 15
( Chai & Webb, 1988 ; Watts et al., 1995 ) and human erythrocytes from Pb

- exposed workers (blood Pb = 

### 16
5.4 to 69.3 μg/dL) ( K.

- Y. Hwang et al., 2002 ) , and decreased activity in mouse macrophages and the rat 

### 17

brain cortex at micromolar concent rations ( Lison et al., 1990 ; Murakami et al., 1993 )

. 

### 18
Calmodulin is another important protein essential for proper Ca 2+

- dependent cell signaling. 

### 19

Calmodulin contains an “EF

- hand” Ca 2+ binding domain, and is dependent on the cation for proper 

### 20
activity ( Garza et al., 2006 ) . Calmodulin regulates events as diverse as cellular structural integrity, ge ne 

### 21
expression, and maintenance of membrane potential ( Saimi & Kung, 2002 ; Vetter & Leclerc, 2003 )

. 

### 22

Haberman et al. ( 1983 ) observed that exposure to Pb altered numerous cellular functions of calmodulin, 

### 23
including activation of calmodulin

- dependent phosphodiesterase activity after 10 minutes incubation 

### 24
(minimal activation at 100 nM, EC 50 = 0.5

- 1.0 μM), stimulation of brain membrane phosphorylation at Pb 

### 25
concentrations greater than 400 nM after 1 minute incubation, and increased binding of calmodulin to 

### 26

brain membrane s at Pb concentrations greater than 1 μM after 10 minutes incubation. Haberman et al. 

### 27
( 1983 ) reported the affinity of Pb for calmodulin’s Ca 2+

- binding sites was approximate to that of Ca 2+ 

### 28
itself (K d ~ 20 μM), whereas Richardt et al. ( 1986 ) observed that Pb was slightly more potent than Ca 2+ at 

### 29
binding calmodulin (IC 50 = 11 and 26 μM, respectively). Both studies indicated that Pb was much more 

### 30
effective at binding calmodulin than any other metal cation investigated (e.g., mercury, cadmium, iron). 

### 31

Kern et al. ( 2000 ) observed that Pb was more potent in binding to, and affecting conformational changes 

### 32
in, calmodulin compared to Ca 2+ (EC 50 values of 400

- 550 pM (threshold = 100 pM) and 450

- 500 nM 

### 33
(threshold = 100 nM), respectively). Pb, in the absence of Ca 2+ , was also observed to activate calmodulin



### 34
dependent cyclic nucleotide phosphodiesterase activity at much lower concentrations compared to Ca 2+ 

### 35
(EC 50 value 430 pM [threshold = 300 pM versus EC 50 1200 nM (threshold = 200 nM; 50

- minute 

### 36May 2011 5

- 10 DRAFT

– DO NOT CITE OR QUOTE incubation]). When incubated with physiological concentrations of Ca 2+ , Pb induced phosphodiesterase 

### 1
activity at concentrations as low as 50 pM. Pb a ctivated calcineurin, a phosphatase with widespread 

### 2
distribution in the brain and immune system, at threshold concentrations as low as 20 pM in the presence 

### 3

of Ca 2+ (incubation time = 30 minutes), but inhibited its activity at concentrations greater than 2 00 pM 

### 4
( Kern & Audesirk, 2000 ) . Thus, picomolar concentrations of intracel lular Pb appear to amplify the 

### 5
activity of calmodulin and thus can be expected to alter intracellular Ca 2+ signaling in exposed cells ( Kern 

### 6
et al., 2000 ) . Mas

- Oliva ( 1989 ) observed that low

- dose (<1 μM, 20

- minute incubation) stimulatory effects 

### 7
of Pb exposure on the activity of Ca 2+

- Mg 2+

- ATPase was due to Pb binding to calmodulin and subsequent 

### 8

activation of the ion pore. Ferguson et al. ( 2000 ) observed that exposure of rat hippocampal neurons to Pb 

### 9

for 1 to 48 hours resulted in increased activation of a calmodulin

- dependent Ca 2+ extrusion mechanism. 

### 10
Pb has also been observed to alter the activity of other proteins that rely on Ca 2+ binding for normal 

### 11
cellular function. Osteocalcin is a matrix protein important in bone resorption, osteoclast differentiation, 

### 12
and bone growth and has three Ca 2+

- binding s ites ( Dowd et al., 2001 ) . Incubation of osteocalcin in 

### 13

solution with Ca 2+ and Pb resulted in the competitive displacement of Ca 2+ by Pb ( Dowd et al., 1994 ) . Pb 

### 14

was found to bind to osteocalcin more than 1000

- times more tightly than Ca 2+ (K d = 1.6 ± 0.42 nM versus 

### 15
0.007 mM, respectively), and analysis with NMR indicated Pb induced simi lar, though slightly different, 

### 16
secondary structures in osteocalcin, compared to Ca 2+ . The authors hypothesized that the observed 

### 17

difference in Pb

- bound osteocalcin structure may explain previous findings in the literature that Pb 

### 18
exposure reduced osteocal cin adsorption to hydroxyapatite ( Dowd et al., 1994 ) . Further research by this 

### 19
group confirmed that Pb bound osteocalcin approximately 10,000

- times more tightly than C a 2+ (K d = 

### 20
0.085 μM versus 1.25 mM, respectively) ( Dowd et al., 2001 ) . However, the authors reported that Pb 

### 21
exposure actually caused increased hydroxyapatite adsorption at concentrations 2

- 3 orders of magnitude 

### 22

lower than seen with Ca 2+ . Additionally, Pb can displace Ca 2+ in numerous other Ca 2+

- binding proteins 

### 23
important in muscle contractions, renal Ca 2+ transport and neurotransmission, including troponin C, 

### 24
parvalbumin, CaBP I and II, phospholipase A 2 , and syntapotagmin I, at concentrations as low as the 

### 25
nanomolar range ( Bouton et al., 2001 ; Ost erode & Ulberth, 2000 ; Richardt et al., 1986 )

. 

### 26
Pb can displace metal cations other than Ca 2+ that are requisite for protein function. One of the most 

### 27
researched targets for molecular toxicity of Pb is the second enzyme in the heme synthetic pathway, 

### 28
aminolevulinic acid dehydratase (ALAD). ALAD contains four zinc

- binding sites and all four need to be 

### 29
occupied to confer full enzymatic activity ( Simons, 1995 ) . ALAD has been identified as the major protein 

### 30
binding target for Pb in human erythrocytes ( Bergdahl, Grubb, et al., 1997 ) , and exposure to Pb results in 

### 31

inhibition of t he enzyme in the erythrocytes of Pb

- exposed workers and adolescents (blood Pb level >10 

### 32
μg/dL) ( Ademuyiwa, Ugbaja, Ojo, et al., 2005 ; Ahamed et al., 2006 ) , in human erythrocytes exposed to 

### 33
Pb for 60 minutes (K i = 0.07 pM) ( Simons, 1995 ) , and in rats exposed to 25 mg/kg Pb once a week for 4 

### 34
weeks (blood Pb level = 6.56 ± 0.98 μg/dL) ( M. K. Lee et al., 2005 ) . Further experiments indicated that 

### 35May 2011 5

- 11 DRAFT

– DO NOT CITE OR QUOTE lower concentrations of zinc result in greater inhibition of enzyme activity by Pb, suggesting a 

### 1
competitive inhibition between zinc and Pb at a single site ( Simons, 1995 )

. 

### 2

Zinc

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

### 3
expression, including GATA proteins a nd transcription factors TFIIIA, Sp1, and Erg

- 1 ( Ghering et al., 

### 4

2005 ; Hanas et al., 1999 ; M. Huang et al., 2004 ; Zawia et al., 1998 ) ( G. R. Reddy & Zawia, 2000 ) . Pb 

### 5 was found to form tight complexes with the cysteine residues in GATA proteins (β Pb = 6.4 × 10 9 M - 1 for 

### 6 single zinc fingers and β 2 Pb2 = 6.4 × 10 19 M - 2 ), and was able to displace bound zinc from the protein under 

### 7
physiologically relevant conditions ( Ghering et al., 2005 ) . Once Pb was bound to GATA proteins, they 

### 8
displayed decreased ability to bind to DNA (Pb concentrations ≥ 1.25 μM) and activate transcription (Pb 

### 9
concentration = 1 M). Pb also binds to the zinc domain of TFIIIA, inhibiting its ability to bind DNA at 

### 10
concentrations as low as 10 μM ( Hanas et al., 1999 ; M. Huang et al., 2004 ) . Huang et al. ( 2004 ) also 

### 11
reported that exposure to Pb caused the dissociation of TFIIIA

- DNA adducts and that NMR spectroscopy 

### 12
indicated that altered TFIIIA activity is the result of a Pb

- induced abnormal protein conformation. 

### 13
Pb exposure modulated the DNA

- binding profiles of the transcription factors Sp1 and Erg

- 1 in rat 

### 14
pups exposed to 0.2% Pb acetate via lactation, resulting in a shift in DNA

- binding towards early 

### 15
development (i.e., the first week following birth) ( G. R. Reddy & Zawia, 2000 ; Zawia et al., 1998 )

. The 

### 16
shifts in Sp1 DNA

- binding profiles were shown to be associated with abnormal expression of genes 

### 17
related to myelin formation (Section 5.2.7.5). Further mechanistic research utilizing a synthetic peptide 

### 18
containing a 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 efficiency of the 

### 20
synthetic peptide ( Razmiafshari et al., 2001 ; Razmiafshari & Zawia, 2000 ) . However, in DNA

- binding 

### 21
assays utilizing recombinant Sp1 (which has three zinc f inger motifs, opposed to only one in the synthetic 

### 22
peptide), incubation with as little as 37 μM Pb resulted in the abolishment of Sp1’s DNA

- binding 

### 23
capabilities ( Razmiafshari & Zawia, 2000 )

. 

### 24
Pb has also been reported to competitively inhibit Mg binding and thus inhibit the activities of 

### 25
adenine and hypoxanthine/guanine phosphorib osyltransferase in erythrocyte lysates of rats exposed to 

### 26
0.1% Pb in drinking water for 9 months (blood Pb = 7.01 ± 1.64 μg/dL) and in human erythrocyte lysates 

### 27
exposed to 100 nM Pb for as little as 5 minutes ( Baranowska

- Bosiacka et al., 2009 ) , and cGMP 

### 28
phosphod iesterase at picomolar concentrations in homogenized bovine retinas ( D. Srivastava et al., 1995 )

. 

### 29
Pb was also reported to inhibit p yrimidine 5’

- nucleotidase through competitive inhibition of magnesium 

### 30
binding, resulting in conformational changes and improper amino acid positioning in the active site ( Bitto 

### 31
et al., 2006 )

. 

### 32
In summary, Pb has the ability to displace metal cations from the active sites of multiple enzymes 

### 33
and proteins, and thus to alter the functions of those proteins. These alterations in protein function have 

### 34
implications for numerous cellular and physiological processes, including cell signaling, growth and 

### 35May 2011 5

- 12 DRAFT

– DO NOT CITE OR QUOTE differentiation, gene expression, energy metabolism, and biosynthetic pathways. Table 5

- 1 provides a list 

### 1

of enz ymes and proteins whose function may be perturbed by Pb exposure. 

### 2

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 Ac tion Metal Cation; Reference

Enzymes Aminolevulinic acid dehydratase ↓ Zn; Simons ( 1 995 )

Ferrochelatase ↓ Fe (2Fe

- 2S Cluster); Crooks ( 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 cyclase ↓ 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 ( 1999 )

Pyrimidine 5 ’

- nucleotidase ↓ Mg, Ca; Bitto et al. ( 2006 ) , Amici ( 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

- dependent 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 )

↑ indicates increased activity; ↓ indicates decreased activity; ↓↑ indicates activity can be alternatively increased or decreased.



### 5.2.2.4. Mitochondrial Abnormality

Alterations in mitochondrial function, including disruptions in ion transport, ultrastructural 

### 3
changes, altered energy metabolism, and perturbed enzyme activities due to Pb intoxication are well 

### 4
documented in the scientific literature. Exposure of rats to Pb in feed (1% Pb for 4, 6, 8, 10, 12, or 20 

### 5
weeks) or drinking water (300 ppm for 8 weeks, 500 ppm for 7 months, or 1% for 9 months) resulted in 

### 6
gross ultrastructural changes in renal tubule and epididymal mitochondria characterized as a general 

### 7

swollen a ppearance with frequent rupture of the outer membrane, distorted cristae, loss of cristae, 

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

### 9

mitochondria ( Goyer, 1968 ; Goyer et al., 1968 ; Marchlewicz et al., 2009 ; Navarro

- Moreno et al., 2009 ; L. 

### 10
Wang et al., 2010 )

. 

### 11May 2011 5

- 13 DRAFT

– DO NOT CITE OR QUOTE Transmembrane mitochondrial ion transport mechanisms are perturbed by exposure to Pb

. Pb 

### 1
inhibits the uptake of Ca 2+ into mitochondria ( Parr & Harris, 1976 ) , while simultaneously stimulat ing the 

### 2

efflux of Ca 2+ out of the organelle ( Simons, 1993a ) , thus disrupting intracellular/mitochondrial Ca 2+ 

### 3
homeostasis. Pb exposure has also been shown to decrease the mito chondrial transmembrane potential in 

### 4
astroglia incubated with 0.1 or 1.0 μM Pb for 14 days ( Legare et al., 1993 ) , proximal tubule cells exposed 

### 5
to 0.25, 0.5, and 1.0 μM for 12 hours ( L. Wang, H. Wang, et al., 2009 ) , and retinal rod photoreceptor cells 

### 6
incubated with 10 nM to 10 μM for 15 minutes ( L. H. He et al., 2000 )

. Further research indicated that 

### 7

observed Pb

- induced mitochondrial swelling and decreased membrane potential is the result of the 

### 8
opening of a mitochondrial transmembrane pore (MTP), possibly by directly binding t o the metal (Ca 2+ )

- 

### 9
binding site on the matrix side of the pore ( Bragadin et al., 2007 ; L. H. He et al., 2000 )

. Opening of the 

### 10
MTP is the first step of the mitochondrial

- regulated apoptotic cascade pathway in many cells ( Lidsky & 

### 11
Schneider, 2003 ; Rana, 2 008 )

. He et al. ( 2000 ) additionally observed cytochrome c release from 

### 12
mitochondria, and caspase

- 9 and

- 3 activation following exposure of rod cells to Pb

. Induction of 

### 13
mitochondrially

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

### 14

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

. 

### 15

Altered Energy Metabolism

Pb has been reported to alter normal cellular bioenergetics. In mitochondria isolated from the 

### 16
kidneys of rats exposed to 1% Pb in feed for 6 weeks, the rate of oxygen uptake during ADP

- activated 

### 17
(st ate 3) respiration was lower compared to controls ( Goyer et al., 1968 ) . The rate of ATP formation in 

### 18
exposed mitochondria was observed to be approximately 50% that of control mitochondria. A decrease in 

### 19
state 3 respiration and respiratory control ratios (state 3/state 4 [succinate or pyruvate/malate

- activated]) 

### 20
was also observed in kidney mitochondria from rats exposed continuously from conception to six or nine 

### 21
months of age (i.e., gestationally, lactationally, and via drinking water after weaning) to 50 or 250 ppm Pb 

### 22
( Fowler et al., 1980 ) . Statistically significant Pb

- induced decreases in ATP a nd adenylate energy charge 

### 23
were observed concurrently with increases in ADP, AMP, and adenosine in rats exposed to 1% Pb in 

### 24
drinking water for 9 months ( Marchlewicz et al., 2009 ) and cellular ATP levels were decreased in 

### 25

differentiated PC

- 12 cells incubated with as little as 1 μM Pb for 48 hours ( Prins et al., 2010 ) . T he 

### 26

observed decrease in cellular ATP levels in Prins et al. ( 2010 ) were correlated with a Pb

- induced decrease 

### 27
in the expression of the voltage

- dependent anion channel (VDAC), which maintains cellular ATP levels in 

### 28

ne urons. Dowd et al. ( 1990 ) reported that oxidative phosphorylation was decreased up to 74% after 

### 29
exposure of osteoblasts to 10 μM Pb. P arr and Harris ( 1976 ) reported that Pb inhibited coupled and 

### 30
uncoupled respiratory oxygen use in mitochondria, and that Pb prevented pyruvate , but not malate, 

### 31
uptake. Mitochondrial levels of ATP were diminished after exposure, and the authors compared the effects 

### 32
of Pb on the energy supply to the actions of classic respiratory inhibitors, low temperature and chemical 

### 33May 2011 5

- 14 DRAFT

– DO NOT CITE OR QUOTE uncouplers. Bragadin et al. ( 1998 ) supported this view by demonstrating that alkylated Pb compounds 

### 1
acted as a chemical uncoupler of respiration by abolishing the pr oton gradient necessary for oxidative 

### 2
phosphorylation. Contrary to the above findings, Rafalowska et al. ( 1996 ) reported that, although ATP 

### 3
levels did decrease, chronic exposure to Pb did not inhibit oxidative phosphorylation in the synaptosomes 

### 4
of rats exposed to 200 mg/L Pb in water for 3 months. Similar effects with regard to the activity of the 

### 5

mitochondrial ox idative chain were observed in rats injected with 15 mg/kg Pb i.p. daily for seven days, 

### 6
as reported by Struzynksa et al. ( 1997 ) , although ATP levels were reported to increase after exposure to 

### 7

Pb. 

### 8
Pb has also been shown to decrease glycolysis in osteo blasts exposed to 10 μM Pb and in human 

### 9
erythrocytes exposed to 30 μg/dL Pb ( Dowd et al., 1990 ; Grabowska & Guminska, 1996 ) . Contrary to 

### 10
these findings, Antonowicz et al. ( 1990 ) observed higher levels of glycolytic enzymes in erythrocytes 

### 11
obtained from Pb workers directly exposed to Pb, compared to controls exposed to lower c oncentrations 

### 12
of Pb (blood Pb level = 82.1 versus 39.9 μg/dL), and suggested that Pb activated anaerobic glycolysis. In 

### 13
vitro exposure of human umbilical cord erythrocytes to 100

- 200 μg/dL Pb for 20 hours was observed to 

### 14
lower the cellular pools of adenine and guanine nucleotide pools, including NAD and NADPH 

### 15
( Baranowska

- Bosiacka & Hlynczak, 2003 ) . These decreases in nucleotide pools were accompanied by an 

### 16
increase in purine degradation products (adenosine, etc.). Similar d ecreases in cellular nucleotide pools 

### 17
were observed when rats were exposed to 1% Pb in drinking water for four weeks ( Baranowska

- Bosiacka 

### 18
& Hlynczak, 2004 ) . In erythrocytes, nucleotides are synthesiz ed via salvage pathways such as the adenine 

### 19
pathway, which requires adenine phosphoribosyltransferase (APRT). The activity of this enzyme is 

### 20
inhibited by exposure to Pb in human and rat erythrocytes (see above for dose and duration) 

### 21
( Baranowska

- Bosiacka et al., 200 9 )

. 

### 22
Disruptions in erythrocyte energy metabolism have been observed in workers occupationally 

### 23
exposed to Pb. Nikolova and Kavaldzhieva ( 1991 ) reported higher ratios of ATP/ADP in Pb

- exposed 

### 24

worker s with an average duration of exposure of 8.4 years (blood Pb not reported). Morita et al

. ( 1997 ) 

### 25

evaluated the effect of Pb on NAD synthetas e in the erythrocytes of Pb

- exposed workers (blood Pb = 34.6 

### 26
± 20.7 μg/dL) and observed an apparent dose

- dependent decrease in NAD synthetase activity with 

### 27

increased blood Pb. The blood Pb associated with 50% inh ibition of NAD synthetase, which requires a 

### 28
magnesium cation for activity ( Hara et al., 2003 ) , was 43 μg/dL

. 

### 29

Altered Heme Synthesis

Exposure to Pb is known to inhibit two key steps in the synthesis of heme: porphobilinogen 

### 30 synthase (i.e., δ

- aminolevulinic acid dehydratase), a cytoplasmic enzyme requiring zinc for enzymatic 

### 31
activity that condenses two molecules of aminolevulinic acid into porphobilinogen, and ferrochelatase, a 

### 32

mitochondrial iron

- sulfur containing enzyme that incorporates Fe 2+ into protoporphyrin IX to create 

### 33May 2011 5

- 15 DRAFT

– DO NOT CITE OR QUOTE heme. Farant and Wigfield ( 1987

, 1990 ) observed that Pb inhibits the activity of porphobilinogen 

### 1
synthase in rabbit and human erythrocytes, and that the effect on the enzyme was dependent on the 

### 2
affinity for thiol groups at its active site. Taketami et al. ( 1985 ) examined the activity of Pb on 

### 3
ferrochelatase in rat liver mitochondria and observed that 10 μM Pb (30 minute incubation) redu ced 

### 4
NAD(P)H

- dependent heme synthesis by half when ferric, but not ferrous, iron was used. Pb inhibits the 

### 5

insertion of Fe 2+ into the protoporphyrin ring and instead, Zn is inserted into the ring creating zinc 

### 6
protoporphyrin (ZPP). While not directly measur ing the activity of ferrochelatase, numerous studies have 

### 7
shown that blood Pb levels are statistically significantly associated with increased erythrocyte ZPP levels 

### 8
in humans (average blood Pb ranging from 21.92 to 53.63 μg/dL) ( Ademuyiwa, Ugbaja, Ojo, et al., 2005 ; 

### 9
Counter et al., 2007 ; Mohammad et al., 2008 ; Patil, Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 

### 10

2006 ) and animals (b lood Pb = 24.7 μg/dL) ( Rendón

- Ramirez et al., 2007 )

. 

### 11



## 5.2.3. Protein B inding

Pb is able to bind to proteins within cells through interactions with side group moieties (e.g., thiol 

### 12
residues) and can potentially disrupt cellular function (Sections 5.2.2.3 and 5.2.2.4). However, some 

### 13
proteins are also able to bind Pb and protec t against its toxic effects through sequestration. The ability of 

### 14
Pb to bind proteins was first reported by Blackmon ( 1936 ) : Pb intoxication was observed to induce the 

### 15

formation of intranuclear inclusion b odies in the liver and kidney. Further research into the composition 

### 16
of intranuclear inclusion bodies and the identification of specific Pb

- binding proteins has been conducted 

### 17

since that time. 

### 18

5.2.3.1. Intranuclear and Cytoplasmic Inclusion Bodies

Goyer ( 1968 ) and Goyer et al. ( 1968 ) observed the formation of intranuclear inclusion bodies in the 

### 19
renal tubules of rats fed 1% Pb in food for up to 20 weeks. The observation of inclusion bodies was 

### 20
accompanied by altere d mitochondrial structure and reduced rates of oxidative phosphorylation. Pb has 

### 21
further been observed to form cytoplasmic inclusion bodies preceding the formation of the intranuclear 

### 22
bodies, and to be concentrated within the subsequently induced intranucl ear inclusion bodies following 

### 23
i.p. injection, drinking water, and dietary exposures ( Choie & Richter, 1972 ; Fowler et al., 1980 ; Goyer, 

### 24
Leonard, et al., 1970 ; Goyer, May, et al., 1970 ; McLachlin et al., 1980 ; Navarro

- Moreno et al., 2009 ; 

### 25
Oskarsson & Fowler, 1985 ) . Inclusion bodies have also been observed in the mitochondria of kidneys and 

### 26
the perinuclear space in the neurons of rats exposed to 500 ppm Pb acetate in drinking water for 60 days 

### 27

or 7 months ( Deveci, 2006 ; Navarro

- Moreno et al., 2009 ) . Intranuclear and cytoplasmic inclusions have 

### 28

also been found in organs other than the kidney, including liver, lung, and glial cells ( Goyer & Rhyne, 

### 29

1973 ; J. Singh et al., 1999 ) . Pb found within nuclei has also been shown to bind to the nuclear membrane 

### 30

and histone fractions ( Sabbioni & Marafante, 1976 )

. 

### 31May 2011 5

- 16 DRAFT

– DO NOT CITE OR QUOTE Upon denaturing intranuclear inclusion bodies with strong denaturing agents, Moore et al. ( 1973 ) 

### 1
observed that proteins included in the bodies were rich in aspartic and glutamic acid, glycine, and 

### 2
cysteine. Further work by Moore and Goyer ( 1974 ) characterized the protein as a 27.5 kDa protein that 

### 3
migrates as a single band on ac rylamide gel electrophoresis. In contrast with Moore and Goyer’s findings, 

### 4
Shelton and Egle ( 1982 ) identified a 32 kDa protein with an isoelectric point of 6.3 from the kidneys of 

### 5
rats exposed to 1% Pb acetate in feed or 0.75% in drinking water. This protein, dubbed p32/6.3, was not 

### 6
found in control rats, indicating that the protein was induced by P b exposure. This finding was in 

### 7
agreement with studies that indicated formation of intranuclear inclusion bodies required protein synthesis 

### 8
( Choie et al., 1975 ; McLachlin et al., 1980 ) . In addition to its presence in kidneys of Pb

- exposed animals, 

### 9
p32/6.3 has been observed to be present and highly conserved in the brains of rats, mice, dogs, chickens, 

### 10

and humans ( Egle & Shelton, 1986 ) . Ex posure of neuroblastoma cells to 50 or 100 μM Pb glutamate for 1 

### 11
or 3 days increased the abundance of p32/6.3 ( Klann & Shelton, 1989 ) . Shelton et al. ( 1990 ) determined 

### 12
that p32/6.3 was enriched in the basal ganglia, dien cephalon, hippocampus, cerebellum, brainstem, spinal 

### 13
cord, and cerebral cortex, and that it contained a high percentage of glycine, aspartic, and glutamic acid 

### 14

residues. Selvin

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

- and postnatal exposure of rats 

### 15

to 0.2

- 1.0% Pb in drinking water increased the levels of another brain protein, glial fibrillary acidic 

### 16
protein (GFAP), in d eveloping astrocytes and that this increase may be indicative of a demand for 

### 17
astrocytes to sequester Pb. 

### 18

5.2.3.2. Cytosolic Lead Binding Proteins

Numerous studies have also identified cytosolic Pb

- binding proteins. Two binding proteins, with 

### 19
molecular weights of 11.5 and 63 kDa, were identified by ( Oskarsson et al., 1982 ) in the kidney 

### 20
postmitochondrial cytosolic fraction after injection with 50 mg Pb. The two proteins were also fou nd in 

### 21
the brain, but not the liver or lung. Mistry et al. ( 1985 ) observed three proteins (MW = 11.5, 63, and > 200 

### 22
kDa) in rat kidney cytosol, and that the 11.5 and 63 kDa proteins were able to translocate into the nucleus. 

### 23
The 11.5 kDa kidney protein was also able to reverse Pb binding to ALAD through chelation of Pb and 

### 24

donation of a zinc cation to ALAD ( Goering & Fowler, 1984

, 1985 ) . Cadmium and zinc, but not Ca 2+ or 

### 25
Fe, prevent the binding of Pb to the 63 and 11.5 kDa cytosolic proteins, which agrees with previous 

### 26
observations that cadmium is able to reduce total kidney Pb and prevent intranuclear inclusion bodies 

### 27
( Mahaffey et al., 1981 ; Mahaffey & Fowler, 1977 ; Mistry et al., 1986 ) . Additional cytosolic Pb

- binding 

### 28
proteins have been identified in the kidneys of Pb

- exposed rats and humans, including the cleavage 

### 29
prod uct of α2

- microglobulin, acyl

- CoA binding protein (MW = 9 kDa), and thymosin β4 (MW = 5 kDa) 

### 30
( Fowler & DuVal, 1991 ; D. R. Smith et al., 1998 )

. 

### 31
Cytosolic Pb

- binding proteins distinct from kidney proteins have also been identified in the brain 

### 32
of exposed rats and human brain homogenates exposed in vitro ( DuVal & Fowler, 1989 ; Goering et al., 

### 33May 2011 5

- 17 DRAFT

– DO NOT CITE OR QUOTE 1986 ; Quintanilla

- Vega et al., 1995 ) . One protein (MW = 12 kDa) was shown to alleviate hepatic ALAD 

### 1
inhibition due to Pb exposure through competitive binding with Pb and donation of zinc to ALAD. 

### 2
Cytosolic Pb

- binding proteins have been shown to be high in gl utamic acid, aspartic acid, and cysteine 

### 3

residues ( D uVal & Fowler, 1989 ; Fowler et al., 1993 ) . Some evidence exists that cytosolic Pb

- bind ing 

### 4
proteins directly target Pb and compartmentalize intracellular Pb as protective measure against toxicity 

### 5
( Y. Qian et al., 2000 ; Y. Qian et al., 2005 )

. 

### 6

5.2.3.3. Erythrocytic Lead Binding Proteins

The majority (94%) of Pb in whole blood is found in erythrocytes ( Ong & Lee, 1980a ) . Originally, 

### 7
the major Pb

- binding protein in erythrocytes was identified as hemoglobin ( B. S. Cohen et al., 2000 ; 

### 8
Lolin & O'Gorman, 1988 ; Ong & Lee, 1980a

, 1980b ; Raghavan & Gonick, 1977 ) . However, Bergdahl et 

### 9

al. ( 1997 ) observed the principal Pb

- binding protein to be 240 kDa and identified it as ALAD. Two 

### 10

smaller Pb

- binding proteins were observed, but not iden tified (MW = 45 and <10 kDa). ALAD levels are 

### 11
inducible by Pb exposure; the total concentration of the enzyme, but not the activity, increases after 

### 12
exposure in both exposed humans (blood Pb = 30

- 75 μg/dL) and rats (Pb exposure = 25 mM in drinking 

### 13
water) ( Boudene et al., 1984 ; Fujita et al., 1981 ; Fujita et al., 1982 )

. 

### 14
ALAD is a polymorphic gene with three isoforms: ALAD 1

- 1, ALAD 1

- 2, or ALAD 2

- 2. Carriers 

### 15

of the ALAD

- 2 allele have been shown to have higher blood Pb levels than carriers of the homozygous 

### 16

ALAD

- 1 allele ( Astrin et al., 1987 ; H.

- S. Kim et al., 2004 ; Pérez

- Bravo et al., 2004 ; Scinicariello et al., 

### 17

2007 ; C. M. Smith, Hu, et al., 1995 ; Wetmur, 1994 ; Wetmur et al., 1 991 ; Y. Zhao et al., 2007 ) . Some 

### 18
newer studies, however, either observed lower blood Pb levels in carriers of the ALAD

- 2 allele or no 

### 19
difference in Pb levels among the different allele carriers ( Y. Chen et al., 2008 ; Chia et al., 2007 ; Chia et 

### 20
al., 2006 ; E. F. Krieg, Jr. et al., 2009 ; Scinicariello et al., 2010 ; Wananukul et al., 2006 )

. 

### 21

The ALAD

- 2 protein binds Pb more tightly than the ALAD

- 1form: in workers carrying the ALAD



### 22
2 gene, 84% of blood Pb was bound to ALAD versus 81% in carriers of the ALAD

- 1 gene (p = 0.03) 

### 23
( Bergdahl, Grubb, et al., 1997 ) . This higher affinity for Pb in ALAD

- 2 carriers may sequester Pb and 

### 24
prevent its bioavailability for reaction with other enzymes or cellular components. This is supported by 

### 25

the observation that carriers of the ALAD

- 2 gene have higher levels of hemoglobin ( Scinicariello et al., 

### 26

2007 ) , decreased plasma l evulinic acid ( B. S. Sc hwartz, Lee, Stewart, Sithisarankul, et al., 1997 )

, 

### 27
decreased levels of zinc protoporphyrin ( H.

- S. Kim et al., 2004 ; Scinicariello et al., 2007 ) , lower cortical 

### 28

bone Pb ( C. M. Smith, Wang, et al., 1995 ) , and lower amounts of DMSA

- chelatable Pb ( B. S. Schwartz et 

### 29

al ., 2000 ; B. S. Schwartz, Lee, Stewart, Ahn, et al., 1997 ; Scinicariello et al., 2007 ) . However, the 

### 30
findings that ALAD

- 2 polymorphisms reduced the bioavailability of Pb are somewhat equivocal. W u et 

### 31

al. ( 2003 ) observed that ALAD

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

### 32

ALAD

- 1 gene (blood Pb level = 6.2 ± 4.1 μg/dL), and that ALAD

- 2 carriers demonstrated decreased 

### 33May 2011 5

- 18 DRAFT

– DO NOT CITE OR QUOTE renal function at lower patellar Pb concentrations than those observed to decrease renal function in 

### 1

ALAD

- 1 carriers. This potentiall y indicates that ALAD

- 2 carriers have enhanced Pb bioavailability. 

### 2

Weaver et al. ( 2003 ) observed that ALAD

- 2 polymorphisms were associated with hig her DMSA



### 3
chelatable Pb concentrations, when normalized to creatinine levels. Further, Montenegro et al. ( 2006 ) 

### 4
observed among individuals with ALAD 1

- 1 or ALAD 1

- 2/2

- 2 genotypes a significant increase in the 

### 5
amount of Pb found in the plasma (0.44 μg/L versus 0.89 μg/L, respectively) and in the % plasma/blood 

### 6
ratio (0.48% versus 1.45%, respectively). This suggests that the increased plasma levels of Pb in subjects 

### 7

with ALAD 1

- 2/2

- 2 genotypes increases the probability of adverse health effects in these individuals. 

### 8
ALAD’s capacity for binding Pb has been estimated at 85 μg/dL in erythrocytes and 40 μg/dL in 

### 9

whole blood ( Bergdahl et al., 1998 ) . The 45 and <10 kDa Pb

- binding proteins bound approximately 12

- 

### 10
26% and <1% of the blood Pb, respectively. At blood Pb concentratio ns greater than 40 μg/dL, greater 

### 11
binding to these components likely would be observed. Bergdahl et al. ( 1998 ) tentatively identified the 45 

### 12

kDa protein as pyrimidine

- 5’

- nucleotidase and the <10 kDa protein as acyl

- CoA binding protein. Smith et 

### 13

al. ( 1998 ) previously identified acyl

- CoA binding protein as a Pb

- bind ing protein found in the kidney. 

### 14
Studies also observed the presence of an inducible, low

- molecular weight (approximately 10 kDa) 

### 15

Pb

- binding protein in workers occupationally exposed to Pb ( Gonick et al., 1985 ; Raghavan et al., 1980

, 

### 16

1981 ; Raghavan & Gonick, 1977 ) . The presence of this low molecu lar weight protein seemed to have a 

### 17
protective effect as workers that exhibited toxicity at low blood Pb concentrations were observed to have 

### 18
lowered expression of this protein or low levels of Pb bound to it ( Raghavan et al., 1980

, 1981 ) . The 

### 19
presence of low molecular weight Pb

- binding proteins in exposed workers was confirmed by Lolin and 

### 20

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

, 1993b ) . Further Lolin and O’Gorman ( 1988 ) reported tha t the 

### 21
observed protein was only present when blood Pb levels were greater than 39 μg/dL, in agreement with 

### 22

ALAD’s Pb

- binding capacity identified by Bergdahl et al. ( Bergdahl et al., 1998 ) . Xie et al. ( 1998 ) 

### 23
confirmed this, observing the presence of a second low molecular weight protein with greater affinity than 

### 24

ALAD onl y at higher blood Pb levels. Church et al. ( 1993a

, 1993b ) observed the presence of a 6

- 7 kDa 

### 25
protein in the blood of 2 Pb workers (blood Pb >160 μg/dL); approximately 67% of Pb was bound to the 

### 26

prot ein in the blood of the asymptomatic worker, whereas only 22% of the Pb was bound to it in the 

### 27
symptomatic worker. The reported protein was rich in cysteine residues and tentatively identified as 

### 28

metallothionein. 

### 29

5.2.3.4. Metallothionein

Metallothionein is a low

- mo lecular metal

- binding protein, most often zinc or copper, that is rich in 

### 30
cysteine residues and plays an important role in the protection against heavy metal toxicity, trace element 

### 31
homeostasis, and scavenging free radicals ( J. Yu et al., 2009 ) . Exposure to Pb acetate induces the 

### 32
production of Pb

- and Zn

- metallothionein in mice exposed via i.p. or i.v. injection at 30 mg/kg ( Maitani et 

### 33May 2011 5

- 19 DRAFT

– DO NOT CITE OR QUOTE al., 1986 ) , in mice exposed via i.p. injection at 300 μmol/kg ( J. Yu et al., 2009 ) , or in rats exposed via i. p. 

### 1
injection at 24 μmol/100g ( Ikebuchi et al., 1986 ) . The induced Pb

- metallothionein consisted of 28% half



### 2
cysteine and reacted with an antibody for Zn

- metallothionein II ( Ikebuchi et al., 1986 ) . In contrast, 

### 3
exposure of rats to Pb via drinking w ater (200 or 300 mg/L) failed to induce metallothionein in the 

### 4
kidneys or intestines ( Jamieson et al., 2007 ; L. Wang, D. W. Chen, et al., 2009 ) . Goering and Fowler 

### 5
( 1987a

, 1987b ) observed that pretreatment of rats with zinc before injection with Pb resulted in Pb and 

### 6

zinc co

- eluting with zinc

- thionein, and that zinc

- thionein I and II were able to bind Pb in vitro ( Goering & 

### 7
Fowler, 1987a

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

- thionein 

### 8
decreased binding of Pb to liver ALAD and was able to donate zinc to ALAD, thus attenuating the 

### 9
inhibition of ALAD due to Pb exposure. These findings are in agreement with Goering et al. ( 1986 ) and 

### 10

DuVal and Fowler ( 1989 ) who demonstrated rat brain Pb

- binding proteins attenuated Pb

- induced 

### 11

inhibition of ALAD. 

### 12
Metallothionein has been reported to be important in the amelioration of Pb

- induced toxicity 

### 13

effec ts. Liu et al. ( 1991 ) reported that zinc

- metallothionein reduced Pb

- induced membrane leakage and 

### 14
loss of potassium in cultured hepatocytes incubated with 600

- 3,600 μM Pb. Metallothionein

- null mice 

### 15
exposed to 1,000, 2,000, or 4,000 ppm Pb for 20 weeks suffered renal toxicity de scribed as nephromegaly 

### 16
and decreased renal function compared to Pb

- treated wild

- type mice ( Qu et al., 2002 ) . Interestingly, 

### 17

metallothionein

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

### 18

Pb than the wild

- type mice ( Qu et al., 2002 ) . Metallot hionein levels were induced by Pb exposure in non



### 19
null mice. Exposure to Pb (1,000, 2,000, or 4,000 ppm), both for 104 weeks as adults and from GD8 to 

### 20
early adulthood, resulted in increased preneoplastic lesions and carcinogenicity in the testes, bladder, and 

### 21
kidneys of metallothionein

- null rats compared to wild type mice ( Tokar et al., 2010 ; Waa lkes et al., 2004 )

. 

### 22
Inclusion bodies were not observed in null mice. The authors concluded that metallothionein is important 

### 23
in the formation of inclusion bodies and mitigation of Pb

- induced toxic effects, and that those with 

### 24
polymo rphisms in metallothionein coding genes may be at greater susceptibility to Pb. In support of this 

### 25
theory, Chen et al. ( 2010 ) observed that Pb

- exposed workers with a mutant metallothionein allele had 

### 26
higher blood Pb levels than carriers of the normal allele (24.17 and 21.27 versus 17.03 μg/dL), and were 

### 27
more susceptible to Pb toxicity. 

### 28



## 5.2.4. Oxidative Stress

Oxidative stres s occurs when free radicals or reactive oxygen species (ROS) exceed the capacity of 

### 29
antioxidant defense mechanisms. This oxidative imbalance results in uncontained ROS, such as 

### 30
superoxide (O 2 - ), hydroxyl radical (OH ̇), and hydrogen peroxide (H 2 O 2 ), that ca n attack and denature 

### 31
functional/structural molecules and, thereby, promote tissue damage, cytotoxicity, and dysfunction. Pb has 

### 32
been shown to cause oxidative damage to the heart, liver, kidney, reproductive organs, brain, and 

### 33May 2011 5

- 20 DRAFT

– DO NOT CITE OR QUOTE erythrocytes, which may be re sponsible for a number of Pb

- induced pathologies ( Gonick et al., 1997 ; 

### 1

Khalil

- Manesh, Gonick, Cohen, Bergamaschi, et al., 1992 ; Khalil

- Manesh et al., 1994 ; Salawu et al., 

### 2

2009 ; Sandhir & Gill, 1995 ; Shan et al., 2009 ; Vaziri, 2008b )

. The origin of ROS produced after Pb 

### 3
exposure is likely a multipathway process, resulting from oxidation of δ

- aminolevulinic acid (ALA), 

### 4
membrane and lipid oxidation, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 

### 5
activation, and antioxidant enzyme depletion, as discussed below. Some of these processes result from the 

### 6
disruption of functional m etal ions within oxidative stress proteins, such as superoxide dismutase (SOD), 

### 7
catalase (CAT), and glutathione peroxidase (GPx). Interestingly, Pb exposure in many species of plants, 

### 8
invertebrates, and vertebrates discussed in the Ecological Effects of Pb results in upregulation of 

### 9
antioxidant enzymes and increased lipid peroxidation (Chapter 7). 

### 10

5.2.4.1. δ

- ALA Oxidation

The majority of Pb present in the blood accumulates in erythrocytes where it enters through passive 

### 11

carrier

- mediated mechanisms including a vanada te

- sensitive Ca 2+ pump. Once Pb enters erythrocytes, it is 

### 12
predominantly found in the protein

- bound form, with hemoglobin and δ

- ALA dehydratase (δ

- ALAD) 

### 13
both identified as targets ( Bergdahl, Grubb, et al., 1997 ) . Through i ts sulfhydryl and metal ion disrupting 

### 14
properties, Pb incorporates with and inhibits a number of enzymes in the heme biosynthetic process, 

### 15 including δ

- ALA synthetase, δ

- ALAD, and ferrochelatase. Pb is able to disrupt the Zn ions requisite for 

### 16
the activity of δ

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

### 17
picomolar concentrations ( Simons, 1995 ) . Additionally, low blood Pb levels (7 μg/dL) have been found to 

### 18 inhibit the activity of δ

- ALAD in humans with a threshold value around 5 μg/dL ( Ahamed et al., 2005 ; 

### 19
Sakai & Morita, 1996 ) . A significant negative correlation (r =

- 0.6) was found between blood Pb levels in 

### 20
adolescents (4

- 20 μg/dL) and blood δ

- ALA activity ( Ahamed et al., 2006 ) . This inhibition of δ

- ALAD 

### 21

results in the accumulation of δ

- ALA in blood and urine, where δ

- ALA undergoes tautomerization and 

### 22

au toxidation. Oxidized δ

- ALA generates ROS through reduction of ferricytochrome c and electron 

### 23
transfer from oxyHb, metHb, and other ferric and ferrous complexes ( Hermes

- Lima et al., 1991 ; Monteiro 

### 24
et al., 1991 ) . The autoxidation of δ

- ALA produces O 2 - , OH ̇, H 2 O 2 , and an ALA radical ( Monteiro et al., 

### 25

1989 ; Monteiro et al., 1986 )

. 

### 26

5.2.4.2. Membrane and Lipid Peroxidation

A large number of studies in humans and experimental animals have found that exposure to Pb can 

### 27

lead to membrane a nd lipid peroxidation. It is possible that ROS produced from δ

- ALA oxidation, as 

### 28
described above, interacts with and disrupts membrane lipids ( Bechara et al., 1993 ; Oteiza et al., 1995 )

. 

### 29
Additionally, Pb has the capacity to stimulate ferrous ion initiated membrane lipid peroxidation serving as 

### 30

a catalyst for these events ( Adonaylo & Oteiza, 1999 ; Quinlan et al., 1988 ) . Increased lipid peroxidation 

### 31May 2011 5

- 21 DRAFT

– DO NOT CITE OR QUOTE measured as TBARS from liposomes, microsomes, and erythrocytes was shown after Pb (Pb(NO3)2) 

### 1 exposure for ≤ 2 hours at concentrations as low as 10 μM in vitro ( Aruoma et al., 1989 ; Quinlan et al., 

### 2

1988 ) . The extent of peroxidation of lipids varies based on the number of double bonds present in 

### 3
unsaturated fatty acids, since double bonds weaken the C

- H bonds on the adjacent carbon, making H 

### 4

removal easier ( Yiin & Lin, 1995 ) . After Pb exposure (4

- 12 μg/dL, 24 hours, in vitro), the production of 

### 5
malondialdehyde (MDA), a marker of oxidative stress and lipid oxidation end product, increased relative 

### 6
to the number of double bonds of the fatty acid. In the absence of Fe 2+ , Pb does not promote lipid 

### 7
peroxidation, however it may accelerate peroxidation by H 2 O 2 ( Quinlan et al., 1988 )

. This cou ld be due to 

### 8
altering membrane structure, restricting phospholipid movement, and facilitating the propagation of 

### 9
peroxidation. 

### 10
Pb induces changes in the fatty acid composition of a membrane, which could lead to oxidative 

### 11
damage. Exposure to Pb (>62.5 ppm in drinking water, 3 weeks) in chicks promoted an increase in 

### 12
arachidonic acid (AA, 20:4) as a percentage of total fatty acids, and decreased the relative proportion of 

### 13
shorter chain fatty acids (linoleic acid, 18:2) ( Lawton & Donaldson, 1991 ) . It is possible that Pb 

### 14
depressed the desaturation of saturated fatty acids to the corresponding monoenoic fatty acids, while 

### 15
stimulating elongation and desaturation of linoleic acid to AA. Since fatty acid chain length and 

### 16

unsaturation are related to the oxidative potential, changes in fatty acid membrane composition may result 

### 17
in enhanced lipid peroxidation. In addition, changes in fatty acids, thus membrane composition, can result 

### 18
in altered membrane fluidity ( Donaldson & Knowles, 1993 ) . Cha nges in membrane fluidity will disturb 

### 19
the conformation of the active sites of membrane associated enzymes, disrupt metabolic regulation, and 

### 20
alter membrane permeability and function. 

### 21
A number of recent studies report increased measures of lipid peroxidati on in various organs, 

### 22
tissues, and species. Occupational Pb exposure resulting in elevated blood Pb levels (>8 μg/dL) in various 

### 23
countries provides evidence of lipid peroxidation, including increased plasma malondialdehyde levels 

### 24
( Dogru et al., 2008 ; Ergurhan

- Ilhan et al., 2008 ; D. A. Khan et al., 2008 ; Mohammad et al., 2008 ; Patil, 

### 25
Bhagwat, Patil, Dongre, Ambekar, & Das, 2006 ; Patil, Bhagwat, Patil, Don gre, Ambekar, Jailkhani, et al., 

### 26

2006 ; Quintanar

- Escorza et al., 2007 ) . One study found a correlation between the MDA levels and blood 

### 27
Pb levels even in the unexposed workers who had lower (i.e., <12 μg/dl) blood Pb l evels ( Quintanar



### 28
Escorza et al., 2007 ) . Other studies found increased evidence of lipid peroxidation among the general 

### 29
population, including children, with elevated blood Pb levels (>10 μg/dL) ( Ahamed et al., 2008 ; Ahamed 

### 30
et al., 2006 ; Y. P. Jin et al., 2006 ) . Ahamed et al. ( 2006 ) found a significant positive correlation (r = 0.7) 

### 31

between blood Pb levels between 4

- 20 μg/dL in adolescents and blood MDA level. Similar results have 

### 32
been shown after Pb exposure in animal studies ( Adegbesan & Adenuga, 2007 ; M. K. Lee et al., 2005 ; 

### 33
Pandya et al., 2010 ; D. Y. Yu et al., 2008 ) . An enhanced rate of lipid peroxidation has been found in Pb 

### 34
treated (50 μg, 1

- 4 hours) rat brain homogenates ( Rehman et al., 1995 ) and in specific brain regions, 

### 35
hippoca mpus and cerebellum, after Pb exposure (500 ppm, 8 weeks) to rats ( Bennet et al., 2007 ) . Overall, 

### 36May 2011 5

- 22 DRAFT

– DO NOT CITE OR QUOTE there was a correlation between the blood Pb level and measures of lipid peroxidation often measured by 

### 1

MDA levels. 

### 2
Interestingly, studies in many species of plants, invertebrates, and vertebrates exhi bit increased 

### 3
lipid peroxidation (Chapter 7.4.4). The increase in lipid peroxidation following Pb exposure observed 

### 4
across species and kingdoms demonstrate an evolutionarily conserved oxidative response following Pb 

### 5

exposure. 

### 6

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 the 

### 7
production of O 2 - from NAD(P)H and molecular oxygen ( Leseney et al., 1999 ) . Two studies provide 

### 8
evidence for increased activation of NAD(P)H oxidase contributing to the production of ROS after Pb 

### 9

exposure ( Ni et al., 2004 ; Vaziri et al., 2003 ) . Vaziri et al. ( 2003 ) found increased protein expression of 

### 10
the NAD(P)H subunit gp 91 phox in the brain, heart, and renal cortex of Pb treated rats (100 ppm in 

### 11
drinking water, 12 weeks). This upregulation was present in Pb

- treated (1

- 10 ppm) human coronary artery 

### 12

endothelial ce lls, but not vascular smooth muscle cells (VSMC), which do not express the protein ( Ni et 

### 13
al., 2004 ) . It is possible that NAD(P)H oxidase serves as a potential source of ROS in cells that express 

### 14
this protein. 

### 15

5.2.4.4. Antioxidant Enzyme Disruption

Oxidative stress will result not only from the increased production of ROS, but also from the 

### 16
decreased activity of antioxidant defense enzymes. P b has been shown to alter the function of several 

### 17
antioxidant enzymes, including SOD, CAT, glucose

- 6

- phosphate dehydrogenase (G6PD), and the 

### 18
enzymes involved in glutathione metabolism, GPx, glutathione

- S

- transferase (GST), and glutathione 

### 19
reductase (GR). T hese changes in the antioxidant defense system could be due to the high affinity of Pb 

### 20
for sulfhydryl groups contained within proteins and its metal ion mimicry, however it could also be a 

### 21
consequence of increased oxidative damage by Pb. 

### 22

Studies of the eff ects of Pb on the activities of SOD and CAT give divergent results. These 

### 23
metalloproteins require various essential trace elements for proper structure and function, making them a 

### 24
target for Pb toxicity. CAT is a heme containing protein that requires iron ions to function ( Putnam et al., 

### 25

2000 ) . SOD exists in multiple isoforms that require copper and zinc (SOD1 and SOD3) ( Antonyuk et al., 

### 26

2009 ) or manganese (SOD2) ( Borgstahl et al., 1992 ) . A num ber of studies have found decreased activity 

### 27
of these enzymes ( Ergurhan

- Ilhan et al., 2008 ; Mohammad et al., 2008 ; Pandya et al., 2010 ; Patil, 

### 28
Bhagwat, Patil, Dongre, Ambekar, & Das, 2006 ; Patil, Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 

### 29

2006 ; D. Y. Yu et al., 2008 ) , whereas others observe increased activity ( Ahamed et al., 2008 ; M. K. Lee et 

### 30
al., 2005 ) . Pb exposure (500 ppm, 1, 4, and 8 weeks) in rats showed that organ SOD and CAT responded 

### 31May 2011 5

- 23 DRAFT

– DO NOT CITE OR QUOTE differently depending on th e dose and tissue investigated. Activity of SOD and CAT varied based on the 

### 1
brain region analyzed and time of exposure ( Bennet et al., 2007 ) . Another study found that the brain had 

### 2
consistently decreased SOD activity, irrespective of dose in prenatally exposed animals (0.3 and 3.0 mg/l, 

### 3

blood Pb le vel 20.4 and 24.5 μg/dL); however hepatic SOD activity increased at low level Pb 

### 4
administration and decreased after high level exposure ( Uzbekov et al., 2007 ) . It is possible that the 

### 5
increased SOD and CAT protein is due to activation by ROS, while decreased enzyme activity is the 

### 6
result of metal ion substitution by Pb causing enzyme inactivation. 

### 7

Glutathione is a tripeptide antioxidant containing a cysteine with a reactive thiol group that can act 

### 8
nonenzymatically as a direct antioxidant or as a cofactor in enzymatic detoxification reactions by GST. 

### 9
Glutathione will donate an electron while in its reduced state (GS H), which leads to conversion to the 

### 10
oxidized form, glutathione disulfide (GSSG). Pb bind s to the thiol and can both interfere with the 

### 11
antioxidant capacity of and decrease levels of GSH. Acute administration of Pb (0.1 μM in vitro, 18 

### 12
μg/dL human) results in decreased blood and organ GSH and cysteine content, which may be due to 

### 13

increased GSH efflux from tissues ( Ahamed et al., 2008 ; Ahamed et al., 2009 ; Ahamed et al., 2005 ; 

### 14
Chetty et al., 2005 ; Flora et al., 2007 ; Nakagawa, 1989

, 1991 ; Pandya et al., 2010 ) . Chronic Pb exposure 

### 15
elicits a compensatory upregulation in the biosynthesis of GSH in the attempt to overcome Pb toxicity, 

### 16
thus often manifesting as an increase in Pb

- induced GSH ( Corongiu & Milia, 1982 ; Daggett et al., 1998 ; 

### 17
J. M. Hsu, 1981 ) . However, other studies have found that chronic Pb exposure resulting in blood Pb level 

### 18
between 6.6 and 22 μg/dL, causes the depletion of GSH ( Ercal et al., 1996 ; M. K. Lee et al., 2005 ; 

### 19
Mohammad et al., 2008 ) . Thus, the time of exposure is important to consider when measuring GSH 

### 20

levels. 

### 21
Glutathione reductase (GR) is able to reduce GSSG back to GSH. Therefore, an increased 

### 22

GSSG /GSH ratio is considered indicative of oxidative stress. Pb exposure has been shown to increase the 

### 23

GSSG/GSH ratio ( Ercal et al., 1996 ; Mohammad et al., 2008 ; Sandhir & Gill, 1995 ) , even at blood Pb 

### 24
level below 10 μg/dL in children ( Diouf et al., 2006 ) . Studies have found mixed effects on GR activation. 

### 25
GR possesses a disulfide at its active site that is a target for inhibition by Pb. Studies have found 

### 26

decreased ( Bokara e t al., 2009 ; Sandhir & Gill, 1995 ; Sandhir et al., 1994 ) , increased ( Howard, 1974 ; 

### 27
Sobekova et al., 2009 ) , and no change ( J. M. Hsu, 1981 ) in GR activity after Pb exposure. This could be 

### 28
because the effect of Pb on GR varies depending on sex ( Sobekova et al., 2009 ) and organ or organ region 

### 29
( Bokara et al., 2009 )

. 

### 30
GSH is used as a cofactor for pe roxide reduction and detoxification of xenobiotics by the enzymes 

### 31
GPx and GST. GPx requires selenium for peroxide decomposition ( Rotruck et al., 1973 ) , whereas GST 

### 32
functions via a sulfhydryl group. By reducing the uptake of selenium, depleting cellular GSH, and 

### 33
disrupting protein thiols, Pb is able to decrease the activity of GPx and GST ( M. K. Lee et al., 2005 ; 

### 34
Nakagawa, 1991 ; Schrauzer, 1987 ; D. Y. Yu et al., 2008 ) . Simil ar to other antioxidant enzymes, 

### 35
compensatory upregulation of these enzymes is described after Pb treatment ( Bokara et al., 2009 ; 

### 36May 2011 5

- 24 DRAFT

– DO NOT CITE OR QUOTE Conterato et al., 2007 ; Daggett et al., 1998 ; Ergurhan

- Ilhan et al., 2008 ) . However, other studies have 

### 1
shown that these enzymes may not be able to compensate for the increased Pb

- induced ROS, further 

### 2
contributing to the oxidative environment ( Farmand et al., 2005 )

. 

### 3 Recently, γ

- glutamyltranferase (GGT) within i ts normal range has been regarded as an early and 

### 4
sensitive marker of oxidative stress. This may be because cellular GGT metabolizes extracellular GSH to 

### 5
be used in intracellular GSH synthesis. Thus, cellular GGT acts as an antioxidant enzyme by increasing 

### 6
the intracellular GSH pool. However, the reasons for the association between GGT and oxidative stress 

### 7
have not been fully realized ( D. H. Lee et al., 2004 ) . Occupational Pb exposure (blood Pb level, 29.1 

### 8
μg/dL) results in increased serum GGT levels ( D. A. Khan et al., 2008 ) . Interestingly, low blood Pb levels 

### 9
found in a sample of the U.S. population (NHANES III) were positively associated with s erum GGT 

### 10
levels showing a dose

- response relationship at levels <7 μg/dL ( D. H. Lee et al., 2006 )

. 

### 11

5.2.4.5. Nitric Oxide Signaling

 ̇NO, also known as endothelium

- derived relaxing factor, is a potent endogenous signaling 

### 12
molecule involved in vasodilation. Acute and chronic Pb exposure d ecreases the biologically active  ̇NO, 

### 13 not through reduction in  ̇NO

- production capacity ( Vaziri & Ding, 2001 ; Vaziri, Ding, et al., 1999 ) , but as 

### 14

a result of inactivation and sequestration of  ̇NO by ROS ( Malvezzi et al ., 2001 ; Vaziri, Liang, et al., 

### 15

1999 ) . Endogenous  ̇NO can interact with ROS, specifically O 2 - , produced following exposure to Pb to 

### 16
form the highly cytotoxic reactive nitrogen species, peroxynitrite (ONOO - ). This reactive compound can 

### 17
damage cellular DNA and proteins, resulting in the formation of nitrotyrosine among other products. 

### 18
Overabundance of nitrotyr osine in plasma and tissues is present after exposure to Pb ( Vaziri, Liang, et al., 

### 19

1999 ) .  ̇NO is also produced by macrophages in the defense against certain infectious agents, including 

### 20
bacteria. Studies have found that Pb exposure c an significantly reduce production of  ̇NO in immune cells 

### 21
( J.

- E. Lee et al., 2001 ; Pineda

- Zavaleta et al., 2004 ; L. Tian & Lawrence, 1995 ) , possibly leading to 

### 22

reduced host resistance ( L. Tian & Lawrence, 1996 )

. 

### 23 Production of  ̇NO is catalyzed by a fam ily of enzymes called nitric oxide synthases (NOS), 

### 24
including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS), that require a 

### 25
heme prosthetic group and a zinc cation for enzymatic activity ( Messerschmidt et al., 2001 )

. 

### 26 Paradoxically, the reduction in  ̇NO availability in vascular tissue following Pb exposure is accompanied 

### 27
by significant upregulation in NOS isotype s ( Gonick et al., 1997 ; Vaziri & Ding, 2001 ; Vaziri, Ding, et 

### 28
al., 1999 ) . A direct inhibitory action of Pb on NOS enzymatic activity has been rejected ( Vaziri, Ding, et 

### 29
al., 1999 ) . Instead, the upregulation of NOS occurs as compensation for the decre ased  ̇NO resulting from 

### 30

ROS inactivation ( Vaziri & Ding, 2001 ; Vaziri et al., 2 005 ; Vaziri & Wang, 1999 )

. 

### 31May 2011 5

- 25 DRAFT

– DO NOT CITE OR QUOTE Soluble Guanylate Synthase

M any 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 substrate 

### 2
guanosine triphosphate (GTP). Soluble guanylate cyclase is a heterodimer requiring one molecule of 

### 3
heme for enzymatic activity ( Boerrigter & Burnett, 2009 ) . In vascular smooth muscle cells (VSMC), sGC 

### 4

serves as th e  ̇NO receptor. Marked reduction in plasma concentrations and urinary excretion of cGMP is 

### 5
observed after Pb exposure (5 ppm for 30 days and 0.01% for 12 months) ( Khalil

- Manesh, Gonick, 

### 6
Weiler, et al., 1993 ; M. Marques et al., 2001 ) . In addition, Pb exposure downregulated the protein 

### 7

abundance of sGC in vascular tissue ( Courtois et al., 2003 ; Farmand et al., 2005 ; M. Marques et al., 

### 8

2001 ) . This downregulation in sGC was prevented by antioxidant therapy suggesting that oxidative stress 

### 9
also plays a role in Pb

- induced downregulation of sGC ( M. Marques et al., 2001 )

. 

### 10



## 5.2.5. Inflam mation

Misregulated inflammation represents one of the major effects of Pb

- induced immunotoxicity. It is 

### 11
important to note that this can manifest in any tissue where immune cell mobilization and tissue insult 

### 12

occurs. Enhanced inflammation and tissue damage occurs throug h the modulation of inflammatory cell 

### 13
function and production of pro

- inflammatory cytokines and metabolites. O verproduction of ROS and an 

### 14
apparent depletion of antioxidant protective enzymes and factors (e.g., selenium) accompany this 

### 15

immunom odulation ( Chetty et al., 2005 )

. 

### 16
Traditional immune mediated inflammation can be seen with bronchial hyper

- responsiveness, 

### 17
asthma, and respiratory infections after exposure to Pb. But it is important to recognize th at any tissue or 

### 18
organ can be affected by immune

- mediated inflammatory dysfunction given the distribution of immune 

### 19
cells as both permanent residents and infiltrating cell populations ( Carmignani et al., 2000 ; Mudipalli, 

### 20

2007 ) . Pb spheres implanted in the brains of rats produced neutrophil

- driven inflammation with apoptosis 

### 21
and indications of neurodegeneration ( Kibayashi et al., 2010 )

. Pb also induces renal tubulointerstitial 

### 22
inflammation (18 μg/dL or 100 ppm for 14 weeks) ( Ramesh et al., 2001 ; Rodriguez

- Iturbe et al., 2005 )

, 

### 23
which has been coupled with activation of the redox sensitive nuclear transcription factor kappa B 

### 24 (NFκB) and lymphocyte and macrophage infiltration (23

- 27 μg/dL, 100 ppm for 14 weeks) ( Bravo et al., 

### 25

2007 )

. These events could be in response to the oxidative environment developed from Pb, since Pb



### 26

induced inflammation and NFκB activation can be ameliorated by antioxidant therapy ( Rodriguez

- Iturbe 

### 27
et al., 2004 )

. 

### 28
Inflammation can be mediated by the prod uction of chemical messengers such as prostaglandins 

### 29
(PG). Pb exposure has been reported to increase arachidonic acid (AA) metabolism, thus elevating th e 

### 30
production of PGE 2 , PGF 2 , and thromboxane in humans (48 μg/dL) and cell models (0.01 μM, 48 h) 

### 31
( Cardenas et al., 1993 ; Chetty et al., 2005 ; Flohe et al., 2002 ; Knowles & Donaldson, 1997 ; J. J. Lee & 

### 32May 2011 5

- 26 DRAFT

– DO NOT CITE OR QUOTE Battles, 1994 ) . Dietary Pb supplementation (5 00 ppm, 19 days) can increase the percentage of cell 

### 1
membrane AA, the precursor of cyclooxygenase and lipoxygenase metabolism to PGs and leukotrienes 

### 2
( Knowles & Donaldson, 1990 ) . Additionally, Pb (1 μM, 20 μg/dL) may promote the release of AA via 

### 3

acti vation of phospholipase A 2 , as shown in isolated VSMC ( Dorman & Freeman, 2002 )

. 

### 4
Inflammation may be the result of increased pro

- inflammatory sig naling or may exacerbate these 

### 5 signaling pathways. Pb can elevate the expression of NFκB, as well as expression of activator protein

- 1 

### 6
(AP

- 1) and cJun ( Bravo et al., 2007 ; Korashy & Ei

- Kadi, 2008 ; Korashy & El

- Kadi, 2008 ; Pyatt et al., 

### 7

1996 ; Ramesh et al., 1999 ) . Pb exposure (25 μM) to dendritic cells stimulate s phosphorylation of the 

### 8
Erk/MAPK pathway, but not p38, STAT3 or 5, or CREB ( D. Gao et al., 2007 ) 

### 9

5.2.5.1. Cytokine Production

There are three modes of major effects of P b on immune cytokine production

. First, Pb can act on 

### 10
macrophages to elevate the production of pro

- inflammatory cytokines such as TNF

- α and IL

- 6 ( S. Chen 

### 11
et al., 1999 ; Y.

- J. Cheng et al., 2006 ; Dentener et al., 19 89 ) . This can result in local tissue damage during 

### 12
the course of immune responses affecting such targets as the liver. Second, when Pb acts on dendritic 

### 13
cells, it skews the ratio of IL

- 12/IL

- 10 such that Th1 responses are suppressed and Th2 responses are 

### 14
promoted ( S. Chen et al., 2004 ; M iller et al., 1998 ) . Third, when acquired immune responses occur 

### 15
following exposure to Pb, Th1 lymphocyte production of cytokines is suppressed (e.g. IFN

- γ ) ( Heo et al., 

### 16

1996 ; Lynes et al., 2006 )

. In contrast, Th2 cytokines such as IL

- 4, IL

- 5

, and IL

- 6 are elevated ( D. Gao et 

### 17
al., 2007 ; D. Kim & L awrence, 2000 )

. The combination of these three mode s of cytokine changes induced 

### 18
by Pb creates a hyperinflammatory state among innate immune cells and acquired immunity is skewed 

### 19
toward Th2 responses. 

### 20

Iavicoli et al. ( 2006 ) reported below

- background blood Pb concentrations produced significant 

### 21
changes in cytokine levels

. At the lowest diet ary Pb concentration (0.8 μ g/dL), IL

- 2 and IFN

- γ were 

### 22
elevated over the controls, indicating an enhanced Th1 response. However, as the dietary and blood Pb 

### 23
concentrations increased (resulting in blood Pb level 12

- 61 μ g/dL), a Th2 phenotype was observed with 

### 24
suppressed IFN

- γ and IL

- 2 and elevated IL

- 4 production. These findings support the notion that the 

### 25
immune system is remarkably sensitive to Pb

- induced functional alterations and that nonlinear effects 

### 26
may occur at extremely low Pb exposures. TGF

- β pro duction is also altered by Pb exposure to cells (1 

### 27
μM, 3 days) ( Zuscik et al., 2007 ) . IL

- 2 is one of the more variable cytokines relative to Pb

- induced 

### 28
changes. Depending upon the protocol it can be slightly elevated in production or unchanged. Recently, 

### 29

Gao et al ( 2007 ) found that Pb

- treated dendritic cells (25 μM) promoted a slight but significant increase in 

### 30

IL

- 2 production among lymphocytes. Proinflammatory cytokines have been measured in other organs and 

### 31
cell types after Pb exposure. Elevation of IL

- 1 β and TNF

- α were observed in the hippocampus after Pb 

### 32
exposure and increased IL

- 6 was found in the forebrain ( Stru zynska et al., 2007 )

. 

### 33May 2011 5

- 27 DRAFT

– DO NOT CITE OR QUOTE Consistent with animal studies, epidemiologic studies have also demonstrated Pb

- associated 

### 1

decreases in Th1

- type cytokines and increases in Th2

- type cytokines. Among adults without occupational 

### 2
Pb exposures in Incheon, Korea with blood Pb levels ranging from 0.337 to 10.47 μ g/dL, Kim et al. 

### 3
( 2007 ) found associations of blood Pb with serum levels of TNF

- α and IL

- 6 that were larger among men 

### 4

wi th blood Pb levels ≥ 2.51 μ g/dL (median). In models that adjusted for age, sex, BMI, and smoking 

### 5
status, a 1 μ g/dL increase in blood Pb was associated with a 23% increase (95% CI: 4,55) in log of TNF

- α 

### 6
and a 26% increase in log of IL

- 6 (95% CI: 0,55). 

### 7

Resu lts from studies of occupationally

- exposed adults also suggested that Pb exposure may be 

### 8
associated with decreases in Th1 cytokines and increases in Th2 cytokines; however, analysis were 

### 9
mostly limited to comparisons of levels among different occupational groups with different mean blood 

### 10

Pb levels ( Di Lorenzo et al., 2007 ; Valentino et al., 2007 ; Yucesoy et al., 1997a )

. The exception was the 

### 11
study of male foundry workers, pottery workers, and unexposed workers by Valentino et al. ( 2007 )

. 

### 12
Multiple regression analyses were performed with age, BMI, smoking, and alcohol consumption included 

### 13
as covariates. Although effect estimates were not provided, statistically significant associations were 

### 14

observed between blood Pb and IL

- 10 and TNF

- α, with R 2 values of 0.249 and 0.235, respectively. 

### 15
Exposed workers also had higher levels of most Th2 cyto kines (IL

- 2, IL

- 6, IL

- 10, and TNF

- α) and lower 

### 16
levels of the Th1 cytokine IFN

- γ. Levels of IL

- 2, IL

- 6, and IL

- 10 showed an increasing trend from the 

### 17
lowest to highest blood Pb group. In contrast with most other studies, both exposed worker groups had 

### 18

lower IL

- 4 levels compared with controls. In a similar analysis, DiLorenzo et al. ( 2007 ) separat ed 

### 19
exposed workers into intermediate (9.1

- 29.4 μ g/dL) and high (29.4

- 81.1 μ g/dL) blood Pb level groups, 

### 20
with unexposed workers comprising the low exposure group (blood Pb levels 1

- 11 μ g/dL). Excluded from 

### 21
this study were exposed workers from the highest en d of the distribution of blood Pb levels in DiLorenzo 

### 22

et al. ( 2006 ) . Mean TNF

- α levels showed a monotonic increase from the low to high blood Pb group, 

### 23
which was suggestive of a concentration

- dependent relationship. Levels of granulocyte colony



### 24
stimulating factor (G

- CSF) did not differ between the intermediate and high blood Pb groups among the 

### 25
Pb recyclers; however, G

- CSF levels were higher in the Pb recyclers than in the unexposed controls. 

### 26
Furthermore, among all subjects, blood Pb showed a st rong, positive correlation with G

- CSF. Yucesoy et 

### 27

al. ( 1997a ) found statistically significant lower serum levels of the Th1 cytokines, IL

- 1β and IFN

- γ, in 

### 28
workers compared with controls; however levels of the Th2 cytokine s, IL

- 2 and TNF

- α levels, were 

### 29
similar between groups. Overall, exposure to Pb increases the production of pro

- inflammatory cytokines, 

### 30
skews the ratio of Th1 and Th2 cytokines to favor Th2 responses, and suppresses lymphocyte cytokine 

### 31
production. 

### 32May 2011 5

- 28 DRAFT

– DO NOT CITE OR QUOTE 

## 5.2.6. Endocrine Disruption

5.2.6.1. Hypothalamic

- Pituitary

- Gonadal Axis

Pb is a potent endocrine disrupting chemical that causes reproductive and developmental effects at 

### 1
moderate levels of exposure in both male and female animal models. Pb may act both at multiple points 

### 2
along t he hypothalamic

- pituitary

- gonadal (HPG) axis and directly at gonadal sites. The HPG axis 

### 3
functions in a closely regulated manner to produce circulating sex steroids and growth factors required for 

### 4
normal growth and development. Chronic Pb exposure has been shown to reduce serum levels of follicle



### 5
stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and estradiol ( Biswas & Ghosh, 

### 6

2006 ; Dearth et al., 2002 ; E. F. Krieg, Jr., 2007 ; Ronis et al., 1998 ; Rubio et al., 2006 ; Sokol & Berman, 

### 7

1991 ) . This is likely through th e inhibition of LH secretion and the reduction in the expression of the 

### 8
steroidogenic acute regulatory protein (StAR) ( B. M. Huang et al., 2002 ; B. M. Huang & Liu, 2004 ; 

### 9
Ronis et al., 1996 ; V. Srivastava et al., 2004 ) . StAR is the rate

- limiting step essential in maintai ning 

### 10
gonadotropin

- stimulated steroidogenesis, which results in the formation of testosterone and estradiol. Pb 

### 11
(prenatal exposure resulting in blood Pb level 3 μg/dL) decreases basal StAR synthesis, but not 

### 12
gonadotropin

- stimulated StAR synthesis, suggestin g that Pb may not directly affect ovarian 

### 13
responsiveness to gonadotropin stimulation ( V. Srivastava et al., 2004 ) . I nstead, Pb may act on the 

### 14
hypothalamic

- pituitary level to alter LH secretion, which is necessary to drive StAR production and 

### 15
subsequent sex hormone synthesis. Release of LH and FSH from the pituitary is controlled by 

### 16
gonadotropin

- releasing hormone (GnRH). Pb exposure (10 μM, 90 min) in rat brain median eminence 

### 17

cells can block GnRH release ( Bratton et al., 1994 ) . Pb may also interfere with release of pituitary 

### 18
hormones through interference with ca tion

- dependent secondary messenger systems, which mediate 

### 19
hormone release and storage. 

### 20
Endocrine disruption may also be a result of a ltered hormone binding to endocrine receptors. 

### 21
Prenatal and postnatal Pb exposure (20 ppm) is able to decrease the number o f estrogen receptors found in 

### 22
the uterus and receptor binding affinity ( Wiebe & Barr, 1988 )

. Altered hormone binding ability may be 

### 23
due to the ion binding properties of Pb, resulting in changes in receptor tertiary structure that will disrupt 

### 24
ligand binding. In addition, Pb

- induced changes in hormone levels that act as inducing agents for receptor 

### 25
synthesis may affect the number of hormone receptors produced. 

### 26
Some of these endocrine disrupting effects of Pb have been related to the generation of ROS. 

### 27
Treatment with antioxidants is able to counteract a number of the endocrine disrupting effects of Pb, 

### 28
including apoptosis and decreased sperm motility and production ( P. C. Hsu, Liu, et al., 1998 ; Madhavi et 

### 29
al., 2007 ; Rubio et al., 2006 ; Salawu et al., 2009 ; Shan et al., 2009 ; C. H. Wang et al., 2006 )

. Direct 

### 30
generation of ROS in epididymal spermatozoa was observed after Pb exposure (i.p. 20 or 50 mg/kg, 6 

### 31May 2011 5

- 29 DRAFT

– DO NOT CITE OR QUOTE wk) ( P. C. Hsu, Hsu, et al., 1998 ) . In addition, lipid peroxidation in the seminal plasma was significantly 

### 1

increased in a group of Pb

- exposed workers with blood Pb >40 μg/dL ( A. Kasperczyk et al., 2008 )

. 

### 2
The liver is often associated with the HPG axis due in part to its production of insulin

- like growth 

### 3
factor 1 (IGF

- 1). Pb exposed humans (>4 μg/dL ), animals (14 μg/dL ), and g onadal cells (0.05 mg/mL) 

### 4
show a decrease in plasma IGF

- 1 ( Dearth et al., 2002 ; Huseman et al., 1992 ; Kolesarova et al., 2010 ; Pine 

### 5
et al., 2006 ) , which may be the result of decreased translation or secretion of IGF

- 1 ( Dearth et al., 2002 )

. 

### 6

IGF

- 1 also induces LH

- re leasing hormone release , such that IGF

- 1 decrements may explain decreased LH 

### 7

and estradiol levels. IGF

- 1 production is stimulated by growth hormone (GH) secreted from the pituitary 

### 8
gland and could be the result of GH depletion. 

### 9
A number of studies have revealed that Pb exposure affects the dynamics of growth. Decreased 

### 10
growth after Pb exposure could be the result of Pb induced decreased GH levels ( Berry et al., 2002 ; 

### 11
Camoratto et al., 1993 ; Huseman et al., 1987 ; Huseman et al., 1992 ) . This decrease in GH could be a 

### 12
result of decreased release of GH releasing hormone (GHRH) from the hypo thalamus or disrupted GHRH 

### 13
binding to its receptor, which has been reported in vitro after Pb treatment (IC 50 free Pb in solution 52 

### 14
pM, 30 minutes) ( Lau et al., 1991 ) . GH secretion may also be altered from decreased testosterone, a result 

### 15
of Pb exposure. 

### 16

5.2.6.2. Hypothal amic

- Pituitary

- Thyroid Axis

The effects of Pb on the hypothalamic

- pituitary

- thyroid (HPT) axis are mixed. Pb exposure impacts 

### 17
a variety of players in the thyroid hormone system. A number of human studies have shown a negative 

### 18

association between elevated b lood Pb and thyroxine (T 4 ) and free T 4 levels without alteration in 

### 19
triiodothyronine (T 3 ), suggesting that long

- term Pb exposure may depress thyroid function ( Dunda r et al., 

### 20

2006 ; Robins et al., 1983 ; Tuppurainen et al., 1988 ) . However, animal studies on thyroid hormones have 

### 21
shown mixed results. Pb exp osed cows were reported to have an increase in plasma T 3 and T 4 levels 

### 22
( Swarup et al., 2007 ) , whereas mice and chickens manifested decreased serum T 3 concentrations after Pb 

### 23
exposure accompanied by increased lipid peroxidation ( Chaurasia et al., 1998 ; Chaurasia & Kar, 1997 )

. 

### 24

Decreased serum T 3 and increased lipid peroxidation were both restored by vitamin E treatment, 

### 25
suggesting the disruption of thyroid hormone homeostasis could be a result of altered membrane 

### 26

architecture and oxidative stress ( Chaurasia & Kar, 1997 )

. 

### 27

Decreased T 4 and T 3 may be the result of altered pituitary release of thyroid stimulating hormone 

### 28
(TSH). However, several studies have reported higher TSH levels in high level Pb

- exposed subjects 

### 29
( Abdelouahab et al., 2008 ; Gustafson et al., 1989 ; Lopez et al., 2000 ; B. Singh et al., 2000 ) , which would 

### 30

result in increased T 4 levels. Overall, results on the effects of Pb on the HPT axis are inconclusive. 

### 31May 2011 5

- 30 DRAFT

– DO NOT CITE OR QUOTE 

## 5.2.7. Cell Death and Genotoxicity

A number of studies have attempted to characterize the ge notoxicity of inorganic Pb in human 

### 1
populations, laboratory animals, and cell cultures. Endpoints investigated include DNA damage (single



### 2

and double

- strand breaks, DNA

- adduct formation), mutagenicity, clastogenicity (sister chromatid 

### 3
exchange, micronucleu s formation, chromosomal aberrations), and epigenetic changes (changes in gene 

### 4
expression, mitogenesis). It is important to note that numerous studies have utilized exposure to Pb 

### 5
chromate to investigate genotoxicity endpoints; some studies have specifical ly attributed the observed 

### 6
increases in DNA damage and clastogenicity to the chromate ion while others have not. Due to the 

### 7
uncertainty whether observed genotoxic effects are due to chromate or Pb in studies using this form of 

### 8
inorganic Pb, only studies ut ilizing other forms of inorganic Pb (e.g., Pb nitrate, acetate) are discussed 

### 9

below. 

### 10

5.2.7.1. DNA Damage

A number of studies in human populations have observed positive associations between Pb 

### 11
exposure and DNA damage, as measured as DNA strand breaks. Most of thes e associations have been 

### 12
observed in occupationally

- exposed populations ( Danadevi et al., 2003 ; de Restrepo et al., 2000 ; Fracasso 

### 13
et al., 2002 ; Grover et al., 2010 ; Hengstler et al., 2003 ; Minozzo et al., 2010 ; Palus et al., 2003 ; Shaik & 

### 14
Jamil, 2009 ) . It is important to note that occupationally

- exposed workers have very high blood Pb levels, 

### 15
and in one study ( de Restrepo et al., 2000 ) the association between blood Pb and DNA damage was only 

### 16
observed in workers with blood Pb greater than 120 μg/dL. Also, the studies were equivocal in regard to 

### 17
how blood Pb levels correlated with DNA damage: Fracasso et al. ( 2002 ) observed that DNA damage 

### 18
increased with increasing blood Pb levels ( blood Pb levels, <25, 25

- 35, and >35 μg/dL), whereas Paulus 

### 19

et al. ( 2003 ) and Minozzo et al. ( 2010 ) observed no correlation (mean blood Pb = 50.4 (range = 28.2

- 

### 20

6 5.5) and 59.43 ± 28.34 μg/dL, respectively). Lastly, workers occupationally

- exposed to Pb are also 

### 21
potentially exposed to other genotoxic materials, making it difficult to rule out confounding co

- exposures. 

### 22
For example, Hengstler et al. ( 2003 ) examined workers exposed to Pb, cadmium, and cobalt and observed 

### 23

that neither blood or air Pb (4.4 (IQR: 2.84

- 13.6) μg/dL; 3.0 (IQR: 1.6

- 50.0) μg/m 3 ) was associated with 

### 24
DNA damage when examined alone, but that blood Pb influenced the occurrence of single strand DNA 

### 25
breaks when included in a multiple regression model along with cadmium in a ir and blood and cobalt in 

### 26
air. Two studies were found that investigated Pb

- induced DNA damage resulting from nonoccupational 

### 27
exposures. Mendez

- Gomez ( 2008 ) observed that children living at close and intermediate distances to a 

### 28

Pb smelt er had blood Pb levels of 19.5 (11.3

- 49.2) or 28.6 (11.4

- 47.5) μg/dL, compared to blood Pb level 

### 29
of 4.6 (0.1

- 8.7) μg/dL for children living distant to the smelter. DNA damage was significantly increased 

### 30
in children living nearest the smelter, compared to t he intermediate distant children, but was not 

### 31
significantly different from children living farthest away from the smelter. Multivariate analysis (which 

### 32May 2011 5

- 31 DRAFT

– DO NOT CITE OR QUOTE considered the children’s urinary As levels, highest in children farthest from the smelter), revealed no 

### 1
significant associations between DNA damage and blood Pb. Further, DNA repair ability was also 

### 2
observed to be nonrelated to blood Pb. Alternatively, Yanez et al. ( 2003 ) observed that children living 

### 3
close to a mining complex (blood Pb level= 11.6, range = 3.0 to 19.5 μg/dL) did have increased levels of 

### 4
DNA damage compared to control children that lived further awa y from the mining facility (blood Pb 

### 5
level = 8.3 (3.0

- 25.0) μg/dL). 

### 6
In mice given 0.7 to 89.6 mg/kg Pb nitrate by gavage for 24, 48, or 72 hours, or 1 or 2 weeks, 

### 7
single strand DNA breaks in white blood cells were observed but did not increase with increas ing dose 

### 8
( K. D. Devi et al., 2000 ) . The three highest doses had responses which were similar in magnitude and 

### 9 were actually lower than the responses to lower doses tested. In mice exposed to Pb (0.68 μg/dL) via 

### 10
inhalation for up to 4 weeks, differential leve ls of DNA damage were observed in different organ systems, 

### 11
with only the lung and the liver demonstrating statistically greater DNA damage compared to the 

### 12
respective organ controls after acute exposure ( Valve rde et al., 2002 ) . Statistically elevated levels of DNA 

### 13
damage were observed in the kidneys, lungs, liver, brain, nasal cavity, bone marrow, and leukocytes of 

### 14
mice exposed over a period of 4 weeks, although variability was high in all groups, and the ma gnitude of 

### 15
the DNA damage was characterized as weak and did not increase with increasing durations of exposure. 

### 16

Xu et al. ( 2008 ) exposed mice to 10

- 100 mg/kg Pb acetate vi a gavage for four weeks and observed a 

### 17

dose

- dependent increase in DNA single strand breaks in white blood cells that was statistically significant 

### 18
at 50 and 100 mg/kg. The authors characterized the observed DNA damage as severe. Pb nitrate induced 

### 19

DNA dama ge in primary spermatozoa in Pb

- exposed rats (blood Pb level = 19.5 and 21.9 μg/dL, 

### 20
respectively) compared to control rats ( Nava

- Hernandez et al., 2009 ) . The level of DNA damage was not 

### 21

dose

- dependent, and was comparable in both exposure groups. Narayana and Al

- Bader ( 2011 ) observ ed 

### 22
no increase in DNA damage in the livers of rats exposed to 0.5 or 1% Pb nitrate in drinking water for 60 

### 23
days. Interestingly, although the results were not statistically significant and highly variable, DNA 

### 24
fragmentation appeared to be lower in the expo sed animals. 

### 25
Studies investigating Pb

- induced DNA damage in human cell cultures were contradictory. Pb 

### 26
acetate did not induce DNA strand breaks in HeLa cells when exposed to 500 μM for 20

- 25 hours or 100 

### 27
μM for 0.5

- 4 hours ( Hartwig et al., 1990 ; R. D. Snyder & Lachmann, 1989 ) . Pb nitrate, administered to 

### 28
lymphoma cells at 1

- 10 mM for 6 hours, did not result in any DNA

- protein crosslinks ( M. Costa et al., 

### 29

1996 ) . Pb acetate was observed by Wozniack and Blasiak ( 2003 ) to result in DNA single and double 

### 30
strand breaks in primary human lymphocytes exposed to 1

- 100 μM for 1 hour, although the pattern of 

### 31
damage was peculiar. DNA damage was greater in cells exposed to 1 or 10 μM, compared to those 

### 32
exposed to 100 μM. DNA

- protein crosslinks were only observed in the 100 μM exposure group, 

### 33
suggesting that the decreased strand breaks observed in the high dose group may be a result of increased 

### 34
crosslinking in this group. Shaik et al. ( 2006 ) also observed DNA damage in human lymphocytes exposed 

### 35

to 2.1

- 3.3 mM Pb nitrate for 2 hours. Although there was a dose

- dependent increase in DNA damage from 

### 36May 2011 5

- 32 DRAFT

– DO NOT CITE OR QUOTE 2.1 to 3.3 mM, no statistics were reported and no unexposed control group was included making it 

### 1
difficult to interpret these results. Gastaldo et al. ( 2009 ) observed that exposure of human endothelial cells 

### 2

to 1

- 1000 μM Pb nitrate for 24 hours resulted in a dose

- dependent increase in DNA double strand breaks. 

### 3
Studies in animal cell lines were equally as ambiguous as those using human cell lines. Zelikoff et 

### 4

al. ( 1988 ) and Roy and Rossman ( 1992 ) reported that Pb acetate (concentration not stated and 1 mM, 

### 5
respectively) did not induce single or double DNA strand breaks or DNA

- protein or DNA

- DNA 

### 6
crosslinks in CHV79 cells exposed to Pb acetate. However, both Xu et al. ( 2006 ) and K ermani et al. 

### 7
( 2008 ) reported Pb acetate induced DNA damage in PC12 cells exposed to 0. 1, 1, or 10 μM for 24 hours 

### 8
and in bone marrow mesenchymal stem cells exposed to 60 μM for 48 hours, respectively. Wedrychowski 

### 9

et al. ( 1986 ) reported that DNA

- protein crosslinks were induced in a dose

- dependent manner in hepatoma 

### 10
cells exposed to 50

- 5000 μM Pb nitrate for 4 hours. Pb acetate and Pb nitrate i ncreased the incidence of 

### 11
nick translation in CHV79 cells when a bacterial DNA polymerase was added. 

### 12 Pb acetate did not induce single strand DNA breaks in HeLa cells exposed to 500 μM for 20

- 25 

### 13

hours ( Hartwig et al., 1990 ) . However, exposure to both Pb acetate and UV light resulted in increased 

### 14
persistence of UV

- induced strand breaks, compared to exposure to UV light alone. Similar effects were 

### 15

seen i n hamster V79 cells: UV

- induced mutation rates and SCE frequency was exacerbated by co



### 16
incubation with Pb acetate. Taken together, these data suggest that Pb acetate interferes with normal DNA 

### 17
repair mechanisms triggered by UV exposure alone. Pb nitrate wa s observed to affect different DNA 

### 18 double strand break repair pathways in human endothelial cells exposed to 100 μM for 24 hours. 

### 19
Exposure to Pb inhibited nonhomologous end joining (NHEJ) repair, but increased two other repair 

### 20
pathways, MRE11

- dependent and Rad51

- related repair ( Gastaldo et al., 2007 ) . Interestingly, expo sure of 

### 21 lung carcinoma cells to 100, 300, or 500 μM Pb acetate for 24 hours resulted in an increase in nucleotide 

### 22
excision repair efficiency ( J. P. Li et al., 2008 ) , though this result is difficult to interpret. Roy and 

### 23

Rossman ( 1992 ) observed an increase in UV

- induced mutagenicity w hen CHV79 cells were co

- exposed 

### 24 to 400 μM Pb acetate (a nonmutagenic dose of Pb acetate), indicating an inhibition of DNA repair. 

### 25
Treatment of Chinese hamster ovary cells to 0.5

- 500 μM Pb acetate resulted in a dose

- dependent 

### 26
accumulation of apurinic/apyrim idinic site incision activity, indicating that DNA repair was adversely 

### 27

affected ( McNeill et al., 2007 )

. 

### 28

5.2.7.2. Mutagenicity

Only one human study was found that investigated Pb

- induced mutagenic ity. Van Larebeke et al. 

### 29
( 2004 ) investigated the frequency of mutations in the hypoxanthine phosphoribosyltransferase (HPR T) 

### 30
gene in Flemish women without oxxupational Pb exposures or a number of other heavy metals and 

### 31
organic contaminants. Blood Pb (range 78.2

- 251.0 nM) was statistically significantly positively 

### 32
associated with HPRT mutation frequency in the total population . Also, women with high blood Pb (i.e., 

### 33May 2011 5

- 33 DRAFT

– DO NOT CITE OR QUOTE greater than the population median, not reported) demonstrated a greater mutation frequency compared to 

### 1

women with lower blood Pb. 

### 2

Pb

- induced mutagenicity was investigated in four studies using human cell cultures. Y e ( 1993 ) 

### 3
exposed human keratinocytes to 0.1

- 100 μM/mL Pb acetate for 2

- 24 hours. This study did not measure 

### 4
HPRT mutations directly, but rather measured the amount of tritium incorporated into DNA as an 

### 5
indicator of mutation. In the presence of 6

- thioguanine, tritium incorporation was increased in exposed 

### 6
cells, indicating weak mutagenicity. Hwua and Yang ( 1998 ) reported th at Pb acetate was not mutagenic in 

### 7
human foreskin fibroblasts exposed to 500

- 2000 μM for 24 hours. Pb acetate remained nonmutagenic in 

### 8
the presence of 3

- aminotriazole, a catalase inhibitor, indicating that oxidative metabolism did not play a 

### 9
part in potent ial mutagenicity of Pb. Exposure to Pb acetate alone did not induce mutagenicity in lung 

### 10
carcinoma cells (100

- 500 μM for 24 hours) or fibroblasts (300

- 500 μM for 24 hours) ( J. P. Li et al., 2008 ; 

### 11
C. Y. Wang et al., 2008 ) . However, pretreatment with PKC inhibitors before Pb treatment did result in 

### 12
statistically significant increases in mutagenicity in both cell lines. 

### 13
Results from investigations into Pb

- induced mutagenicity using animal cell lines were as equivocal 

### 14
as the findings from human cell line studies, although differences in mutagenicity m ay be reflective of 

### 15
specific Pb compounds used. Pb acetate was observed to be nonmutagenic (HPRT assay) in Chinese 

### 16
hamster V79 cells exposed to 1

- 25 μM of the compound for 24 hours ( Hartwig et al., 1990 ) , but elicited a 

### 17
mutagenic response in CHV79 cells (gpt assay) exposed to 1700 μM for 5 days ( N. K. Roy & Rossman, 

### 18

1992 ) . Pb acetate was observed to be nonmutagenic (HPRT assay) in Chinese hamster ovary cells 

### 19
exposed to 5 μ M for 6 hours ( McNeill et al., 2007 ) . The observation of muta genicity in the second study 

### 20
is complicated by the concurrent observation of severe cytotoxicity at the same dose. Pb nitrate was 

### 21
alternatively found to be nonmutagenic in CHV79 cells (gpt assay) exposed to 0.5

- 2000 μM for 5 days 

### 22
( N. K. Roy & Rossman, 1992 ) , or mutagenic in the same cell line (HPRT assay) exposed to 50

- 5000 μM 

### 23
for 5 days ( Zelikoff et al., 1988 ) . However, mutagenicity was only observed at 500 μM, and was higher 

### 24
than that observed at higher doses. Pb sulfate was also observed to be mutagenic in CHV79 cells (HPRT 

### 25
assay) exposed to 100

- 1000 μM for 24 hours, but as with Pb nitrate, it was not dose

- d ependent ( Zelikoff 

### 26
et al., 1988 ) . Pb chloride was the only Pb compound tested in animal cell lines that was consistently 

### 27
mutagenic: three studies from the same laboratory observed dose

- dependent mutagenici ty in the gpt assay 

### 28
in Chinese hamster ovary cells exposed to 0.1

- 1 μM Pb chloride for one hour ( Ariza et al., 1998 ; Ariza & 

### 29
Williams, 1996

, 1999 )

. 

### 30

5.2.7.3. Clastog enicity

Clastogenicity is the ability of a compound to induce chromosomal damage, and is commonly 

### 31
observed as sister chromatid exchange, micronuclei formation, or incidence of chromosomal aberrations 

### 32May 2011 5

- 34 DRAFT

– DO NOT CITE OR QUOTE (i.e., breaks or gaps in chromosomes). The potential for Pb to be clastogenic has been investigated in 

### 1

numerous studies as described below. 

### 2

Sister Chromatid Exchange

An association between blood Pb (mean blood Pb = 10.48

- 86.9 μg/dL ) and sister chromatid 

### 3
exchange (SCE) was observed in a number of occupational studies ( Anwar & Kamal, 1988 ; Bilban, 1998 ; 

### 4
Duydu et al., 2005 ; Duydu et al., 2001 ; X. P. Huang et al., 1988 ; Palus et al., 2003 ; Pinto et al., 2000 ; 

### 5
Wiwanitkit et al., 2008 ) . However, there are numerous methodological issues that limit firm conclusions 

### 6
from being drawn, most notably that occupational co

- exposures to other genotoxic materials were 

### 7
possible, although some studies excluded workers with exposures to known mutagens ( X. P. H uang et al., 

### 8

1988 ; Pinto et al., 2000 ) . In most studies that attempted to investigate the dose

- response relationship in 

### 9
workers, no association was observed between increasing blood Pb levels and the incidence of SCE 

### 10
( Du ydu et al., 2001 ; Palus et al., 2003 ; Pinto et al., 2000 ) . However, Huang et al. ( 1988 ) did observe 

### 11
increased SCE in exposed workers in the two highest bl ood Pb groups (52.1 and 86.9 μg/dL), although 

### 12
the association was only statistically significant in the 86.9 μg/dL group. Pinto et al. ( 2000 ) did report an 

### 13
association with duration of exposure (range of years exposed = 1.6

- 40). Two studies reported no 

### 14
correlation between occupational exposure to Pb and incidence of SCE ( T. Rajah & Ahuja, 1995 ; T. T. 

### 15
Rajah & Ahuja, 1996 ) . However, these two studies may have suffered from l imited statistical power to 

### 16
observe an effect as they only included very small Pb exposed populations. Mielzynska et al. ( 2006 ) 

### 17
investigate d the incidence of SCE in children exposed to Pb and PAHs in Poland. Children had an average 

### 18
blood Pb concentration of 7.69 μg/dL and 7.87 SCEs/cell. Male children had higher blood Pb 

### 19
concentrations compared to females, but lower numbers of SCEs. No contro l population was included in 

### 20
this study, and thus interpretation of these findings is difficult. 

### 21
Pb exposure has been observed to induce SCE in multiple laboratory animal studies. In mice 

### 22
exposed to up to 100 mg/kg Pb acetate intraperitoneally, Pb induced SCE at 50 and 100 mg/kg ( Fahmy, 

### 23

1999 )

. Pb nitrate, also administered i.p., induced the formation of SCE in a dose

- dependent manner (10

- 

### 24
40 mg/kg) in the bone marrow of exposed mice ( Dhir et al., 1993 ) . Nayak et al. ( 1989 ) exposed pregnant 

### 25

mice to 100

- 200 mg/kg Pb nitrate via i.v. injection and observed an increase in SCE in dams at 150 and 

### 26
200 mg/kg; no SCEs were observed in the fetuses. Tapisso et al. ( 2009 ) exposed rats to 21.5 mg/kg Pb 

### 27
acetate (1/10 th the LD 50 ) via intraperitoneal injection on alternating days for 11 or 21 days, for a total of 5 

### 28
or 10 exposures. Induction of SCE in the bone marrow of exposed rats was increased over controls in a 

### 29
significant duration

- dependent manner. It is important to note that all three of these studies utilized an 

### 30
injection route of exposure that may not be relevant to exposures encountered by the general population 

### 31
(e.g., drinking water exposure). 

### 32May 2011 5

- 35 DRAFT

– DO NOT CITE OR QUOTE Only two studies were found that investigated SCE formation in human cell lin es due to Pb 

### 1
exposure. Statistically significant, dose

- dependent increases in SCE were observed in human lymphocytes 

### 2
obtained from a single donor when incubated with 1, 5, 10, or 50 μM Pb nitrate ( Ustundag & Duydu, 

### 3

2007 ) . Melatonin and N

- acetylcysteine were reported to ameliorate these effects, indicating Pb may 

### 4
induce SCE through increased oxidative stress. Pb chloride was also observed to increase SCE levels in 

### 5
human lymphocytes exposed to 3 or 5 ppm ( Turkez et al. )

. 

### 6
Studies investigating SCE in rodent cells were more equivocal than those in human cells. Pb 

### 7
sulfate, acetate, and nit rate were found to not induce SCE in Chinese hamster V79 cells ( Hartwig et al., 

### 8

1990 ; Zelik off et al., 1988 ) . Both of these studies only examined 25

- 30 cells per concentration, reducing 

### 9
their ability to detect Pb

- induced SCE. Cai and Arenaz ( 1998 ) , on the other hand, used 100 cells per 

### 10
treatment and observed that exposure to 0.05

- 1 μM Pb nitrate for 3

- 12 hours resulted in a weak, dose



### 11
dependent increase in SCE in Chinese hamster ovary cells. Lin et al. ( 1994 ) also observed a dose



### 12
dependent increase in SCE in Chinese hamster cells exposed to 3

- 30 μM Pb nitrate for 2 hours. 

### 13

Micronucleus Formation

Micronucleus formation in Pb

- exposed workers was investigated in numerous occupational studies 

### 14
( Bilban, 1998 ; Grover et al., 2010 ; M. I. Khan et al., 2010 ; Minozzo et al., 2004 ; Minozzo et al., 2010 ; 

### 15
Palus et al., 2003 ; Pinto et al., 2000 ; Shaik & Jamil, 2009 ; Vaglenov et al., 1998 ; Vaglenov et al., 2001 )

. 

### 16
The workers in the occupational studies generally had high blood Pb levels (>20 μg/dL) making 

### 17
comparisons to the general population difficult, although Pinto et al. ( 2000 ) observed increased 

### 18
micronuclei in exposed workers with an average blood Pb concentration of only 10.48 μg/dL. Studies that 

### 19
analyzed workers according to the magnitude of their blood Pb levels reported no correlation between Pb 

### 20
exposure and the observation of micronuclei ( Minozzo et al., 2004 ; Minozzo et al., 2010 ; Palus et al.

, 

### 21

2003 ; Pinto et al., 2000 ) , although Pinto et al. ( 2000 ) , Grover et al. ( 2010 ) , and Minozzo et al. ( 2010 ) did 

### 22
report an association between micronuc lei formation and duration of exposure. Only one study was found 

### 23
that investigated micronucleus formation in a nonworker population; Mielzynska et al. ( 2006 ) examined 

### 24
associations of blood Pb and urinary PAH metabolite levels with micronuclei formation in children in 

### 25
Poland. Children, with an average blood Pb concentration of 7.69 μg/dL, were observed to have 4.44 

### 26
micronucleated cells per 100 0 cells analyzed. Although no control group was included in this study, a 

### 27
statistically significant positive correlation was observed between blood Pb concentrations and 

### 28
micronuclei frequency, and children with blood Pb greater than 10 μg/dL had significan tly more 

### 29
micronucleated cells than children with blood Pb less than 10 μg/dL. 

### 30
Micronucleus formation in response to Pb exposure has been observed in rodent animal studies. 

### 31

Celik et al. ( 2005 ) observed that exposure of female rats to 140, 250, or 500 g/kg Pb acetate once per 

### 32
week for 10 weeks resulted in statistically significantly increased numbers of micronucleated 

### 33May 2011 5

- 36 DRAFT

– DO NOT CITE OR QUOTE polychromatic erythrocytes (PCEs) compared to controls. Similarly, Alghazal et al. ( 2008 ) exposed rats to 

### 1

100 mg/L Pb acetate daily for 125 days and observed statistically significant increases in micronucleated 

### 2
PCEs in both sexes. Tapisso et al. ( 2009 ) exposed rats to 21.5 mg/kg Pb acet ate (1/10th the LD50) via i.p. 

### 3
injection on alternating days for 11 or 21 days, for a total of 5 or 10 exposures. Formation of micronuclei 

### 4
in the bone marrow of exposed rats was increased over controls in a significant duration

- dependent 

### 5

manner. Two furthe r studies investigated formation of micronuclei in the bone marrow of exposed mice: 

### 6
Roy et al. ( 1992 ) exposed mice to 10 or 20 mg/k g Pb nitrate i.p. and observed a dose

- dependent increase 

### 7
in micronuclei, whereas Jagetia and Aruna ( 1998 ) observed an increase in micronuclei in mice exposed to 

### 8

0.625

- 80 mg/kg Pb nitrate i.p., though the increase was not dose

- dependent. Mice exposed to 1 g/L Pb 

### 9
acetate via a more environmentally relevant route of exposure, drinking water, for 90 days had 

### 10
statistically significant increases in micronucleated PCEs ( C. C. Marques et al., 2006 )

. 

### 11
Three studies were found that reported increased micronucleus formation in human cell lines 

### 12

treated with Pb. Do se

- dependent micronucleus formation was observed in human lymphocytes when 

### 13
exposed to either 1, 5, 10, or 50 μM Pb nitrate or 3 or 5 ppm Pb chloride ( Turkez et al. ; Ustundag & 

### 14
Duydu, 2007 ) . Gastaldo et al. ( 2007 ) also observed a dose

- dependent increase in micronuclei in human 

### 15
endothelial cells exposed to 1

- 1000 μM Pb nitrate for 24 hours. Only two animal cell culture studies 

### 16
investigating micronuclei formation were found. One study observed that micronuclei were not induced in 

### 17
Chinese hamster cells exposed to 3

- 30 μM Pb nitrate for 2 hours ( R. H. Lin et al., 1994 ) , whereas the 

### 18

other observed that Pb acetate induced a dose

- dependent increase in Chinese hamster cells when 

### 19

administered at 0.05

- 10 μM for 18 hours ( Bonacker et al., 2005 )

. 

### 20

Chromosomal Aberrations

Chro mosomal aberrations (e.g., chromosome breaks, nucleoplasmic bridges, di

- and acentric 

### 21
chromosomes, and rings) were examined in a number of occupational studies ( Bilban, 1998 ; De et al., 

### 22

1995 ; Grover et al., 2010 ; X. P. Huang et al., 1988 ; Pinto et al., 2000 ; Shaik & Jamil, 2009 )

. 

### 23
Methodological limitations outlined in previous sections, including potential for occupational co

- exposure 

### 24
to genotoxic substances and generally high blood Pb levels (> 20 μg/dL) that are difficult to interpret in 

### 25
the context of the general population, also pertain to the present findings. No correlation was observed 

### 26
between increasing blood Pb levels and incidence of chromosomal aberrations, although an association 

### 27

was o bserved between duration of exposure and chromosomal damage ( Grover et al., 2010 ; Pinto et al., 

### 28

2000 ) . Two studies reported no associa tion between occupational exposure to Pb and chromosomal 

### 29

aberrations ( Andreae, 1983 ; Anwar & Kamal, 1988 ) . Smejkalova ( 1990 ) observed chromosomal damage 

### 30
and aberrations in children living in a heavily Pb

- contaminated area of Czechoslovakia and an area with 

### 31
less contamination, although the difference between the two was not statistically significant. Although 

### 32
blood Pb levels were statistically significantly higher in the Pb

- contaminated children , they were 

### 33May 2011 5

- 37 DRAFT

– DO NOT CITE OR QUOTE generally comparable (low 30s versus high 20s μg/dL, respectively), indicating there may not be enough 

### 1
of an exposure difference to detect a significant difference in aberration rates. 

### 2
The majority of animal studies investigating Pb

- induced ge notoxicity focused on the ability of Pb 

### 3
to produce chromosomal damage. Fahmy ( 1999 ) exposed mice to 25

- 400 mg/kg Pb acetate i.p., either as a 

### 4
single dose or repeatedly for 3, 5, or 7 days. Chromosomal damage was observed to increase in bone 

### 5
marrow cells (100

- 400 mg/kg) and spermatocytes (50

- 400 mg/kg) in a dose

- dependent manner after both 

### 6
exposure regimens. Pb nitrate was also observed to produce dose

- dependent chromosomal damage in 

### 7
mice exposed i.p. to a single dose of 5, 10, or 20 mg/kg ( Dhir, Sharma, et al., 1992 ) . In a similar 

### 8
experiment, Dhir et al. ( 1990 ) exposed mice to 10, 20, or 40 mg/kg Pb nitrate and saw an increase in 

### 9
chromosomal aberrations, although there was no dose

- response as the response was similar in all doses 

### 10
tested. Nayak et al. ( 1989 ) exposed pregnant mice to 100

- 200 mg/kg Pb nitrate via i.v. injection and 

### 11
observed no chromosomal gaps or breaks in dams or fetuses, but did report some karyotypic 

### 12
chromosomal damage and weak aneuploidy at the low dose. In a similar experiment, low levels of 

### 13
chromosomal aberrations were observed in dams and fetuses injected with 12.5

- 75 mg/kg Pb nitrate, but 

### 14

there was no dose

- response reported and few cells were analyzed ( Nayak, Ray, & Persaud, 1989 ) . I n rats 

### 15
given 2.5 mg/100 g Pb acetate i.p. daily for 5

- 15 days or 10

- 20 mg/100 g once and analyzed after 15 days, 

### 16

Pb

- induced chromosomal aberrations were observed ( Chakraborty et al., 1987 ) . The above studies all 

### 17

suffer from the use of a route of exposure that may not be relevant to human environmental exposures. 

### 18
However, studies utilizing drinking water or dietary exposures also observed increases in chromosomal 

### 19
damage. Aboul

- Ela ( 2002 ) exposed mice to 200 or 400 mg/kg Pb acetate by gavage for 5 days and 

### 20
reported that chromoso mal damage was present in the bone marrow cells and spermatocytes of animals 

### 21
exposed to both doses. Dhir et al. ( 1992 ) also observed a dose

- dependent increase in chromosomal 

### 22
damage in mice exposed via gavage, albeit at much lower doses: either 5 or 10 mg/kg. Nehez et al. ( 2000 ) 

### 23

observed a Pb

- induced increase in aneuploidy and percent of cells with damage after exposure to 

### 24

10 mg/kg administered by gavage 5 day s a week for 4 weeks. In the only study that investigated dietary 

### 25
exposure, El

- Ashmawy et al. ( 2006 ) exposed mice to 0.5% Pb acetate in feed, and observed an increase in 

### 26
abnormal cells and frequency of chromosomal damage. 

### 27
Only three studies were found that investigated the ability of Pb to induce ch romosomal damage in 

### 28
human cell lines and all three reported negative findings. Wise et al. ( 2004 ; 2005 ) observed that Pb 

### 29
glutamate was not mutagenic in human lung cells exposed to 250

- 2,000 μM for 24 hours. Shaik et al. 

### 30
( 2006 ) observed that Pb nitrate did not increase chromosomal aberrations in primary lymphocytes 

### 31
(obtained from healthy volunteers) when incubated with 1.2 or 2 mM for 2 hours. Four studies util izing 

### 32
animal cell lines generally supported the finding of no Pb

- induced chromosomal damage in human cell 

### 33
lines. Pb nitrate was found to induce no chromosomal damage in Chinese hamster ovary cells exposed to 

### 34

500

- 2000 μM for 24 hours ( J. P. Wise, Sr. et al., 1994 ) , 3

- 30 μM for 2 hours ( R. H. Lin et al., 1994 ) , or 

### 35

0.05

- 1 μM for 3

- 12 hours ( C ai & Arenaz, 1998 ) . Wise et al. ( 1994 ) did observe increased chromosomal 

### 36May 2011 5

- 38 DRAFT

– DO NOT CITE OR QUOTE damage in Chinese hamster ovary cells exposed to 1,000 μM Pb glutamate for 24 hours, but did not see 

### 1
any d amage in cells exposed to higher concentrations (up to 2,000 μM). 

### 2

5.2.7.4. Epigenetic Effects

Epigenetic effects are heritable changes in gene expression resulting without changes in the 

### 3
underlying DNA sequence. A prime example of an epigenetic effect is the abnor mal methylation of DNA, 

### 4
which could lead to altered gene expression and cell proliferation and differentiation. 

### 5

DNA Methylation

A single i.v. injection of 75 μmol/kg Pb nitrate resulted in global hypomethylation of hepatic DNA 

### 6

in rats ( Kanduc et al., 1991 ) . The observed hypomethylation in the liver was associate d with an increase 

### 7
in cell proliferation. Two additional studies in humans observed that DNA methylation patterns in adults 

### 8
and cord blood were inversely correlated with bone Pb levels ( Pilsner et al., 2009 ; R. O. Wright et al., 

### 9

2010 ) . Changes in DNA methylation patterns could potentially lead to dysregulation of gene expression 

### 10

and altered tissue differentiation. 

### 11

Mitogene sis

Only a few studies have investigated the potential epigenetic effects of Pb exposure in human 

### 12
populations. One such epigenetic effect investigated was mitogenesis that induces cells to proliferate 

### 13
when they should not. Three studies ( Minozzo et al., 2004 ; Minozzo et al., 2010 ; T. Rajah & Ahuja, 1995 ) 

### 14
were found that reported that Pb reduced mitogenesis in Pb

- exposed workers (blood Pb = 35.4 μg/dL, 

### 15
59.4 μg/dL, and not reported, respectively). The observation of decreased cell division in exposed 

### 16
workers may indicate that cells suffered DNA damage and d ied during division, or that division was 

### 17
delayed to allow for DNA repair to occur. It is also possible that Pb exerts an aneugenic effect and arrests 

### 18
the cell cycle. 

### 19
Many studies have investigated the ability of Pb to induce mitogenesis in animal models, and have 

### 20
consistently shown that Pb nitrate can stimulate DNA synthesis and cell proliferation in the liver of 

### 21
animals exposed to 100 μM/kg via i.v. injection ( Columbano et al., 1987 ; Columbano et al., 1990 ; Coni et 

### 22
al., 1992 ; Ledda

- Columbano et al., 1992 ; Nakajima et al., 1995 ) . Shinozu ka et al. ( 1996 ) observed that 

### 23

Pb

- induced hepatocellular proliferation was similar in magnitude to that induced by TNF

- α at 100 μM/kg, 

### 24

and Pb was observed to induce TNF

- α in glial and nerve cells and NF

- κB, TNF

- α, and iNOS in liver cells 

### 25
in exposed animals at 12.5 mg/kg and 100 μM/kg, respectively ( Y.

- J. Cheng et al., 2002 ; Menegazzi et al., 

### 26

1997 ) . Only one study was found that observed a mitogenic effect after inhalation: exposure to 0.01M Pb 

### 27
acetate for 4 weeks resulted in increased cellular proliferation in the lungs ( Fortoul et al., 2005 )

. 

### 28May 2011 5

- 39 DRAFT

– DO NOT CITE OR QUOTE A great amount of research has been conducted investigating the potential effects of Pb on 

### 1
mitogenesis in human and animal cell cultures. In human cell cultures, Pb acetate inhibited cell growth in 

### 2
hepatoma c ells (0.1

- 100 μM for 2

- 6 days) ( Heiman & Tonner, 1995 ) and primary oligodendrocyte 

### 3
progenitor cells (1 μM for 24 hours) ( W. Deng & Poretz, 2002 ) , and had no observable effects on growth 

### 4
in gliom a cells (0.01

- 10 μM for 12

- 72 hours) ( M. Y. Liu et al., 2000 ) . Pb glutamate had no effect on cell 

### 5
growth in human lung cells, but did increase the mitotic index (250

- 1,000 μM for 24 hours) ( S. S. Wise et 

### 6
al., 2005 ) . The increase in the mitotic index was attributed to an arrest of the cell cycle at M

- phase, and 

### 7
was not attributed to an actual increase of cell growth and proliferation. Gastaldo et al. ( 2007 ) also 

### 8 reported S and G2 cell cycle arrests in human endothelial cells following exposure to 100 μM Pb nitrate 

### 9
for 24 hours. Conflicting res ults with regard to DNA synthesis were reported, with a dose

- dependent 

### 10
inhibition of DNA synthesis reported in hepatoma cells (1

- 100 μM for 72 hours) ( Heiman & Tonner, 

### 11

1995 ) , but an induction of synthesis observed in astrocytoma cells (1

- 50 μM for 24 hours) ( Lu et al., 

### 12

2002 )

. 

### 13
In rat fibroblasts and epithelial cells, Pb acetate, chloride, oxide, and sulfate were all obse rved to 

### 14
inhibit cell growth (10 μM

- 1 mM for 1

- 7 days and 0.078

- 320 μM for 48 hours, respectively) ( Apost oli et 

### 15
al., 2000 ; Iavicoli et al., 2001 ) . Iavicoli et al. ( 2001 ) observed that in addition to inhibiting cell growth in 

### 16
rat fibroblasts, Pb acetate caused GS/M and S

- phase arrest. Pb acetate decreased cell proliferation in 

### 17
mouse mesenchymal stem cells when administered at 0

- 100 μM for 48 hours ( Kermani et al., 2008 ) . Pb 

### 18
nitrate was alternatively reported to increase ( R. H. Lin et al., 1994 ) and decrease ( Cai & Arenaz, 1998 ) 

### 19
the mitotic index in Chinese hamster ovary cells exposed to 1 μ M Pb nitrate. Lin et al. ( 1994 ) did not 

### 20
consider cell cycle arrest when measuring the mitotic index, and did not observe a decrease at higher 

### 21
concentrations; in fact, th e highest concentration tested, 30 μ M, had a mitotic index equal to the untreated 

### 22

control cells. 

### 23

5.2.7.5. Gene Expression

Two animal studies have investigated the ability of Pb to alter gene expression in regard to phase I 

### 24
and II metabolizing enzymes. Suzuki et al

. ( 1996 ) exposed rats to 100 μg/kg Pb acetate or nitrate via i.p. 

### 25
injection and observed an induction of glutathione transferase P (GST

- P) with both Pb compounds. The 

### 26

induction of GST

- P by Pb was observed to operate on the transcriptional level a nd to be dependent on the 

### 27

direct activation of the cis

- element GPEI enhancer. Degawa et al. ( 1993 ) reported that i.v. exposure to 20, 

### 28 50, or 100 μmol/kg Pb nitrate selectively inhibited CYP1A2 levels. Pb was shown to not inhibit CYP1A2 

### 29
by direct enzyme inhibition, but rather to decrease the amount of Cyp1A2 mRNA. In contrast, Korashy 

### 30

and El Kadi ( 2004 ) observed that exposure of murine hepatoma cells to 10

- 100 μM Pb nitrate for 24 

### 31
hours increased the amount of Cyp1A1 m RNA while not influencing the activity of the enzyme. 

### 32
NAD(P)H:quinone oxidoreductase and glutathione S

- transferase Ya activities and mRNA levels were 

### 33May 2011 5

- 40 DRAFT

– DO NOT CITE OR QUOTE increased after exposure to Pb. Incubation of primary human bronchial epithelial cells with 500 μg/L Pb 

### 1

ac etate for 72 hours resulted in the up

- regulation of multiple genes associated with cytochrome P450 

### 2
activity, glutathione metabolism, the pentose phosphate pathway, and amino acid metabolism ( Glahn et 

### 3
al., 2008 )

. 

### 4
One additional animal study provides further evidence that exposure to Pb compounds can perturb 

### 5
gene expression

. Zawia and Harry ( 1995 ) investigated whether the observed Pb

- induced disruption of 

### 6
myelin formation in rat pups exposed postnatally was due to altered gene expression

. In pups exposed to 

### 7

0.2% Pb acetate via lactation from PND1

- 20, the expression of proteolipid protein (PLP), a major 

### 8
structural constituent of myelin, was statistically significantly elevated at PND20, compared to controls

. 

### 9
The expression of another structural element of myelin, myelin basic protein (MBP), was similarly 

### 10
elevated in exposed animals, although not sig nificantly so

. The expression of both genes returned to 

### 11
control levels 5 days following the termination of exposure

. These data suggest that altered gene 

### 12
expression in structural myelin proteins due to Pb exposure may be responsible for observed alteration s in 

### 13
abnormal conduction of nerve impulses. 

### 14

5.2.7.6. Apoptosis

Occupational exposure to Pb and induction of apoptosis was investigated in three studies. One 

### 15
study directly reported that exposure to Pb increased apoptosis compared to nonexposed controls 

### 16
( Minozzo et al., 2010 ) , whereas the other two reported that two early indicators of apoptosis, karyorrhexis 

### 17
and karyolysis, were elevated in exposed workers ( Grover et al., 2010 ; M. I. Khan et al., 2010 ) . Pb nitrate 

### 18
was also observed to induce apoptosis in the liver of exposed animals ( Columbano et al., 1996 ; Nakajima 

### 19
et al., 1995 ) . Apoptosi s was observed in rat fibroblasts exposed to Pb acetate and rat alveolar 

### 20
macrophages exposed to Pb nitrate ( Iavicoli et al., 2001 ; Shabani & Rabbani, 2000 ) . Observati on of Pb



### 21
induced apoptosis may represent the dysregulation of genetically

- controlled cell processes and tissue 

### 22

homeostasis. 

### 23



## 5.2.8. Summary

The diverse health effects of Pb are mediated through multiple, interconnected modes of action. 

### 24

Each of the modes of action discussed has the potential to contribute to the development of a number of 

### 25

Pb

- induced health effects (Table 5

- 2). Evidence for the majority of these modes of action is observed at 

### 26

low blood Pb level in humans and animals, between 2 and 5 μg/dL, and at dos es as low as the picomolar 

### 27
range in animals and cells. Dose captures Pb exposure concentration in invitro systems or in animal 

### 28
studies when no blood Pb level was reported. These observable effect levels are limited by the data and 

### 29

methods available and do not imply that these modes of action are not acting at lower exposure levels or 

### 30
that these doses represent the threshold of the effect. 

### 31May 2011 5

- 41 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 2

. Related health effects resulting from the MOAs of Pb and the lowest level eliciting the MOA reported as bl ood Pb level and dose delivered

M O A Related Health Effects (Section) ISA Lowest Level at which MOA O bserved Studies

Blood Pb Dose

Altered Ion Status All Heath Effects of Pb 3.5 μg/dL 50 pM Huel et al. ( 2008 ) Kern et al. ( 2000 )
Protein Binding Renal (5.5), Effects on Heme Synthesis and Red Blood Cell Funtion (5. 7 ) 6.4 μg/dL 50 μM Chen et al. ( 2010 ) Klann and Shelton ( 1989 )

Oxidative Stress All Heath Effects of Pb 5

- 10 μg/dL 10 μM Quinlan et al. ( 1988 ) Ah amed et al. ( 2006 ) Yiin and Lin ( 1995 )

Inflammation Neurological (5.3), Cardiovascular (5.4), Renal (5.5), Immune (5.6), Respiratory (5.6.4), Cancer (5. 10 ), Hepatic (5. 9 .1) 3 μg/dL 0.01 μM Kim et al. ( 2007 ) Chetty et al. ( 2005 )

Endocrine Disruption Reproductive Effects and Birth O utcomes (5. 8 ), Bone and Teeth (5. 9 .4), Endocrine System (5. 9 .3) 2 μg/dL 20 ppm Krieg ( 2007 ) Wiebe and Barr ( 1988 )

Cell Death/Genotoxicity Cancer (5. 10 ), Reproductive Effects and Birth Outcomes (5. 8 ), Bone and Teeth (5. 9 .4) 3.1 μg/dL 50 nM Van et al. ( 2004 ) Bonacker et al. ( 2005 )

The alteration of cellular ion status (including disruption of Ca 2+ homeostasis, altered ion transport 

### 1
mechanisms, and perturbed protein function through displacement of metal cofactors) appears to be the 

### 2
major unifying mode of action underlying all subsequent modes of action (Figure 5

- 1). Pb will interfere 

### 3
with endogenou s Ca 2+ homeostasis, necessary as a cell signal carrier mediating normal cellular functions. 

### 4
[ Ca 2+ ] i have been shown to increase in a number of cell types including bone, erythrocytes, brain cells, 

### 5
and white blood cells, due to the increased flux of extracellular Ca 2+ into the cell. This disruption of ion 

### 6
transport is due in part to the alteration of the activity of transport channels and proteins, such as Na +

- K + 

### 7
ATPase and voltage

- sensitive Ca 2+ channels. Pb can interfere with these proteins through direct 

### 8
competition between Pb and the native metals present in the protein metal binding domain or through 

### 9
disruption of proteins important in Ca 2+

- dependent cell signaling, such as PKC or calmodulin. Disruption 

### 10
of ion transport not only leads to altered Ca 2+ homeostasis, it can also result in perturbed neurotransmitter 

### 11

function. Pb

- exposure decreases evoked r elease of neurotransmitters, while simultaneously increasing 

### 12
spontaneous release of neurotransmitters through Ca 2+ mimicry. Pb is able to displace metal ions, such as 

### 13
Zn, Mg, and Ca 2+ , from proteins due to the flexible coordination number of Pb and multipl e ligand 

### 14
binding ability, leading to abnormal conformational changes to proteins and altered protein function. 

### 15
Evidence for this metal ion displacement and protein pertubation has been shown at picomolar 

### 16

concentrations of Pb. Additional effects of altered cellular ion status are the inhibition of heme synthesis 

### 17
and decreased cellular energy production due to perturbation of mitochondrial function. 

### 18
Although Pb will bind to proteins within cells through interactions with side group moieties, thus 

### 19
potentially disrupting cellular function, protein binding of Pb may represent a mechanism by which cells 

### 20
protect themselves against the toxic effects of Pb. Intranuclear and intracytosolic inclusion body 

### 21
formation has been observed in the kidney, liver, lung, and brai n following Pb exposure. A number of 

### 22
unique Pb binding proteins have been detected, constituting the observed inclusion bodies. The major Pb 

### 23May 2011 5

- 42 DRAFT

– DO NOT CITE OR QUOTE binding protein in blood is ALAD with carriers of the ALAD

- 2 allele potentially exhibiting higher Pb 

### 1
binding affini ty. Additionally, metallothionein is a important protein in the formation of inclusion bodies 

### 2
and mitigation of the toxic effects of Pb. 

### 3
A second major mode of action of Pb is the development of oxidative stress, due in many instances 

### 4
to the antagonism of normal metal ion functions. The origin of oxidative stress produced after Pb 

### 5 exposure is likely a multipathway process, resulting from oxidation of δ

- ALA, NAD(P)H oxidase 

### 6
activation, membrane and lipid peroxidation, and antioxidant enzyme depletion. Throug h the inhibition of 

### 7

δ

- ALAD due to displacement of Zn, accumulated δ

- ALA goes through an auto

- oxidation process to 

### 8
produce ROS. Additionally, Pb can induce the production of ROS through the activation of NAD(P)H 

### 9

oxidase. Pb

- induced ROS can interact with mem brane lipids to cause a membrane and lipid peroxidation 

### 10
cascade. Enhanced lipid peroxidation can also result from Pb potentiation of Fe 2+ initiated lipid 

### 11
peroxidation and alteration of membrane composition after Pb exposure. Increased Pb

- induced ROS will 

### 12

a lso sequester and inactivate biologically active ·NO, leading to the increased production of the toxic 

### 13
product nitrotyrosine, increased compensatory NOS, and decreased sGC protein. Pb

- induced oxidative 

### 14
stress not only results from increased ROS production but also through the alteration and reduction in 

### 15
activity of the antioxidant defense enzymes. The biological actions of a number of these enzymes are 

### 16
antagonized due to the displacement of the protein functional metal ions by Pb. 

### 17
In a number of organ syst ems Pb

- induced oxidative stress is accompanied by misregulated 

### 18
inflammation. Pb exposure will modulate inflammatory cell function, production of pro

- inflammatory 

### 19
cytokines and metabolites, inflammatory chemical messengers, and pro

- inflammatory signaling ca scades. 

### 20
Cytokine production is skewed toward the production of pro

- inflammatory cytokines like TNF

- α and IL

- 6 

### 21
as well as toward the promotion of a Th2 response and suppression of a Th1 response accompanied by 

### 22
production of related cytokines. 

### 23
Pb is a potent endocrine disrupting chemical. Pb will disrupt the HPG axis evidenced by a decrease 

### 24
in serum hormone levels, such as FSH, LH, testosterone, and estradiol. Pb interacts with the 

### 25
hypothalamic

- pituitary level hormone control causing a decrease in pituitary h ormones, altered growth 

### 26
dynamics, inhibition of LH secretion

, and reduction in StAR protein. Pb has also been shown to alter 

### 27
hormone receptor binding likely due to interference of metal cations in secondary messenger systems and 

### 28
receptor ligand binding and through generation of ROS. Pb also may disrupt the HPT axis by alteration of 

### 29
a number of thyroid hormones, possibly due to oxidative stress. However the results of these studies are 

### 30
mixed and require further investigation. 

### 31
Genotoxicity and cell death has been investigated after Pb exposure in humans, animals, and cell 

### 32
models. High level Pb exposure to humans leads to increased DNA damage, however lower blood Pb 

### 33
levels have caused these effects in experimental animals and cells. Reports vary on the effect o f Pb on 

### 34
DNA repair activity, however a number of studies report decreased repair processes following Pb 

### 35

exposure . There is evidence of mutagenesis and clastogenicity in highly exposed humans, however weak 

### 36May 2011 5

- 43 DRAFT

– DO NOT CITE OR QUOTE evidence has been shown in animals and cells based systems. Human occupational studies provide limited 

### 1
evidence for micronucleus formation (>10 μg/dL), supported by Pb

- induced effects in both animal and 

### 2
cell studies. Animal studies have also provided evidence for Pb

- induced chromosomal aberrations. The 

### 3

obs erved increases in clastogenicity may be the result of increased oxidative damage to DNA due to Pb 

### 4

exposure, as co

- exposures with antioxidants ameliorate the observed toxicities. Limited evidence of 

### 5
epigenetic effects is available, including DNA methylatio n, mitogenesis, and gene expression. Altered 

### 6
gene expression may come about through Pb displacing Zn from multiple transcriptional factors, and thus 

### 7
perturbing their normal cellular activities. Consistently positive results have provided evidence of 

### 8

increa sed apoptosis following Pb exposure. 

### 9
Overall, Pb

- induced health effects can occur through a number of interconnected modes of action 

### 10
that generally originate with the alteration of ion status. 

### 11



# 5.3. N eurological Effects

5.3.1. Introduction

The central nervous system u ndergoes rapid differentiation in utero and in early life, which makes 

### 12

t he developing fetus and infant particularly susceptible to neurological effects associated with 

### 13
environmental exposures ( Landrigan et al. , 1999 ; Rice & Barone, 2000 ) . Based on evidence from diverse 

### 14
prospective a nd cross

- sectional studies focusing primarily on effects associated with Pb exposure levels 

### 15
less than 10 μg/dL, the 2006 Pb AQCD concluded that the “ overall weight of the available evidence 

### 16
provides clear substantiation of neurocognitive decrements being a ssociated in young children with 

### 17

blood

- Pb concentrations ” ( U.S. EPA, 2006 ) . Several individual studies observed inverse associations 

### 18

between blood Pb levels and IQ that persisted at blood Pb levels in the range of 2

- 8 μg/dL ( Lanphear et 

### 19
al., 2000 ; J. Schwartz, 1994 )

. Th is association was substantiated in a pooled analysis of children, 5 to 10 

### 20

year s of age, participating in s even prospective studies (Boston, MA; Cincinnati, OH; Rochester, NY; 

### 21
Cleveland, OH; Mexico City, Mexico; Port Pirie, Australia; and Kosovo, Yugoslavia) ( Lanphear et al., 

### 22

2005 ) . T he 2006 AQCD described associations of blood Pb with a broad range of addit ional, related 

### 23
neurodevelopmental endpoints, including academic achievement and performance, motor skills, mood, 

### 24
and antisocial and delinquent behavior ( U.S. EPA, 2006 )

. 

### 25
Toxicological studies not only provided coherence with similarly consistent findings for Pb



### 26
induced impairments in learning, behavior and attention, and sensory acuities, but also provided 

### 27
biological plausibility by characterizing mechanisms for Pb

- induced neurological effects. In particular, 

### 28
toxicological evidence for Pb exposure interfering with neuronal metabolism at the cellular and 

### 29
histological level (e.g., synaptic architecture during development , neurotransmitter release, glia, neurite 

### 30
outgrowth, the blood brain barrier, and oxidative stress), provided biological plausibility for blood Pb 

### 31May 2011 5

- 44 DRAFT

– DO NOT CITE OR QUOTE levels of children being associated with deficits in multiple functional domains such as cognitive function, 

### 1
motor function, memory, mood, and behavior. Additional b iological plausibility was provided by 

### 2

observations of associations of childhood blood Pb levels with decreased neuronal density and neuronal 

### 3

loss measured in adulthood , as assessed by magnetic resonance imaging techniques ( Cecil et al., 2005 ; 

### 4
Meng et al., 2005 ; Trope et al., 2001 ; Trope et al., 1998 ; Yuan et al., 2006 )

. 

### 5
A key finding across several epidemiologic studies of children was a larger estimated effect for a 

### 6
given incremental increase i n blood Pb levels on neurocognitive deficits in children with lower blood Pb 

### 7
levels compared with children with higher blood

- Pb levels ( Bellinger & Needleman, 2003 ; Canfield, 

### 8
Henderson, et al., 2003 ; Kordas et al., 2006 ; Lanphear et al., 2005 ; Rothenberg & Rothenberg, 2005 ; 

### 9

Tellez

- Rojo et al., 2006 )

. Among these studies were two analyses of the pooled cohort data, both of wh ich 

### 10
demonstrated a supralinear relationship between blood Pb level and IQ with a steeper negative slope 

### 11

observed at blood Pb levels <10 μg/dL ( Lanphear et al., 2005 ; Rothenberg & Rothenberg, 2005 )

. 

### 12
Consistent with epidemiologic findings, toxicological studies also observed nonlinear Pb exposure



### 13
response relationships for outcomes such as behavioral res ponses, neuronal activation, and dopamine 

### 14

release. 

### 15

Another area of focus included the comparison of various lifestages of Pb exposure in terms of risk 

### 16
of neurodevelopmental deficits. Toxicological studies demonstrated that in utero with or without early 

### 17
postnatal exposure to Pb was the most sensitive window for Pb

- dependent neurological effects. 

### 18
Epidemiologic studies observed neurocognitive deficits in association with prenatal, peak childhood, 

### 19
cumulative childhood, and concurrent blood Pb levels. Among studies of children that examined multiple 

### 20
lifestages of exposure, several found that concurrent blood Pb was associated with the largest decrement 

### 21

in IQ ( Baghurst et al., 1992 ; Canfield, Henderson, et al., 2003 ; A. Chen et al., 2005 ; Lanphear et al., 

### 22

2005 ) , with some studies finding that the magnitude of association increas ed with age ( Dietrich, Berger, 

### 23
Succop, et al., 1993 ; Factor

- Litvak et al., 1999 ; Ris et al., 2004 ; G. A. Wasserman et al., 1994 ) . Several 

### 24
studies of children compared effect estimate s for Pb levels measured in blood, deciduous tooth, or tibial 

### 25

bone and found that compared with blood Pb levels, tooth or bone Pb levels w ere a ssociated with an equal 

### 26
or larger magnitude of neurodevelopmental deficits ( Bellinger et al., 1991 ; Greene & Ernhart, 1993 ; 

### 27
Needleman et al., 1979 ; G. A. Wasserman et al., 2003 ) . These f indings pointed to an effect of cumulative 

### 28
childhood exposure

. A common limitation of epidemiologic studies of children was the high correlation 

### 29
among Pb exposure metrics at different ages, making it difficult to distinguish among effects of Pb 

### 30

exposure at different ages ( Lanphear et al., 2005 ) and to ascertain which developmental periods of Pb 

### 31
exposure were associated with the greatest risk of neurodevelopmental decrements . The issue of 

### 32
persistence of the neuro logical effects of Pb exposure also was considered, with some evidence 

### 33
suggesting that the associations of blood Pb levels with neu rodevelopmental outcomes persisted into 

### 34
adolescence and young adulthood in the absence of marked reductions in blood Pb level ( Needleman et 

### 35
al., 1990 ; Ris et al., 2004 ; Tong et al., 1996 )

. 

### 36May 2011 5

- 45 DRAFT

– DO NOT CITE OR QUOTE In epidemiologic studies of adults, neurological effects (e.g., impaired memory, attention, reaction 

### 1
time, visuomotor tasks and rea soning, alterations in visual or brainstem evoked potentials, postural sway, 

### 2

and nerve conduction ) were mostly clearly indicated in occupationally

- exposed workers in association 

### 3
with blood Pb levels in the range of 14 to 40 μg/d L ( Baker et al., 1979 ; Cantarow & Trumper, 1944 )

. 

### 4

Studies of envi ronmentally

- exposed adults produced mixed findings; however, bone Pb levels ( Weisskopf 

### 5
et al., 2004 ; R. O. Wright et al., 2003 ) were associated with decrements in ne urocognitive function more 

### 6

so than were blood Pb levels ( E. F. Krieg, Jr et al., 2005 ; Nordberg et al., 2000 ) . These findings suggested 

### 7

that cumulative Pb e xposures, including higher past exposures, may be important contributors to 

### 8
neurological effects in adults.In addition to cognitive function, blood and bone Pb levels also were 

### 9
associated with greater self

- reported anxiety and depression scores among envir onmentally

- exposed 

### 10

adults ( Rhodes et al., 2003 ) . These findings in adults were strongly supported by parallel findings in 

### 11

female and male rodents for Pb

- induced depression and emotional changes, respectively. Studies of adults 

### 12
also reported associations of blood Pb with risk of amy otrophic lateral sclerosis (ALS) and essential 

### 13

tremor, although the body of literature was smaller and evidence was less consistent than that for 

### 14

cogn itive function. Whereas toxicological studies demonstrated Pb

- induced neurodegeneration and 

### 15
formation of neurofibrilary tangles commonly associated with Alzheimer’s disease pathophysiology, 

### 16
blood Pb level generally was not associated with Alzheimer’s disea se in adults. 

### 17
As discussed throughout this section, recent epidemiologic and toxicological studies continue to 

### 18
demonstrate associations between exposure to or biomarkers of Pb and neurological effects and expand 

### 19
upon the previous body of evidence by demon strating similar effects (e.g., cognitive function, 

### 20
impairments in behavior) at lower blood Pb levels (1

- 5 μg/dL ). Whereas previous evidence was 

### 21
inconsistent, new studies in children report positive associations between blood Pb levels and attention 

### 22

defici t hyperactivity disorder (ADHD). New toxicological studies expand evidence for prenatal and 

### 23
postnatal Pb exposure effects on learning, memory, and attention and provide insight into the contribution 

### 24
of stress to this paradigm. New or expanded areas of toxi cological research related to Pb exposure include 

### 25
mood disorders, neurofibrilary tangle formation, and adult dementia after early life Pb exposures. 

### 26
Historically important areas of toxicological research are further expanded with recent publications of Pb



### 27
dependent effects on neurotransmitters, synapses, glia, neurite outgrowth, the blood brain barrier, and 

### 28
oxidative stress. The data detailed in the subsequent sections continue to enhance the understanding of 

### 29
neurological and neurobehavioral outcomes associ ated with Pb exposure. 

### 30May 2011 5

- 46 DRAFT

– DO NOT CITE OR QUOTE 

## 5.3.2. Neurocognitive Function and Learning

5.3.2.1. Epidemiologic Studies of Cognitive Function in Children

Full

- scale IQ in Children

Several longitudinal cohort studies were initiated in the 1980s in order to address limitations of 

### 1

cross

- sectional studies, including establishing a temporal association between exposure and outcome, 

### 2
examining persistence of neurocognitive deficits to older ages, and comparing risk estimates among blood 

### 3

Pb levels at different lifestages . Moreover, cooperation among in vestigators to adopt similar assessment 

### 4
protocols facilitated pooled and meta

- analyses and comparison of results across populations that differed 

### 5

in blood Pb levels

, race/ ethnicity, and SES. Individual cohort studies in diverse populations were 

### 6

consistent in demonstrating that blood Pb levels in the range of 5 to 10 μg/dL ( Bellinger et al., 1987 ; 

### 7
Bellinger & Needleman, 2003 ; Bellinger et al., 1991 ; Canfield, Henderson, et al., 2003 ; J. Schwartz, 

### 8

1994 ; Tellez

- Rojo et al., 2006 ) , were associated with losses in full

- scale IQ (FSIQ) points in children. 

### 9
These findings were substantiated in a pooled analysis of seven prospective studies (Boston, MA; 

### 10
Cincinnati, OH; Rochester, NY; Cleveland, OH; Mexico City, Mexico; Port Pirie, Australia; and Kosovo, 

### 11
Yugoslavia) by Lanphear et al. ( 2005 ) as well as multiple meta

- analyses that included both cross

- sectional 

### 12
and prospective studies ( Needleman & Gatsonis, 1990 ; Pocock et al., 1994 ; J. Schwartz, 1994 ) (Figure 5

- 

### 13

2 and Table 5

- 3)

. 

### 14
The analysis p ooling data from seven prospective studies included 1333 children with mean 

### 15
(5 th

- 95th percentile) blood Pb levels of 12.4 μg/dL (4.1

- 34.8 μg/dL) ( Lanphear et al., 2005 ) . In 

### 16
multivariate models that adjusted for study site, maternal IQ, Home Observation for the Measurement of 

### 17
Environment ( HOME ) inventory, birth weight , and maternal educ ation, IQ measured at schoolage (mean 

### 18
6.9 years) was inversely associated with concurrent, peak, average lifetime, and early childhood blood Pb 

### 19
levels, with the largest decrement in IQ estimated for concurrent blood Pb levels (

- 0.23 points [95% CI:

- 

### 20
0.32,

- 0.14] at a blood Pb level of 1 μg/dL )

. 

### 21

In Lanphear et al. ( 2005 ) , various models were investigated to characte rize the shape of the blood 

### 22

Pb dose

- response relationship. Consistent with findings from individual studies , Lanphear et al. ( 2005 ) 

### 23
found that a log

- linear model best fit the data, with a greater decrease in IQ estimated for an increase in 

### 24

concurrent blood Pb from <1

- 10 μg/dL (6.2 points [ 95% CI: 3.8, 8.6 ] ) than an increase from 10 to 20 

### 25
μg/dL (1.9 points [ 95% CI: 1.2, 2.6 ] ). Sensitivity analyses, in which one study was successively excluded, 

### 26

revealed that no single study was responsible for driving the results. Although HOME score was not 

### 27
available in the Rochester study, exclusion of that cohort ’ s data resulted in a less negative effect estimate. 

### 28
However, the change was less than 3%. 

### 29May 2011 5

- 47 DRAFT

– DO NOT CITE OR QUOTE Studies published s ince the 2006 Pb AQCD continue to demonstrate associations between 

### 1
increasing blood Pb level and decrements in FSIQ (Figure 5

- 2 and Table 5

- 3). Whereas most studies 

### 2
demonstrated decrements in FSIQ in association with blood Pb levels ranging from 5 to10 μg/dL

, Kim et 

### 3

al. ( 2009 ) was particularly noteworthy for demonstrating an association in a population with lower blood 

### 4
Pb levels (mean 1.73 μg/dL , range 0.42

- 4.91 μg/dL ) . Children ages 8 to 11 years of age in Korea were 

### 5

tested using the Korean Educational Development Institute

- WIS C

, which assesses vocabulary, arithmetic, 

### 6
picture arrangement, and block design. In a linear regression analysis adjusted for age, sex , maternal and 

### 7
paternal education, yearly income, prenatal smoking, postnatal environmental tobacco smoke exposure, 

### 8

birth weight , and maternal age at birth, a 1 μg/dL increase in blood Pb level was associated with a

- 1.03 

### 9
point decrease (95% CI:

- 1.71,

- 0.36) in FSIQ . Increasing blood Pb levels also was associated in 

### 10
performance and verbal IQ ( PIQ and VIQ, respectively). Alth ough several important confounders were 

### 11
considered, there was no direct assessment of the home environment and the primary caregiver ’ s IQ in 

### 12
this study which are notable limitations. 

### 13

Kim et al. ( 2009 ) also examined effect modification of the blood Pb

- FSIQ relationship by blood 

### 14
manganese ( Mn ) levels . The m ean (range) blood Mn level was 14.3 μg/dL (5.3

- 29.02), respectively

. 

### 15

Blood Pb and Mn leve ls were not correlated (r =

- 0.03, p = 0.64)

. To examine effect modification

, 

### 16
children were divided into two groups : blood Mn level above and below the median ( 14 μg/dL)

. 

### 17
Multivariate linear regression models predicting FSIQ, VIQ, and P IQ used concurrently measured blood 

### 18
Pb level as the predictor variable in the low and high Mn groups . The associations for blood Pb level with 

### 19
FSIQ and VIQ were larger in magnitude for children in t he high Mn group (e.g.,

- 5.3 FSIQ points [95% 

### 20

CI:

- 10.1,

- 5.3] per 1 μg/dL inc rease in blood Pb level), compared with children in the low Mn group 

### 21
(e.g.,

- 4.0 FSIQ points [95% CI:

- 9.9, 1.80] per 1 μg/dL increase in blood Pb level) (Figure 5

- 3 )

. 

### 22May 2011 5

- 48 DRAFT

– DO NOT CITE OR QUOTE Reference Exposure Period Mean ( SD ) B lood Pb (μg/dL) FSIQ A ge ( Yea rs)

Kim et al. ( 2009 ) Concurrent 1.73 (0.80) 8

- 11
Walkowiak et al. ( 1998 ) Concurrent 4.7 (2.3) 6
Chiodo et al. ( 2004 ) Concurrent 5.4 (3.3) 7.5 Bellinger et al. ( 1992 ) Early childhood 6.5 (4.9) 10
Min et al. ( 2009 ) Concurrent 7.0 4
9
11
Canfield et al. ( 2003 ) Cumulative 7.4 (4.3) 3 or 5
Schnaas et a l. ( 2006 ) Prenatal 7.8 6

- 10
Dietrich et al. ( 1993 ) Concurrent NR 6.5
Ris et al. ( 2004 ) Early childhood NR 15

- 17 Baghurst et al. ( 1992 ) Early childhood 21.7 (25 th

- 50 th ) 7

- 8 Tong et al. ( 1996 ) Cumulative 17.8 (5.8) 11

- 3
Factor

- Litvak et al. ( 1999 ) Cumulative 30 and 8 a 7
Wasserman et al. ( 2003 ) Cumulative 31.6 and 6.3 a 10

- 12
Surkan et al. ( 2007 ) Concurrent 5

- 10 vs

. 1

- 2 b 6

- 10
Pooled /M eta

- A nalyses Lanphear et al. ( 2005 ) Concurrent 6.9 (1.2) 4.8

- 10
Schwartz et al. ( 1994 ) Early childhood 6.5

- 23 School age Pocock et al. ( 1994 ) Early childhood 6.8

- 21.2 5

- 14
Concurrent 7.4

- 23.7 5

- 14

Change in FSIQ (95CI)
Note: In general, studies are presented in ascending order of mean blood Pb level, followed by a study analyzing blood Pb level as a categorical variable and then by pooled/meta

- analyses. Effect estimates are standardized to a 1 μg/dL increase in blood Pb. The various tests used to measure IQ are scored on a similar scale (approximately 40

- 160). a Effect estimate represents the loss in FSIQ points in c hildren with blood Pb levels 5

- 10 μg/dL, with children with blood Pb levels 1

- 2 μg/dL serving as the referen ce group. b These values represent the mean blood Pb levels in the two groups from different cities. Figure 5

- 2

. Associations of blood Pb levels with full

- scale IQ (FSIQ) among children.May 2011 5

- 49 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 3. Additional characteristics and quantitative results for studies presented in Figure 5

- 2

Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis FSIQ Assessment Effect Estimate (95% CI) a

Kim et al. ( 2009 ) 279 children in Seoul, Seongnam, Ulsan, and Yeoncheon, Korea, ages 8

- 11 yr in April

- December 2007 Concurrent mean ( SD ): 1.73 ( 0.80 ) 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

- 1.03 (

- 1.71,

- 0.36)

Walkowiak et al. ( 1998 ) 384 children in East Germany, age 6 yr in 1994 Concurrent (age 6 yr) mean (SD): 4.7 (2.3) Log linear regression model adjusted for city, visual acuity, contrast sensitivity, parental education, sex, breastfeeding, height, nationality WISC verbal and block design summed index - 0.51 (

- 1.03, 0.01)

Chiodo et al. ( 2004 ) 246 African

- American children Detroit, MI Concurrent (age 7.5 yr) mean (SD): 5.4 (3.3) 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

- 0.20 (

- 0.35,

- 0.05) b

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) Linear regression model adjusted for HOME score (age 10 and 5), child stress, race, maternal IQ, SES, sex, birth order, marital status WISC

- R at age 10 yr

- 0.58 (

- 0.99,

- 0.18)

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. Concurrent mean (range): 7.0 (1.3

- 23.8) Linea r regression model adjusted for HOME score, cargiver’s vocabulary test, sex, parity, maternal marital status, head circumference at birth WISC

- R at age 4 yr 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,

- 0.1 7)

Canfield et al. ( 2003 ) 172 children born 1994

- 1995 in Rochester, NY followed fr om infancy to age 3

- 5 yr 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

- Binet at age 3 or 5 yr. - 1.37 (

- 2.56,

- 0.17 )

Schnaas et al. ( 2006 ) 175 children in Mexico City, Mexico fol lowed from birth (1987

- 1992) followed until age 10

- 15 yr. Prenatal (3 rd trimester) geometric mean (5

- 95th): 7.8 (2.5

- 24.5) Linear mixed effects regression model adjusted for sex, SES, maternal IQ, HOME score, birth weight, postnatal blood Pb, random slope for subject WISC

- R at ages 6

- 10 yr

- 3.4 (

- 5.6,

- 1.3)

Dietrich et al. ( 1993 ) 253 children in Cincinnati, OH followed from birth (1979

- 1985) to age 20

- 23 yr Concurrent Linear regression model WISC

- R at age 6.5 yr

- 0.33 (

- 0.60,

- 0.06)

Ris et al. ( 2004 ) 195 children in Cincinnati, OH followed from birth (1979

- 1985) to age 20

- 23 yr Early childhood (age 6.5 yr) Linear regression model adjusted for maternal IQ, sex, and average total HOME score WISC

- III indices at age 15

- 17 yr factored into Learning/IQ - 0.08 (

- 0.16, 0.004)

Baghurst et al. ( 1992 ) 494 children in Port Pirie, Australia followed from birth (1979

- 1982) to age 11

- 13 yr. Early childhood (avg of age 0

- 3 yr) 25

- 50 th: 1 7.4, 50

- 75th: 21.7 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 W ISC

- R at age 7

- 8 yr

- 3.3 (

- 6.5,

- 0.2)May 2011 5

- 50 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis FSIQ Assessment Effect Estimate (95% CI) a

Tong et al. ( 1996 ) 375 children in Port Pirie, Australia followed from birth (1979

- 1982) to age 11

- 13 yr. Cumulative 0

- 7 yr mean (SD): 17.8 (5.8) Regression model adjusted for sex, age, schoolgrade, parental occupation al prestige, HOME score, maternal IQ, family functioning score, parental smoking, 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

- 4.3 (

- 8.5,

- 0.14)

Factor

- Litvak et al. ( 1999 ) 577 children in Kosovo, Yugoslavia followed from birth (1985

- 1986) to age 10

- 12 yr Cumulative (4

- 7 yr)

mean c : 30 (K. Mitrovica, 8 (Pristina) Log linear regression model adjusted for HOME score, ethnic group, maternal age, birth weight, maternal Raven’s score, maternal education, birth order, sibship size, sex WISC

- R at age 7 yr

- 8.3 (

- 11.4,

- 5.05)

Wasserman et al. ( 2003 ) 290 children in Kosovo, Yugoslavia followed from birth (1985

- 1986) to age 10

- 12 yr Lifetime avg mean: 31.6 (K. Mitrovica, 6.3 (Pristina) 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

- 2.3 (

- 4.0,

- 0.58)

Surkan et al. ( 2007 ) 389 children ages 6

- 10 yr. from Boston, MA and Farmington, ME Concurrent mean (range): 2.2 (1

- 10) Linear regression model adjusted for caregiver IQ, child age, SES, race, birth weight. WISC

- III at ages 6

- 10 yr

- 6.07 (

- 10.7,

- 1.36), blood Pb = 5

- 10 vs. 1

- 2
Pooled/Meta

- analyses
Lanphear et al. ( 2005 ) 1333 children pooled from Boston, Cincinnati, Cleveland, Mexico City, Port Pirie, Rochester, and Yugoslavia cohorts Concurrent mean (SD): 6.9 (1.2) Log linear regression model adjusted for HOME score, birth weight, maternal IQ, maternal education FSIQ measured at ages 4.8

- 10 yr - 0 .23 (

- 0.32,

- 0.14)

Schwartz et al. ( 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 schoolage - 3.75 (

- 4.91,

- 2.60)

Pocock e t al. ( 1994 ) Meta

- analysis of 5 prospective (over 1,100 children and 14 cross

- sectional studies (3,499 children) Prospective studies mean at age 2 yr: 6.8

- 21.2 Cross

- sectional means: 7.4

- 23.7 Meta

- analysis of combining effect estimates from individual studies FSIQ measured at ages 5

- 14 Prospective studies Cross

- sectional studies - 2.7 (

- 4.1,

- 1.2)

- 3.7 (

- 4.8,

- 2.5)

a Effect estimates are standardized to a 1 μg/dL increase in blood Pb level.
b 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.

c Quantitative data not presented. Means estimated from a figure.May 2011 5

- 51 DRAFT

– DO NOT CITE OR QUOTE Source: Used with permission from Elsevier Science, Kim et al. ( 2009 )

. Fi gure 5

- 3. Effect modification of association between concurrent blood Pb level and IQ by blood Mn level. High and low Mn refers to levels above and below the median of 14 μg/dL, respectively.

Min and colleagues ( 2009 ) examined the relationship between Pb exposure assessed at age 4 years 

### 1

and children ’ s IQ and academic achievement at 4, 9, and 11 years of age in a sample of 278 urban 

### 2
children originally enrolled in a prospective study on the effects of prenatal poly

- drug exposure 

### 3
(determined by assay of infant meconium or urine, maternal urine, or maternal self

- report)

. The study 

### 4
population was primarily African

- American (86%) and low SES (98%) ; 39% of mothers had not finished 

### 5
high school

, and 14% were married at the time of enrollment

. The mean blood Pb level at age 4 years was 

### 6
7.0 μg/dL (SD 4.1, range 1.3

- 23.8); 36% had blood Pb level s <5 μg/dL

, and 19% had levels >10 μg/dL. 

### 7

The researchers utilized restricted cubic spline functions for blood Pb level to test for a nonlinear 

### 8
relationship between blood Pb levels and FSIQ. Although the cubic spline term did not attain statistical 

### 9
significance

, analyses suggested a steeper slope at lower Pb levels (up to 7 μg/dL) These findings were 

### 10

consistent with those from the pooled analysis ( Lanphear et al., 2005 ) and other studies ( Tellez

- Rojo et 

### 11
al., 2006 )

. 

### 12
Also similar to previous studies with repeated assessments of cognitive function over time, Min et 

### 13

al. ( 2009 ) found that the association between concurrent blood Pb level and FSIQ persisted with 

### 14May 2011 5

- 52 DRAFT

– DO NOT CITE OR QUOTE increasing age. The magnitude of the inverse association between blood Pb level an d FS IQ was consistent 

### 1
at ages 4, 9 and 11 years . A 1 μg/dL increase in blood Pb level was associated with a loss in IQ points of 

### 2
0.50, 0.41, and 0.54 at ages 4, 9, and 11 years, respectively (Figure 5

- 2 and Table 5

- 3). Th e findings of 

### 3

this study also indicated that specific cognitive domains may be more sensitive to Pb exposure at different 

### 4
stages of development

. Non

- verbal reasoning decrements assessed using the Wechsler Preschool and 

### 5

Primar y Scales of Intelligence

- Revised ( WPPSI

- R ) Performance IQ (PIQ) and Wechsler Intelligence 

### 6

Scales ( WISC )

- IV Perceptual Reasoning Index were consistently associated with increasing blood Pb 

### 7

level even at younger ages while verbal decrements did not become apparent until assessments at 11 years 

### 8
of age

. Lower reading scores were associated with increased Pb exposure at 9 and 11 years while math 

### 9
scores were not affected until age 11 years . An importa nt consideration that may limit the generalizability 

### 10
of these findings is the high prevalence of prenatal exposure to cocaine (51% of subjects) and alcohol 

### 11
(77% of subjects). H owever, accounting for prenatal drug exposure did not attenuate or modify (i.e., no 

### 12
interaction effects) the negative associations between blood Pb level and cognitive outcomes

. 

### 13

Surkan et al. ( 2007 ) used data originally collected for the New England Children ’ s Amalgam Trial 

### 14
(NECAT), a study of 6 to 10 year old English

- speaking children from urban Boston, Massachusetts and 

### 15
rural Farmington, Maine designed to assess the effect of amalgam dental fillings on children ’ s 

### 16
neurodevelopment

. At baseline (prior to placement of am algam fillings), blood Pb levels were measured, 

### 17

and children were administered an extensive battery of neuropsychological tests including tests of 

### 18
memory, learning, visual

- motor ability, reading, reaction time . In analyses that excluded 3 children with 

### 19

blo od Pb level >10 μg/dL

, children with blood Pb levels of 5 to 10 μg/dL had significantly lower WISC



### 20
III Full FSIQ scores (

- 6.07 points ( 95% CI:

- 10.7,

- 1.36] ) compared with children who had levels of 1 to 2 

### 21
μg/dL (referent group), adjusting for age, race/et hnicity (Black or Hispanic vs. non

- Hispanic white), birth 

### 22
weight

, SES , and primary caregiver IQ. 

### 23

Specific Indices of Cognitive Function in Children

In addition to FSIQ, an index of global cognitive function

, blood Pb levels also were associated 

### 24
with speci fic cognitive abilities, including attention, executive function, language, memory and learning, 

### 25
and visuospatial processing in previous studies of children and adolescents ( Bellinger et al., 1991 ; 

### 26
Bellinger & Stiles, 1993 ; Canfield, Kreher, et al., 2003 ; Chiodo et al., 2004 ; Dietrich et al., 1991 ; Dietrich 

### 27
et al., 1992 ; Kordas et al., 2006 ; Lanphear et al., 2000 ; Needleman et al., 1979 ; Ris et al., 2004 ; Tellez



### 28
Rojo et al., 2006 )

. Studies often find associations with several endpoints, and because several tests of 

### 29
neurocognitive function are interrelated, it is difficult to ascribe the effects of Pb exposure to a specific 

### 30
domain of neurocognitive function. For example, i n U.S. re presentative analysis of NHANES III (1988

- 

### 31
1994) data, which included 4853 children ages 6

- 16 years with a geometric mean blood Pb level of 1.9 

### 32
μg/dL, Lanphear et al. ( 2000 ) found that a 1 μg/dL increase in blood Pb levels was associated with 

### 33May 2011 5

- 53 DRAFT

– DO NOT CITE OR QUOTE decreases in arithmetic (

- 0.70 points [ 95% CI:

- 1.0,

- 0.37]), reading (

- 0.99 points [95% CI:

- 1.4,

- 0.62]), 

### 1

b lock d esign (

- 0.10 points [95% CI:

- 0.18,

- 0.02]), and digit span (

- 0.05 [95% CI:

- 0.09,

- 0.01]) subtests. 

### 2
Finding that blood Pb levels are associated with a spectrum of neurocognitive i ndices provides biological 

### 3
plausibility for associations observed between blood Pb levels and IQ. Furthermore, these tests of 

### 4
attention, learning, and memory in humans have parallel tests in animals, and compared with evidence for 

### 5
IQ, evidence for these sp ecific tests may improve understanding of the coherence between findings in 

### 6

humans and animals ( Rice, 1996 )

. 

### 7
Studies published since the 2006 Pb AQCD continue to observe associations between increasing 

### 8
blood Pb level and decrements in these specific indices of cognitive function. Compared with studies of 

### 9
IQ, studies of specific cognitive indices consistently find associations at lower blood Pb levels 

### 10
(population means: 1.2 to 7 μg/dL and quantities of blood Pb levels as low as 2 μg/dL) (Figure 5

- 4 and 

### 11

Table 5

- 4). Recent studies of cognitive function also expanded on previous evidence by providing 

### 12
information on effect modification by genetic, nutritional, and caregiving quality ( E. F. Krieg, Jr. et al., 

### 13

2010 ; Pilsner et al., 2010 ; Solon et al., 2008 ; Surkan et al., 2008 )

. 

### 14May 2011 5

- 54 DRAFT

– DO NOT CITE OR QUOTE Reference Exposure Period Blood PbLevel (μg/dL) a Outcome

Jedrychowski et al. ( 2009b ) Prenatal 1.23 (0.44 to 5) Bayley MDI

Cho et al. ( 2010 ) Concurrent 1.9 (0.67) No omission errors b

Krieg et al. ( 2010 ) Concurrent 1.95 (0.16) Block design Digit Span Reading score
Math score

Miranda et al. ( 2007 ) Early childhood 2 vs. 1 d E nd of grade score c

3 vs. 1 d

Miranda et al. ( 2009 ) Early childhood 2 vs. 1 d End of grade score
3 vs. 1 d

Froehlich et al. ( 2007 ) Concurrent 6.1 (4.9) Spatial memory b Rule learning Spatial span Problem solving

Surkan et al. ( 2008 ) Concurrent 6.4 (4.3) Bayley MDI, all subjects Bayley MDI, high maternal self

- esteem Bayley MDI, low maternal self

- esteem

Pilsner et al. ( 2010 ) Prenatal 6.7 (3.6) Bayley MDI

Hu et al. ( 2006 ) Prenatal 7.1 (5.1) Bayley MDI

Change in test score (standardize d) per unit increase in blood Pb (95% CI)

Note: Test scores were standardized to their standard deviation to facilitate comparisons among tests with different scales. Studies are presented in ascending order of blood Pb level. Effect estimates are standardized to a 1 μg/dL increase in blood Pb. MDI = Mental Developmental Index.

a Blood Pb level refers to the study mean (SD), unless otherwise specified. b S tandard error was estimated from p

- value

. Effect estimate represents association of blood Pb level with number of correct answers on test. c Standard error was estimated from p

- value. d Effect estimate compares test scores of children in different categories of blood Pb concentration. Figure 5

- 4

. As sociations of blood Pb levels with standardized scores for specific indices of cognitive function in children.

Table 5

- 4. Additional characteristics and quantitative results for studies presented in Figure 5

- 4

Study Population/ Location Blood Pb Levels (μ g/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI) a

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

e ducation, birth order, prenatal ETS, sex Bayley MDI assessed at age 36 mo - 0.26 (

- 0.49,

- 0.02)

Cho et al. ( 2010 ) 667 children ages 8

- 11 yr in 2008 Five Korean cities Concurrent mean (range): 1.9 (0.53

- 6.16) Log linear regression model adjusted for age, sex, paren tal education, maternal IQ, child IQ, birth weight, urinary cotinine No omission errors b No comission errors b Word reading score c using KEDI

- WISC at ages 8

- 11 yr - 0.45 (

- 3.02, 2.06)

- 1.80 (

- 4.33, 0.26)

- 1.37 (

- 3.77, 1.03)May 2011 5

- 55 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Blood Pb Levels (μ g/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI) a

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) 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 - 0.05 (

- 0.08,

- 0.009)

- 0.04 (

- 0.06,

- 0.02)

- 0.05 (

- 0.07,

- 0.02)

- 0.03 (

- 0.05,

- 0.004)

Miranda et al. ( 2007 ) 8603 4 th grade children , 2000

- 2004 7 counties in central N orth Carolina Early childhood (ages 0

- 5 yr.) range: 1

- ≥10 Linear regression model adjusted for sex, race, free/reduced

– price lunch, parental education, daily computer use, charter school, age of blood Pb screening, school system 4 th grade end

- of

grade score c - 0.08 (

- 0.16, 0), blood Pb 2 μ g/dL vs. 1 μ g/dL d

- 0.13 (

- 0.21,

- 0.05), blood Pb 3 μ g/dL vs. 1 μ g/dL d

Miranda et al. ( 2009 ) 57,678 4 th grade children , 2001

- 2005 All 100 North Carolina counties Early childhood (ages 9

- 36 mos.) mean (range): 4.8 (1

- 16) Linear regression model adjusted for race, sex, parental education, free/reduced

- price lunch, charter school, school system 4 th grade end

- of

grade score - 0.04 (

- 0.07,

- 0.001), blood Pb 2 μ g/dL vs. 1 μ g/dL d

- 0.05 (

- 0.09,

- 0.02) , blood Pb 3 μ g/dL vs. 1 μ g/dL d

Froelich et al. ( 2007 ) 174 children age 5 yr Rochester, NY Concurrent mean (SD): 6.1 (4.9) 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 b Rule learning and reversal Spatial span Problem solving using CANTAB at age 5 yr - 0.02 (

- 0.06.

- 0.008)

- 0.03 (

- 0.06,

- 0.001)

- 0.007 (

- 0.01, 0)

- 0.04 (

- 0.09, 0.01)

Surkan et al. ( 2008 ) 309 children ages 12

- 36 mo during 1996

- 2001 or 2004

- 2005 Mexico City, Mexico Concurrent mean (SD): 6.4 (4.3) Linear mixed effects regression model adjusted for sex, maternal age, maternal IQ, maternal education, parity, alcohol consumption, smoking, cohort, maternal self

- esteem Bayley MDI, all subjects Bayley MDI, high maternal self

- esteem Bayley MDI, low maternal self

- esteem assessed at ages 12

- 36 mo - 0.013 (

- 0.033, 0.0007) 0.027 (

- 0.037, 0.0 9)

- 0.02 (

- 0.04,

- 0.001)

Pilsner et al. ( 2010 ) 255 children age 24 mo born 1994

- 1995 Mexico City, Mexico Prenatal (cord blood) mean (SD): 6.7 (3.6) Linear regression model adjusted for maternal age, maternal IQ, marital status, parity, gestational age, inadequate folate intake, MTHFR genotype Bayley MDI assessed at age 24 mo - 0.051 (

- 0.087,

- 0.016)

Hu et al. ( 2006 ) 146 children born 1997

- 1999 followed prenatally to age 24

mo Mexico City, Mexico Prenatal (maternal blood Pb) in 1 st trimester mean (range): 7.1 (1.5

- 43.6) 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 - 0.36 (

- 0.70,

- 0.01)

S tudies not included in figure due to lack of sufficient data to calculate z

- scores

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 Reading score Math score Assessed using WIAT at age 6

- 10 yr - 5.20 (

- 9. 45,

- 0.95)

- 4.02 (

- 7.6,

- 0.43), blood Pb level 5

- 10 μ g/dL vs. 1

- 2 μ g/dL d

Chandramouli et al. ( 2009 ) 488 children born 1991

- 1992 followed from birth to age 7

- 8 y r Avon, U.K. Early childhood (age 30 mos.) mean (SD): 4 .22 (3.12) Log linear regression model adjusted for sex, child IQ, maternal education, home ownership, maternal smoking, HOME score, paternal SES, family adversity index, parenting attitudes at 6 mos. Standardized Assessment Test assessed at age 7

- 8 yr - 0.61 (

- 0.82,

- 0.46), continuous blood Pb

- 1.08 (

- 1.71,

- 0.69), blood Pb 2

- 5 μ g/dL vs. 0

- 2 μ g/dL d

- 0.49 (

- 0.79,

- 0.31), blood Pb 5

- 10 μ g/dL vs. 0

- 2 μ g/dL d

- 0.44 (

- 0.93,

- 0.21), blood Pb >10 μ g/dL vs. 0

- 2 μ g/dL dMay 2011 5

- 56 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Blood Pb Levels (μ g/dL) Statistical Analysis Cognitive Index Effect Estimate (95% CI) a

Solon et al. ( 2008 ) 502 children ages 6

- 35 mo 377 children 3

- 5 yr, 2003

- 2004 Visayas, Phi lippines Concurrent mean (range): 7.1 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 pre mature, region of residence) BSID

- II (ages 6

- 35 mo) WPPSI

- III (ages 3

- 5 yr) - 3.32 (

- 5.02,

- 1.60)

- 2.47 (

- 4.58,

- 0.35)

MDI = Mental Development Index, MTHFR = methylenetetrahydrofolate reductase, WIAT = Weschler Individual Achievement Test

a Effect estimates are transformed to a z

- score and standardized to a 1 μ g/dL increase in blood Pb level.
b 95% CI was constructed using a s tandard error that was estimated for a p

- value of 0.01. Authors specified a p

- value of <0.01. Effect estimate represents association of blood Pb with correct answers on test.

c Standard error was estimated from p

- value.
d Effect estimates compare test performance of children in higher blood Pb groups to children in lowest blood Pb group.

Krieg et al. ( 2010 ) examined an older subset of children (ages 12

- 16 years) from NHANES III 

### 1
(1988

- 1994) that were previously examined in Lanphear et al. ( 2000 ) . Similar to Lanphear et al. ( 2000 )

, 

### 2
Krieg et al. ( 2010 ) found associations of increasing concurrent blood Pb level with decrements in block 

### 3
design, digit span, reading score, and arithmetic score in these older children (Figure 5

- 4 and Table 5

- 4). 

### 4
In another study of Korean children ( ages 8

- 11 years) with similar blood Pb levels (mean: 1.9 μg/dL [SD: 

### 5
0.67]), Cho and colleagues ( 2010 ) found concurrent blood Pb levels to be associated with decreases in in 

### 6
tests of attention (errors in responding to targets); however associations were not statist ically significant 

### 7
in models that adjusted for urinary cotinine levels (Figure 5

- 4 and Table 5

- 4). Although associations of 

### 8
blood Pb levels with word and color naming were negative, effect estimates were associated with wide 

### 9

95% CIs. 

### 10
Krieg et al. ( 2010 ) additionally provided additional information on effect modificati on by vitamin 

### 11
D receptor (VDR) variants. Although there were not differences in blood Pb levels among the various 

### 12
haplotypes of VDR

, various polymorphisms and haplotypes modified the association between blood Pb 

### 13
level and a range of neurocognitive tests. The VDR regulates calcium absorption and metabolism, and 

### 14
effect modification by VDR variants is consistent with the well

- established mode of action of Pb in 

### 15
mimicking calcium in shared transport and metabolic pathways. However, several inconsistencies were 

### 16

observed by Krieg et al. ( 2010 ) in that a particular variant w as associated with a lower Pb

- associated 

### 17
decrement in performance for some tests and a greater Pb

- associated decrement in performance for other 

### 18
tests. For example, among children ages 12

- 16 years , the VDR rs2239185 CC genotype was associated 

### 19
with the large st blood Pb

- associated decrease in digit span score and reading score. The slopes for digit 

### 20
span score ( 95% CI) per 1 μg/dL increase in blood Pb level were

- 1.5 (

- 2.2,

- 0.71 ) for the CC genotype 

### 21

and

- 0. 26 (

- 0.99

, 0.46 ) for the TT genotype. Conversely, the TT genotype was associated with the greatest 

### 22

Pb

- associated decrease in arithmetic score. The slopes (95% CI s ) for math score per 1 μg/dL increase in 

### 23

blood Pb level were

- 8.4 (

- 11.5

, - 5.2 ) for the TT genotype and

- 2.7 (

- 10.1, 4.6 ) for the CC genotype. 

### 24May 2011 5

- 57 DRAFT

– DO NOT CITE OR QUOTE Effec t modification by VDR rs731236 was more consistent across cognitive tests, with larger decrements 

### 1

in blood Pb

- associated cognitive performance among children with the CC genotype

. 

### 2
Multiple studies in different Mexico City mother

- child dyads recently report ed on associations 

### 3
between blood Pb levels (e.g., maternal, cord blood, or child postnatal Pb levels) and mental development 

### 4
in children at age 24 or 36 months ( H. Hu et al., 2006 ; Pilsner et al., 2010 ; Surkan et al., 2008 )

. Hu et al

. 

### 5
( 2006 ) assess ed the impact of timing of exposure prenatally among 146 mother

- child dyads meeting the 

### 6
following criteria: born at 37 weeks or greater gestati onal age, at least one valid Pb measurement during 

### 7
pregnancy, complete information on maternal age and IQ, and child ’ s blood Pb level at 24 months when 

### 8

the 24

- month Bayley Mental Development Index (MDI) was ascertained

. Comparing whole blood and 

### 9
plasma Pb levels collected at each of 3 trimesters, this group found that 1st trimester blood Pb (or plasma 

### 10
Pb) was the best predictor of subsequent 24

- month Bayley Scale MDI scores

. Another study excluding 

### 11

the first trimester demonstrated an inverse association bet ween IQ assessed at age 6

- 10 years and third 

### 12

trimester maternal blood Pb level , but not with maternal blood Pb levels measured at other times during 

### 13
pregnancy or with in child Pb blood levels averaged over 6 to 10 years ( Schnaas et al., 2006 )

. 

### 14

Surkan et al. ( 2008 ) found negative associations (statistically nonsignificant) of concurrent b lood 

### 15
Pb levels with Bayley MDI and Psychomotor Development Index overall in a population of 379 Mexico 

### 16
City children between ages 12 and 36 months. However, when data were stratified by maternal self



### 17
esteem, negative associations were observed among childr en with mothers with in the lowest three 

### 18
quartiles of self

- esteem but not among children with mothers in the highest quartile of self

- esteem (Figure 

### 19

5

- 4 and Table 5

- 4). These findings indicate that higher maternal psychosocial functioning (e.g., stress, 

### 20

an xiety, depression, self

- esteem) may contribute to better caregiving

, which in turn may improve 

### 21
neuropsychological functioning of the child. The se limited data in humans are well

- supported by findings 

### 22

from a nimal studies that have shown that environmental e nrichment can reverse the effects of early stress 

### 23
experiences on reactions such as depressed behavior, HPA activation, and immunosuppression ( Francis et 

### 24
al., 2002 ; Laviola et al., 2008 ; Laviola et al., 2004 ; Moreley

- Fletcher et al., 2003 ) . With specific regards 

### 25
to Pb exposure, Schneider et al. ( 2001 ) and Guilarte et al

. ( 2003 ) demonstrated that the social isolation or 

### 26
enrichment can exacerbate or protect against, respectively, the neurological effects from Pb exposure . It is 

### 27
worth mentioning in this context that the p otential programming effects of stress on childhood health 

### 28
outcomes may occur at an even more fundamental level, i.e., through epigenetic programming ( Dolinoy 

### 29
& Jirtle, 2008 ) . Pb exposure of animals and blood Pb levels in huma ns have been associated with altered 

### 30
DNA methylation patterns which in turn, may be associated with altered gene expression patterns 

### 31
(Section 5.3.6.11 and Section 5.10.4). 

### 32
In a recent study in Mexico City, Mexico, investigators found increasing cord blood Pb levels to be 

### 33
associated with lower MDI scores among children at age 24 months (

- 0.051 points [95% CI:

- 0.087,

- 

### 34
0.016] in standardized score per 1 μg/dL increase in blood Pb level) ( Pilsner et al., 2010 ) . Investigators 

### 35
additionally examined effect modification by variants in the methylenetetrahydrofolate reductase 

### 36May 2011 5

- 58 DRAFT

– DO NOT CITE OR QUOTE (MTHFR) gene. The MTHFR enzyme is involved in folate metabolism, specifically, catalyzing the 

### 1
conversion of 5,10

- methylenetetrahydrofolate to 5

- methylenehydrofolate, which, in turn, is involved in 

### 2
homocysteine methylation to the amino acid methionine. The transfer of methyl groups that results from 

### 3

folate metabolism is important for biological processes including Phase II detoxification reactions and 

### 4
epigenetic regulation of gene expression. The MTHFR gene has common functional variants, including 

### 5
the C677T SNP, which produces an enzyme with lower metabolic activity and is associated with lower 

### 6

serum folate levels ( Kordas et al., 2009 ) . Although Pilsner et al. ( 2010 ) found that both cord blood Pb 

### 7

levels and the MTHFR 6 77T allele were associated with lower child MDI score at age 24 months , they 

### 8

did not find a statistically significant interaction between blood Pb level and the MT HFR 677T allele. 

### 9
Results from stratified analyses were not reported, thus differences in the magnitude of association 

### 10
between genotypes could not be compared. 

### 11
Instead of analyzing MTHFR genetic variants to represent folate metabolism, Solon et al. ( 2008 ) 

### 12
measured red blood cell folate levels among children in the Phillippines. Not only did investigators find 

### 13
an association between increasing blood Pb level and lower cognitive test performanc e (Table 5

- 4), but 

### 14
they found effect modification by red blood cell folate levels . Among children with folate levels less than 

### 15
or equal to 230 μg/m L , blood Pb level had a negative marginal effect on MDI (

- 0.80 to

- 2.44 points), 

### 16
whereas among children with higher folate levels, blood Pb level did not have a negative marginal impact. 

### 17
Thus, in contrast with those from Pilsner et al. ( 2010 ) , findings from So lon et al. ( 2008 ) indicate that 

### 18
children with folate deficiencies may be at increased susceptibili ty to Pb effects on cognitive function. 

### 19

Academic Performance in Children

Although the preponderance of evidence for Pb

- associated neurodevelopmental deficits is for IQ 

### 20

and specific indices of cognitive function , academic achievement and school performance are corollaries 

### 21
to aptitude that may be more objective measure of one ’ s abilities and skills and have important 

### 22
implications for success later in life. Aptitude tests are used to predict future performance of an individual 

### 23

on a task or test. Achievement te sts and school performance, in comparison, assess an individual ’ s actual 

### 24
knowledge in subject areas the individual has studied and measure the acquired knowledge of that subject. 

### 25
Studies reviewed in the 2006 Pb AQCD consistently demonstrated associations o f Pb biomarkers with 

### 26
measures of academic achievement and performance including scores on math or vocabulary tests, class 

### 27
rank, teacher ’ s assessment of academic functioning, and high school completion. Several studies found 

### 28

that blood or dentin Pb levels m easured at an early age (ages 2

- 8 years) were associated with outcomes at 

### 29
older ages (ages 8

- 18 years), suggesting early exposure to Pb may have persistent effects ( Bellinger et al., 

### 30

1992 ; Leviton et al., 1993 ; Needleman et al., 1990 ) . Results from the longitudinal study by Bellinger et al. 

### 31
( 1992 ) was particularly noteworthy for examining associations of blood Pb at several ages with scores on 

### 32
the Kaufman Test of Educational Achievement at age 10 years . Only blood Pb level at age 2 years showed 

### 33May 2011 5

- 59 DRAFT

– DO NOT CITE OR QUOTE a statistically significant association with lower predicted academic achievement. Additionally, the 

### 1
association was robust to adjustment for IQ, indicating that blood Pb levels may be associated with 

### 2
reduced performance on academic tasks not reflec ted in indices of IQ. Several studies also found 

### 3

associations between concurrent blood Pb levels and academic achievement ( Al

- Saleh et al., 2001 ; 

### 4
Kordas et al., 2006 ; Lanphear et al., 2000 ; C.

- L. Wang et al., 2002 )

. Amo ng r ecent studies , academic 

### 5
performance was examined less frequently; however, findings are consistent with the extant body of 

### 6

evidence. 

### 7

Miranda et al. ( 2007 ) lin ked blood Pb surveillance data collected between 0 and 5 years with end



### 8

of

- grade (EOG) testing data at 4 th grade for 7 of the largest counties in North Carolina. Approximately 

### 9

22

- 30% of children in these counties were screened for Pb poisoning, and the tot al sample size in the 

### 10
analysis was approximately 8,600 children for both math and reading achievement tests. For both reading 

### 11
and math, achievement test scores were inversely associated with early childhood blood Pb screening data 

### 12
(Figure s 5

- 4 and 5

- 5 and Table 5

- 4 ). 

### 13

Source: Used with permission from Elsevier Science, Miranda et al. ( 2007 )

. Figure 5

- 5. Comparing model results fo r 4th

- grade EOG mathemati cs
scores. Based on a referent individual who was screened at 2 years of age and is a white female, living in Wake County, NC, parents with a high school education, not enrolled in the school lunch program, and who does not use a computer every day. Baseline score is 262.6.

Similar results were obtained in a subsequent study expanded to the entire state of North Carolina 

### 14
( Miranda et al., 2009 ) (Figure 5

- 4 and Table 5

- 4)

. Investigators additionally used q uantile regres sion to 

### 15May 2011 5

- 60 DRAFT

– DO NOT CITE OR QUOTE estimate effects for conditional percentiles of EOG (e.g., what is the 10th percentile of EOG test scores 

### 1
conditioned on early childhood blood Pb levels) rather than conditional means . Compared with linear 

### 2
regression, quantile regression is more ro bust in response to outliers and predicts outcomes at the top and 

### 3

bottom tails of the distribution of the outcome rather than at the mean. The distributions in EOG scores 

### 4

for children with blood Pb levels greater than or equal to 10 μ g/dL were more spread out than those for 

### 5

children with lower blood Pb levels. With increasing blood Pb levels, the lower tail of the EOG 

### 6

distribution was stretche d out more so than the middle or upper tail of the distribution. For example, in 

### 7
comparisons of children with blood Pb levels of 5 μ g/dL versus children with blood Pb levels of 1 μ g/dL, 

### 8
children in the 5th percentile of EOG have a greater decrease in EOG score compared with children in the 

### 9
95th percentile of EOG ( F igure 5

- 6 ). These findings indicate that children residing at the lowest 

### 10
performance regions of the EOG distribution may be differentially affected by blood Pb levels as 

### 11
compared with children in the middle or higher regions

. Similarly, using quantile regression, Miranda et 

### 12

al. ( 2009 ) s howed that, in addition to elevated blood Pb levels , cumulative social risk (lower parental 

### 13
education, being enrolled in a school lunch program) further enhanced the negative effects on academic 

### 14

achievement in these children. 

### 15

Source: Used with permission from Elsevier Science, Miranda et al. ( 2009 ) Figure 5

- 6. Reduction in EOG achievement test scores at each perce ntile of the test distribution. Note greater effect of Pb at low end of the distribution.May 2011 5

- 61 DRAFT

– DO NOT CITE OR QUOTE Similar to Miranda et al. ( 2009 )

, Chandramouli et al. ( 2009 ) observed associations between early 

### 1

childhood b lood Pb level s ( age 30 months ) and later academic performance ( Standard Assessment Tests a t 

### 2
age 7 years ) among participants of the Avon Longitudinal Study of Parents and Children conducted in the 

### 3

U

. K (Figure 5

- 4 and Table 5

- 4). While these aforementioned recent studies found negative associations 

### 4
for early childhood blood Pb levels, unlike the longitudinal assessment by Bellinger et al. ( 1992 ) , they did 

### 5
not have available blood Pb measurements at other lifestages to compare associations with blood Pb 

### 6
levels at other lifestages. Therefore, results fr om these recent studies do not preclude associations with 

### 7
blood Pb levels at other lifestages. Among children (ages 6

- 10 years) participating in NECAT, increasing 

### 8
concurrent blood Pb level was associated with poorer performance on the Wechsler Indi vidual 

### 9

A chievement Test, even when adjusted for IQ ( Surkan et al., 2007 ) . In analyses adjusted for child IQ, 

### 10
caregiver IQ, age, SES, race, and birth weight, children with concu rrent blood Pb levels 5

- 10 μg/dL 

### 11

scored 5.2 (95% CI: 0.95, 9.45) points and 4.0 ( 95% CI: 0.43, 7.6) points lower o n reading and math 

### 12
composite scores on the respectively, compared to children with levels of 1

- 2 μg/dL . Blood Pb levels 

### 13
were similarly associa ted with other tests of cognitive function (e.g., FSIQ, working memory, cognitive 

### 14
flexibility and a number of executive functioning domains [ i.e., ability to formulate, test, and adapt 

### 15
hypotheses ]). Children with blood Pb levels 3

- 4 μg/dL had lower scores compared with children with 

### 16

blood Pb levels 1

- 2 μg/dL ; however, differences were not statistically significant. 

### 17

Age

- based S usceptibility to Lead

- associated Neurodevelopmental Deficits

Plasticity is a consequence of environmental exposures during critical l ife periods affecting key 

### 18
physiological systems that orchestrat e underlying developmental processes ( Feinberg, 2007 )

. Exposure t o 

### 19
environmental toxins during prenatal and/or early postnatal development may alter the normal course of 

### 20
morphogenesis and maturation that occurs in utero and early in life, resulting in changes that affect 

### 21
structure or function of the central nervous syst em via altered neuronal growth and/or 

### 22
synaptogenesis/pruning structure ( Landrigan et al., 1999 ; Rice & Barone, 2000 )

. This hypothesis is well



### 23
supported by findings in animals that prenatal Pb exposure alters brain development via changes in 

### 24
synaptic architecture (Section 5.3.6.5) and neuronal outgrowth (Section 5.3.6.10) and leads to 

### 25
impairments in memory and learning (Se ction 5.3.2.2) and emotional and depressive changes postnatally 

### 26
(Section 5.3.3.4). Unlike other organ systems, the unidirectional nature of CNS development limits the 

### 27
capacity of the developing brain to compensate for cell loss, and environmentally

- induced cell death can 

### 28

result in a permanent reduction in cell numbers ( Bayer, 1989 ) . Hence, when normal development is 

### 29
altered, the early effects may persist into adult life even in the absence of current exposure, magnifying 

### 30
the public health impact . Supporting evidence is provided by toxicological st udies that find that Pb 

### 31
exposure during neonatal development but not in adulthood leads to neurodegenerative amyloid plaque 

### 32
formation in the brains of aged rodents and monkeys (Section 5.3.5.2). 

### 33May 2011 5

- 62 DRAFT

– DO NOT CITE OR QUOTE With repeated assessment of children prenatally to later chil dhood and early adulthood, t he 

### 1
prospective cohort studies have aimed to distinguish among the effects of blood Pb levels at different 

### 2
periods of development. In the collective body of evidence, neurocognitive decrements have been 

### 3
associated with prenatal, early childhood, childhood average, and concurrent blood Pb levels. In these 

### 4
studies, the identification of developmental periods when children are most sensitive to Pb

- associated 

### 5
neurocognitive decrements has been complicated by the high degree of correla tion in children’s blood Pb 

### 6
levels over time and the confounding of age and peak blood Pb levels ( Dietrich, Berger, & Succop, 1993 ; 

### 7
Lanphear et al., 2005 ; Needleman et al., 1990 )

. 

### 8
As described in detail in the 2006 Pb AQCD, several studies with varying lengths of follow

- up 

### 9
demonstrated associations of prenatal blood Pb levels with neurodevelopmental deficits thoughout 

### 10

c hildhood and into early adulthood ( U.S. EPA, 2006 ) . The prenatal period may be susceptible lifestage of 

### 11
Pb exposure not only because of the nervous system developmental process occurring as described above 

### 12
but also because of factors that result in elevated Pb exposures. S ubstantial fe tal Pb exposure may occur 

### 13
from mobilization of maternal skeletal Pb stores even related to remote exposure s ( Gulson et al., 2003 ; H. 

### 14

Hu & Hernandez

- Avila, 2002 )

. Pb can cross the placenta to affect the developing fetal nervous system 

### 15
( Rabinowitz, 1988 )

. Maternal and umbilical cord blood Pb levels generally are highly correlated, 

### 16
indicating that a newborn infant ’ s blood Pb levels reflects that of the mother ( Schell et al., 2003 )

. 

### 17
Associations with prenatal blood Pb levels were demonstrated most consistently for cognitive 

### 18
function and behavior assessed between infancy and age 3 years (Figures 5

- 7 and 5

- 8 and Table 5

- 5). 

### 19
Among studies examining associations of early

- life blood Pb measures (maternal, cord, or neonatal 

### 20
blood), results were mixed as to whether prenatal ( Bellinger et al., 1984 ; H. Hu et al., 2006 ) or concurr ent 

### 21

blood Pb levels ( G. Wasserman et al., 1992 ; G. A. Wasserman et al., 1998 ) were associated with a greater 

### 22

decrement in cognitive function. Several studies found that prenatal or neonatal blood Pb levels were 

### 23
associated with neurodevelopmental decrements assessed in neonates (within 30 days) or early in infancy 

### 24
(within 3 months), which indicated that relatively short

- durati ons of Pb exposure may be associated with 

### 25
negative neurological effects ( Dietrich et al., 1987 ; Ernhart et al., 1986 ; Rothenberg et al., 1989 ; Shen et 

### 26
al., 1998 )

. 

### 27
A recent analysis of 444 children participating in the Krakow Prospective Cohort Study 

### 28
corroborated previous findings for prenatal exposure and found associations at lower umbilical cord blood 

### 29
Pb levels than those in previous studies. Children in Jedrychowski et al. ( 2009b ) had a median (range) of 

### 30
cord blood Pb levels of 1.23 (0.44

- 6. 9 μg/dL), and increasing umbilical cord blood Pb levels was 

### 31

associated with lower 36

- month Bayley MDI (Figure 5

- 4 and Table 5

- 4). Investigators also observed a 

### 32
larger magnitude of effect in males compared with females (Figure 5

- 9). These findings are consi stent 

### 33
with the hypothesis that the developing male central nervous system may be more vulnerable than 

### 34
females’ to environmental insults resulting in later behavioral problems ( Moffitt et al., 2001 )

. 

### 35May 2011 5

- 63 DRAFT

– DO NOT CITE OR QUOTE References Mean (SD) blood Pb (μg/dL) Outcome Exposure Period
Rothenberg et al. ( 1989 ) 15.5 (5.7) Self

- quieting, 30 days Prenatal

Dietrich et al. ( 1987 ) 6.3 (4.5) MDI, 3 mo Prenatal 5.9 (3.4) Concurrent

Dietrich et al. ( 1986 ) 8.0 (3.8) MDI, 6 mo Prenatal 4.5(2.9) Neonatal

Hu et al. ( 2006 ) 7.1 (5.1) MDI, 24 mo Prenatal (1 st trimester) NR Prenatal (average) 5.2 (3.4) 12 month 4.8 (3.7) Concurrent

Gomaa et al. ( 2002 ) 6.7 (3.4) MDI, 24 mo Prenatal
Jedrychowski et al. ( 2009b ) 1.29 (1.24, 1.34) a MDI, 24 mo Prenatal

Wasserman et al. ( 1992 ) 14.4 (10.4) MDI, 24 mo Prenatal 35.4, 8.5 b Concurrent Bellinger et al. ( 1987 ) ≥15 vs. <3 c MDI, 24 mo Prenatal
Assessment at School Age

Wasserman et al. ( 1994 ) 1 4.4 (10.5) GCI, 4 yr Prenatal 39.9, 9.6 b Concurrent

Bellinger et al. ( 1991 ) 6.8 (6.3) GCI, 57 mo Early childhood, 2 yr 2.8 (1.7) d Early childhood, tooth 6.4 (4.1) Concurrent

Dietrich et al. ( 1992 ) e 8.2 (3.8) FWS, 5 yr Prenatal 4.8 (3.3) Neonatal 11.9 (6.4) Concurrent NR Lifetime avg

Dietrich et al. ( 1993 ) 8.4 (3.8) Coordination, 6 yr Prenatal 4.8 (3.1) Neonatal 10.1 (5.6) Concurrent NR Lifetime avg

Dietrich et al. ( 1993 ) 8.3 ( 3.7) FSIQ, 6.5 yr Prenatal 5.0 (3.4) Neonatal 11.8 (6.3) Concurrent NR Lifetime avg

Schnaas et al. ( 2006 ) NR FSIQ, 6

- 10 yr Prenatal 9.8 (2.8

- 36.4) f Early childhood avg 6.2 (2.2

- 18.6) f Later childhood avg
Baghurst et al. ( 1992 ) 7.4 (2 nd quartile) FSIQ, 7 yr Prenatal 16.6 (2 nd quartile) Early childhood, 2 yr 15.7 (2 nd quartile) Lifetime avg Bellinger et al. ( 1992 ) >10 vs. <3 c FSIQ, 10 yr Prenatal 6.5 (4.9) Early childhood, 2 yr 2.9 (2.4) Concurrent, 10 yr Lanphear et al. ( 2005 ) 12.7 (4.0

- 34.5) a FSIQ, 5

- 10 yr Early childhood 18.0 (6.2

- 47.0) a Peak 12.4 (4.1

- 34.8) a Lifetime average 9.7 (2.5

- 33.2) a Concurrent
Pocock et al. ( 1994 ) NR FSIQ, 5

- 10 yr Around birth 6.8

- 21.2 Early childhood, 2 yr NR Postnatal mean

Ris et al. ( 2004 ) NR Learning, 15

- 17 yr Prenatal NR Early childhood, 6.5 yr NR Early childhood avg

Change in Cognitive Score (95% CI) Note: Effect estimates are standardized to a blood Pb levels of 1 μg/dL. Studies are arranged in order of ascending age of cognitive function assessment. Cognitive function test scores are not standardized to a similar scale because not all studies provided sufficient data.Red = prenatal or neonatal blood Pb levels, Blue = Earl y childhood levels, Black = concurrent or lifetime average levels. MDI = Mental Development Index, Bayley Scales; NR = Not reported; GCI = General Cognitive Index, McCarthy Scales; FWS = Filtered Word Test, Kaufman Assessment Battery for Children, FSIQ = F ull

- scale IQ. a = 95% CI for blood Pb levels. b = values represent mean blood Pb levels in the two towns studied. c = Effect estimate compares children in different categories of blood Pb levels. d = mean (SD) and effect estimate for tooth Pb levels (μg/g) . e = Sufficient data were not provided in order to calculate 95% CIs. f = range of blood Pb levels. Figure 5

- 7. Associations of blood Pb measures at various lifestages with cognitive function in children.May 2011 5

- 64 DRAFT

– DO NOT CITE OR QUOTE Study Mean (SD) Blood Pb (μg/dL) Outcome Exposure Period

Wasserman et al. ( 1998 ) 16.1 (2.6) Anxiety/ Prenatal 25.8 (19.1) Depression, Concurrent

3 yr s

Leviton et al. ( 1993 ) 4.8

- 6.3 (2nd quartile) Hyperactivity, Prenatal

2.0

- 2.9 (2nd quartile) a Girls, 8 yr s Early childhood, 6 yr

Bellinger et al. ( 1994 ) 1.98 (0 .38) Behavioral Prenatal
3.4 (2.4) b P roblems

, 8 yr s Early childhood, 6.5 yr

Ris et al. ( 2004 ) NR Inattention, Prenatal

NR 15

- 17 yr Early childhood, 6.5 yr

NR Early childhood avg

Dietrich et al. ( 2001 ) 8.9 (3.9) Delinquent Prenat al

NR B ehavior, Early childhood, 6.5 yr

NR 15

- 17 yr s Early childhood avg

Change in Behavioral Index per 1 unit increase in Pb biomarker (95% CI)

Note: Positive effect estimates represent an increase in behavioral index. Effect estimates are standardized to a blood Pb levels of 1 μg/dL. Studies are arranged in order of ascending age of behavioral assessment. Behavioral asssessment scores are not standardized to a similar scale because not all studies provided sufficient data. Red = prenatal bl ood Pb levels; Black = blood Pb levels at other lifestages. a = second quartile levels (μg/g) and effect estimate for tooth Pb levels b = mean (SD) in ppm and effect estimate for tooth Pb levels. Figure 5

- 8. Associations of Pb biomarkers at various lifesta ges with behavioral indices in children.May 2011 5

- 65 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 5. Additional characteristics and quantitative results for studies presented in Figures 5

- 7 and 5

- 8

Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) a

Rothenberg et al. ( 1989 ) 42 children followed 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)

Hu et al. ( 2006 ) 146 children born 1997

- 1999 followed prenatally to age 24 mo Mexico City, Mexico Prenatal (maternal blood Pb) in 1 st 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 1 st 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 Prenatal:

- 2.1 (

- 3.9,

- 0.39)

Jedrychowski e t 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)

Wasserman et al. ( 1992 ) 392 childr en followed prenatally to age 24 mo Kosovo, Yugoslavia (K. Mitrovica, Pristina) Prenatal (cord blood) mean (SD): 14.4 (10.4) Concurrent means: K. Mistrovica: 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 Prenatal:

- 3.2 (

- 7.2, 0.86) Concurrent:

- 4.1 (

- 6.2,

- 2.0)

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

a ssessed 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 b

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. Mistrovica: 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 Prenatal:

- 3.2 (

- 5.1,

- 1.2) C oncurrent:

- 4.1 (

- 6.2,

- 2.0)May 2011 5

- 66 DRAFT

– DO NOT CITE OR QUOTE Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) a

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) μ g/g Concurrent mean (SD): 6.4 (4.1) Log l inear 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 orde r 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. ( 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, sociodemographic characteristics, HOME

score Total FWS assessed using KABC at age 5 yr Prenatal:

- 0.25, p ≤ 0. 01 c Neonatal:

- 0.38, p ≤ 0.01 c Concurrent:

- 0.19, p ≤ 0.01 c Lifetime avg:

- 0.16, p ≤ 0.01 c

Dietrich et al. ( 1993 ) 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 adjus ted for obstretric 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 Prenatal:

- 0.04 (

- 0.20, 0.12) Neonatal:

- 0.15 (

- 0.33, 0.03) Concurrent:

- 0.18 (

- 0.26,

- 0.10) Lifetime avg:

- 0.11 (

- 0.19,

- 0.03)

Dietrich et al. ( 1993 ) 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 using WISC

- R at age 6.5 yr Prenatal: 0.15 (

- 0.26, 0.56) Neonatal:

- 0.03 (

- 0.42, 0.36) Concurrent:

- 0.33 (

- 0.60,

- 0.06) Lifetime avg:

- 0.13 (

- 0.35, 0.09)

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): NR Early childhood avg (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 (28

- 36 weeks gestation):

- 3.9 (

- 6.5, 1.4) Early childhood avg: 0.10 (

- 3.9, 4.1) Later childhood avg: 0.17 (

- 1.4, 1.8)

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 duration, parental education, maternal age, parental smoking, SES, quality of home environment, maternal IQ, parents living together FSIQ assessed using WISC

- R at

a ge 7

- 8 yr Prenatal: 0.26 (

- 0.67, 1.5) Early childhood:

- 2.0 (

- 3.8,

- 0.21) Lifetime avg:

- 1.6 (

- 3.7, 0.52)

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 Prenatal:

- 0.48 (

- 5.7, 4.7), blood Pb >10 μ g/dL vs. <3 μ g/dL b Early childhood:

- 0.58 (

- 0.99,

- 0.17) Concurrent:

- 0.46 (

- 1.5, 0.56) Lanphear et al. ( 2005 ) 1333 children pooled from Boston, Cincinnati, Cleveland, Mexico City, Port Pirie, Roches ter, and Yugoslavia cohorts Median (5 th

- 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 Early childhood:

- 0.14 (

- 0.23,

- 0.06) Peak:

- 0.20 (

- 0.29,

- 0.11) Lifetime avg:

- 0.15 (

- 0.22,

- 0.09) Concurrent:

- 0.23 (

- 0.32,

- 0.14)May 2011 5

- 67 DRAFT

– DO NOT CITE OR QUOTE Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI) a

Pocock et al. ( 1994 ) Meta

- analysis of 5 prospective (over 1100 children and 14 cross

sectional studies (3499 children) Early childhood (2 yr) range in means: 6.8

- 2 1.2 Meta

- analysis of combining effect estimates from individual studies FSIQ assessed using various tests at ages 5

- 10 yr Around birth: 0.26 (

- 1.5, 2.0) Early childhood:

- 2.7 (

- 4.1,

- 1.2) Postnatal mean:

- 1.3 (

- 2.9, 0.37) 

### 1

Ris et al. ( 2004 ) 195 children in followed from birth (1979

- 1985) to age 15

- 17 yr Cincinnati, OH NR Linear regression mo del adjusted for maternal IQ, sex, and average total HOME score Learning/IQ composite assessed using WISC

- III indices at age 15

- 17 yr Prenatal:

- 0.08 (

- 0.18, 0.03) Early 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) Prena tal 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 Prenatal: 1.16 (0.0 2, 2.3) Concurrent: 1.45 (0.04, 2.86)

Leviton et al. ( 1993 ) 1923 children followed from birth (1979

- 1980) to age 8 yr Boston area, MA Prenatal blood 2 nd quartile: 4.8

- 6.3 Early childhood (tooth) second quartile: 2.0

- 2.9 μ g/g Log linear regression model adjusted for single

parent family, gestational age <37 wk, mother not a college graduate, self

identification as black, only child, daycare during first 3 yr Hyperactivity

as sessed 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. ( 1994 ) 1,782 children followed from birth (1979

- 1 980) 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 Repo rt Form of the Child Behavior Profile at age 8 yr Prenatal:

- 0.31 (

- 1.7, 1.07) Early childhood: 1.8 (0.49, 3.1)

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 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 f ollowed 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 Prenatal: 0.19 (0.02, 0.37) Early childhood, 6.5 yr: 0.13 (

- 0.01, 0.27) Early 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 Assessm ent Battery of Children, FSIQ = Full

- scale IQ, WISC = Weschler Intelligence Scale for Children, NR = Not reported

a Effect estimates are standardized to a 1 μg/dL increase in blood Pb level in analyses of blood Pb as a continuous variable.
b Effect estimate represent comparisons between children in different categories of blood Pb level, with children in the lower blood Pb categor y serving as the reference group.

c Sufficent data were not provided in order to calculate 95% CI.May 2011 5

- 68 DRAFT

– DO NOT CITE OR QUOTE Source: Used with permission from Elsevier Science, Jedrychowski et al. ( 2009a )

. Figure 5

- 9. Regression of fitted M DI score at 36 months on log

transformed con centration of cord blood Pb level by sex.

In studies that examined cognitive and behavioral indices measured in schoolaged children (ages 4

- 

### 1
17 years), statistically significant, negative associations with prenatal or neonatal (10 days after birth) 

### 2
blood Pb measures were observed less frequently (Figures 5

- 7 and 5

- 8 and Table 5

- 5). Mul tiple studies 

### 3
conducted in the Cincinnati cohort found that blood Pb levels measured 10 days after birth but not 

### 4
maternal blood Pb during pregnancy were associated with impairments in cognitive function, auditory 

### 5
processing, and motor function as well as i ncreased behavioral problems in children between ages 4 and 6 

### 6

years ( Dietrich, Berger, & Succop, 1993 ; Dietrich, Berger, Succop, et al., 1993 ; Dietrich et al., 1991 ; 

### 7
Dietrich et al., 1992 ) . In these Cincinnati studies, concurrent blood Pb levels generally were estim ated to 

### 8
have similar magnitudes of effect. In most of the studies examining associations of prenatal or neonatal 

### 9
blood Pb levels with neurodevelopmental outcomes in later childhood, early or cumulative childhood 

### 10
blood Pb levels were associated with the gre ater decrements in function. A larger effect estimate for peak 

### 11

blood Pb levels was corroborated in meta

- analysis of results from five cohort studies ( Pocock et al., 1994 ) 

### 12
(Figure 5

- 7 and Table 5

- 5). These findings may indicate the lack of persistence of early Pb exposures

. 

### 13
Further, associations of cord blood Pb levels with neurodevelopmental measures in infancy may reflect 

### 14
associations with blood Pb levels in infancy, which are expected to be similar to those during the prenatal 

### 15
period . C ord blood Pb level may be a good surrogate of early postnatal blood Pb leve ls

. 

### 16

E arly childhood blood Pb levels also were associated with diverse neurodevelopmental effects 

### 17

assessed later in childhood and into early adulthood in both recent (reviewed earlier in Section 5.3.2.1, 

### 18
Figure 5

- 4, and Table 5

- 4) and previous studies that did not compare various lifestages of Pb exposure 

### 19May 2011 5

- 69 DRAFT

– DO NOT CITE OR QUOTE ( Cecil et al., 2005 ; Tong et al., 2000 ; Yuan et al., 2006 ) . This lag effect may be the result of a 

### 1
toxicological process in which some period of time is required for past Pb exposure to affect CNS 

### 2
function. Alternatively, Pb exposure may affect higher

- order neurodevelopmental processes that are more 

### 3
reliably assessed at later ages wh en children’s processes modalities are more highly differentiated. Early 

### 4
testing may lead to false

- negative results and fail to identify a child who is at risk for later 

### 5
neurodevelopmental dysfunction. In some studies that have reported associations betwee n early



### 6

childhood blood Pb levels and neurodevelopmental decrements later in life , children’s blood Pb levels had 

### 7
not markedly changed over time Thus, the early

- childhood blood Pb levels may have been serving as 

### 8
surrogates of concurrent or cumulative blood Pb levels. 

### 9
As depicted in Figures 5

- 7 and 5

- 8 and Table 5

- 5, several studies estimated larger decreases in 

### 10
neurodevelopmental endpoints for concurrent or lifetime average blood Pb levels than blood Pb levels at 

### 11
other lifestages. These findings were substa ntiated in the analysis pooling data from seven prospective 

### 12
studies, in which concurrent, peak, average lifetime, and early childhood blood Pb levels were all 

### 13
negatively associated with IQ, with the largest magnitude of decrease associated with concurrent blood Pb 

### 14

levels ( Lanphear et al., 2005 )

. Childhood average blood Pb levels ( Dietrich, Berger, & Succop, 1993 ; 

### 15

Dietrich , Berger, Succop, et al., 1993 ; Lanphear et al., 2005 ) and tooth Pb levels ( Bellinger et al., 1994 ) 

### 16
have been associated with neurodevelopmental eff ects, indicating that biomarkers of cumulative 

### 17
childhood Pb exposure also may contribute to neurodevelopmental effects in children. Associations with 

### 18
concurrent blood Pb level were also demonstrated consistently in studies without comparisons to 

### 19

exposures at other lifestages (Figures 5

- 2 and 5

- 4 and Tables 5

- 3 and 5

- 4). 

### 20

Some studies have aimed to improve assessment of age

- based susceptibility by examining children 

### 21
with different degrees of changes in blood Pb levels over time (i.e., children whose blood Pb level ranking 

### 22
changed over time) ( Bellinger et al., 1990 ; A. Chen et al., 2005 ; Hornung et al., 2009 ; Tong et al., 1998 )

. 

### 23
Except for Tong et al. ( 1998 ) , these studies have demonstrated stronger effects of concurrent blood Pb 

### 24
levels (Figure 5

- 10 and Table 5

- 6). Tong et al. ( 1998 ) found that early

- life blood Pb level was associated 

### 25
with a larger deficit in IQ. As part of the Port Pirie, Australia cohort study, investigators separately 

### 26

examined intellectual attainment in groups of children with different degrees of decline in blood Pb levels 

### 27
between ages 2 and 11

- 13 years . Although the mean blood Pb level in the study population declined 

### 28
overall from 21.2 μg/dL at age 2 years to 7.9 μg/dL at age 11

- 13 years, the magnitude of decline varied 

### 29
among children. In comparisons of tertiles of change in blood Pb level between age 2 and 11

- 13 years, 

### 30
investigators found that intellectual attainment scores at ages 2, 4, 7, and 11

- 13 years did not significantly 

### 31

differ between children with the largest declines (>16 μg/dL ) in blood Pb level and children with a lower 

### 32

decline (<10 μg/dL ). These findings indicated a strong er effect of high er blood Pb levels early in life even 

### 33
among groups with lower concurrent levels

. 

### 34May 2011 5

- 70 DRAFT

– DO NOT CITE OR QUOTE Reference Outcome Blood Pb Variable Examined

Tong et al. ( 1998 ) FSIQ, 11

- 13 yr. < 10.2 decline age 2 to 11

- 13

10.2

- 16.2 decline age 2 to 11

- 13

> 16.2 decline, age 2 to 11

- 13

Bellinger et al. ( 1990 ) GCI, 24 and 57 mo Concurrent, Prenatal < 3 a

Concurrent, Prenatal 3

- 10 a

Concurrent, Prenatal ≥ 10 a

Chen et al. ( 2005 ) FSIQ, 7 yr. Low 2 yr, (<24.9), Low 7 yr, (<7.2)

Low 2 yr, (<24.9), High 7 yr, (≥ 7.2)

High 2 yr, (≥ 24.9), Low 7 yr, (<7.2)

High 2 yr, (≥ 24.9), High 7 yr, (≥ 7.2)

Hornung et al. ( 2009 ) FSIQ, 6 yr. 0.5 ratio age 6 to 2 yr b

2.0 ratio age 6 to 2 yr b

Change in Cognitive Score (95% CI)
Note: Effect estimates represent associations between concurrent blood Pb level and cognitive function (standardized to standard deviation) in children categorized by prenatal blood Pb level. bValues represent the ratio of blood Pb level at age 6 years to that at age 2 years. FSIQ = Full

scale IQ, GCI = General Cognitive Index. Cognitive function scores were standardized to their standard deviation. Effect estimates in red represent blood Pb level variables associated with the greater decrease in cognitive function. Figure 5

- 10. Associations of cognitive function in children with different degrees of changes in blood Pb levels over time.May 2011 5

- 71 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 6. Additional characteristics and qu antitative results for studies presented in Figure 5

- 10

Study Population/ Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Effect Estimate (95% CI)a

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 μ g/dL decline: 0.03 (

- 0.15, 0.21) b 10.2

- 16.2 μ g/dL decline: 0.04 (

- 0.15, 0.23) b >16.2 μ g/dL decline:

- 0.01 (

- 0.20, 0.18) b

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 c Prenatal <3 μ g/dL:

- 0.16 (

- 0.43, 0.11) Prenatal 3

- 10 μ g/dL:

- 0.14 (

- 0.57, 0.29) Prenatal ≥ 10 μ g/dL:

- 0.46 (

- 0.81,

- 0.11)
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 Mean (SD): Age 2 yr: 26.2 (5.1) Age 5 yr: 12.0 (5.2) Age 7 yr: 8.0 (4.0) 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 (<24.9 μ g/dL, Low age 7 (<7.2 μ g/dL): 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)

Hornung et al. ( 2009 ) 462 children followed from birth (1979

- 1984) to age 6 yr Rochester, NY and Cincinnati, OH Geometric mean (5 th

- 95 th ): 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)

a Effect estimates represent the cognitive function score or change in score over time standardized to its standard deviation.
b Investigators estimated changes in IQ in groups of children with different degrees of decline in blood Pb levels over the study period: children with <10.2 μg/dL decline, children with a 10.2

- 16.2 μg/dL decline, and children with > 16.2 μg/dL.

c Effects are estimated for concurrent blood Pb level (continuous variable) in children in different categories of prenatal blo od Pb level: <3 μg/dL , 3

- 10 μg/dL , and ≥ 10 μg/dL

. d Investigators compared IQs among children with different categories of blood Pb level early and later in childhood: low levels at age 2 (<11.4 μg/dL) and age 7 (<7.2 μg/dL), low levels at age 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), a nd 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.

In several different U.S. cohorts of children, larger decrements on neurocognitive function were 

### 1

estimated for concurrent blood Pb levels ( Bellinger et al., 1990 ; A. Chen et al., 2005 ; Hornung et al., 

### 2

2009 ) (Figure 5

- 10 and Table 5

- 6). In the Boston cohort, Bellinger et al. ( 1990 ) found that at age 57 

### 3
months, cognitive performance, as assessed by McCarthy GCI, was similar between children with higher 

### 4 (≥ 10 μg/dL ) and lower (<3 μg/dL ) prenatal blood Pb levels. Additionally, increasing concurrent blood Pb 

### 5
levels (age 57 months) were associated with the largest decline in GCI scores over time (score at age 57 

### 6

months

– score at age 24 months) among children with high pren atal blood Pb levels (≥ 10 μg/dL ), which 

### 7
indicated an effect among children with both high early and concurrent blood Pb levels (Figure 5

- 10 and 

### 8

Table 5

- 6)

. These findings indicated that by age 5 years, children with higher prenatal blood Pb levels 

### 9

appear to recover the Pb

- associated decrements in cognitive function unless concurrent blood Pb levels 

### 10May 2011 5

- 72 DRAFT

– DO NOT CITE OR QUOTE remain high. The investigators also demonstrated that positive home and caregiving environment (e.g., 

### 1
HOME score >52, higher SES, higher maternal IQ) may also p rotect against decrements in cognitive 

### 2
function associated with higher postnatal Pb exposures. 

### 3
As part of the multicenter Treatment of Lead

- Exposed Children ( TLC ) trial, Chen et al. ( 2005 ) 

### 4
evaluated how change in blood Pb over time was related to IQ at later ages. The TLC was a clinical trial 

### 5
designed to examine the effect of chelation using succ imer to prevent cognitive impairment in 780 urban 

### 6
children enrolled at 12 to 33 months of age with elevated blood Pb concent rations (20

- 44 μg/dL). Blood 

### 7
Pb and IQ were assessed at ages 24 and 36 months and 3, 5, and 7 years . C oncurrent blood Pb level above 

### 8
the median level (>7.2 μg/dL ) was associat ed with a larger decrease in IQ, regardless of whether prenatal 

### 9
blood Pb levels were low or high (less than or greater than the median of 11.4 μg/dL , respectively). 

### 10
Pooling the Cincinnati and Rochester cohorts (n = 397), Hornung et al. ( 2009 ) also created a new 

### 11

indicator of Pb exposure : the ratio of blood Pb level at 6 years o f age to that at 2 years of age. The greatest 

### 12

decrease in cognitive and behavioral development was observed for children with blood Pb r atios greater 

### 13
than 1 (indicating an increase in blood Pb level from 2 to 6 year s of age ) (Figure s 5

- 10 and 5

- 11 and Table 

### 14

5

- 6 ). Presumably areas under the curve would be similar among children with blood Pb level ratios of 1, 

### 15
greater than 1, and less than 1, indicating that cumulative blood Pb levels would not be predictive . It is 

### 16
important to note that in these aforementioned studies, blood Pb levels were higher than those curren tly 

### 17
measured in among children in the U.S. Additionally, children in these study populations experienced 

### 18
larger decreases in blood Pb levels over time. It is unclear whether these findings would apply to children 

### 19
Blood Pb levels in the U.S. who currently a re within the same age range and who would be expected to 

### 20

have smaller decreases in blood Pb levels over time. 

### 21May 2011 5

- 73 DRAFT

– DO NOT CITE OR QUOTE Source: Hornung et al. ( Hornu ng et al., 2009 )

Note: All three patterns have an identical mean blood Pb level level of 10 μg/dL

. Figure 5

- 11. Estimated IQ in combined Cincinnati and Rochester cohorts for 3 patterns of blood Pb level levels from 1 through 6 ye ars of age: peak at 2 years ( blue diamonds), peak at 5 years ( black triangles), and constant blood Pb level level ( white squares).

In the collective body of epidemiologic evidence of children, it is difficult to ascertain which 

### 1
lifestage of Pb exposure is associated with the greatest susceptibility to Pb

- associated neurodevelopmental 

### 2
effects. Associations have been observed with prenatal, early

- childhood, lifetime average, and concurrent 

### 3

blood Pb levels as well as childhood tooth Pb levels. The assessment of age

- based susceptibility is 

### 4
complicated further by the fact that b lood Pb levels in children, although highly affected by recent dose, 

### 5

are also influenced by Pb stored in bone due to rapid growth

- related bone turnover in children relative to 

### 6

adults. Thu s, concurrent blood Pb level in children also may reflect cumulative dose (Section 4.3.5)

. 

### 7
Nonetheless, while the evidence indicates that prenatal and early

- childhood blood Pb levels are associated 

### 8
with neurodevelopmental deficits, subsequent exposures tha t are reflected in concurrent, cumulative 

### 9
blood Pb levels or tooth Pb levels also are demonstrated to contribute to neurodevelopmental deficits 

### 10
throughout schoolage and into adolescence. Additional results from Hornung et al. ( 2009 ) and recent 

### 11
studies described earlier in the section support the conclusion from the 2006 AQCD that concurrent blood 

### 12
Pb level appears to be the best predictor of neurodevelopmental effects in child ren. These findings are 

### 13
consistent with the understanding that the nervous system continues to develop throughout childhood. 

### 14
Thus, the course of cognitive development may be modified in children, depending on concurrent blood 

### 15
Pb levels or positive caregivi ng environment. 

### 16May 2011 5

- 74 DRAFT

– DO NOT CITE OR QUOTE Figure 5

- 12. Neurological summary array of toxicological outcomes after Pb exposure. Dosimetric representation reported by blood Pb level. (ID corresponds to Table 5

- 7.)May 2011 5

- 75 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 7. Summary of findings from neurotoxicological exposure

- re sponse array presented in Figure 5

- 12.

Study ID Reference Blood Pb Level (ug/dL) Outcome

1 Beaudin et al. ( 2007 ) 13 & 31 Behavior, neonate: Lactational Pb exposure, offspring deficient in Reward Omission testing.
2 Kishi et al. ( 1983 ) 59 & 186 Behavior, neonate: Pb exposure (oral gavage of pups) during lactational period, Changed emotional behavior, ma les. 3 Stangle et al. ( 2007 ) 13 & 31 C ognition; Developmental Pb exposure (PND1

- 30): Impaired learning with visual discrimination task, heightened response to errors. 6 Gong & Evans ( 1997 ) 38 & 85 Cognition

- Adult male 21 day Pb exposure: Hyperactivity with Habituation to new cage environment.
4 Rice ( 1990 ) 32 & 36 Cognition

- Chronic Pb exposure from birth: Spatial discrimination reversal task impairment.
7 Li et al. ( 2009 ) 80 & 100 Cognition

- Gestational & lactational Pb exposure: Morris water maze performance impaired.
8 Li et al. ( 2010 ) 80 & 102 Cognition

- Gestational & lactational Pb exposure: Morris water maze performance impaired.
5 Overmann ( 1977 ) 33, 174 & 226 Cognition

- Pb exposure (oral gavage of pups) during lactation: Response inhibition impaired.
17 Cory

- Slechta et al. ( 2010 ) 10 & 13 Stress: Corticoste rone

- Lifetime Pb plus stress: Affects FI performance, dopamine and serotonin levels in female offspring. 16 Virgolini

, Rossi

- George

, Weston et al. ( 2008 ) 25 Stress: Corticosterone

- Mat ernal Pb plus stress: Affects FI performance.

16 Virgolini

, Rossi

- George

, Weston et al. ( 2008 ) 19 & 35 Stress.

18 Virgolini et al. ( 2005 ) 15 & 27 Stress: Corticosterone

- Chronic Pb plus stress: Affects neurotransmitters & FI performance.
15 Virgolini

, Rossi

- George

, Lisek et al. ( 2008 ) 11 &/or 31 Stress: Corticosterone

- Maternal Pb plus stress: Affects FI performance, dopamine, serotonin, and NE levels.
9 Hu et al. ( 2008 ) 4 & 12 Morphology; Gestational Pb exposure: Neurite outgrowth marker PSA

- NCAM decreased in rat pups.
10 Wu et al. ( 2008 ) 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). 11 Tavakoli

- Nezhad et al. ( 2001 ) 18, 29, & 54 Morphology; 3 to 6 weeks of Postnatal (starting at PND22) Pb exposure in males: Decreased number of spontaneously active midbrain dopamine neurons.

7 Li et al. ( 2009 ) 40 & 100 Morphology; Gestational & lactational Pb exposure: Increased levels of inflammatory cytokines & exocytosis related proteins in brains of pups at weaning. 8 Li et al. ( 2010 ) 80 & 102 Morphology: Increased levels of Alzhei mer disease

- associated proteins in mice with gestational and lactational Pb exposure. 6 Gong & Evans ( 1997 ) 85 Morphology; 21 day Pb exposure to adult males: Marker of neuronal injury

- elevated hi ppocampal glial fibrillary acidic protein (GFAP). 12 Leasure et al. ( 2008 ) 10 & 42 Motor function; Mouse maternal (dam) Pb exposure: Induced decreased rotarod performance in offspring (1 year

- old male offspring). 13 Bielarczyk et al. ( 1996 ) 1.8, 3.8, 22 Neurotransmitter; Perinatal (GD16

- PND28) Pb exposure: Decreased hippocampal ChAT activity and increased hippocampal tyrosine hydroxylase activity. 14 Fortune & Lurie ( 2009 ) 8 & 43 Neurotransmitter; Mouse maternal (dam) Pb exposure: Affects offspring superior olivary complex (auditory) neurotransmitters. 12 Leasure et al. ( 2008 ) 10 & 42 Neurot ransmitter; Mouse maternal (dam) Pb exposure: Affects 1 year old male offspring dopamine homeostasis. 15 Virgolini

, Rossi

- George

, Lisek et al. ( 2008 ) 31 Neurotransmitter; Gestational and lactational Pb exposure: Induced NE aberrations in adult rat offspring (both sexes).
16 Virgolini

, Rossi

- George

, Weston et al. ( 20 08 ) 19 & 30 Neurotransmitter; Gestational and lactational Pb exposure: Induced DA and 5HT changes in rat offspring.

10 Wu et al. ( 2008 ) 19 & 26 Oxidative stress: Elevated oxidative DNA damage in aged brains of female monkeys (exposed to Pb as infants). 9 Hu et al. ( 2008 ) 15 Physical developmen; t

- Gestational Pb exposure: Early brain synapse development impaired (hippocampal PSA

- NCAM and sialytransferase). 1 Beaudin et al. ( 2007 ) 13 & 31 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 Kishi et al. ( 1983 ) 59 & 186 Physical development; Pb exposure during lactation (oral gavage): Delayed development of righting reflex in male rats. 6 Gong & Evans ( 1997 ) 85 Physical development; Adult male rats (21 day Pb exposure): Neurotoxicity measured with brain glial fibrillary acidic protein (GFAP). 16 Virgolini

, Rossi

- George

, Weston et al. ( 2008 ) 19 & 35 Stress: Corticosterone levels affected.

17 Cory

- Slechta et al. ( 2010 ) 10 & 13 Stress: Corticosterone

- neurotransmitter

- Lifetime Pb exposure in female rats plus stress: Dopamine homeostasis affected.



### 5.3.2.2. Toxicological Studies of Neurocognition, Memory and Learning

The 2006 AQCD reported deficits in the Morris water maze with Pb exposure. The Morris water 

### 1
maze tests memory and learning by having a mouse swim and locate or remember the location of a 

### 2May 2011 5

- 76 DRAFT

– DO NOT CITE OR QUOTE platform submerged in opaque water. New research since the 2006 AQCD continues to show Pb

- induced 

### 1
impaired Morris water maze performance. Data on neurocognition and learning as well as other 

### 2
neurotoxicological endpoints with dose responsive data are shown in Figure 5

- 12 and accompanying 

### 3

Table 5

- 7. Dams received Pb acetate d issolved in drinking water ( 0.1%, 0.5%, and 1% with corresponding 

### 4
blood Pb level of 4, 8 and 10 μg/dL at postnatal day [PND] 21) throughout gestation and lactation. 

### 5
Beginning at weaning, Pb exposed pups were subjected to Morris water maze performance testi ng. Pb 

### 6
exposed pups had significant increases in escape latency and number of crossings of the platform area at 

### 7
0.5% and 1% Pb acetate exposure (blood Pb levels of 8 and 10 μg/dL, respectively, indicating impaired 

### 8
memory and learning ( Li et al., 2009 ) . The pups in Li et al. ( 2009 ) were not separated by sex. Another 

### 9
study found that dietary supplementation with various supplements listed below or with 

### 10
methioninecholine concomitant with Pb exposure in weanling males shortened the escape latency of Pb



### 11
exposed pups to more closely resemble the escape latency of control pups ( G. Fan et al., 2009 ; Fan et al., 

### 12

2010 ) . Zinc or methionine were effective dietary supplements in the G. Fan et al. 2009 study ( 2009 ) ; 

### 13
glycine, taurine, vitamin C, vitamin B1, tyrosine had no effect on the Morris water maze results. These 

### 14
data on the effect of Pb on learning and memory in the Morris water maze confirm findings by two other 

### 15

labs. Jett et al. ( 1997 ) showed increased escape latency in adult rats exposed to Pb via direct injections 

### 16
into the dorsal hippocampus. Kuhlmann et al. ( 1997 ) used maternal Pb diet exposure (gestation a nd 

### 17
lactation), continuous Pb exposure (gestation through adulthood) or post

- weaning Pb exposure and only 

### 18
found only significant impairments in the maternal and continuous exposure groups. This new study 

### 19
confirms the findings of earlier studies that learning and memory are significantly impaired in rodents 

### 20
who are exposed to Pb early in life. 

### 21

Working Memory

Working memory is the ability to temporarily keep information in mind while using the 

### 22
information to perform a related or unrelated task. The Morr is water maze is able to measure working 

### 23
memory in addition to learning. Using this test, the 2006 AQCD found working memory was significantly 

### 24
affected in chronic developmentally exposed (Pb Acetate in feed 10 days prior to mating through PND 

### 25
21) female of fspring at PND 21 ( Jett, Kuhlmann, Farmer, et al., 1997 ) . Delayed spatial altern ation (DSA) 

### 26
is another test used to measure working memory. With DSA, an animal receives rewards based on 

### 27
responses at two separate levers. Work from the 2006 AQCD showed that Pb

- exposed animals had 

### 28
deficits under DSA testing. These deficits included incre ased response errors, decreased percent of correct 

### 29
responses, and perseverance at one lever (repeatedly pressing the same lever without moving between the 

### 30
two locations). These results have been consistently shown with non

- human primate studies (continuous 

### 31
Pb exposure or juvenile to adult exposure) and less consistently shown with rats (juvenile only or juvenile 

### 32
to adult exposure). Working memory is a subcategory of executive function or goal

- oriented problem 

### 33May 2011 5

- 77 DRAFT

– DO NOT CITE OR QUOTE solving. Deficits in working memory are thought to underlie associations between blood Pb levels and 

### 1

ADHD in humans. 

### 2

Response Inhibition

Response inhibition is another measure of executive function and is measured with multiple tests 

### 3
that measure premature responses, decreased pause time between two sc heduled events and increased 

### 4
perseverance. These tests include Differential Reinforcement of Low Rates of Responding (DRL), DSA, 

### 5
Fixed Interval testing (FI), FI with Extinction (FI

- Ext), Fixed Ratio

- FI (FR

- FI), and Signal Detection with 

### 6
Distraction. Multip le studies from the 2006 AQCD and earlier literature have shown that early life Pb 

### 7
exposure contribute to response inhibition across the spectrum of these aforementioned tests. Monkeys 

### 8
with moderate blood Pb levels (11

- 13 μg/dL) learned the DRL task more s lowly but eventually acquired 

### 9
reinforcement rates equal to controls. Newer data from female rats exposed to Pb ( Stangle et al., 2007 ) 

### 10
continued to show animals with premature responses after Pb exposure or response inhibition decrements. 

### 11

Learning Ability, Schedule

- Control led Behavior

The 2006 AQCD discussed learning or cognition as measured with schedule controlled

- behaviors 

### 12
including fixed interval (FI) and fixed ratio (FR) operant conditioning and found that FI response rate was 

### 13

affected differen tially with low level and high level Pb exposures increasing and decreasing FI response 

### 14
rate in females, respectively. This curvilinear response has since been further explored in more recent 

### 15
work, much of which also includes the effect of psychological st ress on Pb exposure. 

### 16

Learning Ability with Stress

The combined paradigm of Pb exposure and stress experienced by a person or a laboratory animal 

### 17
is now being studied by multiple investigators who are focusing on the common pathway of HPA axis 

### 18

alteration a nd altered brain neurotransmitter levels. Data on stress and Pb with dose responsive data 

### 19
endpoints are shown in Figure 5

- 12 and accompanying Table 5

- 7. Cory

- Slechta and colleagues have 

### 20
conducted multiple investigations in this area. Most recently, they ha ve shown enhanced learning deficits 

### 21
in female rats (offspring) following lifetime Pb exposure combined with maternal restraint or prenatal 

### 22

stress ( Cor y

- Slechta et al., 2010 ) . This exposure paradigm used dams who were exposed to Pb for 2 

### 23
weeks prior to mating through lactation and pups from a mixed sex litter received drinking water Pb (50 

### 24
ppm) exposure through the remainder of their lifetime resultin g in blood Pb levels of dams and pups 

### 25
ranging from 5

- 13 μg/dL. 

### 26
Pb plus stress

- related outcomes were followed in female offspring of dams who were exposed to 

### 27
Pb from 2 months prior to mating through lactation, i.e., developmental Pb exposure (2 exposure gro ups: 

### 28May 2011 5

- 78 DRAFT

– DO NOT CITE OR QUOTE 50 or 150 ppb Pb acetate drinking water solutions) ( Virgolini, Rossi

- George, Lisek, et al., 2008 ) . Dams 

### 1
underwent restraint stress at gestational 16

- 17. Marked increases in response rates on FI performance in 

### 2

the Pb

- stress female offspring versus control was found in animals whose mean blood Pb level was 11 

### 3
μg/dL (50 ppb Pb acetate). Because these animals di d not show effects with maternal stress or Pb 

### 4
exposure alone, this effect was potentiated in the animals exposed to Pb plus additional stress. 

### 5
Similarly, lifetime Pb exposure (50 or 150 ppm, blood Pb level 11

- 16 and 25

- 33 μg/dL, 

### 6
respectively) plus stress (maternal or offspring) also induced FI aberrations at the post

- reinforcement 

### 7
pause (PRP) period in female offspring, another potentiated effect ( Rossi

- George et al., 2011 ) (Table 5

- 8). 

### 8
Within the FI schedule, the PRP represents timing capacity or proper temporal discrimination. Namely, 

### 9
the PRP is the period during which the animal must wait or pause before depressing the lever for a 

### 10
reward. In this case, Pb pl us stress exposed animals start responding too early due to a decreased pause or 

### 11
PRP interval. Abberant FI performance in infants and children has been used as a marker for impulsivity. 

### 12
Separately, overall FI response rate was significantly increased in Pb exposure alone and with maternal or 

### 13
offspring stress at the 50 ppm exposure dose. At 150 ppm, stress (maternal or offspring) increased FI 

### 14
response rate but Pb alone had no effect on FI. Biochemical analysis of possible mechanistic contributions 

### 15

to these a berrations revealed alterations in frontal cortex norepinephrine, reductions in dopamine 

### 16
homeostasis in the nucleus accumbens and enhancement of the striatial monoamine system. This study on 

### 17
the effect of lifetime Pb exposure with or without stress on FI t esting itself or during the PRP component 

### 18
of FI testing further confirm learning deficits and provide possible mechanistic explanations. 

### 19
Pb exposure over various exposure windows has been shown to affect corticosterone levels in 

### 20
rodents. Maternal Pb exposu re (150 ppm drinking water from 2 months prior to mating through lactation 

### 21
with restraint stress as detailed above) induced increased basal corticosterone in female and male 

### 22
offspring at 9 months of age; no interactions of Pb and stress were seen in this m odel ( Cory

- Slechta et al., 

### 23

2004 ) . By 14 months of age, these offspring had reduced corticosterone concentrations versus co ntrol 

### 24
animals, indicating a possible acceleration of age

- related decreases in basal corticosterone levels ( Cory



### 25
Slechta et al., 2008 ) that were enhanced with maternal stress. Postnatal exposure of male rodents to Pb 

### 26
(PND 21

- 5 months of age) showed signific ant decrements in baseline corticosterone; this effect produced 

### 27

a U

- shaped concentration

- response curve with significant decrements in basal corticosterone levels in the 

### 28
50 ppm exposure group versus control ( Virgolini et al., 2005 ) . In summary, developmental (gestational 

### 29
and lactational) and post

- weaning exposure to Pb induced permanent changes in the HPA axis 

### 30
(corticosterone levels) in both sexes which are dynamic as the animal ages. 

### 31May 2011 5

- 79 DRAFT

– DO NOT CITE OR QUOTE Source: Rossi

- George et al. ( 2011 ) Figure 5

- 13. Mean basal corticosterone levels of female and male offspring exposed to lifetime Pb (0, 50, 150 ppm) and/or stress (PS (dam stress) or OS (offspring stress)). *denotes significantly diffe rent from 0NS control; # denotes significant ly different from corresponding Pb

- NS value; + differs from 50

- NS.

Mechanistic understanding of the cognitive deficits seen with Pb and/or stress exposure was 

### 1
explored in a recent study. HPA hypofunction followin g dam Pb exposure (pup gestational and lactational 

### 2
Pb exposure) with or without maternal stress was reported ( Rossi

- George et al., 2009 ) . This study used 

### 3
the same model of developmental Pb exposure as is detailed in the preceding paragraph. Outco mes were 

### 4
followed in both male and female offspring. At 5

- 6 months of age, basal corticosterone in females was 

### 5
significantly increased at 150 ppm and unaffected at 50 ppm (Figure 5

- 13). In males, basal corticosterone 

### 6
of the 50 ppm group was significantly d ecreased and 150ppm was unaffected. These authors also 

### 7
explored the function of the glucocorticoid negative feedback loop and found that Pb and/or maternal 

### 8
stress significantly impacted this negative feedback. This negative feedback loop was more greatly 

### 9

i mpacted at the lower dose (50 ppm v 150 ppm Pb acetate, blood Pb level at PND 21 of 19 and 32 μg/dL, 

### 10
respectively). To test the effect of an outside stress on the HPA axis, mice in this study were subjected to 

### 11
vehicle injections. The corticosterone respons e to this vehicle injection stress was prolonged in a non



### 12
linear dose response manner in both sexes with the most profound effects seen at the lower 50 ppm dose. 

### 13
Maternal stress also prolonged the corticosterone stress response to vehicle injection and enh anced the Pb 

### 14
effect in males. To test the negative feedback of the HPA axis, exogenous dexamethasone (DEX) was 

### 15
administered to suppress endogenous corticosterone. The DEX test revealed HPA axis hypofunction. 

### 16May 2011 5

- 80 DRAFT

– DO NOT CITE OR QUOTE Specifically Pb and Pb plus maternal stress init ially reduced the ability of DEX to suppress 

### 1
corticosterone. With time, the effect of this DEX test in males induced prolonged corticosterone 

### 2
suppression or failure to return to baseline as seen in control animals. In summary, dam Pb exposure 

### 3
induced negat ive feedback hypofunction in both sexes with an inverse U dose response function. A 

### 4
similar recent study explored the effect of lifetime Pb exposure on the HPA axis, looking at basal 

### 5
corticosterone levels in male and female offspring at two time periods (2 months old and 10 months old; 

### 6
before and after behavioral testing, respectively) in adulthood ( Rossi

- George et al., 2011 ) . Pb and stress 

### 7

have no effect on ba sal corticosterone levels in males at either time period (Figure 5

- 13). At the first time 

### 8
period, Pb exposure elevated basal corticosterone levels in a dose

- dependent fashion in females, and Pb 

### 9
plus stress attenuated the PB

- dependent elevations in corticos terone to baseline levels (Figure 5

- 13). At 

### 10
the second time period, Pb and stress accelerated the age

- dependent decrease in basal corticosterone 

### 11
levels in females (Figure 5

- 13). 

### 12
These two studies of lifetime exposure ( Rossi

- George et al., 2009 ) r eported different basal stress 

### 13
hormone levels with Pb exposure. Males with lifetime Pb exposure had no significant corticosterone 

### 14
response to Pb exposure; whereas males with dam Pb exposure had significant decreases in corticosterone 

### 15
at 5 months of age in the 50 ppm exposure group only (not seen in 150 ppm Pb exposure group). On the 

### 16
other hand, females had dose

- dependent corticosterone responses to Pb exposure in both exposure models 

### 17
(lifetime Pb exposure and dam Pb exposure. 

### 18May 2011 5

- 81 DRAFT

– DO NOT CITE OR QUOTE Source: Used with permission from Elsevier Science, Virgolini, Rossi

- George, Weston, et al. ( 2008 )

. Figure 5

- 14. Changes in FI performance (FI overall performance, run rate, PRP) in female offspring with maternal Pb exposure with various s tressors in adulthood (restraint, cold, novelty).

Source: Used with permission from Elsevier Science, Virgolini, Rossi

- George, Weston, et al. ( 2008 )

. Figure 5

- 15. Changes in FI performance (FI overall performance, run rate, PRP) in male offspring with maternal Pb exposure with various stressors in adulthood (restraint, cold, novelty).May 2011 5

- 82 DRAFT

– DO NOT CITE OR QUOTE Maternal stress alone also led to HPA axis negative feedback hypofunction. Pb plus maternal stress 

### 1
enhanced negative fe edback in males and attenuated this effect in females. Pb exposure with or without 

### 2
maternal stress prolonged the effect of DEX

- dependent corticosterone supression in males. These data 

### 3
together show that HPA axis alterations could be an underlying mechanism linking commonalities 

### 4

between the contribution of Pb and stress to adverse health outcomes. 

### 5

Schedule

- control behavior is often measured using FI or FR testing. Because the FI animals are 

### 6
regularly handled by laboratory personnel and participate in tests of cognition, their baseline level of 

### 7
stress may be skewed from that of a laboratory animal that constantly remains in a cage without daily 

### 8
handling. Because effects on the HPA axis are of interest to Pb researchers, the baseline corticosterone 

### 9

levels of a nimals who have participated in behavior testing (FI) and those who have not (NFI) have been 

### 10
compared. Specifically, the corticosterone differences between FI and NFI animals after developmental 

### 11
Pb exposure (dam

- only Pb exposure) have been measured. Virgol ini et al. ( 2008 ) found that basal 

### 12
corticosterone levels were significantly different between FI and NFI animals. Also, the combination of 

### 13
dam Pb exposure with maternal stress was explored in FI and NFI animals . At the baseline age of 4

- 5 

### 14
months, NFI animals who were not behaviorally trained displayed significant differences from FI 

### 15
animals. Pb exposure with or without stress did not induce differences in corticosterone levels in FI 

### 16

females. The corticosterone l evel of male FIs was affected by Pb and stress exposure (Figure 5

- 13). In the 

### 17
FI males, the 50 ppb Pb exposure group (50Pb) had decreased corticosterone versus control (no Pb 

### 18
exposure) and the 150 ppb Pb exposure group (150Pb) had elevated corticosterone v ersus control. Male 

### 19
NFI animals demonstrated a U shaped dose response corticosterone curve with 50Pb significantly less 

### 20
than control or 150Pb. In the NFI males, stress did not affect corticosterone levels or interact with the 

### 21
effect of Pb. NFI females expo sed to 150Pb had significantly elevated corticosterone versus control (no 

### 22
Pb exposure). When drawing conclusions about the effects of Pb exposure, these data demonstrate that 

### 23
behaviorally trained animals have an altered HPA axis and response to Pb exposure versus animals who 

### 24
are housed under conditions without daily handling by caregivers. 

### 25
Another study looked at female rats with lifetime Pb exposure combined with prenatal stress and 

### 26
found enhanced learning deficits(drinking water 50 ppm Pb acetate, offspr ing blood Pb 7

- 13 μg/dl) 

### 27
( Cory

- Slechta et al., 2010 ) . Learning was evaluated with multiple schedule of repeated learning (RL) and 

### 28
performance testi ng. Repeated learing was impaired but performance was not affected with Pb exposure. 

### 29
The impaired RL was further enhanced with prenatal stress. There were significant associations between 

### 30
Pb/stress and coticosterone concentration, dopamine from the frontal cortex, dopamine turnover in the 

### 31
nucleus accumbens, and total number of responses required to learn a sequence. Also Pb exposed 

### 32
offspring with and without maternal stress exposure had significant decreases in hippocampal nerve 

### 33
growth factor (NGF) versus c ontrol. Thus, this study demonstrates that lifetime Pb exposure with or 

### 34
without prenatal stress induced learning deficits in female mice. In a similar study, the authors proposed 

### 35
that associations of Pb and stress with learning deficits (F 1 testing in females) may be related to 

### 36May 2011 5

- 83 DRAFT

– DO NOT CITE OR QUOTE aberrations in corticosterone and dopamine ( Rossi

- George et al., 2011 )

. Earlier work has shown that dam 

### 1
or prenatal str ess (PS) affects the HPA axis of the offspring. A newer study was conducted to determine 

### 2
the influence of low level dam Pb exposure and prenatal stress on offspring stress challenge responsivity 

### 3
(intermittent stress as an adult) ( Rossi

- George et al., 2011 )

. In a similar study, the authors proposed that 

### 4
associations of Pb and stress with learning deficits (F1 testing in females) may be related to aberrations in 

### 5
corticosterone and dopamine ( Rossi

- George et al., 2011 )

. Dam Pb exposure (50 or 150 ppm Pb acetate) 

### 6
followed by intermittent stressors (cold, novelty or restr aint) to offspring as adults induced significant 

### 7
changes in FI response rate. Females were more sensitive to the adult intermittent stressors at the higher 

### 8
dose of Pb (150 ppm) with significant increases in FI response rate and decreased PRP, i.e. increase d 

### 9
impulsivity (Figure 5

- 14) . Males were more sensitive (decreased FI response rate due to decreased run 

### 10
rate) to the restraint stress at the lower Pb dose (50 ppm). At the higher dose of Pb, males were more 

### 11
sensitive to the cold stress (increased FI respon se rate and increased run rate) (Figure 5

- 15). 

### 12
Corticosterone levels were followed in this study and showed dose dependent correlations with FI 

### 13
outcomes in females but were independent of dose in males. 

### 14

Table 5

- 8. Summary of effects of maternal and lifetim e Pb exposure on FI performance water a

.

Pb (ppm) Maternal Pb b Lifetime Pb c

Overall rate PRP Overall rate PRP

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 EFFECTMay 2011 5

- 84 DRAFT

– DO NOT CITE OR QUOTE 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 indu ced learning deficits in fe male mice. Mechanistically, these authors propose that associations of Pb and stress with learning deficits may be related to aberrations in corticosterone and dopamine.

a Based on calculation of group mean values across session block post

- stress challenge for both maternal and lifetime Pb exposure studies. All calculations represent percent of 0

- NS control values; ↑, represents increase; ↓, represents decrease.
b Data from Virgolini et al. ( 2005 )

. * Denotes significant effect versus 0ppm control (p<0.05).

c Data from current study. * Denotes significant effect versus 0ppm control (p<0.05).

Source: Used with permission from Elsev ier Science, Rossi

- George et al. ( 2011 ) (Table 1).

Cognitive Flexibility



### Cognitive flexibility is the ability to reallocate mental resources when situations 1 change ( Monsell, 2003 ) . Discrimination reversal learning is used to measure cognitive 2 flexibility, which is a subclass of executive function. The 2006 AQCD reported reversal 3 learning deficits in monkeys with blood Pb of 11

- 20 μg/dL. Rats also showed similar 4

deficits but the aut hors attributed the changes to learning related problems instead of 5 cognitive flexibility ( Garavan et al., 2000 ; Hilson & Strupp, 1997 ) . Interestingly, recent 6 work has shown that NMDA receptors and D2

- like receptors, two known targets of Pb, are 7 involved in discrimination reversal learning ( Herold, 2010 ) . Another test of cognitive 8 flexibility is called concurrent random interval (RI

- RI) scheduling in which depression on 9 two response levers is reinforced at different frequencies. The 2006 AQCD reported 10 monkeys with cognitive flexibilit y impairment under RI

- RI ( Newland et a l., 1994 )

. 11

Selective Attention

Few animal toxicology studies measure selective attention. Those that do employ signal detection 

### 12
with distraction, a test looking for increased omissions after exposure to an external distraction. The 

### 13
newest publication in this area showed no effect with t his test after juvenile through adolescence exposure 

### 14

in female rats ( Stangle et al., 2007 ) . The two dose groups yielded blood Pb levels of 13 μg/dl and 31ug/dl 

### 15
( Stangle et al., 2007 )

. 

### 16

5.3.2.3. Toxicological Studies on the Effects of Chelation

Earli er work in the animal toxicology literature has shown that succimer or chelation treatment of 

### 17
Pb exposed lab animals was able to normalize various aberrant Pb

- induced behaviors including activity 

### 18
level, habituation ( Gong & Evans, 1997 ) and forced

- swim immobility ( P. W. Stewart et al., 1996 ) . A more 

### 19
recent study looked at the effect of succimer treatment on various neurobehaviroal and neurocognitive 

### 20
outcomes in control and neonatally Pb

- exposed female animals (PND 1

- 30 Pb acetate exposure, 300 ppm 

### 21
dam through lactation and either 30 or 300 ppm pup water) by drinking water, generating a moderate Pb 

### 22
(m

- Pb) exposure and a high Pb (h

- Pb) exposure group. Pb blood levels at PND52 in the control, m

- Pb, h

- 

### 23May 2011 5

- 85 DRAFT

– DO NOT CITE OR QUOTE Pb, m

- Pb+succimer, and h

- Pb+succimer are 1.5, 12.6, 31, 2.8, an d 8.5 μg/dL, respectively; brain Pb levels 

### 1
at the same time for the same groups are 41, 1040, 3690, 196, and 1370 ng/g dry weight, respectively. 

### 2
Succimer treatment significantly attenuated the m

- Pb induced impaired learning ability. Effects on arousal 

### 3

that were significantly affected in h

- Pb rats were significantly attenuated with succimer treatment. 

### 4

Succimer treatment in the h

- Pb animals only slightly improved learning ability and did not improve the 

### 5
impaired inhibitory control ( Stangle et al., 2007 ) . These are important findings because they provide 

### 6
evidence that certain adverse neurobehavioral or cognitive outcomes associated with Pb exposure appear 

### 7
to be reversible with chelation therapy. 

### 8

A 3

- week course of Pb acetate ( PND 1

- 17, dam drinking wate r) plus or minus succimer /chelator 

### 9
(PND 31

– 52) treatment was performed to determine if succimer could alleviate behavioral deficits in rats 

### 10
exposed to Pb for the first 4 weeks of life. Pb

- exposed animals had altered reactivity and increased reward 

### 11

omission and errors. Pb

- exposed animals receiving chelation therapy had normalize d reactivity to reward 

### 12

omission and errors ( Beaudin et al., 2007 )

. Adverse Pb

- induced behavioral outcomes were attenuated with 

### 13
chelation therapy in Pb

- exposed animals. 

### 14
Another study ( G. Fan et al., 2009 ) looked at methionine choline supplementation in Pb exposed 

### 15

an imals to understand its effect on Pb disposition in various tissues (blood, bone, brain) and how this 

### 16
might contribute to neurocognitive or neurobehavioral changes. As a sulfur source, methionine is a 

### 17

chelator and a free

- radical scavenger. Choline is impor tant for cell membranes and neurotransmitter 

### 18
synthesis ( Zeisel & Blusztajn, 1994 ) . In this model, methionine choline attenuated Pb

- dependent memory 

### 19
and learning defecits (Section 5.3.2.2). Exposure of weanling male rats to Pb acetate in drinking water 

### 20
(300 mg/L) out to PND60 produced a blood Pb level of 60 μg/dL, bone Pb of 165 μg/g and brain Pb of 

### 21
0.63 μg/g. Methionine choline supplementation significantly attenuated blood Pb and bone Pb but 

### 22
produced a non

- significant attenuation of brain Pb (0.51 μg/g) in mice that had significant improvements 

### 23
in learning and memory (Section 5.3.2.2). 

### 24
Also, in another study the metal chelators DP

- 109 and DP

- 460 are neuroprotective in the ALS 

### 25
mouse neurodegenerative model or Tg(SOD1

- G93A) ( Petri et al., 2007 )

. 

### 26
In summary, suc cimer or chelation therapy appears to be able to restore Pb

- dependent impairments 

### 27
of learning and arousal as well as being neuroprotective in a dose dependent fashion. In these studies 

### 28
succimer use was most efficacious at lower doses of Pb exposure. C helat ion does not restore Pb



### 29
dependent impaired inhibitory control . Chelation with the supplement methionine choline affected the 

### 30
disposition of Pb in various tissues, significantly attenuating blood and bone Pb levels and non



### 31
significantly attenuating brain Pb

. 

### 32May 2011 5

- 86 DRAFT

– DO NOT CITE OR QUOTE 

### 5.3.2.4. Epidemiologic Studies of C ognitive Function in 

### Adults

Adults without Occupational Lead Exposures

The 2006 AQCD cited more consistent associations of bone Pb levels but not concurrent blood Pb 

### 1
levels with cognitive performance in environmentally

- exposed adults ( U.S. EPA, 2006 ) . Studies published 

### 2

s ince the 2006 Pb AQCD continue to support the previous findings. Several studies analyzed data from 

### 3
NHANES III (1991

- 1994) and investigated effect modification by age and genetic variants. Krieg et al. 

### 4
( 2009 ) overall did not find blood Pb level to be associated consistently with poorer performance on 

### 5
cognitive testing among 2,090 ad ults 20

- 59 years of age nor among 1,796 adults 60 years of age and 

### 6

older. There were also few statistically significant differences in the association with blood Pb between 

### 7
ALAD genotypes ( E. F. Krieg, Jr. et al., 2009 ) . A mong the 20

- 59 year

- old adults, a borderline significant 

### 8

di fference was observed for mean reaction time, however, among subjects in the CC and CG ALAD 

### 9
genotype groups combined (ALAD2 carriers in the terminology above) (n=161) , reaction time improved 

### 10
(i.e., faster reaction time) with increasing blood Pb level. In c ontrast, ALAD2 subjects had a greater 

### 11
increase in the number of errors on a symbol

- digit substitution task (p = 0.07) in association with 

### 12
increasing blood Pb level. In the same study, contrasting observations were made in adolescent NHANES 

### 13
participants. Ef fect modification by ALAD2 is not entirely clear as it may increase susceptibility to Pb



### 14
associated health effects by increasing blood Pb levels or diminish Pb bioavailability by maintaining it in 

### 15
a sequestered state in the bloodstream. Krieg et al. ( 2010 ) also found differences in the association 

### 16

between blood Pb lev el and scores on a symbol

- digit substitution test by the VDR variants, rs731236 and 

### 17

VDR rs2239185

, as well as VDR haplotype . Similar to observations in children (Section 5.3.2.1), results 

### 18

were not consistent across the various tests . However, blood Pb leve l generally was associated with 

### 19
greater decrements in cognitive performance among adults with the CC genotypes of VDR variants. 

### 20

Shih et al. ( 2006 ) studied participants in the Baltimore Memory Study (BMS), a longitudinal study 

### 21

of men and women 50

- 70 years of age residing in Baltimore, MD with a mean (SD) blood Pb level of 

### 22 3.46 (2.23) μg/dL

. A total 1,140 out o f 2,351 (48.5%) subjects participated from neighborhoods that 

### 23
represented a diversity of race and SES. Of these, 991 had complete data for blood and tibia bone Pb

, 

### 24
cognitive testing, and covariates

. After excluding the three participants with the highest bone Pb values, 

### 25

the two participants with t he two most negative bone Pb values, and one person with a blood Pb level 10 

### 26
standard deviations greater than the mean, the analytic sample was 985

. Scores on individual cognitive 

### 27
tests from a battery of 20 in

- person administered tests were grouped into di fferent cognitive domains 

### 28
(language, processing speed, eye

- hand coordination, executive function, verbal memory and learning, 

### 29
visual memory, and visuoconstruction) by transforming individual test scores into z

- scores and averaging

. 

### 30
Negative associations we re observed more consistently for tibia Pb levels than for blood Pb levels (Figure 

### 31

5

- 16 and Table 5

- 9). Tibia bone Pb levels were associated with worse performance on tests in all domains 

### 32May 2011 5

- 87 DRAFT

– DO NOT CITE OR QUOTE in models adjusted for age, sex, testing technician, and presence of the apolipoprotein ( APO ) E

- ε4 allele

. 

### 1

The magnitudes and statistical significance of associations were attenuated with additional adjustment for 

### 2
education, race, and wealth

. In these fully

- adjusted models, all domains except language and processing 

### 3
speed we re negatively associated with tibia bone Pb , but only the association with visuoconstruction was 

### 4
borderline statistically significant . Analysis of a quadratic term for tibia Pb indicated no e vidence of 

### 5
nonlinearity , thus results for linear models were presented. In linear models, vi suoconstruction scores 

### 6
decreased by 0.0044 SDs (95% CI:

- 0.0091, 0.0003) per 1 μg/g bone increase in tibia Pb level. The mean 

### 7
( SD ) tibia Pb in this group was 18.7 (11.2) μg/g bone

. Of particular note in this study is that it was the 

### 8
first such study with a large proportion of African

- Americans (n=395). In a subsequent analysis, 

### 9
increasing tibia Pb levels were associated with a greater decrease in cognitive performance among 

### 10
subjects living in neighborhood s with a greater numbe r psychosocial hazards ( Glass et al., 2009 ) (Figure 

### 11

5

- 16 and Table 5

- 9). 

### 12May 2011 5

- 88 DRAFT

– DO NOT CITE OR QUOTE Reference Da ta (μg/dL) Outcome Subgroup/Bone type
Blood Pb Krieg and Butler ( 2009 ) 2.88 (6.91) Symbol digit substitution Ages 20

- 39 yrs Ages 40

- 59 yrs Serial digit learning Ages 20

- 39 yrs Ages 40

- 59 yrs Krieg et al. ( 2009 ) 2.85 (7.31) Symbol digit substitution Ages 20

- 59 yrs, ALAD GG Ages 20

- 59 yrs, ALAD CC/CG Serial digit learning Ages 20

- 59 yrs, ALAD GG Ages 20

- 59 yrs, ALAD CC/CG 4.02 (3.56) Word recall Ages ≥60 yrs, ALAD GG Ages ≥60 yrs, ALAD CC/CG Story recall Ages ≥60 yrs, ALAD GG Ages ≥60 yrs, ALAD CC/CG Krieg et al. ( 2 010 ) 2.85 (7.32) Symbol digit substitution Ages 20

- 59 yrs, VDR hap CC Ages 20

- 59 yrs, VDR hap CT Ages 20

- 59 yrs, VDR hap TC Ages 20

- 59 yrs, VDR hap TT

Shih et al. ( 2006 ) a 3.5 (2.2) Language Eye

- hand coordination Executive functioning Visuoconstruction

Rajan et al. ( 2008 ) 5.3 (2.9), 4.8 (2.7) b Visuospatial construction Executive functioning Verbal memory Perceptual speed

Weuve et al. ( 2009 ) 2.9 (1.9) Composite cognitive score Composite, except letter

Gao et al. ( 2008 ) 0.39 (0.63) (plasma) Composite cognitive score

Bone Pb

Shih et al. ( 2006 ) a 18.7 (11.2) Language Eye

- hand coordination Executive functioning Visuoconstruction

Bandeen

- Roche et al. ( 2009 ) c 18.8 (11.6) Eye

- hand coordination African

- Americans, ≤ 15 African

- Americans, > 15 White, ≤ 15 White, > 15 Verbal memory African

- Americans, ≤ 15 African

- Americans, > 15 White, ≤ 15 White, > 15 Glass et al. ( 2009 ) 18.8 (11.1) Language Middle NPH d High NPH d Eye

- hand coordination Middle NPH d High NPH d

Rajan et al. ( 2008 ) 21.9 (13.8), 21.2

e Visuospatial construction Tibia Executive functioning Tibia Verbal memory Tibia Perceptual speed Tibia 29.3 (19.1), 27.9
f Visuospatial construction Patella Executive functioning Patella Verbal memory Patella Perceptual speed Patella

Weuve et al. ( 2009 ) 10.5 (9.7) Composite cognitive score Tibia Composite, except letter Tibia 12.6 (11.6) Composite cognitive score Patella Composite, except letter Patella

Change in Cognitive Score (95% CI)

Note: B lack diamonds = blood Pb

, blue circles = tibia Pb , wh ite circles = patella Pb . ALAD = aminolevulinate dehydratase, VDR = vitamin D receptor, NPH = neighborhood psychosocial hazard. a Effect estimates for Model B are presented. b Blood Pb levels represent levels in ALAD wildtype a nd ALAD2 carriers, respectively. c Effect estimates for the following strata: African

- Americans with tibia Pb levels ≤ 15 μg/dL, African

- Amer icans with tibia Pb levels > 15 μg/dL, whites with tibia Pb levels ≤ 15 μg/dL, and whites with tibia Pb levels > 15 μg /dL. d Effect estimates for the interaction between tibia Pb levels and NPH tertile, with the lowest NPH tertile serving as the reference group. e Tibia Pb levels represent levels in ALAD wildtypes and ALAD2 carriers, respectively. f Patella Pb levels represe nt levels in ALAD wildtypes and ALAD2 carriers, respectively.
Figure 5

- 16

. Associations of blood and bone Pb levels with cognitive function among adults without occupational exposures to Pb.May 2011 5

- 89 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 9. Additional characteristics and quantitative results for studies presented in Figure 5

- 16

Study Population/ Location Pb Biomarker Data Statistical Analysis Cognitive Test Subgroup/ Model Effect Estimate (95% CI) a

Blood Pb Studies
Krieg and Butler ( 2009 ) 2823 adults, ages 20

- 59 yr, U.S. NHANES III (1991

- 1994) Blood mean (SD): 2.88 (6.91) μg/dL 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 Symbol Digit Substitution (mean total latency, sec) Serial digit learning score Assessed using the Neurobehavioral Evaluation System 2 Ages 20

- 39 yr Ages 40

- 59 yr Ages 20

- 39 yr Ages 40

- 59 yr - 0.014 (

- 0.061, 0.032)b

- 0.042 (

- 0.087, 0.0009)b

- 0.017 (

- 0.067, 0.034)b 0. 058 (

- 0.028, 0.143)b

Krieg et al. ( 2009 ) 2090 adults, ages 20

- 59 yr 1976 adul ts, ages ≥60 yr

U.S. NHANES III (1991

- 1994) Blood mean (SD): 20

- 59 yr: 2.85 (7.31) μg/dL ≥60 yr : 4.02 (3.56) μg/dL 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 (≥60 yr ) Symbol Digit Substitution (mean t otal latency, sec)
Serial digit learning score

Word recall

Sto ry recall Assessed using the Neurobehavioral Evaluation System 2 Ages 20

- 59 yr ALAD GG ALAD CC/CG Ages 20

- 59 yr ALAD GG ALAD CC/CG Ages ≥60 yr ALAD GG ALAD CC/CG Ages ≥60 yr ALAD GG ALAD CC/ CG - 0.018 (

- 0.049, 0.013)b

- 0.072 (

- 0.153, 0.008) b

- 0.003 (

- 0.072, 0.067)b 0.056 (

- 0.088, 0.201) b

- 0.021 (

- 0.08, 0.034 ) 0.007 (

- 0.114, 0.129)

0.024 (

- 0.028, 0.077 )

- 0.131 (

- 0.301, 0.039) Krieg et al. ( 2010 ) 2093 adults, ages 20

- 59 yr 1799 adults, ages ≥60 yr U.S. NHANES III (1991

- 1994) Blood mean (SD): 20

- 59 yr: 2.85 ( 7.32) μg/dL ≥60 yr : 4.02 (3.39) μg/dL 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, educati on, family income, race

- ethnicity, test language ( ≥60 yr ) Symbol Digit Substitu tion (mean total latency, sec)
Serial digit learning score (# errors, pos is bad)

Word recall
Story recall Assessed using the Neurobehavioral Evaluation System 2 Ages 20

- 59 y r 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 ≥60 yr VDR haplotype CC VDR haplotype CT VDR haplotype TC VDR haplotype TT Ages ≥60 yr VDR haplotype CC VDR haplotype CT VDR haplotype TC VDR haplotype TT - 0.535 (

- 1.18, 0.107)b 0.019 (

- 0.038, 0.077)b

- 0.069 (

- 0.140, 0.002)b

- 0.095 (

- 0.192, 0.001)b

- 0.346 (

- 0.665, 0.026)b

- 0.044 (

- 0.126, 0.039)b 0.061 ( 0.074, 0.135)b 0. 006 (

- 0.107, 0.119)b

- 0.226 (

- 0.536, 0.087)

- 0.028 (

- 0.118, 0.067)

- 0.010 (

- 0.139, 0.115)

- 0.028 (

- 0.264, 0.209)

0.043 (

- 0.494, 0.581) 0.001 (

- 0.057, 0.060) 0.010 (

- 0.095, 0.116)

- 0.049 (

- 0.128, 0.064)
Shih et al. ( 2006 ) 985 adults, mean age: 59 yr Baltimore Memory Study, B altimore, MD Blood mean (SD): 3.5 (2.2) μg/dL Linear regression adjusted for : Model A: age, sex, technician, presence of APOE

- ε4 allele Model B: Model I, years of education, race/ethnicity, wealth Language

Eye

- hand coordination
Executive functioning

Visuoconstruction

Assessed using Raven ’ s Colored Progressive Matrices Model A Model B Model A Model B Model A Model B Model A Model B - 0.0060 (

- 0.029, 0.017)

- 0.0019 (

- 0.020, 0.016)

- 0.0110 (

- 0.032, 0.01)

- 0.0076 (

- 0.026, 0.011)

- 0.0143 (

- 0.034, 0.005)

- 0.0101 (

- 0.027, 0.007)

- 0.0191 (

- 0.046, 0.008)

- 0.0143 (

- 0.038, 0.009)

Rajan et al. ( 2008 ) 720 males, ages ≥45 yr Normative Aging Study, Boston, MA Blood m ean (SD): 5.3 (2.9) μg/dL (ALAD wildtype) 4.8 (2.7) μg/dL (ALAD2 carriers) L inear regression adjusted for blood Pb main effect, ALAD genotype, age at cognitive test, education, alcohol consumption, cumulativ e smoking, English as first language Visuospatial, constructional praxis Executive function verbal fluency Verbal memory, word recall Perceptual speed, mean latency
Assessed using CERAD, Neurobehavioral Evaluation Sysem, WIAS

R - 0.017 (

- 0.077, 0.043) c

- 0.01 (

- 0.073, 0.053) c 0.001 (

- 0.06, 0.062) c

- 0.06 (

- 0.14, 0.02) cMay 2011 5

- 90 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Pb Biomarker Data Statistical Analysis Cognitive Test Subgroup/ Model Effect Estimate (95% CI) a

Weuve et al. ( 2009 ) 587 females, ages 47

- 74 yr Nurses’ Health Study, Boston, MA Blood mean (SD): 2.9 (1.9 ) μg/dL 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, aspirin use, ibuprofen use, use of Vitamin E

s upp lements, menopausal status and postmenopausal hormone use Composite cognitive score Composite except letter fluency

Assessed using Telephone Interview for Cognitive Status and East Boston Memory Test - 0.008 (

- 0.036, 0.020) 0.008 (

- 0.037, 0.054)

Gao et al. ( 2008 ) 188 adults, mean age 69.2 yr Sichuan and Shandong Provinces, China Plasma mean (SD): 0.39 (0.63) μg/dL ANCOVA adjusted for age, sex, education, BMI, APOE ε4 Composite cognitive score Assessed using CERAD, CSID, IU story recall, Animal fluency test, IU token test - 0.009 (

- 0.025, 0.007)

Excluded From Figure Due Insufficient Data To Standardize Test Scores

Weuve et al. ( 2006 ) 720 males, ages ≥45 yr Normative Aging Study, Boston, MA Blood mean (range): 5.2 ( ≤1

- 28) μg/dL Linear mixed effects regression adjusted for smoking stat us, alcohol consumption, calorie adjusted calcium intake, regular energy expenditure on leisure time physical activity, diabetes MMSE score ALAD wildtypes ALAD2 carriers - 0.013 (

- 0.053, 0.027)

- 0.087 (

- 0.180, 0.007)

Bone Pb Studies

Shih et al., ( Shih et al. ) 985 adults, mean age: 59 yr Baltimore Memory Study, Baltimore, MD Tibia mean (SD): 18.7 (11.2) μg/g Linear regression adjusted for : Model A: age, sex, technician, presence of APOE

- ε4 allele Model B: Model I, years of education, race/ethnicity, wealth Language

Eye

- hand coordination
Executive functioning

Visuoconstruction

Assessed using Raven ’ s Colored Progressive Matrices Model A Model B Model A Model B Model A Model B Model A Model B - 0.08 (

- 0. 13,

- 0.04) 0.00 6 (

- 0.03, 0.04)

- 0.0 8 (

- 0.12,

- 0.04)

- 0.0 3 (

- 0.06, 0.01)

- 0.0 8 (

- 0.11,

- 0.04)

- 0.0 14 (

- 0.05, 0.02)

- 0.0 22 (

- 0.17,

- 0.07)

- 0.0 44 (

- 0.09, 0.003)

Bandeen

Roche et al. ( 2009 ) 1140 adults, ages 50

- 70 yr Baltimore Mem ory Study cohort Baltimore, MD Tibia mean (SD): 18.8 (11.6) μg/g Marginal longitudinal linear regression models adjusted for age, household wealth, education, race/ethnicity Demographic characteristics, socioeconomic status, race/ethnicity Eye hand coordin ation African

- Americans

White

Verbal memory African

- Americans

White

Visual memory African

- Americans

White Pb ≤ 15 μg/g Pb >15 μg/g Pb ≤ 15 μg/g Pb >15 μg/g
Pb ≤ 15 μg/g Pb >15 μg/g Pb ≤ 15 μg/g Pb >15 μg/g

Pb ≤ 15 μg/g Pb >15 μg/g Pb ≤ 15 μg/g Pb >15 μg/g - 0. 04 (

- 0.112, 0.03) 0.0 2 (

- 0.05, 0.10)

- 0. 02 (

- 0.06, 0.01) 0.0 4 (

- 0.002, 0.08)

- 0.0 08 (

- 0.08, 0.06) 0.00 2 (

- 0.09, 0.09) 0.0 4 (

- 0.003, 0.09)

- 0. 07 (

- 0.13,

- 0.005)

0.001 (

- 0.007, 0.0 9)

- 0.0009 (

- 0.011, 0. 09) 0.003 (

- 0.003, 0.0 8)

- 0.004 (

- 0.011, 0.03)
Glass et al. ( 2009 ) 1001 adults, mean age 59 yr Baltimo re Memory Study, Baltimore, MD Tibia Pb mean (SD): 18.8 (11.1) μg/g Multilevel hierarchical regression model adjusted for age, sex, race/ethnicity, education, testing technician, time of day Language

Eye

- hand coordination
Executive functioning

Visuoconstruction

Assessed using Raven ’ s Colored Progressive Matrices Middle NPH High NPH Middle NPH High NPH Middle NPH High NPH Middle NPH High NPH 0.0 1 (

- 0.08, 0.09)d

- 0. 09 (

- 0.17,

- 0.001)d

- 0. 04 (

- 0.12, 0.05)d

- 0. 06 (

- 0.15, 0.02)d

- 0. 02 (

- 0.10, 0.06)d

- 0. 10 (

- 0.18,

- 0.02)d

- 0. 003 (

- 0.14, 0.08)d

- 0. 006 (

- 0.17, 0.05)dMay 2011 5

- 91 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Pb Biomarker Data Statistical Analysis Cognitive Test Subgroup/ Model Effect Estimate (95% CI) a

Rajan et al. ( 2008 ) 720 males, ages ≥45 yr Normative Aging Study, Boston, MA 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) Linear regression adjusted for bone Pb main effect, ALAD genotype, age at cognitive test, education, alcohol consumption, cumulative smoki ng, English as first language Visuospatial, constructional praxis

Executive function verbal fluency

Verbal memory, word recall

Percep tual speed, mean latency
Assessed using CERAD, Neurobehavioral Evaluation System, WIAS

- R Tibia Patella Tibia Patella Tibia Patella Tibia Patella - 0. 17 (

- 0.33,

- 0.007) 0.0 1 (

- 0.10, 0.12)

- 0. 07 (

- 0.23, 0.08)

- 0. 01 (

- 0.12, 0.10) 0.0 5 (

- 0.10, 0.21) 0.07 (

- 0. 4 0, 0.18)

- 0. 12 (

- 0.39, 0.06)

- 0. 08 (

- 0.22, 0.06)

Weuve et al. ( 2009 ) Nurses’ Health Study cohort 587 subjects Age range: 47

- 74 yr All females Boston, MA Tibia mean (SD): 10.5 (9.7) μg/g Patella mean (SD): 12.6 (11.6) μg/g 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, aspirin use, ibuprofen use, use of Vitamin E supplements, menopausal status and postmenopausal hormone use Composite cognitive score

Composite except lette r fluency

Assessed using Telephone Interview for Cognitive Status and East Boston Memory Test Tibia Patella Tibia Patella - 0.0 4 (

- 0.09, 0.005)

- 0.0 1 (

- 0.05, 0.03)

- 0.0 5 (

- 0.10,

- 0.003)

- 0.0 3 (

- 0.07, 0.01)

Excluded From Figure Due Insufficient Data To Standardize Test Scores Or Calculate Effect Estimates
Weisskopf et al. ( 2007 ) 1089 males, mean age 68.7 yr Normative Aging Study, Boston, MA Mean (IQR): 20 (13

- 28) μg/g (Tibia) 25 (17

- 37) μg/g (Patella) Linear repeated measures analysis adjusted for age, age squared, education, smoking, alcohol intake, yr between bone Pb

meas urement and first cognitive test, yr between cognitive tests Visuospatial, pattern compar ison (pos is bad, latency) Executive function verbal fluency

Short

- term memory, word list
Assessed using CERAD, Neurobehavioral Evaluation System, WIAS

- R, MMSE, VMI Tibia Patella Tibia Patella Tibia Patella 0. 79 (0.40, 1.2)e 0. 73 (0.40, 1.2)e

- 0. 40 (

- 1.6, 0.80)e

- 0. 86 (

- 2.00, 0.30)e

- 0. 28 (

- 1.2, 0.60)e

- 0. 81 (

- 1.7, 0.05)e

Weuve et al. ( 2006 ) 720 males, ages ≥45 yr Normative Aging Study, Boston, MA Median (1 st

- 3rd quartile): Tibia: 19 (13

- 28) μg/g Patella: 27 (18

- 39) μg/g Linear mixed effects regression adjusted for smoking status, alcohol consumption, calorie adjusted calcium intake, regular energy expendit ure on leisure time physical activity, diabetes MMSE score Tibia ALAD wildtype ALAD2 carrier Patella ALAD wildtype ALAD2 carrier - 0. 03 (

- 0.14, 0.07)

- 0. 11 (

- 0.30, 0.06)

- 0.0 3 (

- 0.11, 0.04)

- 0. 12 (

- 0.30, 0.06)

Wang et al. ( 2007 ) 358 males, median ages: 67.2 yr (HFE wild

- type) 67.7 yr (HFE variant) Normative Aging Study, Boston, MA Median : 19 μg/g (Tibia), 23 μg/g (Patella) Linear regression adjusted for age, years of education, nonsmoker, former smoker, pack

- years, nondrinker, alcohol consumption, English as first language, computer experience, diabetes MMSE HFE wildtype One HFE variant Two HF E variants - 0. 20 (

- 1.0, 0.70)e

- 1.40 (

- 3.3, 0.40)e

- 6.3 (

- 10.4,

- 2.1)eMay 2011 5

- 92 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Pb Biomarker Data Statistical Analysis Cognitive Test Subgroup/ Model Effect Estimate (95% CI) a

Van Wijngaarden et al. ( 2009 ) 47 adults, mean age 61.5 yr Rochester, NY Mean (SD): 2.0 (5.2) μg/g (Tibia) 6.1 (8.5) μg/g (Calcaneus) Linear regression adjusted for age, gender, educational level, history of hypertension Delayed matching, % correct Lowest tertile Medium tertile Highest tertile Lowest tertile Medium tertile Highest tertile Total tri als Lowest tertile Medium tertile Highest tertile Lowest tertile Medium tertile Highest tertile Assessed using CANTAB and Montreal Cogn itive Assessment Calcaneus

Tibia

Calcaneus

Tibia 87.56 f 86.67 80.67, p = 0.027 85.42 f 87.08 82.44, p = 0.25

2.54 f 2.61 2.72, p = 0.21 2.62 f 2.59 2.66

a Effect estimates have been standardized to the standard deviation of the cognitive test scores and standardized to a 1 μg/dL increase in blood Pb and 10 μg/g increase in bone Pb. b The directions of effect estimates were changed to indicate a negative slope as a decrease in cognitive performance.

c Effect estimates indicate interactions between Pb and ALAD genotype. d Effect estimates indicate interactions between Pb and category of neighborhood psychosocial hazard (NPH), with the lowest ter tile of NPH serving as the reference group.

e Effect estimate refers to the change in cognitive function sco re over time. f Results refer to mean cognitive function scores among tertiles of bone Pb.

Weuve et al. ( 2009 ) studied the association of blood and bone Pb levels with cognitive function 

### 1
among a subset of 587 women from the Nurses Health Study from whom blood and bone Pb 

### 2

measurement s were taken between the ages of 47 a nd 74 years. The mean (SD) blood Pb level in this 

### 3 group was 2.9 (1.9) μg/dL

. The women had had Pb measured as part of their participation in two separate 

### 4

sub

- studies, one of osteoporosis and the other of hypertension

. The inclusion criteria included reside nce in 

### 5

the Boston area and absent of major chronic diseases at the time of Pb measurement

. Cognitive function 

### 6
was assessed via a battery of telephone administered tests (Telephone Interview of Cognitive Status

, digit 

### 7
span backwards, alphabetizing span, ani mal naming [category fluency], “ f ” naming [letter fluency], and a 

### 8
composite verbal memory score) an average of 5 years after Pb measurement . As in the aforementioned 

### 9
studies of adults, negative associations were observed more consistently for tibia and pat ella Pb levels 

### 10
than for blood Pb levels (Figure 5

- 16 and Table 5

- 9). Contrary to expectation, scores on the “ f ” nam ing 

### 11

test were positively associated with patella and tibia bone Pb levels. In separate models , the “f” naming 

### 12

test was omitted from a composite index of all cognitive tests , and a one SD (10 μg/g bone) increase in 

### 13

tibia Pb level was associated with 0.051

- point decrease (95% CI:

- 0.010,

- 0.003) in the standardized 

### 14
composite score

. This effect size of tibia bone Pb level was approximately equivalent to the effect size for 

### 15
3 years of age among these women. The magnitude of effect was smaller for patella Pb levels (

- 0.033 

### 16
[95% CI:

- 0.080, 0.014) per 1 SD unit increase in patella Pb level) 

### 17
Several analyses of Normative Aging Study ( NAS) have c ontributed to greater understanding of the 

### 18
relationship between biomarkers of Pb dose and neurocognitive effects in adults. The NAS is conducted 

### 19
at the VA Outpatient Clinic in Boston, MA and is a multidisciplinary longitudinal investigation of the 

### 20May 2011 5

- 93 DRAFT

– DO NOT CITE OR QUOTE aging pr ocess that was established in 1961 and originally involv ed 2,280 men residing in the Greater 

### 1
Boston area aged 21 to 80 years with no current or past chronic medical conditions

. Participants are 

### 2
evaluated every 3

- 5 years with in

- person clinical assessments, self

- administered questionnaires

. In

- person 

### 3
administered cognitive tests included those in the general domains of attention/executive function, short



### 4
term memory, visuospatial, and verbal/language

. About 60

- 70% of participants, depending on the specific 

### 5

c ognitive test, took individual tests twice approximately 3.5 years apart

. The average (SD) age of the men 

### 6
at baseline was 69 ( ± 7) years

, and the median blood Pb level was 5 μg/dL

. Beginning i n 1991

, 

### 7

measurement of bone Pb levels began , with 68% of active NAS subjects participating. A ll analyses were 

### 8
adjusted for age, age squared, education, smoking, and alcohol intake

. In one study of Mini

- Mental State 

### 9
Examination (MMSE) tests scores among 72 0 men 45 years of age and older, blood Pb levels were 

### 10
negatively associated with MMSE score among ALAD2 carriers ( Weuve et al., 2006 ) . A 3 μg/dL 

### 11

i ncrement in blood Pb level (the interquartile range) was associated with a 0.26 point lower mean MMSE 

### 12
score (95% CI:

- 0.54,

- 0.01) among ALAD2 carriers and a 0 .04 point lower score (95% CI:

- 0.16,

- 0.07) 

### 13
among noncarriers. A subsequent study in the same cohort considered performance on a battery of oher 

### 14

cog nitive tests and did not find a consistent pattern of modification of the association between blood or 

### 15

bone Pb levels and cognitive function by ALAD genotype ( Rajan et al., 2008 ) . Nonetheless, Rajan et al. 

### 16
( 2008 ) found tibia Pb levels to be associated more consistently with lower cognitive performance 

### 17
compared with blood or patella Pb levels (Figure 5

- 16 and Table 5

- 9). 

### 18

The NAS also examined effect modification by hemochromatosis (HFE) gene variants. I n models 

### 19
adjusted for age, years of education, nonsmoker, former smoker, pack

- years, nondrinker, alcohol 

### 20
consumption, English as first language, computer experience, and diabetes

, an interquartile range increase 

### 21

in tibia bone Pb level (15μg/g) was associated with a 0.22 point steeper annual decline (95% CI:

- 0.39,

- 

### 22
0.05) in MMSE score a mong men with any variant HFE allele (either H63D or C282Y) . The magnitude 

### 23
of association was less negative among those with only HFE wildtype alleles ( F. T. Wang et al., 2007 ) 

### 24
(Figure 5

- 17 ). As indicated in Figure 5

- 17, a nonlinear association was observed between tibia Pb levels 

### 25
and change in MMSE score, with a steeper decline per unit increase in tibia Pb level at higher tibia Pb 

### 26
levels. Effect modification by n ongenetic factors also were e xamined in the NAS cohort. Increasing bone 

### 27

Pb levels were associated with greater decreases in c ognitive function among individuals with higher 

### 28

individual

- level perceived stress ( Peters et al., 2007 )

. 

### 29May 2011 5

- 94 DRAFT

– DO NOT CITE OR QUOTE Source: Wang et al. ( 2007 )

. Note: The lines indicate curvilinear trends estimated from the penalized spline method. Among HFE wild

- types, the optimal degree of smoothing was 1, meaning that the association between tibia Pb and annual cognitive decline was nearly linear, but amon g variant allele carriers, the association tended to deviate from linearity (p = 0.08), with an optimal 1.68 degree of smoothing. The model was adjusted for age, years of education, nonsmoker, former smoker, pack

- years, nondrinker, alcohol consumption, Eng lish as first language, computer experience, and diabetes. Figure 5

- 17. Exploration of nonlinear association of tibia Pb concentration with annual rate of cognitive decline, by class of HFE genotype.

Two large NAS studies examined the relation ship of blo od and bone Pb levels with the change in 

### 1
cognitive function over time

. Weisskopf et al. ( 2007 ) expanded on an earlier study of Pb biomarkers and 

### 2
cognitive function ( Payton et al., 1998 ) The average (SD) age of the men at baseline was 69 ( ± 7) years, 

### 3

and the median blood Pb concentration was 5 μg/dL . Two measurements of c ognitive function, collected 

### 4
approxi mately 3.5 years apart, were available 60

- 70% of participants. A ll analyses were adjusted for age, 

### 5
age squared, education, smoking, and alcohol intake

. There was little association between blood Pb levels 

### 6
and cognitive test scores, except possibly for voca bulary scores, although this association was greatly 

### 7
weakened when the five men with the highest blood Pb levels (>15 μg/dL ) were excluded

. For bone Pb 

### 8
analyses, the authors used repeated measures analysis with an interaction term between bone Pb level and 

### 9

time in order to estimate the association between bone Pb level and decline in cogniti ve test score over 

### 10

time. There were no main effect associations with bone Pb levels; however, increases in patella and tibia 

### 11

bone Pb levels were associated with decrease s in cognitive performance over time. The only test for 

### 12
which this reached statistical significance (p<0.05), however, was increased response latency on a pattern 

### 13
comparison test . Contrary to expectation, bone Pb levels were associated with fewer errors on the same 

### 14
pattern comparison test . The authors proposed that t his may be related to slowing reaction time to 

### 15
improve accuracy

. When the 9 men with the highest bone Pb levels were removed, the association was no 

### 16
longer statistically significant. 

### 17May 2011 5

- 95 DRAFT

– DO NOT CITE OR QUOTE In an exami nation of the patella Pb concentration

- reaction time relationship, Weisskopf et al. 

### 1
( 2007 ) found a statistically significant non linear association, with latency times on the pattern comparison 

### 2
test becoming worse over time (i.e., larger values or slower response latencies) up to approximately 60 

### 3

μ g/g bone mineral, but the change over time level ing off at higher concentrations (Figure 5

- 18 )

. Below 60 

### 4

μ g/g

, a 20 μ g/g difference in patella Pb level was associated with a decrease in pattern comparison test 

### 5
score of approximately 0.15 ms

. Intriguingly, however, the cognitive tests where the associati on with 

### 6

bone Pb was significantly worse among ALAD

- 2 carriers (constructional praxis and pattern comparison) 

### 7
were the same tests that were significantly associated with bone Pb in an earlier study ( Weisskopf, 

### 8
Proctor, et al., 2007 )

. 

### 9

Source: Used with permission from Williams & Wilkins, Weisskopf et al. ( 2007 )

. Note: Models are a djusted 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 removed. The estimate is indicated by the solid line and the 95% confidence interval by the dashed lines. Patella Pb concentrations of all individual s ubjects are indicated by short vertical lines on the abscissa

. Figure 5

- 18. Nonlinear association between patella bone Pb concentration and the relative change in response latency over time on the pattern comparison test (reference = 0 at mean of patella P b concentration)

.

In a somewhat similar approach to that taken in the NAS, BMS investigators took advantage of 

### 10
repeat cognitive testing of study subjects ( 91% of the original cohort returned for a second round of 

### 11
testing and 83% for a third round each at approximately 14

- month intervals ) to analyze associations of 

### 12
blood and bone Pb levels with changes in cognitive performance over time cohort ( Bandeen

- Roche et al., 

### 13

2009 ) . An interquartile rang e increase in tibia Pb level (12.7 μ g/g) was associated with a 0.019 units per 

### 14May 2011 5

- 96 DRAFT

– DO NOT CITE OR QUOTE year decrease in eye

- hand coordination z

- score

. The association was somewhat stronger among African



### 1
Americans than it was among whites (Figure 5

- 16 and Table 5

- 9). Of the other tests, only change in 

### 2
verbal memory and learning suggested some association with tibia Pb levels. In analyses of what they 

### 3

ter m persistent effects (analogous to the main effects analyses of longitudinal data in the NAS) , a similar 

### 4
pattern was found as that reported by Shih et al. ( 2006 ) in the original BMS cross

- sectional analyses

. 

### 5

I ncreasing tibia Pb levels were associated with decreases in cognitive performance; however, the effect 

### 6

sizes decrease d as more covariates were added to models. In models adjusted for age, sex, race, and 

### 7
education, performance on executive function, verbal memory and learning, and visual memory test 

### 8

decreased w ith increasing tibia Pb levels, with p

- values ranging from 0.16 to 0.26 . In contrast to the 

### 9
results of the longitudinal analyses described above, race

- stratified analyses of persistent effects indicated 

### 10

that tibia Pb levels we re associated with greater decreases in performance on tests of eye

- hand 

### 11
coordination, executive functioning, and verbal memory and learning among whites compared with 

### 12

African

- Americans. 

### 13
Other studies generally confirm the same pattern as described in the larger studies above

. One 

### 14
study of 188 rura l Chinese men and women found a weakly negative association between plasma Pb 

### 15

levels and a composite cognitive score based on a battery of in

- person administered tests ( S. J. Gao et al., 

### 16

2008 ) . It should be noted, though, that Pb in plasma makes up a very small fraction of all Pb in blood and 

### 17
is a different, and much less use d, biomarker than Pb in whole blood. The relevance of this Pb fraction is 

### 18
not entirely clear. Pb in plasma is not bound to erythrocytes, as is about 99% of blood Pb. Thus, it has 

### 19
been postulated that plasma Pb may be more toxicologically active ( Chuang et al., 2001 ; Hernandez

- Avila 

### 20
et al., 1998 )

. In a nother study of 47 men and women in Rochester, NY ( 55

- 67 years of age ), subjects in 

### 21
the higher two tertiles of calcaneal bone Pb level had lower scores on a delayed matching

- to

- sample task 

### 22
( van Wijngaarden et al., 2009 ) ( Table 5

- 9 )

. The pattern was similar for a paired associates learning task, 

### 23
although the results did not reach statistical sign ificance. In analyses of tibia Pb levels, subjects in the 

### 24
highest tertile of tibia Pb level performed worse on cognitive tests (Table 5

- 9). 

### 25
In summary, among adults without occupational exposures to Pb, there is weak e vidence for an 

### 26

association between blo od Pb levels and cognitive function. The strongest evidence of association 

### 27
between blood Pb levels and cognitive function in adults was provided by the various NHANES analyses 

### 28
of various age and genetic variant subgroups ( E. F. Krieg, Jr. & Butler, 2009 ; E. F. Krieg, Jr. et al., 2009 ; 

### 29
E. F. Krieg, Jr. et al., 2010 ) . These NHANES analyses did not have bone Pb measurements for 

### 30
comparison. Consistent with the conclusion of the 2006 Pb AQCD , recent studies continued to 

### 31

demonstrate associations between bone Pb levels and cognitive deficits in adults (Figure 5

- 16 an d Table 

### 32

5

- 9). Recent studies also demonstrated that bone Pb levels may be associat ed specifically with cognitive 

### 33

decline over time. Among recent studies that analyzed both blood and bone Pb levels, bone Pb levels, in 

### 34
particular tibia Pb levels, were associ ated with greater decreases in cognitive performance than were 

### 35
blood Pb levels across of the various cognitive tests that were performed. The discrepant findings for 

### 36May 2011 5

- 97 DRAFT

– DO NOT CITE OR QUOTE blood and bone Pb levels indicate that biomarkers of cumulative Pb exposure , including hig her levels in 

### 1
the past, may the best predictors of neurocogn itive function in adults

. The effects associated with 

### 2
cumulative Pb exposure are also demonstrated by observations that tibia Pb levels were associated with 

### 3
larger decreases in cognitive performan ce than were patella Pb levels. Tibia bone has a slower rate of 

### 4
turnover compared with patella bone and is an indicator of longer

- term Pb exposure. 

### 5

Adults with Occupational Lead Exposures

The 2006 Pb AQCD concluded that the evidence for a n association betw een blood Pb levels and 

### 6
cognitive function in adults was most consistent among adults occupational ly exposed to Pb. Results from 

### 7

a few recent studies of occupationally

- exposed adults support the previous conclusions. Dorsey et al. 

### 8
( 2006 ) followed

- up on a cohort of Pb

- e xposed workers in Korea with a mean age of 43.4 years, on whom 

### 9
patella bone Pb measurements were made

. This group represented a typically high ly

- exposed 

### 10
occupational group with an average blood Pb level of 30.9 μg/dL . In this cohort, both blood and tibia Pb 

### 11
levels previously were associated with poorer performance on a battery of neurocognitive tests ( B. S. 

### 12
Schwartz et al., 2005 ; B. S. Schwartz et al., 2001 ) . Dorsey et al. ( 2006 ) found patella Pb levels to be 

### 13
associated with poorer performance in the domains of manual de xterity, sensory PNS function, and 

### 14
depression symptoms

. In this occupational cohort, however, the as sociations between patella Pb levels 

### 15
and cognitive function were not as strong as the associations with either blood or tibia Pb levels

. 

### 16

A follow

- up study o f the original 1982 Lead Occupational Study was conducted in 2001

- 2004 

### 17
among 83 of the original 288 Pb

- exposed workers and 51 of the original 181 controls ( Khalil, Morrow, et 

### 18
al., 200 9 )

. Those originally in the exposed workers group had last worked in a job with Pb exposure from 

### 19
0.02 to 16 years (median = 6) prior to follow

- up testing

. While the follow

- up participation was somewhat 

### 20
low, par ticipants did not appear to differ from nonparticipants on most baseline cognitive tests except for 

### 21
performing slightly better on aspects of the grooved pegboard test and recall on a paired associates 

### 22
learning task. This suggests that the follow

- up participation was not biased to poor performers

. At follow



### 23
up, the former Pb

- exposed workers performed significantly (p <0.05) worse than the controls in total 

### 24
cognitive score and in the spatial and general intelligence domains. They also performed worse in all 

### 25
other domains ( e.g., motor, executive, an d memory) although not significantly so

. A similar pattern was 

### 26
observed in analyses using tibia Pb levels measured at the follow

- up visit as the exposure variable

. 

### 27

Associations also were seen with blood Pb levels (median among the exposed: 12 μg/dL ) , altho ugh these 

### 28
generally did not reach statistical significance. Among the former Pb workers, tibia Pb levels were 

### 29
associated with a greater decrease in total score and scores for spatial and executive domains between 

### 30

baseline and follow

- up . Tibia Pb level were associated inversely with o ther domains as well, although 

### 31
they were not statistically significant. 

### 32May 2011 5

- 98 DRAFT

– DO NOT CITE OR QUOTE Two additional studies aimed to characterize factors that either mediate or modify the association 

### 1

between Pb biomarkers a nd cognitive function

. A study amo ng 61 current Pb smelter workers with an 

### 2
average age of 40 years and blood Pb of 29.1 μg/dL found that both a time

- weighted integrated blood Pb 

### 3
measure (p = 0.09) and tibia Pb level (p = 0.08) were associated with longer times to complete the 

### 4
grooved pegboard test ( Bleecker, Ford, Vaughan, et al., 2007 ) . In another study to examine the modifyi ng 

### 5

effects of c ognitive reserve ( assessed by performance on the Wide Range Achievement Test

- R for 

### 6
reading ) on the Pb

- cognitive function association among 112 Pb smelter workers, a time

- weighted 

### 7
integrated blood Pb measure (an index of cumulative exposure ) was associated with decrements in motor 

### 8
performance (p<0.05), and among those with low cognitive reserve, tests of attention tasks and a digit 

### 9
symbol task as well ( Bleecker, Ford, Celio, et al., 2007 )

. 

### 10

Iwata et al. ( 2005 ) examined the association between blood Pb level and aspects of postural sway 

### 11
among 121 Pb

- exposed workers in Japan with blood Pb levels between 6 and 89 μg/dL (mean : 40 μg/dL)

. 

### 12
In multiple regression analyses adjusted for age, height, and smoking and drinking status

, increasing 

### 13

blood Pb level was a ssociated with increases in sagittal sway with eyes open (p <0.05) and eyes closed (p 

### 14
<0.01) and transversal sway with eyes closed (p <0.05)

. The authors calculated a benchmark dose level 

### 15
( Budtz

- Jorgensen et al., 2001 ; NRC, 2000 ) of 14.3 μg/dL from a linear dose

- respon se model of their data

. 

### 16
Although the data were slightly better fit with a supralinear dose

- response function, the linear function 

### 17
was also statistically significant. 

### 18
Apolipoprotein E is a transport protein for cholesterol and lipoproteins. The gene appears to 

### 19
regulate synapse formation (connections between neurons) and may be particularly critical in early 

### 20

childhood

. A genetic variant, called the ApoE

- ε4 allele is a haplotype between 2 exonic SNPs and is 

### 21
perhaps the most widely studied genetic variant with respect to increasing risk of neurologic disease. 

### 22

Carriers of the E4 variant are at two fold increased risk of developing Alzheimer ’ s disease, although the 

### 23
majority of such individuals still do not develop the disease. A study of occupationally

- exposed adul ts 

### 24
found that among individuals with at least one ApoE

- ε4 allele, Pb was associated with greater decrements 

### 25
in tests such as digit symbol, pegboard assembly, and complex reaction time ( W. F. Stewart et al., 2002 )

. 

### 26



## 5.3.3. Neurobehavioral Effects

5.3.3.1. Epidemiologic Studies of Behavioral Effects in Children

Several epidemiologic studies reviewed in the 2006 Pb AQCD reported a ssociations be tween blood 

### 27

Pb levels and problems with behavior and social conduct that ranged from inattentiveness to self

- reported 

### 28
delinquent behaviors to criminal activities ( Bellinger et al., 1994 ; Bellinger & Rappaport, 2002 ; Burns et 

### 29
al., 1999 ; Dietrich et al., 2001 ; Needleman et al., 2002 ; Needleman et al., 1996 ; G. A. Wasserman et al., 

### 30

1998 )

. Recent studies continue to demonstrate similar associations and provide new evidence for 

### 31May 2011 5

- 99 DRAFT

– DO NOT CITE OR QUOTE associations of blood Pb levels with ADHD diagnosis and diagnostic indices (Figure 5

- 19 and Table 5

- 

### 1
10). Noncog n itive effects of Pb are more complex to study relative to IQ tests

. However, domain

- specific 

### 2
neuropsychological assessments are advantageous as they may provide greater insight into the underlying 

### 3
CNS damage that may be associated with exposures (e.g., st ructural, neural system, neurotransmitter) ( R. 

### 4

F. White et al., 2009 ) . Most of these studies found that blood or dentin Pb levels measured at an early age 

### 5
(e.g., 2

- 6 years of age) were associated with behavioral problems later in childhood and early adulthood 

### 6
(7

- 22 years of age). Most of these studies examined associations with blood Pb levels assessed at one 

### 7
time point; however, even the prospective studies with serial measurements of blood Pb levels , found 

### 8

associations w ith both prenatal and early childhood blood Pb levels ( Dietrich et al., 2001 ; J. P. Wright et 

### 9
al., 2008 ) and lifetime average blood Pb levels ( Burns et al., 1999 )

. Therefore, uncertainty remained over 

### 10
what was the critical time period of Pb exposure for increasing risk of behavioral problems and 

### 11

misconduct. In many of the studies noted above, blood Pb level also was associated with IQ and other 

### 12
endpoints of cogniti ve function , thus it was unclear whether blood Pb

- associated neurocognitive deficits, 

### 13
poor school performance, and attention problems, in turn, progressed to antisocial and delinquent 

### 14

behavior later in life. 

### 15 16May 2011 5

- 100 DRAFT

– DO NOT CITE OR QUOTE Reference Mean (SD) Blood Pb (μg/dL) Exposure Period Outcome Chen et al. ( 2007 ) 8 Concurrent Behavioral symptoms 8 Concurrent Externalizing problems 8 Concurrent School problems

Nicolescu et al. ( 2010 ) 3.7 (IQR: 2.6) Concurrent Distractibility 3.7 (IQR: 2.6) Concurrent Inattention

Roy et al. ( 2009 ) 11.4 (5.3) Concurrent Anxiety 11.4 (5.3) Concurrent Inattention 11.4 (5.3) Concurrent Hyperactivity

Nicolescu et al. ( 2010 ) 3.7 (IQR: 2.6) Concurrent ADHD score

Roy et al. ( 2009 ) 11.4 (5.3) Concurrent ADH D index

Braun et al. ( 2006 ) a 1.1

- 1.3 (3 rd quintile) Concurrent ADHD Dx 0.8

- 1.0 (2 nd quintile) Concurrent ADHD Dx
1

. 4

- 2 .0 ( 4th quintile) a Concurrent ADHD Dx >2.0 (5 th quintile) a Concurrent ADHD Dx

Froehlich et al. ( 2009 ) >2.0 a Concurrent ADHD Dx, prenatal smoke exposure

Braun et al. ( 2008 ) 0.8

- 1.0 (2 nd quartile) a Concurrent Conduct disorder 1.0

- 1.4 (3 rd quartile) a Concurrent Conduct disorder 1.5

- 10 (4 th quartile) a Concurrent Conduct disorder

Dietrich et al. ( 2001 ) NR Early childhood Delinquent behavior

Wright et al. ( 2008 ) 8.3 (3.8) Prenatal Criminal arrests 8.3 (4.8) Early childhood avg Criminal arrests

Odds ratio (95% CI) Note: Studies are presented in order of increasing severity of behavioral outcome. Odds ratios are standardized to a 1 μg/dL increase in blood Pb level in analyses of blood Pb level as a continuous variable . IQR = Interquartile range, ADHD = attention de ficit hyperactivity disorder, NR = Not reported. a Effect estimate compares children in higher quantiles of of blood Pb level, with children in the lowest blood Pb quantile serving as the reference group

. Figure 5

- 19

. Associations of blood Pb levels with be havioral indices in children.

Table 5

- 10. Additional characteristics and quantitative results for studies presented in Figure 5

- 19.

Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Odds ratio (95% CI)a

Chen et al. ( 2007 ) 780 children participating in TLC trial followed between ages 2

- 7 yr Baltimore, MD; Cincinnati, OH; Newark, NJ; Philadelphia, PA Concurrent mean (range): 8 (0

- 26) Linear regression model adjusted for city, race, sex, language, parental eduction, parental employment, single parent, age at blood Pb measurement, caregiver IQ Behavioral problems Externalizing problems School problems At age 5

- 7 yr assessed using CPRS

- R, BASC

- TRS, and BASC

- PRS 1.02 (0.99, 1.06) 1.04 (1.00, 1.07) 1.03 (1.00, 1.06)

Nicolescu et al. ( 2010 ) 83 children ages 8

- 12 yr tested in 2007 Bucharest and Pantelimon, Romania Concurrent mean (IQR): 3.7 (2.6) Log

- linear regression model adjusted for city, sex, age, computer experience, handedness, eye problems, number of siblings, parental education, prenatal smoking, family psychopathology Distractibility Inattention At age 8

- 12 yr assessed using German version of Teacher’s Conner’s scales 1.35 (0.99, 1.85) 1.14 (0.95, 1.37)

Roy et al. ( 2009 ) 756 children ages 3

- 7 yr tested 2005

- 2006 Chennai, India Concurrent mean (SD): 11.4 (5.3) Linear regression model adjusted for age, sex, hemoglobin, average monthly income, parental education, number of other children, clustering in school and classroom Anxiety Inattention Hyperactivity At age 3

- 7 yr assessed

us ing Conners’ ADHD/DSM

IV Scales and Behavior Rating Inventory of Executive Function 1.19 (1.03, 1.36) 5.31 (1.46, 19.3) 2.39 (0.77, 7.39)May 2011 5

- 101 DRAFT

– DO NOT CITE OR QUOTE Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Odds ratio (95% CI)a

Nicolescu et al. ( 2010 ) 83 children ages 8

- 12 yr tested in 2007 Bucharest and Pantelimon, Romania Concurrent mean (IQR): 3.7 (2.6) Log

- linear regressio n model adjusted for city, sex, age, computer experience, handedness, eye problems, number of siblings, parental education, prenatal smoking, family psychopathology ADHD score At age 8

- 12 yr assessed using German version of Conner’s scales 1.16 (0.98, 1.37 )

Roy et al. ( 2009 ) 756 children ages 3

- 7 yr tested 2005

- 2006 Chennai, India Concurrent mean (SD): 11.4 (5.3) Linear regression model adjusted for age, sex, hemoglobin, average monthly income, parental education, number of other children, clust ering in school and classroom ADHD index At age 3

- 7 yr assessed using Conners’ ADHD/DSM

IV Scales and Behavior Rating Inventory of Executive Function 4.26 (0.90, 20.08)

Braun et al. ( 2006 ) 4,704 children ages 4

- 15 yr U.S. NHANES 1999

- 2002 Concurrent 3 rd 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 μ g/dL vs. blood Pb <0.8 μ g/dL b 2.1 (0.7, 6.8), blood Pb 1.1

- 1.3 μ g/dL vs. blood Pb <0.8 μ g/dL b 2.7 (0.9, 8.4), blood Pb 1.4

- 2.0 μ g/dL vs. blood Pb <0.8 μ g/dL b 4.1 (1.2, 14.0), blood Pb > 2.0 μ g/dL vs. blood Pb <0.8 μ g/dL b

Froehlich et al. ( 2007 ) 2,588 children, ages 8

- 15 yr U.S. NHANES 2001

- 2004 2nd quartile: 0.9

- 1.3 Logistic regression model adjusted for current household ETS 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 μ g/dL plus prenatal ETS exposure vs. blood Pb level <0.8 μ g/dL and no prenatal ETS exposure b

Braun et al. ( 2008 ) Children ages 8

- 15 yr U.S. NHANES 2001

- 2004 2 nd 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 at age 8

- 15 yr 7.24 (1.06, 49.47), blood Pb level 0.8

- 1.0 μ g/dL vs. blood Pb <0.8 μ g/dL b 12.37 (2.37, 64.56), blood Pb level 1.1

- 1.4 μ g/dL vs. blood Pb <0.8 μ g/dL b 8.64 (1.87, 40.04), blood Pb level 1.5

- 10 μ g/dL vs. blood Pb <0.8 μ g/dL b

Dietrich et al. ( 2001 ) 195 children followed from birth (1979

- 1985) to age 15

- 17 yr Cincinnati, OH Early childhood (0

- 6 yr avg): NR Linear regression model adjusted for HOME score, parental IQ, current SES Delinquent behavior assessed at ages 15

- 17 yr using the Self

- Report of Delinquent Beha vior 1.21 (1.08, 1.37)

Wright et al. ( 2008 ) 250 adults followed from birth (1979

- 1985) to age 19

- 24 yr Cincinnati, OH Early childhood mean (SD): 8.3 (4.8) Negative binomial regression models adjusted for maternal IQ, sec, SES, maternal education Criminal arrests at ages 20

- 23 yr 1.05 (1.00, 1.09)

a Effect estimates are standardized to a 1 μg/dL increase in blood Pb level.
b Odds in higher quantile of blood Pb level compared to that in lowest quantile of blood Pb level

.

B urns et al. ( 1999 ) and Silva et al. ( 1988 ) aimed t o characterize the direct association of blood Pb 

### 1

level with behavior by adjusting for child IQ in their models. They found positive associatio ns, suggesting 

### 2

that blood Pb level may have an i ndependent effect on behavior. However, because a decrement in IQ 

### 3
may on the causal pathway to behavioral problems, including both IQ and behavioral problems may result 

### 4
in an underestimate of the effect on behavior. Hence, to account for the relationship between IQ and 

### 5
behavior, Chen an d colleagues ( 2007 ) used structural equation modeling (specifically path analysis) to 

### 6

estimate the direct effects of blood Pb level on behavioral problems as well as indirect effects mediated 

### 7May 2011 5

- 102 DRAFT

– DO NOT CITE OR QUOTE through child ’ s IQ (assessed u sing the WPPSI

- R at age 5 years and the WISC

- III) at age 7 years). Among 

### 1

5

- and 7

- year

- old participants from the TLC trial (described in Section 5.3.2.1), c onduct problems were 

### 2
assessed using the Conners ’ Parent Rating Scale

- Revised, and other behaviors we re assessed with the 

### 3
Behavior Assessment System for Children

- T eacher R ating S cale ( BASC

- TRS) and Behavior Assessment 

### 4
System for Children

- P arent R atings S cale ( BASC

- PRS)

. In models that adjusted for sex , race, clinical 

### 5
site, language (English or Spanish), p arent ’ s education, parent ’ s employment, age at blood Pb testing, 

### 6
caregiver ’ s IQ, and treatment group, peak blood Pb levels at age 2 years were not associated with conduct 

### 7
problems at age 5 years or any of the BASC scores at age 7 years

. Co ncurrent blood Pb levels had 

### 8
statistically significant direct effects on behavioral symptoms index, externalizing problems and school 

### 9
problems as assessed by BASC

- TRS and externalizing problems (outbursts of behavior) as assessed by 

### 10

BASC

- PRS (Figures 5

- 20 and 5

- 21). 

### 11

Chen e t al. ( 2007 ) also found significant indirect effects (mediated through child ’ s IQ) of blood Pb 

### 12
levels at age 7 years with all measurements except the BASC

- TRS externalizing problems and BASC



### 13
PRS internalizing problems (rep ressing problems)

. However, because blood Pb level was estimated to 

### 14
have direct effects on several behavioral endpoints and, in for some endpoints, a larger magnitude of 

### 15
direct effect, the authors inferred IQ to only partially mediate associations between blood Pb levels and 

### 16
behavioral outcomes (Figures 5

- 20 and 5

- 21 ). The s tudy also had limitations, including the lack of 

### 17
adjustment for HOME score and lack of information on other co varying family and neighborhood 

### 18
characteristics that may be relevant (i.e., social stressors). Also

, these findings may not be generalizable to 

### 19
the general population given that the children in the study population had been referred for chelation 

### 20
therapy at enrollment because of high blood levels . In this study, only concurrent bl ood Pb levels were 

### 21
examined, and it is uncertain whether the observed associations were due to the residual effect of high 

### 22
blood Pb levels (20

- 44 μg/dL) four years earlier. 

### 23May 2011 5

- 103 DRAFT

– DO NOT CITE OR QUOTE Source: Used with permission from the American Academy of Pediatrics, Chen et al. ( 2007 )

Note: V alues represent change in behavioral score per 10 μg/dL increase in blood Pb level. Figure 5

- 20. Estimates and 95% CI of direct and indirect effects of concurrent blood Pb concentrations at age 7 on BASC

- TRS

.

Source: Used with permission from the American Academy of Pediatrics, Chen Chen et al. ( 2007 )

Note: V alues represent change in behavioral score per 10 μg/dL increase in blood Pb level. Figure 5

- 21. Estimates and 95% CI of direct and indirect effects of concurrent blood Pb conc entrations at age 7 on BASC

- PRS.

The 2006 Pb AQCD summarized indirect evidence for associations of blood Pb level with 

### 1
behavioral features of ADHD including distractibility, poor organization, lack ing persistence in 

### 2
completing tasks, and daydreaming ( Bellinger et al., 1994 ; Fergusson et al., 1993 ; Needleman et al., 1 979 ; 

### 3May 2011 5

- 104 DRAFT

– DO NOT CITE OR QUOTE G. A. Wasserman et al., 2001 ; G. A. Wasserman et al., 1998 )

. Prior studies examining the association of 

### 1

blood Pb level w ith a diagnosis of ADHD were limited by small sample size

, and results were 

### 2

inconclusive ( David et al., 1972 ; Gittleman & Eskenazi, 1983 ) . In addition, many earlier studies of 

### 3

inattention and impulsivity included children with higher blood Pb level s than those observed in 

### 4
contemporary children

. 

### 5
A recent analysis of data from NHANES (1 999

- 2002) found a positive relationship between blood 

### 6

Pb level and ADHD ( parent

- report of a diagnosis of ADHD or use of stimulant medication ) ( Braun et al., 

### 7

2006 ) . This analysis included 4,704 children aged 4

- 15 years

. These authors reported a monotonic 

### 8

increase in odds of ADHD from the lowe st to highest quintile of blood Pb level (Figure 5

- 22 ) . C hildren i n 

### 9
the fifth quintile of blood Pb level (>2.0 μg/dL ) had the highest odds of A DHD compared with those in 

### 10
the lowest quintile (<0.8 μg/dL ) ( OR : 4 .1 [95% CI: 1.2

, 14 ] ) . Children in the other three higher quintiles 

### 11
of blood Pb level also had increased odds of ADHD relative to the reference group; however, the 

### 12
associations were not statistically significant (Tables 5

- 19 and 5

- 10). 

### 13
In the same NHANES dataset, however, restricted to children ages 8

- 15 years, Froehlich and 

### 14
colleagues ( 2009 ) demonstrated the joint effects of prenat al tobacco smoke (m aternal report) and blood 

### 15

Pb levels. They found independent effects on ADHD for prenatal tobacco smoke exposure (OR : 2.4 [ 95% 

### 16

CI : 1.5 , 3.7]) and concurrent blood Pb levels (OR : 2.3 [ 95% CI : 1.5

, 3.8 ] ) in children with blood Pb levels 

### 17
>1.3 μg/dL compared wi th children with blood Pb levels < 0.8 μg/dL. A statistically significant 

### 18

interaction also was found. Compared to children in the lowest tertile of blood Pb levels with no exposure 

### 19
to prenatal tobacco smoke, children in the highest tertile of blood Pb leve l with exposure to prenatal 

### 20

tob acco smoke had the greatest odds of ADHD (OR : 8.1 [ 95% CI : 3.5 , 18.7]). 

### 21

Source: Braun et al. ( 2006 )

Note: Adju sted for child ’ s age, gender, race/ethnicity, preschool attendance, serum ferritin, prental tobacco smoke exposure, smoker in the household, and insurance status (p for trend = 0.012). Figure 5

- 22. Adjusted odds ratio for ADHD among U

. S

. children (ages 4

- 15 years) from NHANES 1999

- 2002 by quintile of blood Pb level

.May 2011 5

- 105 DRAFT

– DO NOT CITE OR QUOTE Pb has long been known to impact dopaminergic neurons. Pb will inhibit depolarization

- evoked 

### 1
neurotransmitter release and stimulate spontaneous neurotransmitter release, including effects spec ific to 

### 2
dopaminergic neurons (Section 5.3.6.9). This background would make dopamine related genes strong 

### 3

candidates as effect modifiers of Pb

- associated neurodevelopmental effects . There are several known 

### 4
functional variants in dopamine related genes, incl uding the dopamine 2 receptor, dopamine 4 receptor 

### 5
(DRD4), and the dopamine transporter. The DRD4 gene has a 48 base pair long repeat in exon 3. The 

### 6
longer repeat alleles (DRD4.7) appear to have less binding affinity for dopamine and have been 

### 7

associated w ith ADHD. However, DRD4.7 also has been associated with sustained attention, response 

### 8
inhibition, and quicker response time. Thus, it is not clear whether DRD4.7 would increase or decrea se 

### 9
susceptibility to Pb

- associated ADHD . Froehlich et al. ( 2007 ) studied 174 children from the Rochester 

### 10
prospective birth cohort and measured DRD4 genotype, blood Pb level , and several functional domains 

### 11
using th e Cambridge Neuropsychological Testing Automated Battery . Increasing blood Pb levels were 

### 12
associated with poorer rule learning and reversal, spatial span, and planning. These negative associations 

### 13
were larger in magnitude among boys and those lacking DRD4. 7. 

### 14
In a population of children in a similar age range (8

- 11 years) and similar blood Pb levels (mean 

### 15
1.9 μg/dL) as in the NHANES studies, Cho et al. ( 2010 ) found a statistically s ignificant relationship of 

### 16

blood Pb levels with parent

- and teacher

- rated ADHD sympto ms ( i.e., inattentiveness, hyperactivity, and 

### 17
total score)

. 

### 18

Nicolescu et al. ( 2010 ) examined the relationship between blood Pb level and ADHD

- related 

### 19
behaviors among 83 children, ages 8

- 12 years, living near a metal

- processing plant in Romania . T hese 

### 20
authors examined associations of Pb, mer cury, and aluminum biomarkers with performance on four 

### 21
different attention tasks on the computerized German Kinder

- KITAP as well as behavior ratings using the 

### 22
parent and teacher report s on the Connors scale

. Only con current blood Pb levels, and not other m et als, 

### 23
were consistently associated with increased odds for ADHD

- related behaviors (OR: 1.16 [95% CI: 0.98, 

### 24
1.37] per 1 μg/dL increase in blood Pb level . Notably, blood Pb levels ranged from 1.1 to 14.3 μg/dL, and 

### 25
when analyses were repeated taking out the 5 children with blood Pb levels at or above 10 μg/dL, 

### 26

association s with both questionnaire

- base d ADHD ratings and KITAP

- performance remained statistically 

### 27
significant. 

### 28
Roy and colleagues ( 2009 ) examined associations between blood Pb level and a range of 

### 29
behavioral problems in 756 children, age s 3

- 7 year s, in Chennai, India . In this population, m ean (SD) 

### 30

blood Pb level was 11.4 ( 5.3 ) μg/dL

. Anxiety, social problems, inattention, hyperactivity, and ADHD as 

### 31

well as executive function were assessed based on the teacher’s report of the Conners ’ Teac her Rating 

### 32

Scales

- 39, Conners ’ ADHD/Diagnostic and Statistical Manual for Mental Disorders, 4th Edition Scales, 

### 33
and the Behavior Rating Inventory of Executive Function questionnaire . In generalized estimating 

### 34
equations, increasing blood Pb level was associ ated with higher anxiety , social problems, and AD HD 

### 35
index scores (Figure 5

- 19 and Table 5

- 10). 

### 36May 2011 5

- 106 DRAFT

– DO NOT CITE OR QUOTE Recent studies add to the collective body of evidence demonstrating associations between blood Pb 

### 1

level and a variety of conduct problems including conduct disor der ( Braun et al., 2008 ) , oppositional 

### 2

defiant disorder ( Nigg et al., 2008 ) and more serious behaviors such as criminal behavior ( J. P. Wright et 

### 3
al., 2008 ) . These findings were corroborated in a recent meta

- analysis on Pb an d conduct problems 

### 4
( Marcus et al., 2010 ) that included 19 studies with a total of 8,561 children and adolescents (mean ages 

### 5
ranging from 3.5 years to 18.4 years). An overall medium effect size was estimated (r across studies was 

### 6
0.19, p<0.001)

. Moreover, the meta

- analysis demonstrated a consisten t relationship between increasing 

### 7

blood Pb levels and conduct problems despite considerable heterogeneity across studies (e.g., ways in 

### 8
which conduct problems were defined and assessed, participant ages, participant blood Pb levels) . In the 

### 9

meta

- analysis, covariates such as SES, birth weight , parental IQ, and family environment did not 

### 10

attenu ate the relation s hip between blood Pb level and conduct problems

. Interestingly, a larger magnitude 

### 11

of effect w as estimated for hair Pb levels compared with bone or blood Pb levels. Although the authors 

### 12
suggested that hair Pb may be a better indicator of cumulative Pb expo sure compared to bone Pb levels, 

### 13
due to the high turnover of bone in throughout childhood and into adolescence, an empirical basis for 

### 14
interpreting hair Pb measurements in terms of body burden or exposure has not been firmly established 

### 15
(Section 4.3.4.2). 

### 16
In addition to examining ADHD in the NHANES population, Braun and colleagues ( 2008 ) also 

### 17

examined conduct disorder defined using DSM

- IV criteria. T he prevalence of conduct disorder was 

### 18
higher among male s, older children ( 13

- 15 years

, relative to those less than 13 years), and children with 

### 19
higher blood Pb levels. In analyses adjusted fo r child ’ s age, gender race/ ethnicity, SES, mother ’ s age at 

### 20

child ’ s birth, prenatal tobacco smoke exposure (based on maternal report), and child ’ s cotinine levels

, 

### 21
children in the highest quartile of blood Pb level (> 1.5 μg/dL ) had increased odds of conduct disorder 

### 22
relative to children in the lowest quartile (0.2

- 0.7 μg/dL ) ( OR : 8. 7 [ 95% CI : 1.9, 40 ] )

. Poisson regression 

### 23

models showed that compared with children in the lowest quartile of blood Pb level, children in the 

### 24
highest quartile had 1.73 (95% CI : 1.2 , 2.4) times as many conduct disorder symptoms

. 

### 25
In their longitudinal study of children in the U

. K

. , Chandramouli et al. ( 2009 ) found associations of 

### 26

blood Pb level at age 30 months not only with hyperreactivity but also with antisocial behavior at ages 7 

### 27
and 8 years. Child behavior was assessed using three methods: parent

- and teacher

- report on the Strengths 

### 28
and Difficulties Questionnaire at age 7 years

, the Develop ment and Well

- being Assessment at age 8 years, 

### 29

and the Anti

- social Behaviour Interview at age 8 years. Attention was measured using the Test of 

### 30
Everyday Attention for Children at age 8 years . B lood Pb levels showed statistically significant positive 

### 31

associ ation with antisocial behavior. Similar to Chen et al. ( 2007 ) and Burns et al. ( 1999 )

, increasing 

### 32

blood Pb level was assoc iated with antisocial behavior, independent ly of IQ. 

### 33
Wright et al. ( 2008 ) recently examined the relationship between prenatal and postnatal blood Pb 

### 34

levels and arrests for criminal offenses at ages 19

- 24 years in the CLS. In this birth cohort

, prenatal and 

### 35
postnatal blood Pb levels p reviously have been reported to be associated with self

- and parent

- reported 

### 36May 2011 5

- 107 DRAFT

– DO NOT CITE OR QUOTE delinquent and social acts at ages 16

- 17 ( Dietrich et al., 2001 ) . Data on criminal arrests for participants 

### 1
and their mothers were obtained from a computer search of Hamilton County, Ohio criminal justice 

### 2

records

. They also examined the absolute change in arrest rates between participants with higher levels of 

### 3
blood Pb compared to those with lower blood Pb leve ls defining attributable risk as the average difference 

### 4
in annual arrest rates between participants at the 95th percentile of blood Pb and those at the 5th 

### 5
percentile. Mean blood Pb concentrations were 8.3 μg/dL (range 1 to 26) for the prenatal period (mat ernal 

### 6
blood) , 13.4 μg/dL (range 4 to 37) for the average between birth and age 6 years , and 8.3 μg/dL (range 2 

### 7

to 33) at age 6 yr

. In models that adjusted for maternal IQ, sex, SES score, and maternal education , t he 

### 8
relative risks ( RRs ) for total arrests p er 1 μg/dL increment in blood Pb level were 1.07 ( 95% CI: 1.01, 

### 9

1.13 ) for prenatal blood Pb level, 1.01 (95% CI : 0.97, 1.05 ) for average childhood blood Pb level, and 

### 10

1.05 (95% CI : 1.01, 1.09 ) for blood Pb level at age 6 years. Relative risks for violent c riminal arrests were 

### 11
also larger for prenatal blood Pb levels and age 6

- year blood Pb levels. The RRs for arrests involving 

### 12

violent crimes per 1 μg/dl increment in blood Pb level were 1.06 (95% CI : 0.97, 1.15 ) for prenatal blood 

### 13

Pb level, 1.05 (95% CI : 1.0 1, 1 .10 ) for average childhood blood Pb level, and 1.08 (95% CI : 1.03, 1.14 ) 

### 14

for blood Pb level at age 6 years . Although i nteractions terms of blood Pb by sex were not statistically 

### 15
significant, the attributable risk for males was considerably higher for males ( 0.85 arrests/year [ 95% CI : 

### 16

0.48

, 1.47 ] ) than for females (0.18 [ 95% CI : 0.09

, 0.33 ] ) . Similar to findings from Dietrich et al. ( 2001 )

, 

### 17
prenatal and early chil dhood blood Pb levels were associated positively with risk of criminial behavior in 

### 18
early adulthood. Results from the two studies combined suggest that in addition to the prenatal period, 

### 19
early childhood blood Pb levels may also predict criminal behavior i n adulthood. However, it is important 

### 20
to note that in these CLS studies, concurrent blood Pb levels were not analyzed. 

### 21

5.3.3.2. Epidemiologic Studies of Behavior, Mood, and Psychiatric Effects in
Adults

Effects of blood Pb levels on emotional regulation in adults h ave received far less attention than 

### 22

that in children and cognitive function in adults. Nonetheless, evaluation of mood states is an integral part 

### 23
of the World Health Organization ’ s (WHO) neurocognitive test battery and, indeed, it has been suggested 

### 24

that the assessment of mood with the Profile Of Mood States may be particularly sensitive to toxicant 

### 25

exposures ( 1987 )

. With respect to Pb exposure , several early studies of Pb

- exposed worke rs (mean blood 

### 26
Pb levels ranging from 23.5 to 64.5 μg/dL) found higher prevalence of symptoms of mood disorders and 

### 27
anxiety among Pb

- exposed workers than unexposed controls (mean blood Pb levels ranging from 15

- 38 

### 28
μg/dL) ( Baker et al., 1984 ; Baker et al., 1985 ; Lilis et al., 1977 ; Maizlish et al., 1995 ; Parkinson et al., 

### 29

1986 ; B. S. Schwartz et al., 2005 )

. In one of the few previous studies of adults without occupational 

### 30
exposure to Pb and more relevant to blood and bone Pb levels measured currently in the U.S. among 

### 31
adults without occupational exposures, a n association was observed between both blood (mean: 6.3 μg/dL 

### 32May 2011 5

- 108 DRAFT

– DO NOT CITE OR QUOTE [SD: 4.16] and bone (tibia mean: 2 1.9 μg/g [SD: 13.5]) Pb levels and depression and anxiety symptoms 

### 1
( Brief Symptom Inventory , BSI) among men in the NAS ( Rhodes et al., 2003 )

. 

### 2
More recently, subsequent assessme nts of the N AS men have provided understanding of effect 

### 3

modificat ion of the associations of blood, patella, and tibia Pb levels with psychiatric symptom 

### 4
dimensions by ALAD genotype ( Rajan et al., 2007 ) . In addition to corroborating associations of Pb with 

### 5
BSI symptoms reorted by Rhodes et al. ( 2003 ) , Rajan et al. ( 2007 ) found that o f eight symptom 

### 6
dimensions considered, associati ons of tibia bone Pb levels with phobic anxiety

, positive symptom total

, 

### 7
and anxiety scale were modified by ALAD genotype, with interaction terms at taining statistical 

### 8
significance for phobic anxiety and positive symptom total. For all psychiatric symptoms, the association 

### 9

with tibia Pb was worse among ALAD 1

- 1 carriers, which was the opposite genotype observed to have 

### 10
larger Pb

- associated decrements in cognitive performance (Section 5.3.2.4). 

### 11
A study of 1,987 adults age 20

- 39 years participating in NHANES 1999

- 2004 was the largest study 

### 12
of psychiatric disorders and the study with the lowest blood Pb levels (mean 1.61 μg/dL [SD: 1.72]) 

### 13
( Bouchard et al., 2009 ) . Investigators examined cases of major depressive disorder (MDD), panic disorder 

### 14
and generalized anxiety disorder (GAD) as determined using the WHO Composite International 

### 15
Diagnostic Interview , which follows criteria defined in DSM. Compared with those in the lowest quintile 

### 16 of blood Pb level (<0.7 μg/dL), adults in the highest quintile (≥ 2.11 μg/dL) had increased odds of MDD 

### 17
( OR: 2.32 [95% CI: 1.13, 4.75]) and panic disorder ( OR: 4.94 [95% CI: 1.32, 18.48]) . Odds ratios were 

### 18
even larger in analyses excluding current smokers. While those in higher quintiles of blood Pb level (>0.7 

### 19
μg/dL) had increased odds to GAD, the associations were not statistically significant. A lthough studies in 

### 20

adults without occupation Pb exposure are sparse, consistent with studies of occupationally

- exposed 

### 21
adults and experimental evidence, they demonstrate associations of blood (as low as 2.11 μg/dL) and 

### 22
bone Pb level (population mean tibia Pb level 22 μg/g) with psychiatric outcomes. 

### 23

5.3.3.3. Toxicological Studies of Neurobehavioral Outcomes

Pb is a known risk factor for neurobehavioral changes with preferentially targeted sites including 

### 24
the prefrontal cerebral cortex, cerebellum, and hippocampus; affected functions include cognitio n, 

### 25
execution of motor skills, and memory/behavior, respectively. As discussed in earlier AQCDs, young 

### 26
animals are especially susceptible to the effects of Pb due to differences in structure of the nervous system 

### 27
and to the ongoing development with greater Pb absorption and retention. Pb exposure has been 

### 28
documented to induce neurobehavioral changes in exposed animals including effects on learning, social 

### 29
behavior, memory, attention, motor function, locomotor ability and vocalization. At the cellular level, Pb 

### 30
impairs axon and dendritic development and contributes to neurochemical changes in proteins, 

### 31
membranes, redox/antioxidant balance, and neurotransmission through a multitude of mechanisms, many 

### 32
of which involve the ability of Pb to mimic calcium. Very ea rly research on neurobehavioral endpoints 

### 33May 2011 5

- 109 DRAFT

– DO NOT CITE OR QUOTE failed to capture the disposition of Pb and its resulting body burden or blood Pb and is thus difficult to use 

### 1

in risk assessment. 

### 2
Since then, the 1986 AQCD reported the literature on rodent and nonhuman primate P b

- induced 

### 3
aberrant operant conditioning tasks in rodents and non

- human primates (blood Pb 11 μg/dL to 15 μg/dL) 

### 4
with other studies yielding hyperactive or inappropriate Pb

- mediated responses ( U.S. EPA, 1986 ) possibly 

### 5
of hippocampal origin with a curvilinear response, decreasing at higher doses possibly due to impairment 

### 6
of motor function at the high doses ( Crofton et al., 1980 ; Ma et al., 1999 ) . Pb exposure in lab animals 

### 7
contributed to distractibility, reduced adaption capacity to changes in behavior, impaired ability to inhibit 

### 8
inappropriate responses and p erservation ( U.S. EPA, 2006 ) . Pb is known to impair learning (blood Pb 11 

### 9
μg/dL) measure d using Fixed Interval tasks (FI) defined as scheduled reinforcement delivered after a 

### 10
fixed period since the last reward, with premature responses not rewarded. The 2006 AQCD found FI 

### 11
response rates (blood Pb 58 to 94 μg/dL) were sensitive to Pb exposure, which was primarily accounted 

### 12
for by decreased interresponse times. Inter

- response rates and overall run rate are the two subcomponents 

### 13
of FI response rate. S patial and non

- spatial discrimination reversal or reversal of a previously learned 

### 14
habit is signi ficantly affected after developmental Pb exposure and is exacer bated with distracting stimuli; 

### 15
discrimination reversal has been shown to be especially sensitive to Pb exposure. Repeat

- acquisition 

### 16
testing revealed that these deficits are likely not due to s ensory or motor impairment at this dose. The 

### 17
results from different studies testing the effect of Pb on memory are mixed with impaired memory shown 

### 18
at blood Pb level of 10 μg/dL and improved memory in other studies. Low dose Pb does not appear to 

### 19

affect sh ort

- term memory. Memory tests may give incorrect results when opportunities exist for impaired 

### 20

attention to contribute to test results ( U.S. EPA, 2006 ) . Together, the data from the 2006 AQCD showed 

### 21
that social behavior and learning in rodents and nonhuman primates is significantly affected by Pb 

### 22
exposure (blood Pb 15

- 40 μg/dL). 

### 23
In the new literature, gestationally

- Pb exposed (GLE) male mice (low and high dose Pb, 10 μg/dL 

### 24
and 42 μg/dL blood Pb at PND10) were significantly less active than control mice and low dose GLE 

### 25
mice were significantly less active than high dose GLE mice, demonstrating a non

- linearity of GLE dose 

### 26
responsiveness ( Leasure et al., 2008 ) . A similar Pb dose response non

- linearity (baseline corticosterone) 

### 27
was seen in male mice exposed post

- weaning to Pb ( Virgolini et al., 2005 ) . Activity level of GLE female 

### 28

mice versus control was unaffected ( Leasure et al., 2008 ) . Amphetamine induced motor activity was 

### 29
monitored in male and female GLE mice at 1 year of age. Amphetamine

- induced activity of male low and 

### 30
high dose GLE offspring was significantly elevated versus control; GLE females had no chan ge in 

### 31
sensitivity to amphetamine

- induced motor activity ( Leasure et al., 2008 )

. 

### 32
Rotarod performance measures endurance, balance and coordination in mice. GLE male mice had 

### 33
significantly shorter mean latencies to fall from the rotarod compared with controls; females were 

### 34
unaffected. Further, low dose GLE male mice had significantly poorer rotarod performance than high 

### 35
dose GLE male mice (fell off quicker, 10 μg/dL and 42 μg/dL blood Pb at PND10), showing non

- linearity 

### 36May 2011 5

- 110 DRAFT

– DO NOT CITE OR QUOTE of dose

- responsiveness to G LE ( Leasure et al., 2008 ) . Other rotarod experiments at higher doses of Pb 

### 1
exposure and at various speeds of rotarod rotation yielded mixed results ( Grant et al., 1980 ; Kishi et al., 

### 2

1983 ; Overmann, 1977 )

. 

### 3
Herring gull chicks exposed to a single IP bolus dos e (100 mg/kg Pb acetate) of Pb on post

- natal 

### 4
day two at a dose created to be similar to that which wild herring gulls are exposed to in the wild were 

### 5
found to have neurobehavioral deficits, including learning deficits. Pb

- exposed chicks displayed multiple 

### 6
deficits related to impaired survival skills including decreased time spent begging the parent for food, 

### 7
decreased accuracy at pecking for food in the parent bird’s mouth, decreased time spent in the shade 

### 8
(behavioral thermoregulation), decreased learning in food location testing, decreased recognition of 

### 9
familiar individuals (caretaker or sibling), and slower development of motor skills (treadmill test) versus 

### 10

control birds ( Burger & Gochfeld, 2005 ) . The impaired thermoregulation with Pb exposure agrees with 

### 11

earlie r work in rat pups who also showed impaired thermoregulatory behavior, i.e., impaired ultrasonic 

### 12

vocalization ( Davis, 1982 ) . These studies in herring gull chicks demonst rate that a single early life 

### 13
exposure to Pb can induce neurobehavioral deficits that affect survival skills. 

### 14

5.3.3.4. Toxicological Studies of Mood Alterations

Neurotoxicological studies often focus on motor, sensory, behavioral or cognitive outcomes and 

### 15
often fail to evaluate psychological pathologies. Recent epidemiologic studies have reported that prenatal 

### 16
or early life blood Pb levels or ALAD changes, a bioma rker of Pb exposure, may be a risk factor for 

### 17
development of mood disorders in adulthood (i.e. schizophrenia, major depression or panic disorders) 

### 18
( Bouchard et al., 2009 ; Opler et al., 2004 ) . With this in mind, animal studies of Pb exposure during 

### 19

pregna ncy and lactation and outcomes in offspring look to address the role of developmental Pb exposure 

### 20

on emotional state and mood disorder

- like behavior in adult offspring. Wistar rats exposed to 10mg of Pb 

### 21
acetate daily by gavage during pregnancy (G) or pregn ancy plus lactation (G+L) produced pups that were 

### 22
then tested in the open field test or the forced swimming test also known as Porsolt’s test. Blood Pb levels 

### 23
in the pups at PND70 was 5

- 7 μg/dL. The open field test can measure emotion and exploratory behav ior; 

### 24
Pb (G+L) treated male rats had increased emotionality with the open field test. Pb exposed (G+L) female 

### 25
offspring had a significant increased depressive phenotype in the forced swim test ( de Souza Lisboa et al., 

### 26

2005 ) . It is interesting to note that this is one of many Pb

- induced changes that seem to be sex

- specific. 

### 27
Depression m ay seem initially like an unexpected comorbidity for immune inflammatory 

### 28
dysfunction, but many forms of depression are li n ked with the same cytokine imbalances that occur with 

### 29

Pb

- induced innate immune dysfunction ( Maes ; T. W. W. Pace & Miller, 2009 )

. Some researcher use 

### 30
sickness behavior and its associated malaise as a model for depression. Examples from animal models 

### 31
include the study by Dyatlov and Lawrence ( 2002 ) with Pb exposure in mice

. In thi s study, sickness 

### 32
behavior, which is due to an interaction of the immune system and the CNS in Pb

- exposed mice, was 

### 33May 2011 5

- 111 DRAFT

– DO NOT CITE OR QUOTE potentiated by Pb

- exposure ( blood Pb level 17 μg/dL ) and this correlated with depletions in specific 

### 1
thymic T

- cell populations. Pb

- exposure also potentiated the infection

- dependent elevation in IL

- 1 β , a 

### 2
cytokine which has been shown to inhibit hippocampal glutamate release in young and not aged animals

. 

### 3
Sickness behavior was induced with Listeria monocytogenes infection; Pb exposure was from b irth 

### 4
through lactation and continued for a brief period after weaning until the experiment was terminated. 

### 5
Sickness behavior is characterized by overall malaise, decreased food intake, immobility and changes in 

### 6
core body temperature. Pb potentiated the sic kness behavior in exposed animals. 

### 7

Figure 5

- 23. Animal toxicology evidence of possible Pb

- dependent contributors to the development of mood disorders.



### Schizophrenia is associated with a shortened lifespan in humans as reflected by 8

increased standardized mortality ratio ( McGrath et al., 2008 ) . An environmental origin of 9 schizophrenia was proposed years ago ( Tsuang, 2000 ) but the specific link between prenatal 10 Pb exposure, using ALAD as a biomarker, and schizophrenia is just beginning to appear in 11

the literature and has been described in two publications ( Opler et al., 2004 ; Opler et al., 12

2008 ) . Because of this, the animal toxicology literature is beginning to explore the 13 mechanisms that may contribute to schizophrenia d evelopment and has proposed two 14 explanations. These are Pb

- induced NMDA receptor (NMDAR) hypofunction and Pb

15 induced decreases in hippocampal neurogenesis (Figures 5

- 23, 5

- 24, and 5

- 25). Pb may bind 16 a divalent cation site in the NMDAR and allosterically in hibit glycine binding 17 ( Hashemzadeh

- Gargari & Guilarte, 1999 ) ; human studies of patients with schizophrenia 18

have shown aberrations at this site ( Coyle & Tsai, 2004 ) . These findings are consistent with 19 the glutamatergic hypothesis of schizophrenia which shows that NMDAR noncompetitive 20 antagonist use in patients with schizophrenia exacerbates their psychotic symptoms and that 21 Epidemilogic

associations 

## Immune

Elevated Cytokines Sickness Behavior NMDA Receptor

Hypofunction Decreased hippocampal neurogenesis 

## Structural Neuro

transmitters

Behavioral

Humans Rodents

More emotional DepressionMay 2011 5

- 112 DRAFT

– DO NOT CITE OR QUOTE 

### administration of antagonist to non

- psychotic subjects can induce a schizophrenic 1 phenotype. The second mechanistic hypothesis for Pb

- associated schizophrenia induction is 2 decreased hippocampal degenerate gyrus (DG) neurogenesi s, which is seen in patients with 3 schizophrenia ( Kempermann et al., 2008 ; Reif et al., 2006 ) , in animal models of 4

schi zophrenia {M, 2007, 670321} and in animal models with developmental Pb exposure 5 ( Jaako

- Movits et al., 2005 ; Verina et al., 2007 ) (Figures 5

- 23, 5

- 24, and 5

- 25). Animal 6 models of schizophrenia (i.e., phencyclidine administration) show decreased hippocampal 7 DG neurogenesis that can be reversed by treatment with clozapine, which is often used in 8 schizop hrenia ( Maeda et al., 2007 ) . These DG pathways ar e also NMDAR

- dependent. 9 Studies cited in this section are further detailed in other sections of the ISA. Thus, a Pb

10 dependent contribution to mood disorders exists in the toxicology literature with support 11 from behavioral, neurochemical and ultrastructural data. 12

Figure 5

- 24. Schematic representation of the contribution of Pb exposure to the development of a phenotype consistent with schizophrenia.

As recently reviewed by Wright ( 2009 )

, social stress and physical environmental toxins impact 

### 13
overlapping biological processes which determine adaptive plasticity in early neurodevelopment

. 

### 14
Development of CNS organization into functional neuronal and synaptic networks can be determined by 

### 15

envi ronmental signals which modify neuronalgenesis, synaptic formation and synaptic pruning ( LeDoux, 

### 16

2003 )

. Environmental factors can promote or disrupt this process depending on whether they are positive 

### 17
(social supports, good nutrition, etc.) or negative (psychosocial stress, chemical toxicants, malnutrition, 

### 18

trauma, e tc.)

. While plasticity allows recovery from short

- term toxic exposures, the neural mechanisms 

### 19 

### 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 AntagonismMay 2011 5

- 113 DRAFT

– DO NOT CITE OR QUOTE underlying the plasticity of the developing brain exposed to chronic toxic exposures could induce 

### 1
permanent structural or organizational changes via altered neuro nal growth and/or 

### 2
synaptogenesis/pruning. While historically research has focused on how social and physical 

### 3
environmental factors independently affect children ’ s health, evolving theory and methodologies 

### 4
underscore the importance of studying integrated ef fects ( L. D. White et al., 2007 ; R. J. Wright, 2009 )

. 

### 5
Recent studies highlight how social conditions influence susceptibility to future environmental exposures 

### 6
and, w hen contemporaneously exposed, how social

- physical environmental interactions may account for 

### 7
more variance in explaining risk than main effects of either factor alone ( R. J. Wright, 2009 )

. 

### 8

Source: Used with permission from Elsevier Scienc e, Fox et al. ( 2010 ) Figure 5

- 25. Neurogenesis (production of new cells) in the rat dentate gyrus after postnatal Pb exposure. (A) Control ; (B) Pb exposed light micrograph pictures of Brd

- U positive cells ; (C) Counts of Brd

- U positive cells (proliferating cells); and (D) volume of dentate gyrus. *p<0.05 v. control.May 2011 5

- 114 DRAFT

– DO NOT CITE OR QUOTE 

## 5.3.4. Sensory Acuity

5.3.4.1. Epidemiologic Studies of Children

Although not as widely examined as cognitive and behavioral outcomes, several studies 

### 1

demonstrat ed associations of blood Pb level with increased hearing thresholds and decrements in auditory 

### 2
processing in children ( U.S. EPA, 2006 ) . Such evidence has been limited largely to studies described in 

### 3
the 2006 Pb AQCD, including large U.S. studies, including NHANES II ( J. Schwartz & Otto, 1987 ) and 

### 4
the Hispanic Health and Nutrition Examination Survey (HHANES) ( J. Schwartz & Otto, 1991 ) . In these 

### 5
studies, concurrent blood Pb level (median 8 μg/dL) from 6 to 18 μg/dL was associated with a 2

- db loss 

### 6
in hearing and an increase in the percentage (15%) of children with a substandard hearing threshold 

### 7
(2,000 Hz). Blood Pb leve l also was associated with increases in hearing threshold across several 

### 8
frequencies in a population of children in Poland with similar blood Pb levels (median 7.2 μg/dL [range: 

### 9
1.9 to 28]) ( Osman et al., 1999 ) . In the HHANES and Polish studies, associations persisted in analyses 

### 10
restricted to subjects with blood Pb levels below 10 μg/dL. In the CLS, blood Pb level was associated 

### 11
with impaired auditory processing, albeit at higher concentra tions. In this cohort, the mean (SD) lifetime 

### 12
average blood Pb level was 17.4 μg/dL ( 8.8 ), and a 1 μg/dL increase in lifetime average blood Pb was 

### 13

associated with a 0.07

- point (p <0.05) lower score on the Filtered Word test, indicative of incorrect 

### 14

identif ication of filtered or muffled words ( Dietrich et al., 1992 ) . Despite the higher blood Pb levels in the 

### 15
CLS, the observed associations with auditory function were consistent with those with related endpoints, 

### 16
including c ognitive deficits (Section 5.3.2 .1) and be havioral problems (Section 5.3.3 .1)

. 

### 17

5.3.4.2. Epidemiologic Studies of A dults

Rather than evidence for effect on hearing thresholds, among adults, evidence of association 

### 18

between blood Pb levels and auditory function comprised changes in auditory evoked brainstem 

### 19
potentials ( U.S. EPA, 2006 ) . Two studies of hearing thresholds came to somewhat differen t conclusions

. 

### 20

One study examined 183 Pb

- workers with blood Pb levels from 1 to 18 μg/dL and among multiple 

### 21
frequencies examined, found correlations between increasing blood Pb level and increased hearing 

### 22

thr eshold at 4 kHz ( Forst et al., 1997 )

. A sec ond study included 220 Pb

- battery workers with hi gher blood 

### 23

Pb levels (mean : 56.9 μg/dL [SD : 25.3 ] ) ( Wu et al., 2000 )

. Although hearing impairment was associated 

### 24

with a measure of cumulative Pb exposure based on years of work and ambient Pb measurements, no 

### 25

association was found with blood Pb levels at the time of hearing testing in analyses adjusted for age

, 

### 26
gender, and duration of employment . These findings may indicate that in an occupational setting with 

### 27
high Pb exposure , any one blood Pb measure may not be the best biomarker f or cumulative expo sure over 

### 28

the duration of work. 

### 29May 2011 5

- 115 DRAFT

– DO NOT CITE OR QUOTE Studies published since the 2 006 Pb AQCD have produced findings consistent with previous 

### 1

studies. While studies of Pb

- exposed workers continued to dominate, an NAS study provided new 

### 2
evidence for increasing bone Pb levels being associated with hearing loss in adult males without 

### 3

occup ational Pb exposures ( Park et al., 2010 )

. A hospital

- based case

- control study recrui ted workers 

### 4
referred for hearing testing (average hearing thre s holds a bove 25 dB) as cases and workers with normal 

### 5
hearing thresholds who were having occupational health examinations for other reasons as controls 

### 6
( Chuang et al., 2007 ) . The 121 cases had a geometric mean blood Pb level of 10.7 μg/dL

, and the 173 

### 7

controls had a geometric mean blood Pb level of 3.9 μg/dL . In models that adjusted for age, smoking, 

### 8
alcohol consumption, years of noise exposure, as well as Mn, As, and Se levels in blood , blood Pb levels 

### 9
was associated with a statistically significan t higher average hea ring threshold (0.5

- 6 kHz). 

### 10
Similar findings were reported in a s tudy of 259 steel plant workers with no parental history of ear



### 11
related problems, no congenital abnormalities, no occupational organic solvent exposure, and hearing loss 

### 12

difference no more than 15 dB between both ears ( Y.

- H. Hwang et al., 2009 )

. The participants had an 

### 13
average b lood Pb level of 54.3 μg/dL ( SD: 34.6) . A verage noise levels also were measured in work areas 

### 14

and dichotomized at 80dB . In analyses adjusted for age and work area noise (dichotomized at 80dB)

, 

### 15

workers with blood Pb ≥ 7 μg/dL had a statistically significant increased odds (range of OR s : 3 .06 to 

### 16
6.26) of hearing loss at frequencies of 3, 4, 6, and 8 kHz compared to workers with blood Pb levels ≤ 4 

### 17 μg/dL. 

### 18

Park et al. ( 2010 ) analyzed data from 448 men in the NAS with an audiometric hearing test within 

### 19
5 years of bone Pb measurements (all but 5 of these men had audiometric testing before the bone Pb 

### 20

measuremen t) and who did not have unilateral hearing loss . In cross

- sectional analyses adjusted for age, 

### 21
race, education, body mass index, pack

- years of cigarettes, diabetes, hypertension and occupational noise 

### 22
(based on a job

- exposure estimate from occupations), an d presence of a noise notch (indicative of noise



### 23
induced hearing loss) , higher patella bone Pb level was associated with a statistically significant higher 

### 24
hearing thresholds for frequencies greater than 1 kHz . In analyses of pure tone average hearing loss , a 21 

### 25 μg/g increase in patella bone Pb level (interquartil e range ) was associated with an OR of 1.48 (95% CI: 

### 26

1.14

, 1.91) after controlling for all covariates

. Similar, but slightly weaker associations were seen with 

### 27

tibia bone Pb levels. A udiometric data collected from the same men approximatly 20 years earlier 

### 28
(median observations/participant: 5; median follow

- up: 23 years) were used to assess the association 

### 29

between tibia bone Pb levels and the change in hearing thresholds over that time. Increasing tib ia Pb level 

### 30

was associated with faster rate of increase in hearing threshold for frequencies of 1, 2, and 8 kHz and a 

### 31

pure tone average

. For the pure tone average , a 15 μg/g increase in tibia bone Pb level (interquartile 

### 32

range ) was associated with an incre ased in hearing threshold of 0.05 dB per year

. Blood Pb was not 

### 33

examined in this study. Together with those from studies of Pb

- exposed workers, findings from the NAS 

### 34
study primarily indicate that biomarkers of cumulative Pb exposure are associated with increased hearing 

### 35
thresholds in adults. Because only bone Pb levels were examined in the NAS study, further investigation 

### 36May 2011 5

- 116 DRAFT

– DO NOT CITE OR QUOTE is required to characterize potential differences between measures of cumulative and recent Pb exposures 

### 1
in the effects on hearing in adults without occupational exposures. 

### 2

5.3.4.3. Toxicological Studies of Sensory Organ Function

Pb affects multiple aspects of the nervous system including the sensory organs. The 1986 and 2006 

### 3
Pb AQCDs detailed the effects of Pb on the retina, CNS visual processin g areas, and the auditory system 

### 4
and described possible or known mechanisms of action where available. The new research examines 

### 5
effects on these systems at even lower Pb exposures and blood Pb levels. 

### 6

The Effect of Lead on Vision

The selective action of Pb on retinal rod cells and bipolar cells is well documented in earlier 

### 7
AQCDs and research in this area continues to date ( Fox et al., 1997 ; Fox & Sillman, 1979 ) . Pb exposure 

### 8
during perinatal development and adulthood has also been shown to affect the visual cortex ( L. G. Costa 

### 9
& Fox, 1983 ) and subcortical neurons ( Cline et al., 1996 ) . The 1986 AQCD mentioned that neonatal rats 

### 10
exposed to Pb out to PND21, through gestational and lactational exposure, had significant alterations in 

### 11
visual evoked responses and impaired visual acuity; it was hypothesized that a decreased number of 

### 12
cholinergic receptors and alterations in the ratio of inhibito ry to excitatory systems in the cerebrospinal 

### 13
axis may be the underlying mechanisms leading to these retinal changes ( U.S. EPA, 1986 ) . A 1996 

### 14
publication detailing environmentally relevant doses of Pb administered to tadpoles showed that P b 

### 15
inhibited the growth of developing neurons in the subcortical retinotectal pathway, the main efferent from 

### 16

the retina ( Cline et al., 1996 ) . The 2006 AQCD evolved to detail Pb

- induced impairment of retinal 

### 17

function in non

- human primates as well as focusing on mechanisms of action f or specific physiological 

### 18
retinal changes using both in vitro and in vivo evidence, where available. With Pb

- induced retinal effects, 

### 19
decreased maximal ERG amplitude or sensitivity and increased mean ERG latency was linked to 

### 20

increased retinal cGMP both in vitro and in vivo. Delayed dark adaptation and increased response 

### 21
thresholds at scotopic backgrounds were linked to in vivo apoptotic endpoints including rod bipolar cell 

### 22
death, increased Bax (apoptosis protein) translocation, increased cytochrome c relea se (apoptosis trigger), 

### 23
and decreased rhodopsin; in vitro evidence also included retinal apoptosis from the calcium/Pb signal 

### 24
localized to the mitochondrial permeability transition pore. Other endpoints seen in Pb

- induced impaired 

### 25

retinal function included competitive inhibition of cGMP phosphodiesterase (PDE) in vitro and decreased 

### 26
stimulated cGMP PDE in vivo; also decreased retinal Na+/K+ ATPase activity has been reported both in 

### 27
vivo and in vitro. The effects of early life Pb exposure on the retina in mo nkeys was detailed in work by 

### 28

Reuhl et al. ( 1989 ) in the 2006 AQCD. Chronic Pb exposure from birth to age 6 produced 

### 29
cytoarchitectural changes in vis ual projection areas of the brain of rodents; maximum blood Pb level in 

### 30
the low and high dose group reached 20 μg/dL and 220μg/dL, respectively. Liliental et al. ( 1988 ) found 

### 31May 2011 5

- 117 DRAFT

– DO NOT CITE OR QUOTE decreased amplitudes and increased latencies in visual evoked potentials from electroretinograms. Pb 

### 1
affected amplitude under dark conditions (dark adaptation, B waves affected) and latencies under bright 

### 2
conditions; blood Pb levels were 40 and 50 μg/dL in the 350 or 600 ppm Pb groups, respectively. Earlier 

### 3
work in rodents found that a moderate to high postnatal Pb exposure induced ERG subnormality ( Fox & 

### 4
Chu, 1988 ; Fox & Farber, 1988 ; Otto & Fox, 1993 ) . Thus, the historical animal toxicology literature 

### 5
shows multiple effects on vision from Pb exposure (Table 5

- 11, high dose). 

### 6

Table 5

- 11. Summary of toxicological studies of P b on the r etina

.

High Dose Pb Low Dose Pb Gestational (GLE) or Postnatal Pb exposure Persistent subnormal scotopic ERG Delayed Dark Adaptation Decreased thickness of ONL INL (GLE) Dose dependent decreased DA homeostasis (GLE) Increased retinal cell apoptosis (postnatal Pb) No increased rod neurogenesis (GLE) No increased progenitor cell proliferation (GLE) Gestational Pb exposure Persistent scotopic ERG supeanormality Increased thickness of the ONL Dose dependent decreased DA homeostasis Males affe cted, females not affected No increase in retinal apoptosis Increased progenitor cell proliferation Increased neurogenesis of rods Larger size of retina

The current literature for this ISA has more work by the Fox lab showing retinal effects in rodents 

### 7
after human equivalent gestational Pb exposure (GLE, gestationally out to PND10); this is 

### 8
developmentally equivalent to the in utero retinal development per iod in humans. Pb exposure during 

### 9
various developmental windows and at specific doses has been shown to significantly affect 

### 10
electroretinographs (ERG) both in Pb

- exposed humans and rodents ( Fox et al., 2008 ; Rothenberg et al., 

### 11

2002 ) (Table 5

- 11 and Figure 5

- 26). Consis tent with low dose Pb exposure associated ERG 

### 12
supernormality in children ( Rothenberg et al., 2002 ) , Fox et al. ( 2008 ) found low and moderate ose 

### 13
gestational Pb

- exposure (GLE) induced persistent supernormal scotopic ERGs in rodents. Low and 

### 14
moderate GLE also induced increased rod and rod bipolar cell ne urogenesis (proliferation) and increased 

### 15
thickness and cell number of the outer and inner neuroblastic layers of the retina (ONL and INL) ( Fox et 

### 16
al., 2008 ; Giddabasap pa et al., 2011 ) . Rodents with moderate dose GLE (bl ood Pb level 25 μg/dL) had 

### 17

27

- fold increased retinal progenitor cell proliferation ( Giddabasappa et al., 2011 ) . Extension of the in vivo 

### 18
studies to isolated cultured cells showed GLE increased and prolonged proliferation of retinal prog enitor 

### 19

cells ( Giddabasappa et al., 2011 ) . Nitric oxide has been shown to regulate retinal progenitor cell 

### 20
proliferation in chick embryos ( Magalhaes et al., 2006 )

. 

### 21

Because Pb exposure has been shown to impair NOS activity in other organs ( S ection 5.2.4.5 ), 

### 22
these authors postulate that impaired NO production m ay be linked to aberrant retinal cell proliferation 

### 23
( Giddabasappa et al., 2011 ) . GLE did not significantly affect apoptosis during retinal development 

### 24
( Giddabasappa et al., 2011 ) but it did contribute to increased p roliferation of retinal cells. GLE induced 

### 25
decreased DA synthesis and use in a dose

- dependent fashion ( Fox et al., 2008 ) (Figure 5

- 27). 

### 26May 2011 5

- 118 DRAFT

– DO NOT CITE OR QUOTE Note: *p <0.05

Source: Fox et al. ( 2008 ) Figure 5

- 2 6

. Retinal a

- w ave and b

- wave ERG amplitude in GLE adult males.

Note: *p <0.05

Source: Fox et al. ( 2008 ) Figure 5

- 27. Retinal do pamine metabolism in adult control and GLE rats.

Many outcomes in this study showed an inverse U Pb dose

- response curve as is shown with the 

### 1
high dose exposures having vastly different effects from low dose GLE. GLE exposure at high doses 

### 2
produced ERG sub normality, rod cells loss, and decreased rod neurogenesis ( Fox et al., 2008 ) (Table 5

- 

### 3
11). The high dose GLE rodents showed dose

- dependent decreased DA synthesis and use (Figure 5

- 27). 

### 4
These new animal toxicology data confirm the epidemiologic data showing ERG supernorm ality at low 

### 5
dose GLE. They provide further insight into retinal changes showing increased proliferation of Pb

- 

### 6May 2011 5

- 119 DRAFT

– DO NOT CITE OR QUOTE exposed retinal progenitor cells without changes in apoptosis. GLE induced dose

- dependent decrements 

### 1
in retinal DA use and synthesis. 

### 2

Lead

- Induce d Auditory Effects

The 2006 Pb AQCD mentioned auditory effects on non

- human primates who were exposed to Pb 

### 3
throughout gestation and out to age 8

- 9 (blood Pb levels 33

- 56 μg/dL during Pb exposure period). 

### 4
Auditory evoked potentials, which are used as a gen eral test to assess neurological auditory function, 

### 5
revealed Pb related effects that persisted even after Pb exposure had ceased and blood Pb level had 

### 6

returned to baseline levels. In Pb

- exposed animals, half of the pure tone detection thresholds were outs ide 

### 7
of the control range at certain frequencies ( Rice, 1997 ) , which is consistent with data from humans 

### 8
developmentally exposed to Pb. Thus, these authors found that early life Pb exposure impaired auditory 

### 9
function. The cochlear nerve in both developing and mature humans appears to be especially sensitive to 

### 10
the Pb insult. At low to moderate Pb exposures, elevated thresholds and increased latencies are seen in 

### 11
brainstem auditory evoked potentials. There is coherence between the animal and the human literature on 

### 12

the effects of chroni c Pb exposure on auditory function. 

### 13



## 5.3.5. Neurodegenerative Diseases

5.3.5.1. Epidemiologic Studies of Adults

The 2006 Pb AQCD described several studies examining associations of blood and bone Pb levels 

### 14
with neurodegenerative diseases such as Alzheimer ’ s disease and dementia . T wo NAS studies found 

### 15

associations between increasing bone Pb levels and decreasing M MSE scores ( Weisskopf et al., 2004 ; R. 

### 16
O. Wright et al., 2003 )

, which pointed to an association with dementia, given that t he MMSE is widely 

### 17
used as a screening tool for dementia. Overall, studies had s ufficient limitations, and findings were 

### 18

inconclusive ( U.S. EPA, 2006 ) . New studies on dementia are not available to assess further the 

### 19

association of Pb biomarke rs with dementia. Similarly, new studies examining Alzheimer’s disease are 

### 20
not available, and as in 2006, the evidence is inconclusive regarding the association between Pb 

### 21

biomarkers and Alzheimer ’ s disease

. In contrast, there has been additional investiga tion of ALS, 

### 22

Parkinson ’ s disease (PD), and essential tremor , which is described below. 

### 23

Amyotrophic Lateral Sclerosis

Most studies of the association between Pb and ALS have relied on indirect methods of assessing 

### 24

Pb exposure and overall, have produced inconsistent results. The 2006 Pb AQCD r eviewed two case



### 25

control studies that measured blood Pb levels. One study found no difference in mean blood Pb levels 

### 26May 2011 5

- 120 DRAFT

– DO NOT CITE OR QUOTE between the 16 ALS cases (mean: 12.7 μg/dL) and 39 controls (mean: 10. 8 μg/dL) ( Vinceti e t al., 1997 )

. 

### 1
Another study that examined blood and bone Pb levels in a New England

- area population found increased 

### 2 odds of ALS among subjects with blood Pb levels ≥ 3 μg/dL ( Kamel et al., 2002 ) . In analyses of tibia or 

### 3 patella Pb tertiles, subjects in the highest two tertiles (≥ 10 μg/g patella Pb and ≥ 8 μg/g tibia Pb) had 

### 4
elevated odds of ALS; however, associations were not statistically significant. In analyses of Pb 

### 5
biomarkers as continuous variables, odds ratios for all three biomarkers were similar; however, only 

### 6

associations for blood Pb level were statistically significant. Kamel et al. ( 2002 ) also found that an 

### 7

estimate of cumulative Pb exposure based on occupational history to be significantly associated with ALS 

### 8
( Kamel et al., 2002 ) . Thus, the stronger findings for blood Pb level were surprising given that bone Pb 

### 9

level is a better biomarker of cumulative Pb exposure than is blood Pb level. One explanation for these 

### 10

findi ngs is that the association could be the result of reverse causality since the half

- life of blood Pb is 

### 11
only about 30 days

, and blood was collected from people who already had ALS

. If, for example, reduced 

### 12
physical activity among those with ALS led to more bone turnover, then more Pb would be released from 

### 13

bones into circulation leading to elevations in blood Pb levels among cases as a result of effects of th e 

### 14

disease. 

### 15

Since the 200 6 Pb AQCD, additional studies have been conducted in the New England

- area case



### 16
control study. One study indicated that the association between blood Pb level and ALS was not modified 

### 17
by the ALAD genotype ( Kamel et al., 2005 )

. Another study examined survival with ALS among 100 of 

### 18
the original 110 ALS cases ( Kamel et al., 2008 ) . Higher tibia Pb levels were associated with longer 

### 19

s urvival time. Findings were similar for patella and blood Pb levels, however, they were associated with 

### 20

smaller increases in surviv al time. These paradoxical finding s raise the concern that in a case

- control 

### 21
study of ALS, the association between bone Pb levels and ALS may be biased because the case group 

### 22
may comprise more individuals with longer survival time. Consequently, their bone Pb levels may be 

### 23
higher because they reflect a longer period of cumulative exposure. However, because the strongest 

### 24
findings for survival were found for tibia Pb , it might be expected a bias would be most apparent in a 

### 25
study examining associations of tibi a Pb levels and ALS incidence . However, this was not observed in the 

### 26
one study that had bone and blood Pb biomarkers ( Kamel et al., 2002 )

. 

### 27

A nother case

- control study examined blood Pb levels and odds of ALS among 184 cases (33 were 

### 28
either progressive muscular atrophy or primary lateral sclerosis , mean blood Pb level: 2.41 μg/dL ) and 

### 29

194 controls (mean blood Pb level: 1.76 μg/dL) ( Fang et al., 2010 )

. The cases were recruited from the 

### 30
National Registry of U

. S

. Veterans with ALS

, and controls were recruited from among U

. S

. Veterans 

### 31
without ALS frequ ency matched by age, gender, race, and past use of the Veterans Administration system 

### 32

for health care

. A doubling of blood Pb levels was associated with an OR (95% CI) of 2.6 (1.9, 3.7). 

### 33

Associations did not differ sub stantially by indicators of bone turno ver but were slightly higher among 

### 34

ALAD 1

- 1 carriers

. The association with blood Pb level was similar in analyses that excluded the 

### 35
progressive muscular atrophy and primary later al sclerosis cases. The similar results by degree of bone 

### 36May 2011 5

- 121 DRAFT

– DO NOT CITE OR QUOTE turnover suggests th at reverse causation is not likely driving the association between blood Pb level and 

### 1

ALS. Whether other types of reverse causality are occurring, however, cannot be ruled out

. This study did 

### 2

not have measures of bone Pb and therefore could not assess the association with biomarkers of 

### 3

cumulative Pb exposure. 

### 4
In summary, several studies have found associations of blood and bone Pb levels with ALS ; 

### 5
however, the issues of reverse causality and bias due to survival time make it difficult to draw firm 

### 6

conclusio ns. 

### 7

Parkinson ’ s Disease

A few studies, some ecological ( Aquilonius & Hartvig, 1986 ; Rybicki et al., 1993 ) and some case



### 8
control relying on questionnaire data or occupational history ( Gorell et al., 1997 ; Gulson et al., 1999 ; 

### 9

Tanner e t al., 1989 ) have indicated associations between expos ure to heavy metals, particularly Pb

, and 

### 10
risk of PD, although the evidence is limited a nd far from conclusive

. Coon et al. ( 2006 ) expanded on 

### 11

ear lier studies that had found more than a two fold increased risk of PD among adults occupationally



### 12
exposed to Pb for more than 20 years ( Gorell et al., 1997 ; Gulson et al., 1999 ) by examining associations 

### 13

with bone and whole

- body Pb levels. Coon et al. ( 2006 ) included 121 PD patients and 414 age

- , sex

- , and 

### 14

race

- , frequency

- matched controls all receiving health care services from the Henry Ford Health System

. 

### 15
Subjects i n the highest quartile of both tibia (OR: 1.62 [ 95% CI: 0.83

, 3.17 ] for levels ≥ 15 μg/g ) and 

### 16
calcaneus (OR: 1.50 [ 95% CI: 0.75

, 3.00 ] for levels ≥ 25.29 μg/g ) bone Pb levels were at elevated odds of 

### 17
PD compared to those in the lowest quartile

. The highest OR for PD was estimated for subjects in the 

### 18
highest quartile of whole

- body lifetime ex posure to Pb , compared to the lowest quartile of exposure (OR: 

### 19

2.27 [ 95% CI:1.13

, 4.55 ] for levels ≥ 80.81 μg/g ). 

### 20

The second study to explore the association between bio markers of Pb and PD was published 

### 21
recently ( Weisskopf et al., 2010 ) . This study was based in the Boston, MA area and had more than twice 

### 22

as many cases as Coon et al. ( 2006 ) : 330 cases and 308 controls . Subjects in the highest quartile of tibia 

### 23

Pb level (>16.0 μg/g) had elevated odds of PD compared to t hose in the lowest quartile (OR: 1.91 [ 95% 

### 24

CI: 1.01

, 3.60 ] )

. In this study

, cases and controls were recruited from several different sources including 

### 25

movement disorder clinics and community

- based cohorts, which could have introduced some biases. 

### 26

However, when analyses were restricted to cases recruited from movement disorder clinics and to their 

### 27
spouse, in

- law, or friend as controls, the results were even stronger (OR: 3.21 [ 95% CI: 1.17

, 8.83 ] )

. 

### 28
Although the use of spouse, in

- law, and friend controls can introduce bias, this is expected to be towards 

### 29
the null as these groups are likely to share many exposures. 

### 30May 2011 5

- 122 DRAFT

– DO NOT CITE OR QUOTE Essential Tremor

The 2006 Pb AQCD described t wo studies that found associations between blood Pb levels and 

### 1
odds of PD in New York City

- based popula tions ( Louis et al., 2005 ; Louis et al., 2003 )

. Since then

, Dogu 

### 2

et al. ( 2007 ) reported on a case

- control study of 105 essential tremor cases from a movement disorder 

### 3
clinic in Turkey a nd 105 controls (69 spouses and 36 other relative s living in th e same district). Blood Pb 

### 4

levels in this study were comparable to those found in the earlier New York based studies: the median 

### 5

blood Pb was 2.7 μg/dL among cases and 1.5 μg/dL among controls. After adjusting for age, sex, 

### 6

educa tion, cigaret te smoking (yes versus no), cigarette pack

- years, and ethanol use (yes vs. no), a 1 μg/dL 

### 7

increase in blood Pb level was associated with more than a four fold increase in odds of essential tremor 

### 8
(OR : 4.19 [ 95% CI : 2.59

, 6.78 ] )

. This OR was much larger than that obtained in the New York study (OR: 

### 9

1.19 [ 95% CI: 1.03

, 1.37 ] ) ( Louis et al., 2003 ) . The magnitude of association in Dogu et al. ( 2007 ) is even 

### 10
more striking because so ma ny of the controls were spouses who are expected to share many 

### 11
environmental exposures as cases. Most of the essential tremor cases were retired at the time of the study, 

### 12
but past occupation that could have contributed to Pb exposure ( possibly stored in bo ne from where it 

### 13

could have contributed to blood Pb levels at the time of the study ) was not assessed

. One of the earlier 

### 14

New York studies also fo und a very high OR for essential tremor among ALAD2 carriers (per 1 μg/dL 

### 15

increase in blood Pb level, OR: 71.8 [95% CI: 1.08, 4789.68); however, the very wide 95% CI indicated 

### 16
lack of precision in the effect estimate

. 

### 17

Several studies of essential tremor h ave reported a very strong association between blood Pb levels 

### 18

and essential tremor, although studies employing biomarkers of Pb have had relatively small sample sizes 

### 19
and have produced imprecise effect estimates. 

### 20

5.3.5.2. Toxicological Studies

In epidemiological studies, Pb level (bone and blood) is associated with increased odds of ALS 

### 21
( Kamel et al., 2005 ) and paradoxically with longer survival time in patients diagnosed with ALS ( Kamel 

### 22
et al., 2008 ) . Chronic Pb exposure (Pb acetate in drinking water at 200 ppm from weaning onward, blood 

### 23
Pb level 27 μg/dL) reduced astrocyte reactivity and induced increased survival time in the superoxide 

### 24
dismutase transgenic (SOD1 Tg) mouse model of severe ALS ( Barbeito et al., 2010 ) . In this model, Pb 

### 25
exposure does not significantly increase the onset of the ALS disease in SOD1 Tg mice, but Pb exposure 

### 26
was associated with longer survival time in SOD1 Tg mice ( Barbeito et al., 2010 ) . Baseline levels of 

### 27
VEGF are elevated in astrocytes from the ventral spinal cord of untreated SOD1 Tg control mice versus 

### 28
control, non

- transgenic animals. VEGF was not induced in Pb treated non

- transgenic astrocytes. Further, 

### 29

Pb

- exposed SOD1 Tg mice had s ignificant elevations of astrocyte VEGF versus vehicle treated SOD1 

### 30

animals ( Barbeito et al., 2010 ) . Control animals exposed to Pb showed no elevation in VEGF expression 

### 31

above control vehicle

- treated animals ( Barbeito et al., 2010 ) . Other research has suggested that ALS 

### 32May 2011 5

- 123 DRAFT

– DO NOT CITE OR QUOTE initiation is dependent on motor neuron function and ALS progression is depende nt on astrocyte and 

### 1
microglia function ( Boillee et al., 2006 ; Yamanaka et al., 2008 )

. 

### 2
Consistent these findings, other have reported that VEGF administration to the SOD1 Tg mice 

### 3

si gnificantly reduced glial reactivity, a marker or neuroinflammation ( Zheng et al., 2007 ) . Using a cell 

### 4

based co

- culture system of neurons and astrocytes, Barbeito et al ( 2010 ) found that an up

- regulation of 

### 5
VEGF production by astrocytes in the Pb

- exposed SOD1 Tg mice is protective against motor neuron 

### 6
death in the SOD1 Tg cells ( Barbeito et al., 2010 ) . Chronic Pb exposure in a mouse model of ALS wa s 

### 7
associated with increased survival time and correlated with higher spinal cord VEGF levels, making 

### 8
astrocytes less cytotoxic to surrounding motor neurons ( Barbeito et al., 2010 ) . Also, in another study the 

### 9

metal chelators DP

- 109 and DP

- 460 are neuroprotective in the ALS mouse model or Tg(SOD1

- G93A) 

### 10
( Petri et al., 2007 )

. 

### 11
Improper activation of microglia and release of inflammatory cytokines and metabolites can 

### 12
contribute to neurodegeneration ( L. Qian & Flood, 2008 ; D. Zhang et al., 2010 )

. These two cell types are 

### 13
known to accumulate or sequester Pb in the nervous system. R esearchers have implicated dysfunctional 

### 14
astrocytes as playing an important role in the chain of misregulated inflammation lead ing to 

### 15
neurodegenerative conditions ( Barbeito et al., 2010 ; De Keyser et al., 2008 )

. 

### 16

Cell Death Pathways

Earlier work has documented that Pb exposure could induce cell death or apoptosis in various 

### 17
models including rat brain ( Tavakoli

- Nezhad et al., 2001 ) , retinal rod cells ( L. H. He et al., 2000 ; 2003 )

, 

### 18

cerebellar neur ons ( Oberto et al., 1996 ) , and PC12 cells ( Sharifi & Mousavi, 2008 ) . This st udy addresses 

### 19
chronic (40 days) Pb exposure

- induced hippocampal apoptosis in young (exposure starting at 2

- 4 weeks 

### 20
of age) and old (exposure starting at 12

- 14 weeks of age) male rats exposed to 500 ppm Pb by drinking 

### 21
water (blood Pb, 98 μg/dL); apoptosis w as verified by light and electron microscopy, and increased pro



### 22
apoptotic Bax protein levels ( Sharifi et al., 2010 ) . Another study followed the developmental profile of 

### 23
changes in various apoptotic factors in specific brain regions of animals exposed to Pb acetate (0.2% dam 

### 24
drinking water) during lactation. Male offspring blood Pb at the end of lactation or PND20 was 80 μg/dL. 

### 25

T hese data showed hippocampal mRNA for various apoptotic factors including caspase

- 3, Bcl

- x and 

### 26

Brain

- derived neurotrophic factor (BDNF) was significantly upregulated on PND12, PND15 and PND20. 

### 27
The cortex of these male pups also showed upregulation of Bcl

- x and BDNF on PND 15 and PND20 

### 28
( Chao et al., 2007 ) . The cerebellum did not have elevated apoptotic mRNA levels in this model. This 

### 29
study shows temporal and regional changes in activation of death protein message levels in male 

### 30
offspring. Thus, the new data continue to confirm that Pb exposure induced apoptosis in brains of exposed 

### 31

animals. 

### 32May 2011 5

- 124 DRAFT

– DO NOT CITE OR QUOTE Lead

- Induced Neuronal Pl aque Formation

Studies from the 2006 Pb AQCD highlighted the importance of Pb exposure in early life in 

### 1
promoting Alzheimer’s like pathologies in the adult brain. Pb has been recognized for decades as having a 

### 2
more profound effect on children than adults w ho receive the same exposure. In the last decade, the 

### 3
developmental origins of adult disease (DoAD) paradigm and the similar Barker hypothesis have reported 

### 4
that early life exposures can result in aberrant adult outcomes. Epidemiology data show that blood Pb 

### 5
level effects on neurological outcomes fit this paradigm. Recent evidence in the toxicological literature 

### 6
points to latent effects in rodent and non

- human primate models of gestational and/or early life exposure 

### 7
to Pb including neurodegeneration similar to Alzheimer’s like pathologies, obesity (in males only), retinal 

### 8
aberrations (male only), and neurobehavioral aberrations. Immune outcomes fitting the DoAD paradigm 

### 9
involve tissue inflammation and loss of organ function. Bolin et al. ( 2006 ) demonstrated the connection 

### 10
between developmental exposure to Pb in the rat with early life programming and the resulting 

### 11

inflammation

- associated DNA damage with neurodegenerative loss in the adult brain. Wu and colleagues 

### 12
( 2008 ) had similar findings in a study using infantile exposure to Pb in monkeys. The investigations 

### 13

reinforce the need t o directly examine the long

- term effects of developmental exposure to xenobiotics 

### 14
rather than relying on adult exposure information to predict probable health risks from prenatal, neonatal 

### 15
or juvenile exposure ( Dietert & Piepenbrink, 2006 )

. Mechanistically, some of these pathologies have been 

### 16
associated with changes in the epigenome. Multiple Pb studies point to sensitive windows of exposure; 

### 17
early life or developmental exposures are far more sens itive than adult exposures. Alzheimer’s disease is 

### 18
characterized by amyloid

- beta peptide (Ab) accumulation, hyper

- phosphorlyation of the tau protein, 

### 19
neuronal death and synaptic loss. The toxicological evidence for Pb in contributing to the AD pathology 

### 20

fo llows. 

### 21
The fetal basis of amyloidogenesis has been explored extensively by Zawia’s laboratory in both 

### 22

rodents and non

- human primates. Amyloid deposits in the brain are seen in patients with Alzheimer’s 

### 23
disease and in the aged brain of individuals with Down Syndrome who display an Alzheimer’s

- like 

### 24
pathology. Mechanistically, amyloid plaques originate from the cleavage of the amyloid precursor protein 

### 25
(APP) to Ab, which comprises the plaque. By exposing rodents to Pb as neonates or as aged animals, it 

### 26

was det ermined that neonatal Pb exposure is a sensitive window for induction of the amyloidogenesis in 

### 27
the aged animal brains and that exposure to Pb in old animals did not contribute to plaque formation. 

### 28
Following cortical APP gene expression over the lifetime o f male rodents exposed neonataly via lactation 

### 29
to Pb (PND1

- PND20 exposure, dam drinking water Pb acetate 200 ppm, pup PND20 blood Pb level 46 

### 30
μg/dL and cortex 0.41 μg /g wet w eight of tissue ), one sees a biphasic significant increase above control 

### 31
animals in APP expression with the first increased phase manifesting neonatally and the second phase 

### 32
manifesting in old age (82 weeks of age) ( M.R. Basha et al., 2005 ) . A concomitant biphasic response is 

### 33
seen in specificity protein 1 (Sp1), a transcription factor known to be related to APP expression. Ab, t he 

### 34May 2011 5

- 125 DRAFT

– DO NOT CITE OR QUOTE amyolid plaque constituent, was also significantly elevated in these aged animals developmentally 

### 1
exposed to Pb. A separate subset of rodents exposed to Pb only as aged adults (18

- 20 weeks of age) were 

### 2
unresponsive in APP or Sp1 expression or Ab product ion after Pb exposure, indicating the developmental 

### 3
window and not adult exposure as the susceptible period for Pb

- dependent amyloidogenesis. The Zawia 

### 4

lab ( J. Wu et al., 2008 ) has confirmed similar amyloid findings in brains of monkeys that were exposed to 

### 5
Pb as infants (PND1

- PND400), i.e., significantly higher message levels of APP, and Sp1 and significantly 

### 6
higher protein expression of APP and Ab in aged female monkey cortex tissue (23 year old Macaca 

### 7

fascicularis ) from a cohort of animals established in the 1980s ( Rice, 1990

, 1992 ) . After weaning and 

### 8
when still being dosed with Pb, the monkeys had blood Pb level of 19

- 26 μg/dL. As a caveat, by the time 

### 9
neonatally Pb

- exposed animals bec ome aged and manifest with amyloid plaques, blood Pb level and brain 

### 10
cortex Pb levels have returned to control or baseline levels. Thus, the rodent and non

- human primate 

### 11
toxicology studies concur and show that developmental Pb exposure induced elevations i n neuronal 

### 12
plaque proteins in the aged animals. 

### 13
Transcription factors are essential in the regulation of the developing brain. Pb exposure is known 

### 14
to perturb DNA binding of transcription factors including SP1 at essential sites like zinc finger proteins. 

### 15
In Long

- Evans hooded rat pups exposed during lactation, these Pb

- induced developmental perturbations 

### 16
of SP1 DNA binding can be ameliorated by exogenous zinc supplementation ( M. R. Basha et al., 2003 )

. 

### 17
The mechanism by which Ab peptide formation is affected by Pb exposure has been explored by 

### 18
Behl et al. It has been shown that the choroid plexus is able to remove beta

- amyloid peptides from the 

### 19
brain extracellular matrix and that Pb impairs this function and may be mediated by the 

### 20
metalloendopeptidase , insulin

- degrading enzyme (IDE), which metabolizes Ab ( Behl et al., 2009 )

. 

### 21
Further studies with developmental Pb exposure (gestational plus lactational, dam drinking water 

### 22
solutions of 0.1%, 0.5% or 1%, blood Pb level 400, 800 and 1,000 μg/L) showed that the hippocampus 

### 23
contained neurofibriallary changes as early as PND21. These changes manifested with Tau hyper



### 24
phosphorylation, and increased tau and beta amyloid hippo campal protein levels in Pb

- exposed offspring 

### 25
( Li et al., 2010 ) . The multiple new studies on Pb

- dependent changes in the neurofibrillary proteins show 

### 26
that developmental Pb exposure induced significant increases in neuronal plaque associated proteins, 

### 27
indicating that early life Pb exposure s may contribute to dementia in adulthood. 

### 28
Data from the animal toxicology literature point to an early life window in which Pb exposure can 

### 29
contribute to pathological brain changes consistent with those seen in Alzheimer’s disease including Ab 

### 30
peptide ac cumulation and activation of Ab supporting transcription factors as well as tau 

### 31
hyperphosphorylation. 

### 32May 2011 5

- 126 DRAFT

– DO NOT CITE OR QUOTE 

## 5.3.6. Studies of Mechanisms of the Neuro logical Effects of Lead

5.3.6.1. Effects on Brain Physiology and Activity

A growing body of epidemiologic evidence demonstrates associations of Pb biomarkers with 

### 1
electrophysiologic changes in the brain. By providing insight into the underlying mechanisms by which 

### 2

Pb exposure may disrupt brain function, findings from these studi es have provided biologically plausible 

### 3
evidence for the effects of Pb exposure on cognitive, psychological and behavioral consequences 

### 4

observed in children and adults. Much of the early work was conducted by Otto and colleagues ( Otto et 

### 5
al., 1985 ; Otto & Fox, 1993 ) , which found associations of blood Pb level with auditory and visual evoked 

### 6
potentials. Rothenberg et al. ( 1994 ) and Rothenberg et al. ( 2000 ) reported similar findings; however, the 

### 7

direction of associatio n differed between prenatal (maternal) and postnatal (ages 1

- 4 years) blood Pb 

### 8

level . Postnatal blood Pb level was associated with a decrease in interpeak intervals in auditory evoked 

### 9
potentials at age 5

- 7 years. Prenatal blood Pb level showed a biphasic r elationship, with a negative 

### 10

association at blood Pb levels of 1

- 8 μg/dL and a positive association at blood Pb levels of 8

- 3 0 μg/dL. 

### 11
These findings provide mechanistic support for observations of Pb

- associated changes in sensory acuities 

### 12
(Section 5.3.4

. 1)

. 

### 13
Studies using magnetic resonance imaging (MRI) or spectroscopy (MRS) as clinical outcome 

### 14
measures have been limited in number and sample size, but have shown associations of blood Pb level 

### 15
with alterations in brain physiology such as reduced levels of N

- acetylaspartate, creatine, or choline in 

### 16
young adults ( Cecil et al., 2005 ; Meng et al., 2005 ; Trope et al., 2001 ; Trope et al., 1998 ; Yuan et al., 

### 17

2006 )

. These changes have been linked to decr eased neuronal density or loss and alteration in myeline. 

### 18
Notably, Trope et al. ( 2001 ; 1998 ) and Meng et al. ( 2005 ) reported that all subjects had normal MRIs with 

### 19
no evidence of structural abnormalities. Thus, the clinical significance of the observed physiological 

### 20
changes is unclear. Additionally, these studies compared su bjects with relatively high childhood blood 

### 21
levels (23

- 65 μg/dL) to those with childhood blood Pb levels < 10 μg/dL. Therefore, it is unclear whether 

### 22
physiological changes would be observed in association with lower blood Pb levels. Cecil et al. ( 2005 ) 

### 23

and Yuan et al. ( 2006 ) conducted functional MRI in 42 adult (ages 20

- 23 years) participants from the CLS 

### 24
cohort during a verb generation language task and found that mean childhood blood Pb level was 

### 25

associated with decreased activation in the left frontal gyrus and left middle temporal gyrus, regions 

### 26
traditionally associated with semantic language function. Although these findings were in adults, they 

### 27
were consistent with findings in the same cohort of associations of blood Pb level with other indic es of 

### 28
language in childhood. 

### 29
Since the 2006 Pb AQCD, studies examining MRI data have been limited to CLS cohort 

### 30
participants as adults (ages 19

- 24), and recent results continue to support associations of childhood blood 

### 31May 2011 5

- 127 DRAFT

– DO NOT CITE OR QUOTE Pb levels with physiological change s in the brain in adulthood. These recent studies expanded on previous 

### 1
studies by including larger sample sizes and aiming to characterize Pb effects more precisely by 

### 2
examining blood Pb levels at different periods in childhood and aiming to link changes i n brain activity to 

### 3
neurodevelopmental deficits. Brubaker et al. ( 2009 ) used di ffusion tensor imaging to examine 

### 4
associations between mean childhood blood Pb and white matter integrity in 91 young adults

, 

### 5
hypothesizing that childhood Pb exposure may alter adult white matter architecture via deficits in axonal 

### 6
integrity and myelin org anization. Fractional anisotropy (F r A), mean diffusivity (MD), axial diffusivity 

### 7
(AD), and radial diffusivity (RD) were measured on an exploratory voxel

- wise basis. In adjusted 

### 8
analyses, mean childhood blood Pb levels were associated with decreased F r A thr oughout white matter. 

### 9
Regions of the corona radiata demonstrated highly significant Pb

- associated decreases in F r A and AD and 

### 10
increases in MD and RD. The genu, body, and splenium of the corpus callosum demonstrated highly 

### 11
significant Pb

- associated decrease s in RD, smaller and less significant decreases in MD, and small areas 

### 12
with increases in AD. The results of this analysis suggest multiple insults appear as distinct patterns of 

### 13
white matter diffusion abnormalities in the adult brain which may be indicativ e of altered myelination and 

### 14
axonal integrity. Additionally, childhood blood Pb levels appear to differentially affect neural elements

, 

### 15

which may be related to the stage of myelination development at various periods of exposure. 

### 16
Another study of 157 CLS pa rticipants provided evidence of region

- specific reductions in adult 

### 17
gray matter volume in associatio n with childhood blood Pb levels and by examining associations between 

### 18

MRI

- assessed brain volume and historic neur opsychological assessments, provided insig ht into the 

### 19
potential clinical significance of changes in brain physiology associat ed with blood Pb levels ( Cecil et al., 

### 20

2008 ) . Using conservative, minimum contiguous cluster size and statistical criteria (700 voxels, 

### 21
unadjusted p < 0.001), approximately 1.2% of the total gray matter was significantly and in versely 

### 22

associated with mean childhood blood Pb level . The most affected regions included frontal gray matter, 

### 23
specifically the anterior cingulate cortex and the ventrolateral prefrontal cortex (i.e., areas traditionally 

### 24
related to executive functions, moo d regulation, and decision

- making). Comparing neuropsychological 

### 25
factor scores with gray matter volume, investigators found that fine motor factor scores positively 

### 26
correlated with gray matter volume in the cerebellar hemispheres ; adding blood Pb level as a variable to 

### 27
the model attenuated this correlation. These findings are notable in light of other studies linking brain 

### 28
volume changes with altered function and suggest that MRI changes association with blood Pb levels m ay 

### 29

be indicative of decrements in ne urocognitive and neurobehavioral function . Schwartz and colleagues 

### 30
( 2007 ) showed that larger RO1 volumes were associated with better cognitive function in 5 or 6 cognitive 

### 31
domains (visuoconstruction, processing, speed, visual memory, executive functioning, and eye

- hand 

### 32
coordination)

. More recent studies by Raine and colleagues suggest that deficits in cortical volume or 

### 33
activity found in select brain regions, inclu ding the prefrontal gray matter, may predispose individuals to 

### 34
impulsive, aggressive, or violent behavior

. 

### 35May 2011 5

- 128 DRAFT

– DO NOT CITE OR QUOTE In a subsequent analysis, Brubaker et al. ( 2010 ) investigated the developmental trajectory of 

### 1
childhood blood Pb levels on adult gray matter. Adjuste d voxel

- wise regression analyses were performed 

### 2
for associations between adult gray matter volume loss and yearly mean blood Pb levels from 1 to 6 years 

### 3
of age in the entire cohort and by sex. Investigators observed significant inverse associations between 

### 4
gray matter volume loss and yearly mean blood Pb levels from 3 to 6 years of age. The extent of 

### 5
prefrontal gray matter loss associated with yearly mean blood Pb levels increased with advancing age of 

### 6

the subjects. These results indicate that blood Pb leve ls later in childhood are associated with greater 

### 7

loss es in gray matter volume than are childhood mean or maximum blood Pb levels . This study 

### 8
demonstrates that maximum blood Pb levels do not fully account for gray matter changes, particularly in 

### 9

the fronta l lobes of young men

. Notably, although they did not consider Pb, Yang et al. ( 2005 ) reported 

### 10
volume reduction in gray matter in psychopaths, adding additional evi dence that these physiological 

### 11
changes may be related to overt deleterious outcomes. Consistent with Wright et al. ( 2008 ) , Cecil et al. 

### 12
( 2008 ) found that gray matter volume loss associated wi th childhood blood Pb levels was much larger in 

### 13

CLS male adults than female adults. In an expanded analysis of the developmental trajectory of childhood 

### 14
blood Pb levels on adult gray matter, Brubaker et al. ( 2010 ) found that the inverse associations between 

### 15
gray matter volume loss and yearly mean blood Pb levels were more pronounced in the frontal lobes of 

### 16

men than women for blood Pb levels measured at all ages. 

### 17
Whereas the aforementioned CLS studies examined associations of childhood blood Pb levels, a 

### 18

rece nt analysis of the NAS participants indicated that biomarkers of cumulative, long

- term Pb exposure 

### 19
also may be associated with changes in brain structure and function in older adults. Weisskopf et al. 

### 20
( 2007 ) found increasing bone Pb level to be associated with incr eased myoinositol (mI)/Cr ratio with 

### 21
increasing bone Pb concentration among 31 elderly men from the NA S. A higher mI/Cr ratio may be 

### 22
indicative of glial activation and is a signal reportedly seen in the early stages of HIV

- related dementia 

### 23

and Alzheimer ’ s disease. 

### 24

Studies of Pb

- workers also found associations of blood and bone Pb levels with changes in brain 

### 25
structure and p hysiology. Stewart et al. ( 2006 ) studied 532 former organolead workers with a mean age of 

### 26
56 ye ars and found that an estimate of past peak tibia bone Pb (mean: 23.9 μg/g) was significantly 

### 27
associated with more white matter lesions (WML)

. Higher estimated peak tibia Pb also was associated 

### 28

with smaller total brain volume and volumes of frontal and tot al gray matter, parietal white matter, the 

### 29
cingulate gyrus

, and insula

. In this same group, Caffo et al. ( 2008 ) found evidence that the association 

### 30

between tibia Pb level and cognitive function

, in particular

, visuo

- construction domain tasks

, and to a 

### 31
lesser degree

, executive function and eye

- hand coordination , were mediated b y the association between 

### 32

tibia Pb levels and brain region volume changes

. In a similar study of 61 current Pb smelter workers with 

### 33
an average age of 40 years, higher estimates of cumulative Pb exposure were also associated with WML, 

### 34

and there was evidence that this association accounted for , in part, the association between higher 

### 35

cumulative Pb exposure and worse performance on the grooved pegboard test ( Bleecker, Ford, Vaughan, 

### 36May 2011 5

- 129 DRAFT

– DO NOT CITE OR QUOTE et al., 2007 )

. Another small study of 15 current occupationally Pb

- exposed workers (mean blood Pb level: 

### 1
63.5 μg/dL) and 19 non

- Pb exposed controls (mean blood Pb level: 8.7 4 μg/dl) found smaller 

### 2
hippocampal volumes in MRI scans among the Pb workers ( Jiang et al., 2008 )

. A lower ratio of the brain 

### 3

metabolites N

- acetyl

- aspartate (NAA) and creatine (Cr) , indicative of neuronal density

, was also found 

### 4
among the Pb workers, as well as an increased lip id to creatine ratio, indicative of cell membrane or white 

### 5
matter damage

. Similarly, a study of 22 Pb paint factory workers and 18 controls found lower NAA/Cr 

### 6
ratios among the Pb exposed workers as well as lower choline/ Cr, possibly indicative of reduced cell 

### 7

membrane turnover ( Hsieh et al., 2009 )

. 

### 8

5.3.6.2. Cholesterol and Lipid Homeostasis

Various pathological conditions are associated with elevated plasma free fatty acids or elevated 

### 9
cholesterol. Adult male rats exposed to Pb acetate (200, 300 or 400 ppm) in their drinking water for 12 

### 10

weeks manifested with Pb

- induced cholesterogenesis and phospholipidosis in brain tissue ( Ademuyiwa et 

### 11
al., 2009 ) . Pb

- dependent changes in brain cholesterol produced an inverse U dose response curve with the 

### 12
highest brain cholesterol at 200 ppm followed by 300 ppm Pb. Animals exposed to 400 ppm Pb did not 

### 13
have significant changes in brain cholesterol. Mechanistically , Pb exposure has been shown to depress the 

### 14
activity of cholesterol

- 7

- a

- hydroxylase, an enzyme involved in bile acid biosynthesis ( Kojima et al., 

### 15

2005 ) ; bile acids are the route by which cholesterol is eliminated from the body. Pb exposure produced 

### 16
significant increases in brain triglycerides with an 83% increase at 300 ppm and a 108% increase at 400 

### 17
ppm. At 200 ppm, Pb exposure induced a non

- significant decrease in brain triglycerides. Pb exposure 

### 18
across all three dose groups induced significantly increased brain phospholipids. Interestingly, plasma free 

### 19
fatty acids were significantly elevated in a dose

- dependent fashion; plasma triglycerides and cholesterol 

### 20

were una ffected by Pb exposure. The molar ratio of brain cholesterol to phospholipids, an indicator of 

### 21
membrane fluidity ( Abe et al., 2007 ) , was significantly increased at 200 and 300 ppm Pb exposure 

### 22
indicating increased membrane fluidity. Brain Pb in all dose groups was be low the limit of detection (0.1 

### 23
ppm). Blood Pb at 0, 200, 300, and 400 ppm were 7, 41, 61, and 39 μg/dL, respectively. In summary, Pb 

### 24
exposure significantly increased brain cholesterol, triglycerides, and phospholipids as well as significantly 

### 25
increased pl asma free fatty acids. These effects were sometimes more prominent at lower doses of Pb. 

### 26
Future characterization of molecular and cellular pathways affected by Pb exposure may bring insight to 

### 27

this Pb

- dependent phospholipidosis and cholesterogenesis. 

### 28

5.3.6.3. Oxid ative Stress

Pb has been shown to induce oxidative stress in multiple animal models and this oxidative stress 

### 29
can contribute to DNA damage, which can be measured with the biomarker 8

- hydroxy

- 2’

- 

### 30
deoxyguanosine (8

- oxo

- dG). The contribution of reactive oxygen or nitrogen species to these Pb induced 

### 31May 2011 5

- 130 DRAFT

– DO NOT CITE OR QUOTE changes was assayed by looking at the ratio of 8

- oxo

- dG to 2

- deoxyguanosine (2

- dG). 2

- dG is a DNA 

### 1
nucleoside enzyme that can generate 8

- oxo

- dG from a parent compound forming a DNA adduct or 

### 2
biomarker during conditio ns of nitrosative or oxidative stress. The 8

- oxo

- dG to 2

- dG ratio data from 

### 3
rodent male offspring recapitulated the amyloid data with significant biphasic elevations in 

### 4
developmentally Pb

- exposed animals (0.2% Pb acetate in dam drinking water from PND1

- 20) versus 

### 5
control, non

- Pb exposed animals at early (PND5) and late life time points (80 weeks of age) ( Bolin et al., 

### 6

2006 ) . Activity of the base

- excision DNA re pair enzyme o xoguanine glycosylase or Ogg1 was unaffected 

### 7
by Pb exposure ( Bolin et al., 2006 ) . Interestingly, the monkey data were the same as the rodent data . The 

### 8

ratio of 8

- oxo

- dG to 2

- dG in the brains of aged monkeys (23 years) after being exposed as infants, was 

### 9
significantly elevated above controls ( J. Wu et al., 2008 ) . Similar to the amyloid data, the oxidative stress 

### 10
markers showed no significant changes above baseline when animals were exposed to Pb as aged adults 

### 11
( Bolin et al., 2006 ; J. Wu et al., 2008 ) . Thus, the data for biomarkers of oxidative stress concur with the 

### 12
amyloidogenesis data with both demonstrating kinetically similar biphasic significant elevations in 

### 13
markers of oxidative stress and amyloidogenesis with early life Pb exposure. 

### 14
Because the brain has the highest energy deman d and metabolism of any organ, energy homeostasis 

### 15
is of utmost importance. Pb has been shown to inhibit various enzymes involved in energy production or 

### 16
glucose metabolism including glyceraldehydes

- 3 phosphate dehydrogenase, hexokinase, pyruvate kinase, 

### 17

an d succinate dehydrogenase ( Regunathan & Sundaresan, 1984 ; S terling et al., 1982 ; Verma et al., 2005 ; 

### 18
Yun & Hoyer, 2000 ) . Mitochondria produce ATP or energy throu gh oxidative phosphorylation. Aberrant 

### 19
mitochondrial function can decrease the energy pool and contribute to ROS formation via electron 

### 20
transport chain disruption

. ATP depletion can also affect synaptic and extracellular neuotransmission

. The 

### 21

mitochondrial Na/K ATPase is important in maintaining the inner mitochondrial membrane potential 

### 22
(delta omega sub m) and the health of the mitochondria. To address the effect of Pb exposure on these 

### 23
mitochondrial parameters, mice were mated, produced offspring and nurs ed the offspring until PND8 at 

### 24
which time the brains were collected from the pups ( Baranowska

- Bosiacka et al., 2011 ) . Cerebellar 

### 25
granular cells were harvested from these PND 8 control and Pb

- exp osed animals (0.1% Pb acetate in dam 

### 26
drinking water, blood Pb level 4 μg/dL and cerebella Pb 7.2 μg /g dry weight). These neuronal cells were 

### 27
cultured for 5 days in vitro, at which point various mitochondrial parameters were measured

. With Pb 

### 28

exposure, reac tive oxygen species were significantly increased in both the cortical granule cells and in the 

### 29

mitochondria

. Intracellular ATP concentration and adenylate energy charge values were significantly 

### 30

decreased in cells of Pb

- exposed mice versus control

. Neurona l Na/K ATPase activity was significantly 

### 31
lower in cortical granule cells from Pb

- exposed mice versus controls. Mitochondrial mass was unaffected 

### 32
with Pb treatment, but mitochondrial membrane potential was significantly decreased with Pb exposure. 

### 33

Pb

- expose d crayfish who are placed under hypoxic conditions adapt to the situation by decreasing their 

### 34

metabolism ( Morris et al., 2005 ) , manifesting with whole organism findings consistent with these cell 

### 35

data. These data show impaired mitochondrial function and energy production in neuronal cells from mice 

### 36May 2011 5

- 131 DRAFT

– DO NOT CITE OR QUOTE with gestational and lactational Pb exposure with concomitant increases in mitochondrial and cellular 

### 1
ROS production

. 

### 2

5.3.6.4. Nitrosative Signaling and Nitros ative Stress

The nitric oxide system is increasingly being recognized as a signaling system in addition to its 

### 3
more classical role as a marker of cellular stress. In studies of learning and memory using the Morris 

### 4
water maze, hippocampal changes in NO were noted after completion of the test. Pb exposure has been 

### 5
repeatedly shown to increase the escape latency in Pb

- exposed animals (Section 5.3.2.2). Chetty ( 2001 ) 

### 6
initially reported decreased hippocampal nNOS with perinatal Pb exposure. Namely, with repeated swim 

### 7
tests, control animals more quickly find a submerged platform, i.e. escape, than do Pb

- exposed animals. 

### 8
After either 4 or 8 weeks of Pb exposure to weanli ng male rats (blood Pb level 0.3 umol/L), hippocampal 

### 9
NOS and NO are significantly decreased. Dietary supplementation concomitant with 8 weeks of Pb 

### 10
exposure induced significant increases in hippocampal NOS (taurine or glycine) and decreases in 

### 11
hippocampal NOS (vitamin C, methionine, tyrosine, or vitamin B1). These animals also had significant 

### 12
changes in hippocampal NO with supplementation. NO increased with taurine and decreased with vitamin 

### 13
C, tyrosine or glycine co

- exposure with Pb. Dietary supplementati on after 4 weeks of Pb exposure in 

### 14
weanling males (4 week blood Pb level 2.3 umol/L & 8 week Blood Pb level 0.39 umol/L), induced 

### 15
significant increases in NO with the supplements tyrosine, methionine, or ascorbic acid. Zinc 

### 16
supplementation in this model ha d no effect on the NO system. The conclusions of this study are that 

### 17
various combinations of nutrients significantly attenuate Pb

- dependent decreases in NO/NOS. 

### 18
Specifically, nutrients prevented (8 weeks Pb plus concomitant exposure to methionine, zinc, as corbic 

### 19
acid, and glycine) or restored (4 weeks Pb exposure followed by 4 weeks nutrient exposure, taurine and 

### 20
thiamine) Pb

- dependent decrements in NO/NOS concentrations ( G. Fan et al., 2009 )

. 

### 21

5.3.6.5. Synaptic Changes

Work in earlier criteria documents as well as earlier publications in the scientific literature point to 

### 22
an effect of developmental Pb exposure on synapse development, which mechanistically may contribute 

### 23
to multiple Pb related aberrant outcomes, including changes in long

- term potentiation (LTP) and 

### 24
facilitation. Earlier work has shown that developmental Pb exposure is responsible for altered density of 

### 25
dendritic hippocampal spines ( Király & Jones, 1982 ; Petit & LeBoutillier, 1979 ) , aberrant synapse 

### 26

elimination ( Lohmann & Bonhoeffer, 2008 ) and abnormal long

- term and short

- term plasticity 

### 27
( MacDonald et al., 2006 ) . Newer research using the Drosophila larval neuromuscular junction model has 

### 28
shown that stimulation with multiple action potentials (also called AP trains) induced significant increases 

### 29

in intracel lular calcium and significant delays in calcium decays back to baseline levels at the pre



### 30
synaptic neuronal bouton in developmentally Pb exposed larvae versus control. Pb

- exposed larvae had 

### 31May 2011 5

- 132 DRAFT

– DO NOT CITE OR QUOTE reduced activity of the plasma membrane calcium ATPase, which is r esponsible for extravasations of 

### 1
calcium from the synaptic terminal ( T. He et al., 2009 ) . Intracellular calcium in Pb exposed larvae was no 

### 2
different from controls under resting conditions or in neurons with stimu lation by a single action 

### 3
potential. Pb media concentrations in these experiments were 100 or 250 μM with the low dose body 

### 4
burden (100 μM) of Pb calculated to be 13

- 48 μM per larvae. Facilitation of a neuronal terminal is 

### 5
defined as the increased ability to transmit an impulse down a nerve due to prior excitation of the nerve. 

### 6
After stimulation of the axon, facilitation of the excitatory post

- synaptic potential, which is dependent on 

### 7
residual terminal calcium, was significantly elevated in Pb exposed larva e versus control ( T. He et al., 

### 8

2009 ) . The data from this synapse study demonstrate that developmental Pb exposure affected the plasma 

### 9
membrane calcium ATPase, induced changes in the intracellular calcium levels d uring impulse activation, 

### 10
and produced changes in facilitation of the neuronal networks of Drosophila. Thus, the neuromuscular 

### 11
junction is a potential site of Pb interaction. 

### 12
A study by Li et al. ( 2009 ) focused on inflammatory endpoints and syna ptic changes after 

### 13
gestational plus lactational dam drinking water Pb exposure (solutions of 0.1%, 0.5% or 1%, offspring 

### 14
blood Pb level 40, 80 and 100 μg/dL, respectively at PND 21). Hippocampal TNF

- alpha was significantly 

### 15
elevated with Pb exposure and pro teins that comprise the SNARE complex were all changed with Pb 

### 16
exposure. The SNARE complex of synaptic proteins includes SNAP

- 25, VAMP

- 2 and Syntaxin 1a and is 

### 17
essential in exocytotic neurotransmitter release at the synapse ( Li et al., 2009 ) . Thus, Li et al. ( 2009 ) 

### 18
found significant difference in hippocampal synaptic protein composition and increased pro

- inflammatory 

### 19
cytokine levels in the brains of Pb

- exposed offspring. 

### 20
Neurotransmission is an energy

- dependent p rocess with calcium

- dependent ATP releases found at 

### 21
the synaptic cleft. At the synapse, ATP is metabolized by ecto

- nucleotidases. In heme synthesis, Pb is 

### 22
known to substitute for the cation zinc in another nucleotidase, p yrimidine 5′

- nucleotidase , and is t hus 

### 23
used as a biomarker of Pb exposure. Acute exposure (96h) of male and female zebrafish to Pb acetate (20 

### 24
μg/L) in their water induced significant decreases in ATP hydrolysis in brain tissue. This dose is deemed 

### 25
to be an environmentaly relevant dose. Wit h a chronic exposure ( 30 days ) , Pb acetate promoted the 

### 26
inhibition of ATP, ADP and AMP hydrolysis ; these data are consistent with findings in rodents 

### 27
( Baranowska

- Bosiacka et al., 2011 )

. The auth ors hypothesize that at 30 days, this change in nucelotide 

### 28
hydrolysis was likely due to post

- translational modification because message level of enzymes 

### 29
responsible for the hydrolysis, NTPDase1 and 5′

- nucleotidase , were unchanged ( Senger et al., 2006 )

. 

### 30
Thus, Pb is shown to affect nucleotidase activity in the central nervous system of zebrafish, p ossibly 

### 31
contributing to aberrant neurotransmission. 

### 32
Another enzyme important in synaptic transmission at cholinergic junctions in the CNS and at 

### 33
neuromuscular junctions peripherally is acetylcholinesterase (AChE). After 24 hours of exposure to 

### 34

environmenta lly relevant concentrations of PbAcetate (20 ug/L water), AChE activity was significantly 

### 35

inhibited in zebrafish brain tissue. In Pb

- exposed fish, AChE activity returned to baseline by 96 hours and 

### 36May 2011 5

- 133 DRAFT

– DO NOT CITE OR QUOTE maintained baseline activity after chronic exposure of 30 days. Thus, Pb is also able to affect synaptic 

### 1
homeostasis of AChE in the brain of exposed zebrafish ( Richetti et al., 2010 )

. 

### 2

Pb is known t o act as an antagonist of the NMDA receptor. The NMDAR is essential for proper 

### 3

pre

- synaptic neuronal activity and function. Primary cultures of mouse hippocampal cells were exposed to 

### 4
Pb (10 or 100 μM solutions in media) during the period of synaptogenesis ( Neal et al., 2010 ) . This 

### 5
exposure induced the loss of two proteins necessary for presynaptic vesicular release, synaptophys in 

### 6
(Syn) and synaptobrevin (Syb), without affecting a similar protein synaptotagmin (Syt). This deficit is 

### 7
seen in both GABAergic and glutamatergic neurons. Pb also induced an increase in number of 

### 8
presynaptic contact sites. But, these sites may be non

- fun ctional as they lack the protein receptor 

### 9
complexes necessary for proper vesicular exocytosis. Another factor involved in growth and signaling of 

### 10

pre

- synaptic neurons is BDNF, which is synthesized and released by post

- synaptic neurons. BDNF is 

### 11
regulated by the NMDAR and acts in a retrograde fashion, participating in pre

- synaptic maturation. In 

### 12
this model, both pro

- BDNF and BDNF release were significantly attenuated with Pb exposure. Further, 

### 13
exogenous BDNF administration rescued the aforementioned Pb

- depend ent pre

- synaptic effects. Thus, 

### 14

this cell culture model shows that Pb

- dependent pre

- synaptic aberrations are controlled by NMDAR



### 15
dependent BDNF effects on synaptic transmission. 

### 16

5.3.6.6. Blood Brain Barrier

Two barrier systems exist in the body to separate the brai n or the central nervous system from the 

### 17
blood. These two barriers are the blood brain barrier (BBB) and the blood cerebrospinal fluid barrier 

### 18
(BCB). The blood brain barrier, formed by tight junctions at endothelial capillaries forming the zonulae 

### 19

occluden s (occludins, claudins, and cytoplasmic proteins), separates the brain from the blood and its 

### 20
oncotic and osmotic forces, allowing for selective transport of materials across this barrier. Pb exposure 

### 21
during various developmental windows is known to affect the blood brain barrier even at low Pb 

### 22
concentrations resulting in increased permeability ( Dyatlov et al., 1998 ; Moorhouse et al., 1988 ; 

### 23
Struzynska, Walski, et al., 1997 ; Sundstrom et al., 1985 ) . Because the BBB is under

- developed early in 

### 24
life, prenatal and perinatal Pb exposure results in higher brain Pb accumulation than does similar 

### 25
exposures later in life ( Moorhouse et al., 1988 ) . Earlier AQCDs have shown that the chemical form of Pb, 

### 26
and its ability to interact with proteins and other blood components affects its ability to penetrate the BBB 

### 27
( U.S. EPA, 2006 ) . P b compromises the function of the BCB, and decreased the CSF level of transthyretin

, 

### 28
a thryoid binding protein made in the choroid plexus. The choroid plexus and cerebral endothelial cells 

### 29
that form these BBB and BCS tight junctions are known to accumulate Pb more than other cell types and 

### 30
regions of the CNS. Hypothyroid status can contribute to impaired learning and IQ deficits. More recent 

### 31
research with weanling rats fed Pb through Pb acetate drinking water exposure manifested histologically 

### 32
with leaky ce rebral vasculature as detected with lanthum nitrate staining of the brain parenchyma, an 

### 33May 2011 5

- 134 DRAFT

– DO NOT CITE OR QUOTE indication of BBB impairment, that was ameliorated or that resembled controls after iron 

### 1
supplementation. These weanlings also had significant Pb

- induced decreases in the BBB tight junction 

### 2
protein occludin in the hippocampus, brain cortex, and cerebellum that were rescued to control levels with 

### 3
iron supplementation ( Q. Wang et a l., 2007 ) . These data demonstrate that Pb induced a leaky BBB in 

### 4
weanling rats with associated decreases in the junctional protein occludin; dietary supplementation with 

### 5

iron was able to ameliorate these Pb

- induced impairments of the BBB in male rats. 

### 6

5.3.6.7. Cell Adhesion Molecules

Classic cell adhesion molecules including NCAM and the cadherins are junctional or cell surface 

### 7
proteins that are critical for cell recognition and adhesion. Cell adhesion molecules, particularly the 

### 8
cadherins, are calcium

- dependent and thus interaction from competing cations like Pb can contribute to 

### 9
nervous system barrier function disruption, tissue development dysregulation, immune dysfunction, and 

### 10
affect learning and memory ( Prozialeck et al., 2002 )

. 

### 11

5.3.6.8. Glial Effects

Astroglia and oligodendroglia are supporting cells in the nervous system that maintain the 

### 12
extracellular space in the brain and provide support and nutrition to neurons via nutrient transport, 

### 13
strucutral support to neurons, and myelination, among other effects. Glial cells are known to serve as Pb 

### 14
sinks in the developing and mature brain ( Tiffany

- Castiglioni et al., 1989 ) by sequestering Pb. This glial 

### 15
sequestration of Pb has been shown to decrease brain glutamine concentrations at a dose of 0.25 ± 1.0 μ M 

### 16

Pb acetate via Pb

- dependent reduction in glutamine synthestase activity in the astroglia; astroglia take up 

### 17
glutamate after its release and convert it to glutamine. Pb causes hypomyelinati on and demyelination ( F. 

### 18
Coria et al., 1984 ) mediated through the oligodendrocytes with younger animals being more susceptible to 

### 19

the effects of ( Tiffany

- Castiglioni et al., 1989 ) . Unfortunately, Pb accumulation in young glial cells may 

### 20
contribute to a lifelong exposure to this Pb sink in the brain as it is released over time where it can 

### 21
damage surrounding neurons ( Holtzman et al., 1987 )

. 

### 22

Glial transmitters

To determine the contribution of the gliotransmitter serine to Pb mediated changes in long

- term 

### 23
potentiation (LTP), Sun et al. ( 2007 ) performed in vitro patch clamp monitoring of rat hippocampal CA1 

### 24
section LTPs collected from pups exposed to Pb Acetate in utero, lactationally and through drinking water 

### 25

out to PND28. D

- serine supplementation relieved the chronic Pb exposure dependent impaired magnitude 

### 26
of hippocampal LTP ( H. Sun et al., 2007 ) , which is known to be regulated by the NMDAR ( Bear & 

### 27
Malenka, 1994 ) . The use of 7

- chlorokynurenic acid, an antagonist of the glycine binding site of the 

### 28

NMDAR

- the binding site of D

- serine, effectively abolished D

- serine’s rescue of the LTP. NMDAR

- 

### 29May 2011 5

- 135 DRAFT

– DO NOT CITE OR QUOTE independent LTP hippocampal neurotransmissi on with slices from Pb

- exposed mossy

- CA3 synapses was 

### 1
not rescued by exogenous D

- serine supplementation. These data indicate that glial transmission may 

### 2
provide promising therapeutic targets for intervention after Pb exposure or with other affective or 

### 3

cog nitive disorders known to manifest with aberrant NMDAR

- dependent neurotransmission. 

### 4

5.3.6.9. Neurotransmitters

Pb has been shown to compete with calcium for common binding sites and second messenger 

### 5

activation. When Pb activates a calcium

- dependent system in the ne rvous system, it can contribute to 

### 6
aberrant neurotransmitter regulation and release because this system intimately relies on calcium signals 

### 7

for its homeostasis. Pb

- dependent alterations in neurotransmission are discussed in further detail below. 

### 8

Monoamine Neurotransmitters and Stress

Combined exposures of maternal stress and Pb exposure can synergistically enhance behavioral 

### 9
and neurotoxic outcomes in exposed offspring, sometimes even potentiating an effect that would 

### 10

otherwise be sub

- threshold. Virgolini et al. ( 2008 ) found that effects on the central nervous system by 

### 11
developmental Pb exposure (2 months prior to mating through lactation, 50 or 150 ppm Pb aceta te 

### 12
drinking water exposure, blood Pb level 11 μg/dL and 35 μg/dL, respectively) are enhanced by combined 

### 13
maternal and offspring stress. Offspring neurotransmitter concentrations were significantly affected with 

### 14
Pb exposure, but the most interesting finding s were those of potentiated effects or effects that were not 

### 15
seen with Pb exposure alone or stress alone. These potentiated effects were only seen when Pb was 

### 16
combined with stress (maternal [MS] and/or offspring stress [OS]). Potentiation of serotonin (5HT ) levels 

### 17
in females was significant in the frontal cortex in females and in the nucleus accumbens (NAC) in the 

### 18
male offspring (50 and 150 ppm Pb drinking water exposure) ( Cory

- Slechta et al., 2009 ) . Regional 5HT 

### 19
levels were unaffected in offspring with no stressors and Pb exposu re. Thus, Pb alone did not significantly 

### 20
affect 5HT levels. 5HIAA concentration was significantly increased with Pb exposure alone in the 

### 21
striatum of male offspring at 150 ppb Pb exposure; with the remaining exposures, Pb plus stress 

### 22
potentiated striatal a nd frontal cortex 5HIAA in males. Potentiated 5HIAA levels in females were 

### 23
significant in the NAC at both Pb doses; stress alone also significantly increased 5HIAA levels in females 

### 24
with no Pb exposure. Pb

- induced changes in brain neurochemistry with or wi thout concomitant stress 

### 25
exposure are complex with differences varying by brain region, neurotransmitter type and sex of the 

### 26

animal. 

### 27May 2011 5

- 136 DRAFT

– DO NOT CITE OR QUOTE Monoamine Neurotransmitters and Auditory Function

The monoamine neurotransmitters include DA, 5HT, and NE. Earlier work ha s shown that perinatal 

### 1
rat Pb exposure induced increased tyrosine hydroxylase, increased DA and increased cerebral cortex 

### 2

catecholamine neurotransmission ( Bielarczyk et al., 1996 ; C. B. Devi et al., 2005 ; Leret et al., 2002 )

. 

### 3
Earlier publications detailing the window of exposure, duration of exposure and dose of Pb used have 

### 4
varying effects on monoamine transmitters. In more recent work, these neurotransmitters, among others, 

### 5
have been implicated in auditory function in the b rainstem in various integration centers there including 

### 6
the lateral superior olive (LSO), and the superior olivary complex (SOC). The SOC is vital for sound 

### 7
detection in noisy settings among other functions. Low level Pb exposure has been associated with a ltered 

### 8
processing of auditory temporal signals in animal studies ( Finkelstein et al., 1998 ; Lurie et al., 2006 )

. 

### 9
Because Pb alters auditory processing, the monoamine system is a potential target for Pb

- mediated 

### 10

interactions. Blood Pb levels for control, very low Pb (VLPb) and low Pb (LPb) exposure groups are 1.4, 

### 11
8.0, and 42.2 μg/dL, respectively. Developmental Pb exposure from the formation of breeding pairs to 

### 12
PND21, which is at the end of auditory development in the mouse, led to significant decreases in 

### 13
immunostaining of LSO and SOC brainstem sections for monoamine vesicular transporter VMAT2, and 

### 14
for 5HT and dopamine beta

- hyrdoxylase (DbH), a marker for NE. This immunochemistry was significant 

### 15
for both VLPb and LPb exposure for VMAT2 and DbH but 5HT only had significant decrements with 

### 16
VLPb. Immunostaining for TH and transporters including VGLUT1, VGAT, VAChAT indicated that they 

### 17
were unaffected by developmental Pb exposure. These data provide evidence that specific regions of th e 

### 18
brainstem relating to auditory integration with interaction from the monoamine neurotransmitter system 

### 19
are affected by developmental Pb exposure ( Fortune & Lurie, 2009 )

. 

### 20

Dopamine

The 2006 AQCD detailed low dose P b

- dependent decreased dopaminergic cell activity in the 

### 21
substantia nigra and ventral segmental areas. Earlier studies with moderate to high dose postnatal or adult 

### 22
Pb exposure have reported changes in dopamine (DA) metabolism, DA and DOPAC, a DA metabolite

. 

### 23
Thus, these were measured in various brain regions of year old males to determine if GLE affected DA 

### 24
metabolism. Low and high dose GLE in male rodents induced significant elevations in the DOPAC to DA 

### 25
ratio, and DOPAC concentration in the forebrain. In t he forebrain, DA was significantly decreased in low 

### 26
dose GLE males and significantly elevated in GLE high dose males compared to controls. In the striatum, 

### 27
DOPAC was significantly elevated in both low and high dose GLE exposed males, but DA concentration 

### 28

w as only significantly elevated in high dose GLE males. The striatum ratio of DOPAC to DA was not 

### 29
significantly different from control. These new data expand upon the monoamine literature base which 

### 30
reports perinatal rat low concentration Pb exposure induce d increased sensitivity of the dopamine 

### 31
receptors (D2 and D3) ( Cory

- Slechta et al., 1992 ; Gedeon et al., 2001 ) , produced higher DA levels ( C. B. 

### 32May 2011 5

- 137 DRAFT

– DO NOT CITE OR QUOTE Devi et al., 2005 ; Leret et al., 2002 ) , and enhanced catecholamine neurotransmission in the cerebral 

### 1
cortex, cerebellum, and hipp ocampus ( C. B. Dev i et al., 2005 )

. 

### 2
The interaction of dopamine and the nitric oxide system in the striatum was studied after prenatal 

### 3
Pb exposure. Blood Pb was not reported in this study, but similarly treated Wistar rat pups in other studies 

### 4
report blood Pb levels at p arturition in range of 50

- 100 μg/dL ( Grant et al., 1980 ) . 7

- nitroinidazole (7

- NI), 

### 5

a selec tive inhibitor of nNOS, enhanced amphetamine

- evoked dopamine release in the rat striatum 

### 6
( Nowak et al., 2008 ) . Prenatal P b exposure attenuated 7

- NI’s facilitatory effect on dopamine release in the 

### 7

striatum. This interaction is ROS

- independent; using spin trap measurements, there were no significant 

### 8
concentration changes in hydroxyl radical with Pb exposure ( Nowak et al., 2008 ) . Thus, the neuronal NO 

### 9
system appears to be involved in specific aspects of Pb

- dependent dopaminergic changes. 

### 10

Dopami ne and Vision

In various experimental animal models, the loss of retinal dopamine or zinc is associated with 

### 11
supernormal rod

- mediated scotopic electroretinograms (ERGs), pointing to the retina as a sensitive target 

### 12
of low dose Pb exposure (19 μg/dL). In th e human and non

- human primate literature, GLE is associated 

### 13
with increased amplitude (supernormality) of ERGs ( Lilienthal et al., 1994 ; Lilienthal et al., 1988 ; 

### 14
Rothenberg et al., 2002 ) and in the animal toxicology literature postnatal Pb exposure induces 

### 15
subnormality of the ERGs. New research in the animal toxicology literature recapitulated the low dose 

### 16
human literature, showing that low

- and moderate (LPb or MPb) level gestational Pb e xposure in the rat 

### 17
produced supernormal ERGs with associated significant increases in retinal neurogenesis and significant 

### 18
decreases in retinal dopamine use and dopamine turnover, DA and DOPAC:DA ratio, respectively. High 

### 19
gestational Pb exposure (HPb) prod uced significant subnormal ERGs, similar to the findings with 

### 20
postnatal human Pb exposures. Rats (dams) were exposed to Pb acetate in drinking water starting 2 weeks 

### 21
prior to mating and throughout gestation and lactation until PND 10, a period of developme ntal exposure 

### 22
that is equivalent to gestational exposure in humans with peak blood Pb PND1

- 10 of 12, 24, and 46 μg/dL 

### 23
in LPb, MPb, and HPb, respectively. LPb and MPb gestational exposure induced increased cellularity or 

### 24

retinal thickness in the outer nucle ar layer, inner nuclear layer and total retina ( Leasure et al., 2008 ) . In 

### 25
conclusion, the retina is a sensitive site to low

- dose Pb exposure and gestational Pb exposure produced 

### 26

dose

- dependent decreases in DA use and turnover; inverted U shaped Pb dose response curves were 

### 27
reported for retinal endpoints including ERG and retinal thickness. 

### 28

NMDA

NMDA receptors (NMDAR) have been shown to contribute to synaptic plasticity and Pb

- exposure 

### 29
at different developmental stages is known to contribute to a berrations in LTP or LTD in the hippocampus 

### 30
via reduced NMDA current, among other mechanisms ( L. Liu et al., 2004 ) . The 2006 AQCD detailed that 

### 31May 2011 5

- 138 DRAFT

– DO NOT CITE OR QUOTE Pb induced decreases in stimulated gluta mate release that affected LTP. Further, it detailed that the Pb



### 1
dependent decreased magnitude and increased threshold of the LTP in the hippocampus is biphasic or 

### 2

non

- linear. NMDAR subtypes have been shown to be significantly decreased with developmental Pb 

### 3

exposure ( Guilarte & McGlothan, 1998 ) . Recent work looking at supplement use, found Pb

- dependent 

### 4
decreases in message and protein level of NMDAR subunit NR1 was rescued with methioninecholine 

### 5
coexposure in these weanling male rats ( Fan et al., 2010 ) . Fan et al. ( Fan et al., 2010 ) found that Pb



### 6
dependent suppression of the NMDAR subunits NR2A and NR2B were not rescued with 

### 7

methioninecholine treatment. Other recent mechanistic studies have found that pretreatment of primary 

### 8
fetal brain neuronal rat cultures with glutamic acid, a NMDAR agonist, reversed Pb

- dependent reductions 

### 9

in NMDAR subunits ( S.

- Z. Xu & Rajanna, 2006 ) whereas pretreatment with the NMDA antagonist MK



### 10

801 exacerbated Pb

- induced NMDAR defecits ( S.

- Z. Xu & Rajanna, 2006 ) . Thus, glutamic a cid or 

### 11
methioninecholine may offer therapeutic possibilities for Pb

- induced neuronal NMDAR decrements. 

### 12
The Guilarte lab has made extensive contributions to the Pb animal toxicology literature and a 

### 13
recent publication details the effect of low dose develop mental/lifetime Pb exposure on changes in 

### 14
hippocampal neurogenesis in adulthood ( Verina et al., 2007 ) , an emerging area of research affecting long



### 15
term potentiation, spatial learning, neuronal outgrowth, and possibly mood disorders like schizophrenia. 

### 16
NMDAR mediates the integration of new neurons into existing neuronal pathways in the adult 

### 17
hippocampal DG, which is important to learning and memory. Lifetime Pb chow exposure (dam Pb 

### 18

a cetate exposure 10 days pr ior to mating through pregnancy out to PND50 or PND78) induced significant 

### 19
decrements in hippocampal granule cell neurogenesis or proliferation of new cells in adult rats. Also, Pb 

### 20
exposed animals had significant decreases in brain volume in the stratum or iens (SO) region of the 

### 21
hippocampus, specifically significant decreases in the mossy fiber terminals of the SO. A marker for 

### 22
immature or newly formed neurons showed a significant decrease in the length

- density of these cells in 

### 23
the outer portion of the DG in Pb

- exposed animals. These findings show that exposure to environmentally 

### 24
relevant doses of Pb induced significant aberrations in adult hippocampus granule cell neurogenesis and 

### 25
morphology, providing mechanistic explanations for Pb

- induced neuronal aberr ations. Guilarte et al. 

### 26
( 2003 ) demonstrated that Pb exposure of rats from an enriched environment was associated with reversal 

### 27
of learning impairment, increased expression of hippocampal NMDA receptor subunit 1, and increased 

### 28
induction of brain derived neurotrophic factor mRNA ( Guilarte et al., 2003 )

. 

### 29

5.3.6.10. Neurite Outgrowth

The 2006 AQCD reported Pb decreased neurite outgrowth at 20μg/dL noting that Pb interfered 

### 30
with neurite outgrowth via protein kinase mediated pathways (MAPK/ERK); earlier work has 

### 31
documented decreased primary DA neuron outgrowth with 0.001 μM Pb exposure ( Lidsky & Schneider, 

### 32

2004 ) . More recent studies have shown that dam exposure to low dose Pb (blood Pb level 4 μg/dL) of 

### 33May 2011 5

- 139 DRAFT

– DO NOT CITE OR QUOTE dams significantly decreased pup hippocampal neurite outgrowth (pup blood Pb level 12 μg/dL) and 

### 1
reduced the expression of hippocampal polysialylated neural cell adhesion molecule (PSA

- NCAM), 

### 2
NCAM, and sialytransferase; PSA

- NCAM is transiently expressed in newly formed neurons ( Q. S. Hu et 

### 3
al., 2008 ) during the period of neurite outgrowth from embyrogenesis until the early postnatal period and 

### 4

is down

- regulated in the adults e xcept in areas known to exhibit synaptic plasticity ( Seki & Arai, 1993 )

. 

### 5
NCAM is important for memory formation, plasticity and synapse formation and early life Pb exposure in 

### 6
laboratory rodents affects its expression. 

### 7

5.3.6.11. Epigenetics

DNA methyltransferase activity was significantly decreased in co rtical neurons from both monkey 

### 8
(aged animals) and mouse brains (fetal cells exposed to Pb in culture, 0.1 μM Pb) after Pb exposure ( J. F. 

### 9
Wu et al., 2008 ) . DNA methyltransferases catalyze the transfer of a methyl group to DNA and are 

### 10
impor tant in epigenetics (i.e., silencing of genes like tumor suppressors) and imprinting. 

### 11



## 5.3.7. Examination of the Lead Concentration

- Response Relationship

With each successive Pb AQCD and supplement, epidemiologic and toxicological studies find that 

### 12
progressively lower blood Pb levels are associated with cognitive deficits and behavioral impairments. 

### 13
For example, among children, such decrements were observed in association with blood Pb levels in the 

### 14
range of 10

- 15 μg/dL in the 1986 Addendum and 1990 Supplement an d 10 μg/dL and lower in the 2006 

### 15
AQCD ( U .S. EPA, 2006 ) . Furthermore, in the 2006 AQCD, several individual studies, pooled analyses, 

### 16

and meta

- analyses estimated a supralinear blood Pb concentration

- response relationship in children, i.e., 

### 17
greater decrements in cognitive function per incrementa l increase in blood Pb level among children in 

### 18
lower strata of blood Pb levels compared with children in higher strata of blood Pb levels (Figure 5

- 28 

### 19

and Table 5

- 12). While the majority of epidemiologic evidence indicated differences in effect estimates 

### 20

a bove and below 10 μg/dL, several studies of children with mean blood Pb levels less than 5 μg/dL 

### 21
estimated larger effects for children with <5 μg/dL (compared with children with blood Pb levels 5

- 10 

### 22
μg/dL, and >0 μg/dL) ( Tellez

- Rojo et al., 2006 ) , <2.5 μg/dL (compared with children with blood Pb 

### 23

levels <5 μg/dL, <.5 μg/dL, <10 μg/dL, and all subjects) ( Lanphear et al., 2000 ) , and <1.2 μg/dL 

### 24
(maternal plasma Pb, compared with plasma Pb levels >1.2 μg/dL) ( H. Hu et al., 2006 ) . Using data from 

### 25

NHANES 1999

- 1994, Lanphear et al. ( 2000 ) examined differences in effect estimates among multiple 

### 26
strata of blood Pb levels and found the largest deficit in reading score per 1 μg/dL increment in blood Pb 

### 27
among children with blood Pb levels less than 1 μg/dL. As lower concentrations of Pb exposure are being 

### 28
used experimentally, the toxicological literature reports nonlinear concentration

- response relationships for 

### 29May 2011 5

- 140 DRAFT

– DO NOT CITE OR QUOTE some endpoints and similar to the epidemiologic literature, shows larger effects in lower Pb exposure 

### 1

groups. 

### 2 3May 2011 5

- 141 DRAFT

– DO NOT CITE OR QUOTE Reference Exposure Period Outcome Blood Pb strata Lanphear et al. ( 2000 ) Concurrent Reading score All <10 <7.5 <5 <2.5

Bellinger and Needleman ( 2003 ) Early childhood FSIQ >10 <10

Canfield et al. ( 2003 ) Lifetime avg FSIQ All <10

Tellez

- Rojo et al. ( 2006 ) Concurrent Bayley MDI ≥10 <10
5

- 10
<5

Hu et al. ( 2006 ) Prenatal Bayley MDI/10 >1.226 a <1.226 a

Kordas et al. ( 2006 ) Concurrent Math score All
<10

Lanphear et al. ( 2005 ) Concurrent F SIQ ≥10
<10 ≥7.5
<7.5

Schwartz ( 1994 ) Early childhood FSIQ >15 ≤15

Change in Cognitive Score (95% CI)

Note: a = Pb levels measured in plasma of maternal blood during 1 st trimester of pregnancy. FSIQ = full

- scale IQ, MDI = mental development index. Effect estimates are standardized to a 1 μg/dL increase in blood Pb level. Black symbols represent effect estimates among all subjects or in highest blood Pb stratum. Red symbols represent effect estimates in lower blood Pb strata. Effect estimates without error bars are from studies that did not provide sufficient information in order to calculate 95% CIs. Figure 5

- 28. Comparison of associations between blood Pb and cognitive function among various blood Pb strata.

Table 5

- 12. Additional characteristics and quan titative results for studies presented in Figure 5

- 28

Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Blood Pb stratum (μg/dL) Effect Estimate (95% CI) a

La nphear et al. ( 2000 ) 4853 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)

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 mod el adjusted for HOME score (age 10 and 5), child stress, race, maternal IQ, SES, sex, birth order, marital status WI SC

- R at age 10 yr >10 <10 - 0.58 b

- 1.56 bMay 2011 5

- 142 DRAFT

– DO NOT CITE OR QUOTE Study Population/Location Blood Pb Levels (μg/dL) Statistical Analysis Outcome Blood Pb stratum (μg/dL) Effect Estimate (95% CI) a

Canfield et al. ( 2003 ) 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

- Binet at age 3 or 5 yr All <10 - 0.87 (

- 1.19,

- 0 .55)

- 1.37 (

- 2.56,

- 0.17)

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 (p = 0.84) b

- 1.04 (p <0.01) b

- 0.94 (p = 0.12) b

- 1.71 (p = 0.01) b

Hu et al. ( 2006 ) 146 children followed prenatally (1997

- 1999) to age 24 mo Mexico City, Mexico Maternal 1 st trimester plasma Pb median: 1.226 Linear regression model adjusted for plasma Pb in 2 nd trimester, plasma Pb in 3 rd trimester, child 24 mo blood Pb, sex, maternal age, height

- for

- age Z score, maternal IQ Bayley MDI at age 24 mo >1.226 <1.226 - 4.0 b

- 15.0 b

Kordas et al. ( 2006 ) 602 children in 1 st 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, test er, school Math achievement test in 1 st grade all <10 - 0.17 (

- 0.28,

- 0.06)

- 0.42 (

- 0.92, 0.08)

Lanphear et al. ( 2 005 ) 1333 children pooled from Boston, Cincinnati, Cleveland, Mexico City, Port Pirie, Rochester, and Yugoslavia cohorts Median (5 th

95th) Peak: 18.0 (6.2

- 47.0) 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)

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 effec t estimates from individual studies FSIQ measured at schoolage Studies with mean >15 Studies with mean ≤15 - 2.32 (

- 3.10,

- 1.54)

- 3.23 (

- 5.70,

- 0.76)

a Effect estimates are standardized to a 1 μg/dL increase in blood Pb level.
b Investigators did not provide sufficient information in order to calculate 95% CIs.

Using data pooled from seven prospective studies, Lanphear et al. ( 2005 ) fit various types of 

### 1
models to the data and observed that a cubic spline, log

- linear model, and piece

- wise linear model all 

### 2
supported a more negative concentration

- response relationship at lower blood Pb levels. A linear model 

### 3

was found to be inadequate as the polynomial terms for concurrent blood Pb were statistically significant. 

### 4
These findings were corroborated by a separate analysis by Rothenberg and Rothenberg ( 2005 ) w hich 

### 5
found that the log

- linear model fit the relationship between blood Pb level and IQ better than did a linear 

### 6

model. 

### 7
Studies of adults have not widely examined the shape of the relationship between blood or bone Pb 

### 8
level and cognitive performance. In th e various NHANES analyses, only log

- linear models were used to 

### 9

fit the data ( E. F. Krieg, Jr. & Butler, 2009 ; E. F. Krieg, Jr. et al., 2009 ; E. F. Krieg, Jr. et al., 2010 ) . Other 

### 10

st udies examined non linear ity with the use of quadratic terms, penalized splines, or visual inspection of 

### 11
bivariate plots ( Bandeen

- Roche et al., 2009 ; Shih et al., 2006 ; Weisskopf, Proctor, et al., 2007 )

. While 

### 12

there was some evidence for non linearity for some cognitive tests (Figure s 5

- 1 7 and 5

- 18 ), the majority of 

### 13
results suggested linear associations. Shih et al. ( 2006 ) found that a quadratic term for tibia Pb was not 

### 14May 2011 5

- 143 DRAFT

– DO NOT CITE OR QUOTE statistically signif icant and found a linear model fit adequately the relationship between tibia Pb level and 

### 1
various tests of cognitive performance. In contrast to most studies, Wang et al. ( 2007 ) found that among 

### 2

HFE variant carriers , there was a steeper decline in MMSE score at higher tibia Pb levels ( 20

- 25 μg/g

, 

### 3
Figure 5

- 17 ). 

### 4

Attenuation of the concentration

- response relationships at higher exposure or dose levels has been 

### 5
reported in the occupational literature, and explanations have included greater ex posure measurement 

### 6
error, competing risks, saturation of biological mechanisms at higher levels, larger proportion of 

### 7
susceptible populations at lower exposure levels, and variations in other risk factors among exposure 

### 8

levels ( Stayner et al., 2003 ) . Other explanations for nonlinearity include different mechanisms operating 

### 9

at different exposure levels , confounding by omitted or misspecified variables, and the lower incremental 

### 10
effect of Pb due to covarying risk factors such as low SES, poor caregiving environment, and higher 

### 11
exposure to other environmental factors. 

### 12
The contribution of these factors to the supralinear r elationship between blood Pb levels and 

### 13
neurocognitive function has not been examined widely in epidemiologic studies to

- date. However, in 

### 14
several populations, higher blood Pb levels have been measured in susceptible groups such as those with 

### 15
higher povert y, greater exposure to tobacco smoke, lower parental education, and lower birth weight, 

### 16
which argues against a larger proportion of susceptible populations at lower blood Pb levels ( Lanphear et 

### 17
al., 2000 ; Lanphear et al., 2005 ) . It has been suggested that in populations of low SES, poorer caregiving 

### 18

environmen t, and greater social stress, the incremental effect of Pb exposure may be attenuated due to the 

### 19
overwhelming effects of these other risk factors ( J. Schwartz, 1994 ) . Several studies have found 

### 20
significant associations of these sociodemographic risk factors with neurocognitive deficits, an d Miranda 

### 21

et al. ( Miranda et al., 2009 ) found that indicators of SES (i.e., parental education and enrollment in a 

### 22
free/reduced fee lunch program) accounted for larger decrements in EOG scores than did blood Pb level 

### 23
(Figu re 5

- 6). Few studies have compared Pb effect estimates among groups in different sociodemographic 

### 24
strata, and the limited data are mixed. Greater Pb

- associated neurocognitive deficits in low

- SES groups 

### 25
were reported by Bellinger et al. ( 1990 ) . In a meta

- analysis of eight studies, Schwartz ( 1994 ) found a 

### 26
smaller decrement in IQ per 1 μg/dL increase in blood Pb level for studies in disadvantage d populations 

### 27
(

- 2.7 points [95% CI:

- 5.3,

- 0.07]) than for studies in advantaged populations (

- 4.5 points [95% CI:

- 5.6,

- 

### 28
2.8]). It is important to note that blood Pb is associated with deficits in neurocognitive function in both 

### 29
higher and lower SES group s; however, it is unclear what differences there are between groups in the 

### 30
decrement per unit increase in blood Pb and whether these differences can explain the nonlinear dose



### 31
response relationship. 

### 32
Rothenberg and Rothenberg ( 2005 ) formally assesed the influence of residual confounding on the 

### 33

nonlinear blood Pb concentration

- response relationship by comparing model fit between linear and spline 

### 34
transformations (df = 2) of covariates such as maternal IQ, HOME score, and maternal education. 

### 35
Inclusion of covariates as spline functions did not significantly improve model fit either with a linear 

### 36May 2011 5

- 144 DRAFT

– DO NOT CITE OR QUOTE blood Pb term or log blood Pb term, which indicated that their inclusion as linear functions was adequate. 

### 1

The se findings demonstrate that the improved model fit with log

- specification of blood Pb level was not 

### 2
due to residual confounding by covariates. 

### 3
Consistent with the epidemiologic literaure, toxicological studies also find nonlinear relationships 

### 4

between Pb exposure and neurological effects in animals. In particular, multiple studies have shown U

- or 

### 5

inverse U

- shaped curves with lower exposures of Pb having different or often the opposite effect from 

### 6
higher doses. U

- shaped Pb exposure

- responses include rotoar od performance, adult forebrain dopamine 

### 7
levels, amphetamine

- induced motor activity, and latency to fall from rotarod ( Leasure et al., 2008 )

. 

### 8

Inverted U

- shaped Pb dose

- responses include histological findings such as the numbers of rod 

### 9
photorecep tors and bipolar cells, forebrain dopamine use, activity level, and adult body weight ( Leasure 

### 10
et al., 2008 ) as well as ERG wave amplitudes ( Fox et al., 2008 ) and hippocampal neurogenesis ( Fox et al., 

### 11

2008 ; Gilbert et al., 2005 )

. 

### 12
Because toxicological studies typically do not have confounding, exposure measurement error or 

### 13
other epidemiologic influences, i.e., susceptibility, they have permitted assessment of a mechanistic basis 

### 14
for nonlinear Pb exposure

- or dose

- response relationships. Several lines of evidence support the 

### 15
possibility of low

- dose and high dose

- Pb acting thr ough differential activation of mechanisms. For 

### 16
example, in mice, lower Pb exposure (50 ppm) is associated with differential responses of the 

### 17
neurotransmitters dopamine and norepinephrine compared with control treatment and higher doses (150 

### 18
ppm) ( Leasure et al., 2008 ; Virgolini et al., 2005 ) . These differenti al responses of neurotransmitter 

### 19
systems to lower versus higher Pb exposures may provide mechanistic understanding of the nonlinearity 

### 20

of Pb

- induced behavioral changes in animals and may also explain the nonlinear blood Pb

- neurocognitive 

### 21

and neurobehaviora l associations reported widely among children. Additional evidence points to 

### 22
differences in hormonal homeostasis by Pb exposure level. In male mice with chronic Pb exposure 

### 23
(PND21

- 9 months of age), basal corticosterone levels are significantly lower in the 50 ppm exposure 

### 24
group than in the control or 150 ppm Pb exposure group. 

### 25
Additional mechanistic understanding comes from differences in histological changes found in Pb



### 26
exposed animals. Compared with higher Pb exposure, lower Pb exposure stimulates greater induction of 

### 27

c

- fos, a marker of neuronal activation and action potential firing ( Lewis & Pitts, 2004 ) . These findings 

### 28
may underlie the nonlinear association between Pb exposure and learning and the U

- shaped behavioral 

### 29

dose

- responsiveness seen with amphetamine

- induced motor activity in males after GLE ( Leasure et a l., 

### 30

2008 )

. 

### 31
Sensory organ findings in animals also show vastly different outcomes with low versus higher Pb 

### 32
exposure. Higher Pb exposure produces subnormal retinal ERGs and lower Pb exposure produces 

### 33
supernormal ERGs in both children ( Rothenberg et al., 2002 ) and rodents ( Fox & Chu, 1988 ; Fox & 

### 34
Farber, 1988 ; Fox et al., 1991 ) . Inverted U

- shaped dose

- response curves have been seen for rod 

### 35
photor eceptor numbers or neurogenesis ( Giddabasappa et al., 2011 ) and retinal thickness ( Fox et al., 

### 36May 2011 5

- 145 DRAFT

– DO NOT CITE OR QUOTE 2010 ) . Thus, these dichotomous histological findings are coherent with the fun ctional retinal test or the 

### 1
ERG where higher Pb exposure produces subnormal ERGs and lower exposure Pb produces supernormal 

### 2

ERGs. 

### 3
Hierarchical enzyme activity also may explain nonlinear Pb concentrations

- response relationships. 

### 4
The phosphatase enzyme calci neurin has been shown to be inhibited by higher Pb exposure and 

### 5
stimulated by lower Pb exposure ( Kern & Audesirk, 2000 ) . At lower Pb exposure, Pb displaces calcium at 

### 6
its binding sites on calmodulin and by acting as a calmodulin agonist at calcineurin’s catalytic A subunit, 

### 7
stimulates calcineurin activity. At higher Pb exposure , Pb can bind directly to a separate calcium

- binding 

### 8
B subunit, overriding the calmodulin

- dependent effect and turning off the activity of calcineurin. 

### 9
Interestingly, mice with modulated calcineurin expression exhibit aberrant behavior related to 

### 10
schizophr enia or impaired synaptic plasticity and memory ( Zeng et al., 2001 ) . This example of the 

### 11
stimulatory effects of Pb at lower exposure and inhibitory effects at higher exposure gives another 

### 12
example of bio logical plausibility for the nonlinear concentration

- response relationship reported for Pb in 

### 13
multiple studies. 

### 14
The supralinear concentration

- response relationship widely documented for Pb is consistent with 

### 15

the lack of a threshold for Pb

- associated neurol ogical effects as a smaller effect estimate would be 

### 16
expected at lower blood Pb levels if a threshold existed. Schwartz ( 1994 ) explicitly assessed evidence for 

### 17
a threshold in the Boston prospective cohort data by regressing IQ and blood Pb level on potential 

### 18
confounders including age, race, maternal IQ, SES, an d HOME score and fitting a nonparametric 

### 19
smoothed curve to the residuals of both regression models (variation in IQ or blood Pb level not explained 

### 20
by covariates). A 7

- point decrease in IQ was observed over the range of blood Pb residuals below 0, 

### 21

which co rresponds to the mean blood Pb level in the study (6.5 μg/dL). Thus, in the Boston study, the 

### 22
association between blood Pb level and IQ was clearly demonstrated at blood Pb levels below 5 μg/dL. 

### 23
An important limitation of previous studies in terms of chara cterizing the concentration

- response 

### 24
relationship, in particular, identifying whether a threshold exists, has been the limited examination of 

### 25
effects in populations or blood Pb strata with blood Pb levels more comparable to the current U.S. 

### 26
population mean . While Schwartz ( 1994 ) did not find evidence for a thre shold in the Boston study data, 

### 27
the mean blood Pb in that population was 6.5 μg/dL, and 56% of subjects had a blood Pb level >5 μg/dL. 

### 28
Recent studies indicate a downward shift in the distribution of blood Pb levels (i.e., 50% of subjects in the 

### 29

2001

- 2004 N HANES population had a blood Pb <1 μg/dL ( Braun et al., 2008 )

. Additionally, more 

### 30
sensitive quantification methods have improved the detection limits, for example, from 0.6 μg/dL to 0.025 

### 31
μg/dL in NHANES. This has allowed categorization of children in mulitple blood Pb quantiles below 1 

### 32
μg/dL ( Braun et al., 2008 ) . Consequently, the examination of populations with large proportions of 

### 33
subjects at very low blood Pb levels has improved the ability to discern a threshold for Pb

- associated 

### 34
neurological effects. Several recent studies reported associations between blood Pb levels and deficits in 

### 35
neurocognitive and neurobehavioral endpoints in populations with mean blood Pb levels <2 μg/dL ( Braun 

### 36May 2011 5

- 146 DRAFT

– DO NOT CITE OR QUOTE et al., 2008 ; Braun et al., 2006 ; Cho et al., 2010 ; E. F. Krieg, Jr. et al., 2010 )

. In comparisons of various 

### 1
quantiles of blood Pb, Chandramouli et al. ( 2009 ) observed a lower SAT score among children in the U.K. 

### 2

with blood Pb levels 2

- 5 μg/dL compared with children with blood Pb levels 0

- 2 μg/dL. Likewise, 

### 3

Miranda et al. ( 2009 ) re ported lower EOG scores in children in North Carolina with blood Pb levels of 2 

### 4
μg/dL compared with children with blood Pb levels of 1 μg/dL. In the 2001

- 2004 NHANES population, 

### 5

Braun et al. ( 2008 ) found higher odds ratios for conduct disorder and ADHD among children with blood 

### 6

Pb levels 0.8

- 1.0 μg/dL (2nd quartile) compared with childr en with blood Pb levels 0.2

- 0.7 μg/dL (1st 

### 7
quartile). Collectively, these new findings in children, as summarized in this document, do not provide 

### 8

evidence for a threshold for the neurological effects of Pb in the ranges of blood Pb levels examined to



### 9

date

. 

### 10
It is important to note, however, that the lack of a reference population with blood Pb levels 

### 11
reflecting pre

- industrial Pb exposures limits the ability to identify a threshold. Esti mates of “background” 

### 12

blood Pb levels have been garnered from the analysis of ancient bones in pre

- industrialized societies. 

### 13

These studies suggest that the level of Pb in blood in preindustrial humans was approximately 0.016 

### 14
μg/dL ( Flegal & Smith, 1992 ) , approximately 65

- fold lower than that currently measured in U.S. 

### 15
populations and lower than the levels at which neurological effects have been observed (1 μg/dL). Thus, 

### 16
the current evidence does not prec lude the possibility of a threshold existing in the large range of blood 

### 17
levels between 1 μg/dL and preindustrial “background” levels. 

### 18



## 5.3.8. Summary and Causal Determination

The 2006 Pb AQCD concluded that t he collective body of epidemiologic studies provides cl ear and 

### 19
consistent evidence for the effects of Pb exposure on neuro cognitive function in children. This conclusion 

### 20
was substantiated by the coherence of findings across studies of diverse design and populations (varying 

### 21

distribution s of blood Pb levels, SE S, parental intelligence, and quality of caregiving ) that blood Pb 

### 22
levelswere associated with a broad spectrum of neurocognitive and neurobehavioral indices, including 

### 23
cognitive function (IQ)

, higher

- order processes such as language and memory , academic ac hieve ment, 

### 24
behavior and conduct, delinquent and criminal activity , sensory acuities, and changes in brain structure 

### 25
and activity as assessed by MRI or MRS (Figure 5

- 29). Toxicological studies not only provided coherence 

### 26
with similarly consistent findings f or Pb

- induced impairments in parallel tests of learning, behavior and 

### 27
attention, and sensory acuities, but also provided biological plausibility by characterizing mechanisms for 

### 28

Pb

- induced neurological effects (Figure 5

- 29). These mechanisms included Pb

- in duced inhibition of 

### 29
neurotransmitter release, decline in synaptic plasticity, decreases in neuronal differentiation, and 

### 30
decreases in the integrity of the blood

- brain

- barrier. Both epidemiologic studies in children and 

### 31
toxicological studies reviewed in the 2006 Pb AQCD demonstrated neurocognitive deficits in association 

### 32

with blood Pb levels at or below 10 μg/dL , and evidence from both disciplines also supported a nonlinear 

### 33May 2011 5

- 147 DRAFT

– DO NOT CITE OR QUOTE concentration

- response relationship, with greater cognitive or behavioral decrements per unit increase in 

### 1

blood Pb level estimated for lower blood Pb levels or estimated for lower Pb exposures

. Among adults, 

### 2
although associations of blood Pb level with the spectrum of neurological effects (e.g., impairments in 

### 3
memory, attention, mood, bala nce, and motor function) were most consistently observed in 

### 4
occupationally

- exposed adults with blood Pb levels ≥ 14 μg/dL

, studies of adults without occupational Pb 

### 5
exposures indicated associations between biomarkers of cumulative Pb exposure, serial blood Pb or bone 

### 6
Pb measurements, and decrements in cognitive function. 

### 7
Building on the strong body of evidence presented in the 2006 AQCD, recent studies continue to 

### 8
support associations of Pb biomarkers or exposures with neurological effects (Figure 5

- 29). Although 

### 9
fewer in number, recent studies in children corroborate find ings from the several previous longitudinal 

### 10

and cross

- sectional studies in demonstrating associations between blood Pb levels and FSIQ. In the 

### 11
cumulative body of evidence, negative associations between blood Pb level and IQ are best substantiated 

### 12

at mean b lood Pb levels in the range of 5

- 10 μg/dL ; however, an association was observed in a recent 

### 13
study with a mean blood Pb level of 1.73 μg/dL . A majority of recent epidemiologic studies in children 

### 14
has focused on examining specific indices of neurocognitive f unction such as reading and verbal skills, 

### 15
memory, learning, and visuospatial processing and has demonstrated associations with blood Pb levels as 

### 16

low as 2 μg/dL (population mean or quantiles). The consistently positive associations observed between 

### 17

blood Pb levels and this diverse set of neurocognitive indices provides coherence with findings for IQ, a 

### 18
global measure of cognitive function that reflects the integration of these individual domains. Additional 

### 19
coherence for findings in children is derived fro m evidence in animals that blood Pb levels of 11.6 μg/dL 

### 20
and higher are associated with changes in learning and memory (Figure 5

- 29). Recent toxicological 

### 21
studies continue to demonstrate that in utero and early postnatal exposure to Pb is the most sensitiv e 

### 22

window for Pb

- dependent neurological effects. In the 2006 Pb AQCD, uncertainty was noted among 

### 23

studies in children regarding the relative importance of prenatal, early life, concurrent, and lifetime 

### 24

measures of Pb exposures. It also was noted that distin guishing among the effects of Pb exposures at 

### 25
different lifestages is difficult in epidemiologic studies due to the high correlations among children’s 

### 26
blood Pb levels over time. Although blood Pb levels at all of these lifestages are associated with 

### 27

neuroc ognitive deficits in children, stronger effects generally are estimated for concurrent blood Pb 

### 28
levels, and recent evidence indicates that among both children with relatively lower or higher early 

### 29

childhood blood Pb levels

, concurrent blood Pb levels are more strongly associated with neuro cognitive 

### 30

deficits . In addition to performance on various neurocognitive tests, recent epidemiologic studies in 

### 31
children link blood Pb levels (in quantiles as low as 2 μg/dL ) with factors that may be indicators of 

### 32

childr en’s life success, including the level of educational attainment and end

- of

- grade score. In particular, 

### 33

observations of lower 4 th grade end

- of

- grade score among children with blood Pb levels of 2 μg/dL 

### 34
compared with children with blood Pb levels of 1 μg/dL indicate that a threshold may not exist for the 

### 35
neurodevelopment effects of Pb in children. 

### 36May 2011 5

- 148 DRAFT

– DO NOT CITE OR QUOTE Recent studies in children continue to support associations of blood Pb levels (population means or 

### 1
quantiles of 3

- 11 μg/dL ) with a range of behavioral problems fr om anxiety and distractability to conduct 

### 2
disorder and delinquent behavior (Figure 5

- 29). Whereas previous evidence was not compelling, new 

### 3
evidence indicates associations between blood Pb levels and ADHD diagnosis and contributing diagnostic 

### 4
indices. In p articular, a recent NHANES analysis demonstrated associations at blood Pb levels between 1 

### 5

and 2 μg/dL ). These findings for ADHD are well

- supported by observations in animals of Pb

- induced 

### 6
increased response rates and impulsivity. Additional coherence is p rovided by evidence in aquatic and 

### 7
terrestrial species for Pb affecting behaviors that decrease the ability of organisms to escape predators or 

### 8
capture prey (Chapter 7.1, 7.2). Both epidemiologic studies in children and adults as well as toxicological 

### 9

stud ies demonstrate associations of Pb biomarkers or exposure with deficits in visual acuity and hearing 

### 10
and auditory processing. New evidence from toxicological studies demonstrates the effects of lower blood 

### 11
Pb levels (<15 μg/dL) with retinal changes in male offspring. Combined evidence for Pb

- associated 

### 12
neurocognitive deficits, inattention, conduct disorder, and effects on sensory function provides plausible 

### 13
mechanisms by which Pb exposure may contribute to academic underachievement and to more serious 

### 14
probl ems of delinquent behavior. 

### 15
Studies in adults without occupational exposure to Pb have not provided consistent evidence for 

### 16
associations or blood or bone Pb levels with the range of neurological effects. Levels of Pb in bone, 

### 17
particularly in tibia, which i s an indicator of cumulative Pb exposure, including higher exposures in the 

### 18
past, are better predictors of cognitive performance rather than a single blood Pb measurement. One 

### 19
explanation for the overall weaker body of evidence may be that cognitive reserv e may compensate for 

### 20
the effects of Pb exposure on learning new information. Compensatory mechanisms may be overwhelmed 

### 21
with age, which may provide an explanation for more consistent associations between biomarkers of 

### 22
cumulative Pb exposure (serial blood m easurements, tibia Pb levels) and neurocognitive deficits. Among 

### 23
recent studies of adults, blood Pb levels and bone Pb levels have been associated with essential tremor 

### 24
and Parkinson’s Disease, respectively. These findings are well

- supported by toxicologic al evidence for 

### 25

Pb

- induced decreased dopaminergic cell activity in the substantia nigra, which contributes to the primary 

### 26
symptoms of Parkinson’s disease. Biological plausibility also is provided by observations of 

### 27
developmental Pb exposures of monkeys and rats resulting in neurodegeneration in aged brains. Recent 

### 28
evidence also indicates associations between early

- life ALAD activity, a biomarker of Pb exposure, and 

### 29
schizophrenia later in adulthood. Consistent with these findings, toxicological studies have observed Pb



### 30
induced emotional changes in males and depression changes in females. It is not surprising that Pb 

### 31
exposure may increase the risk of different neurological endpoints in children and adults given the 

### 32
predominance of different neurological proces ses operating at different ages, in particular, neurogenesis 

### 33
and brain development in children and neurodegeneration in adults. 

### 34
Several host and environmental factors may modify the association between Pb exposure and 

### 35
neurological effects in children. I nt eract ions of blood Pb levels with race/ethnicity and SES continue to be 

### 36May 2011 5

- 149 DRAFT

– DO NOT CITE OR QUOTE poorly characterized . Although the 2006 Pb AQCD cited mixed epidemiologic evidence for effect 

### 1
modification by sex , recent epidemiologic evidence points to males having increased suscep tibility for 

### 2

Pb

- associated neuro logical effects

. Toxicological studies continue to demonstrate increased susceptibility 

### 3
of males for endpoints such as sensory function, balance, stress hormone homeostasis, and brain 

### 4
membrane composition. Although limited, evidence suggests that risk of Pb

- associated neuro cognitive 

### 5

deficits in children also may be modified by variants in genes for apolipoprotein E and dopamine 

### 6
receptors. In addition to host factors, recent studies suggested th at associations between blood Pb levels 

### 7
and neurological effects in children are greater with coexposures to environmental tobacco smoke and 

### 8

manganese. Historical animal toxicology findings demonstrate interactions between Pb exposure and 

### 9
stress. Namely, Pb

- exposed animals reared in cage s with enriched environments (toys) perform better in 

### 10

the Morris water maze than their Pb

- exposed littermates who were reared in isolation. New findings 

### 11
indicate a potentiating effect of stress on behavior and memory with lower Pb exposures. In comparison

, 

### 12
epidemiologic evidence for such interactions has been sparse. However, consistent with historical animal 

### 13
studies, a recent study indicated that positive social environment of children as characteri zed by maternal 

### 14

self

- esteem, attenuates the negative assoc iation between blood Pb level and cognitive function . While 

### 15
effect modification by these host and environmental factors has not been examined widely in 

### 16
epidemiologic studies, new studies provide information on potentially susceptible populations that may 

### 17

b enefit from early intervention to reduce the risk of neurological effects

. Furthermore, the robust evidence 

### 18
for varying susceptibilities to Pb

- induced neurological effects provides a basis to integrate 

### 19
mechanistically the findings of toxicology and epidemi ology. 

### 20
Extensive evidence from toxicological studies clearly substantiates the biological plausibility for 

### 21
epidemiologic findings by characterizing mechanisms underlying neurological effects. Pb exposure of 

### 22
animals induces dopamine changes in animals (Figu re 5

- 29). Dopamine plays a key role in cognitive 

### 23
functions mediated by the prefrontal cortex and also motor functions mediated by the substantia nigra, 

### 24
and animal toxicological findings provide mechanistic support for associations in humans between blood 

### 25

P b levels and neurocognitive deficits and in adults for associations with Parkinson’s Disease. Current 

### 26
toxicological research has been expanded to document that early

- life Pb exposure can contribute to 

### 27
neurodegeneration and neurofibrillary tangle formation in the aged brain. Pb induces complex 

### 28
neurochemical changes in the brain that differ by region of the brain, neurotransmitter type, age and sex 

### 29
of the organism. These changes remain aberrant over time but are dynamic in nature. The effect of Pb on 

### 30

NMDA rec eptors and the contribution of this paradigm to mood disorders is detailed. Synapse formation, 

### 31
adhesion molecules, and nitrosive stress continue to be areas of research with known Pb

- associated 

### 32
adverse outcomes. Finally, the new area of epigenetics details that Pb exposure affects methylation 

### 33
patterns in rodent brains. These toxicological data complement the expanding epidemiologic data and 

### 34
often provide coherence between the two fields. 

### 35May 2011 5

- 150 DRAFT

– DO NOT CITE OR QUOTE In summary, recent evidence substantiates and expands upon the establi shed epidemiologic and 

### 1
toxicological literature of neurologic effects associated with Pb exposure. In epidemiologic studies of 

### 2
children, consistently positive associations of blood Pb levels with deficits in neurocognitive function, 

### 3
attention, and sensory acuities support observed associations with school performance as assessed by end



### 4

of

- grade scores and level of educational attainment, which in turn, may explain associations with 

### 5
delinquent and criminal behavior. In particular, observations of lower acade mic achievement and ADHD 

### 6
among children in quantiles of blood Pb levels in the range of 1 to 2 μg/dL have not indicated that a 

### 7
threshold exists for the neurodevelopment effects of Pb in children. Epidemiologic findings are 

### 8
strengthened by the coherence and biological plausibility provided by toxicological findings for similar or 

### 9
parallel endpoints and for the mechanisms underlying the neurological effects (Figure 5

- 29). The 

### 10
collective body of evidence integrated across epidemiologic and toxicological studie s and across the 

### 11
spectrum of neurological endpoints is sufficient to conclude that there is a causal relationship between 

### 12

Pb exposure s and neurological effects. 

### 13May 2011 5

- 151 DRAFT

– DO NOT CITE OR QUOTE Figure 5

- 29

. Snapshot of evidence for the spectrum of effects to the nervous system associated with Pb exposure. Green=animal toxicological studies (left side); purple=epidemiological studies (right side).May 2011 5

- 152 DRAFT

– DO NOT CITE OR QUOTE 

# 5.4. Cardiovascular Effects

5.4.1. Introduction

Both human and animal studies provide consistent evidence for an association of increased BP and 

### 1

a rterial hypertension with chronic exposure to Pb resulting in adult blood Pb levels below 5 μg/dL. In 

### 2
addition, studies have suggested a connection between measures of Pb exposure and other cardiovascular 

### 3
diseases in adults such as ischemic heart disease, cerebrovascular disease, peripheral vascular disease, and 

### 4
cardiovascular disease related mortality. Toxicological studies explore the underlying mechanisms by 

### 5
which Pb exposure can lead to human cardiovascular health outcomes. Such studies have demonstrate d 

### 6

that the Pb content in heart tissue reflects the increases in blood Pb levels ( Lal et al., 1991 )

. In general, 

### 7

assoc iations between blood Pb and bone Pb (particularly in the tibia) with health outcomes in adults 

### 8
indicate acute effects of recent dose and chronic effects of cumulative dose, respectively. In some 

### 9
physiological circumstances of increased bone remodeling or loss (e.g., osteoporosis and pregnancy), Pb 

### 10
from bone of adults may also contribute substantially to blood Pb concentrations. Additional details on 

### 11
the interpretation of Pb in blood and bone are provided in Section 4.3.5. Additionally, as the 

### 12

cardiovascula r and renal system s are intimately linked , cardiovascular effects can arise secondarily to Pb



### 13
induced renal injury (Section 5.5). 

### 14
The previous Pb AQCD ( U.S. EPA, 2006 ) concluded that both epidemiologic and animal 

### 15
toxicological s tudies support the relationship between increased Pb e xposure and increased cardiovascular 

### 16
outcome, including increased BP , increased incidence of hypertension , and cardiovascular morbidity and 

### 17
mortality. Meta

- analysis of these human studies found that e ach doubling of blood Pb level (between 1 

### 18

and > 40 μg/dL ) was associated with a 1 mmHg increase in systolic BP and a 0.6 mmHg increase in 

### 19

diastolic BP ( Nawrot et al., 2002 )

. On a population

- wide bas is, the measured effect size translates into a 

### 20
large number of events for a moderate population size and thus has important health consequences for the 

### 21
occurrence of stroke, myocardial infarction, and sudden death. It was also noted that most of the review ed 

### 22
studies using cumulative Pb exposure measured by bone Pb also showed increased BP ( Y. Cheng et al., 

### 23

2001 ; H. Hu et al., 1996 ) or increased hypertension with increas ing bone Pb ( B.

- K. Lee et al., 2001 )

. Over 

### 24

a range of bone Pb concentration s ( <1.0 to 96 μg/g ) , every 10 μg/g increase in bone Pb was associated 

### 25

with increased odds ratio of hypertension between 1.28 and 1.86, depending upon the study. Two studies 

### 26

observed averaged increased systolic BP of ~0.75 mmHg for every 10 μg/g increase in bone Pb 

### 27 concentration over a range of <1 to 52 μg/g. Since bone Pb measures Pb accumulation over time, duration 

### 28
of past exposure to Pb plays a role in increased BP. 

### 29
The previous Pb AQCD also provided compelling evidence for a number of mechanisms leading to 

### 30
increased BP, and the development of hypertension and other cardiovascular diseases observed after Pb 

### 31
exposure. The strongest evidence supported the role of oxidative stress in the pathogenesis of Pb

- induced 

### 32May 2011 5

- 153 DRAFT

– DO NOT CITE OR QUOTE hypertensi on. Additionally, several studies focused on other pathways or cellular, molecular, and tissue 

### 1
events promoting the Pb

- induced increase in BP. These mechanisms include inflammation, adrenergic and 

### 2
sympathetic activation, renin

- angiotensin

- aldosterone syste m (RAAS) activation, vasomodulator 

### 3
imbalance, and vascular cell dysfunction. Studies continue to support the observed increase in BP and 

### 4
hypertension development following Pb exposure, as well as build on the evidence for the biological 

### 5
pathways of these e ffects. This section reviews the published studies pertaining to the cardiovascular 

### 6
effects of Pb exposure in experimental animals, isolated vascular tissues, cultured vascular cells, and 

### 7
humans. Emphasis has been placed on studies published since the 2006 AQCD ( U.S. EPA, 2006 )

, 

### 8

howeve r the large body of evidence that existed prior to that review has been summarized and 

### 9
incorporated into the current review. 

### 10



## 5.4.2. Blood Pressure and Hypertension

5.4.2.1. Epidemiology

The most commonly used indicator of cardiovascular morbidity is increased BP and its derived 

### 11
index, hypertension. Hypertension diagnoses in these studies require that the patient or subject have 

### 12
diastolic and/or systolic BP above certain cut

- points or be taking anti

- hypertensive medicines. These BP 

### 13

cut

- points have historically been establi shed by reference to informed medical opinion and as medical 

### 14
knowledge improves BP cut

- points defining hypertension have been lowered over time. Consequently, 

### 15
different studies using “hypertension” as a cardiovascular outcome may assign different cut

- point s, 

### 16
depending on the year and location of the study and the individual investigator. All of the new studies in 

### 17
the current review used the same criteria for hypertension (e.g., systolic BP at or above 140, diastolic at or 

### 18
above 90 or taking anti

- hypertensiv e medications). Studies in the medical literature show that increasing 

### 19
BP is associated with increased rates of cardiovascular disease including coronary disease, stroke, 

### 20
peripheral artery disease, and cardiac failure. Coronary disease (i.e. myocardial inf arction, angina 

### 21
pectoris, sudden death) is the most lethal sequela of hypertension ( Chobanian et al., 2003 ; Ingelsson et al., 

### 22

2008 ; Kannel, 2000a

, 2000b ; Neaton et al., 1995 ; Pastor

- Barriuso et al., 2003 ; Prospective Studies 

### 23
Collaboration, 2002 )

. 

### 24
Several recent general population and occupational cohort studies examined the associations of 

### 25

blood Pb and/or bone Pb with BP ( Figure 5

- 30 and Table 5

- 13) as well as the associations of these Pb 

### 26
exposure metrics with hypertension ( Figure 5

- 31 and Table 5

- 14) . In a cross

- sectional analysis, Martin et 

### 27

al. ( 2006 ) examined the association of blood and tibia Pb with BP and hypertension in a community

- based 

### 28

cohort study of older adults (n = 964)

. Four models evaluated associations for BP and hypertension 

### 29
considering SES and race/ethnicity. Blood Pb but not tibia Pb was a strong and significant predictor of BP 

### 30
in all models with an approximately 1 mmHg increase in systolic BP with each 1 μg/dL increase in blood 

### 31May 2011 5

- 154 DRAFT

– DO NOT CITE OR QUOTE Pb level and an approximately 0.5 mmHg increase in diastol ic BP per 1 μg/dL increase in blood Pb level

. 

### 1

Tibia Pb but not blood Pb was associated with hypertension in logistic regression models. T he authors 

### 2
applied propensity analysis to their models to better account for the effect of risk factors such as 

### 3

race/et hnicity, age and SES that were strongly associated with tibia Pb level

. T he propensity score 

### 4
analysis and model adjustment did not substantially change the numerical findings and conclusion s (e.g. 

### 5

tibia Pb and hypertension were positively associated indepe ndent of race/ethnicity and socioeconomic 

### 6

status ). No evidence for effect modification by race/ethnicity was found. Overall, the results suggest that 

### 7
Pb has an acute effect on BP as a function of recent dose and a chronic effect on hypertension risk as a 

### 8

f unction of cumulative exposure. 

### 9

Reference Strata Pb Distribution a Exposure Metric b

Martin et al. ( 2 006 ) 2.9 (2.0, 4.4) Blood Pb Scinicariello et al. ( 2010 ) NHANES III

– Whites 1.6 (0.8, 3.3) Blood Pb NHANES III

– Blacks 1.4 (0.6, 3.6) Blood Pb NHANES III

– Mexicans 2.0 (1.0, 3.9) Blood Pb Weaver et al. ( 2008 ) Korean Workers 27.2 (19.3, 28.3) Blood Pb Glenn et al. ( 2006 ) Korean Workers Blood Pb (concurrent) Blood Pb (longitudinal) Martin et al. ( 2006 ) 15.7 (10.5, 23.5) Tibia Pb Peters et al. ( 2007 ) High Stress 18.1 (12.2, 26.9) Tibia Pb Low Stress 18.1 (12.2, 26.9) Tibia Pb Glenn et al. ( 2006 ) Korean Workers Tibia Pb (historical) Weaver et al. ( 2008 ) Korean Workers 74.3 (67.3, 82.0) Patella Pb Peters et al. ( 2007 ) High Stress 26.9 (18.4, 39.3) Patella Pb

Scinicariello ( 2010 ) NHANES III

– Whites 1.6 (0.8, 3.3) Blood Pb NHANES III

– Blacks 1.4 (0.6, 3.6) Blood Pb NHANES III

– Mexicans 2.0 (1.0, 3.9) Blood Pb Martin et al. ( 2006 ) 2.9 (2. 0, 4.4) Blood Pb 15.7 (10.5, 23.5) Tibia Pb

Zhang et al. ( 2010 ) HFE Wild

- type 18 (12, 27) Tibia Pb H63D variant 19 (14, 26) Tibia Pb C282Y variant 20 (14, 27) Tibia Pb Any HFE variant 19 (14, 27) Tibia Pb HFE Wild

- type 26 (17, 34) Patella Pb H63D variant 27 (19, 37) Patella Pb C282Y variant 25 (17, 37) Patella Pb Any HFE variant 26 (18, 37) Patella Pb

Slope of BP (mmHg) per μg/dL BLL at 1 μg/dL or per μg/g bone Pb

Note: a Pb distribution is the median (IQR) estimated to make comparable. b Effect estimates were standardized to 1 μ g/dL blood Pb or 10 μ g/g bone Pb. Figure 5

- 30. Slope of BP (mmHg) per μ g/dL blood Pb level at 1 μ g/dL or per 10 μ g/g bone Pb (95% CI) for associatio ns of blood Pb (closed circles) and bone Pb (open circles) with systolic BP (SBP; blue), diastolic BP (DBP; red), and pulse pressure (PP; purple).May 2011 5

- 155 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 13. Additional characteristics and quantitative data for associations of blood and bone Pb with BP me asures for results presented in Figure 5

- 30

Study Population /Location Parameter Pb Data Statistical Analysis Effect Estimate (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 techn ician, serum total cholesterol. SES, race/ ethnicity also included in select model s that are presented in Figure 5

- 30 and tabulated here.) Blood Pb SBP: β=1.05 (0.53,1.58) DBP; β=0.53 (0.25, 0.81)

Tibia Pb: SBP: β=0.07 (

- 0.05, 0.14) DBP: β=0.05 (

- 0.02, 0.08)

mmHg per μg Pb /dL blood mmHg per μg Pb/g bone

Glenn et al. ( 2006 ) 575 Pb exposed workers, age 18

- 65 yr, in South Korea (10/1997

- 6/2001) BP Blood Pb mean (SD): Vis it 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, base line lifetime alcohol consumption, baseline body mass index, sex, baseline BP lowering medication use, alcohol consumption, body mass index, sex, BP lowering medication use. Model 1 (short

- term) Blood Pb concurrent β=0.08 (

- 0.01, 0.16) Blood Pb (longitudin al) β=0.09 (0.01, 0.16)

Model 4: short and longer

- term) Blood Pb concurrent β=0.10 (0.01, 0.19) Blood Pb longitudinal: β=0.09 (0.01, 0.16)

Per 10 μg/dL blood Pb

Weaver et al. ( 2008 ) 652 current and former Pb workers in South Korea (12/1999

- 6/2001) BP Blood Pb: Mean (SD): 3 0.9 (16.7) μg/dL

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 β=0.0059 (p=0.41) Blood Pb β=0.1007 (p=0.01)

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 Normative Aging Study 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 of smoking, alcohol consumption, and physical activity SBP Tibia Pb/ High Stress: β =3.57 (0.39, 6.75) Low Stress: β = - 0.21 (

- 1.70, 1.29) per SD increase in tibia Pb Patella Pb/ High Stress: β =2.98 (

- 0.12, 6.08) per SD increase in tibia Pb Patella Pb/ Low Stress: NRMay 2011 5

- 156 DRAFT

– DO NOT CITE OR QUOTE Study Population /Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI)

Scinicariello et al. ( 2010 ) 6,016 NHANES III participants ≥ 17 y r BP Blood Pb: Overall Mean (SE): 2.99 (0.09) μg/dL Non

- Hispanic Whites: 2.8 7 (0.09) Non

- Hispanic Blacks 3.59 (0.20) Mexican American 3.33 (0.11) Multivariable linear regression of log

- transformed b lood Pb level adjusted for age, sex, education, smoking status, alcohol intake, BMI, serum creatinine levels, serum calcium, glycosyla ted hemoglobin, and hematocrit Ln blood Pb SBP Non

- Hispanic whites: β ±SE=1.05±0.37 (p=0.01) Non

- H ispanic blacks: β ±SE=2.55±0.49 (p=0.001) Mexican Americans: β ±SE=0.84±0.46 (p=0.08)

DBP Non

- Hispanic whites: β ±SE=

- 0.14±0.49 (p=0.77) Non

- Hispanic blacks: β ±SE=1.99±0.44 (p=0.0002) Mexican Americans: β ±SE=0.74±0.38 (p=0.06)
Significant i nteractions with blood Pb and ALAD observed in relation to SBP for non

- Hispanic whites and non

- Hispanic blacks

Zhang et al. ( 2010 ) 619 older adult males (mean 67 y r ) enr olled in the VA

- NAS in Greater Boston, MA

area PP Wild type Tibia Pb: Med (IQR):8(12

- 27) μg/g Patella Pb: Med(IQR):26(17

- 37) μg/g C282Y Tibia Pb: Med(IQR):20 (14

- 27) μg/g Patella Pb: Med(IQR):25(17

- 37) μg/g H63D Tibia Pb: Med(IQR):19(14

- 26) μg/g Patella Pb: Med(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, sodium, and potassium; total calories; family history of hypertension; diabetes; he ight; heart rate; high

- density lipoprotein (HDL); total cholesterol:HDL ratio; and waist circumference PP Tibia Pb per 13 μg/g: Wild Types: β=0.38 (0,1.96) H63D: β=3.30 (0.16, 6.46) C282Y: β=0.89 (0, 5.24) Any HFE: β=2.90 (0.31, 5.51)

Patella Pb per 19 μ g/g: Wild Type: β=0.26 (0, 1.78) H63D: β=2.95 (0, 5.92) C282Y: β=0.55 (0, 1.66) Any HFE: β=2.83 (0.32,5.37)

Perlstein et al. ( 2007 ) 593 predominantly white men from VA

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 rate, waist circumference, diabetes, family history of hypertension, education level achieved, smoking, alcohol intake, fasting plasma glucose, and ratio of total cholesterol to HDL cholesterol Tibia Pb: SBP r=0.06 (p=0.15) DBP r=

- 0.02 (p=0.63) Blood Pb : SBP r=0.05 (p=0.28) DBP r=0.12 (p=0.01)

Pulse Pressure Tibia Pb: >Median: 4.2 (1.9, 6.5) mmHg (mean higher than men below the median) <Median: Referent 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 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: ReferentMay 2011 5

- 157 DRAFT

– DO NOT CITE OR QUOTE Study Population /Location Parameter Pb Data Statistical Analysis Effect Estimate (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 I nverse variance weighted random

- effects meta

- analyses Pooled Estimates per 10 μg/g increase in Tibia Pb Prospective/SBP β =0.33 (

- 0.44, 1.11) X

- sectional SBP β =0.26 (0.02, 0.50) X

- sectional DBP β =0.02 (

- 0.15, 0.19

x

- Sectional hypertension OR=1.04 (1.01, 1.07) Pooled Estimates per 10 μg/g increase in patella Pb hypertension OR=1.04 (0.96, 1.12)

Gump et al. ( 2007 ) 122 children age 9.5 yr in Oswego, NY BP, TPR (total peripheral vascular resistance) Blood Pb: Mean (SD): 4.6 (2.5) μg/dL Pb is a me diator/modifier and moderator in the analysis, no effects presente d

Yazbeck et al. ( 2009 ) 971 pregnant women, age 18

- 45 yr, in France BP Blood Pb: PIH group mean (SD): 2.2 (1.4) No PIH group mean (SD): 1.9 (1.2) Multivariable l ogistic 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 consumption before and durin g 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 ) 471 elderly men (mean 67 yr) from Normative Aging Study 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: β=0.40.(0.11, 0.70)

Low calcium group (<800 mg/d): SBP: β=0.19 (0.01, 0.37)
mmHg per μg/g tibia Pb

References not included in Figure 5

- 30 are included in this table.May 2011 5

- 158 DRAFT

– DO NOT CITE OR QUOTE Reference Study Strata Blood Pb (μg/dL) Comparison

Scinicariello et al. ( 2010 ) NHANES III Non

- H ispanic Whites 0.7

- 1.4 Q1 Reference 1988

- 1994 1.5

- 2.3 Q2 v Q1 age ≥ 17 y 2.4

- 3.7 Q3 v Q1 Mean blood Pb = 3.8

- 52.9 Q4 v Q1 2.99 μg/dL 2.4

- 3.7 ALAD2 v 1 b
Non

- H ispanic Blacks 0.7

- 1.4 Q1 Reference 1.5

- 2.3 Q2 v Q1 2.4

- 3.7 Q3 v Q1 3.8

- 52.9 Q4 v Q1 2.4

- 3.7 ALAD2 v 1 b
Mexican Americans 0.7

- 1.4 Q1 Reference 1.5

- 2.3 Q2 v Q1 2.4

- 3.7 Q3 v Q1 3.8

- 52.9 Q4 v Q1 2.4

- 3.7 ALAD2 v 1 b
Park et al. ( 2009 ) NHANES III Overall 3.52 (0.10) White Men Mean blood Pb = 3.52 μg/dL Black Men White Women Black Women Men <50 Men > 50 Women < 50 Women >50 Muntner et al. ( 2005 ) NHANES III Non

- H ispanic Whites <1.06 Q1 Reference Mean blood Pb = 1999

- 2002 1.06

- 1.63 Q2 v Q1 1.64 μg/dL 1.63

- 2.47 Q3 v Q1 >2.47 Q4 v Q1 Non

- H ispanic Blacks <1.06 Q1 Reference 1.06

- 1.63 Q2 v Q1 1.63

- 2.47 Q3 v Q1 >2.47 Q4 v Q1 Mexican Americans < 1.06 Q1 Reference 1.06

- 1.63 Q2 v Q1 1.63

- 2.47 Q3 v Q1 >2.47 Q4 v Q1 Martin et al. ( 2 006 ) c Baltimore, MD 3.5 (2.3) Yazbeck et al. ( 2009 ) France Pregnant Women Q1 Reference 1.2

- 1.7 Q2 v Q1 1.71

- 2.30 Q3 v Q1 >2.30 Q4 v Q1

Tibia Pb ( μg/g )

Martin et al. ( 2006 ) c Baltimore, MD 18.8 (12.4) Peters et al. ( 2007 ) d Boston, MA High Stress 21.5 (13.4)

Patella Pb (μg/g)

High Stress 31.5 (19.3)

Odds Ratio (95% CI)

Note: a The outcomes plotted are hypertension with the exception of Yazbeck et al. ( 2009 ) which measured pregnancy induced hypertension and Peters et al. ( 2007 ) which measured hypertension incidence. b ALAD2 v 1 indicates comparison between ALAD 2 carriers (e.g. ALAD1

- 2 and ALAD2

- 2) and ALAD 1 homozyg otes (e.g., ALAD1

- 1). c Effect estimates were standardized to 1 μ g/dL blood Pb. d Effect estimates were standardized to 1 μ g/g bone Pb. Figure 5

- 31. Odds ratio (95% CI) for associations of blood and bone Pb with hypertension measures.May 2011 5

- 159 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 14. Additional characteristics and quantitative data for associations of blood and bone Pb with hypertension measures for results presented in Figure 5

- 31

Study Population/ Location Parameter Pb Data S tatistical Analysis Effect Estimate (95% CI)

Scinicariello et al. ( 2010 ) 6,016 NHANES III participants ≥ 17 y Hypertension (cur rent use of antihypertensive medication, SBP ≥ 140 mmHg, or DBP ≥ 90 mmHg) 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 Whites: 2.87 (0.09) Non

- Hispanic Blacks 3.59 (0.20) Mexican American 3.33 (0.11) Multivariable l ogistic 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 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

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

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

POR for hypertension with ALAD2 carriers across quartiles of blood Pb level also reported. ALAD2 carriers associated with hypertension in non

- Hispanic whites.
Park et al. ( 2009 ) 12,500 NHANES III participants Hypertension NHAN ES III 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) L ogistic 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’s 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) M en < 50 yr 0.98 (0.80, 1.22) M en > 50 yr 1.20 (1.02, 1.41), W omen < 50 yr 1.23 (1.04, 1.46), W omen > 50 yr 1.09 (1.94, 1.26).May 2011 5

- 160 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Parameter Pb Data S tatistical 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) 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 logistic regression models adjust ed for age, sex, diabetes mellitus, BMI, cigarette smoking, alcohol consumption, high school education, and health insurance status Adjusted OR of having a blood Pb level of 10 μg/dL Non

- Hispanic white: Q1 reference 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 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 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)
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 antihy pertensive medication, mean SBP ≥ 140 mmHg or DBP ≥ 90 mmHg) Blood 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 homocysteine Blood Pb level OR=1.02 (0.87, 1.19) Tibia Pb OR=1.24 (1.05, 1.47) mmHg per μg Pb /dL blood mmHg per μg Pb/g bone

Peters et al. ( 2007 ) 513 elderly men (mean 67 y) from Normative Aging Study 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

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: 1.2 0

- 1.70 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, pari ty, hematocrit, BMI, gestational diabetes, educational levels, SES, geographic residence, and smoking status during pregnancy PIH Log Blood Pb OR=3.29 (1.11, 9.74) per 1 μg/dL maternal blood Pb level

Q1: reference Q2: OR 1.84 (0.77, 4.41) Q3: OR=2.07 (0.83, 5.13) Q4: OR=2.56 (1.05, 6.22)

Weaver et al. ( 2008 ) 652 current and former Pb workers in South Kore a (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 f or age, gender, BMI

, diabetes, antihypertensive and analgesic medication use, Pb job duration, work status, tobacco and alcohol use No ne of the Pb exposure metrics examined were (blood, patella, and ln patella) w ere significantly associated with hypertens ion (results not reported)

Chen et al. ( 2006 ) 2,994,072 pregnant women in United States (1998) PIH (gestational hypertension as increased SBP of ≥ 30 mmHg or DBP of ≥ 15 mmHg after 20th wk of gestation) Pb in TSP Seasonal mean at conception: 0.0940 μg/m 3 Seasonal mean at birth: 0.0950 μg/m 3 Generalized estimating equations (GEEs) adjusted for maternal age, race, education, marital status, parity, adequacy of care, and tobacco use OR at conception Q1 Referent Q2 1.07 (1.05, 1.08) Q3 1.22 (1.20, 1.25) Q4 1.16 (1.15, 1.18) 0.05 μg/m 3 increase:1.04 (1.03, 1.04)

OR at birth Q1 Referent Q2 1.07 (1.05, 1.09) Q3 1.21 (1.19, 1.23) Q4 1.15 (1.13, 1.17) 0.05 μg/m 3 increase:1.04 (1.04, 1.05)May 2011 5

- 161 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Parameter Pb Data S tatistical Analysis Effect Estimate (95% CI)
Elmarsafawy et al. ( 2006 ) 471 elderly men (mean 67 y) from Normative Aging Study 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 model s 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)

References not included in Figure 5

- 31 are included in this table.

In another cross

- sectional analysis, Perlstein et al. ( 2007 ) examined the association of BP and pulse 

### 1
pressure (PP) among predominan tly white older adults in the greater Boston area. The subjects in this 

### 2
study had at least one bone Pb measurement during the years 1991

- 1997 and were not on antihypertensive 

### 3

medication at the time of the measurement. A statistically significant associatio n between blood Pb and 

### 4
DBP was observed in adjusted models but the correlations of BP with tibia Pb were not significant. Men 

### 5
with tibia Pb above the median had a significantly higher mean PP compared to men with tibia Pb below 

### 6
the median (4.2 mmHg [95%CI: 1.9, 6.5]). The trend toward increasing PP with increasing tibia Pb was 

### 7
significant although none of the confidence intervals for PP referenced to the lowest quintile of tibia Pb 

### 8

excluded the null value

. 

### 9

Peters et al. ( 2007 ) examined the modification by self

- report ed stress of the associations of Pb 

### 10
exposure (tibia and patella Pb) with BP and hypertension in a cohort of subjects enrolled in the VA

- NAS. 

### 11

Cross

- sectional analyses of the effect of bone Pb and stress on BP and hypertension were conducted. 

### 12

Increased but n onsignificant associations between hypertension status and SBP with bone Pb were 

### 13 observed. Interaction of stress with tibia Pb (tibia Pb β=3.77 [CI: 0.46, 7.09]) and stress with patella Pb 

### 14 was significant (patella Pb β=2.60 [CI:

- 0.95, 6.15]) in systolic B P models (neither bone, self

- reported 

### 15
stress, nor the interaction predicted DBP). Figure 5

- 3 2 shows the association between SBP and tibia Pb, 

### 16
comparing those with high and low self

- reported stress. Peters et al. ( 2007 ) also used Cox proportional 

### 17

hazards models to ass ess the interaction of stress and bone Pb level on the association of bone Pb with the 

### 18
development of hypertension among those free of hypertension at baseline. The results of this analysis 

### 19
showed interactions between both tibia and patella Pb and the inci dence of hypertension (RR of 

### 20
developing hypertension among those with high stress: 2.66 [CI: 1.43, 4.95] per SD increase in tibia Pb 

### 21
and 2.64 [CI: 1.42, 4.92] per SD increase in patella Pb). These provide information regarding factors that 

### 22

moderate or modi fy Pb effects on cardiovascular health. Gump et al. ( 2005 ) described significantly 

### 23
greater total peripheral vascular resistance (TPR) associated with increased blood Pb among 122 children 

### 24
(mean age 9.5 years) under acute stres s, assessed by minor tracing or reaction time tasks which are 

### 25 consistent with α and β adr energic activation. In a new analysis ( Gump et al., 2007 ) significant effects of 

### 26May 2011 5

- 162 DRAFT

– DO NOT CITE OR QUOTE SES on stress

- induced react ivity of BP and TPR are reported. A significant SES by blood Pb level 

### 1
interaction is also reported suggesting possibly heightened effects of blood Pb level on stress changes in 

### 2
TPR and BP in low SES groups. 

### 3

Source: Peters et al. ( 2007 ) Figure 5

- 32. The relationship between tibia Pb and estimated SBP for those with high self

- reported stress versus those with low self

reported stress.

Elmarsafawy et al. ( 2006 ) examined the modif ication of Pb effect by dietary calcium, with 467 

### 4
subjects from the VA

- NAS. Responses on a semi

- quantitative dietary frequency questionnaire with one



### 5
year recall were converted to estimated calcium intake . Hypertension was modeled using logistic 

### 6
regression and included interaction terms between Pb (tibia, patella and blood Pb) and a dichotomized 

### 7
calcium intake variable (split at 800 mg/day). They also constructed alternative models stratified on the 

### 8

calcium variable. Nonsignificant increases in hypertension associated with elevated blood, tibia, and 

### 9
patella Pb were observed in both low and high calcium groups. The only significant interaction reported 

### 10

was between BMI and calcium in a tibia Pb model. The authors report that in linear regression models of 

### 11

BP s tratified by calcium status, S BP increased 0.40 (95% CI: 0.11, 0.70) mmHg for every 1 μg/g increase 

### 12

in tibia Pb concentration in the high calcium group, and 0.19 ( 95% CI: 0.01, 0.37) mmHg in the low 

### 13
calcium group. 

### 14

Glenn et al. ( 2006 ) simultaneously modeled the multiple Pb dose measures of individuals over 

### 15
repeated time periods, assessing cross

- sectional as well as longitudinal relationships. The initial blood Pb 

### 16

level was used as a baseline covariate and the difference in blood Pb level between visits were computed 

### 17
for subsequent visits. The bone Pb measures were used to indicate historical exposure and cumulative 

### 18
exposure. Four models were specified: Model 1 was conceptualized to reflect short

- term exposure; Model 

### 19
2 to reflect longer

- term exposure controlling for recent dose; Model 3 to reflect longer

- term exposure 

### 20
controlling for cross

- sectional influence of cumulative dose; and Model 4 to reflect both short

- term 

### 21May 2011 5

- 163 DRAFT

– DO NOT CITE OR QUOTE change with rece nt dose and longer

- term change with cumulative dose. Concurrent and longitudinal 

### 1
measures of blood Pb were associated with SBP in Model 1 (short

- term exposure) and Model 4 (short



### 2
and longer

- term exposures). No associations with tibia Pb at baseline were o bserved while historical tibia 

### 3
Pb metric was negatively associated with SBP in each of the models. This study suggests that Pb exposure 

### 4
may act continuously on systolic BP and reduction in exposure may contribute to reductions in BP, while 

### 5

cumulative Pb bu rden may contribute to hypertension incidence by other mechanisms over longer time 

### 6
periods. Elevated BP may reflect an immediate response to Pb at a biochemical site of action as a 

### 7
consequence of recent dose or a persistent effect of cumulative doses over a lifetime

. 

### 8
In a separate analysis of the third year cross

- sectional results of the same occupationally

- exposed 

### 9

group , Weaver et al. ( 2008 ) examined associations between patella Pb and blood Pb level and SBP, DBP, 

### 10

and hypertension to determine interactions of the patella Pb effects with ALAD and vitamin D receptor 

### 11
(VDR) polymorphisms. None of the Pb exposure metrics were associated with DBP. Patella Pb alone was 

### 12
not significantly associated with SBP, while blood Pb, either alone or with patella Pb was positively and 

### 13
significantly associated with SBP. The patella Pb

- age and blood Pb

- age interactions were not significant. 

### 14

T here were no significant effects of blood Pb or patella Pb on hypertension status, or effect modification 

### 15
by age or sex. Further, interactions between polymorphisms of the VDR and of ALAD with blood Pb and 

### 16
patella Pb on S BP were not significant

. Mean blood Pb level was high (30.9 μg/dL ) compared to non



### 17
occupational groups

. 

### 18

Weaver et al. ( 2010 ) provided the results of further analysis of the Korean worker cohort ( V. M. 

### 19
Weaver et al., 2008 ) , with a focus on determining functional form of the concentration

- response 

### 20
relationships. The coefficient indicates that every doubling of blood Pb level is associated with a systolic 

### 21

BP increase of 1.76 mmH g. The J test, a statistical test for determining which, if either, of two functional 

### 22
forms of the same variable provides superior fit to data in non

- nested models ( Davidson & MacKi nnon, 

### 23

1981 ) returned a p

- value of 0.013 in favor of the natural log blood Pb level over the linear blood Pb level 

### 24
specification. This analysis indicates that systolic BP increase in this cohort is better described as a 

### 25
logarithmic function of blood Pb l evel within the blood Pb level range of the study than by a linear 

### 26

function. 

### 27

Yazbeck et al. ( 2009 ) conducted a cross

- sectional study examining a community

- based group of 

### 28

pregnant wo men to determine the association of Pregnancy Induced Hypertension ( PIH ) with blood Pb 

### 29
level and unlike most other studies adjusted their model for metal blood concentrations of cadmium, 

### 30
manganese, and selenium. PIH was defined as systolic BP >140 mmHg and /or diastolic BP >90 mmHg 

### 31
during at least two clinic visits after week 22 of gestation

. Patients with pre

- existing chronic hypertension 

### 32

were excluded. An association between blood Pb and PIH was observed (OR 3.29 [95% CI: 1.11, 9.74]) 

### 33
between PIH cases (2. 2 ± 1.4 μg/dL blood Pb) and normotensive patients (1.9 ± 1.2 μg/dL blood Pb). 

### 34
Cadmium and selenium concentrations were comparable between PIH and no PIH groups. Adjustment for 

### 35
the metals slightly attenuated but did not eliminate the association between blo od Pb levels and the risk of 

### 36May 2011 5

- 164 DRAFT

– DO NOT CITE OR QUOTE PIH. They observed no significant interactions among blood Pb level, any of the other elements, and 

### 1
maternal characteristics in predicting the risk of PIH. Interaction between selenium and Pb concentrations 

### 2
was not significant, and the putative protection effects of selenium through antioxidative properties were 

### 3
not confirmed in this study. 

### 4

Chen et al. ( 2006 ) reported an ecological study of air Pb concentration and PIH, agg regated by all 

### 5

50 U

. S

. states and the District of Columbia. The PIH data were taken from the live births and infant 

### 6
deaths up to 1 year of age compiled by the National Center for Health Statistics ( CDC, 2000 ) for the year 

### 7

1998. Associations between state level air Pb and state level PIH were reported (OR 1.04 [95% CI: 1.03, 

### 8
1.04] per 0.05 μg/m 3 Pb)

. No individual level data were used in the analysis. Wells et al. ( 2011 ) measured 

### 9
the influence of cord blood Pb on BP in 285 women at admission to the Johns Hopkins Hospital in 

### 10
Baltimore, MD, during labor and delivery. Women in the fourt h quartile of blood Pb elevations (>0.96 

### 11
μg/dL) had significantly higher systolic and diastolic BP (upon admission and for maximum BP) 

### 12
compared to women in the first quartile (<0.46 μg/dL). The authors used Benchmark Dose Software 

### 13
V2.1, developed by the EP A, to estimate benchmark dose (BMD) and the associated lower confidence 

### 14
limit for benchmark dose (BMDL) for one standard deviation (SD) increase in BP, which is 

### 15
approximately equivalent to a 10% increase above the mean for the first quartile blood Pb “ cont rols ” . The 

### 16
BMD approach is used here only as a means of characterizing the exposure level where effects might be 

### 17

found. These BMDL results indicate that the 95% lower bound confidence limit on the venous blood Pb 

### 18

level that is associated with a 1 SD increa se is about 1.85 μg/dL for all BP outcomes. While these reported 

### 19
results are similar to those in the 2006 Pb AQCD as well as those found 25 years ago but with blood Pb 

### 20

level s an order of magnitude lower, the authors did not provide enough information to al low for 

### 21
verification of the BMD analysis. 

### 22
Zhang et al. ( 2010 ) examined the effect of polymorphisms of the hemochromatosis gene (HFE) on 

### 23

the bone Pb effect on PP among older adult men participating in the VA

- NAS . Subjects had up to three PP 

### 24

measurements during the 10 year stud y period

. The overall results demonstrated a strong relationship 

### 25

between bone Pb and PP in this study, similar to the cross

- sectional PP study of many of the same subjects 

### 26

of the VA

- NAS group, without genotyping, reviewed above ( Perlstein et al., 2007 )

. T he effect of bone Pb 

### 27
(tibia and patella) among those with the H63D variant was greater compared to those with the wild

- type 

### 28
or the C282Y variant. In another gene

- environment interaction analysis, Scinicariello et al. ( 2010 ) used 

### 29

NHANES III (1988

- 1994) data to examine the interaction between ALAD genotype and blood Pb in 

### 30

relation to BP in a cross

- se ctional analysis. A significant interaction between log blood Pb level and 

### 31

ALAD1

- 2/2

- 2b among non

- Hispanic whites and non

- Hispanic blacks was observed. In addition, 

### 32
associations of blood Pb with SBP and DBP across race/ethnicity strata were presented. The strongest 

### 33
associations were observed among non

- Hispanic blacks. Scinicariello et al. ( 2010 ) also examined the 

### 34
association of blood Pb level with hypertension. Significant associations between blood Pb level and 

### 35
hypertension were observed among non

- Hispanic blacks and nonsignificant increases were observed 

### 36May 2011 5

- 165 DRAFT

– DO NOT CITE OR QUOTE among non

- H ispanic whites and Mexican Americans (with the exception of Q2 association for Mexican 

### 1
Americans.) In addition, non

- Hispanic white ALAD2 carriers in the highest blood Pb level quartile had a 

### 2
significantly higher association with hypertension compared with ALAD1 homozygous individuals

. 

### 3

Muntner et al. ( 2005 ) also used the NHANES data ( 1999

- 2002 ) to examine the cross

- sectional 

### 4

effect of blood Pb on hypertension , peripheral artery disease (PAD), and chronic kidney disease. T he PAD 

### 5

results are discussed later in Section 5.4.3.4 and chronic kidney disease results are discussed in Section 

### 6

5.5.2.2 )

. Blood Pb increased regularly with age among those with blood Pb measurements ( 1.28 μg/dL 

### 7
[ 95% CI: 1.23, 1.33 ] in the 18

- 39 age group to 2.32 μg/dL [ 95% CI: 2.20, 2.44 ] in the 75 and older age 

### 8

group. ) Associations were observed between blood Pb level and hypertension across race/ethnicity groups 

### 9
with significant trends observed for non

- Hispanic blacks and Me xican Americans. Park et al. ( 2009 ) 

### 10
examined the association of blood Pb as well as bone Pb, which was predicted from blood Pb, with 

### 11
hypertension. The predicted bone Pb metrics were derived from m odels using VA

- NAS data and applied 

### 12
to the NHANES (1988

- 1994) population. Blood Pb was associated with hypertension overall in the 

### 13
NHANES part of this study, with larger associations among black men and women as well as older adults. 

### 14

Associations with esti mated bone Pb were also observed. 

### 15

5.4.2.2. Toxicology

An array of studies have provided evidence that extended exposure to low levels of Pb (<5 μg/dL) 

### 16
can result in delayed onset of hypertension in experimental animals that persists long after the cessation of 

### 17

Pb exposure ( U.S. EPA, 2006 ) . Tsao et al

. ( 2000 ) found significantly increased systolic and diastolic BP 

### 18
in rats with blood Pb levels relevant to human exposure (2.15 μg/dL). As this was the lowest Pb level 

### 19
tested, no evidence of a threshold was evident. After Pb exposure is removed, blood, heart, aorta, and 

### 20
kidney Pb levels decreased quickly within the first three months ( H.

- R. Chang et al., 2005 ) . Pb

- induced 

### 21
elevated systolic BP persisted for one month following Pb exposure cessation, followed by obvious 

### 22
decreases in BP until 4 months after Pb exposure. Between 4 and 7 months after Pb exposure, the still



### 23
elevated BP did not decrease further, thus never returning to control BP levels. Decreases in BP were 

### 24
closely correlated with decreases in blood Pb level after exposure cessation. Prenatal Pb exposure in rats 

### 25
given a low calcium diet also resulted in increased arterial pressure ( Bogden et al., 1995 )

. 

### 26
Experimental animal studies continue to provide evidence to conclude that Pb exposure results in 

### 27
delayed, yet sustained arterial hypertension. Increased systolic BP developed in rats after exposure to 90

- 

### 28
10,000 ppm Pb (as Pb acetate in drinking water) for various time periods resulting in blood Pb level 

### 29

between 19.3

- 240 μg/dL ( Badavi et al., 2008 ; Bagchi & Preuss, 2005 ; Bravo et al., 2007 ; Grizzo & 

### 30
Cordellini, 2008 ; Heydari et al., 2006 ; Reza et al., 2008 ; Vargas

- Robles et al., 2007 ; L.

- F. Zhang et al., 

### 31

2009 ) . However, past studies have shown statistically significant elevations in BP in rats with lower blood 

### 32
Pb level ( Figure 5

- 3 3). Consistent w ith measurements of systolic BP, Pb exposure (100 ppm for 14 

### 33May 2011 5

- 166 DRAFT

– DO NOT CITE OR QUOTE weeks; blood Pb level 24 μg/dL) also caused an increase in intra

- aortic mean arterial pressure ( Bravo et 

### 1
al., 2007 ) . One study tested low levels of Pb exposure (30 ppm; blood Pb level 7.6 μg/dL) and did not 

### 2
find a statistically significant increase in systolic BP despite elevated blood Pb level after 8 weeks of 

### 3
treatment, however the data do represent a trend of increasing BP ( Rizzi et al., 2009 ) . Additionally, pups 

### 4
of Pb exposed dams (1,000 ppm through pregnancy and lactation) exhibit ed increased blood Pb level 

### 5
(58.7 μg/dL) and increased arterial systolic BP after weaning ( Grizzo & Cordellini, 2008 ) suggesting a 

### 6
role for childhood Pb exposure leading to adult disease. 

### 7May 2011 5

- 167 DRAFT

– DO NOT CITE OR QUOTE Reference Duration n ΔSBP

Chang et al. ( 2005 ) 5 13.81

Tsao et al. ( 2000 ) 8 weeks 10 13

Rizzi et al. ( 2009 ) 8 weeks 11 13.3

Chang et al. ( 1997 ) 8 weeks 10 58

Carmignani et al. ( 2000 ) 12 weeks 12 17

Heydari et al. ( 20 06 ) 12 weeks 6 25.8

Reza et al. ( 2008 ) 12 weeks 6 28.5

Bravo et al. ( 2007 ) 14 weeks 12 30

Zhang et al. ( 2009 ) 40 week s 8

- 10 15.3

Blood Pb (μg/dL)



### Note: Red square = blood Pb level where no statistically significant change in BP was observed; Blue circles = lowest blood Pb level reported with statistically significant changes in BP; Arrow line = higher blood Pb level reported in the same study with significant changes in SBP; Δ SBP = the change in SBP from control to first statistically significant blood Pb level in mmHg; n = number of animals in treatment group.
Figure 5

- 33. Rat blood Pb levels reported to be a ssociated with changes in SBP from the current ISA and 2006 Pb AQCD.

Pb induced hypertension persists long after cessation of Pb exposure. Bagchi and Preuss ( 2005 ) 

### 1
found that elevated systolic BP was maintained for 210 days aft er Pb exposure cessation. Chang et al. 

### 2
( 2005 ) reported a partial reversibility of effect after cessation of Pb exposure, where Pb

- induced elevated 

### 3
BP decreased but did not return to control levels 7 months post Pb exposure. However, chelation therapy 

### 4
using Na 2 CaEDTA was able t o return systolic BP to levels comparable to untreated rats ( Bagchi & Preuss, 

### 5

2005 ) . Studies reporting the effect of Pb (as blood Pb level) on systolic BP in unanesthetized adult rats 

### 6
since 1992 repor t a positive increase in BP with increasing blood Pb level (Figure 5

- 34 ). 

### 7 0 5 10 15 20 25 30 35 Dose with Statistically Significant Effect
Dose with No Statistically Significant EffectMay 2011 5

- 168 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 reported. Arrows represent higher doses tested.
Figure 5

- 34. Changes in BP after Pb exposure (as blood Pb level) in unanesthetized adult rats across studies.



### 5.4.2.3. Hypertension Mechanisms

The previous Pb AQCD examined a number of mechanisms leading to Pb

- induced hypertension, 

### 1
including oxidative stress, hormonal and blood pressure regulatory system dysfunction, vasomodulation, 

### 2
and cellular alterations. Further examination of these possible mechanisms of hypertension from Pb 

### 3
exposure in experimental animals and cells has been conducted and are provided below. 

### 4

Oxidativ e Stress Response

**Reactive Oxygen Species and Nitric Oxide**

Studies discussed in the previous Pb AQCD suggest a role for oxidative stress in the pathogenesis 

### 5

of Pb

- induced hypertension, mediated by the inactivation of nitric oxide ( ̇NO) and downregulation of 

### 6
soluble guanylate cyclase (sGC) ( Attri et al., 2003 ; Dursun et al., 2005 ; Gonick et al., 1997 ; Khalil



### 7
Manesh et al., 1994 ; Khalil

- Manesh, Gonick, Weiler, et al., 1993 ; Vaziri et al., 1997 ) . Pb

- induced 

### 8 reduction of biologically active  ̇NO is not due to a reduction in  ̇NO

- production capacity ( Vazir i & Ding, 

### 9

2001 ; Vaziri, Ding, et al., 1999 ) ; instead it is a result of inactivation and se questration of  ̇NO by ROS 

### 10 100 120 140 160 180 200

0 5 10 15 20 25 30 35 40Blood Pressure (mmHg)

Blood Pb Level (μg/dL)

Bravo et al. 2007 Chang et al. 1997 Chang et al. 2005 Heydari et al.2006 Nakhoul et al. 1992

Rizzi et al. 2009 Rizzi et al. 2009 Tsao et al. 2000 Zhang et al. 2009May 2011 5

- 169 DRAFT

– DO NOT CITE OR QUOTE ( Malvezzi et al., 2001 ; Vaziri, Liang, et al., 1999 ) . Oxidative stress from Pb exposure in animals may be 

### 1
due to upregulation of NAD(P)H oxidase ( Ni et al., 2004 ; Vazi ri et al., 2003 ) , induction of Fenton and 

### 2

Haber

- Weiss reactions ( Ding et al., 2000 ; Ding et al., 2001 ) , and failure of the antioxidant enzymes, CAT 

### 3
and GPx, to compensate for the increased ROS ( Farmand et al., 2005 ; Vaziri et al., 2003 )

. M any 

### 4

bi ological actions of  ̇NO, such as vasorelaxation, are mediated by cGMP, which is produced by sGC 

### 5
from the substrate GTP. Oxidative stress also plays a role in Pb

- induced downregulation of sGC ( Courtois 

### 6
et al., 2003 ; Farmand et al., 2005 ; M. Marques et al., 2001 )

. The reduction of the vasodilator  ̇NO leads to 

### 7

increased vasoconstriction and BP. 

### 8

Pb

- induced oxidative stress also induces renal tubulointerstitial inflammation which plays a c rucial 

### 9
role in models of hypertension ( Rodriguez

- Iturbe et al., 2005 ; Rodriguez

- Iturbe et al., 2004 )

. 

### 10
Tubulointerstitial inflammation from treatment with Pb has been coupled with activation of the redox 

### 11

sensitive NFκB ( Ramesh et al., 2001 ) . Pb

- induced hypertension, inflam mation, and NFκB activation can 

### 12
be ameliorated by antioxidant therapy ( Rodriguez

- Iturbe et al., 2004 ) . There is m ixed evidence to suggest 

### 13

that Pb

- induced hypertension may also be promoted by activation of PKC leading to enhanced vascular 

### 14
contractility ( Valencia et al., 2001 ; Watts et al., 1995 )

. 

### 15 Recent studies continue to provide evidence for the role of ROS and  ̇NO metabolism in Pb



### 16
induced hypertension and vascular disease. Increased SBP after Pb exposure has been a ccompanied by 

### 17
increased superoxide (O 2 - ) and O 2 - positive cells ( Bravo et al., 2007 ; Vargas

- Robles et al., 2007 ) , elevated 

### 18
urinary malondialdehyde (MDA) ( Bravo et al., 2007 ) , and increased 3

- nitrotyrosine ( Vargas

- Robles et al., 

### 19

2007 ) . Inhibition of NAD(P)H oxidase, an enzyme that generates O 2 - and hydrogen peroxide, was able to 

### 20

block Pb

- induced (1 ppm) aortic contraction to 5

- HT ( L. F. Zhang et al., 2005 ) . Increased SBP, intra



### 21
aortic mean arterial pressure, and MDA after Pb exposure (100 ppm; blood Pb level 23.7

- 27 μg/dL) were 

### 22
also prevented by treatment with the immunosuppressive, mycophenolate mofetil (MMF) ( Bravo et al., 

### 23

2007 ) . MMF has been shown to inhibit endothelial NAD(P)H oxidase, which could explain the decrease 

### 24
in oxidative stress and BP. Red grape seed extract was also able to protect rats from Pb

- induced (100 

### 25
ppm) increased BP and heart rate, perhaps through the antiox idant properties of the extract ( Badavi et al., 

### 26

2008 )

. 

### 27
Exposure to Pb can also affect the activity and levels of antioxidant enzymes. Male and female rats 

### 28
exposed to Pb for 18 weeks (100

- 1,000 pp m) had altered responses in antioxidant enzyme found in heart 

### 29

tissue ( Alghazal, Lenártová, et al., 2008 ; Sobekova et al., 2009 ) . Pb exposure (>100ppm) in female rats 

### 30
increased the activity of cardiac SOD, GST, GR, and GPx and increased cardiac TBARS (1,000 ppm). Pb 

### 31
exposure in male rats did not affect the activity of SOD or production of TBARS, however decreased the 

### 32
activity of GST and GR (>100 ppm). Male and female rats also accumulated different amounts of Pb in 

### 33 the cardiac tissue after similar exposure (♂ 100 ppm: 205% of con trol, 1,000 ppm: 379%; ♀ 100 ppm: 

### 34
246%, 1,000 ppm: 775%), which could explain the sex differences observed. 

### 35May 2011 5

- 170 DRAFT

– DO NOT CITE OR QUOTE Oxidative stress can trigger a cascade of events that promote cellular stress, renal inflammation, 

### 1
and hypertension. As was shown previously ( Rodriguez

- Iturbe et al., 2004 ) , Pb exposure can increase 

### 2 renal NFκB, which was associated with tubulointerstitial damag e and infiltration of lymphocytes and 

### 3
macrophages ( Bravo et al., 2007 ) . These events could also be ablated by MMF treatment, likely due to its 

### 4

anti

- inflammatory and antioxidant properties. Pb is also able to induce inflammation in human endothelial 

### 5

cells as a model for vessel intima hyperplasia ( Zeller et al., 2010 ) . The proinflammatory cytokine, 

### 6

interleukin

- 8 (IL

- 8) protein and mRNA were increased, dose and time dependently, after in vitro Pb 

### 7
exposure (5

- 50 μM). Enhanced IL

- 8 production was mediated through activation of the transcription 

### 8 factor Nrf2 (but not NFκB, hypoxia inducible factor

- 1, or aryl hydrocarbon receptor), as shown through 

### 9

increased nuclear transl ocation and Nrf2 cellular knockdown experiments. Additionally, measures of 

### 10
endothelial stress, NQO1 and HO

- 1 protein, were induced by Pb exposure ( Zeller et al., 2010 )

. 

### 11 Oxidative stress affects vascular reactivity and tone through inactivation and sequestration of  ̇NO, 

### 12 causing a reduction in biologically active  ̇NO. Recent studies confirm these past conclusions on the 

### 13 interplay of ROS and  ̇NO metabolism in the cardiovascular effects of Pb. Elevated SBP and altered 

### 14
vasorelaxation after Pb exposure is accompanied by a decrease in total nitrates and nitrites (NOx) 

### 15
( Heydari et al., 2006 ; L. F. Zhang et al., 2007 ) . Serum NOx levels in Pb

- treated rats remained depressed 

### 16
for 8 weeks and then reversed after 12 weeks, despite continued elevation in SBP ( Heydari et al., 2006 )

. 

### 17
This return of serum NOx levels could be a result of compensatory increases in endothelial NOS (eNOS) 

### 18
attempting to replenish an over

- sequestered  ̇NO supply. With this in mind, studies have shown increased 

### 19
eNOS protein expression after chronic Pb exposure in kidney ( L. F. Zhang et al., 2007 ) and isolated 

### 20

cultured aorta ( Vargas

- Robles et al., 2007 ) . No change in inducible NOS was observed in isolated 

### 21
cultured aorta after 1 ppm Pb exposure ( L. F. Zhang et al., 2007 )

. 

### 22
 ̇NO, also known as endothelium

- derived relaxing factor, is a potent endogenous vasodilator. 

### 23 Studies continue to investigate the effects of Pb on  ̇NO dependent vascular reactivity. Perinatal Pb 

### 24
exposure (10 00 ppm through pregnancy and lactation, blood Pb level 58.7 μg/dL) resulted in a greater 

### 25

increase in maximal contraction to L

- NAME, which decreases  ̇NO production, with a greater effect in 

### 26

the endothelium than the smooth muscle cells ( Grizzo & C ordellini, 2008 ) . Additionally, blocking NOS 

### 27

with L

- NAME abolished the relaxant response evoked by ACh, which triggers the release of  ̇NO from 

### 28
the endothelial cell, in aortic rings of perinatally exposed rats. Cyclooxygenase (COX) inhibition 

### 29

decreased t he EC50 of the ACh response in Pb treated animals. This study suggests that pups exposed to 

### 30
Pb through pregnancy and lactation have an altered vascular reactivity that is endothelium dependent and 

### 31

occurs due to the altered release of  ̇NO and a COX

- derived vasoconstrictor ( Grizzo & Cordellini, 2008 )

. 

### 32
Similarly, a recent study provides evidence that acute Pb exposure increases rat tail artery reactivity 

### 33
in an endothelium dependent manner due to a COX

- derived vasoconstrictor and in part free rad icals and 

### 34

 ̇NO ( Silveira et al., 2010 ) . Acute exposure of rat tail artery to Pb (100 μM, 1 h) increased reactivity to 

### 35
phenylephrine. Pb exposure decreased ACh induced relaxation, suggesting damage to the endothelium. Pb 

### 36May 2011 5

- 171 DRAFT

– DO NOT CITE OR QUOTE did not affect smooth muscle integrity si nce sodium nitroprusside (SNP)

- induced vasorelaxation was 

### 1
unchanged. Inhibition of NOS increased the Pb pressor response whereas COX inhibition eliminated the 

### 2
response to PHE. Treatment with the SOD mimetic Tempol decreased, but did not eliminate, the Pb 

### 3

p ressor response ( Silveira et al., 2010 ) . A second study showed that Pb (90 ppm) exposure did not change 

### 4
the rat thoracic aortic ring relaxation response curves to the  ̇NO donor, SNP ( Rizzi et al., 2009 )

. 

### 5
Conversely, Skoczynska and Stojek ( 2005 ) found that Pb exposure (50 ppm; blood Pb level 11.2 

### 6 μg/dL) enhanced  ̇NO

- mediated vasodilation by ACh in rat mesenteric arteries and NOS inhibition 

### 7
enhanced the ACh relaxant response. In rat renal interlobar arteries, Pb expos ure blunted the increase in 

### 8
AngII mediated contraction from NOS inhibition by L

- NAME ( Vargas

- Robles et al., 2007 )

. 

### 9

Vascular Reactivity

Alteration of the adrenergic system from Pb exposure, which can increase peripheral vascular 

### 10
resistance, and thereby arterial pressure, may be one cause of P b

- induced hypertension. Pb exposure in 

### 11
animals can increase stimulation of the sympathetic nervous system (SNS), as shown by increased plasma 

### 12
norepinephrine and plasma catecholamines ( Carmignani et al., 2000 ; H.

- R. Chang et al., 1997 ) , and 

### 13 decreased β adrenergic receptor dens ity and β agonist

- stimulated cAMP production in the aorta and heart 

### 14
( H.

- R. Chang et al., 1997 ; Tsao et al., 2000 ) . These stimulatory effects on the SNS paralleled effects on 

### 15
BP, cardiac contractility, and carotid blood flow. Increased Pb induced arterial pressure and heart rate 

### 16
were abrogated by ganglionic b lockade ( C.

- C. Lai et al., 2002 ) and gradually decreased 7 months after Pb 

### 17
exposure cessation along with Pb

- induced SNS alterations ( H.

- R. Chang et al., 2005 )

. 

### 18
Increased BP can be caused by vascular narrowing leading t o increased total peripheral resistance, 

### 19
resulting from activation of the SNS. In this neural mechanism, activation of the SNS leads to 

### 20
vasoconstriction, whereas inhibition leads to vasodilation. It has been suggested that Pb leads to increased 

### 21

vascular re activity to catecholamines (i.e. epinephrine, norepinephrine (NE), and dopamine), hormones of 

### 22
the SNS. Indeed, the isolated mesenteric vessel bed from Pb treated rats (50 ppm blood Pb level: 11.2 

### 23
μg/dL, but not 100 ppm blood Pb level: 17.3 μg/dL) exhibited increased reactivity to NE ( Skoczynska & 

### 24
Stojek, 2005 ) . Similarly, 100 ppm Pb did not affect the NE induced contractile response after 10 months 

### 25
of exposure ( L.

- F. Zhang et al., 2009 ) , suggesting a small range of doses affecting pressor response to NE. 

### 26
Catecholamines act primarily through the adrenergic and dopaminergic receptor s. Antagonists of α1

- 

### 27 adrenergic, α2

- adrenergic, β

- adrenergic, and dopamine D1 receptors abolish Pb

- induced aortic contraction 

### 28
( Fazli

- Tabaei et al., 2006 ; Heydari et al., 2006 ) . Phenylephrine

- induced aortic contractions were enhanced 

### 29 by treatment with Pb (100 ppm; blood Pb level: 26.8 μg/dL), indicating a specific role for the α1

- 

### 30
adrenergic receptor. Additionally, Pb blunted the isoproterenol

- induced relaxation, supporting a role for 

### 31 the β

- adrenoceptors ( Heydari et al., 2006 ; Vassallo et al., 2008 )

. 

### 32May 2011 5

- 172 DRAFT

– DO NOT CITE OR QUOTE Recently, there has been mixed evidence for Pb disrupting vascular reactivity to other pressor 

### 1
agents. One study found that Pb (50 ppm; 12 weeks; blood Pb level : 11.2 μg/dL) increased acetylcholine 

### 2
(ACh) induced re laxation in rats ( Skoczynska & Stojek, 2005 ) . Additionally, studies have shown no 

### 3
change in ACh induced vasorelaxation after Pb exposure ( Grizzo & Cordellini, 2008 ; Rizzi et al., 2009 )

. 

### 4
However, Zhang et al. ( 2007 ) and Silveira et al. ( 2010 ) found that Pb (1 ppm and 100 μM, 1 h ) blunted 

### 5

ACh induced relaxation in isolated rat thoracic aorta and tail artery, respectively . Another study 

### 6
investigated the influence of Pb on vasoconstriction from 5

- hydroxytryptamine (5

- HT). Pb (1 ppm) 

### 7

treatment of isolated rat thoracic aorta increased 5

- HT induced contraction, which was endothelium 

### 8
dependent, but not due to 5

- HT2B receptor expression ( L. F. Zhang et al., 2005 ) . Follow

- up of this study 

### 9
in whole animals found, on the contrary, that Pb (100 ppm; blood Pb level : 28.4 μg/dL) decreased the 

### 10

maximum contractile response to 5

- HT, but did not affect 5

- HT plasma levels or 5

- HT2B receptor 

### 11
expression ( L.

- F. Zhang et al., 2009 )

. 

### 12 Studies continue to investigate the role of  ̇NO, also known as endothelium

- d erived relaxing factor, 

### 13
in Pb induced changes in vascular reactivity. A recent study provides evidence that acute Pb exposure 

### 14
increases rat tail artery reactivity in an endothelium dependent manner due in part to free radicals and 

### 15

 ̇NO ( Silveira et al., 2010 ) . Acute exposure of rat tail artery to Pb (100 μM, 1 hour) increased reactivity to 

### 16
phenylephrine. Pb exposure decreased ACh induced relaxation, suggesting damage to the endothelium. 

### 17
However, Pb did not affect smooth muscle integrity since SNP

- induced vasor elaxation was unchanged. 

### 18
Similarly, Pb (90 ppm) exposure did not change the rat thoracic aortic ring relaxation response curves to 

### 19
the  ̇NO donor, SNP ( Rizzi et al., 2009 )

. Inhibition of NOS increased the Pb pressor response to PHE 

### 20
( Silveira et al., 2010 ) . Another study showed that blocking NOS w ith L

- NAME, abolished the relaxant 

### 21
response evoked by ACh, which triggers the release of  ̇NO from the endothelial cell, in aortic rings of 

### 22
perinatally exposed rats (1,000 ppm through pregnancy and lactation, blood Pb level 58.7 μg/dL) ( Grizzo 

### 23

& Cordellini, 2008 )

. Additionally, perinatal Pb exposure resulted in a greater increase in maximal 

### 24

contraction to L

- NAME, which decreases  ̇NO production, with a greater effect in the endothelium than 

### 25
the smooth muscle cells. This study suggests that pups exposed to Pb through pregnancy and lactation 

### 26
have an altered vascular reactivity that is endothelium dependent and occurs due in part to the altered 

### 27

release of  ̇NO ( Grizzo & Cordellini, 2008 ) . In addition, Pb exposure (100 ppm, 12 weeks) inc reased the 

### 28
renal vascular response to AngII in isolated perfused kidneys from Pb exposed rats ( Vargas

- Robles et al., 

### 29

2007 ) . NOS inhibition by L

- NAME increased AngII

- induced vasoconstriction in control but not Pb



### 30 exposed arteries, suggesting impaired  ̇NO availability ( Vargas

- Robles et al., 2007 ) . Conversely, 

### 31
Skoczynska and Stojek ( 2005 ) found that Pb exposure (50 ppm; blood Pb level 11.2 μg/dL) enhanced 

### 32

 ̇NO

- mediated vasodilation by ACh in rat mesenteric arteries and NOS inhibition enhanced the ACh 

### 33
relaxant response. 

### 34May 2011 5

- 173 DRAFT

– DO NOT CITE OR QUOTE Renin

- Angiotensin

- Aldosterone and Kininergic Systems

The adrenergic sys tem also affects the renin

- angiotensin

- aldosterone system (RAAS), which is 

### 1
responsible for fluid homeostasis and blood pressure regulation, and has been shown to be affected by Pb 

### 2
exposure. Meta

- analysis found that Pb exposure (30

- 40 μg/dL) increases plasm a renin activity and renal 

### 3
tissue renin in young rats, but not old ( Vander, 1988 ) . Exposure of ex perimental animals to Pb also 

### 4
induced increases in plasma, aorta, heart, and kidney angiotensin converting enzyme (ACE) activity; 

### 5
plasma kininase II, kininase I, and killikrein activities; and renal angiotensin II (AngII) positive cells 

### 6
( Carmignani et al., 1999 ; Rodriguez

- Iturbe et al., 2005 ; Sharifi et al., 2004 ) . ACE activity declined over 

### 7

tim e while arterial pressure stayed elevated, suggesting that the RAAS may be involved in the induction, 

### 8

but not the maintenance of Pb

- induced hypertension in rats. 

### 9
Recent studies continue to implicate the RAAS in the development of Pb

- induced hypertension, 

### 10

e specially during early exposure in young animals. Low level Pb (100 ppm, 14 weeks; blood Pb level 

### 11

23.7

- 27 μg/dL) exposure increased renal cortical AngII content and the number of tubulointerstitial 

### 12
AngII

- positive cells ( Bravo et al., 2007 ) . This heightened in trarenal angiotensin corresponded with 

### 13
sodium retention and increased SBP and was ablated by the anti

- inflammatory antioxidant, MMF. 

### 14
Similarly, early high level Pb (1% Pb, 40 days; blood Pb level >240 μg/dL after exposure, 12

- 13 μg/dL 

### 15

after chelation after 1 year) accumulation resulted in sustained hypertension ( Bagchi & Preuss, 2005 )

. 

### 16
Treatment with the AngII receptor blocker, Losartan, resulted in a greater decrease in SBP in Pb exposed 

### 17

rats than con trol rats that continued into later periods of follow

- up (day 283). Increased SBP after early 

### 18
exposure to Pb corresponded with increased water intake, urine output, potassium excretion, and 

### 19
decreased urinary sodium and urine osmolality. These functional ch anges in renal behavior are consistent 

### 20
with the actions of a stimulated RAAS. AngII, a main player in the RAAS, induces arteriolar 

### 21
vasoconstriction leading to increased BP. Pb exposure increased the vascular reactivity to AngII ( Vargas



### 22
Robles et al., 2007 ) . These studies point to the activatio n of the RAAS in the course of Pb

- induced 

### 23
hypertension. 

### 24

Vasomodulators

The balance between production of vasodilators and vasoconstrictors is important in the regulation 

### 25
of blood pressure and cardiovascular function. The previous AQCD reported that the ef fects of Pb on 

### 26
vasomodulators are contradictory. Urinary excretion of the vasoconstrictor, thromboxane (TXB 2 ), and the 

### 27
vasodilatory prostaglandin, 6

- keto

- PGF1α, were unchanged in rats with Pb

- induced hypertension ( Gonick 

### 28
et al., 1998 ) . However, in vitro Pb exposure promoted the release of the prostaglandin substrate, 

### 29
arachidonic acid, in vascular smooth muscle cells (VSMC) via activation of phospholipase A 2 ( Dorman & 

### 30
Freeman, 2002 ) . Plasma concentration and urinary excretion of the vasoconstrictive peptide, endothelin 

### 31
(ET) 3 was increased after low (100 ppm), but not high level (5000 ppm) Pb exposure in rats ( Gonick et 

### 32May 2011 5

- 174 DRAFT

– DO NOT CITE OR QUOTE al., 1997 ; Khalil

- Manesh et al ., 1994 ; Khalil

- Manesh, Gonick, Weiler, et al., 1993 ) . Antagonism of the ET 

### 1
receptor A bl unted the downregulation of sGC and cGMP production by Pb in isolated rat artery 

### 2
segments, suggesting that some of the hypertensive effects of Pb exposure may be mediated through ET 

### 3
( Courtois et al., 2003 ) . Additionally, Pb

- exposed animals exhibite d fluid retention and a dose dependent 

### 4
decline in the vasodilator, atrial natriuretic factor (ANF) ( Giridhar & Isom, 1990 ) . These studies suggest 

### 5
that Pb may interfere with the balance between vasodilators and vasoconstrictors forming the hormonal 

### 6
regulation of vascular contraction and blood pressure

. 

### 7
The imbalance in vasomodulators is one explanation for the concentration

- dependent 

### 8
vasoconstriction observed after Pb exposure ( Piccinini et al., 1977 ; Valencia et al., 2001 ; Watts et al., 

### 9

1995 ) . Vasoconstriction after Pb exposure was not reported in all studies ( Shelkovnikov & Gonick, 2001 ) 

### 10
and is likely varied depending on the type of vessel used, th e Pb concentration employed, and the animal 

### 11
species being studied. Studies have reported attenuation of acetylcholine

- and NO

- mediated vasodilation 

### 12
( M. Marques et al., 2001 ; Oishi et al., 1996 ) in some, but not all vascular tissues and in some, but not all 

### 13

studies ( Purdy et al., 1997 ) . These effects have been variably attributed to Pb mediated activation of PKC 

### 14
and direct action on the VSMCs through the Ca 2+ mimetic properties of Pb among other possibilities 

### 15
( Piccinini et al., 1977 ; Valencia et al., 2001 ; Watts et al. , 1995 )

. 

### 16
One recent study investigated the role of the endothelial derived vasoconstrictor, ET

- 1, in Pb



### 17
induced hypertension. ET

- 1 from the endothelium acts on the ET A - type receptor s located on the vascular 

### 18 smooth muscle layer and may be involved in vascular reactivity by  ̇NO and COX derivatives. Pb 

### 19
exposure (1 ppm, 24 hours) to rat aortic segments decreased expression of sGC

- β1 subunit, an enzyme 

### 20

involved in  ̇NO

- induced vasodilation , and increased expression of COX

- 2 in an endothelium dependent 

### 21

manner ( Molero et al., 2006 ) . Even though Pb treatment did not alter ET

- 1 or ET A - type receptor protein 

### 22
expression in this system, blocking the ET A - type receptors partially reversed Pb

- induced changes in sGC 

### 23

and COX

- 2 in vascular tissue. This study suggests that the endothelium and ET

- 1 may contribute to Pb



### 24
induced hypertension through activation of ET A - type receptors that alter expression of COX

- 2 and sGC



### 25 β1 subunit, which affects  ̇NO signaling. 

### 26

COX

- 2 blockade ha s been shown to prevent Pb

- induced downregulation of sGC expression 

### 27
( Courtois et al., 2003 ) . Inhibition of COX

- 2 also decreases the Pb induced pressor response to ACh 

### 28
( Grizzo & Cordellini, 2008 ) and PHE ( Silveira et al., 2010 ) in experime ntal animals. These studies 

### 29
suggest that Pb induced vascular reactivity may depend on the participation of a COX

- derived 

### 30
vasoconstrictor, such as prostaglandins, prostacyclins, or thromboxanes. 

### 31

5.4.2.4. Summary

The 2006 Pb AQCD reported a clear positive association between blood Pb level and BP. The effect 

### 32
was modest, but highly significant, as determined by a meta

- analysis ( Nawrot et al., 2002 ) of over 30 

### 33May 2011 5

- 175 DRAFT

– DO NOT CITE OR QUOTE studies comprising over 40,000 subjects ( Figure 5

- 3 5 ), reporting that each doubling of blood Pb was 

### 1
associated with a 1 mmHg increase in systolic BP and a 0.6 mmHg increase in diastolic BP. Recent 

### 2
studies support this conclusion at lower blood Pb level s (<2 μg/dL) and add to the evidence base on 

### 3
susceptibility factors and bone Pb associations with BP and hypertension at levels <20 μg/g

. Associations 

### 4

of bone and/or blood Pb with systolic BP and hypertension were higher among non

- whites, those 

### 5
reporting h igh stress

, and those with the HFE H63D and ALAD genotypes

. 

### 6
A recent study in an e thnically diverse community

- based cohort of women and men aged 50

- 70 

### 7
years of age suggests that Pb has an acute effect on BP as a function of recent dose measured by blood Pb 

### 8

and a chronic effect on hypertension risk as a function of cumulative exposure measured by tibia Pb 

### 9
( Martin et al., 2006 ) . This study verified other studies by demonstrating that with each increase of 1 

### 10
μg/dL blood Pb level, systolic BP would increase 1 mm Hg and diastolic BP would increase 0.5 mmHg. 

### 11
Additionally, recent epidemiologic stu dies provided evidence for associations between blood Pb and BP 

### 12
and hypertension at relatively low blood Pb level; a positive relationship was found in the NHANES data 

### 13
(1999

- 2002) at a geometric mean blood Pb level of 1.64 μ g/dL ( Muntner et al., 2005 )

. Animal 

### 14
toxicological studies also provide support for effects of low blood Pb level on increased BP with 

### 15
statistically significant increases shown as low as 2 μg/dL ( Tsao et al., 2000 ) . Collectively, all animal 

### 16
toxicological studies providing blood Pb level and BP measurements report positive increases in BP with 

### 17
increasing blood Pb level ( Figure 5

- 3 4). New studies also demonstrate reversibility of Pb

- induced 

### 18

increased BP following Pb exposure cessation or chelation. 

### 19May 2011 5

- 176 DRAFT

– DO NOT CITE OR QUOTE Source: Used with permission from 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.
Figur e 5

- 35. Change in SBP, association size in mmHg with 95% CI, associated with a doubling in the blood Pb concentration.

Epidemiologic studies continue to investigate the relationship between bone Pb and increased BP. 

### 1
A recently published meta

- analysis of e pidemiological studies used bone Pb as an exposure index and BP 

### 2
or hypertension as the outcome ( Figure 5

- 3 6) ( Navas

- Acien et al., 2008 )

. The eight studies (three 

### 3

p rospective, five cross

- sectional) showed positive relationships between tibia Pb and systolic BP but not 

### 4May 2011 5

- 177 DRAFT

– DO NOT CITE OR QUOTE diastolic BP. A pooled estimate for an increase in systolic BP of 0.26 mmHg ( 95% CI: 0.02, 0.50) for the 

### 1

cross

- sectional studies was reported. The esti mate for the longitudinal studies was 0.33 mmHg (95% CI:

- 

### 2
0.44, 1.11). With the exception of one study, positive associations of bone Pb with hypertension were also 

### 3
reported. Pooled estimates of 1.04 ( 95% CI: 1.01, 1.07) per 10 μg/g increase in tibia Pb an d 1.04 ( 95% 

### 4

CI: 0 .96, 1.12) per 10 μg/g increase in patella Pb were reported

. 

### 5
Recent epidemiological studies have also emphasized the interaction between long

- term Pb 

### 6
exposure and factors that moderate or modify the Pb effect, like chronic stress and meta bolic syndrome, 

### 7
on BP and hypertension. Bone Pb coupled with high stress was associated with a strong and reliable 

### 8
increased risk of developing hypertension in an originally nonhypertensive group ( Peters et al., 2007 )

. 

### 9
Also, long duration Pb exp osure interacted with components of the metabolic syndrome to drive HRV in 

### 10

directions associated with increased cardiovascular events ( Park et al., 2006 )

. 

### 11May 2011 5

- 178 DRAFT

– DO NOT CITE OR QUOTE Sour ce: Used with permission from Elsevier Publishers, Navas

- Acien et al. ( 2008 )

In the Normative Aging Study, Hu et al ( 1996 ) reported the cross

- sectional association between bone P b levels and the prevalence of hypertension and Cheng et al ( 2001 ) reported the cross

sectional association between bone Pb levels and SBP in study par ticipants 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 the 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 descriptive purposes. RR indicates risk ratio. Figure 5

- 36. Prospective and cross

- sectional increase in SBP and DBP and relative risk of hypertension per 10 μ g/g increase in bone Pb levels.

Recent epidemiologic studies investigated the interaction of genotypes with effects of Pb on the 

### 1
cardiovascular system. Significant evidence was presented for modification of the effect of blood Pb level 

### 2
on BP by ALAD genotype ( Scinicariello et al., 2010 ) . Additionally, polymorphisms in the 

### 3
hemochromatosis gene modified the pulse pressure response to bone Pb exposure, where pulse pressure 

### 4
represents a good predictor of cardiovascular morbidity and mortality and an indicator of arterial stiffness 

### 5
( A. Zhang et al., 2010 ) . Park et al. ( 2009 ) provided further evidence of gene variants, sp ecifically those 

### 6
related to iron metabolism, impacting the effect of long

- term Pb exposure on the cardiovascular system, 

### 7
evaluated by QT interval changes. 

### 8May 2011 5

- 179 DRAFT

– DO NOT CITE OR QUOTE Not only has Pb exposure been shown to increase BP and hypertension, but Pb exposure can 

### 1

contribute t o the development of other cardiovascular diseases. Recent epidemiologic and toxicological 

### 2
studies provide evidence for increased atherosclerosis, thrombosis, ischemic heart disease

, p eripheral 

### 3
artery disease , arrhythmia, and cardiac contractility. 

### 4

Animal toxicological evidence continues to build on the evidence supporting the mechanisms 

### 5
leading to these cardiovascular alterations. Enhanced understanding of Pb

- induced oxidative stress 

### 6 including  ̇NO inactivation, endothelial dysfunction leading to altered v ascular reactivity, activation of the 

### 7
RAAS, and vasomodulator imbalance provides biological plausibility for the consistently positive 

### 8

associations observed between blood and bone Pb and cardiovascular effects. 

### 9



## 5.4.3. Vascular Effects and Cardiotoxicity

Not only has Pb exposure been shown to increase BP and alter vascular reactivity, but Pb can alter 

### 10
cardiac function, initiate atherosclerosis, and increase cardiovascular mortality. Past toxicological studies 

### 11
have reported that Pb can increase atheromatous plaque f ormation in pigeons, increase arterial pressure, 

### 12
decrease heart rate and blood flow, and alter cardiac energy metabolism and conduction ( Prentice & 

### 13
Kopp, 1985 ; Revis et al., 1981 ) . Epidemiologic studies discussed in the previous AQCD provided limited 

### 14
evidence to support the association of ischemic heart disease (IHD) and perip heral artery disease (PAD) 

### 15

with increased blood Pb. 

### 16

5.4.3.1. Effects on Vascular Cell Types

The endothelium layer is an important constituent of the blood vessel wall, which regulates 

### 17
macromolecular permeability, vascular SMC tone, tissue perfusion, and blood fluid ity. Damage to the 

### 18
endothelium is an initiating step in development of atherosclerosis, thrombosis, and tissue injury. Given 

### 19
that chronic Pb exposure promotes a number of these diseases, numerous studies have investigated the 

### 20
role of Pb on endothelial dysf unction. The endothelial layer makes up only a small part of the vascular 

### 21
anatomy; the majority of the vessel wall is composed of vascular SMC, which work in concert with the 

### 22
EC in contraction and relaxation of the vessel, local BP regulation, and atherosc lerotic plaque 

### 23
development. Since Pb has been shown repeatedly to result in hypertension and vascular disease, studies 

### 24
continue to investigate the effects of Pb on SMC. 

### 25
Pb exposure (50 μM, 2 weeks) stimulated SMC invasiveness in isolated human arteries leading to 

### 26
the invasion of medial SMC into the vessel intima and development of intimal hyperplasia, a key step in 

### 27
atherosclerotic progression ( Zeller et al., 2010 ) . In addition, Pb exposure (50 μM, 12 hours) promoted 

### 28
SMC elastin expression and increased arterial extracellular matrix in isolated human arteries. SMC 

### 29

invasiveness was also increased in culture by treatment with supernatant of Pb

- treated human EC (50 

### 30
μM), suggesting that Pb

- exposed EC secrete an activating compound. This compound was confirmed to 

### 31May 2011 5

- 180 DRAFT

– DO NOT CITE OR QUOTE be interleukin

- 8 (IL

- 8). Pb exposure (5

- 50 μM) was able to dose

- depende ntly increase IL

- 8 synthesis and 

### 1
secretion in human umbilical vein EC cultures through activation of the transcription factor Nrf2. 

### 2

Neutralization of IL

- 8 could block SMC invasion and arterial intima thickening ( Zeller et al., 2010 ) . This 

### 3
study provides evidence that Pb exposure stimulates EC to secrete IL

- 8 in an Nrf2

- dependent manner that 

### 4
stimulates SMC invasion from the vessel media to intima leading to a va scular thickening and possibly 

### 5
atherogenesis. 

### 6
A number of cardiovascular diseases, including atherosclerosis, are characterized by increased 

### 7
inflammatory processes. Numerous studies have shown that Pb exposure is able to induce an 

### 8
inflammatory environment in humans and animals by increasing inflammatory mediators like 

### 9
prostaglandin E 2 (PGE 2 ). Human aortic vascular SMC treated with Pb (1 μM, 1

- 12 hours) exhibited 

### 10

increased secretion of PGE 2 time

- dependently through enhanced gene transcription ( W. C. Chang et al. )

. 

### 11
This was preceded by a Pb

- induced increase in gene expression of the rate limiting enzymes in the 

### 12
regulation o f prostaglandins, cytosolic phospholipase A 2 (cPLA 2 ) and COX

- 2. The induction of these 

### 13
enzymes was mediated by activation of ERK1/2, MEK1, and MEK2. Further investigation into the 

### 14

entrance of Pb into the cell revealed that inhibition of the store

- operated calcium channels (SOC) could 

### 15
only partially suppress cPLA 2 and COX activation by Pb; however inhibition of epidermal growth factor 

### 16
receptor (EGFR) attenuated Pb

- induced cPLA 2 and COX activation and PGE 2 secretion. Overall this 

### 17
study suggests that Pb can in duce proinflammatory events in vascular SMC in the form of increased PGE 2 

### 18

secretion and cPLA 2 and COX

- 2 expression through activation of EGFR via ERK1/2 pathways. 

### 19
Damage to the endothelium is a hallmark event in the development of atherosclerosis. Past stu dies 

### 20
have shown that Pb exposure results in de

- endothelialization, impaired proliferation, and inhibition of 

### 21
endothelium repair processes after injury ( Fujiwara et al., 1997 ; Kaji et al., 1995 ; Kishimoto et al., 1995 ; 

### 22
Ueda et al., 1997 ) . However, Pb exposure does not lead to nonspecific cytotoxicity at low exposure levels 

### 23
(2

- 25 μM) as shown by the lack of release of lactate dehydrogenase (LDH) from Pb

- treated bovine aortic 

### 24

EC ( Shinkai et al., 2010 ) . Instead, Pb results in specific cytotoxicity (caspase3/7 activation) through 

### 25
endoplasmic reticulum (ER) stress that can be protected against by the ER chaperones glucose

- regulated 

### 26
protein 78 (G RP78) and glucose

- regulated protein 94 (GRP94). GRP78 and GRP94 play key roles in the 

### 27
adaptive unfolded protein response that serves as a marker of and acts to alleviate ER stress. Exposure of 

### 28

Pb to EC induces GRP78 and GRP94 gene (2

- 25 μM) and protein (GRP78 (5

- 25 μM) and GRP94 (10

- 25 

### 29
μM)) expression through activation of the IRE1

- JNK

- AP

- 1 pathways ( Shinkai et al., 2010 ) . This study 

### 30
suggests that the functional damage caused by Pb exposure to EC may be partly attributed to induction of 

### 31

ER stress. 

### 32May 2011 5

- 181 DRAFT

– DO NOT CITE OR QUOTE 

### 5.4.3.2. Cholesterol

As blood cholesterol rises so does the risk of coronary heart disease. Ea rly occupational studies 

### 1
( Ademuyiwa, Ugbaja , Idumebor, et al., 2005 ; Bener et al., 2001 ; Kristal

- Boneh et al., 1999 ) at higher than 

### 2

current blood Pb level s reported higher total cholesterol levels related to Pb exposure , and mixed results 

### 3
for HDL, LDL, and triglycerides. More recently

, Poreba et al. ( 2010 )

, in an occupational study, reports no 

### 4
significant differences in parameters of lipid metabolism between Pb exposed and unexposed 

### 5
hyper tensive patients. Other Pb studies ( Menke et al., 2006 ) adjust models for total cholesterol to control 

### 6
for this coronary heart disease risk factor and note that mean total cholesterol was higher at higher blood 

### 7

Pb levels . In developing models to predict bone Pb levels, Park et al. ( 2009 ) note d that total and high



### 8
density lipoprotein cholesterol were selected as 2 of 18 predictors for the bone Pb level model. Their 

### 9
findings suggested that total and HDL cholesterols, as part of a larger groups of predictors, may be critical 

### 10
contributors to such p rediction models. The major risk factor that lipids represent for heart disease make 

### 11
relating lipid levels to lead exposures an interesting but challenging hypothesis to test. 

### 12

5.4.3.3. Heart Rate Variability

Pb has been shown to not only affect vascular contractil ity but also cardiac contractility. Park et al. 

### 13
( 2006 ) investigated the interac tion of key markers of the metabolic syndrome and bone Pb effect on heart 

### 14
rate variability (HRV) in a group of 413 older adults with patella Pb measurements from the VA

- NAS. 

### 15
Metabolic syndrome was defined to include three or more of the following: waist ci rcumference >102 cm

, 

### 16
hypertriglyceridemia (>150 mg/dL), low HDL cholesterol (<40 mg/dL in men), high blood pressure 

### 17

>130/85 mmHg , and high fasting glucose (>110 mg/dL). Those using antihypertensive medication or 

### 18
diabetes medications were counted as high BP or high fasting glucose, respectively. The strongest 

### 19
relationships with bone Pb were among those with three or more metabolic abnormalities. Tests for trend 

### 20
by number of metabolic abnormalities was significant at p<0.05 for patella Pb. These results sugge st 

### 21
multiplicative effects of long duration Pb exposure on key predictors of CVD. Park et al. ( 2006 ) also 

### 22

report ed the penalized spline fits to bone Pb in models assessing only main effects of bone Pb . The 

### 23
optimal degree of smoothing determined by the generalized cross

- validation criterion for all HRV 

### 24
measures was 1, which indicate d t hat the associations were nearly linear. The spline fits and associated 

### 25

statistics show ed that the (nonsignificant) bone Pb main effects on HRV measures were linear. However, 

### 26
the relationship with LF/HF was linear in log(LF/HF). 

### 27

Park et al. ( 2009 ) followed up a previous report ( Y. Cheng et al., 1998 ) , which found prolongation 

### 28 of corrected QT interval (QTc) with increasing bone Pb in men <65 years, but not in men ≥ 65. In the 

### 29

recent work, authors stratified multiple regression models on polymorphisms in three genes known to 

### 30
alter iron metabolism, hemochromatosis (HFE), transferrin (TF), and heme oxygenase

- 1 (HMOX

- 1), and 

### 31
related QTc intervals to blood, tibia and patella Pb level. The y also used interaction models with cross

- 

### 32May 2011 5

- 182 DRAFT

– DO NOT CITE OR QUOTE product terms between genotype and the Pb biomarker. The distributions of all genotypes but the HFE 

### 1
mutant, H63D, were in Hardy

- Weinberg equilibrium. Subjects homozygous for the other HFE mutant, 

### 2
C282Y, had higher bone Pb concentrations and those homozygous for H63D and heterozygous for both 

### 3

C282Y and H63D had lower bone Pb. The HMOX

- 1 variant alone, compared to the wild type, showed a 

### 4
significant interaction with tibia Pb (increased QTc [11.35 msec] for each 13 μg/ g increase in bone Pb in 

### 5

L

- allele variants). No other gene variant alone showed significant QTc differences from wild types, either 

### 6
for tibia and patella Pb or for (linear) blood Pb. Lengthening of QTc with increased tibia and blood Pb 

### 7
was more pronounced with an increase in the total number of gene variants, driven by a joint effect 

### 8

between HFE variant and HMOX

- 1 L allele. Tests for linear trend in QTc by increasing number of gene 

### 9
variants from 0 to 3 were significant for both tibia and blood Pb. This stud y provided further suggestive 

### 10
evidence of gene variants impact on long

- term Pb exposure on the cardiovascular system. 

### 11
Increased incidence of arrhythmia and atrioventricular conduction block increased in rats after 12 

### 12
weeks of Pb exposure (100 ppm; blood P b level 26.8 μg/dL) ( Reza et al., 2008 ) . Also, Pb exposure after 8 

### 13
weeks increased heart rate and systolic BP. These corresponded with increased cardia c contractile force 

### 14
and prolonged ST interval, without alteration in QRS duration or coronary flow. In contrast, another study 

### 15
found that Pb (100 μM) exposure dose

- dependently reduced myocardial contraction using rat right 

### 16
ventricular strips by reducing sa rcolemmal Ca 2+ influx and myosin ATPase activity ( Vassallo et al., 2008 )

. 

### 17
This study also found that Pb exposure could change the response to inotropic agents and blunted the 

### 18
force produced during contraction. Conversely, past studies have found that Pb exposure incre ases 

### 19

intracellular Ca 2+ content ( Favalli et al., 1977 ; Lal et al., 1991 ; Piccinini et al., 1977 ) , which could result 

### 20
in increased cardiac output and hypertension. 

### 21

5.4.3.4. Peripheral Artery Diseas e

P eripheral artery disease (P AD ) is an indica tor of atherosclerosis and measured by the ankle 

### 22
brachial index, which is the ratio of BP between the posterior tibia artery and the brachial artery

. PAD is 

### 23
typically defined as an ankle brachial index of less than 0.9. Muntner et al. ( 2005 ) , whose results 

### 24
describing the association of blood Pb and hypertension in the NHANES 1999

- 2002 dat a set were 

### 25

discussed previously , also examined the association of blood Pb with PAD. They observed an increasing 

### 26
trend in the prevalence of PAD with increasing Pb level. The OR for PAD comparing the fourth quartile 

### 27
of blood Pb (>2.47 μg/dL) to the first qu artile of blood Pb (<1.06 μg/dL) was 1.92 (95% CI: 1.02, 3.61). 

### 28
These results are consistent with a previous NHANES analysis of the association of blood Pb with PAD 

### 29
conducted by Navas

- Acien et al. ( 2004 )

. 

### 30

Navas

- Acien et al. ( 2004 ) reported a significant trend of increasing OR with increasing quartile of 

### 31
blood Pb or Cd. However, the authors reported a significant association of blo od Cd with PAD and a non



### 32
significant association with blood Pb. These authors tested both Pb and Cd in separate models

, tested the 

### 33May 2011 5

- 183 DRAFT

– DO NOT CITE OR QUOTE metals simultaneously , and tested the interaction between the metals . The correlation coefficient between 

### 1
natural log Pb and natural log Cd was 0.32 (p <0.001). When blood Pb and blood Cd were in the same 

### 2

model together, the ORs were diminished slightly but both had significant trends of increasing OR with 

### 3
increasing quartile of the metal. Their interaction was not significant. In a later analysis, Navas

- Acien et 

### 4

al. ( 2005 ) used the same 1999

- 2000 NHANES data set that they used in their 2004 paper, but constructed 

### 5

PAD models u sing a suite of urine metal concentrations. Power was reduced in this study because only 

### 6

659

- 736 subjects (compared to 2,125) had spot urine metal tests in the data set. Urine Cd, but not urine 

### 7
Pb, was reliably elevated in all models in PAD subjects, while there were indications of elevations in 

### 8
antimony and tungsten. Spot urine Pb measurements are less reliable compared to blood Pb 

### 9
measurements. In another NHANES analysis Navas

- Acien et al. ( 2005 ) reported associations between 

### 10
PAD and urinary Pb level that were sensitive to adjustment for urinary creatinine

. 

### 11

5.4.3.5. Ischemic Heart Disease

A few studies discussed in the previous Pb AQCD reported association between Pb exposure and 

### 12
increased risk of cardiovascular outcomes associated with IHD, including left ventricular hypertrophy ( J. 

### 13
Schwartz, 1991 ) and myocardial infarction ( Gustavsson et al., 2001 ) . Recently, Jain et al. ( 2007 ) reported 

### 14
the incidence of IHD (physician confirmed MI, angina pectoris) among older adult males enrolled in the 

### 15

VA

- NAS during the period of September, 1991 to December

, 2001. All subjects had blood Pb and bone 

### 16

Pb measurements with no IH D at enrollment

. Fatal and nonfatal cases were combined for analysis. Blood, 

### 17
tibia, and patella Pb levels were log

- transformed. Significant hazard ratios wer e reported for the 

### 18
association of IHD with blood Pb level and patella Pb level. When blood Pb and patella Pb were included 

### 19
simultaneously in the model, HR were only moderately attenuated (HR = 1.24 [95% CI: 0.80, 1.93 ] per 

### 20

SD increase in blood Pb and HR = 2 .62 [95% CI: 0.99, 6.93 ] per SD increase in patella Pb)

. When blood 

### 21

Pb and tibia Pb were in cluded simultaneously in the model the estimates were only moderately attenuated 

### 22
(HR = 1.38 [95% CI: 0.89, 2.13 ] per SD increase in blood Pb and HR = 1.55 [95% CI: 0 .44, 5.53 ] per SD 

### 23
increase in tibia Pb)

. This suggests that both contribute independently to IHD. In an ecological study, 

### 24

Marchwinska

- Wyrwal et al. ( 2010 ) report simple associations between 15

- year air Pb averages in 15 

### 25

Silesian cities in Poland with similar health and socioeconomic conditions but widely varying air 

### 26
contamination, including air Pb and air Cd

. They report associations between air Pb and CVD by city 

### 27
when each city was weighted equally. 

### 28
IHD, characterized by reduced blood supply to the heart, may result from increased thrombosis. A 

### 29
recent study suggests that Pb exposure promotes a procoagulant state that would contribute to thrombus 

### 30

formation ( Shin et al., 2007 ) . Pb exposure (i.v. 25 mg/kg, 1 hour) in a rat model of venous thrombosis 

### 31
resulted in increased thrombus formation. Additionally, i ncreased blood coagulation (5 μM) and thrombin 

### 32
generation (2

- 5μM) was observed in a dose

- dependent manner after Pb exposure to human erythrocytes. 

### 33May 2011 5

- 184 DRAFT

– DO NOT CITE OR QUOTE This enhanced procoagulant activity in Pb

- treated erthyrocytes was the result of increased outer cell 

### 1

membran e phosphatidylserine (PS) exposure (human RBC: 2

- 5 μM; rat RBC: 5 μM). Similar to these in 

### 2
vitro results, PS externalization on erythrocytes was increased in Pb exposed rats (50

- 100 mg/kg). 

### 3
Increased PS exposure was likely the result of increased intracell ular calcium (5 μM), enhanced 

### 4
scramblase activity (5

- 10 μM), inhibited flippase activity (5

- 10 μM), and ATP depletion (1

- 5 μM) after Pb 

### 5

exposure ( Shin et al., 2007 )

. 

### 6

5.4.3.6. Atherosclerosis

Studies provide evidence for increased atherosclerosis and intimal medial thickening (IMT) after 

### 7
Pb exposure. The association between stroke su btypes and severity of cerebral atherosclerosis was 

### 8
examined in relation to a single blood Pb level and total 72

- hour urine Pb amount (body Pb store

– EDTA 

### 9
mobilization test) in a cross

- sectional study of 153 patients (mean age 63.7) receiving digital subt raction 

### 10
angiography in Chang Gung Memorial Hospital in Taiwan from 2002 to 2005 ( T.

- H. Lee et al., 2009 )

. 

### 11
Body Pb stores were positively associated with the severity of artery stenosis in the intracranial carotid 

### 12
system but not the ext racranial and vertebrobasilar systems. As development of atherosclerosis is a 

### 13
lifelong process, cumulative body Pb stores may be more sensitive than single blood Pb in the prediction 

### 14

of atherosclerosis. 

### 15

Zeller et al. ( 2010 ) examined human radial and internal mammary arteries exposed to Pb in culture 

### 16
and reported a dose

- dependent increase in arterial intimal thickness (nonsignificant at 5 μM, significant at 

### 17
50 μM, 2 weeks) and intim al extracellular matrix accumulation (50 μM). Also, Pb exposure promoted 

### 18
endothelial cell (EC) proliferation (5 and 50 μM, 72 hours) and SMC elastin expression (50 μM, 12 

### 19
hours), as discussed above (Section 5.4.3.1) ( Zeller et al., 2010 ) . A second study showed that Pb exposure 

### 20
(100 ppm, 10 months; blood Pb level 28.4 μg/dL) to rats also increased the aortic media thickness, media



### 21
lumen ratio, and medial collage n content ( L.

- F. Zhang et al., 2009 ) . These morphological changes to the 

### 22
vessel do not only imply initiation of arteriosclerosis. These vascular changes could be to blame for 

### 23

decreased contrac tile response of the vessel due to altered visco

- dynamic vessel properties or could be an 

### 24

effect of Pb

- induced hypertension. 

### 25May 2011 5

- 185 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 15. Characteristics and quantitative data for associations of blood and bone Pb with other CVD measures.

Study Population/ Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI) a

Ischemic Heart Disease

Jain et al. ( 2007 ) 837 men from VA

NAS in Greater Boston, MA area (1991

- 2001) IHD ( MI or angina pectoris) Blood Pb: Non

- cases 6.2 (4.3) μg/dL; Cases 7.0 (3.8) μg/dL Patella Pb: Non

- case s 30.6 (19.7) μg/dL; Cases 36.8 (20.8) μg/dL

Tibia Pb: 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’s proportional hazards models adjusted for age, BMI, education, race, smoking status, pack

- years smoked, alcohol intake, history 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= 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 metric

Marchwinska

- Wyrwal et al. ( 2010 ) Residents f rom 13 Silesian cities, Poland CVD Air Pb (avg yearly rate): 127.8

- 359.9 ng/m 3 Linear regression models β= 1.52 (0.76)

Peripheral artery disease

Muntner et al. ( 2005 ) 9,961 NHANES (1999

- 2002) participants PAD Blood Pb: Q 1: <1.06 μg/dL, Q 2: 1.06

- 1.63 μg/dL Q 3: 1.63

- 2.47 μg/dL Q 4: >2 .47 μg/dL Logistic regression models adjusted for age, race/ethnicity, sex, diabetes mellitus, BMI, ciga rette smoking, alcohol consumption, high school education, healt h insurance status OR (95% CI): 1.00 (Reference ), 1.00 (0.45, 2.22), 1 .21 (0.66, 2.2 3), 1.92 (1.02, 3.61)

Navas

- Acien et al. ( 2005 ) 790 participants, age ≥ 40 y, from NHANES (1999

- 2000) PAD Urine 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 Model 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 of urinary Pb
Array of metals in urine also evaluated. Heart rate variability

Park et al. ( 2006 ) 413 men from Normative Aging Study in Greater Boston, MA area (11/14/2000

- 12/22/2004) HRV (SDNN, HF, HF norm , LF, LF norm , LF/HF) Patella Pb: Median (IQR): 23.0 (15

- 34) μg/g Estimated a : Median (IQR): 16.3 (10.4

- 25.8) μg/g

Tibia Pb: Median (IQR): 19.0 (11

- 28) μg/g Linear regression models adjusted for age, cigarette smoking, alcohol consumption, room temperature, season (model 2) BMI, fasting blood glucose, HDL cholesterol, 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) LF: 0.9 (

– 2.0, 3.9) L og 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) LF: 0.6 (

– 1.9, 3.1) Log LF/HF: 3.0 (

– 8.7, 16.2) Per 15.4 μg/g patella Pb

Among those with metabolic syndrome the strongest effects were observed.May 2011 5

- 186 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Parameter Pb Data Statistical Analysis Effect Estimate (95% CI) a

Park et al. ( 2009 ) 613 men from Normative Aging Study in Greater Boston, MA area (8/1991

- 12/1995) QTc b interval Blood Pb: Median (IQR): 5 (4

- 7) μg/dL

Patell a Pb: Median (IQR): 26 (18

- 37) μg/g

Tibia Pb: Median (IQR): 19 (14

- 27) μg/g Linear regression models adjusted for age, BMI, smoking status, serum calcium, and diabetes. Blood Pb: β: 1.3 (

- 0.76, 3.36) IQR: 3 μg/dL

Patella Pb: β: 2.64 (0.13, 5.15) IQR: 19 μg/g

Tibia Pb: β: 2.85 (0.29, 5.40) IQR: 13 μg/g

a Estimated patella Pb accounts for declining trend in patella Pb levels between analysis of bone Pb and HRV.
b Heart

- rate

- corrected QT interval calculated by Bazett ’ s formula



### 5.4.3.7. Summary

There are a limited number of studies that investigate the association between Pb exposure and 

### 1
cardiovascular effects other than hypertension (Table 5

- 15). These studies have presented associations 

### 2
between various measures of Pb, representing distinct expo sure time periods, and atherosclerosis, IHD, 

### 3
PAD, and HRV. Also, limited, mixed evidence of occupational exposure to Pb and altered cholesterol 

### 4
have been reported. Additionally, studies in isolated vascular tissues and cells provided mechanistic 

### 5

evidence t o support the biological plausibility of these other vascular effects and cardiotoxicity. A recent 

### 6
study provided evidence for the interaction between Pb exposure and gene variants for iron metabolism 

### 7
on the prolonged QT interval ( Park, Hu, et al., 2009 ) . Blood Pb (>2.5 μg/dL) was associated with greater 

### 8

risk for P AD in two NHANES analyses ( Muntner et al., 2005 ; Navas

- Acien et al., 2004 ) . In addition, in 

### 9

the VA

- NAS cohort of older adult men, blood Pb (≥ 5 μg/dL) and patella Pb levels were associated with 

### 10
increased morbidity from IHD ( Jain et al., 2007 ) . A recent study involving both human and toxicological 

### 11

studies eluc idated the mechanisms for observed Pb

- mediated arterial IMT, an early event in Pb

- induced 

### 12
atherogenesis ( Zeller et al., 2010 ) . Studies in isolated tissues and cells found that Pb stimulated the 

### 13
synthesis and secretion of IL

- 8 in EC, which was responsible for stimulating SMC invasion into the 

### 14
vessel intimal layer. Pb exposure also increased extracellular matrix and elastin, primary sites for lipid 

### 15
deposition in the vessel wall. Overall, the relatively few studies that investigate associations between 

### 16
biomarkers of Pb exposure and other cardiovascular events provide supportive evidence for the role of Pb 

### 17
in the development of these diseases, yet further research i s needed to understand these relationships. 

### 18



## 5.4.4. Mortality Associated with Long

- Term Lead Exposure

The previous Pb AQCD ( U.S. EPA, 2006 ) stated that collectively the then available analyses of 

### 19
NHANES II and III data suggest a significant effect of Pb on cardiovascular mortality in the gener al U.S. 

### 20
population but cautioned that these findings should be replicated before these estimates for Pb

- induced 

### 21
cardiovascular mortality could be used for quantitative risk assessment purposes. This involved two 

### 22
NHANES analyses that examined the associatio n of blood Pb with all cause and cause

- specific mortality 

### 23May 2011 5

- 187 DRAFT

– DO NOT CITE OR QUOTE ( Lustberg & Silbergeld, 2002 ; Schober et al., 2006 ) . As bl ood Pb levels in adults are comprised of both 

### 1
recent Pb exposure and Pb mobilization from bone, it is unclear whether the mortality associated with 

### 2
blood Pb levels are due to current or cumulative Pb exposures. Given the decline in ambient air Pb 

### 3

concentra tions and population blood Pb levels, it is likely that study subjects had a much higher Pb 

### 4
exposure in their past than what they are experiencing currently. Using NHANES II data, Lustberg and 

### 5
Silbergeld ( 2002 ) found significant increases in all

- cause, circulatory and cancer mortality, comparing 

### 6

those with blood Pb levels from 20

- 29 μg/dL to those with blood Pb levels l ess than 10 μg/dL . Using 

### 7
NHANES III data, Schober et al. ( 2006 ) found significant associations for all cause mortality, 

### 8
cardiovascular, and cancer mortality comparing those with blood Pb levels from 5

- 9 μ g/dL and above 10 

### 9
μg/dL to those with blood Pb levels less than 5 μ g/dL

. 

### 10

Three new studies make substantial additions to the evidence base. A furt her analysis of the 

### 11
NHANES III database by a different research group using different methods provides further information 

### 12
addressing uncertainties from other earlier analyses. Two longitudinal prospective studies in different 

### 13
cohorts conducted by differen t researchers with different methods in different parts of the U.S. provide 

### 14

coherence to the evidence base. Menke et al. ( 2006 ) examined the associations of all

- cause and cause



### 15
specific mortality using NHANES III data

. Subjects at least 18 years of age were followed up to 12 years 

### 16
after they were surveyed and 1,661 deaths were identified. Those with blood Pb levels from 3.63 to 10 

### 17
μg/dL had significantly higher risks of all

- cause (1.25 [95% CI: 1.04, 1.51]), cardiovasc ular (1.55 [95% 

### 18
CI: 1.08, 2.24]), MI (1.89 [95% CI: 1.04, 3.43]), and stroke (2.51 [95% CI: 1.20, 2.26]) mortality 

### 19
compared to those with blood Pb levels less than 1.93 μg/dL and non

- significantly increased risk of 

### 20
cancer mortality. Hazard ratios were not higher comparing those with blood Pb levels from 1.94 to 3.62 

### 21
μg/dL to those with blood Pb levels <1.93 μg/dL. However, t rends of increasing hazard with increasing 

### 22

blood Pb tertile were significant (p <0.017) for all models of all CVD presented. Menke et al. ( 2006 ) 

### 23
evaluated several of the model covariates (e.g. diabetes, hypertension, and GFR) in a subgroup analysis 

### 24
( Figure 5

- 3 7). The authors reported that there were no interactions between blood Pb and oth er adjusted 

### 25

variables and found a high consistency of HRs across models with a varying number of control variables 

### 26
(indicating little residual confounding). In the previous NHANES III analysis of the association of blood 

### 27
Pb with mortality, Schober et al. ( 2006 ) included participants greater than 40 years of age (N = 9,686) and 

### 28
adjusted for covariate s including age, sex, ethnicity and smoking rather than the full suite of covariates 

### 29
evaluated by Menke et al. ( 2006 ) . Schober et al. ( 2006 ) reported significant associations comparing those 

### 30 with blood Pb levels ≥ 10 μg/dL to those with blood Pb levels <5 μg/dL for all

- cause (1.59 [95% CI: 1.28, 

### 31
1.98]), CVD (1. 55 [95% CI: 1.16, 2.07]), and cancer (1.69 [95% CI: 1.14, 2.52]) mortality and generally 

### 32

non

- significant increases comparing those with blood Pb levels from 5

- 9 μg/dL to those with blood Pb 

### 33
levels <5 μg/dL. 

### 34May 2011 5

- 188 DRAFT

– DO NOT CITE OR QUOTE Source: Used with permission from Lippincott W iliams & 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 8 0th and 20th percentiles of the blood Pb distribution (4.92 μg/dL versus 1.46 μg/dL, respectively). Figure 5

- 37. Multivariate adjusted relative hazards of all

- cause and cardiovascular mortality.

Both Menke et al. ( 2006 ) and Schober et al. ( 2006 ) present mortality curves that plot the hazard 

### 1

ra tios against blood Pb level. Figure 5

- 3 8 shows the mortality curves for both stroke and MI reported by 

### 2

Menke et al. ( 2006 ) , which reach a peak around 6

- 7 μg/dL. The curves were fitted by restricted quadratic 

### 3
splines w ith knots at the 10th percentile (1.00 μg/dL ; 0.048 μM/L ), the 50th percentile (2.67 μg/dL ; 0.287 

### 4
μM/L )

, and the 80th percentile (5.98 μg/dL) blood Pb levels. 

### 5May 2011 5

- 189 DRAFT

– DO NOT CITE OR QUOTE Source: Used with permission from Lippincott Wiliams & Wilkins, Menke et al. ( 2006 )

Note: A h istogram of blood Pb levels is superimposed in the background and displayed on the right axis. Figure 5

- 38. Multivariate adjusted relative hazard (left axis) of mortality associated with blood Pb lev el between 1 μ g/dL and 10 μ g/dL.

Schober et al. ( 2006 ) examined proportional hazard assumption s, test ed for linear trend across 

### 1
blood Pb tertiles, and evaluat ed log

- transformed continuous blood Pb level as a 5

- knot cubic spline 

### 2
(position of knots not stated). The test for linear trend by blood Pb tertile was significant at the p<0.01 

### 3

level . The results of the spline fit of the continuous blood Pb level term to relative hazard of all 

### 4

cardiovas cular diseases reported by Schober et al. ( 2006 ) is shown in Figure 5

- 39

. In contrast to the curve 

### 5
presented by Menke et al. ( 2006 ) the relative hazard axis and the blood Pb axis are both linear

. Dashed 

### 6

line s are 95% CIs and the referent blood Pb level was 1.5 μg/dL. Despite differences in the age groups 

### 7
included, follow

- up time, and categorization of blood Pb levels, results reported by Menke et al. ( 2006 ) 

### 8

and Schober e t al. ( 2006 ) consistently conclude that higher blood Pb is associated with increased CVD 

### 9

mortal ity. 

### 10May 2011 5

- 190 DRAFT

– DO NOT CITE OR QUOTE 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

- 39. Relative risk of all cause mortality for different blood Pb levels compared with referent level of 1.5 μ g/dL (12.5 th percentile).

In addition to the NHANES analyses de scribed above, two studies of older adult males ( Weisskopf 

### 1
et al., 2009 ) and older adult females ( Khalil, 2010 ; Khal il, Wilson, et al., 2009 ) were conducted. 

### 2
Weisskopf et al. ( 2009 ) used data from the VA

- NAS to determine the association of blood, tibia, and 

### 3
patella Pb with mortality

. The authors identified 241 deaths over an average observation period of 8.9 

### 4
years (7

, 673 person

- years) in study su bjects. The strongest associations were observed between mortality 

### 5
and patella Pb concentration. Non

- significant increases in CVD mortality with t ibia Pb and no association 

### 6
between blood Pb and mortality were observed. Tibia bone Pb concentration is though t to reflect a longer 

### 7
cumulative exposure period than patella bone Pb because the residence time of Pb in trabecular bone is 

### 8
shorter than in cortical bone. Ischemic heart disease contributed most to the relationship between patella 

### 9

Pb and all CVD death wit h HR of 2.69 (95% CI: 1.42, 5.08). Although there was high correlation between 

### 10
tibia and patella Pb (Pearson r = 0.77), trabecular bone Pb may have more influence on circulating blood 

### 11
Pb level, and thus organ concentration of Pb, than cortical bone Pb beca use of its shorter residence time in 

### 12
bone. In contrast to the NHANES analyses, blood Pb was not significantly related to cardiovascular 

### 13
mortality in this study. This discrepancy may be related to differences in sample size and resulting power, 

### 14
modeling str ategies (e.g. linear versus log

- linear blood Pb level terms), or age range of the study 

### 15
populations

. T he youngest subjects at baseline in the Weisskopf et al. ( 2009 ) study were approximately 

### 16

50

- 55 years old, compared to the youngest in the Menke et al. ( 2006 ) and Schober et al. ( 2006 ) studies, 

### 17

which were 17 and 40 years , respectively

. Further the blood Pb tertile analysis of th e Weisskopf study 

### 18May 2011 5

- 191 DRAFT

– DO NOT CITE OR QUOTE could have been affected if the majority of a hypothesized non

- linear effect was contained largely in the 

### 1
lowest (reference) blood Pb tertile. 

### 2
Weisskopf et al. ( 2009 ) also conducted an exposure

- response analysis. The test for linear trend of 

### 3
HR by bone Pb tertile was significant in both tibia and patella Pb models. The linear relationship using 

### 4
tertile patella Pb was confirmed in other models using continuous patella Pb and non

- linear penalized 

### 5
spline terms, where higher order components were non

- significant. The number of knots and their 

### 6
placement within th e Pb variable, which can influence these results though the number and placement of 

### 7
knots of the penalized spline, were determined by an iterative best fit procedure to the data. 

### 8

Concentration

- response relationships shown in Figure 5

- 40 are approximately l inear for patella Pb on the 

### 9
log hazard ratio scale for all CVD, but appear non

- linear for ischemic heart disease (p <0.10). Peak HR is 

### 10
shown around 60 μg/g, beyond which HR tends to decrease, though the confidence limits are wide

. 

### 11

Source: Used with perm ission from Lippincott Wiliams & 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 dashed 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 participants are indicated by short vertical lines on the absicissa. Figure 5

- 40. Nonlinear association between patella bon e Pb concentratio n and the log of HR (logHR) for all

- cause, cardiovascular, and ischemic heart disease adjusted for age, education, smoking status, and pack

- years of smoking among participants without isch emic heart disease at baseline.May 2011 5

- 192 DRAFT

– DO NOT CITE OR QUOTE The association of Pb with mortalit y has also been examined among women enrolled in the Study 

### 1
of Osteoporotic Fractures (SOF) ( Khalil, Wilson, et al., 2009 ) . This prospective cohort (N = 533) enrolled 

### 2

female volunteers from two locations across the U.S. (Baltimore, MD and Monongahela Valley, PA). All



### 3
cause mortality comparing those with blood Pb levels >8 μg/dL to those with blood Pb levels <8 μg/dL 

### 4
was significantly increased (1.59 [95% CI: 1.02, 2.49])

. Combined cardiovascular disease mortalit y (1.78 

### 5
[95% CI: 0.92, 3.45]), c oronary heart disease mortality (3.08 [95% CI: 1.23, 7.70]), but not s troke 

### 6
mortality (1.13 [95% CI: 0.34, 3.81]) HR was increased among the women enrolled in this study. In 

### 7
addition, Khalil et al. ( 2010 ) provided both tertile and quintiles analyses, as well as exposure

- response 

### 8

results shown in Figure 5

- 41. 

### 9

Source: Khalil et al. ( 2010 ) Figure 5

- 41. Multivariate adjusted relative hazard (left axis) of mortality associated with blood Pb levels between 1 μ g/d L and 15 μ g/dL.



### 5.4.4.1. Summary

The mortality results in this review support and expand upon findings from the previous Pb AQCD 

### 10
( U.S. EPA, 2006 ) , which included two NHANES mortality studies ( Lustberg & S ilbergeld, 2002 ; Schober 

### 11
et al., 2006 ) . The recent NHANES mortality study discussed above ( Menke et al., 2006 ) addressed many 

### 12May 2011 5

- 193 DRAFT

– DO NOT CITE OR QUOTE of the limitations of the these earlier studies, including control for a wider range of potential confounders, 

### 1
testing for interactions with Pb, consideration of exposure

- response relationships, extensive model 

### 2
evaluations, and including sub

- categories of CVD. Further, an assoc iation with increased mortality was 

### 3
observed at lower population blood Pb concentrations. The mean blood Pb level of the NHANES III 

### 4
population was 2.58 μg/dL . The Pb attributable risk of increased cardiovascular mortality in the 

### 5

NHANES III analysis of Menk e et al. ( 2006 ) reached its maximum at blood Pb levels between of 6 and 7 

### 6
μg/dL. In addition, the first evidence that bone Pb, a metric of cumulative Pb exposure, is associated with 

### 7
increased mortality was reported. S everal studies report associations between the accumulated Pb in bon e 

### 8
and higher CVD morbidity, which are consistent with the mortality findings. 

### 9
Quantitative differences in Pb

- associated hazard for death between studies may be influenced by 

### 10
age range of the study groups, follow up time to death, variation in model adjustment, central tendency 

### 11
and range of the Pb exposure measure, assumptions of linearity of the Pb exposure term, and choice of 

### 12
exposure metric. Q uantitative differences in Pb

- associated mort ality across NHANES II and NHANES III 

### 13

studies or between different NHANES III may be explained by the u se of continuous or ordered blood Pb 

### 14
exposure variables and different data selection strategies. Further, s tudies using ordered categories of 

### 15

blood Pb le vel may obtain different results , as the range of blood Pb level represented in the reference 

### 16

category will affect the calculated coefficients of the remaining percentiles or groups. 

### 17
Specifically, Menke et al. ( 2006 ) is the strongest study presently published for estimating the 

### 18

national effects of Pb on cardiovas cular disease

- related mortality

. The study uses the nationally 

### 19
representative NHANES III (1988

– 1994) sample allowing results to be generalized to the segmen t of the 

### 20
US population included in the sample. The results provide confirmation of earlier published studies and 

### 21
address some of the key weaknesses noted in those studies. Weisskopf et al. ( 2009 ) is the first published 

### 22
mortality study using bone Pb as an exposure index. The study is a prospective study with nearly 100% 

### 23

succes sful follow

- up of deaths. This rigorous study found increased cardiovascular disease mortality with 

### 24

trabecular bone Pb. The Khalil et al. ( 2010 ; 2009 ) Study of Osteopor otic Fractures provides supporting 

### 25
results in a different study cohort consisting of white female s aged 65

- 87 years. Further, a number of prior 

### 26
studies have already found association between accumulated Pb reflected in bone Pb measurements and 

### 27
higher CVD m orbidity (see CVD morbidity section), to which is added new findings of increased 

### 28
mortality due to CVD from long

- term Pb exposure (bone Pb). Despite the differences in design and 

### 29
methods across studies, effects on mortality were consistently observed ( Figu re 5

- 42 and Table 5

- 16 ). In 

### 30

studies that broke out CVD

- related mortality into sub

- categories, MI, stroke, and IHD mortality, death



### 31
causes related to higher BP and hypertension were all significantly elevated as Pb exposure increased. 

### 32May 2011 5

- 194 DRAFT

– DO NOT CITE OR QUOTE Reference Outcome Study N Blood Pb Comparison

Menke et al. ( 2006 ) All Cause NHANES III 13,946 ≥3.63 Q3 v Q1
1988

– 1994 1.94

– 3.62 Q2 v Q1
Mean age = 44 ≤1.93 Q1 Reference
Mean blood Pb = 2.58 CVD ≥3.63 Q3 v Q1
1.94

– 3.62 Q2 v Q1
≤1.93 Q1 Reference
MI ≥3.63 Q3 v Q1
1.94

– 3.62 Q2 v Q1
≤1.93 Q1 Reference
Stroke ≥3.63 Q3 v Q1
1.94

– 3.62 Q2 v Q1
≤1.93 Q1 Reference
Schober et al. ( 2006 ) All Cause 9757 ≥ 10 Q3 v Q1
5

- 9 Q2 v Q1 age > 40 y r < 5 Q1 Reference
CVD ≥ 10 Q3 v Q1
5

- 9 Q2 v Q1
< 5 Q1 Reference Lustbert & Silbergeld ( 2002 ) All Cause NHANES II 4190 20

- 29 Q3 v Q1
1976

- 1980 10

- 19 Q2 v Q1
Mean age = 54 y r < 10 Q1 Reference
Mean blood Pb = 14.0 CVD 20

- 29 Q3 v Q1
10

- 19 Q2 v Q1
< 10 Q1 Reference
Khalil et al. ( 2009 ) All Cause SOF 533 ≥8 Dichotom ous

women CVD 1986

– 1988 ≥8 Dichotomous
Mean age = 70 y r CHD ≥8 Dichotomous
Mean blood Pb = 5.3 Stroke ≥8 Dichotomous
<8 Q1 Reference Weisskopf et al. ( 2009 ) All Cause VA

- NAS 868 >6 Q3 v Q1
1991

– 1999 4

- 6 Q2 v Q1
M en <4 Q1 Reference
Mean age = 67.3 y r CVD >6 Q3 v Q1
Mean blood Pb = 5.7 4

- 6 Q2 v Q1
<4 Q1 Reference
IHD >6 Q3 v Q1
4

- 6 Q2 v Q1
<4 Q1 Reference

Tibia Pb (μg/g)
All Cause Mean = 30.6 Q3 v Q1
CVD Mean = 30.6 Q3 v Q1
IHD Mean = 30.6 Q3 v Q1
Mean = 13.9 Q1 Reference

Patella Pb (μg/g)
All Cause Mean = 49.6 Q3 v Q1
Mean = 28.8 Q2 v Q1
Mean = 14.5 Q1 Reference
CVD Mean = 49.6 Q3 v Q1
Mean = 28.8 Q2 v Q1
Mean = 14.5 Q1 Reference
IHD Mean = 49.6 Q3 v Q1
Mean = 28.8 Q2 v Q1
Mean = 14.5 Q1 Reference

Hazard Ratio (95% CI) Figure 5

- 42. Hazard ratios between blood Pb (closed markers), bone Pb (open markers), all

- cause mortality (diamonds), and cardiovascular mortality (circles).May 2011 5

- 195 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 16. Additional characteristics and quantitative data for associations of blood and bone Pb with CVD mortality for results presented in Figure 5

- 42.

Study Population/ Location Parameter Pb Data Statistical Analys is Effect Estimate (95% CI)

Menke et al. ( 2006 ) 13,946 adult participants of NHANES III

, ≥ 17 y r (1988

- 1994) All cause and cause

- specific mortality (through 2000) CVD:ICD

- 9 390

- 434; ICD

- 10 I0 0

- I99), MI (ICD

- 9 410

- 414 and 429.2; ICD

- 10 I20

- I25), stroke (ICD

- 9 430

- 434 and 436

- 438; ICD

- 10 I60

- I69). Blood Pb: 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, Mod el 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 ( 2006 ) 9686 adult participant of NHANES III, ≥ 40 y r All cause and cause

- specific mortality Ordered categorical blood Pb level:

< 5 μg/dL 5

- 9 μg/dL ≥10 μg/dL Also ln(b lood Pb level) Survey

- design adjusted Cox proportional hazard adjusted for sex, age, race/ethnicity, smoking, education level, median follow

- up time = 8.55 y 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) Weisskopf et al. ( 2009 ) 868 men

, > 55 y r , 95% white, from Normative Aging Study in Greater Boston area, MA All cause and cause

- specific mortality 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 haz ard 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

- cause (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. ( 2009 ) 533 women, 65

- 87 y, from Study of Osteoporotic Fractures cohort in Baltimore, MD and Monongahela Valley, PA All cause and cause

- specific mortality Blood Pb: Mean (SD; range): 5.3 (2.3; 1

- 21) μg/dL Cox proportional hazards regression analysis adjusted for age, clinic, BMI, education, smokin g, alcohol intake, estrogen use, hypertension, total hip BMD, walking for exercise, and diabetes, , mean follow

- up time = 12 ± 3 y. All cause ( ≥ 8 μg/dL vs.

< 8 μg/dL): 1.59 (1.02, 2.49)

CVD: 1.78 (0.92, 3.45) Coronary Heart Disease: 3.08 (1.23, 7.70) Str oke: 1.13 (0.34, 3.81) Cancer: 1.64 (0.73, 3.71)May 2011 5

- 196 DRAFT

– DO NOT CITE OR QUOTE Study Population/ Location Parameter Pb Data Statistical Analys is Effect Estimate (95% CI)
Neuberger et al. ( 2009 ) Residents at or near Tar Creek Superfund si te, Ottawa County

, OK (exposed pop. 5,852, unexposed pop. 16,210) Cause

- specific mortality Not reported Standardized m ortality ratio based on 2000 US Census data Heart disease: Both sexes : 114.1 (113.1, 115.2) Men: 118 (116.4, 119.6) Women: 111 (109.5

, 11 2.5)

Stroke: Both sexes: 121.6 (119.2, 123.9) Men: 146.7 (107.4, 195.7) Women: 106.5 (80.2, 138.6)
Cocco et al. ( 2007 ) 933 male Pb smelter workers from Sardinia, Italy (1973

- 2003) All cause and cause

- specific mortality Not reported Standardized mortality ratio All cause: 56 (46, 68)
CVD: 37 (25, 55)

References not included in Figure 5

- 4 2 are included in this table.



## 5.4.5. Air Lead

- PM Studies

5.4.5.1. Hospital Admissions

In addition to blood Pb, some recent epidemiologic studies have used Pb measured in PM 10 and 

### 1

PM 2.5 air samples to represent Pb exposures. Some studies have analyzed Pb individually, whereas others 

### 2
have applied source apportionment techniques to analyze Pb as part of a group of correlated components. 

### 3

A common limitation of air

- Pb studies is the variable size distribution of Pb

- bearing PM (Section 3.5.3) 

### 4
and its relationship with blood Pb level s. Relative to studies of Pb biomarkers, time

- series studies provide 

### 5

weak evidence for association between PM 2.5 - Pb concentrations and cardiovascular hospital admissions 

### 6
and mortality in older adults. In a time

- series study of 106 U.S. counties, Bell et al

. ( 2009 ) found that an 

### 7
increase in lag 0 PM 2.5 - Pb was associated with an increased r isk of cardiovascular hospital admissions. 

### 8
Quantitative results were not presented; however, the 95% CI was wide and included the null value. Ostro 

### 9

et al. ( 2007 ) found that a 5 ng/m 3 (interq uartile range) increase in lag 3 PM 2.5 - Pb was associated with a 

### 10
1.89% increase (95% CI:

- 0.57, 4.40%) in cardiovascular mortality in six California counties in the cool 

### 11
season. In both of these studies, statistically significant positive associations were observed for other PM 

### 12
components such as nickel, vanadium, and zinc also. In the absence of detailed data on correlations 

### 13
among components or results adjusted for copollutants, it is difficult to exclude confounding by these 

### 14
other components. 

### 15

To address c orrelations among PM chemical components, some studies have applied source 

### 16
apportionment techniques to group components into common source categories. In these source

- factor 

### 17
studies, it is difficult to attribute findings to a particular component in a grou p. In a study of 20 counties 

### 18
near Atlanta, GA, Sarnat et al. ( 2008 ) found that PM 2.5 - Pb mass was explained by a “woodsmoke” factor, 

### 19
which was associated with a 2.4% (95% CI: 1.5, 3.3%) increased risk of cardiovascular hospital 

### 20May 2011 5

- 197 DRAFT

– DO NOT CITE OR QUOTE admission (l ag 0, per interquartile range increase). Less than 10% of variation in PM 2.5 - Pb mass was 

### 1
explained by “woodsmoke,” thus the association may not be attributable specifically to Pb. Andersen et 

### 2

al. ( 2007 ) found that in Copenhagen, Denmark, variation in PM 10 - Pb was explained by a “vehicle” factor 

### 3
that also included copper and iron. A 0.6 μg/m 3 increase in the 3

- day lagged sum of “vehicle” factor 

### 4
pollutants was not associated with increased risk of cardiovascular hospital admissions among adults age 

### 5
65 years and older (RR: 0.999, [95% CI: 0.993, 1.004]). 

### 6

5.4.5.2. Mortality

Time

- series studies of PM 2.5 - Pb have reported positive associations with all

- cause mortality. In the 

### 7
Harvard Six Cities Study, Laden et al. ( 2000 ) found a 1.16% (95% CI: 0.20, 2.9%) increased risk in all



### 8
cause mortality per 461.4 ng/m 3 (5th

- 9th percentile) increase i n PM 2.5 - Pb. In six California counties, 

### 9

Ostro et al. ( 2007 ) found that a 5 ng/m 3 (interquartile range) increase in PM 2.5 - Pb was associated with a 

### 10
1.74% (95% CI: 0.24, 3.26%) increased risk i n all

- cause mortality during the cool season. The limitations 

### 11

of air

- Pb studies have been d escribed previously (Section 5.5 .5.1), and are relevant to the interpretation of 

### 12
these findings for all

- cause mortality. 

### 13



## 5.4.6. Summary and Causal Determination

The 2006 Pb AQCD concluded that the re was a relationship between increased blood Pb and bone 

### 14

Pb and increased adverse cardiovascular outcomes in adults, including increased BP and increased 

### 15

incidence of hypertension ( U.S. EPA, 2006 ) . This was substantiated by the coherence of effects observed 

### 16

acr oss epidemiologic and toxicological studies. The large evidence base of epidemiologic studies 

### 17
conducted by many researchers in many locations using diff erent designs found a clear positive 

### 18

association between blood Pb level and BP

. Meta

- analysis of these s tudies found that e ach doubling of 

### 19

blood Pb level (between 1 and > 40 μg/dL ) was associated with a 1 mmHg increase in systolic BP and a 

### 20
0.6 mmHg increase in diastolic BP ( Nawrot et al., 2002 )

. In addition, most of the reviewed studies using 

### 21
cumulative Pb exposure measured by bone Pb levels also showed increased BP. Similarly, toxicological 

### 22
studies provided evidence for exposure to low levels of Pb resulting in increased BP in experimental 

### 23
animals that persists long after the cessation of Pb exposure. Also, animal toxicological studies provided 

### 24
mechanistic evidence to support the biological plausibility of Pb

- induced hypertension, including 

### 25

ox idative stress, altered sympathetic activity, and vasomodulator imbalance. Studies in the 2006 Pb 

### 26
AQCD also suggested a connection between Pb exposure and other cardiovascular diseases such as 

### 27
ischemic heart disease, cerebrovascular disease, peripheral vas cular disease, and cardiovascular disease 

### 28
related mortality, however this evidence was limited. 

### 29
Building on the strong body of evidence presented in the 2006 Pb AQCD, recent studies continue 

### 30
to support associations between long

- term Pb exposure and cardio vascular effects with recent 

### 31May 2011 5

- 198 DRAFT

– DO NOT CITE OR QUOTE epidemiologic studies informing past uncertainties (e.g. confounding, low Pb exposures). A recent study 

### 1

in an e thnically diverse community

- based cohort of women and men aged 50

- 70 years of age suggests 

### 2

that Pb has an acute effe ct on BP as a function of recent dose measured by blood Pb and a chronic effect 

### 3

on hypertension risk as a function of cumulative exposure measured by tibia Pb ( Martin et al., 2006 ) . This 

### 4
study verified other studies by demonstrating that with each increase of 1 μg/dL blood Pb level, systolic 

### 5

BP would increase 1 mm Hg and diastolic BP would increase 0.5 mmHg. Additionally, recent 

### 6
epidemiologic studies provided evidence for associations between blood Pb and hypertension at relatively 

### 7
low blood Pb levels; a positive relationship was found in the NHANES data set at a geometric mean 

### 8

blood Pb lev el of 1.64 μ g/dL ( Muntner et al., 2005 )

. Animal toxicological studies also provide support for 

### 9
effects of low blood Pb level on increased BP with statistically signifi cant increases shown as low as 2 

### 10
μg/dL ( Tsao et al., 2000 ) . Collectively, all animal toxicological studies providing blood Pb level and BP 

### 11
measurements report positive increases in BP with increasing blood Pb level ( Figure 5

- 3 4). New studies 

### 12
also demonstrate reversibility of Pb

- induced increased BP following Pb exposure cessation or chelation. 

### 13
Epidemiologic studies continue to investigate the relationship between bone Pb, representing 

### 14
cumulative Pb exposure, and increased BP. Navas

- Acien et al. ( 2008 ) published a meta

- analysis of 

### 15
epidemiological studies examining this association

. S tudies passing the detailed inclusion criteria all 

### 16
showed positive relationships between bone Pb measures and BP and all but one that characterized 

### 17
hypertension showed positive risk or odds ratios associated with bone Pb

. Recent epidemiological studies 

### 18

hav e also emphasized the interaction between long

- term Pb exposure and factors that moderate or modify 

### 19
the Pb effect, like chronic stress and metabolic syndrome, on BP and hypertension. Bone Pb coupled with 

### 20
high stress was associated with a strong and reliabl e increased risk of developing hypertension in an 

### 21
originally nonhypertensive group ( Peters et al., 2007 ) . Also, long duration Pb exposure interacted with 

### 22
components of the metabolic syndrome to drive HRV in directions associated with increased 

### 23

c ardiovascular events ( Park et al., 2006 )

. 

### 24
Recent epidemiologi c studies investigated the interaction of genotypes with effects of Pb on the 

### 25
cardiovascular system. Significant evidence was presented for modification of the effect of blood Pb level 

### 26
on BP by ALAD genotype ( Scinicariello et al., 2010 ) . Additionally, polymorphisms in the 

### 27

hemochrom atosis gene modified the pulse pressure response to bone Pb exposure, where pulse pressure 

### 28
represents as a good predictor of cardiovascular morbidity and mortality and an indicator of arterial 

### 29

stiffness ( A. Zhang et al., 2010 ) . Park et al. ( 2009 ) provided further evidence of gene variants, specifically 

### 30

those related to iron metabolism , impacting the effect of long

- term Pb exposure on the cardiovascular 

### 31
system, evaluated by QT interval changes. 

### 32
Not only has Pb exposure been shown to increase BP and hypertension, but Pb exposure can 

### 33
contribute to the development of other cardiovascular d iseases. Recent epidemiologic and toxicological 

### 34
studies provide evidence for increased atherosclerosis, thrombosis, ischemic heart disease

, p eripheral 

### 35
artery disease , arrhythmia, and cardiac contractility (blood Pb levels >2.5 μg/dL). 

### 36May 2011 5

- 199 DRAFT

– DO NOT CITE OR QUOTE Animal toxicological evidence continues to build on the evidence supporting the mechanisms 

### 1
leading to these cardiovascular alterations. Enhanced understanding of Pb

- induced oxidative stress 

### 2 including  ̇NO inactivation, endothelial dysfunction leading to altered vascular reacti vity, activation of the 

### 3
RAAS, and vasomodulator imbalance provides biological plausibility for the consistently positive 

### 4

associations observed between blood and bone Pb and cardiovascular effects. 

### 5
New evidence extends the potential continuum of Pb

- related cardiovascular effects by 

### 6
demonstrating associations between Pb exposure and both cardiovascular and all

- cause mortality. All



### 7
cause mortality was positively associated with increased blood Pb level. The recent analysis of the 

### 8
nationally representative NHAN ES III (1988

- 1994) sample reported positive associations with 

### 9
cardiovascular mortality, with stronger associations with myocardial infarction and stroke mortality 

### 10
( Menke et al., 2006 ) . These findin gs were supported by a community

- based cohort of women age 65

- 87 

### 11
years, in which effect estimates were increased for mortality from cardiovascular disease and coronary 

### 12

heart disease ( Khalil, Wilson, et al., 2009 ) . Weisskopf et al. ( 2009 ) published the first mortality study 

### 13
using bone Pb as an exposure index. This prospective study found that trabecular bone Pb levels were 

### 14
associated with increased mortality from cardiovascular disease and ischemic heart disease with hazard 

### 15
ratios of 5.6 and 8.4, respectively. 

### 16
In summary, new studi es evaluated in the current review support or expand upon the strong body of 

### 17
evidence presented in the 2006 AQCD that Pb exposure is causally associated with cardiovascular health 

### 18
effects. Both epidemiologic and toxicological studies continue to demonstrat e a consistently positive 

### 19
relationship between Pb exposure and increased BP or hypertension development in adults and this 

### 20 relationship is observed at adult blood Pb levels (mean 2 μg/dL) lower than that reported in the 2006 

### 21
AQCD. While some studies evalua te exposure

- response relationships, the information is inconclusive. 

### 22
Recent studies investigate cumulative Pb exposure measures and suggest that bone Pb related strongly to 

### 23
hypertension risk. Evidence of Pb increasing the risk of developing other cardiovas cular diseases has also 

### 24
been shown. By demonstrating Pb

- induced oxidative stress including  ̇NO inactivation, endothelial 

### 25
dysfunction leading to altered vascular reactivity, activation of the RAAS, and vasomodulator imbalance, 

### 26
toxicological studies have cha racterized the mode of action of Pb and provided biological plausibility for 

### 27
the consistently positive associations observed in epidemiologic studies between blood and bone Pb and 

### 28
cardiovascular effects. These observed associations between Pb exposure and cardiovascular morbidity 

### 29
are supported by recent reports of increased cardiovascular mortality. Collectively, the evidence integrated 

### 30
across epidemiologic and toxicological studies as well as across the spectrum of cardiovascular health 

### 31
endpoints is suffic ient to conclude that there is a causal relationship between Pb exposure s and 

### 32

cardiovascular health effects

. 

### 33May 2011 5

- 200 DRAFT

– DO NOT CITE OR QUOTE 

# 5.5. Renal Effects

5.5.1. Introduction

This section summarizes key findings with regard to effects of Pb on the kidney in animal 

### 1
toxicology and epidemiologic studies

. After chronic Pb exposure, pathological changes in the renal 

### 2

system include proximal tubule (PT) cytomegaly, renal cell apoptosis, mitochond rial dysfunction, 

### 3
aminoaciduria, increased electrolyte excretion, ATPase dysf unction, oxidant redox imbalance, altered 

### 4
glomerular filtration rate (GFR)

, chronic kidney disease (CKD) development, and altered  ̇ NO 

### 5

homeostasis with ensuing elevated BP

. 

### 6
The cardiovascular and renal system s are intimately linked. Homeostatic control at the kidney level 

### 7
functions to regulate water and electrolyte balance via filtration, re

- absorption and excretion and is under 

### 8
tight hormonal control. Pb exposure damages the kidneys and its vasculature and systemic hypertension 

### 9

ensues with effects on the cardiovascular and renal systems (Section 5.4). Chronic increases in vascular 

### 10
pressure can contribute to glomerular and renal vasculature injury, which can lead to progressive renal 

### 11
dysfunction and kidney failure

. In this manner, Pb

- induced hypertension has been noted as one cause of 

### 12

Pb

- induced renal disease. However, the relationship between BP and renal function is more complicated. 

### 13
Not only does hypertension cont ribute to renal dysfunction but damage to the kidneys can also cause 

### 14
increased BP. Long

- term control of arterial pressure is affected by body fluid homeostasis which is 

### 15
regulated by the kidneys. In examining the physiological definition of BP ( i.e., mean B P equates to 

### 16
cardiac output multiplied by total peripheral resistance [ TPR ]) the role of the kidneys in BP regulation is 

### 17
highlighted

. Cardiac output is driven by left ventricular and circulating blood volume

. TPR is driven by 

### 18
vasomodulation and electrolyte balance. Thus , it is possible to dissect the causes of hypertension from 

### 19
features of primary kidney disease. Increased extracellular fluid volume results in increased blood volume 

### 20

which enhances venous return of blood to the heart and increases cardiac ou tput. Increased cardiac output 

### 21
not only directly increases BP, but also increases TPR due to a compensatory autoregulation or vessel 

### 22
constriction. In addition, damage to the renal vasculature will alter the intra

- renal vascular resistance 

### 23
thereby altering kidney function and affecting the balance between renal function and BP. The interrelated 

### 24
nature of these systems can lead to further exacerbation of vascular and kidney dysfunction following Pb 

### 25
exposure. As kidney dysfunction can increase BP and increased BP can lead to further damage to the 

### 26
kidneys, Pb

- induced damage to both systems may result in a cycle of further increased severity of disease. 

### 27
In general, associations between blood Pb and bone Pb (particularly in the tibia) with health 

### 28

outcomes in adult s indicate acute effects of recent dose and chronic effects of cumulative dose, 

### 29
respectively. In some physiological circumstances of increased bone remodeling or loss (e.g., osteoporosis 

### 30
and pregnancy), Pb from bone of adults may also contribute substantia lly to blood Pb concentrations. 

### 31
Blood Pb in children, although highly affected by recent dose, is also influenced by Pb stored in bone due 

### 32May 2011 5

- 201 DRAFT

– DO NOT CITE OR QUOTE to rapid growth

- related bone turnover in children relative to adults. Thus, blood Pb in children is also 

### 1

reflective o f cumulative dose. Additional details on the interpretation of Pb in blood and bone are 

### 2
provided in Section 4.3.5. 

### 3

5.5.1.1. Kidney Outcome Measures

The primary function of the kidneys is to filter waste from the body while maintaining appropriate 

### 4

levels of water an d essential chemicals, such as electrolytes, in the body. Therefore, the gold standard for 

### 5
kidney function assessment involves measurement of the GFR through administration of an exogenous 

### 6
radionuclide or radiocontrast marker (e.g., 125I

- iothalamate, iohex ol) followed by timed sequential blood 

### 7
samples or, more recently, kidney imaging, to assess clearance through the kidneys. This procedure is 

### 8

invasive and time

- consuming. Therefore, serum levels of endogenous compounds are routinely used to 

### 9

estimate GFR in large epidemiology studies and clinical settings. Creatinine is the most commonly used 

### 10
endogenous compound; blood urea nitrogen (BUN) has also been used. Increased serum concentration or 

### 11
decreased kidney clearance of these markers both indicate decreased k idney function. The main limitation 

### 12
of endogenous compounds identified to date is that non

- kidney factors impact their serum levels. 

### 13
Specifically, since cre atinine is metabolized from creatine in muscle, muscle mass and diet affect serum 

### 14

levels resulting i n variation in different population subgroups, e.g., women and children compared to 

### 15

men, which are unrelated to kidney function. Measured creatinine clearance, involving measurement and 

### 16
comparison of creatinine in both serum and urine, can address this pro blem. However, measured 

### 17

creatinine clearance utilizes timed urine collections, traditionally over a 24

- hour period, and the challenge 

### 18
of complete urine collection over an extended time period makes compliance difficult. 

### 19
Therefore equations to estimate kid ney filtration that utilize serum creatinine but also incorporate 

### 20
age, sex, race, and, in some, weight

, in an attempt to adjust for differences in muscle mass have been 

### 21
developed. Although these are imperfect surrogates for muscle mass, s uch equations are currently the 

### 22
preferred outcome assessment method. Traditionally, the Cockcroft

- Gault equation ( Cockcroft & Gault, 

### 23

1976 ) , which estimates creatinine clearance, a GFR surrogate, has been used. In the last decade, the 

### 24
abbreviated Modification of Diet in Kidney Disease (MDRD ) Study equation ( Levey et al., 1999 ; Levey 

### 25
et al., 2000 )

, which estimates GFR, has become the standard in the kidney epidemiology and clinical 

### 26

communities. With widespread use of the MDRD equation, it became clear that it underestimates GFR at 

### 27
levels in the normal range. Therefore, the CKD

- E pidemiology Collaboratio n (CKD

- EPI) equation was 

### 28
recently developed to be more accurate in this range ( Levey et al., 2009 ) . This is a decided advantage in 

### 29
nephrotoxicant research since most participants in occupational and many even in general population 

### 30
studies have GFRs in a range that is underestimated by the MDRD equation. 

### 31

Both the M DRD and CKD

- EPI equations use serum creatinine and the inability to adjust for 

### 32

muscle mass has led to evaluation of alternative serum biomarkers such as cystatin C, a cysteine protease 

### 33May 2011 5

- 202 DRAFT

– DO NOT CITE OR QUOTE inhibitor that is filtered, reabsorbed, and catabolized in the kidney ( Fried, 2009 ) . It is produced and 

### 1
secreted by all nucleated cells thus avoiding the muscle mass confou nding with serum creatinine ( Fried, 

### 2

2009 )

. Despite this, recent research indicates that serum cystatin C varies by age, sex, and race ( Kottgen 

### 3
et al., 2008 ) a nd a cystatin C

- based eGFR equation that includes age, sex, and race was recently 

### 4
developed ( Stevens et al., 2008 )

. 

### 5

Most of the kidney outcome measures discussed above were developed for use in the clinical 

### 6
setting. Unfortunately, they are insensitive for detection of early kidney damage, as evidenced by the fact 

### 7
that serum creatinine remains normal after kidney donation. Th erefore, in the last two decades, the utility 

### 8
of early biological effect (EBE) markers as indicators of preclinical kidney damage has been of interest. 

### 9
These can be categorized as markers of function (i.e., low molecular weight proteins that should be 

### 10

reab sorbed in the PT such as β2

- microglobulin and retinol

- binding protein [RBP]); biochemical alteration 

### 11
(i.e., urinary eicosanoids such as prostaglandin E2, prostaglandin F2 alpha, 6

- keto

- prostaglandin F 1 alpha, 

### 12
and thromboxane B2); and cytotoxicity (e.g., N

- acetyl

- β

- D

- glucosaminidase [NAG]) ( Cardenas et al., 

### 13

1993 ) . Elevated leve ls may indicate an increased risk for subsequent kidney dysfunction. However, most 

### 14
of these markers are research tools only, and their prognostic value remains uncertain since prospective 

### 15
studies of most of these markers in nephrotoxicant

- exposed populatio ns are quite limited to date. 

### 16
Recently, microalbuminuria has been identified as a PT marker, not just glomerular as previously thought 

### 17
( Comper & Russo, 2009 ) . Kidney EBE markers are a major recent focus for research in patients with 

### 18
acute kidney injury (AKI) and markers such as neutrophil gelatinase

- associated lipocalin (NGAL) and 

### 19
kidney injury molecule

- 1 (Kim

- 1), developed in AKI re search, may prove useful for chronic 

### 20
nephrotoxicant work as well ( Devarajan, 2007 ; M. A. Ferguson et al., 2008 )

. 

### 21



## 5.5.2. Nephrotoxicity and Renal Pathology Related to Lead Effects

5.5.2.1. Toxicology

Renal Function and Interstitial F ibrosis

Past studies have shown that c hronic continuous or repeated Pb

- exposure can result in interstitial 

### 22
nephritis and focal or tubular atrophy. After an initial 3 months of Pb exposure ( in a longitudinal 12

- 

### 23
month exposure study to either 0.01 % [ low dose ] or 0.5% [ high dose ] Pb acetate in drinking water, male 

### 24
rats), elevated GFR, consistent with hyperfiltration, and renal hypertrophy were observed ; high dose 

### 25

animals also had increased NAG and GST ( Khalil

- Manesh, Gonick , Cohen, Bergamaschi, et al., 1992 ; 

### 26

Khalil

- Manesh, Gonick, & Cohen, 1993 ; Khalil

- Manesh, Gonick, Cohen, Alinovi, et al., 1992 ) . At 6 

### 27

mont hs of exposure, GFR decreased , albuminuria was present , and pathology ensue d with focal tubular 

### 28May 2011 5

- 203 DRAFT

– DO NOT CITE OR QUOTE atrophy and interstitial fibrosis formation. This pathology remained consistent out to 12 months, and at 12 

### 1
months glomeruli developed focal and segmental scler osis . Similarly, GFR remained decreased after 12 

### 2
months of exposure

. The evidence provided by toxicological studies that showed a difference in GFR 

### 3
with acute Pb exposure (hyperfiltration) versus chronic exposure (decreased GFR) provided biological 

### 4
plausib ility to epidemiological studies that observed a similar phenomenon by age in humans with Pb 

### 5
exposure. These dichotomous changes in GFR (acute versus chronic Pb exposure) are consistent between 

### 6
the toxicological and epidemiology literature. 

### 7

Biomarkers of Pb

- induced renal toxicity have been developed including the enzymes lysosomal 

### 8
NAG, GST, brush border antigens (BB50, BBA, HF5), and Tamm

- Horsfall protein. GST functions as a 

### 9

biomarker since r enal ALAD is protected by the kidney antioxidant GSH . Urinary NAG and GST levels 

### 10
increase after 3 months of high dose Pb exposure (blood Pb level 125 μg/dL) ( Dehpour et al., 1999 ; 

### 11

Khalil

- Manesh, Gonick, Cohen, Alinovi, et al., 1992 ) , whereas only urinary NAG was increased 

### 12
following low dose Pb exposure (blood Pb level 29 μg/dL) ( Khalil

- Manesh, Gonick, & Cohen, 1993 )

. 

### 13
Occupational stu dies found that urinary NAG correlated best with recent blood Pb changes. 

### 14
The adverse effects of chronic Pb exposure as detailed above are partially rescued with chelation 

### 15
therapy such as DMSA ( Khalil

- Manesh, Gonick, Cohen, Bergamaschi, et al., 1992 ) . Improvements 

### 16

include increased GFR, decreased albuminuria, and decreased inclusion body numbers but little change in 

### 17
tubulointerstitial scarring. Administration of an Indian herb to Pb exposed mice, as is discussed in further 

### 18
detail in the antioxidant section ( Section 5.5.5 ), produced similar findings. There was a fun ction rescue 

### 19

however Pb

- induced pathology remained ( Jayakumar et al., 2009 )

. Thus

, administra tion of various 

### 20
compounds (chelators, antioxidants) to Pb

- exposed animals produced hemodynamic rescue. 

### 21
Recent studies have confirmed the previously observed increase in serum creatinine following 

### 22
chronic Pb exposure in rats. Annabi Berrahal et al

. ( 2011 ) reported on the effects of age

- dependent 

### 23

exposure to Pb on nephrotoxicity in male rats. Pups were exposed to Pb lactationally (as a result of dams 

### 24
consuming water containing 50 ppm Pb acetate ) until weaning. Thereafter the offspring were exposed to 

### 25
the same solution from weaning (day 21) until sacrifice. Male pups were sacrificed at age 40 days 

### 26
(puberty ; blood Pb level 12.7 μg/dL) and at age 65 days (post

- pube rty ; blood Pb level 7.5 μg/dL). Serum 

### 27
creatinine was elevated at both 40 days and 65 days (0.54 and 0.60 mg/dL compared to control values of 

### 28
0.45 mg/dL [ p < 0.001 ])

. Various parameters of Pb

- dependent renal dysfunction are listed in Table 5

- 17 

### 29

below. Other in vestigators have also shown that chronic Pb exposure has adverse effects on renal 

### 30
function. Pb exposed male rats (500 ppm Pb acetate in drinking water for 7 months) had elevated urinary 

### 31
pH, proteinuria, as well as glucose and blood in the urine ( Navarro

- Moreno et al., 2009 )

. 

### 32
Qiao et al

. ( 2006 ) measured the effect of Pb o n the expression of the renal fibrosis

- relate d nuclear 

### 33

factor

- kappa B (NFκB), transforming growth facto r (TGF

- β ) and fibronectin in Sprague

- Dawley rat 

### 34
kidney. Pb was administered at a dose of 0.5% Pb acetate, continuously for either one, two or three 

### 35

months. All growth factors increased by the end of three months of treatment but only NF κB increased 

### 36May 2011 5

- 204 DRAFT

– DO NOT CITE OR QUOTE progres sively at each time period. These changes were hypothetically related to the development of Pb



### 1
induced renal fibrosis in rats, but, no histolog y was performed

. 

### 2

Roncal et al

. ( 2007 ) found that Pb accelerate d arteriolopathy and tubulointerstitial injury in non

- Pb



### 3

related CKD . Sprague

- Dawley rats were administered Pb acetate at 150 ppm for 4 weeks, followed by 

### 4
remnant kidney surgery (left kidney mass reduced by 2/3 and right kidney removed) and then 

### 5

continuatio n of Pb exposure for an additional 12 weeks. Pb

- treated rats had higher systolic BP , lower 

### 6
creatinine clearance, and higher proteinuria than controls. Most striking was development of worse 

### 7
arteriolar disease, peritubular capillary loss, tubulointerstitial damage, and macrophage infiltration. Pb 

### 8
treatment was associated with significant worsening of pre

- glomerular vascular disease, as characterized 

### 9
by an increase in the media

- to

- lumen ratio. There was also a higher percentage of segmental sclerosis 

### 10
within g lomeruli and a tendency for a higher number of sclerotic glomeruli. Additionally, a loss of 

### 11
peritubular capillaries, as reflected by a reduction in thrombomodulin staining , was observed

. This was 

### 12
associated with worse tubular injury ( osteopontin staining ) due to more interstitial fibrosis (type III 

### 13
collagen staining) and a greater macrophage infiltration in the interstitium. The increase in macrophages 

### 14
was associated with higher renal MCP

- 1 mRNA . Low level Pb exposure concomitant with existing renal 

### 15

insuffi ciency due to surgical kidney resection accelerated vascular disease and glomerular pathology. 

### 16
These findings are consistent with the previous work of Bagchi et al. ( 2005 ) also showing that Pb exposed 

### 17

animals with non

- Pb

- related CKD (remnant surgery) had kidney dysfunction including impairment of the 

### 18

renin

- angiotensin system (Losartan challenge), elevated systolic BP, and a lterations in renal excretion of 

### 19
Pb, K + , and Na + . Thus, this model shows that low blood Pb level may exacerb ate pre

- existing underlying 

### 20
kidney disease. 

### 21

Table 5

- 17. Indicators of renal damage in male rats exposed to 50 ppm Pb for 40 and 65 days, starting at parturition

Biomarker PND40 Control PN D 40 Pb PND65 Control PND65 Pb

Blood Pb level (μg/dL) 1.8 12.7 2.1 7.5

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: Used with permission from John Wiley & Sons, Berrahal et al. ( 2011 )May 2011 5

- 205 DRAFT

– DO NOT CITE OR QUOTE Histological C hanges

Historical studies discussed in previous Pb AQCDs have identified Pb

- dependent renal damage by 

### 1
the presence of dense intranuclear inclusion bodies, which are capable of sequestering Pb ( Goyer, May, et 

### 2
al., 1970 ) . Chelators like CaNa 2 EDTA removed these inclusion bodies from affected nuclei ( Goyer et al., 

### 3

1978 ) . Multiple endpoints indicate dysfunction in the PT after Pb exposure. Pb

- induced formation of 

### 4

intranuclear inclusion bodies in the PT is protective; Pb is sequestered such that it is not in its 

### 5
bioavailable, free, toxicologically active form. Intranuclear inclusion bodies are seen in the kidney with 

### 6
acute Pb exposures but present to a lesser degree with chronic exposures (See Section 5.2.3 for further 

### 7
discussion)

. Other PT ultrastructural changes in Pb

- induced nephropathy include changes to the PT 

### 8
epithelium, endoplasmic reticulum dilation, nuclear membrane blebbing, and autophagosome enlargement 

### 9
( Fowler et al., 1980 ; Goyer , Leonard, et al., 1970 ) . Symptoms similar to the PT transport

- associated 

### 10
Fanconi syndrome appear with Pb exposure, albeit often at high doses of Pb . These symptoms, which 

### 11
include increased urinary electrolyte excretion (zinc), decreased Na

- K

- ATPase activity, mitochondrial 

### 12
aberrations, and aminoaciduria, have also been reported in Pb exposed children. 

### 13
New studies since the 2006 Pb AQCD are consisten t with the historical findings and build upon the 

### 14
literature base by including the role of antioxidants in histological preservation. Massanyi et al

. ( 2007 ) 

### 15
reported on Pb induced alterations in male Wistar ra t kidneys after single i.p. doses of Pb acetate ( 50, 25, 

### 16
and 12.5 mg/kg ); kidneys were removed and analyzed 48 hours after Pb administration

. Qualitative 

### 17
microscopic analysis detected dilated Bowman ’ s capsules and dilated blood vessels in the interstitium 

### 18
with evident hemorrhagic alterations. Quantitative histomorphometric analysis revealed increased relative 

### 19
volume of interstitium and increased relative volume of tubules in the experimental groups. The diameter 

### 20
of renal corpuscles and the diameter of glome ruli and Bowman ’ s capsule were significantly increased. 

### 21
Measurement of tubular diameter showed dilatation of the tubule with a significant decrease of the height 

### 22
of tubular epithelium compatible with degenerative renal alterations. These findings extend th e 

### 23

observations of Fowler et al

. ( 1980 ) and Khalil

- Manesh et al. ( 1992 ; 1992 ) ; in particular, the enlarged 

### 24
glomeruli are consistent with the early hyperfiltration caused by Pb

. 

### 25
A recent study has also reported inclusion body formation in the nuclei

, cytoplasm , and 

### 26

mitochondria of PT cells of Pb exposed rats ( 50 mg Pb/kg bw i.p., every 48 h for 14 days) ( Navarro



### 27
Moreno et al., 2009 ) . These inclusion bodies were not observed in chronically Pb

- exposed rats (500 ppm 

### 28
Pb in drinking water, 7 months). However, chronic Pb exposure resulted in morphological alterations 

### 29
including loss of PT apical me mbrane brush border, collapse and closure of the PT lumen, and formation 

### 30
of abnormal intercellular junctions. 

### 31
Vogetseder et al

. ( 2008 ) examined the proliferative capacity of the renal PT (particularly the S3 

### 32
segment) following i.v. administration of Pb to juvenile and adult male Wistar rats. Proliferation induction 

### 33

was examined by detection of Bromo

- 2 ’

- deoxyuridine (BrdU)

, Ki

- 67 ( labels S, G2, and M phase cells)

, 

### 34May 2011 5

- 206 DRAFT

– DO NOT CITE OR QUOTE and cyclin D1 ( a n essential cell cycle progression protein ) . Th e cycling marker Ki

- 67 revea led a much 

### 1
higher proliferation rate in the S3 segment in control juvenile rats (4.8 ± 0.3%) compared with control 

### 2
adult rats (0.4 ± 0.1%). Pb administration ( 3.8 mg /100 g bw) increased the proportion of Ki

- 67

- positive 

### 3

cells to 26.1 ± 0 .3% in juvenile rat s and 31.9 ± 0 .3% in adult rats. Thus

, the increased proliferation caused 

### 4
by Pb was age independent. The proliferation induction caused by Pb administration may be a result of 

### 5
reduced cell cycle inhibition by p27 kip

- 1 . Acute Pb treatment increased the inci dence of cyclin D1 in the 

### 6

BrdU

- positive cells suggesting Pb was able to accelerate reentry into the cell cycle and cause proliferation 

### 7

in the PT. Pb

- dependent proliferation has also been reported in the retina with gestational Pb exposure 

### 8
( Giddabasappa et al., 2011 )

. 

### 9
Ademuyiwa et al

. ( 2009 ) examined Pb

- induced phospholipidosis and cholesterogenesis in rat 

### 10
tissues. Sprague

- Dawley rats were exposed to 200, 300 and 400 ppm Pb acetate for 12 weeks. The Pb 

### 11
exposure resulted in induction of phospholipidosis in kidney tissue, accompanied by depletion of renal 

### 12
cholesterol. The authors suggested that induction of cholesterogenesis and phospholipidosis in kidney 

### 13
may be responsible fo r some of the subtle and insidious cellular effects of Pb

- mediating nephrotoxic 

### 14

manifestations. Drug

- induced PT phospholipidosis is seen clinically with use of the potentially 

### 15
nephrotoxic aminoglycoside drugs, including gentamicin ( Baronas et al., 2007 )

. 

### 16 Various antioxidants have been shown to attenuate histopathological changes to the 17 kidney. Ozsoy et al. ( I n Press ) found L

- carnitine to be protective in a model of experimental 18 Pb toxicity in female rats. Markers of histopathological change in the kidney, including 19

tubule dilatati on, degeneration, necrosis, and interstitial inflammation were rescued by L

20 carnitine treatment in females. Male rats exposed to Pb (0.2% for 6 weeks) also displayed 21 tubular damage, whereas concomitant treatment with Pb and an extract of Achyranthes 22

aspera ameliorated the observed damage ( Jayakumar et al., 2009 ) . El

- Nekeety et al. ( 2009 ) 23 found an extract of the folk medicine plant Aquilegia vulgaris to be protective against Pb 24

acetate

- induced kidney injury in Sprague

- Dawley rats. Rats w ere treated with Pb (20 ppm; 2 25 weeks) and extract (administered before, during, or after Pb). Pb treatment resulted in 26 tubular dilatation, vacuolar and cloudy epithelial cell lining, interstitial inflammatory cell 27 infiltration, hemorrhage, cellular debris, and glomerulus hypercellularity. Concomitant 28 exposure of Pb and extract produced histology indiscernible from control. Post treatment 29 with extract partially rescued the Pb induced histopathology. El

- Neweshy and El

- Sayed 30 studied the influence of vitamin C supplementation (20 mg/kg pretreatment every other day) 31

on Pb

- induced histopathological alterations in exposed male rats (20 mg/kg by intragastric 32 feeding onc e daily for 60 days). Control rats showed normal histology, while Pb

- treated rats 33 exhibited karyomegaly with eosinophilic intranuclear inclusion bodies in the epithelial cells 34 of the proximal tubules. Glomerular damage and tubular necrosis with invading 35

in flammatory cells were also seen. Rats treated with Pb acetate plus vitamin C exhibited 36May 2011 5

- 207 DRAFT

– DO NOT CITE OR QUOTE 

### relatively mild or no karyomegaly with eosinophilic intranuclear inclusion bodies in the 1 proximal tubules. Normal glomeruli were noted in animals exposed to Pb and vitam in C. 2 These findings are presented in more detail in Section 5.5.5 but they consistently show that 3 some antioxidants are capable of preventing or rescuing Pb

- dependent histopathological 4 changes. 5

Alteration of R enal V asculature and R eactivity

As discussed i n Section 5.5.1, changes in renal vasculature function or induction of hypertension 

### 6
can contribute to further renal dysfunction. Pb will increase BP through the promotion of oxidative stress 

### 7
and altered vascular reactivity. Also, Pb has been shown to act o n known vasomodulating systems i n the 

### 8
kidney. In the kidney, two vascular tone mediators

,  ̇ NO and ET

- 1

, are affected by Pb exposure

. 

### 9

A ntioxidant s attenuated Pb

- dependent oxidative/nitrosative stress in the kidney and abrogate d the Pb



### 10

induced increased BP ( Vaziri, Ding, et al., 1999 ) . Administration of the vasoconstri ctor endothelin

- 1 (ET



### 11
1) affected mean arterial pressure (MAP) and decrease d GFR ( Novak & Banks, 1995 )

. Acute high dose 

### 12
Pb exposure completely blocked the ET

- 1

- dependent GFR decrease but had no effect on MAP. Depletion 

### 13
of the endogenou s antioxidant glutathione using the drug buthionine sulfoximine, a GSH synthase 

### 14
inhibitor, increased BP and increased kidney nitr o tyrosine formation without Pb exposure, demonstrating 

### 15
the importance of GSH in maintenance of BP ( Vaziri et al., 2000 ) . Multiple studies have shown that Pb 

### 16
exposure deplet es GSH stores. Catecholamines are vascular moderators that are also affected by Pb 

### 17

exposure ( Carmignani et a l., 2000 ) . The effect on BP with Pb exposure is especially relevant to the kidney 

### 18
because it is both a target of Pb deposition and a mitigator of BP. These historic data detail the interaction 

### 19

of known modulators of vascular tone with Pb. 

### 20
Recently, Varg as

- Robles et al

. ( 2007 ) examined the effect of Pb exposure (100 ppm Pb ace tate for 

### 21
12 weeks) on BP and angiotensin II vasoconstriction in isolated perfused kidney and interlobar arteries. 

### 22
Vascular reactivity was evaluated in the presence and absence of L

- NAME in both Pb

- treated and control 

### 23

animals. Pb exposure significantly incr eased BP (134 ± 3 v ersus 100 ± 6 mm Hg), eNOS protein 

### 24
expression, oxidative stress, and vascular reactivity to angiotensin II. L

- NAME potentiated the vascular 

### 25
response to angiotensin II in the control group, but had no effect on the Pb

- treated group. Conve rsely

, 

### 26
passive microvessel distensibility, measured after deactivation of myogenic tone by papaverine, was 

### 27
significantly lower in the arteries of Pb

- exposed rats. Nitrites released from the kidney under the influence 

### 28
of angiotensin II in the Pb group were lower as compared to the control group whereas 3

- nitrotyrosine 

### 29
was higher in the Pb group. The authors conclude that Pb exposure increases vascular tone through nitric 

### 30

oxide

- dependent and independent mechanisms, increasing renal vascular sensit ivity to vasoconstrictors. 

### 31May 2011 5

- 208 DRAFT

– DO NOT CITE OR QUOTE Apoptosis and/or I schemic N ecrosis of T ubules and G lomeruli

Apoptosis or programmed cell death in excess can cause cell atrophy while an insufficien cy can 

### 1
lead to uncontrolled cell proliferation, such as cancer. Pb exposure has b een shown to cause 

### 2
morphological changes to the kidney structure. Some of these Pb

- induced changes are a result of cellular 

### 3
apoptosis or necrosis. Past studies have shown Pb

- induced necrosis in proximal tubule cells ( Fowler et al., 

### 4

1980 ) . Pb

- induced apoptosis is known to act through the mitochondria ( Rana, 2008 )

. Pb

- induced c alcium 

### 5
overload may depolarize the mitochondria, resulting in cytochrome c release, caspase activation

, and 

### 6
apoptosis. The apoptosis is mediated by Bax translocation to the mitochondria and can be blocked by 

### 7
overexpression of Bcl

- xl. Also, Pb

- induced ALA accumulation can generate ROS , which may damage 

### 8

DNA leading to apoptosis

. 

### 9
Mitochondria are targets of Pb toxicity and often involved in apoptosis

. Pb can induce uncoupling 

### 10
of oxidative phosphorylation, decreased substrate u tilization, and modification of mitochondrial ion 

### 11
transport. ATP energetics are affected when ATP

- Pb chelates are formed and ATPase activity is decreased. 

### 12

ROS formation can contribute to these mitochondrial changes and to other changes within the kidney. 

### 13

A ntioxidant supplementation after Pb exposure can remedy some adverse outcomes. All of these 

### 14
outcomes, in conjunction with Pb

- dependent depletion of antioxidants (e.g. GSH) and elevation of lipid 

### 15
peroxidation point to possible susceptibility of the kidney t o apoptosis or necrosis. Literature in this area 

### 16
is emerging. 

### 17
Rodriguez

- Iturbe et al

. ( 2005 ) reported that chronic exposure to low doses of Pb (100 ppm in 

### 18
drinking water for 14 weeks) results in renal infiltration of immune cells, apoptosis, NFκB activation and 

### 19
overexpression of tubulointerstitial angiotensin II. 

### 20

Navarro

- Moreno et al

. ( 2009 ) examined the effect of 500 ppm Pb in drinking water over 7 months 

### 21
on the structure (including intercellular junctions), function

, and biochemical properties of PT cells of 

### 22

Wistar rats. Pb effects in epithelial cell s consisted of an e arly loss of the apical microvillae, followed by a 

### 23
decrement of the luminal space and the respective apposition and proximity of apical membranes, 

### 24
resulting in the formation of atypical intercellular contacts and adhesion structures. Inclusion bodies were 

### 25

found in nuclei

, cytoplasm , and mitochondria. Lipid peroxidation (TBARS measurement ) was increased 

### 26

in the Pb

- treated animals as compared to controls. Calcium uptake was diminished and neither proline nor 

### 27
serine incorporation that was present in controls wa s noted in the PT of Pb

- exposed animals . The authors 

### 28
speculate that Pb may compete with calcium in the establishment and maintenance of intercellular 

### 29
junctions. 

### 30
Tubular necrosis was also observed in rats exposed to Pb acetate (100 ppm s.c.) for 30 days ( El



### 31
Sokkary et al., 2005 )

. Histological section s of kidneys from Pb treated rats showed tubular degeneration 

### 32

with some necrotic cells . Similarly, El

- Neweshy and El

- Sayed reported g lomerular damage and tubular 

### 33
necrosis with invading inflammatory cells after Pb treatment ( 20 mg /kg by intragastric feeding once daily 

### 34May 2011 5

- 209 DRAFT

– DO NOT CITE OR QUOTE for 60 days ) to male rats. The incidence of necrosis was decreased in both of these studies by pretreatment 

### 1
with either melatonin or vitamin C. Pretreatment with melatonin (10 mg/kg), an efficacious free radical 

### 2

scave nger and indirect antioxidant, resulted in a near normal tubular structure. The authors conclude that 

### 3
melatonin protect ed the liver and kidneys from the damaging effects of exposure to Pb through inhibition 

### 4
of lipid peroxidation and stimulation of endogenous antioxidative defense systems ( El

- Sokkary et al., 

### 5

2005 )

. V itamin C supplementation (20 mg/kg pretreatment every other day) protected the renal 

### 6
architecture and histology ( El

- Neweshy & El

- Sayed )

. 

### 7
Wang et al

. ( 2009 ) examined the effect of Pb ace tate ( 0.25, 0.5 and 1 μM ) on cell death in cultured 

### 8

rat primary PT cells. A progressive loss in cell viability, due to both apoptosis and necrosis, were seen in 

### 9
cells exposed to Pb . Apoptosis predominated and could be ameliorated with concomitant N

- acetylc ysteine 

### 10

exposure , whereas necrosis was unaffected. E levation of ROS levels and intercellular calcium, depletion 

### 11
of mitochondrial membrane potential

, and intracellular glutathione levels were seen durin g Pb exposure. 

### 12

Pb

- dependent a popto sis was demonstrated morphological ly ( Hoechst 33258 staining ) with 

### 13
condensed/fragmented chromatin and apoptotic body formation

. CAT and SOD activities were 

### 14
significantly elevated , reflecting the response to accumulation of ROS

. 

### 15

Table 5

- 18 presents the acute and chronic renal effects of Pb exposure observed in recent and past 

### 16
animal toxicology studies. 

### 17

Table 5

- 18. Acute and chronic effects of Pb on the kidney/renal system

– evidence from animal toxicology studies.

Acute Chronic

Mitochondri al 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 p ressure Decreased GFR



### 5.5.2.2. Epidemiology in Adults

A number of significant advances in research on the impact of Pb on the kidney in the 20 years 

### 18
following the 1986 Pb AQCD ( U .S. EPA, 1986 ) were noted in the 2006 Pb AQCD ( U.S. EPA, 2006 )

. 

### 19
These included research in general and CKD patient populations at much lower levels (5

- 10 μg/dL) of Pb 

### 20
exposure than previously studied. Furthermore, chelation therapy in CKD patients was evaluated, also at 

### 21

levels of ex posure not previously thought to be amenable to chelation. Through these lines of research, it 

### 22
became clear that chronic Pb nephropathy, characterized by tubulointerstitial nephritis due to chronic, 

### 23
high

- level Pb exposure ( Bonucchi et al., 2007 )

, is a small portion of the contribution that Pb makes to 

### 24May 2011 5

- 210 DRAFT

– DO NOT CITE OR QUOTE kidney dysfunction overall in the population . Pb, at much lower doses than those causing chronic Pb 

### 1
nephropathy, acts as a cofactor with other more established kidney risks to increase the risk for CKD and 

### 2
disease progression in susceptible patients. The populations at risk for kidney dysfunction (diabetics and 

### 3
hypertensives) ar e increasing worldwide, particularly in countries where obesity is epidemic. Pb exposure 

### 4
continues to decline in many industrialized countries, although less so among minority populations, 

### 5
which, notably, are also higher risk groups for CKD. Thus, the exte nt of the public health impact of Pb on 

### 6
the kidney depends on the balance of these two factors. 

### 7

In the 2006 Pb AQCD ( U.S. EPA, 2006 ) , several key issues could not be completely resolved based 

### 8

on the Pb

- kidney literature published to date. These included the lowest Pb dose at which adverse kidney 

### 9
effects occur, whether associations at current Pb levels are due to higher past exposures, the impacts of Pb 

### 10
on the kidney in children, the use of paradoxical Pb

- kidney associations on risk assessment in the 

### 11
occupational setting, and the impact of co

- exposure to other environmental nephrotoxicants, such as 

### 12
cadmium. In the intervening five years, relevant data for several of these challenges have been published. 

### 13

General Population Studies

The 2006 Pb AQCD reported studies that examined the effect of Pb exposure on kidney function in 

### 14
general populations. This was a new approach to Pb

- kidney resear ch in the two decade time period 

### 15
covered by the 2006 Pb AQCD. The studies in this category provided critical evidence that the effects of 

### 16
Pb on the kidney occur at much lower doses than previously appreciated based on occupational exposure 

### 17

data. The landma rk Cadmibel Study was the first large environmental study of this type that adjusted for 

### 18
multiple kidney risk factors ( Staessen et al., 1992 ) . It included 965 men and 1,016 women recruited from 

### 19
cadmium exposed and control areas in Belgium. Mean blood Pb was 11.4 μg/dL (range 2.3

- 72.5) and 7.5 

### 20 μg/dL (range 1. 7

- 60.3) in men and women, respectively. After adjustment, log transformed blood Pb was 

### 21
negatively associated with measured creatinine clearance. A 10

- fold increase in blood Pb was associated 

### 22

with a decrease in creatinine clearance of 10 and 13 mL/min in me n and women, respectively. Blood Pb 

### 23
was also negatively associated with estimated creatinine clearance. 

### 24
Four studies assessing the kidney impact of Pb exposure have been published in the Normative 

### 25
Aging Study (NAS) population ( R. Kim et al., 1996 ; Payton et al., 1994 ; Tsaih et al., 2004 ; M. T. Wu et 

### 26
al., 2003 ) . Participants in t his study were originally recruited in the 1960s in the Greater Boston area. 

### 27
Inclusion criteria included male gender, age 21 to 80 years, and absence of chronic medical conditions. 

### 28
Longitudinal data contained in two NAS publications remain essential, parti cularly in light of the dearth 

### 29
of prospective data on the kidney effects of Pb. The first of these included 459 men whose blood Pb levels 

### 30
from periodic exa minations, conducted every 3 to 5 years during 1979

- 1994, were estimated based on 

### 31

measurements in sto red packed red blood cell samples adjuste d for hematocrit level ( R. Kim et al., 1996 )

. 

### 32
Participants were randomly selected to be representative of the entire NAS population in terms of age and 

### 33May 2011 5

- 211 DRAFT

– DO NOT CITE OR QUOTE follow

- up. Kidney status was assessed with serum creatinine. Data from four evaluations were available 

### 1
for the majority of participants. Mean (SD) a ge, blood Pb level, and serum creatinine, at baseline, were 

### 2 56.9 (8.3) years, 9.9 (6.1) μg/dL, and 1.2 (0.2) mg/dL, respectively. In the longitud inal analysis, using 

### 3

random

- effects modeling, ln

- tran sformed blood Pb was associated with change in serum creat inine over 

### 4
the subsequent follow

- up period in the 428 participants whose highest blood Pb level was ≤ 25 μg/dL (β= 

### 5
0.027 [95% CI: 0.0, 0.054]); associations in the entire group and subsets with different peak blood Pb 

### 6
levels ( ≤ 10 or 40 μg/dL) had p

- values between 0.07 and 0.13. 

### 7
This study made two other k ey contributions. In order to address the question of whether 

### 8
nephrotoxicity observed at current blood Pb levels is due to higher blood Pb levels from past exposure, 

### 9

these aut hors performed a sensitivity analysis in participants whose peak blood Pb levels, dating back to 

### 10
1979, were ≤ 10 μg/dL. A significant positive association between blood Pb and concurrent serum 

### 11

creatinine remained. These authors also addressed reverse causa lity, which attributes increased blood Pb 

### 12
levels to lack of kidney excretion rather than as a causative factor for CKD, by showing in adjusted plots 

### 13

that the association between blood Pb and serum creatinine occurred over the entire serum creatinine 

### 14

range , including the normal range where reverse causality would not be expected

. 

### 15

Cortical and trabecular bone Pb measurements were obtained in addition to whole blood Pb in 

### 16
evaluations performed in the Normative Aging Study between 1991 and 1995. Associations be tween 

### 17
baseline blood, tibia, and patella Pb and change in serum creatinine over an average of 6 years in 448 men 

### 18
were reported in a subsequent NAS publication ( Tsaih et al., 2004 ) . At baseline 6 and 26% of subjects had 

### 19
diabetes and hypertension, respectively. Mean blood Pb levels and serum creatinine decreased 

### 20
significantly over the follow

- up period in the group. Pb dose was not associated with change in creatinine 

### 21
in all participants. However, diabetes was observed to be an effect modifier of the relations of blood and 

### 22
tibia Pb with change in serum creatinine. For ln blood Pb, the p ositive association with serum creatinine 

### 23 was substantially stronger in diabetics (β = 0.076 [95% CI: 0.031, 0.121]) compared to non

- diabetics (β = 

### 24
0.006 [95% CI:

- 0.004, 0.016]). A similar relationship was observed for tibia Pb. An interaction was also 

### 25

ob served between tibia Pb and hypertension, although it is possible that m any of the 26 diabetics were 

### 26
also included in the hypertensive group and were influential there as well. Reverse causality was 

### 27
addressed in a sensitivity analysis of participants whose serum creatinine was <1.5 mg/dL; the authors 

### 28
reported that longitudinal associations did not materially change. These studies are depicted in either 

### 29
Figure 5

- 4 3 and Table 5

- 19. 

### 30 31May 2011 5

- 212 DRAFT

– DO NOT CITE OR QUOTE 

### 1

Study/Age (yr) Pb Level a Populations CROSS

- SECTIONAL RESULTS
Serum Creatinine = Scr, mg/L

Kim et al. ( 1996 ) 8.4 (5.7, 12.4) Blood Pb <40 μg/ dL from NAS Avg age (SD) 58.9 (12.4) Blood Pb <25 μg/dL from NAS Blood Pb <10 μg/dL from NAS
Tsiah et al. ( 2004 ) Diabetic from NAS
Baseline blood Pb 5.5 (3.7, 8.1) Nondiabetic from NAS Avg age (SD) 66 (6.6) Hypertensive from NAS Nonhypertensive from NAS
Follow

- up Blood Pb 3.9 (2.8, 5.6) Diabetic from NAS Avg age (SD): 72 (6.5) Nondiabetic from NAS Hypertensive from NAS Nonhypertensive from NAS Creatinine Clearance, mL/min divided by 100

Akesson et al. ( 2005 ) 2.2 (1.7, 3.0) Swedish women Median age (95 th percentile) 58 (54,63) Staessen et al. ( 1992 ) 7.5 (5.2, 10.9) Belgian women Avg age (SD) 48 (16) log e Creatinine Clearance, mL/min

Payton et al. ( 1994 ) 7.3 (5.4, 9.9) Men in NAS Avg age (SD) 64 (7.4) Glomerular filtration rate, mL/min divided by 100

Akesson et al. ( 2005 ) 2.2 (1.7, 3.0) Swedish women GFR mL/min/1.73 m 2

Fadrowski et al. ( 2010 ) 1.4 (0.7, 2.9) Adolescents from NHANES III Age range (12 to 20)
Navas

- Acien et al. ( 2009 ) geomean 1.58 NHANES 1999

- 2006 Age range >20

LONGITUDINAL RESULTS Change in Scr, mg/L per yrs of follow

- up

Tsiah et al. ( 2004 ) Diabetic from NAS
Baseline Blood Pb 3.9 (2.8, 5.6) Nondiabetic from NAS Hypertensive from NAS Nonhypertensive from NAS
Baseline Tibia Pb 18.2 (12.3, 26.9) Diabetic from NAS Nondiabetic from NAS Hypertensive from NAS Nonhypertensive from NAS Change in Scr, mg/L

- adj. for time between visits

Kim et al. ( 1996 ) 8.4 (5.7, 12.4) Blood Pb <40 μg/dL from NAS Blood Pb <25 μg/dL from NAS
Blood Pb <10 μg/dL from NAS Change in eGFR, mL/min over 4 yrs divided by 10

Yu et al. ( 2004 ) 3.2 (2.5, 4.1) CKD patients Age range (25 to 82)

Change in kidney metric per μg/dL blood Pb, at 1 μg/dL

a 

### Pb level presented as median blood Pb level and (IQR) in μ g/dL or bone Pb level (IQR) in μ g/g unless otherwise noted.

Note: The kidney function/blood Pb curves fit by a log

- linear model are depicted by their slopes at a blood Pb level of 1 μg/dL. Comparisons of the magnitude of the effect should not be made between effects having different kidney metrics. For uniform presentation, blood Pb level distributional statistics were converted to median and IQR by assuming that blood Pb is normally distributed

. The white shaded areas include kidney function tests where an increase is considered impaired function. The gray shaded areas include kidney function tests where a decrease is considered impaired function.
Figure 5

- 43. Kidney metric slopes on blood Pb or bone Pb.

NHANES data analyses benefit from a number of strengths including large sample size, ability to 

### 2
adjust for numerous Pb risk factors, and the fact that the study po pulation is representative of the U.S. 

### 3

non

- institutionalized, civilian population. As a result, the impact of Pb on the kidney has been examined 

### 4
in multiple NHANES datasets obtained over the last few decades (Figure 5

- 4 4 and Table 5

- 19). The 

### 5 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6May 2011 5

- 213 DRAFT

– DO NOT CITE OR QUOTE results of the se publications, covering different time frames, have been consistent in providing support for 

### 1
Pb as a CKD risk factor, including NHANES III, conducted from 1988

- 1994, where hypertensives and 

### 2
diabetics were observed to be susceptible populations ( Muntner et al., 2003 ) and NHANES 1999

- 2002 

### 3
( Muntner et al., 2005 )

. 

### 4

A recent publication examined this relationship in NHANES data collected from 1999 through 

### 5

2006 ( Navas

- Acien et al., 2009 )

. The geometric mean blood Pb level was 1.58 μ g/dL in 14,778 adults 

### 6
aged ≥ 20 years. After adjustment for survey year, sociodemographic factors, CKD risk factors, and blood 

### 7 cadmium, the odds ratios for albuminuria (≥ 30 mg/g creatinine), reduced eGFR (<60 mL/min/1.73 m 2 ), 

### 8
and both albuminuria and reduced eGFR were 1.19 (95% CI: 0.96, 1.47), 1.56 (95% CI: 1.17, 2.08), and 

### 9
2.39 (95% CI: 1.31, 4.37), respectively, comparing the highest to the lowest blood Pb quartiles. Th us, in 

### 10
the subset of the population with the most severe kidney disease (both reduced eGFR and albuminuria), 

### 11
the risk from Pb was greater. Cadmium was included as a covariate and Pb remained significantly 

### 12
associated. In fact, the most important contributio n of this recent NHANES analysis was the evaluation of 

### 13
joint Pb and cadmium exposure (discussed below). 

### 14
An important contribution of all three NHANES publications is that they provide evidence that 

### 15
blood Pb remains associated with reduced kidney function ( <60 mL/min/1.73 m 2 as estimated with the 

### 16
MDRD equation cross

- sectionally) despite steadily declining Pb levels during the time periods covered. 

### 17
Additional studies in this category have also reported worse kidney function related to Pb dose ( Goswami 

### 18
et al., 2005 ; Hernandez

- Serrato et al., 2006 ; L. H. Lai et al., 2008 )

. 

### 19May 2011 5

- 214 DRAFT

– DO NOT CITE OR QUOTE5102 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 Pb



### Note: To express these odds 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 was the exponentiated quantity (log(OR) divided by the differ ence in the medians of the groups compared). The resulting odds ratio is presented in terms of percent change=100*(OR

- 1). The blood Pb distribution of the reference group is shaded gray and the other group is shaded black. These articles reported ORs of ki dney function measures by grouping by quartiles of blood Pb and then comparing each group to the quartile with the lowest blood Pb (reference group).
Figure 5

- 44. Percent change for kidney outcomes associated with blood Pb

.May 2011 5

- 215 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 19. Additional characteristics and quantitative data for associations of blood and bone Pb with kidney outcomes for results presented in Figures 5

- 43 and 5

- 44

Reference Population Study Location; Time Period n Pb Level Outcome Model Change or % C hange in K idney M etric (95% CI) Cross

- Sectional
Muntner et al. ( 2003 ) NHANES III, adults US; 1988

- 1994 4813 Mean (SD) blood Pb Hypertensives: 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

Normotensives: 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) Logistic regression Hypertensives Q1: Referent Q2: 28% (2, 54) Q3: 8% (4, 12) Q4: 5% (3, 6) Normotensives Q2: 10% (

- 57, 78) Q3: 3% (

- 9, 15) Q4: 1% (

- 4, 5)

Akesson et al. ( 2005 ) WHILA, adult

women Sweden; 6/1999

- 1/2000 820 Median (5

- 95%) = 2

. 2 (1.1

- 4.6) μ g/dL Cystatin C

- based eGFR (Larsson et al. 2004) Multiple Linear regression - 2.0 (

- 3.2,

- 0.9)

Creatinine clearance Linear regression - 1 .8 (

- 3.0, 0.7 )

Navas

- Acien et al. ( 2009 ) NHANES III

, adults US; 1999

- 2006 14,778 Geometric mean = 1.58 μ g/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 Q1: Referent Q2: 19% (

- 44, 83) Q3: 28% (0.0, 56) Q4: 19% (7, 31) Albuminuria and eGFR <60 mL/minute/1.73 m 2 Logistic regression Q1: Referent Q2:

- 37% (

- 83, 8) Q3:

- 8% (

- 25, 10) Q4: 7% (

- 2, 16) Fadrowski et al. ( 2010 ) NHANES, adolescents US; 1988

- 1994 769 Median = 1.5 μ g/dL Q1: <1.0 Q2: 1.0 to 1.5 Q3: 1.6 to 2.9 Q4: >2.9 Cystatin C

- based eGFR (mL/min/1.73 m 2 ; calculated using the Filler and Lepage equation) Linear regression Q4:

- 0.42 (

- 0.73,

- 0.11)

Kim et al. ( 1996 ) Adul t males Boston, MA; 1979

- 1994 459 Median = 8.6 μ g/dL Serum creatinine concentrations Random

effects model ≤ 40 μ g/dL blood Pb: 0.016 (0.004, 0.028)
≤ 25 μ g/dL blood Pb: 0.019 ( 0.006, 0.032 )
≤ 10 μg/dL blood Pb : 0.0 30 ( 0.011, 0.049 ) Payton et al. ( 1994 ) Adult males Boston, MA; 1988

- 1991 744 Mean (SD) = 8.1 (3.9) μ g/dL Ln creatinine clearance Multiple linear regression Ln blood Pb

- 4.0 (

- 8.0,

- 0.1) Tsaih et al. ( 2004 ) Adult males Boston, MA; 8/1991

- 1995 with mean 6 yr follow

- up 448 Mean (SD) Blood Pb = 6.5 (4.2)
μ g/dL Tibia Pb = 21.5 (13.5) μ g/g Patella Pb = 32.4 (20.5) μ g/g S erum creatinine Multiple linear regression Baseline blood Pb Diabetic :

- 0.05 (

- 0.23, 0.12) Nondiabetic :

- 0.02 (

- 0.06, 0.02) Hypertensive:

- 0.01 (

- 0.09, 0.07) Nonhypertensive :

- 0.03 (

- 0.07, 0.01) Follow

- up blood Pb Diabetic: 0.22 (

- 0.14, 0.58) Nondiabetic: 0.14 (0.03, 0.26) Hypertensive: 0.35 (0.16, 0.54) Nonhypertensive : 0.06 (

- 0.07, 0.19)May 2011 5

- 216 DRAFT

– DO NOT CITE OR QUOTE Reference Population Study Location; Time Period n Pb Level Outcome Model Change or % C hange in K idney M etric (95% CI)
Staesson et al. ( 19 92 ) Adults Belgium; 1985

- 1989 1,981 Blood Pb Mean (SD) Males: 11.4 μg/dL Females: 7.5 μg/dL Creatinine clearance Multiple linear regression - 52.9 (

- 84, 21)

Longitudinal Kim et al. ( 1996 ) Adult males Boston, MA; 1979

- 1994 459 Median = 8.6 μ g/dL Change in s erum creatinine concentrations Random

effects model ≤ 40 μ g/dL blood Pb: 0.01 (

- 0.0, 0.02 ) ≤ 25 μg/d L blood Pb : 0.01 (

- 0.0, 0.03 ) ≤ 10 μg/dL blood Pb: 0.0 2 (

- 0.0, 0.04 ) Tsaih et al. ( 2004 ) Adult males Boston, MA; 8/1991

- 1995 with mean 6 yr follow

- up 448 Mean (SD) Blood Pb = 6.5 (4.2)
μ g/dL Tibia Pb = 21.5 (13.5) μ g/g Patella Pb = 32.4 (20.5) μ g/g Change in serum creatinine Multiple linear regression Blood Pb Diabetics (n=26): 0.076 (0.03, 0.12) Nondiabetic (n=422): 0.006 (

- 0.004, 0.02) Hypertensive (n=115): 0.008 (

- 0.01, 0.03) Normotensive (n=333): 0.009 (

- 0.003, 0.021)

Tibia Pb Diabetics (n=26): 0.082 (0.03, 0.14) Nondiabetic (n=422): 0.005 (

- 0.01, 0.02) Hypertensive (n=115): 0.023 (0.003 , 0.04) Normotensive (n=333): 0.0004 (

- 0.01, 0.01) Yu et al. ( 2004 ) Adult CKD patients Taipei, Taiwan; 48 month longitudinal study period 121 Mean (SD) = 4.2 (2.2) μ g/dL Change in MDRD eGFR Generalized estimating equations - 4.01 ml/min/1.73 m2 body surface area (p=0.0148) in the GFR over the follow

up period for each 1 μg/dL increment of blood PbMay 2011 5

- 217 DRAFT

– DO NOT CITE OR QUOTE Occupational Studies

The vast majority of studies in the literature on the impact of Pb on the kidney have been 

### 1
conducted in the occupational setting. In general, study size and extent of statistical analysis are much 

### 2
more limited than for general population studies. P ublications in only three populations have reported 

### 3
adjusted results in occupationally exposed workers in the five years since the 2006 Pb AQCD. In a two 

### 4
year prospective cohort study

, generalized estimating equations were used to model change in kidney 

### 5

fu nction between each evaluation in relation to tibia Pb and concurrent change in blood Pb in 537 current 

### 6

and former Pb workers ( V. M. Weaver et al., 2009 ) . Tibia Pb was evaluated at the beginning of each 

### 7

follow

- up period and Pb measures were adjust ed for baseline Pb dose and other covariates. In males, 

### 8

serum creatinine decreased and calculated creatinine cleara nce increased over the course of the study; 

### 9
these changes were largest in participants whose blood Pb declined concurrently or whose tibia Pb was 

### 10
lower. In females, decreasing serum creatinine was associated with declining blood Pb (as in males)

, 

### 11
however, increasing blood Pb was associated with a concurrent increase in serum creatinine. Women 

### 12
(25.9% of the study population ) were older and more likely to be former Pb workers than men which may 

### 13
have been important factors in the effect modification observed b y sex. 

### 14
Chia and colleagues observed a significant, positive association between blood Pb and urine NAG 

### 15
in linear regression models after adjustment for age, gender, race, exposure duration, ALAD G177C 

### 16
polymorphism and the interaction between ALAD and blood Pb ( Chia et al., 2006 )

. Similar positive 

### 17
associations were observed between blood Pb and a wider range of EBE markers in models that adjusted 

### 18
for age, gender, race, exposure duration, and the HpyCH4 ALAD SNP (discussed below ) ( Chia et al., 

### 19

2005 )

. A study of 155 male worker s reported significant, positive correlations between blood and urin e Pb 

### 20

and urine NAG and albumin after controlling for age and job duration ( Y. Sun, Sun, Zhou, Zhu, Lei, et al., 

### 21

2008 )

. An important additional study that analyzed occupational Pb exposure is discussed below under 

### 22
patient population studies ( Evans et al., 2010 )

. 

### 23

Two studies have performed benchmark dose calculations for the effect of Pb on the kidney. Both 

### 24
used only EBE markers and found NAG to be the most sensitive outcome; reported lower confidence 

### 25 limits on the benchmark doses were 10.1 μ g/ dL ( Y. Sun, Sun, Zhou, Zhu, Lei, et al., 2008 )

, and 25.3 

### 26

μ g/dL ( T. A. Lin & Tai

- yi, 2007 )

. 

### 27
A number of other publications in the five years since the 2006 Pb AQCD have reported 

### 28
significantly worse kidney outcomes in unadjusted analyses in occupationally exposed workers compared 

### 29
to unexposed controls ( Patil et al., 2007 ) and/or significant correlations between higher Pb dose and 

### 30
worse kidney function ( Alinovi et al., 2005 ; Garcon et al., 2007 ; D. A. Khan et al., 2008 ; T. A. Lin & Tai



### 31
yi, 2007 ; Y. Sun, Sun, Zhou, Zhu, Lei, et al., 2008 )

. One small study found no significant differences 

### 32
( Orisakwe et al., 2007 )

. 

### 33May 2011 5

- 218 DRAFT

– DO NOT CITE OR QUOTE Overall, th e occupational literature published in the last five years on the kidney impact of Pb 

### 1
exposure has been more consistent in reporting significant associations than data reviewed for the 2006 

### 2

Pb AQCD . This may reflect increased reliance on EBE markers as mor e sensitive outcome measures

, 

### 3
pub lication bias , or multiple comparisons due to a greater number of outcomes assessed. 

### 4

Publications that include dose

- response information provide evidence of Pb

- related nephrotoxicity 

### 5
in the occupational setting across the Pb dose ranges analyzed ( Ehrlich et al., 1998 ; V. M. Weaver, Lee, et 

### 6
al., 2003 ) . Data in 267 Korean Pb workers in the oldest age tertile ( mean age = 52 years ) reveal no 

### 7
threshold for a Pb effect (beta = 0.0011, p = <0.05 ; regression and lowess lines shown) ( V. M. Weaver, 

### 8
Lee, et al., 2003 ) (added variable plot shown in Figure 5

- 4 5 ). 

### 9

Source: Used with permission from the BMJ Publishing Group, Weaver et al. ( 2003 )

Note: Both the ad justed regression line and the line estimated by the smoothing method of the S

PLUS statistical software function lowess 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, rather than zero. Figure 5

- 45. A dded variable plot of a ssociation 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 t he potential for Pb

- related 

### 10
hyperfiltration. Hyperfiltration involves an initial increase in glomerular hypertension which results in 

### 11

increase d GFR . If persistent, increase d risk for subsequent CKD occurs. This pattern has been observed in 

### 12
diabetes, hypert ension, and obesity ( Nenov et al., 2000 )

. As discussed in the 2006 Pb AQCD, findings 

### 13
consistent with hyperfiltration have been observed in three occupational populations ( Hsiao et al., 2001 ; 

### 14 Adjusted blood Pb level ( μ g/dL)Adjusted serum creatinine (mg/dL)May 2011 5

- 219 DRAFT

– DO NOT CITE OR QUOTE Roels et al., 1994 ; V. M. Weaver, Lee, et al., 2003 ) , a study of adults who were Pb poisoned as children 

### 1
( H. Hu, 1991 ) , and a study in European children ( De Burbure et al., 2006 ) . Longitudinal data in Pb 

### 2
exposed rodents provide evidence of a hyperfiltration pattern of increased, followed by decreased GFR, 

### 3
associated with Pb exposure and are critical in interpretation of the human Pb

- kidney literature ( Khalil



### 4
Manesh, Gonick, Cohen, Alinovi, et al., 1992 ) . Pb could induce glomerular hyperte nsion resulting in 

### 5
hyperfiltration by several mechanisms including increased ROS , changes in eicosanoid levels, and/or an 

### 6
impact on the renin

- angiotensin s ystem ( Roels et al., 1994 ; Vaziri, 2008b )

. Whether hyperfiltration 

### 7
contributes to pathology in humans is unclear; longitudinal studies are needed. 

### 8
Regardless, significant findings could be obscured if opposite direction associations are present in 

### 9
different segments o f the study population and interaction models are not performed to address this. In the 

### 10

Korean Pb workers ( V. M. Weaver, Lee, et al., 2003 ; V. M. Weaver, Schwartz, et al., 2003 ) , significant 

### 11
associations in opposite directions were observed only when relevant effect modifiers were included in 

### 12

the model. This is a valid concern for risk assessment, since the factors involved in these inverse 

### 13

associations in Pb

- exposed populations are not well defined at present. 

### 14

Patient Population Studies

CKD as defined by t he National Kidney Foundation

- Kidney Disease Outcomes Quality I nitiative 

### 15
(NKF

- K/DOQI) workgroup ( National Kidney Foundation, 2002 ) is the presence of mark ers of kidney 

### 16
damage or GFR <60 mL/min/1.73 m 2 for ≥ 3 months . The MDRD equation is the most common one used 

### 17

in the eGFR determination for this definition. Notably, decreased GFR is not required for the first criteria 

### 18

and markers of kidney damage are not r equired for the second criteria. 

### 19
Several key studies in CKD patient populations have been published in the last five years ( Table 5

- 

### 20

20 )

. One CKD patient study

, discussed in the 2006 Pb AQCD

, remains the hallmark for prospective 

### 21

evaluation of susceptible pa tient populations to determine if CKD progression (kidney function decline ) is 

### 22
greater in participants with higher baseline Pb dose . Yu et al. ( 2004 ) followed 121 patients over a four 

### 23
year period. Eligibility required well

- controlled CKD with serum creatinine between 1.5 and 3.9 mg/dL. 

### 24
Importantly, EDTA

- chelatable Pb <600 μg/72 h , a level below that traditionally thought t o indicate risk 

### 25

for Pb

- related nephrotoxicity, was required at baseline. Patients with potentially unstable kidney disease 

### 26
were excluded (i.e., due to systemic diseases such as diabetes). Mean blood Pb and EDTA

- chelatable Pb 

### 27 levels were 4.2 μg/dL and 99.1 μg/72 h ours , respectively. In a Cox multivariate regression analysis, 

### 28
chelatable Pb was significantly associated with overall risk for the primary endpoint (doubling of serum 

### 29

creatinine over the 4

- year study period or need for hemodialysis). When the group was dichotomized by 

### 30

EDTA chelatable Pb level, Kaplan

- Meier analysis demonstrated that significantly more patients (15/63) in 

### 31
the high

- normal group (EDTA chelatable Pb level ≥ 80 but <600 μ g/72 hours) reached the primary end 

### 32
point than in the lower EDTA ch elatable Pb levels (<80 μ g Pb /72 hours) group (2/58). Associations 

### 33May 2011 5

- 220 DRAFT

– DO NOT CITE OR QUOTE between baseline chelatable or blood Pb level and change in eGFR (estimated by the MDRD equation 

### 1
( Levey et al., 1999 ) were modeled separately using GEE. Based on these models, a 10 μg higher 

### 2 chelatable Pb level or 1 μg/dL higher blood Pb level reduced the GFR by 1.3 and 4.0 ml/min/1.73 m 2

, 

### 3
respectively, during the 4

- year study period. Two recent studies expanded the CKD patient populations in 

### 4
which this effect was observed to those with diabetic nephropathy ( J.

- L. Lin, Lin

- Tan, Yu, et al., 2006 ) 

### 5

and with t he lowest Pb body burdens studied to date ( J.

- L. Lin, Lin

- Tan, Li, et al., 2006 )

. Results of these 

### 6

observation al studies have been summarized ( V. Weaver & Jaar, 2010 )

. 

### 7

Table 5

- 20. 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) B aseline 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. ( 2006 ) 87 6.5 (3.4) 108.5 (53.8) 35.1 (9.0) 1 3.87 Type II diabetics with nephropathy
Lin et al. ( 2006 ) 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: Used with permission from UpToDate.com, Weaver et al. ( 2010 )

A recent population

- based case

- control st udy examined occupational Pb exposure as a risk factor 

### 8

for severe CKD ( Evans et al., 2010 )

. The study included 926 cases with first time elevations of serum 

### 9
creatinine >3.4 mg/dL for men and >2.8 mg/dL for women and 998 population

- based controls. 

### 10
Occupational Pb exposure was assessed using an expert rating method based on job histories. Eighty

- one 

### 11
cases and 95 controls were judged to have had past occupational Pb exposure. Of those, 23 cases and 32 

### 12
controls were thought to have been exposed to Pb levels ≥ 30 μg/m 3 (the current US OSHA limit is 50 

### 13 μg/m 3 ). Using multivariable logistic regression modeling, the adjusted OR for CKD was 0.97 (95% CI : 

### 14

0.68

- 1.38) in Pb

- exposed compared to non

- exposed participants. No significant increased odds were 

### 15
observed for low, mediu m or high exposed groups using either average or cumulative exposure metrics. 

### 16

In addition , the CKD patients were followed prospectively for a mean of 2.5 years for the 70 Pb exposed 

### 17
patients and 2.4 years for the 731 patients without past occupational Pb e xposure. Mean eGFRs (using the 

### 18
MDRD equation) were 16.0 and 16.6 ml/min/1.73 m 2 in exposed and non

- exposed patients, respectively, 

### 19
indicating severe disease in both groups . Using mixed

- effects multivariable models

, eGFRs declined by 

### 20

4.27 and 3.39 mL/min/1. 73 m 2 /y in ever and most Pb

- exposed CKD patients, respectively, compared with 

### 21May 2011 5

- 221 DRAFT

– DO NOT CITE OR QUOTE 4.55 mL/min/1.73 m 2 /y in patients without occupational Pb exposure. Thus, in this study, no adverse 

### 1
kidney effect of occupational Pb exposure was evident. 

### 2
Strengths noted by the authors included virtually complete case ascertainment and minimal loss to 

### 3

follow

- up. Exposure assessment was listed as both a strength and a limitation. Expert rating methods are 

### 4
commonly used when biological monitoring is not an option and in case

- contro l studies where many 

### 5
occupational exposures a re considered. In Pb

- kidney research, this approach is uncommon except in the 

### 6

case

- control setting. However, given the challenges of interpreting blood Pb in dialysis patients (discussed 

### 7
below), this approach ma y have advantages in this study of such severe CKD. Two case

- control studies 

### 8
examining occupational risk factors for CKD have been published; one found Pb exposure to be a risk 

### 9

factor ( Nuyts et al., 1995 )

, the other did not although moonshine alcohol consumption was a risk 

### 10
presumably due to Pb level ( Steenland et al., 1990 )

. The prospective observational aspect of this study is 

### 11
similar in design to the work of Lin and colleagues but differs in several important respects. In this study, 

### 12
only occupational Pb exposure was considered whereas the work in Taiwan excludes occupational 

### 13
exposure and uses Pb dose measures. In the past in developed countries, envir onmental exposures were 

### 14
substantial. For example, mean tibia Pb levels were 21.5 and 16.7 μ g/g bone mineral, in environmentally 

### 15
exposed 50

- to 70

- year

- old African

- Americans and whites, respectively, in Baltimore ( Martin et al., 

### 16

2006 )

. In Korean Pb workers, mean baseline tibia Pb level was only twofold higher (35.0 μ g/g) ( V. M. 

### 17

Weav er, Lee, et al., 2003 ) which illustrates the substantial body burden in middle

- and older

- aged 

### 18
Americans from lifetime Pb exposure. Declines in blood Pb levels in Sweden have been reported and 

### 19
attributed to the leaded gasoline phase

- out ( Elinder et al., 1986 ; Strömberg et al., 1995 ) , although blood 

### 20
Pb levels were lower than those noted during the U

. S

. phase

- out. Finally, the severe degree of CKD in this 

### 21
population creates a survivor bias at enrollment and limits the eGFR decline possible during follow

- up, 

### 22
thus limiting the ability to identify factors that influence that decline. 

### 23

ESRD Patient Studies

End stage renal disease (ESRD) is a well established public health concern, which is characterized 

### 24
by the use of dialysis t o perform the normal functions of the kidney. Incidence and prevalence in the US 

### 25
continue to increase resulting in rates that are the third highest among nations reporting such data ( U.S. 

### 26
Renal Data System, 2009 ) . S tudies in patients with CKD requiring chronic hemodialysis (ESRD) have 

### 27

also be en published in the pa st five years. One stud y reported much higher blood Pb levels than had been 

### 28
appreciated by the treating clinicians ( Davenport et al., 2009 )

. O f 271 adult patients on regular thrice 

### 29

week ly dialysis

, blood Pb levels ranged from 3 to 36

. 9 μg/dL ; 25.5% had level s >20 μg/dL

, 59% had 

### 30

values of 10

- 20 μg/dL , and 15.5% were <10 μg/dL . Few details on the statistical analysis were provided 

### 31
which complicates interpretation of the findings. However, blood Pb was positively correlated with 

### 32

h emodialysis vintage ( months on dialysis; Spearman’s r = 0.38, p

- value <0.001); negatively correlated 

### 33May 2011 5

- 222 DRAFT

– DO NOT CITE OR QUOTE with urine output (r =

- 0.44, p

- value <0.001) and higher in patients using single carbon filter and reverse 

### 1
osmosis water purif ication device s. Another recent publication reported higher Pb in dialysate than in the 

### 2
tap water used in its preparation ( B. Chen et al., 2009 )

. A systematic review of a wide range of trace 

### 3
elements in hemodialysis patients report ed higher Pb levels in patients compared to co nt rols although the 

### 4
difference was not large ( Tonelli et al., 2009 )

. These data su ggest that blood Pb monitoring in dialysis 

### 5
patients may be useful. 

### 6

I nterpretation of Pb dose in patients on dialysis is challenging for several reasons. F irst

, renal 

### 7
osteodystrophy , the bone disease related to kidney disease, may result in increased release of Pb from 

### 8

bone stores . Thus, interpretation of blood and even bone Pb levels may req uire adjustment with one or 

### 9
more of a range of osteoporosis variables. Secondly, as observed above ( Davenport et al., 2009 ) , residual 

### 10
kidney function may have a su bstantial impact on blood Pb levels i n populat ion s with such minimal 

### 11
excretion. Third, as illustrated in the stud ies cited above ( B. Chen et al., 2009 ; Davenport et al., 2009 )

, 

### 12

water and concentrates used in dialysis may be variable sources of Pb. A recent study reported decreased 

### 13
blood Pb in post

- dialysis compared to pre

- dialysis samples ( Kazi et al., 2 008 ) . Thus, substantial 

### 14
fluctuations in blood Pb are possible while on dialysis. Finally, anemia is common in CKD and Pb is 

### 15
stored in red blood cells. Thus, measurement of blood Pb in anemia may require adjustment for 

### 16
hemoglobin; no standardized approac h to this currently exists. 

### 17

Given the se caveats, a pilot study observed significantly higher median blood Pb levels in 55 

### 18

African

- American dialysis patients compared to 53 age

- and sex

- matched controls (6 and 3 μg/dL 

### 19
respectively; P < 0.001) ( Muntner et al., 2007 )

. This study was unique in that tibia Pb levels were 

### 20

assessed. Median tibia Pb was higher in ESRD patients although t he difference did not reach statistical 

### 21 significance (17 and 13 μg/g bone mineral, respectively ( p = 0.13). In order to determine the potential 

### 22
impact of renal osteodystrophy

, median blood and tibia Pb levels in the dialysis patients were examined 

### 23
by level s of serum parathyroid hormone, calcium, phosphorus, and albumin and were not found to be 

### 24
significantly different ( Ghosh

- Narang et al., 2007 )

. A study of 211 diabetic patients on hemodialysis ( J.

- L. 

### 25
Lin et al., 2008 ) found par a thyroid hormone and serum creatinine to be associated with blood Pb level in 

### 26

crude but not a djusted associations . In contrast, a study of 315 patients on chronic peritoneal dialysis 

### 27
observed parathyroid hormone to be positively correlated and residual renal function negatively correlated 

### 28
with logarithmic

- transformed blood Pb level s after adjustme nt ( J.

- L. Lin et al., 2010 )

. I n the prospective 

### 29
portion of this study, blood Pb levels at baseline were categorized by tertile (range of 0.1 to 29.9 μ g/dL 

### 30
with cut points of 5.62 and 8.66 μ g/dL). Cox multivariate analysis, after adjustment for parathyroid 

### 31
hormone level, residual renal function, and 20 other variables, showed increased all

- cause mortality in the 

### 32
middle and highest compared to the l owest tertiles (hazard ratio= 2.1 [ 95% CI: 2.0

- 2.2] and 3.3 [ 95% CI: 

### 33

1.3

- 13.5], respectively). Given other recent publications in hemodialysis patients by this group, it would 

### 34
be valuable to examine this risk after adjustment for serum ferritin ( Jenq et al., 2009 )

, hemoglobin A1C 

### 35
( Lin

- Tan, Lin, Wang, et al., 2007 )

, and blood cadmium ( C. W. Hsu et al., 2009 )

. 

### 36May 2011 5

- 223 DRAFT

– DO NOT CITE OR QUOTE Clinical Trials in Chronic Kidney Disease Patients

Randomized chelation trials in CKD patients, uncommon in nephrotoxicant research, provide 

### 1
unique information on the kidney impact of Pb. These studies have been performed by Li n and colleagues 

### 2

in Taiwan and involve similar study designs. Initially, patients are observed in order to compare CKD 

### 3
progression prior to chelation. Then, CKD patients whose diagnostic EDTA chelatable Pb levels are 

### 4
within certain ranges (generally 60

- 600 μ g/72 hours and thus below the level commonly considered for 

### 5
chelation) are randomized. The treated group receives weekly chelation with 1 g EDTA intravenously for 

### 6
up to 3 months. The control group receives placebo infusions. In the follow

- up period, chel ation is 

### 7
repeated for defined indications such as increased serum creatinine or chelatable Pb levels above specified 

### 8

cut

- offs. Placebo infusions are repeated in the controls as well. The results of the most recent of these 

### 9

trials are summarized in Table 5

- 21 below. 

### 10

Table 5

- 21. 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 Largest study to date
Control 32 5.9 (3.0) 144.5 (87.9) 31.5 (9.0)

- 2.7
Lin et al. ( 2006 ) Chelated 15 7.5 (4.6) 148.0 (88.6) 22.4 (4.4) 15

- 3.5 Type II diabetics with nephropathy
Control 15 5.9 (2.2) 131.4 (77.4) 26.3 (6.2)

- 10.6

Lin et al. ( 2006 ) Chelated 16 2.6 (1.0) a 43.1 (13.7) 41.2 (11.2) 27 +3.0 Lowest Pb exposed and treated range BLB ≥ 20

- <80 μ g)
Control 16 3.0 (1.1) 47.1 (15.8) 42.6 (9.7)

- 2.0
Lin

- Tan et al. ( 2007 ) Chelated 58 5.0 (2.2) 164.1 (111.1) 36.8 (12.7) 51

- 0.3 Non

- diabetic

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 

### 11
confirmed, the effect may be due to removal of Pb. However, chelation may also have a direct beneficial 

### 12
effect on kidney function, regardless of Pb exposure. Antioxidant effects of Ca Na 2 EDTA which may 

### 13
improve kidney functi on directly via improved blood flow to the kidneys have been reported ( Jacobsen et 

### 14
al., 2001 ; Saxena & Flora, 2004 )

. EDTA benefits in a Pb rodent model appeared to occur via reduced 

### 15

oxidation ( Saxena & Flora, 2004 )

. EDTA administration reduced kidney damage in a rat model of acute 

### 16
renal failure induced by ischem ia ( Foglieni et al., 2006 )

. Similarly DMSA has been reported to prevent 

### 17
renal damage when co

- administered during induction of nephrosclerosis in a non

- Pb expose d rat model 

### 18
( Gonick et al., 1996 )

. Benefits from chelation reported in rodent models of Pb

- related nephrotoxicit y 

### 19May 2011 5

- 224 DRAFT

– DO NOT CITE OR QUOTE ( Khalil

- Manesh, Gonick, Cohen, Bergamaschi, et al., 1992 ; Sanchez

- Fructuoso, Blanco, et al., 2002 ; 

### 1

Sanchez

- Fructuoso, Cano, et al., 2002 ) did not appear to occur via reversal of structural damage ( Khalil



### 2
Manesh, Gonick, Cohen, Bergamaschi, et al., 1992 ) ; again suggesting that improved hemodynamics from 

### 3
reduction of reactive oxidant species, which could be du e to reduced Pb and/or directly from the chelating 

### 4
agent, may be a mechanism ( Gonick et al., 1996 )

. However, the most parsimonious explanation for the 

### 5

combination of Lin ’ s observational and experimental chelation work is that Pb is the underlying reason. 

### 6
Moreover, if the benefit can be replicated, this could be a valid treatment regardless of the mechanism or 

### 7

whethe r Pb is involved. 

### 8
The unique body of work in patient populations by Lin and co

- workers, both observational and 

### 9
experimental, has numerous strengths including prospective study design, randomization, Pb dose 

### 10
assessment that includes bioavailable body burden , longitudinal statistical analysis, and control for 

### 11
multiple kidney risk factors. However, the generalizability of the results to broader populations is 

### 12
unknown. In addition, the observed effect of Pb on decline in GFR has been variable; the annual d eclin e 

### 13
in eGFR per standard deviation (SD) higher Pb dose at baseline was much lower in the 2003 study than in 

### 14
subsequent publications (see Table 5

- 21 above). Small sample sizes and differences in renal diagnoses 

### 15
between groups may be factors in this variabilit y. However, if confirmed in large populations at multiple 

### 16
centers and shown not to worsen cognition or other effects through Pb mobilization, chelation could yield 

### 17
important public health benefits. 

### 18

5.5.2.3. Epidemiology in Children

Lead Nephrotoxicity in Children

B oth the 2006 and 1986 Pb AQCDs noted that the degree of kidney pathology observed in adult 

### 19
survivors of untreated childhood Pb poisoning in the Queensland, Australia epidemic ( Inglis et al., 1978 ) 

### 20

has not been observed in other studies of childhoo d Pb poisoning. Recent publications remain consistent 

### 21
with that conclusion; a recent study observed an impact of childhood Pb poisoning on IQ but not kidney 

### 22

outcomes ( C. Coria et al., 2009 ) . Chelation was raised as a potential explanation for this discrepancy in 

### 23
the 2006 Pb AQCD. 

### 24
With declining Pb exposure levels, recent wor k has focused on studies in children at much lower 

### 25

environmental exposure levels. However, insensitivity of the clinical kidney outcome (i.e., GFR) 

### 26
measures for early kidney damage is a particular problem in children who do not have many of the other 

### 27
kidney risk factors that adults do , such as hypertension and diabetes . As a result, such studies have 

### 28
utilized EBE markers. However, data to determine the predictive value of such biomarkers for subsequent 

### 29
kidney function decline in Pb exposed populations are extremely limited ( Coratelli et al., 1988 ) and may 

### 30
pose particul ar challenges in children due to puberty related biomarker changes ( Sarasua et al., 2003 )

. 

### 31May 2011 5

- 225 DRAFT

– DO NOT CITE OR QUOTE Three studies that included analysis of clinical kidney outcomes were discussed in the 2006 Pb AQCD. 

### 1

One found no difference in mean serum creatinine between 62 exposed and 50 control children ( Fels et 

### 2
al., 1998 ) . T wo larger studies observed significant associations that were in opposite directions; blood Pb 

### 3
was positively associated with serum cystatin

- C in 200 17

- year

- old Belgian adolescents but negatively 

### 4
associated with serum creatinine and cy statin C in 300

- 600 European children (n varied by outcome) 

### 5
( De Burbu re et al., 2006 ; Staessen et al., 2001 )

. 

### 6
Therefore, o ne of the key gaps identified in the 2006 Pb AQCD w as limited data in children and 

### 7
adolescents particularly with respect to GFR measures. A recently published NHANES analysis in 

### 8
adolescents begins to fill this gap ( Fadrowski et al., 2010 )

. Associations between blood Pb and kidney 

### 9
function were investigated in 769 adolescents aged 12

- 20 years in the US NHANE S III, conducted from 

### 10

1988

- 1994. Kidney function was assessed with two eGFR equations. One utilized serum cystatin C and 

### 11
the other used the more traditional marker, serum creatinine. Median blood Pb and cystatin C

- based eGFR 

### 12 levels were 1.5 μg/dL and 112.9 mL/min/1.73 m 2 , respectively. Cystatin C

- based eGFR was lower (

- 6.6 

### 13

mL/min/1.73 m 2 [95% CI :

- 0.7,

- 12.6]) in participants with Pb levels in the highest quartile (≥ 3.0 μg/dL) 

### 14
compared with those in the lowest (< 1 μg/dL). A doubling of blood Pb level was associated with a

- 2.9 

### 15

mL/min/1.73 m 2 (95% CI:

- 0.7 to

- 5.0) lower eGFR. In contrast, the association between blood Pb and 

### 16

creatinine

- based eGFR, although in the same direction, was not statistically significant. Additional 

### 17
research in children, including with longitudinal follow

- up, a range of outcome assessment methods, and 

### 18
with exposure only after Pb was banned from gasoline, is warranted

. 

### 19

5.5.2.4. Associations between Lead Dose and New Kidney Outcome Measures

As noted above, in an effort to more accurately estimate kidney outcomes, new equations to 

### 20
estimate GFR based on serum creatinine have been developed, and the utility of other biomarkers, such as 

### 21
cystatin C, as well as equations based on them, are being studied. Ho wever, few publications have utilized 

### 22

these state

- of

- the

- art techniques when evaluating associations between Pb or cadmium dose and renal 

### 23

function. In addition to the study in NHANES adolescents discussed above ( Fadrowski et al., 2010 )

, a 

### 24

cross

- sectional study of Swedish women reported that higher blood Pb (median = 2.2 μg/dL) and 

### 25
cadmium (median = 0.38 μg/L) levels were associated with lower eGFR based on serum cystatin C alone 

### 26
(without age, sex, and race) after adjustment for socio

- demographic and CKD risk factors ( Akesson et al., 

### 27

2005 )

. Associations were comparable to tho se using creatinine clearance as the kidney outcome for Pb; 

### 28
however associations between cadmium dose measures were stronger for the cystatin C based outcome. 

### 29

Staessen et al

. ( 2001 ) found a significant association between blood Pb level and serum cystatin C in a 

### 30

cross

- sectional study of adolescents; creatinine based measures were not reported. However, in a cross



### 31
sectional study of European children, higher bloo d Pb levels were associated with lower serum cystatin C 

### 32
and creatinine; these inverse associations were attributed to hyperfiltration ( De Burbure et al., 2006 )

. A 

### 33May 2011 5

- 226 DRAFT

– DO NOT CITE OR QUOTE very recent publication compared associations of blood Pb and eGFR using the traditional MDRD 

### 1
equation to those with four new equations: CKD

- EPI, and cystatin C singl e variable, multivariable, and 

### 2
combined creatinine/cystatin C, in 3941 adults who participated in the 1999

- 2002 NHANES cystatin C 

### 3
subsample ( Spector et al., 2011 ) . Similar to the NHANES adolescent analysis, associations with the 

### 4
cystatin C outcomes were stronger

. Af ter multivariable adjustment, differences in mean eGFR for a 

### 5
doubling blood Pb w ere

- 1.9 (95% CI:

- 3.2,

- 0.7),

- 1.7 (95% CI:

- 3.0,

- 0.5), and

- 1.4 (95% CI:

- 2.3,

- 0.5) 

### 6

mL/min/1.73 m 2 , using the cystatin C single variable, multivariable and combined creatinine/cystatin C 

### 7
equations, respectively , reflecting lower eGFR with increased b lood Pb . The corresponding differences 

### 8

were

- 0.9 (95% CI:

- 1.9, 0.02) and

- 0.9 (95% CI:

- 1.8, 0.01) using the creatinine

- based CKD

- EPI and 

### 9
MDRD equations, respectively. 

### 10



## 5.5.3. Mechanisms of Lead Nephrotoxicity

5.5.3.1. Altered Uric Acid

Individuals who have been heavily e xposed to Pb are at increased risk for both gout and kidney 

### 11

disease ( Batuman, 1993 ; Shadick et al., 2000 ) . Pb is thought to increase serum uric acid by decreasing its 

### 12
kidney excretion ( Ball & Sorensen, 1969 ; Emmerson, 1965 ; Emmerson & Ravenscroft, 1975 )

. R esearch 

### 13
during th e past decade indicates tha t uric acid is nephrotoxic at lower levels than previously recognized 

### 14
( R. J. Johnson et al., 2003 ) . Therefore, the 2006 Pb AQCD reviewed literature implicating increased uric 

### 15

acid as one mechanism for Pb

- related nephrotoxicity ( Shadick et al., 2000 ; V. M. Weaver et al., 2005 )

. 

### 16
However, this is not the only mechanism, since associations between blood Pb and serum creatinine 

### 17
remained significant even after adjustment for uric aci d ( V. M. Weaver et al., 2005 ) . These mechanistic 

### 18
relations have more than just theoretical importance. Clinically relevant therapies may be possible since 

### 19
EDTA chelation has been reported to improve both kidney function and urate clearance in patients with 

### 20
kidney insufficiency and gout, even when EDTA

- chelatable Pb body burdens were low ( J.

- L. Lin et al., 

### 21

2001 )

. 

### 22

Conterato et al

. ( 2007 ) followed various parameters of kidney function after acute or chronic Pb 

### 23
exposure in rats. Acute exposure to Pb acetate consisted of a single i.p. injection of 25 or 50 mg/kg Pb 

### 24
acetate, while chronic exposure was one daily i.p. injection of either vehicle or Pb acetate (5 or 25 mg/kg 

### 25
) for 30 days. Acute and c hronic exposure at both dose level s increased plasma uric acid levels. 

### 26
Conversely, Annabi Berrahal et al

. ( 201 1 ) found that plasma uric acid levels decreased after 65 days of Pb 

### 27

exposure (post

- puberty ; blood Pb 7.5 μg/dL) ( Table 5

- 17 )

. Plasma urea levels increased after 40 days of 

### 28

exposure (puberty ; blood Pb 12.7 μg/dL) . Changes in plasma urea are used as an ac ute renal marker of 

### 29
injury. 

### 30May 2011 5

- 227 DRAFT

– DO NOT CITE OR QUOTE 

### 5.5.3.2. Oxidative Damage

A role for ROS in the pathogenesis of experimental Pb

- induced hypertension and renal disease has 

### 1

been well established ( Vaziri, 2008a

, 2008b ; Vaziri & Khan, 2007 )

. The production of oxidative stress 

### 2
following Pb exposure is detailed in respect to modes of act ion of Pb (Section 5.2.4). Past studies have 

### 3

shown that a cute Pb exposure can elevate kidney GST levels , affecting glutathione metabolism ( Daggett 

### 4
et al., 1998 ; Moser et al., 1995 ; Oberley et al., 1995 )

. 

### 5

Annabi Berrahal et al

. ( 2011 ) reported on the effects of age

- dependent ex posure to Pb on 

### 6
nephrotoxicity in male rats ( Table 5

- 17 )

. Pups were exposed to Pb lactationally (as a result of dams 

### 7
consuming water containing 50 ppm Pb acetate ) until weaning. Thereafter the male pups were exposed to 

### 8
the same solution from weaning (day 21) until sacrificed at age 40 days (puberty ; blood Pb 12.7 μg/dL) 

### 9
and at age 65 days (post

- puberty ; blood Pb 7.5 μg/dL). MDA concentration in kidney was significantly 

### 10

increased relative to controls to t he same degree at both 40 and 65 days, while total sulfhydryl group s 

### 11
were significantly decreased only at 65 days. These changes reflect an increase in oxidative stress after 

### 12

exposure to Pb. 

### 13

Conterato et al

. ( 2007 ) examined the effect of Pb acetate on the cytosolic thioredoxin reductase 

### 14
activity and oxidative stress parameters in rat kidneys. Acute exposure to Pb acetate consisted of a single 

### 15
i.p. injection of 25 or 50 mg/kg Pb acetate, while chronic exposure con sisted of one daily i.p. injection of 

### 16

Pb acetate (5 or 25 mg/kg ) for 30 days. Measured were thioredoxin reductase

- 1, a selenoprotein involved 

### 17
in many cellular redox processes, SOD , δ

- ALAD

, GST

, GPx

, non protein thiol groups (NPSH)

, CAT , as 

### 18
well as plasma creatinine, uric acid

, and inorganic phosphate levels. Acute exposure at the 25 mg Pb dose 

### 19

level resulted in increased SOD and thioredoxin reductase

- 1 activity, while exposure to the 50 mg dose 

### 20

level increased CAT activity and inhibited δ

- ALAD activity in the kidney. Chronic exposure at the 5 mg 

### 21

dose level of Pb inhibited δ

- ALAD and increased GST

, NPSH

, CAT

, and thioredoxin reductase

- 1. 

### 22
Chronic exposure to the 25

- mg dose level reduced δ

- ALAD , but increased GST

, NPSH , and plasma uric 

### 23

acid levels. No changes were observed in TBARS

, GPx , creatinine or inorganic phosphate levels after 

### 24
either acute or chronic exposure. As both acute and chronic exposure to Pb increased thioredoxin 

### 25

reductase

- 1 activity, the authors suggest that this enzyme may be a sens iti ve indicator to exposure at low 

### 26

Pb dosage. 

### 27

Jurczuk et al

. ( 2006 ) published a study of the involvement of some low molecular weight thiols in 

### 28
the peroxidative mechanisms of action of Pb in the rat kidney. Wistar rats were fed a diet contai ning 500 

### 29

ppm Pb acetate for a period of 12 weeks and were compared to a control group receiving distilled water 

### 30
for the same time period. GSH , metallothionein (MT ), total and nonprotein SH groups (TSH and NPSH ) 

### 31

were m easured

, as well as the blood activity and urinary concentration of δ

- ALA. The concentrations of 

### 32
GSH and NPSH were decreased by Pb administration, while M T concentration was unchanged. δ

- ALAD 

### 33

in blood was decreased, whereas urinary δ

- ALA was increased by Pb administration. Negative 

### 34May 2011 5

- 228 DRAFT

– DO NOT CITE OR QUOTE correlations were found between the kidney GSH concentrations and previously reported concentrations 

### 1

of Pb and MDA in kidneys of these rats. I t is apparent from graphical presentation of the data that GSH 

### 2
was reduced by more than 50% following Pb administration, while TSH was reduced by approximately 

### 3

15%. No values for either blood or kidney Pb levels or kidney MDA were reported in this article. In 2007

, 

### 4

the same authors ( Jurczuk et al., 2007 ) reported on the renal concentrations of the antioxidants, vitamins 

### 5
C and E, in the kidneys of the same Pb treated and control rats. Exposure to Pb significantly decrease d 

### 6

vitamin E concentration b y 13% and vitamin C concentration by 26%. The kidney concentration of 

### 7
vitamin C negatively correlated with MDA concentration. The authors concluded that vitamins E and C 

### 8
were involved in the mechanism of peroxidative action of Pb in the kidney, and their p rotective effect 

### 9
may be related to scavenging of free radicals 

### 10

El

- Neweshy and El

- Sayed s tudied the influence of vitamin C supplementation on Pb

- induced 

### 11
histopathological alterations in male rats. Rats were given Pb acetate, 20 mg/kg by intragastric feeding 

### 12
once daily for 60 days . Control rats were given 15 mg of sodium acetate per kg once daily, and an 

### 13
additional group w as given Pb acetate plus vitamin C (20 mg/kg every other day) 30 minutes before Pb 

### 14
feeding . Control rats showed normal histology, while Pb

- treated rats exhibited karyomegaly with 

### 15
eosinophilic intranuclear inclusion bodies in the epithelial cells of the proximal tubules. Glomerular 

### 16
damage and tubular necrosis with invading inflammatory cells were also seen. Rats treated with Pb 

### 17
acetate plus vitamin C exhibited relat ively mild karyomegaly and eosinophilic intranuclear inclusion 

### 18
bodies of proximal tubules in 5 rats, while an additional 5 rats were normal. Normal glomeruli were noted 

### 19
in all. Thus vitamin C could be shown to ameliorate the renal histopathological effects of Pb intoxication. 

### 20

Masso

- Gonzalez and Antonio

- Garcia ( 2009 ) studied the protective effect of natural antioxidants 

### 21
(zinc, vitamin A, vitamin C, vitamin E, and vitamin B6) against Pb

- induced damage during pregnancy 

### 22
and lactation in rat pups. At weaning, pups were sacrificed and kidneys analyzed. Pb

- exposed pups had 

### 23
decreased body weights. Blood Pb level in the control group was 1.43 μg/dL, in the P b group it was 22.8 

### 24 μg/dL, in the Pb plus zinc plus vitamins it was 21.2 μg/dL, and in the zinc plus vitamin group blood Pb 

### 25 was 0.98 μg/dL. The kidney TBARS were significantly elevated in Pb exposed pups , while treatment with 

### 26

vitamins and zinc returned TBARS to control levels. Kidney c atalase activity was significantly increased 

### 27

above control with Pb treatment; however supplement with zinc and vitamins reduced catalase activity 

### 28

towards normal . Pb exposure i nhibit ed kidney Mn

- dependent SOD but not Cu

- Zn

- d ependent SOD 

### 29
activity. Thus

, supplementation with zinc and vitamins durin g gestation and lactation is effective in 

### 30
attenuating the redox imbalance induced by developmental, chronic low

- level Pb exposure

. 

### 31

Bravo et al

. ( 2007 ) reported further that mycophenolate mofetil (an immuno suppressive agent used 

### 32
in renal transplantation which inhibits T and B cell proliferation ) administration reduces renal 

### 33
inflammation, oxidative stress and hypertension in Pb

- exposed rats. Thus, an inflammatory immune and 

### 34

oxidative stress component can be s een as contributing to Pb

- induced renal effects and hypertension. 

### 35May 2011 5

- 229 DRAFT

– DO NOT CITE OR QUOTE Although the majority of studies of the effects of Pb exposure ha ve been on male rats, two studies 

### 1
have appeared which compare the response of male rats with female rats ( Alghazal, Lenártová, et al., 

### 2

2008 ; Sobekova et al., 2009 ) . Sobekova et al. ( 2009 ) contrasted the activity response to Pb o n the 

### 3
antioxidant enzymes, GPx and GR

, and on TBARS in both male and female Wistar rats of equal age. 

### 4
Males weighing 412 ± 47 g and females weighing 290 ± 19 g were fed diets containing either 100 ppm or 

### 5

1

, 000 ppm Pb acetate for 18 weeks. In the male rats

, kidney Pb content increased by 492% on the 100 

### 6

ppm Pb diet and by 7

, 000% on the 1000 ppm Pb diet. In the female rats

, kidney Pb content increased by 

### 7
410% on the 100 ppm Pb diet and by 23,000% on the 1

, 000 ppm Pb diet. There was virtually no change 

### 8
in GPx in the kidney of male rats given the 100 ppm Pb diet but there was a significant reduction in GPx 

### 9
in the female rats on both the 100 ppm diet and 1000 ppm diet. In male rats, GR was increased from 182 

### 10
units/gram of protein in control kidneys to 220 units on the 100 ppm Pb diet and 350 units on the 1

, 000 

### 11
ppm diet. In female rats
, kidney GR decreased from 242 units in control animals to 164 units in animals 

### 12
on the 100 ppm Pb diet and 190 units in animals on the 1

, 000 ppm diet. In male rats

, kidney TBARS 

### 13

content increased from 7.5 units/gram protein to 10.0 units ( 1

, 000 ppm Pb diet group )

. In female rats, 

### 14
there was a reduction in TBARS from 14.4 units per gram protein to 10.0 units in rats on the 100 ppm Pb 

### 15

diet and to 11 units in rats on the 1

, 000 ppm Pb diet. 

### 16
Alghazal et al

. ( 2008 ) compared the activity responses of the antioxidant enzyme, SOD and the 

### 17
detoxifying enzyme, GST, of the same rats exposed to 100 ppm or 1

, 000 ppm Pb acetate for 18 weeks. 

### 18
Similar to the previous study, k idney TBARS w ere increased only in male r ats given the higher dose of 

### 19

Pb . Kidney SOD activity, on the other hand, was increased in both males and females at the higher dose 

### 20

of Pb , while GST activity was increased in kidney of males at the higher dose of Pb and decreased at the 

### 21
lower dose, but was decreased at both doses of Pb in females. Thus there were significant differences in 

### 22
the response of male and female rats to Pb exposure. Differences could be accounted for in part due to the 

### 23
greater deposition of Pb in female rat kidneys. Another explana tion, offered by the authors, is that male 

### 24
rats are known to metabolize some foreign compounds faster than females, so that the biological half

- life 

### 25

of xenobiotics in the females is longer. 

### 26

5.5.3.3. Lead Effect on Renal Gangliosides

Gangliosides are constituents of the plasma membrane that are important for control of renal GFR 

### 27
because they can act as receptors for various molecules and have been shown to take part in cell

- cell 

### 28
interactions, cell adhesion, recognition and signal transduction. Perez Aguilar et al

. ( 2008 ) studied 

### 29
changes in renal gangliosides following Pb exposure ( 600 ppm Pb acetate in their drinking water for 4 

### 30

months ) in adult male Wistar rats. Pb exposure caused an increase in blood Pb from 2.1 to 35.9 μg/dL. 

### 31

There was no ch ange in serum creatinine or in hemoglobin, but there was an increase in urinary δ

- ALA. 

### 32
The following renal gangliosides were measured by immunohistochemistry and by thin layer 

### 33May 2011 5

- 230 DRAFT

– DO NOT CITE OR QUOTE chromatography: GM1, G M2 , GM4, and 9

- O

- acetylated modified form of the GD3 ganglioside ( 9

- O

- Ac



### 1

GD3 ) . The gan glioside pattern was mainly characterized by a decrease in the GM1 ganglioside as well as 

### 2
by a mild increase in GM4 and G M2 gangliosides, while the strongest alteration was observed in the 9

- O



### 3

Ac

- GD3, which was overexpressed. The latter was observed only i n the glomerular zone. This was 

### 4
associated with a decrease in apoptotic glomerular cells, as assessed by the TUNEL assay. The authors 

### 5
hypothesized that the increase in GD3

- O

- acetylation could represent a strategy to attenuate the normal 

### 6
renal apoptotic pro cess and therefore contribute to cell survival during Pb exposure. 

### 7

5.5.3.4. Role of M etallothionein

Yu et al

. ( 2009 ) described dichotomous effects of Pb acetate on the expression of MT in the liver 

### 8
and kidney of mice. Male mice were i.p. injected with Pb acetate in doses of 100, 200, and 300 μmol/kg 

### 9
and sacrificed 4, 8

, and 24 hours after Pb treatment. Administration of Pb increased the levels of MT

- 1 

### 10
mRNA in the liver and kidneys, but increased MT protein only in the liver. Treatment of mouse PT cells 

### 11

in vitro with Pb also resulted in an increase in MT mRNA, but little increase in MT protein. Thus Pb 

### 12
exerts a dual effect on MT expression in the kidney: enhancement of MT gene transcription but 

### 13
suppression of MT mRNA translation. 

### 14

Zuo et al

. ( 2009 ) explored the potential role of α

- Synuclein (Scna) and MT in Pb induced inclusion 

### 15
body formation. They used MT

- I/II double knockout (MT

- null) and parental wild type (WT) cell lines to 

### 16
explore the formation process of Pb

- induced inclusion bo dies. Unlike WT cells, MT

- null cells did not 

### 17

form inclusion bodies after Pb exposure. Western blot of the cytosol showed that soluble MT protein in 

### 18
WT cells was lost during Pb exposure as inclusion bodies formed. However, transfection of MT

- 1 into 

### 19

MT

- null cells allowed inclusion body formation after Pb expos ure. As Scna is a protein with a natural 

### 20
tendency to aggregate into oligomers, Scna was measured in WT cells and MT

- null cells after Pb 

### 21
exposure. Scna protein showed poor basal expression in MT

- null cell s, and Pb exposure increased Scna 

### 22
expression only in WT cells. MT transfection increased Scna transcript to WT levels. In both of these cell 

### 23

lines Pb

- induced Scna expression rapidly increased and then decreased over 48 hours as Pb

- induced 

### 24

inclusion bodies were formed. A direct interaction between Scna and MT was confirmed ex vivo by an 

### 25
antibody pull down assay, where the proteins co

- precipitated with an antibody to MT . Pb exposure caused 

### 26
increased colocalization of MT and Scna proteins. In archival kidney s amples of renal cortex from WT 

### 27
mice chronically treated with Pb , MT was localized to the surface of inclusion bodies. Thus

, Scna may be 

### 28
a component of Pb

- induced inclusion bodies and

, with MT , may play a role in inclusion body formation. 

### 29
Figure 5

- 4 6 (and T able 5

- 22) presents the recent animal toxicological data for studies investigating 

### 30
the effects of Pb (as blood Pb level) on various measures of kidney health and function. Dysfunction in 

### 31
kidney function measures, including urinary flow, ALP, microalbumin, and NAG, was observed at blood 

### 32
Pb concentrations above 19.7 μg/dL ( L. Wang et al., 2010 )

. 

### 33May 2011 5

- 231 DRAFT

– DO NOT CITE OR QUOTE 1 10 100 Blood Pb Level (μg/dL) Biomarker ( L. Wang et al., 2010 )

Inflammation ( Roncal et al., 2007 )

Oxidative Stress

– Redox ( Navarro

Moreno et al., 2009 )

Oxidative Stress

– Redox ( L. Wang et al., 2010 )

Oxidative Stress

– Redox ( Massó

González & Antonio

- García, 2009 )

Morphology ( Roncal et al., 2007 )

Morphology ( Navarro

- Moreno et al., 2009 )

Morphology ( L. Wang et al., 2010 )

Morphology ( Massó

- González & Antonio

- García, 2009 )

Kidney function ( Navarro

- Moreno et al., 2009 )

Kidney function ( Roncal et al., 2007 )

Kidney function ( L. Wang et al., 2010 )

Kidney function ( Ademuyiwa et al., 2009 )

Figure 5

- 46

. D ose - responsive representation of the effect of Pb on renal outcomes in animal toxicology studies. Highest Concentration Lowest Conc. with Response Highest Conc. with No Response Lowest ConcentrationMay 2011 5

- 232 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 22. Additional characteristics for results of toxicological studies presented in Figure 5

- 46

Reference Blood Pb Level with Response ( μg/dL) Outcome

W ang et al. ( 2010 ) 20 Biomarker

- Aberrant NAG, GGT, β2

- microgobulin expression in adult female mice with chronic Pb exposure.

Roncal et al. ( 2007 ) 26 Inflammation

- Elevation in number of macrophages & marker MCP

- 1 in Pb exposed kidneys with remnant kidney surgery.

Navarro

- Moreno et al. ( 2009 ) 43 Oxidative stress

- Chronic Pb exposure in males increased kidney lipid peroxidation (i.e. TBARS)

Wang et al. ( 2010 ) 20 Oxidative Stress

- Pb caused increased lipid peroxidation (i.e. MDA production), elevated kidney antioxidant enzymes (SOD, GPx, CAT), and depleted GSH in immature female rats.
Massó

- González et al. ( 2009 ) 23 Oxidative stress

- Elevated TBARS and catalase activity in weanling pups exposed to Pb during gestation and lacta tion

Roncal et al. ( 2007 ) 26 Morphology

- Pb induced pre

- glomerular vascular disease of kidney (i.e. sclerosis, fibrosis, peritubular capillary loss)

Navarro

- Moreno et al. ( 2009 ) 43 Morphology

- Electron micrography of chronic Pb exposure in male rats showed lumen reduction, microvill i loss, brush border loss, and mitochondrial damage Wang et al. ( 2010 ) 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 ) 23 Morphology

- Pb elevated relative kidney weight at PND21 in animals with neonatal Pb exposure.

Navarro

- Moreno et al. ( 2009 ) 43 Kidney function

- Pb exposed males had elevated urinary pH and protein, and glucose and blood in the urine. Roncal et al. ( 2007 ) 26 Kidney function

- Remnant kidney surgery and Pb exposure induced decreased creatinine clearance and proteinuria. Wang et al. ( 2010 ) 20 Kidney function

- Elevated urinary total protein, ur inary albumin, and serum urea nitrogen in immature female rats exposed to Pb. Ademuyiwa et al. ( 2009 ) 39 and 61 Kidney Function

- Renal phospholipidosis and depletion of renal cholesterol i n male pups after gestational Pb exposure.



## 5.5.4. Effects of Exposure to Lead Mixtures

The effect of Pb on other cations, specifically calcium, is well established in the kidney literature. 

### 1

Calcium

- mediated processes involving receptors, transport proteins, and second messenger signaling 

### 2
among other endpoints are significantly affected by Pb exposure. The disposition of Pb in the soft tissues 

### 3
(kidney and spleen) can change with exposure to Pb and other compounds. Pb plus Cd exposure change d 

### 4
Pb disposition with increased blood Pb (versus Pb alone group) and decreased metal concentration in the 

### 5
kidney and liver (versus Pb alone). An i ron deficient diet significantly increased Pb deposition in adult 

### 6

animals ( Hashmi et al., 1989 ) , pregnant dams, and maternally exposed fetuses ( U. S. Singh et al., 1991 )

. 

### 7
Dietary t hiamine plus zinc slightly reduced blood and kidney Pb in exposed animals ( Flora et al., 1989 )

. 

### 8
Selenium, a cofactor for GPx , attenuated Pb

- dependent lipid peroxidation and abrogates the Pb

- dependent 

### 9
attenuation of GR and SOD. Concomitant exposure to the cations aluminum and Pb protects Pb

- exposed 

### 10

animals from ensu ing nephropathy ( Shakoor et al., 2000 ) . In summary, Pb i s known to affect processes 

### 11
mediated by endogenous divalent cations. In addition, exposure to other metals or divalent cations can 

### 12
modulate Pb disposition and its effects in the body. 

### 13May 2011 5

- 2 33 DRAFT

– DO NOT CITE OR QUOTE 

### 5.5.4.1. Lead and Cadmium

Cd shares many s imilarities with Pb; it is a ubiquitous PT nephrotoxicant at high exposure levels 

### 1
and accumulates in the body. Despite this, few studies have evaluated association s between low

- level Cd 

### 2
exposure and CKD or the impact of joint exposure of these or other metals on CKD

. As discussed in the 

### 3
2006 Pb AQCD, Cd, at the lower exposure levels common in the U.S. and other developed countries, has 

### 4
a substantial impact on associations between Pb exposure and the kidney EBE marker, NAG, even in the 

### 5
presence of occupat ional level Pb exposure. In one report, mean NAG, although higher in the Pb

- exposed 

### 6
group compared to controls, was correlated with urine Cd and not blood or tibia Pb ( Roels et al., 1994 ) . In 

### 7 another occupational population where both metals were significantly associated with NAG, a 0.5 μg/g 

### 8

creatinine increase in Cd had the same ef fect on NAG as a 66.9 μg/g bone mineral increase in tibia Pb ( V. 

### 9
M. Weaver, Lee, et al., 2003 )

. 

### 10

The 2006 Pb AQCD noted that data examining the dose

- response relation between environmental 

### 11
Cd and the kidney were too scarce to determine the impact of Cd exposure on relations between Pb 

### 12
exposure and other kidney outcomes. A recent publication in NHANES data collected from 1999 through 

### 13

2006 addresses this need; (results pertaining solely to Pb were discussed in Section 5.5.2.2 ) ( Navas

- Acien 

### 14
et al., 2009 ) . G eometric mean blood Cd level was 0.41 μg/L in 14,778 adults aged ≥ 20 years. After 

### 15
adjustment for survey year, sociodemographic factors, CKD risk factors, and blood Pb , the odds ratios for 

### 16 albuminuria (≥ 30 mg/g creatinine), reduced eGFR (<60 mL/min/1.73 m 2 ), and both albuminuria and 

### 17
reduced eGFR were 1.92 (95% CI: 1.53, 2. 43), 1.32 (95% CI: 1.04, 1.68), and 2.91 (95% CI: 1.76, 4.81), 

### 18
respectively, comparing the highest with the lowest blood Cd quartiles. Both Pb and Cd remained 

### 19
significantly associated after adjustment for the other although effect modification was not obse rved. 

### 20

However, t he odds ratios comparing participants in the highest with the lowest quartiles of both metals 

### 21
were 2.34 (95% CI: 1.72, 3.18) for albuminuria, 1.98 (95% CI: 1.27, 3.10) for reduced eGFR, and 4.10 

### 22
(95% CI: 1.58, 10.65) for albuminuria and reduced eGFR together. These findings are consistent with 

### 23
other recent publications ( Akesson et al., 2005 ; Hellstrom et al., 2001 )

, support consideration of both 

### 24
metals as CKD risk factors in the general population

, and provide novel evidence o f increased risk in 

### 25
those with higher environmental exposure to both metals. 

### 26
However, a very recent study suggests that interpretation of low

- level Cd associations with GFR 

### 27
measures may be much more complex. Conducted in Pb workers to address the fact that f ew studies have 

### 28
examined the impact of low

- level Cd exposure in workers who are occupationally exposed to other 

### 29
nephrotoxicants such as Pb, Cd dose was assessed with urine Cd, which is widely considered the optimal 

### 30
dose metric of cumulative Cd exposure. In 712 Pb workers, mean (SD) blood and tibia Pb , urine Cd , and 

### 31
eGFR using the MDRD equation were 23.1 (14.1) μ g/dl, 26.6 (28.9) μ g /g, 1.15 (0.66) μg/g creatinine, 

### 32
and 97.4 (19.2) ml/min/1.73 m 2 , respectively ( V. M. Weaver et al. ) . After adjustment for age , sex, BMI

, 

### 33
urine creatinine, smoking, alcohol, education, annual income, diastolic BP , current or former Pb worker 

### 34May 2011 5

- 234 DRAFT

– DO NOT CITE OR QUOTE job status, new or returning study participant, and blood and tibia Pb , higher ln

- urine Cd was associated 

### 1
with higher calculated creatinine clearance, eGFR ( β = 8.7 ml/min/1.73 m 2 [ 95% CI : 5.4, 12.1 ] ) and ln



### 2

NAG

, but lower serum creatinine. These unexpected paradoxical association s have been reported in two 

### 3

other publication s ( De Burbure et al., 2006 ; Hotz et al., 1999 ) a nd have been observed in two other 

### 4
populations. Potential explanations for these paradoxical results include d a normal physiologic response 

### 5

in which urine Cd levels reflect renal filtration; the impact of adjustment for urine dilution with creatinine 

### 6
in models of kidney outcomes; and Cd

- related hyperfiltration. 

### 7
Wang et al

. ( 2009 ) studied the effects of Pb and/or Cd on oxidative damage to rat kidney cortex 

### 8
mitochondria. In this study young female Sprague Dawley rats were fed for 8 weeks with either Pb 

### 9

acetate ( 300 ppm )

, Cd chloride ( 50 ppm ) , or Pb and Cd together in the same dosage. Lipid peroxidation 

### 10

was assessed as MDA content. Renal cortex pieces were also processed for ultrastructural analysis and for 

### 11
quantitative rtPCR to identify the mitochondrial damage and to quantify the relative expression levels of 

### 12
cytochrome oxidase subunits (COX

- I/II/III). Cytochrome oxidase is the mar ker enzyme of mitochondrial 

### 13
function, and COX

- I, II, and III are the three largest mitochondrially encoded subunits which constitute 

### 14
the catalytic functional core of the COX holoenzyme. Mitochondria were altered by either Pb or Cd 

### 15
administration, but more strikingly by Pb plus Cd administration, consisting of disruption and loss of 

### 16
mitochondrion cristae. Kidney cortex MDA levels were increased significantly by either Pb or Cd , given 

### 17
individually, but more so by Pb plus Cd . COX

- I/II/III were all reduced by e ither Pb or Cd administration, 

### 18
but more prominently by Pb plus Cd administration. This study adds to our knowledge of the synergistic 

### 19

effects of Pb and Cd on kidney mitochondria. 

### 20

5.5.4.2. Lead , Cadmium, and Arsenic

Wang and Fowler ( 2008 ) present a general review of the roles of biomarkers in evaluating 

### 21
interactions among mixtures of Pb

, Cd, and arsenic. Past studies have found that that addition of Cd to 

### 22

treatment of rats with Pb or Pb and As significantly reduced the histological signs of renal toxicity from 

### 23
each element alone; on the other hand, animals exposed to Cd in addtion to Pb or Pb and As showed an 

### 24
additive increase in the urinary excretion of porphyrins, indicating that, although me asured tissue burdens 

### 25

of Pb were reduced, the biologically available fraction of Pb is actually increased ( Mahaffey et al., 1981 ; 

### 26
Mahaffey & Fowler, 1977 )

. 

### 27
Stress proteins were examined after exposure to mixtures of Pb and other metals. Induction of MT 

### 28

was strongest in groups with Cd treatment. However, co

- exposure to Pb and As induced higher levels of 

### 29

MT protein tha n either Pb or As exposure alone in kidney tubule cells. Heat shock proteins (Hsps) are 

### 30
commonly altered under the situation of exposure to metal mixtures. Both in vitro low dose studies and in 

### 31
vivo studies showed that Hsps were induced in a metal/metalloid, dose and time

- specific manner ( G. 

### 32May 2011 5

- 235 DRAFT

– DO NOT CITE OR QUOTE Wang et al., 2005 )

. Additive or more than additive interactions occur among Pb

, Cd and As under 

### 1
combined exposure conditions. 

### 2

5.5.4.3. Lead and Zinc

Zinc has been investigated as a protective compound against the effects of Pb. Pb treatment (35 

### 3
mg/kg i

. p . for 3 days) caused a significant fall in hemoglobin content, significant increases in lipid 

### 4
peroxidation and decreased level of reduced glutathione i n liver, together with diminished total protein 

### 5
content in liver and kidney. Zinc (10 mg/kg i

. p

. ) and ascorbic acid (10, 20 and 30 mg/kg i

. p

. ) treatment 

### 6
showed a moderate therapeutic effect when administered individually, but more pronounced protective 

### 7

eff ects after combined therapy ( Upadhyay et al., 2009 )

. 

### 8

Jamieson et al

. ( 2008 ) studied the effect of dietary zinc content on renal Pb deposition. Weanling 

### 9
Sprague Dawley rats were assigned to marginal zinc (MZ, 8 mg Zn/kg diet), zinc adequate control (CT, 

### 10

30 m g Zn/kg )

, zinc

- adequate diet

- restricted (DR, 30 mg Zn/kg), or supplemental zinc (SZn, 300 mg 

### 11
Zn/kg) groups, with or without Pb acetate, 200 ppm for 3 weeks. Pb exposure did not result in 

### 12
nephromegaly or histological alterations. The marginal zinc rats had higher renal Pb (35%) and lower 

### 13
renal zinc (16%) concentrations than control rats. On the other hand, supplemental zinc was more 

### 14
protective than the control diet against renal Pb accumulation (33% lower). Standard procedures for 

### 15
indirect immunoperoxidase staining were used to determine MT localization in the kidney. Pb had no 

### 16

effect on MT staining intensity, distribution, or relative protein amounts. Western blot analysis confirmed 

### 17

that MT levels were responsive to dietary zinc but not to Pb exposure. 

### 18

5.5.4.4. Lead and Mercury

Stacchiotti et al

. ( 2009 ) studied stress proteins and oxidative damage in a renal derived cell line 

### 19
exposed to inorganic mercury and Pb

. The time course of the expression of several heat shock proteins, 

### 20
glucose

- regulating proteins and metallothioneins in a rat proximal tubular cel l line (NRK

- 52E) exposed to 

### 21
subcytotoxic doses of inorganic mercury (HgCl 2 , 1

- 40 μM) and Pb ( PbCl 2 , 2

- 500 μM) were analyzed. 

### 22
Reactive oxygen and nitrogen species were detected by flow cytometric analysis. Endogenous total GSH 

### 23
content and the enzymatic acti vity of GST were determined in cell homogenates. Western blot analysis 

### 24
and immunohistochemistry were used for quantification of heat shock proteins and metallothionein. 

### 25
Reverse transcription PCR was used for quantification of metallothionein. The higher do ses of mercury 

### 26
(20 μM and 40 μM) were shown to markedly inhibit growth of the cell line while the higher doses of Pb 

### 27 (60 μM to 500 μM) inhibited cell growth to a lesser degree. After 24 hours of exposure at 20 μM mercury, 

### 28
the cells presented abnormal size and py kn otic nuclei, swollen mitochondria and both apoptosis and overt 

### 29

necrosis. In the presence of 60 or 300 μM Pb , the cells lost cell

- cell and cell

- matrix contacts, showed a 

### 30
round size, irregular nuclear contour and often mitotic arrest, but no apoptosi s or overt necrosis at 24 

### 31May 2011 5

- 236 DRAFT

– DO NOT CITE OR QUOTE hours. Mercury induced a significant increase in both and reactive nitrogen species, the reactive nitrogen 

### 1
species maximal at 24 hours, and the ROS at 48 hours. Pb ( 60 or 300 μM ) did not cause an increase in 

### 2
reactive oxygen or rea ctive nitrogen species beyond the levels seen in control cells. Total GSH 

### 3
significantly increased in cells grown in the presence of Pb ; the effect was dose

- dependent and GSH 

### 4
reached its maximal value at a dose of 300 μM Pb . The effect of mercury was biphas ic, 10 μM 

### 5
significantly enhancing GSH by 600%, while the amount of GSH detected after 20 μM mercury only 

### 6
increased by 50% compared to control. GST activity was enhanced by both Pb and mercury. Heat shock 

### 7
proteins Hsp25 and Hsp72 were up

- regulated by mercur y but there was no effect on Grp78 as compared 

### 8

to control. On the contrary, Pb treatment only upregulated Grp78. Mercury induced a time

- dependent 

### 9
effect on metallothionein mRNA expression, which reached its maximal value 3 hours after beginning 

### 10

treatment a nd reverted to control values at 24 hours. With Pb , on the other hand, mRNA transcription was 

### 11

dose

- and time

- dependent. The transcripts remained overexpressed compared to controls up to 72 hours. 

### 12
The results of this study with regard to the Pb effect on me tallothionein synthesis clearly differ from the 

### 13
study of Jamieson et al. ( 2008 ) that found no increase in metallothionein following Pb exposure . This 

### 14
discrepancy remains to be clarified. 

### 15



## 5.5.5. Impact of Treatment with Antioxidants on Renal Lead Accumulation and Pathology

5.5.5.1. Treatment with Antioxidants

Wang et al

. ( 2010 ) assessed the protective effect of N

- acetylcysteine (NAC) on experimental 

### 16

chronic Pb nephrotoxicity in immature female rats. NAC is a poten t oxygen free radical scavenger, a 

### 17

metal chelator , and the precursor to the antioxidant glutathione . Sprague

- Dawley rats received Pb acetate 

### 18
(300 ppm in drinking water) and/or NAC (100 mg/kg/day, by i.p. injection) for 8 weeks to investigate the 

### 19
protective effect of NAC on Pb

- induced renal damage and oxidative stress. Serum and renal cortical Pb 

### 20
levels were markedly increased in the Pb treated animals, but reduced in the Pb plus NAC treated 

### 21

animals. There were time

- related increases in urinary alkaline phosphatase, urinary GGT, urinary NAG, 

### 22
urinary total protein, urinary β

- 2 microglobulin, and urinary microalbumin, which were all decreased by 

### 23
NAC. Serum urea nitrogen was significantly increased by Pb administration and reduced towards normal 

### 24
by Pb plus NAC. Alterati ons in proximal tubular structures were observed in most kidney samples from 

### 25

Pb

- treated rats, but animals treated with combination Pb plus NAC showed well

- preserved cell structures 

### 26
and organelles. Indices of oxidative stress (MDA, SOD, GSH, GPx, and CAT) w ere altered by Pb 

### 27
treatment and restored to or towards normal by Pb plus NAC treatment (MDA increased and the 

### 28
remainder decreased). Thus NAC can be shown to have both an anti

- oxidative and a chelator effect on Pb 

### 29

intoxication. 

### 30May 2011 5

- 237 DRAFT

– DO NOT CITE OR QUOTE Saxena et al

. ( 2005 ) investigated the beneficial role of monoesters of meso

- 2, 3

- 

### 1
dimercaptosuccinic acid in the mobilization of Pb and recovery of tissue oxidative injury in rats. 

### 2
Dimercaptosuccinic acid (DMSA) is known as a Pb chelator and as an antioxidant by v irtue of its 

### 3
possession of thiol groups. In this study, DMSA, and two of its analogues, monomethyl 

### 4
dimercaptosuccinic acid (MmDMSA) and mono

- cyclohexyl dimercaptosuccinic acid (MchDMSA) were 

### 5
assessed as to their capacity to reduce Pb concentration in blood and soft tissues and to recover Pb



### 6
induced oxidative stress in male Wistar rats who were exposed to Pb acetate (0.1% in drinking water) for 

### 7

20 weeks. Rats were then treated orally with five days of DMSA or its two analogues at a dose of up to 

### 8
100 mg/kg once daily. Exposure to Pb caused a rise in blood Pb levels to approximately 25 μg/dL. 

### 9
Exposure to Pb also caused a significant decrease in blood ALAD ac tivity and GSH levels, accompanied 

### 10 by inhibition of kidney ALAD and an increase in δ

- aminolevulinic acid synthatase (ALAS) activity in 

### 11
liver and kidneys. Pb exposure also resulted in increase d blood and soft tissue (brain, liver, and kidney) 

### 12

Pb and TBARS l evels and decrease d GSH levels. These were restored by treatment with DMSA and its 

### 13
analogues, particularly MchDMSA. 

### 14

Abdallah et al

. ( 2010 ) explored the effect of Pb toxicity on coenzyme Q levels in rat tissues. 

### 15
Coenzyme Q acts as an electron and pro ton carrier in mitochondria and functions as an antioxidant in its 

### 16
reduced form (ubiquinol). Both coenzyme Q9 and coenzyme Q10 were measured in rat tissues as 

### 17
coenzyme Q9 is the predominant form found in the rat. Male albino rats were injected i.p. with Pb acetate 

### 18
in a dose of 5 mg/kg daily for 6 weeks. No blood Pb levels were reported. TBARS were elevated above 

### 19
controls in serum, liver, kidney and brain while non

- protein sulfhydryl groups (indicative of GSH) were 

### 20
decreased in serum and kidney. Both oxidize d and reduced coenzyme Q9 levels were significantly 

### 21
reduced in kidneys from Pb

- treated rats as contrasted to controls (4 8.6 ± 5.6 versus 95.5 ± 10.1 nmol/g 

### 22
tissue, oxidized, and 35.4 ± 3.0 v ersus 61.4 ± 5.1 nmol/g tissue, reduced). On the other hand, level s of 

### 23
oxidized and reduced coenzyme Q10 were unchanged. Thus the reduced levels of coenzyme Q 

### 24

attributable to Pb intoxication may participate in the diminished antioxidant defen s e mechanism. 

### 25

El

- Sokkary et al

. ( 2005 ) evaluated the effect of melatonin against Pb

- induced hepatic and renal 

### 26
toxicity in male rats. Melatonin is known to be efficacious as a free radical scavenger and indirect 

### 27
antioxidant. Three groups of animals were used: control, Pb acetate

- treated (100 ppm) and Pb acetate and 

### 28
melatonin (10 mg/kg) given sub cutaneously f or 30 days. Lipid peroxidation was measured as the sum of 

### 29
MDA plus 4

- hydroxyalkenals (4

- HAD). Pb increased kidney lipid peroxidation products, but these were 

### 30
reduced towards normal by melatonin. Both SOD and GSH levels were reduced by Pb , and were 

### 31

increase d by melatonin. Histological section of kidneys of Pb treated rats showed tubular degeneration 

### 32
with some apparently necrotic cells, while melatonin treated rats demonstrated a near normal structure. 

### 33
The authors conclude that melatonin protect ed the liver and kidneys from the damaging effects of 

### 34

exposure to Pb through inhibition of lipid peroxidation and stimulation of endogenous antioxidative 

### 35
defense systems. 

### 36May 2011 5

- 238 DRAFT

– DO NOT CITE OR QUOTE Ozsoy et al

. s tudied the protective effects of L

- carnitine on experimental Pb toxicity in rats. Female 

### 1

two

- month old rats were fed 0.5 mg/kg Pb acetate alone or wit h daily injections of 0.5 mg/kg L

- carnitine 

### 2
for 60 days. Control animals were injected with physiological saline. Pb caused an increase in serum 

### 3
creatinine and histopathological changes in the kidney, consisting of tubule dilatation, degeneration and 

### 4

necro sis and interstitial inflammation. In the Pb and L

- carnitine group serum creatinine was reduced to 

### 5
control values and the histopathological changes were reversed. Immunological staining for Cu/Zn

- SOD 

### 6
was elicited by Pb feeding and reduced by L

- carnitine. T he authors attribute the beneficial effects of L



### 7

carnitine to its antioxidant effect. 

### 8
Reddy et al

. ( 2010 ) used S prague

- Dawley rats that were treated with 10 mg/kg/day of Pb acetate 

### 9

and/or thiamine ( 25 mg/kg/day ) for 7 weeks. Thiamine treatment normalized the Pb

- induced alterations in 

### 10
blood ALAD activity a nd urinary NAG activity. 

### 11

K haroubi et al. ( 2008 ) described the prophylactic effects of Wormwood ( Artemisia absinthium L. ) 

### 12
plant extracts on kidney function on Pb

- exposed animals. M ale Wistar rats were exposed to Pb acetate 

### 13
(750 ppm in drinking water) for 11 weeks, and then received Wormwood extract (200 mg/kg) for 4 

### 14
weeks. Significant differences in blood and urinary Pb concentration were observed between the Pb group 

### 15

and the Wormwo od group (blood Pb 55.6 μg/dL compared to 22.3 μg/dL). Pb induced lipid peroxidation 

### 16
(TBARS and protein carbony ls in the kidney ) , but these levels were reduced by Wormwood extract. 

### 17

Wormwood extract also attenuated the effects of Pb on renal function. These results indicated that 

### 18
Wormwood extract had significant a ntioxidant activity and protected the kidney from Pb

- induced toxicity. 

### 19
Jayakumar et al

. ( 2009 ) evaluated the effect of a methanolic extract of the Indian herb, Achyranthes 

### 20

aspera , in preventing Pb

- induced nephrotoxicity in rats. Male albino Wistar rats, received Pb acetate 

### 21
( 0.2% for 6 weeks ) or Pb acetate plus A. aspe ra ( 200 mg/kg for 6 weeks ) simultaneously. A. aspera 

### 22
partially prevented the increases in kidney weight, BUN , serum uric acid, and serum creatinine caused by 

### 23

Pb administration. The levels of urinary marker enzymes, GGT, β

- glucuronidase, NAG, Cathepsin D, a nd 

### 24
LDH, which were reduced by Pb administration, were increased to or towards normal by A. aspera

. 

### 25
Kidney histology revealed that Pb

- treated animals showed tubular damage, whereas the Pb plus A. aspera 

### 26

- treated animals showed a reduction in tubular damage. 

### 27
The effectiveness of various plant or bacterial extracts as antioxidants in the kidney was explored 

### 28
in two separate publications. El

- Nekeety et al

. ( 2009 ) evaluated the protective effect of an extract of the 

### 29

folk medicine plant Aquilegia vulgaris against Pb acetate

- induced oxidative stress in Sprague

- Dawley 

### 30
rats. The experimental group was treated with Pb acetate, 20 ppm, and/or an extract of A. vulgaris , 100 

### 31
ppm, for 2 weeks prior to Pb acetate. Pb acetate increased serum urea, and decreased serum total protein 

### 32

an d albumin

. These changes were reversed by treatment with the extract

. Histolo gical examination of 

### 33
kidneys of rats treated with Pb showed tubular dilatation, interstitial inflammatory cells, hemorrhage, 

### 34
cellular debris, and hypercellularity in the glomerulus, with apoptotic nuclei in renal tubular epithelial 

### 35

cells. The rats treated simultaneously with Pb and the extract showed essentially normal renal tubules and 

### 36May 2011 5

- 239 DRAFT

– DO NOT CITE OR QUOTE glomeruli while rats treated with Pb and then the extract showed improvement in tubular structure, but 

### 1
interstitial fibrosis was still present. This experiment confirmed tha t exposure to Pb generates free 

### 2
radicals, and that an extract of A. vulgaris resulted in restoration of the dif ferent parameters tested. The 

### 3

second experiment in this group was by Ponce

- Canchihuaman et al

. ( 2010 ) who evaluated the antioxidant 

### 4
activity of the cyanobacterium Spirulina maxima against Pb acetate

- induced hyperlipidemia and oxidative 

### 5
damage in the liver and k idney of male rats. Male Wistar rats were exposed to Pb acetate by i .p. injection 

### 6
( 25 mg/rat on a weekly basis for 3 weeks and a 5% supplement of Spirulina w as given in food)

. The 

### 7
findings in the kidney were similar to those in the liver (see Section 5.9.1) . Thus Pb

- induced oxidative 

### 8
stress and renal damage can be attenuated by treatment with Spirulina extract. 

### 9
Finally, there is a need to examine whether the chelator, CaNa 2 EDTA , acts also as an antioxidant 

### 10
and promotes increased vasodilat ation and thus increased renal blood flow by enhancing the delivery of 

### 11
NO. This question arises because of the observations of Lin et al

. ( 2006 ) that repeated injections of 

### 12

CaNa 2 EDTA lead s to improvement in kidney function in patients with chronic renal failure, even in 

### 13
individuals with very low body Pb stores. Jacobsen et al

. ( 2001 ) ,examined the anti

- oxidative effects of 

### 14
Gallic acid, EDTA, and an emulsifier in mayonnaise enriched with 16% fish oil. EDTA was shown to be 

### 15
an efficient antioxidant in the fish oil enriched mayonnaise as it strongly inhibited the formation of free 

### 16
radicals and volatile oxidation compounds. T he authors suggest that the antioxidative effect appears to be 

### 17
due to its ability to chelate free iron in egg yolk at the oil

- water interface. 

### 18

5.5.5.2. Treatment with Antioxidants plus Chelators

Santos et al

. ( 2006 ) assessed the potentiating effects of chelators (2,3

- dimercaptopropanol [BAL], 

### 19
2,3

- dimercaptopropane

- l

- sulfonic acid [DMPS], and meso

- 2,3

- dimercaptosuccinic acid [DMSA]) given 

### 20

simul taneously with Pb acetate on δ

- ALAD activity, both in vivo and ex vivo. Ex vivo, human blood was 

### 21

pre

- incubated with BAL or DMSA (10 μ M) or DMPS (1 μ M) then Pb acetate added to the reaction 

### 22
mixture. In vivo, mice were given daily injections of 50 mg/kg Pb a cetate for 15 days and then injected 

### 23
with 1/3 of LD50 of the chelating agents. In human blood the inhibitory effect of Pb acetate (1 and 100 

### 24

μ M) was markedly increased in the presence of BAL and DMPS, whereas DMSA ameliorated the enzyme 

### 25

inhibition caused b y 1 μ M Pb acetate. In vivo Pb acetate inhibited δ

- ALAD activity by 42%. Parallel to 

### 26
the ex vivo results, BAL and DMPS, but not DMSA, increased the inhibitory potency of Pb in blood. In 

### 27

the kidney

, BAL and DMSA but not DMPS increased inhibitory activity. Th e authors conjecture that the 

### 28 chelators may deplete the cells of zinc, an essential element for δ

- ALAD activity. Supporting the 

### 29
chelation effect seen is the Santos study is work by Bradberry and Vale ( 2009 )

, Hamidinia et al

. ( 2006 )

, 

### 30

and Aslani et al

. ( Aslani et al., 2010 ) who found decreased kidney Pb content post

- chelation. 

### 31May 2011 5

- 240 DRAFT

– DO NOT CITE OR QUOTE 

## 5.5.6. Summa ry and Causal Determination

The 2006 Pb AQCD concluded that “in the general population, both circulating and cumulative Pb 

### 1
was found to be associated with a longitudinal decline in renal function,” evidenced by increased serum 

### 2

creatinine and decreased crea tinine clearance or eGFR associated with blood and bone Pb levels ( U.S. 

### 3
EPA, 2006 ) . Data in general and patient populations provided consistent evidence of low

- level Pb 

### 4
nephrotoxicity ( Akes son et al., 2005 ; R. Kim et al., 1996 ; Tsaih et al., 2004 ; C.

- C. Yu et al., 2004 ) ; effects 

### 5
on eGFR were observed in human hypertensives at mean blood Pb level o f 4.2 μg/dL ( Muntner et al., 

### 6

2003 ) . These findings were substantiated by the coherence of effects observed across epidemiologic and 

### 7
toxicological studies. B oth human and animal studies have observed hyperfiltration; in animals during the 

### 8
first 3 months after Pb exposure, effects were characterized by increased GFR and increased kidney 

### 9
weight due to glomerular hypertrophy. However, chronic exposure resulted in decreased GFR, interstitial 

### 10
fibrosis, and kidney dysfunction. Additionally, toxicological studies found that early effects of Pb on 

### 11
tubular cells were generally reversible, but continued exposure resulted in chronic irreversible damage. 

### 12
Toxicological stud ies provided mechanistic evidence to support the biological plausibility of Pb

- induced 

### 13 renal effects, including oxidative stress leading to  ̇NO inactivation. Despite the strong body of evidence 

### 14
presented in the 2006 Pb AQCD, uncertainty remained on the pub lic health significance of such effects in 

### 15
the general population, the implications of hyperfiltration, and reverse causality. 

### 16

Recent epidemiologic studies in adult general and patient populations, with few exceptions, 

### 17

continue to be consistent in observin g associations between blood and bone Pb levels and worse kidney 

### 18
function and provide important evidence that nephrotoxicity occurs at current population levels of 

### 19

biomarkers of Pb exposure. These studies benefit from a number of strengths that vary by stu dy but 

### 20
include comprehensive assessment of Pb dose with bone Pb as a measure of cumulative body burden and 

### 21
chelatable Pb as a measure of bioavailable Pb; prospective study design; and statistical approaches that 

### 22
utilize a range of exposure and outcome meas ures, while adjusting for numerous kidney and Pb risk 

### 23

factors. Large sample sizes provide strength to the general population studies. Reexamination of a study 

### 24
from the 2006 Pb AQCD provided data to conclude that a 10

- fold increase in blood Pb (e.g., from 1 to 10 

### 25 μg/dL) would result in an 18 mL/min decrease in estimated creatinine clearance or a 25% decrease from 

### 26

the mean , and that a n increase in blood Pb from the 5th to the 95th percentile (3.5 μg/dL) had the same 

### 27
adverse impact on eGFR as an increase of 4. 7 years in age or 7 kg/ m 2 in body mass index ( Akesson et al., 

### 28

2005 ) . In populations with lower blood Pb levels , a downward shift in kidney function of the entire 

### 29
population due to Pb may not result in CKD in identifiable individuals; however, that s egment of the 

### 30
population with the lowest kidney reserve may be at increased risk for CKD when Pb is combined with 

### 31
other kidney risk factors . At blood Pb levels that are common in the general U.S. population, Pb increases 

### 32

the risk for clinically relevant ef fects particularly in susceptible populations such as those with underlying 

### 33May 2011 5

- 241 DRAFT

– DO NOT CITE OR QUOTE chronic medical diseases that increase CKD risk such as diabetes mellitus and hypertension and co



### 1
exposure to other environmental nephrotoxicants. 

### 2
Absence of impact in the occupat ional setting cannot be used as a rationale for discounting Pb



### 3
related nephrotoxicity at lower environmental levels. Research in the occupational setting has 

### 4
traditionally been far less consistent than in environmentally exposed populations. A number of 

### 5

ex planatory factors for this inconsistency, all due to limitations of the occupational literature, were 

### 6
discussed in the 2006 Pb AQCD. The observation of paradoxical or inverse associations (higher Pb dose 

### 7
with lower serum creatinine, and/or higher eGFR or c alculated or measured creatinine clearance) in 

### 8
several of these studies cannot be resolved solely by utilizing stronger research techniques. Irrespective of 

### 9
the mechanism, these associations have risk assessment implications. If associations are in opposit e 

### 10
directions in different subgroups of the population and the relevant effect modifier is not considered, null 

### 11

associations will be observed. 

### 12
Important data on the kidney effects of Pb on children were reported in a recent NHANES analysis 

### 13

in adolescents th at observed an association between higher blood Pb and lower cystatin C

- based eGFR 

### 14
( Fadrowski et al., 2010 )

. These findings are consistent with a rodent model in which a low dose of Pb (50 

### 15
ppm) administered from birth resulted in renal impairment (elevated serum creatinine as compared to 

### 16
control rats)

, but these observations require confirmation by measurement of GFR and renal pathology 

### 17
( Berrahal et al., 2011 )

. These limited studies add to t he strength of the association between blood Pb and 

### 18
altered renal function in children despite the need for additional research. 

### 19

CKD results in substantial morbidity and mortality

, and, even at earlier stages than those requiring 

### 20
kidney dialysis or transpl antation, is an important risk factor for cardiac disease

. As kidney dysfunction 

### 21
can increase BP and increased BP can lead to further damage to the kidneys, Pb

- induced damage to either 

### 22
or both renal or cardiovascular systems may result in a cycle of furthe r increased severity of disease. Pb 

### 23
exposure has been causally linked to both increased BP and other cardiovascular effects (Section 5.4). 

### 24
Interestingly, animal studies have shown Pb

- induced vascular injury in the kidney associated with 

### 25
increased glomerula r sclerosis, tubulointerstitial injury

, increased collagen staining

, and an increase in 

### 26
macrophages associated with higher levels of MCP

- 1 mRNA ( Roncal et al., 2007 )

. It is possible that the 

### 27
cardiovascular and renal effects of Pb observed are mechanistically linked and are contributing to the 

### 28
progression of the diseases. 

### 29
Recently available animal toxicological studies strengthen the evidence regarding the mechanisms 

### 30

l eading to these renal alterations, especially, similar to the cardiovascular system, the influence of Pb



### 31
induced oxidative stress. The mode of action of Pb in the kidneys has been extended to the field of 

### 32
immunology, where it was shown that low Pb exposure results in infiltration of lymphocytes and 

### 33
macrophages associated with increased expression of NF κ B in proximal tubules and infiltrating cells. 

### 34
Additionally, recent evidence expands on the evidence of acute effects of Pb, including mitochondrial 

### 35May 2011 5

- 242 DRAFT

– DO NOT CITE OR QUOTE dysfuncti on, renal cell apoptosis, and glomerular hypertrophy. These mechanisms are useful in 

### 1
understanding the occurrence of acute hyperfiltration followed by chronic kidney dysfunction. 

### 2

Current evidence does not allow for the identification of a threshold for Pb

- related nephrotoxicity; 

### 3

increased odds of CKD (characterized by eGFR <60 mL/min/1.73 m 2 ) were apparent in NHANES 

### 4
analyses that included data collected as recently as 2006 ( Navas

- Acien et al., 2009 )

. The odds of reduced 

### 5
eGFR increased by 36% (95% CI: 0.99, 1.85) at blood Pb levels as low as 1.6

- 2.4 μg/dL and by 56% 

### 6
(95% CI: 1.17, 2.08) at blood Pb > .4 μg/dL

. 

### 7
In summary, new studies evaluated in the current review support or expand upon the strong body of 

### 8
evidence presented in the 2006 Pb AQCD that biomarkers of Pb exposure are associated with renal health 

### 9
effects. Epidemiologic stu dies continue to demonstrate a consistently positive relationship between blood 

### 10
Pb level and kidney dysfunction at blood Pb levels (mean <2 μg/dL) comparable to those occurring in the 

### 11
current U.S. population with no evidence for a threshold across the rang e of levels studied. By 

### 12
demonstrating Pb

- induced oxidative stress and describing mechanisms of acute changes following Pb 

### 13
exposure, toxicological studies provide biological plausibility for the associations observed in 

### 14
epidemiologic studies between Pb and kidney dysfunction. Collectively, the evidence integrated across 

### 15
epidemiologic and toxicological studies as well as across the spectrum of kidney health endpoints is 

### 16

sufficient to conclude that there is a causal relationship between Pb exposure s and renal health 

### 17

effects

. 

### 18



# 5.6. Immune System Effects

5.6.1. I ntroduction

With respect to studies conducted in laboratory animal and in vitro models, Pb is one of the most 

### 19
extensively researched and studied immunotoxicants. Experimental studies of the effects of Pb exposure 

### 20

on h ost resistance date back to the 1960s while those focusing on Pb

- induced immune functional 

### 21
alterations, including developmental immunotoxicity (DIT), were first conducted during the 1970s. 

### 22
Despite the long history of Pb

- associated immunotoxicity research, the immune

- based effects in animals 

### 23
with blood Pb levels in the range of current U.S. population levels (i.e., <10 μg/dL), particularly early in 

### 24
life, are a relatively recent finding from within the last 10

- 15 years ( Dietert & McCabe, 2007 )

. Over the 

### 25
same time period, similar advances in the understanding of Pb

- associated changes in immunological 

### 26
parameters in humans without occupation Pb exposures have substantiated the immunomodulatory effects 

### 27

of Pb

. 

### 28

In the 2006 Pb AQCD ( U.S. EPA, 2 006 )

, both toxicological studies in animals and epidemiologic 

### 29
studies of humans provided strong evidence that the immune system was one of the more sensitive 

### 30
systems affected by Pb exposure. However, rather than producing overt cytotoxicity or pathology , Pb 

### 31May 2011 5

- 243 DRAFT

– DO NOT CITE OR QUOTE exposure of experimental models and blood Pb levels in humans were found to be associated with 

### 1
alterations in the abundance and function of a variety of immune cells ( Figure 5

- 47). In both toxicological 

### 2
and epidemiologic studies, macrophages and T lym phocytes were observed to be particularly sensitive to 

### 3
Pb, but Pb

- associated changes were also reported in B lymphocytes and neutrophils. Several of these 

### 4
changes were observed at blood Pb levels <10 μ g/dL or the equivalent in humans and experimental 

### 5

model s, levels at which neurological effects also were observed. 

### 6
Alterations in these aforementioned immune cells can lead to changes changes in cell

- to

- cell 

### 7
interactions, multiple signaling pathways, and inflammation that affect both innate and acquired 

### 8

immun ity, that in turn, influence risk of developing infectious, allergic and autoimmune diseases as well 

### 9
as exacerbating inflammatory responses in other organ systems ( Figure 5

- 47). 

### 10

Figure 5

- 47. Immunological pathways by which Pb exposure may increase risk o f immune

- related diseases.

Studies conducted in animal and in vitro models have provided consistent evidence for Pb inducing 

### 11
effects on the range of immune effects presented in this continuum. Among the hallmarks reported for Pb



### 12
induced changes in function al pathways are: (1) a suppression of T

- derived lymphocyte helper (Th)1

- 

### 13 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 diseasesMay 2011 5

- 244 DRAFT

– DO NOT CITE OR QUOTE driven cell

- mediated immunity (as measured by a delayed

- type hypersensitivity [DTH] response); (2) an 

### 1

increase in Th2

- driven immunoglobulin E (IgE) antibody production; and (3) a proin flammatory shift in 

### 2
macrophage function. The latter was characterized by increased production of reactive oxygen 

### 3
intermediates reactive oxygen species (ROS), prostaglandin E 2 (PGE 2 ), and inflammatory cytokines such 

### 4

as tumor necrosis factor

- α (TNF

- α) and in terleukin (IL)

- 6 and decreased production of IL

- 12 and nitric 

### 5
oxide (NO). In animal studies, these effects of Pb were more prominent with in utero Pb exposures and in 

### 6
males. In the 2006 Pb AQCD, epidemiologic evidence was available for relatively fewer imm une 

### 7
endpoints (e.g., proinflammatory shifts in immune cell function); however, coherence was observed 

### 8
between toxicological and epidemiologic findings for Pb

- associated changes in circulating IgE levels and 

### 9

T and B cell abundance and activation ( U.S. EPA, 2006 ) . Although many epidemiologic st udies indicated 

### 10
associations between blood Pb levels and immune system effects, several limitations of study design and 

### 11
analytic methods were noted, including cross

- sectional analyses; small sample size; inconsistent 

### 12
adjustment for potential confounders su ch as age, sex, smoking, and comorbid conditions; and limited 

### 13
assessment of the magnitude of association between blood Pb levels and changes in immune function. 

### 14
Consistent with inhibition of Th1 activity, toxicological evidence presented in the 2006 Pb AQC D 

### 15
linked Pb exposure of animals to impaired host resistance and increased risk of certain infections ( U.S. 

### 16
EPA, 2006 ) . Consistent with inducing a hyperinflammatory state and local tissue damage, Pb exposure 

### 17
was found to induce generation of autoantibodies, indicating an elevated risk of autoi mmune reactions. 

### 18
Additionally, the demonstrated shift toward a Th2 response suggested that Pb could elevate the risk of 

### 19
atopy and allergic responses. While toxicological studies provided the evidence for biological plausibility, 

### 20
the epidemiologic evidence was too sparse to draw conclusions regarding associations between blood Pb 

### 21
levels and these broader indicators of immune dysfunction in humans. 

### 22
Studies published since the 2006 AQCD support the previous findings of Pb

- induced immune 

### 23

effects and demonstrat e similar effects at lower blood Pb levels (<2

- 5 μ g/dL). Recent studies also expand 

### 24
on the array of immunological parameters affected by Pb exposure as presented in Figure 5

- 47. For 

### 25
example, new toxicological evidence indicates that Pb modulates function o f dendritic cells. Results from 

### 26
new toxicological and epidemiologic studies strengthen the link between Pb

- associated effects on immune 

### 27

cells and immune

- and inflammatory

- based diseases by providing evidence for changes in intermediary 

### 28
signaling and inflam matory pathways ( Figure 5

- 47). New epidemiologic studies examine signaling 

### 29
molecules such as proinflammatory cytokines and NO to produce findings parallel with toxicological 

### 30
studies. Another important advance is the increasing knowledge of the broader role of Pb

- associated 

### 31
immune modulation in mediating Pb effects in nonlymphoid tissues (e.g., in the neurological, 

### 32
reproductive, and respiratory systems). Although primarily cross

- sectional in design, recent epidemiologic 

### 33
studies address many limitations of ea rlier studies through greater examination of children and adults 

### 34
without occupation Pb exposures with blood Pb levels more comparable to those currently measured in 

### 35May 2011 5

- 245 DRAFT

– DO NOT CITE OR QUOTE the U.S. population and greater consideration of confounding by age, sex, smoking, and como rbid 

### 1

conditions. 

### 2



## 5.6.2. Cell

- Mediated Immunity

5.6.2.1. T C ells

Toxicological and epidemiologic evidence reviewed in the 2006 Pb AQCD consistently 

### 3
demonstrated Pb effects on decreasing T cell populations ( U.S. EPA, 2006 ) , which mediate responses to 

### 4
antigens and infectious agents

. Consistent with previous f indings, in a recent study of Wistar rats 

### 5
administered 200 ppm Pb acetate, the percentages of CD4+ (T helper) and CD8+ (cytotoxic T) T cells 

### 6
were decreased (with CD4

- CD8

- cells elevated) in the submaxillary lymph nodes (p <0.05), with 

### 7
intraperitoneal (i.p. ) exposure but not oral exposure ( Teijon et al., 2010 )

. The 2006 Pb AQCD also 

### 8

described numerous toxicological studies in which Pb exposure shift ed the development and/or activation 

### 9

of CD 4+ T cell population s such that production of Th2 cytokines was favored and production of Th1 

### 10

c ytokines was suppressed ( U.S. EPA, 2006 )

. Recent studies expand information on the potential 

### 11
mechanisms underlying T cell activation. In cultures of human CD4+ T cells, Pb (1 μM, 30 minutes) has 

### 12
been shown to activate transcription factor NF κ B (regulates T cell activation) ( Pyatt et al., 1996 ) and to 

### 13
increase, in a dose

- dependent manner (10 and 50 μM PbCl 2 , 24 hours), the expression of MHC class II 

### 14

surf ace antigens (HLA

- DR), which mediates the CD4+ response to exogenous antigens ( Guo et al., 

### 15

1996b )

. Heo et al. ( 2007 ) provide evidence for the direct effects of Pb on T cells by showing that Pb (25 

### 16
μM) blocked production of the Th1 cytokine interferon

- γ (IFN

- γ) in cultures of stimulated mouse T cells 

### 17
by suppressing translation of the protein. This blockage was rescued wi th the addition of IL

- 12. Kasten



### 18
Jolly et al. ( 2010 ) found that Pb may not necessarily skew towards a Th2 phenotype via a direct effect on 

### 19
T cells. In this study, developmental Pb exposure of mice ( 0.1mM Pb acetate in drinking water of dams 

### 20
from GD8 to PND21, resulting in pup blood Pb levels 10

- 30 μ g/dL) induced gene expression of IL

- 4 and 

### 21
suppressed production of IFN

- γ in splenic cells. These changes occurred in the absence of STAT4 or 

### 22
STAT6, the pr eferential signaling pathways for T cells and occurred with concomitant increases in 

### 23
adenylate cyclase 8 and phosphatidylinositol 3

- kinase, indicating that Pb may promote Th2 activity via T 

### 24

cell

- independent pathways. 

### 25
Epidemiologic studies provide evidence for blood Pb levels being associated with a shift in 

### 26
production from Th1 to Th2 cytokines in humans ( Section 5.6.5.4 ); however, the extant evidence for 

### 27
effects on T cells in humans is derived largely from older studies describing changes in the abundance o f 

### 28
several T cell subtypes, including CD3+, CD4+, and CD8+, that may affect cell

- to

- cell interactions 

### 29
required in acquired immunity responses to antigens. Among stu dies of subjects without occupational Pb 

### 30

exposures , decreases in the abundance of CD3+ ( Figur e 5

- 48 and Table 5

- 23), CD4+, and CD8+ T cells 

### 31May 2011 5

- 246 DRAFT

– DO NOT CITE OR QUOTE often were observed among children with blood Pb level s in the range of 5 to 44 μg/dL ( Lutz et al., 1 999 ; 

### 1
Sarasua et al., 2000 ; Z. Y. Zhao et al., 2004 )

. However, Karmaus et al. ( 2005 ) observed de creases in 

### 2
several T cell subtypes in children with blood Pb level s 2.2

- 2.8 μg/dL compared with children with blood 

### 3

Pb level s <2.2 μg/dL, indicating that decreases in T cell abundance also may occur at blood Pb levels that 

### 4

are more comparable to those of t he current U.S. general population. In association with higher blood Pb 

### 5
levels ( ≥ 10 μg/dL ), Zhao et al. ( 2004 ) , Fischbein et al. ( 1993 ) , and Mishra et al. ( 2010 ) observed a 

### 6
decrease in the ratio of CD4+/CD8+ cells, indicative of a compromised response to v iral infections. These 

### 7
findings are consistent with the documented effects of Pb exposure on diminished host resistance and 

### 8

associations with bacterial and viral infections (Section 5.6.4.1)

. Changes in the CD4+/CD8+ ratio have 

### 9
not been examined in populat ions with lower blood Pb levels. In a large multicity U.S. study conducted in 

### 10
subjects living near a Pb smelting operation plus demographically

- matched controls , Sarasua et al. ( 2000 ) 

### 11

indicated that the associations between blood Pb levels and T cells may vary with age. Investigators 

### 12
analyzed blood Pb level as a continuous variable and found that among children 6

- 35 months in age, a 1 

### 13
μg/dL increase in blood Pb level was associated with decreases in the percentage of CD3+ (

- 0.18 [95% 

### 14

CI:

- 0.34,

- 0.02]), CD4+ (

- 0.10 [95% CI:

- 0.24, 0.04]), and CD8+ (

- 0.04 [95% CI:

- 0.15, 0.07]) T cells; 

### 15
however, in older children (36

- 71 months, 6

- 15 years) and adults (16

- 75 years), many effect estimates 

### 16
were positive. 

### 17
Despite the consistency o f findings across studies, it is not clear what may be the impact of the 

### 18
small magnitudes of change in T cell abundance on the cell

- to

- cell interactions that mediate downstream 

### 19
acquired immune responses. For example, in populations without occupational Pb exposures, Pb



### 20
associated decreases in the relative abundance of CD3+ cells range between 1 and 9% ( Table 5

- 23). 

### 21
Larger decreases (20

- 35%) are observed in studies of occupationally

- exposed males with higher blood Pb 

### 22
levels than those expected in the general population (>25 μg/dL ) ( Fischbein et al., 1993 ; Undeger et al., 

### 23

1996 )

. 

### 24May 2011 5

- 247 DRAFT

– DO NOT CITE OR QUOTE 0 0.2 0.4 0.6 0.8 1 1.2 Relative CD3+ cell abundance (normalized to lowest blood Pb group) Study Population Blood Pb Group

Karmaus et al. ( 2005 ) Children, ages 7

- 10 yr. <2.2 2.2

- 2.83 2.84

- 3.4 >3.4

Sarasua et al. ( 2000 ) Children, ages 6

- 35 mo. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥15
Sarasua et al. ( 2000 ) Children, ages 36

- 71 mo. 0.6

- 4.9
5

- 9.9 10

- 14.9 ≥15
Sarasua et al. ( 2000 ) Children, ages 6

- 15 yr. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥15

Lutz et al. ( 1999 ) Children, ages 9mo.

- 6 yr. <10 10

- 14 15

- 19 20

- 44

Zhao et al. ( 2004 ) Children, ages 3

- 6 yr. <10 ≥10

Sarasua et al. ( 2000 ) Subjects, ages 16

- 75 yr. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥15

Pinkerton et al. ( 1998 ) Controls <2 Pb workers 39

Fischbein et al. ( 1993 ) Controls NR Pb workers <25 Pb workers ≥25 Underger et al. ( 1996 ) Controls 16.7 Pb workers 74.8

Note: Bars represent the abundance of CD3+ cells normalized to the level measured in the lowest blood Pb group (depicted as black or dark blue). Bars in black or gray represent res ults in subjects without occupational Pb exposures , and bars in dark or light blue represent results in subjects w ith occupational Pb exposures

. Figure 5

- 48

. Comparisons of the relative abundance of CD3+ T cells among groups with increasing blood Pb level (μ g/dL).

Table 5

- 23. Comparison of serum abundance of T

- cell subtypes a among various blood Pb groups.

Study Population Blood Pb group (μg/dl) CD3+ CD4+ CD8+ CD4+:CD8+ CD45RO+ CD45RA+

Children

Karmaus et al. ( 2005 ) b 331 children, ages 7

- 10 yr Hesse, Germany <2.2 2.21

- 2.83 2.84

- 3.41 >3.41 2118 1919 1979 2184 1214 1106 1128 1123 712 634 661 662 358 321 348 351
Sarasua et al. ( 2000 ) c 382 children, ages 6

- 30

mo Multiple U.S. locations 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 67.8 66.6 67.1 65.1 46.6 45.5 47.1 45.2 19.9 20.2 19.1 18.8
Sarasua et al. ( 2000 ) c 562 children, ages 36

- 71

mo Multiple U.S. locations 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 68.1 69.4 68.6 76.1 42.0 44.0 44.2 41.6 23.5 23.1 21.4 24.4
Sarasua et al. ( 2000 ) c 675 children ages 5

- 16 yr Multiple U.S. locations 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 69.2 69.5 71.1 66.3 43.0 43.0 44.5 44.0 24.7 25.4 24.5 20.1
Lutz et al. ( 1999 ) d 279 children, ages 9 mo

6 yr Springfield, MO <10 10

- 14 15

- 19 20

- 44 68.4 69.7 64.6 65.3May 2011 5

- 248 DRAFT

– DO NOT CITE OR QUOTE Study Population Blood Pb group (μg/dl) CD3+ CD4+ CD8+ CD4+:CD8+ CD45RO+ CD45RA+

Zhao et al. ( 2004 ) 75 children, ages 3

- 6 yr Zhejiang Province, China <10 ≥ 10 55.2 54.6 27.1 23.7 e 20.6 23.2 e 1.41 1.09 e

Adults without Occupational Pb Exposures

Sarasua et al. ( 2000 ) c 433 c hildren and adults, ages 16

- 75 yr Multiple U.S. locations 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 71.6 73.4 78.5 70.9 50.3 53.3 54.9 49.6 24.9 23.9 24.1 25.3

Adults with Occupational Pb Exposures

Fischbein et al. ( 1993 ) 36 unexposed controls, mean age 47 yr 51 firearms instructors, mean age 48 yr NR <25 ≥ 15 72.6 57 46.9 e 45.6 29.6 21.4 e 25.7 23 25.4 1.95 1.38 0.95 e

Pinkerton et al. ( 1998 ) f 84 unexposed controls, mean age 30 yr 145 Pb smelter workers, mean age 33 yr <2 39 73.1 74.9 44.7 44.6 23.0 23.3 1.9 1.9 45.6 44.1

Underger et al. ( 1996 ) g 25 unexposed controls, ages 22

- 56 yr 25 Pb battery plant workers, ages 22

- 55 yr 16.7 74.8 2044.7 1644 1140.3 858.8 e 977.6 829.1 1.3 1.1

Yucesoy et al. ( 1997b ) 10 unexposed controls, ages 25

- 42 yr 47 Pb battery plant workers, ages 19

- 49 yr 4.0 59.4, 58.4 30.8 30.1

Mishra et al. ( 2010 ) 21 unexposed controls, median age 27 yr 26 three

- wheel drivers, median age 31 yr 33 Pb battery workers, median age 27 yr 4.5 6.7 132 (103) 55 37 31 e 26 27 26 2.6 1.4 1.3 e 44 43 40 61 73 70 e

a Data are presented as the percentage of T cell subtype among all T cells unless otherwise specified.
b Data represent the number of cells/μL serum. Means are adjusted for age, sex, ETS, number of infections in the previous 12 months, serum lipid concentration, and organochlorine exposures.

c Means are adjusted for age, sex, and location of study.
d Means are adjusted for age.

e p<0.05 for difference among groups.
f Means adjusted for age, race, current smoking status, and workshift.
g Data represent the number of cells/μL serum.



### 5.6.2.2. Lymphocyte A ctivation

Pb exposure produce s an e xpansion of alloreactive T and B lymphocytes . This occurs as a result of 

### 1
reversing the normal suppression that is mediated by a macrophage

- like subpopulation. As discussed in 

### 2

Section 5.6.5.2 , changes in NO production appear to be involved in this process ( Farrer et al., 2008 )

. 

### 3
Additionally, Pb alters antigen processing that occurs in antigen presenting cells (APCs, e.g., primarily 

### 4
dendritic cells and macrophages), which appear to shift signals to T cells skewing both the nature of the 

### 5
subsequent response and the spectrum of ac tivities among those expanded populations of lymphocytes 

### 6
( Farrer et al., 2005 ) . Gao et al. ( 2007 ) reported that dendritic cells that matured in the presence of Pb 

### 7
promoted enhanced alloreactive T cell proliferation compared to control dendritic cells. An add itional 

### 8

effect of Pb has been described relative to activation of T cells. Using the local lymph node assay 

### 9
(LLNA) , Carey et al. ( 2006 ) found that PbCl 2 was able to provide a costimulatory signal to antigens that 

### 10

could activate T cell s. The exact mech anistic basis for this is not known. 

### 11May 2011 5

- 249 DRAFT

– DO NOT CITE OR QUOTE Epidemiologic findings are mostly limited to studies of occupationally

- exposed adults and to 

### 1

associations with blood Pb level >10 μ g/dL. In the only study of children in Missouri, the percentage of 

### 2
activated T cells (as indicated by the cell surface marker HLA

- DR) was higher in children with blood Pb 

### 3

level 15

- 19 μ g/dL than in children with blood Pb level <15 μ g/dL; however, activated cells were not 

### 4

elevated in children with blood Pb levels 20

- 44 μ g/dL ( Lutz et al., 1999 ) . In contrast with toxicological 

### 5
findings, the direction of lymphocyte proliferation (in response to mitogens and/or to specific antigens) in 

### 6
association with occupat ional Pb exposure is unclear. Pb promotes the activation of Th2 cells and 

### 7
suppresses Th1 cells, therefore, the differential activation of specific subtypes may be not discernable in 

### 8
studies that measure overall lymphocyte proliferation. Whereas some studie s have reported similar levels 

### 9 of lymphocyte proliferation (≤ 1% difference) between Pb

- exposed workers and unexposed controls ( N. 

### 10
Cohen et al., 1989 ; Queiroz, Perlingeiro, et al., 1994 ) , others have reported lower lymphocyte 

### 11
proliferation among Pb

- exposed workers (8

- 25%) ( Alomran & Shleamoon, 1988 ; Fischbein et al., 1993 ; 

### 12
Kimber et al., 1986 ; Mishra et al., 2003 ) . With the exception of Fischbein et al. ( 1993 ) , these latter studies 

### 13
included subjects with high blood Pb levels (>60 μ g/dL). Additionally, with much of the evidence limited 

### 14

to com parisons of mean proliferative responses between Pb

- exposed and

- unexposed workers, it is 

### 15
difficult to apply findings to populations with lower blood Pb levels. In one of the few analyses of 

### 16
correlation between blood Pb levels and lymphocyte proliferation, Mishra et al. ( 2003 ) found a lack of 

### 17
correlation, suggesting the influence of another occupationally

- related factor on lymphocyte proliferation. 

### 18

5.6.2.3. Delayed

- type H ypersensitivity

The DTH assay is commonly used as an indicator of the T cell

- mediated adaptive immune 

### 19
response, i.e., induration and erythema resulting from the activation of T cells and recruitment of 

### 20
monocytes to the site of antigen deposition. The DTH response is largely T h1

- dependent in that Th1 

### 21
cytokines drive the production of antigen

- specific T cells directed against the antigen (sensitizing phase) 

### 22
and the recruitment of antigen

- specific T cells and monocytes to the site of antigen deposition (elicitation 

### 23
phase). In the 2006 Pb AQCD and several recent reviews, a suppressed DTH response was identified as 

### 24
one of the most consistently observed and well

- established immunomodulatory effects of Pb exposure in 

### 25

animal models ( Dietert & McCabe, 2007 ; Mishra, 2009 ; U.S. EPA, 2006 ) . A majority of the evidence for 

### 26
Pb suppressing the DTH response is pro vided by the historical literature in which effects were observed in 

### 27
association with both gestational ( Bunn, Ladics, et al., 2001 ; Bunn, Parsons, et al., 2001a

, 2001b ; S. Chen 

### 28
et al., 2004 ; S. Chen et al., 1999 ; Faith et al., 1979 ; J.

- E. Lee et al., 2001 ; Miller et al., 1998 ) and postnatal 

### 29
( Laschi

- Loquerie et al., 1984 ; M. J. McCabe, Jr. et al., 1999 ; Muller et al., 1977 ) Pb exposures of animals. 

### 30
In some studies, the suppressed DTH response was accompanied by a decreased production of IFN

- γ ( S. 

### 31
Chen et al., 1999 ; J.

- E. Lee et al., 2001 ) , which is the primary cytokine that stimulates recruitment of 

### 32
macrophages, a key component of the DTH response. Th e concomitant decrease in IFN

- γ demonstrated 

### 33May 2011 5

- 250 DRAFT

– DO NOT CITE OR QUOTE further that Pb

- induced suppression of the DTH response reflects the inhibition of Th1 functional 

### 1

activities. 

### 2
One of the most salient findings collectively was that DTH was suppressed in animals with blood 

### 3

Pb le vels ranging from <2 to 5 μ g/dL ( Bunn, Ladics, et al., 2001 ; Bunn, Parsons, et al., 2001a

, 2001b ; 

### 4
Miller et al., 1998 ; Muller et al., 1977 ) . In some studies that examined Pb exposures at different stages of 

### 5
gestation, exp osures later in gestation suppressed DTH ( Bunn, Parsons, et al., 2001b ; J.

- E. Lee et al., 

### 6

2001 ) . These latter findings may reflect the status of thymus and T cell development. A recent study 

### 7
contributed to the robust evidence by indicating a role for dendritic cells in the Pb

- induced suppression of 

### 8
the DTH response. Gao et al. ( 2007 ) exposed b one marrow

- derived dendritic cells in vitro to PbCl 2 (25 

### 9
μM, 10 days) then the antigen ovalbumin (OVA) and injected the cells into naïve mice. The Pb

- exposed 

### 10

dendritic cells inhibited the OVA

- specific DTH footpad response in mice compared with mice exposed to 

### 11

control dendritic cells. 

### 12
The capacity of Pb to suppress the DTH response is strongly supported by mechanistic studies in 

### 13
which Pb suppresses Th1 cytokine production ( Section 5.6.5.4 ). Further, coherence is provided by 

### 14

associations observed between Pb e xposure or blood Pb levels and other responses related to the 

### 15

inhibition of Th1

- driven adaptive immune responses, including increased susceptibility to developing 

### 16
certain infections and tumors ( Section 5.6.4.1 ). 

### 17

5.6.2.4. Macrophages and Monocytes

M acrophages and m onocytes, the blood form of tissue macrophages, are among the most sensitive 

### 18
targets of Pb

- induced immune effects. The 2006 Pb AQCD emphasized the large number of toxicological 

### 19
studies showing the effects of Pb on a wide range of alterations in macrophage function to promote a 

### 20
hyperinflammatory phenotype ( U.S. EPA, 2006 ) . These changes include enhanced production of ROS, 

### 21
suppressed production of NO, enhanced production of TNF

- α, excessive metabolism of arachidonic acid 

### 22
into immunosuppressive metabolites (e.g., prostaglandin E 2 ), impaired phago cytic activity and lysosomal 

### 23
function, and potentially altered receptor expression [e.g., toll

- like receptors]). These studies are 

### 24

described in detail in Section 5. 6.5 .2 . Because macrophages are major resident populations in most tissues 

### 25
and organs and are also highly mobile in response to microbial signals and tissue alterations, their 

### 26
functional impairment in response to Pb exposure may serve as a link between Pb

- induced immune 

### 27
effects and impaired host defense, tissue integrity, and organ homeostasis in numerous physiological 

### 28
systems and organs ( Section 5.6. 4.5). Recent studies by Bussolaro et al. ( 2008 ) , Kasten

- Jolly et al. ( 2010 )

, 

### 29

and Mishra et al. ( 2006 ) using mouse macrophages ex posed in vitro to Pb reinforced the capacity of Pb to 

### 30
induce a broad spectrum of functional alterations in macrophages such as lipopolysaccharide (LPS)

- 

### 31
induced production of NO. Although not examined widely , epidemiologic evidence suggests that Pb 

### 32

exposure may be associated with changes in macrophage function in humans. Pineda

- Zavaleta et al. 

### 33May 2011 5

- 251 DRAFT

– DO NOT CITE OR QUOTE ( 2004 ) examined children in Lagunera, Mexico, attending schools at varying distances from an active Pb 

### 1
smelter (range of blood Pb levels: 3.5

- 47.5 μ g/dL) and found that blood Pb levels were associated with a 

### 2
hyperinflammatory state in macrophages as indicated by a decrease in NO production and increase in 

### 3
superoxide anion production ( Section 5. 6.5 .2 ). Stud ies of occupationally

- exposed adults mostly observe 

### 4
that Pb exposure is associated with impaired function of macrophages. Adjusting for age, race, smoking, 

### 5
and workshift, Pinkerton et al. ( 1998 ) found lower abundanc e of monocytes among Pb smelter workers 

### 6
(7.8%) than among unexposed controls (8.5%). Fischbein et al. ( 1993 ) found lower abundance of HLA



### 7

DR+ cells in firearms instructors with blood Pb level <25 μ g/dL (8.8%) and ≥ 2 5 μ g/dL (8.7%) than 

### 8
among unexposed controls (15.2%). HLA

- DR+ is an indicator of activated function state of APCs and is 

### 9
upregulated in response to cell signaling. 

### 10

5.6.2.5. Neutrophils

Although neutrophils were found not to be a significant direct target of Pb in t he 2006 Pb AQCD 

### 11
( U.S. EPA, 2006 ) , the modulation of their activity by Pb may have important consequences on the 

### 12
dysregulation of inflammation and ability to response to infectious agents. Studies of cultured human 

### 13
polymorphonuclear cells (PMNs) ( Governa et al., 1987 ) and occupationally

- exposed adults ( Bergeret et 

### 14
al., 1990 ; Queiroz et al., 1993 ; Queiroz, Costa, et al., 1994 ; Valentino et al., 1991 ) have found reduced 

### 15
PMN functionality, as in dicated by reduced chemotactic response, phagocytic activity, respiratory 

### 16
oxidative burst activity, or reduced ability to kill ingested antigen, among Pb workers compared with 

### 17
controls. Important limitations to applying these epidemiologic findings broadly include male

- only study 

### 18
populations, relatively high blood Pb levels of workers (range of mean levels: 33.1

- 71 μ g/dL) and the 

### 19
lack of direct examination of associations between blood Pb level and neutrophil function. 

### 20
In both studies of animals and occupat ionally

- exposed adults, Pb exposure has been associated 

### 21
with an increase in neutrophil counts, which has been interpreted as a compensatory response to Pb



### 22
induced impairment in neutrophil chemotactic activity and a hyperinflammatory response. In a study by 

### 23
Kibayashi et al. ( 2010 ) to investigate host responses to gunshot wounds in the brain, neutrophils were a 

### 24
major responding cell. Implantation of Pb spheres (compared with glass spheres in the controls) in male 

### 25
Wistar rats led to major neutrophil infiltration with inflammatory

- related damage that included apoptosis 

### 26
and indications of neurodegeneration. 

### 27
In a group of 68 ceramic, Pb recycling, or bullet manufacturing workers and 50 controls selected 

### 28
among food plant workers, DiLorenzo et al. ( 2006 ) observed that a 1 μ g/dL increase in blood Pb level 

### 29

was associated with an increase in ANC of 21.8 cells/ μ L (95% CI: 11.2, 32.4 cells/ μ L) adjusted for age, 

### 30
BMI, and smoking status. The geometric mean (range) of blood Pb levels was 20.5 μ g/dL (3.2

- 120) 

### 31
among workers and 3.5 μ g/dL (1

- 11) among controls. Eight workers with medium to high Pb exposures 

### 32
(exact blood Pb levels not reported) had neutrophilia (n >7,500 cells/mm 3 ) versus no controls, suggesting 

### 33May 2011 5

- 252 DRAFT

– DO NOT CITE OR QUOTE that chronic, higher

- level Pb exposures can lead to a biologically meaningful excess of circulating 

### 1
neutrophils. Additionally, in analyses comparing three blood Pb level groups, controls, workers with 

### 2

blood Pb levels ≤ 30 μ g/dL, and workers with blood Pb levels >30 μ g/dL, ANC was observed to increase 

### 3
monotonically, supporting a blood Pb dose

- dependent relationship. When the three blood Pb groups were 

### 4
further stratified by current smoking, two

- way ANOVA indicated positive int eraction between blood Pb 

### 5
level and current smoking. Higher blood Pb level was associated with higher ANC only when current 

### 6
smokers were compared. Among nonsmokers, ANCs were similar across blood Pb groups. 

### 7
Coherence for the effects of Pb exposure on neutr ophils is provided by findings that blood Pb level 

### 8
is associated with mediators of neutrophil proliferation, survival, maturation, and functional activation. 

### 9
These mediators include cytokines such as TNF

- α ( Section 5.6.5.4 ) and complement. The complement 

### 10

s ystem is a component of the innate immune system that controls various cell

- mediated immune 

### 11
responses such as chemotaxis of macrophages and neutrophils and phagocytosis of antigens. The effects 

### 12
of Pb exposure on complement have not been widely examined; ho wever the limited data suggest Pb may 

### 13
suppress complement activity. Both Ewers et al. ( 1982 ) and Undeger et al. ( 1996 ) measured lower 

### 14
complement C3 protein among Pb

- exposed workers compared with unexposed controls, with Ewers et al. 

### 15
( 1982 ) additionally observing an inverse association between blood Pb level and C3 among wor kers. 

### 16
However, the implications of these findings are limited due to the high blood Pb levels in these 

### 17
occupationally

- exposed groups (range of blood Pb levels: 18.6

- 85.2 μ g/dL and 38

- 100 μ g/dL, 

### 18
respectively) and by the lack of adjustment for potential conf ounding variables. 

### 19

5.6.2.6. Dendritic C ells

Since the 2006 Pb AQCD ( U.S. EPA, 2006 )

, new evidence from both an ex vivo and in vitro model 

### 20
suggests that the effects of Pb exposure on suppressing Th1 activity and promoting Th2 activity may be a 

### 21
consequence of the direct action of Pb on the function of d endritic cell s (a major APC)

. Prior research on 

### 22
the effects of Pb in favoring Th2 over Th1 activity emphasized the direct measurement of Th1 vs. Th2 T



### 23
cell populations and cytokine profiles. But new research techniques ( D. Gao & Lawrence, 2010 ) have 

### 24
provided an opportunity to look upstream at how dendritic cells may be involved in mediating the effects 

### 25
of Pb on acquired immunity. Gao et al. ( 2007 ) use d bone marrow cultures exposed to Pb to examine the 

### 26
impact of Pb on dendritic cell maturation and function. They found that Pb (25 μM, 10 days) altered the 

### 27
course of dendritic cell maturation by changing the ratio of cell surface markers, such as the CD86/ CD80 

### 28
ratio, that promote Th2 cell development. Additionally, upon activation with LPS, Pb

- matured dendritic 

### 29
cells produced less IL

- 6, TNF

- α, and IL

- 12 (stimulates growth and differentiation of T cells) than control 

### 30

cells but the same amount of IL

- 10 (inhib its production of Th1 cytokines). The effect of Pb in altering the 

### 31
cytokine expression profile of dendritic cells, in particular, the lower IL

- 12/IL

- 10 ratio, may serve as an 

### 32
important signal to shift naïve T cell populations towards a Th2 phenotype. Stren gthening the role of 

### 33May 2011 5

- 253 DRAFT

– DO NOT CITE OR QUOTE dendritic cells in mediating Pb immune effects were ex vivo results from the same study that Pb

- naïve 

### 1
Balb/c mice implanted with Pb

- treated dendritic cells were skewed towards Th2 activity as indicated by 

### 2
inhibited DTH ( Section 5.6.2.3 ) and IgG2a antibody ( Section 5.6.3 ) responses ( D. Gao et al., 2007 )

. 

### 3

5.6.2.7. Natural K iller (NK) C ells

The collective body of toxicological and epidemiologic evidence indicates that the innate immune 

### 4
NK cells are not affected to a large extent by Pb exposure. This conclusion is underscored by 

### 5
epidemiologic observations that blood Pb levels are associated wi th T or B cell abudance but are not 

### 6
associated with NK cell abundance or the level of functional activity ( Karmaus et al., 2005 ; Pinkerton et 

### 7
al., 1998 ; Sarasua et al., 2000 ) . Likewise, similar means of NK cell abundance or functional activity have 

### 8

been observed in Pb

- exposed workers and unexposed controls ( Fischbein et al., 1993 ; Kimber et al., 

### 9

1986 ; Mishra et al., 2003 ; Pinkerton et al., 1998 ; Undeger et al., 1996 ; Yucesoy et al., 1997b )

. Consistent 

### 10
with epidemiologic findings, i n a recent in vitro study comparing th e toxicity of metals for different 

### 11
populations of immune cells, Fortier et al. ( 2008 ) found that PbCl 2 (7.5

- 20.7 μ g/dL), did not significantly 

### 12
affect NK cytotoxicity compared with t he DMSO vehicle; however, PbCl 2 did not affect other immune 

### 13
parameters (e.g., monocyte phagocytic activity or lymphocyte proliferation as well. 

### 14



## 5.6.3. Humoral Immun ity

The 2006 Pb AQCD indicated that Pb exposure was associated with enhanced humoral immune 

### 15

respons es as characterized by the proliferation of B cells and increased production of Ig antibodies ( U.S. 

### 16
EPA, 2006 ) . Both toxicological and epidemiologic studies (Figure 5

- 49 and Table 5

- 24 ) have 

### 17

demonstrated Pb

- associated increases in IgE production, which is strongly implicated in mediating 

### 18
allergic responses and inflammation in allergic asthma. In animal studies, Pb exposures induced 

### 19
concomitant increases in IgE and IL

- 4 production by T ce lls ( S. Chen et al., 1999 ; J. E. Snyder et al., 

### 20

2000 ) , consistent with the hypothesis that Th2

- mediated mechanisms can induce class switching of B 

### 21
cells for the production of IgE. Additional coherence and biological plausibility for Pb effects on humoral 

### 22
immunity have been provided by epidemiologic observations for increases in B cell abund ance in 

### 23
association with increasing blood Pb level or occupational Pb exposure ( Figure 5

- 50 and Table 5

- 24). 

### 24
Earlier toxicological and epidemiologic studies also found similar associations of Pb with increases in 

### 25
other classes of Igs including IgG, IgM, an d IgA. 

### 26
Recent toxicological evidence continues to support the role of T cell

- mediated mechanisms in Pb



### 27
induced activation of B cells and production of Ig antibodies. Carey et al. ( 2006 ) treated Balb/c mice with 

### 28
subsensitizing doses of a T cell

- indepe ndent [Trinitrophenyl

- Ficoll (TNP

- Ficoll)] or T cell

- dependent 

### 29
[TNP

- ovalabumin (TNP

- OVA)] hapten

- protein conjugate with or without co

- exposures to PbCl 2 . Seven 

### 30
days later, they examined the effects of PbCl 2 on the LLNA response to TNP

- Ficoll or TNP

- OVA. Pb Cl 2 

### 31May 2011 5

- 254 DRAFT

– DO NOT CITE OR QUOTE exposure (25

- 50 μg, injected) increased the numbers of T and B cells in the lymph node against both 

### 1

TNP

- Ficoll and TNP

- OVA. Further, in a dose

- dependent manner, PbCl 2 induced statistically significant 

### 2
elevations of IgM, IgG2a, and IgG1 antibody produci ng cells in the lymph node. While the increase in 

### 3
IgM

- producing cells against TNP

- Ficoll indicated a T

- cell independent mechanism, the increases in 

### 4
IgG2a

- and IgG1

- producing cells against both antigens indicated a Th1

- and Th2

- mediated mechanism, 

### 5
respecti vely. Despite seeing increases in both IgG1

- and IgG2a

- producing cells, the authors concluded 

### 6
that Pb skewed the response toward Th2 and had considerable potential for promoting allergic 

### 7
sensitization against T

- dependent antigens. 

### 8
Other animal studies pro vided strong support for Pb exposure stimulating humoral responses 

### 9
preferentially via Th2

- mediated mechanisms. Gao et al. ( 2006 ) found that Pb exposure (50 μg, i.p., 

### 10

3 times/week, 3 weeks) of Balb/c mice elevated IgG1 over that of Th1

- driven IgG2a. Similar results were 

### 11

repo rted when Pb

- exposed dendritic cells were used to initiate antibody production ( D. Gao & Lawrence, 

### 12

2010 ) . In a highly

- specialized strain of IFN

- γ knockout mice (lacking the capacity to produce IFN

- γ), Pb 

### 13
exposure had the reverse effect of increasing the IgG2a/IgG1 ratio. These results were surprising given 

### 14

evidence that IFN

- γ usually directs secretion of IgG2a; however, the authors suggested that in these 

### 15

knockout

- mice, Pb may initiate a Th1 response via an IFN

- γ independent pathway to enhance IgG2a 

### 16
production ( D. Gao et al., 2006 ) . In a microarray s tudy examining the DIT of Pb in Balb/c mice, Kasten



### 17
Jolly et al. ( 2010 ) found that early

- life Pb exposure (0.1mM Pb acetate in drinking water of dams from 

### 18
GD8 to PND21, resulting in pup blood Pb levels 10

- 30 μ g/dL) produced statistically significant increases 

### 19
in the expression of genes encoding Ig antibodies or those involved in B lymphocyte function and 

### 20
activation. These genes included those for the heavy chain of IgM, IL

- 4, IL

- 7 and IL

- 7 receptor, IL

- 21, 

### 21

RAG

- 2, CD antigen 27, B

- cell leukemia/lymphoma 6, RNA binding motif protein 24, Histocompatibility 

### 22
class II antigen A (beta 1), Notch gene homolog 2, and histone deacetylase 7A. 

### 23
In epidemiologic studies, associations between increasing blood Pb level and increasing serum IgE 

### 24
level are demonstrated primarily in children ( Annesi

- Maesano et al., 2003 ; Karmaus et al., 2005 ; Lutz et 

### 25
al., 1999 ; L. Sun et al., 2003 )

. Most studies indicate that t he association between blood Pb level and IgE is 

### 26
nonmonotonic ( Figure 5

- 49 and Table 5

- 24). Whereas many studies were focused on examining 

### 27

differences between children with blood Pb levels below and above 10 μ g/dL (Figure 5

- 49 and Table 5

- 

### 28

24 ) , Karmaus et al. ( 2005 ) demonstrated 28% increases in IgE among children with blood Pb levels 2.84

- 

### 29

3.41 and >3.4 μ g/dL compared with children with b lood Pb level <2.2 μ g/dL. Another strength of this 

### 30
study was the adjustment for potential confounding by various organochlorine compounds, age, number 

### 31
of infections in the previous 12 months, and serum lipids. A clear blood Pb dose

- dependent relationship 

### 32

w as not observed as the mean IgE was lower among children in the second quartile of blood Pb levels 

### 33
(2.2

- 2.8 μ g/dL) than among children in the lowest quartile (<2.2 μ g/dL). Annesi

- Maesano et al. ( 2003 ) 

### 34
provided additional information on potential critical developmental windows of Pb exposure. Cord blood 

### 35
IgE was associated with infant hair Pb level but not with cord or placental Pb level. F rom these findings, 

### 36May 2011 5

- 255 DRAFT

– DO NOT CITE OR QUOTE the authors inferred a stronger effect of Pb exposure integrated over the entire gestational period rather 

### 1
than exposures closer to birth. Additionally, the magnitude of association was larger in the subgroup with 

### 2
nonallergic mothers, p ointing to the possible stronger effect of family history of allergy on IgE levels at 

### 3
birth compared with blood Pb level. Sarasua et al. ( 2000 ) was unique among studies of children in 

### 4
examining associations of blood Pb leve l with IgA, IgG, and IgM. Associations were consistently positive 

### 5
(0.8 [95% CI: 0.2, 1.4], 4.8 [95% CI: 1.2, 8.4], and 1.0 [95% CI: 0.1, 1.9] mg/dL increase in IgA, IgG, 

### 6
and IgM, respectively, per 1 μ g/dL increase in blood Pb level, adjusted for age, sex, and location) and 

### 7
tended to be larger in magnitude in the youngest age group (6

- 35 months), suggesting an increased 

### 8
susceptibility of exposure at younger ages. In this study, associations with IgE were not examined. 

### 9
In studies of adults, one without ( Pizent et al., 2008 ) and one with occupational Pb exp osure ( Heo 

### 10
et al., 2004 ) , investigators reported higher IgE levels in association with higher blood Pb levels. In a study 

### 11
of urban adults in Zagreb, Croatia with blood Pb l evels between 0.56 and 7.4 μ g/dL, a statistically 

### 12
significant, positive association between blood Pb level and IgE was found in women but not men ( Pizent 

### 13
et al., 2008 ) . Several covariates were considered in a stepwise multiple regression, including age, 

### 14
smoking intensity, and alcohol consumption. Among women not on hormone replacement therapy or oral 

### 15
contraceptives, a 1 μ g/dL increase in blood Pb level was associated with a 0.60 increase in log IgE (95% 

### 16
CI: 0.58, 1.18). Investigators did not report an effect estimate in men because it did not attain stat istical 

### 17
significance. The study included 166 women and 50 men, thus, it was difficult to ascertain whether there 

### 18
was suggestion of association in men but lack of power to indicate statistical significance. Although blood 

### 19
Pb levels were lower in women (mean : 2.16 μ g/dL, range 0.56

- 7.35 μ g/dL) than in men (mean: 3.17 

### 20

μ g/dL, range 0.99

- 7.23), both groups had levels similar to those reported in studies of children. Among 

### 21
battery manufacturing workers (mostly males), Heo et al. ( 2004 ) found a monotonic increase in IgE levels 

### 22
among workers with blood Pb levels <0, 10

- 29, and ≥ 30 μ g/dL. 

### 23
A majority of the epidemiologic evidence for the effects of Pb on humoral immunity comprises 

### 24
comparisons of seru m IgA, IgG, and IgM levels between Pb

- exposed and

- unexposed workers ( Alomran 

### 25
& Shleamoon, 1988 ; Anetor & Adeniyi, 1998 ; Ewers et al., 1982 ; Kimber et al., 1986 ; Pinkerton et al., 

### 26

1998 ; Queiroz, Perlingeiro, et al., 1994 ; Sarasua et al., 2000 ; Undeger et al., 1996 ) . In contrast with 

### 27
toxicological findings, epidemiologic evidence reflects more mixed associations, with studies reporting 

### 28
higher, lower, and similar Ig levels in Pb

- exposed workers compared with

- unexposed controls. Among 

### 29
studies reporting lower Ig levels in Pb

- exposed workers were a few with high mean blood Pb levels 

### 30
among exposed workers (56.3 and 74.8 μ g /dL) ( Anetor & Adeniyi, 1998 ; Undeger et al., 1996 )

. 

### 31May 2011 5

- 256 DRAFT

– DO NOT CITE OR QUOTE 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Study Population Blood Pb Group

Karmaus et al. Children, ages 9 mo.

- 6yr. < 2.2 ( 2005 ) 2.2

- 2.83

2.84

- 3.4

> 3.4

Sun et al. ( 2003 ) Children, ages 3

- 6 yr. <9.9

≥ 9.9

Lutz et al. ( 1999 ) Children, ages 9 mo.

- 6 yr. <10

10

- 14

15

- 19

20

- 44

Heo et al ( 2004 ) Pb

- exposed workers <10

10

- 29

≥ 30

Relative IgE concentration (normalized to lowest blood Pb group)

Note: Bars represent the IgE normalized to the level measured in the lowest blood Pb group (depicted as black). Figure 5

- 49. Comparison of IgE levels among groups with increasing blood Pb level ( μ g/dL).

Study Population Blood Pb Group

Karmaus et al. ( 2005 ) Children, ages 7

- 10 yr. <2.2 2.2

- 2.83 2.84

- 3.4 >3.4

Sarasua et al. ( 2000 ) Children, ages 6

- 35 mo. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥15

Sarasua et al. ( 2000 ) Children, ages 36

- 71 mo. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥15

Sarasua et al. ( 2000 ) Children, ages 6

- 15 yr. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥15

Zhao et al. ( 2004 ) Children, ages 3

- 6 yr. <10 ≥10

Sarasua et al. ( 2000 ) Subjects, ages 16

- 75 yr. 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥15

Pinkerton et al. ( 1998 ) Controls <2 Pb workers 39

Fischbein et al. ( 1993 ) Controls NR Pb workers <25 Pb workers ≥25
Underger et al. ( 19 96 ) Controls 16.7 Pb workers 74.8

Relative B cell abundance ( normalized to lowest blood Pb group)

Note: Bars represent the abundance of B cells normalized to the level measured in the lowest blood Pb group (depicted as black or dark blue). Bars in black or gray represent results in subjects without occupational Pb exposures, and bars in dark or light blue represent results subjects with occupational Pb exposures. Figure 5

- 50. Comparison of the relative abundance of B cells among groups with incre asing blood Pb level ( μ g/dL).May 2011 5

- 257 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 24

. Additional characteristics and quantitative data a for results presented in Figures 5

- 49 and 5

- 50

Study Population Blood Pb group (μg/dL) IgE IgG IgM IgA B cells

Children

Karmaus et al. ( 2005 ) b 331 children, ages 7

- 10 yr Hesse, Germany <2.2 2.21

- 2.83 2.84

- 3.41 >3.41 46 30 59 59 c 1210 1214 1241 1201 150 143 153 148 123 121 133 136 418 353 389 393
Sun et al. ( 2003 ) Zeijiang Province, China 217 children, ages 3

- 6 yr. <9.9 ≥ 9.9
Lutz et al. ( 1999 ) 279 children, ages 9 mo

- 6 yr Springfield, MO <10 10

- 14 15

- 19 20

- 44 51.8 74.0 210.7 63.7 d 13.4 12.6 16.9 11.1
Sarasua et al. ( 2000 ) e 382 children, ages 6

- 30 mo Multiple U.S. locations 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 609 666 f 680 f 630 103 108 105 124 f 50.1 55.0 58.2 61.4 f 19.1 20 20.4 22.2
Sarasua et al. ( 2000 ) e 562 children, ages 36

- 71 mo Multiple U.S. locations 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 17.6 19.2 18.6
Sarasua et al. ( 2000 ) e 675 children ages 5

- 16 yr Multiple U.S. locations 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 1031 1094 f 1048 1221 128 131 136 106 140 143 140 108 16.1 15.8 15.3 20.1
Zhao et al. ( 2004 ) 75 children, ages 3

- 6 yr Zhejiang Province, China <10 ≥ 10 16.58 16.82

Adults without Occupational Pb Exposures

Sarasua et al. ( 2000 ) e 433 children and adults, ages 16

- 75 yr Multiple U.S. locations 0.6

- 4.9 5

- 9.9 10

- 14.9 ≥ 15 1099 1085 1231 1169 175 175 262 f 139 252 242 283 193 13.9 13.0 12.4 14.8

Adults with Occupational Pb Exposures

Heo et al. ( 2004 ) Korea 606 Pb battery plant workers <10 10

- 29 ≥ 30 112.5 223.3 535.8 g
Pinkerton et al. ( 1998 ) h 84 unexposed controls, mean age 30 yr 145 Pb smelter workers, mean age 33 yr <2

39 1090

1110 94.5

106.2 180

202 14.6

13.2

Fischbein et al. ( 1993 ) 36 unexposed controls,

mean age 47 yr 51 firearms instructors, mean age 48 yr NR <25 ≥ 15 8.6 10.5 11.2 g

Underger et al. ( 1996 ) 25 unexposed controls, ages 22

- 56 yr 25 Pb battery plant workers, ages 22

- 55 yr 16.7

74.8 1202.1

854.6 g 140.4

93.3 g 210.3

168.1 635.9 i

545.5

a IgE data are presented as IU/m L. Other Ig data are presented as mg/dL. B cell data are presented as the percentage of B cells among all lymphocytes unless otherwise specified.
b All means are adjusted for age, sex, ETS, number of infections in the previous 12 months, serum lipid concentration, and orga nochl orine exposures. B cell data represent the number of c ells/μL serum.

c p<0.05 for difference among groups.
d p<0.05 for differences among groups adjusted for age.

e Means are adjusted for age, sex, and location of study.
f p≤0.05 in comparison with mean in lo west blood Pb group.
g p<0.05 for differences among groups.
h Means adjusted for age, race, current smoking status, and workshift.
i Data represent the number of cells/μL serum.May 2011 5

- 258 DRAFT

– DO NOT CITE OR QUOTE 

## 5.6.4. Immune

- based Diseases

5.6.4.1. Host Resistance

The capacity of Pb to reduce host resistance to bacteria has been known for almost 40 years and 

### 1
was supported by several toxicological studies described in the 2006 Pb AQCD ( U.S. EPA, 2006 )

. 

### 2
Biological plausibility has been provided by observations of Pb affecting mechanisms underlying 

### 3

dimini shed host resistance, e.g., decreased capacity for Th1

- driven acquired immune antiviral responses 

### 4
(indicated by the overproduction of PGE 2 ) and increased inflammatory responses in target tissue resulting 

### 5
in further damage to host protective barriers. Gupta et al. ( 2002 ) demonstrated that elevated Pb exposure 

### 6
of mice (>125 mg/kg Pb, 13.0 μg/dL blood Pb) reduce d host resistan ce to vira l infections as indicated by 

### 7
an increased viral titre and increased mortality. Host resistance to bacteria such as Listeria requires 

### 8

effecti ve Th1

- driven responses including the production of IL

- 12 and IFN

- γ ( Lara

- Tejero & Pamer, 2004 )

, 

### 9
but these are suppressed by Pb ( D. Gao et al ., 2007 ) . The lack of IFN

- γ can inhibit appropriate and timely 

### 10
macrophage activation. However, beyond this, suppression of NO production and along with it, the 

### 11
microbicidal metabolite peroxynitrite, can compromise host resistance to some bacteria ( U.S. EPA, 2006 )

. 

### 12
Recently, mechanisms thro ugh which Pb impacts both innate immune cells and natural host defense 

### 13

barriers to increase the likelihood of serious bacterial infections have been delineated further. Kasten

- Jolly 

### 14

et al. ( 2010 ) sh owed that developmental exposure of mice to Pb resulted in an upregulation of splenic 

### 15
RNA of caspase

- 12, a cysteine protease that inhibits the clearance of bacteria both systemically and in the 

### 16

gut mucosa ( Saleh et al., 2006 )

. 

### 17
With limited investigation, the effect of Pb on host resistance to parasitic agents is uncertain. The 

### 18

2006 Pb ACQD described one study in which Pb

- exposed (30

- 100 mM) mouse macrophages had 

### 19
diminished ability to kill Leishmania enrietti parasites ( Mauel et al., 1989 ) ; however, given the well



### 20
established effect of Pb in promoting Th2 activity, it is plausible that Pb could enhance host resistance to 

### 21
parasites that require robust Th2 responses (e.g., helmi n th s) ( U.S. EPA, 2006 )

. In a recent study , Pb 

### 22

enhanced host resistance to m alaria ( Koka et al., 2007 )

. However

, this was related to the capacity of Pb to 

### 23
induce eryptosis and the rapid remova l of malaria

- infected erythrocytes and not to Pb

- induce d alterations 

### 24

in immune function. 

### 25
The collective body of epidemiologic data is sparse; however, several studies have found an 

### 26
association between blood Pb levels and respiratory infections. In a Boston

- area study of 1978 children 

### 27
(ages 4

- 8 years), Rabinowi tz et al. ( 1990 ) reported that compared with children with b lood Pb levels <10 

### 28

μ g/dL, children with blood Pb levels ≥ 10 μ g/dL were more likely to have parental report “other 

### 29
respiratory infections” (not defined by authors) (OR: 1.5 [95% CI: 1.0, 2.3]), severe ear infections (OR: 

### 30
1.2 [95% CI: 1.0, 1.4]), and illnes ses other than cold or influenza (OR: 1.3 [95% CI: 1.0, 1.5]) (Figure 5

- 

### 31

51 and Table 5

- 25). Analyses did not adjust for any potential confounders. Likewise, without considering 

### 32May 2011 5

- 259 DRAFT

– DO NOT CITE OR QUOTE confounders, Karmaus et al. ( 2005 ) found that children with more than 10 infections in the previous 12 

### 1
months had higher blood Pb levels (mean: 3.3 μ g/dL) compared with children with 1 to <5 infections 

### 2
(mean: 2.8 μ g/dL) or 5

- 10 infections (mean: 2.6 μ g/dL) in the same time period. A unique study was 

### 3
conducted in children in Cordoba, Argentina, in which Pb exposure was assessed by measuring Pb in t otal 

### 4
deposition samples and in lichen biomonitors from sites near 4 city health clinics ( Carreras et al., 2009 )

. 

### 5
Lichen has been recognized for the uptake, accumulation, a nd sequestration of environmental chemicals 

### 6
from the air. Pb content in lichen and colocated total deposition samples were highly correlated, 

### 7
indicating that the lichen was a suitable indicator of environmental concentrations. In an ecologic analysis 

### 8

that did not consider potential confounders, clinics near sites with higher levels of Pb in total deposition 

### 9
and lichen samples had higher frequency of visits by children for pharyngitis, tonsilitis, and laryngitis. 

### 10
Because other metals, including manganese, ir on, copper, and nickel were also associated with respiratory 

### 11
illnesses, it was difficult to characterize the independent effects of Pb. Similar to studies in children, 

### 12
among adults, frequency of self

- reported colds or influenza was greater among Pb battery or smelter plant 

### 13
workers (28.8%) than among unexposed controls (16.1%); however, a statistical analysis was not 

### 14
performed on the data ( Ewers et al., 1982 ) . Thus, while several studies in humans indicate associations 

### 15

between indicators of Pb exposure and infectious illnesses, they are limited by weak analytic methods and 

### 16
lack of consideration for potential confounders. Evaluation of the relationship between Pb exposure and 

### 17
host resistance in humans would be improved by more longitudinal in vestigation and rather than group 

### 18
comparisons, analyses of associations between blood Pb levels and viral or bacterial infections. 

### 19

5.6.4.2. Asthma and A llergy

Toxicological studies and to varying degrees, epidemiologic studies, have demonstrated Pb effects 

### 20

on multi ple immunological pathways, including elevated production of Th2 cytokines such as IL

- 4, 

### 21
increased IgE antibody production (Figure 5

- 49), and Pb

- induced inflammatory cell dysfunction. These 

### 22

are well

- recognized pathways in the development of allergy and all ergic disease, including asthma. It has 

### 23
been suggested that low exposure to Pb exerts immunostimulating effects in contrast to higher exposure, 

### 24
which has been implicated in suppressing immune function ( Mishra et al., 2006 ) . Coherent with the 

### 25
mechanistic evidence, several epidemiologic studies indicate associations between blood Pb levels and 

### 26
asthma and allergy ( Figure 5

- 51 and Table 5

- 25). In univariate analyses of children near B oston, MA, 

### 27

Rabinowitz et al. ( 1990 ) reported a positive asso ciation between high blood Pb level (>10 μ g/dL) and 

### 28
asthma (RR: 1.3 [95% CI: 0.8, 2.0]) but not eczema (RR: 1.0 [95% CI: 0.6, 1.6]). 

### 29 30May 2011 5

- 260 DRAFT

– DO NOT CITE OR QUOTE Reference Population Outcome Blood Pb Level Mean or Group

Rabinowitz et al. ( 1990 ) Children Other respiratory infection ≥10 vs. <10

Severe ear infection

Rabinowitz et al. ( 1990 ) Children Eczema ≥10 vs. <10

Jedrychowski et al. ( 2011 ) Children Positive SPT Prenatal: 1.16

Concurrent: 2.02

Pizent et al. ( 2008 ) Adult males Positive SPT 21.6

Rabinowitz et al. ( 1990 ) Children Prevalent asthma ≥10 vs. <10

Joseph et al. ( 2005 ) Children, Caucasian Incident asthma requiring ≥5 vs. Caucasian<5
Children, African American medical care ≥5 vs. African American<5
Children, African American ≥10 vs. African American<5
Children, African American <5 vs. Caucasian<5
Children, African American ≥5 vs. Caucasian<5
Children, African American ≥10 vs. Caucasian<5

Min et al. ( 2004 ) Adults BR 2.9

Odds ratio (95% CI)

Note: Odds ratios are are standardized to a 1 μg/dL increase in blood Pb level or 1 unit in log of blood Pb. SPT = skin prick test, BR = bronchial responsiveness.
Figure 5

- 51

. Associations of blood Pb levels with immune

- based conditions.May 2011 5

- 261 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 25. Additional characteristics and quantitative data for results presented in Figure 5

- 51.

Reference Population B lood Pb level mean or group (μg/dL) Outcome Odds ratio (95% CI) a

Rabinowitz et al. ( 1990 ) Boston area, MA 1768 children ≥ 10 vs. <10 b Other respiratory infection Severe ear infection 1.5 (1.0, 2.3) 1.2 (1.0, 1.4)
Rabinowitz et al. ( 1990 ) Boston area, MA 1768 children. ≥ 10 vs. <10 b Eczema 1.0 (0.6, 1.6)

Jedrychowski et al. ( 2011 ) Krakow, Poland 224 children followed prenatally to age 5 yr Prenatal: 1.16 Concurrent: 2.02 Positive SPT 2.3 (1.1, 4.6) 1.1 (0.7, 1.6)

Pizent et al. ( 2008 ) Zagreb, Croatia 50 males, ages 21

- 67 yr 21.6 Positive SPT 0.92 (0.86, 0.98)
Rabinowitz et al. ( 1990 ) Boston area, MA 1768 children. ≥ 10 vs. <10 b Prevalent asthma 1.3 (0.8, 2.0)

Joseph et al. ( 2005 ) Southeastern MI 4634 children followed prospectively from age 1

- 3 yr Caucasian ≥5 vs. Caucasian <5 c African American ≥5 vs. African American <5 d African American ≥10 vs. African American <5 d African American < 5 vs. Caucasian <5 c African American ≥5 vs. Caucasian <5 c African American ≥10 vs. Caucasian <5 c Incident asthma requiring medical care 2.7 (0.9, 8.1) 1.1 (0.8, 1.7) 1.3 (0.6, 2.6) 1.8 (1.3, 2.4) 1.5 (1.2, 1.8) 3.0 (1.2, 7.1)

Zhao et al. ( 2004 ) 75 children, ages 3

- 6 yr Zhejiang Province, China <10 ≥ 10 BR 1.02 (1.00, 1.03)

SPT = skin prick test, BR = bronchial responsiveness.

a Odds ratios are standardized to a 1 μg/dL increase in blo od Pb level, except in studies in which Blood Pb is analyzed as a categorical variable.
b Odds ratio in children with blood Pb level ≥ 10 μg/dL with children with blood Pb level < 10 μg/dL as the reference group

. No additional covariates included in model.

c Relative risk in each specified subgroup with Caucasian children with blood Pb level <5 μg/dL as the reference group

. Model covariates include sex, birth weight, and annual income.
d Relative risk in each specified subgroup with African American children wi th blood Pb level <5 μg/dL as the reference group

. Model covariates include sex, birth weight, and annual income.

Positive associations between blood Pb level and asthma

- and allergy

- related conditions also were 

### 1
observed in large studies with multivariate analyses. An additional common strength of these studies was 

### 2
the prospective follow

- up of subjects that allowed in vestigators to establish temporality between the 

### 3
measurement of blood Pb level and onset of disease. In a study of 4,634 children (ages 1

- 3 years) in 

### 4
southeastern Michigan that controlled for annual income, birth weight, and sex, an elevated risk of 

### 5

incide nt asthma requiring a doctor visit or medication (indicator of severe asthma) was reported in 

### 6
association with blood Pb levels ≥ 5 μ g/dL most strongly among Caucasian children (relative risk [RR]: 

### 7
2.7 [95% CI: 0.9, 8.1]) ( Joseph et al., 2005 ) ( Figure 5

- 51 and Table 5

- 25). In analyses restricted to African 

### 8
American children, blood Pb level was weakly associated with asthma requiring medical care ( Figure 5

- 

### 9

51 and Table 5

- 25). In analyses that used Caucasian children with blood Pb level <5 μ g/dL as the 

### 10
reference group, blood Pb level was associated with statistically significant incre ases in the risk of asthma 

### 11
among African children in all blood Pb level categories, which indicated a racial/ethnic effect rather than 

### 12 a Pb effect. However, among African American children, the RR was much higher in the ≥ 10 μ g/dL 

### 13
blood Pb category (RR: 3. 0 [95% CI: 1.2, 7.1]) than in the ≥ 5 μ g/dL (RR: 1.5 [95% CI: 1.2, 1.8]) or <5 

### 14May 2011 5

- 262 DRAFT

– DO NOT CITE OR QUOTE μ g/dL blood Pb categories (RR: 1.8 [95% CI: 1.3, 2.4]), which pointed to a possible race/ethnicity by 

### 1
blood Pb level interaction. These findings should be interpreted with cauti on due to the small number of 

### 2
asthmatics requiring medical care in the high blood Pb level categories (5 Caucasian children with blood 

### 3

Pb ≥ 5 μ g/dL and 9 African American children with blood Pb level ≥ 10 μ g/dL). 

### 4

While the aforementioned studies examined c oncurrent blood Pb levels, findings from a recent 

### 5
prospective birth cohort study demonstrated that prenatal blood Pb levels (cord blood Pb level mean: 1.16 

### 6

μ g/dL [95% CI: 0.12, 1.22]) was associated with an increased risk of allergic sensitization at age 5 years 

### 7
( Jedrycho wski et al., 2011 ) . In models that adjusted for sex, parity, maternal age, maternal education, 

### 8
maternal atopy, and environmental tobacco smoke exposure, measures of prenatal Pb exposure (cord or 

### 9
maternal blood Pb) were associated with greater risk of po sitive skin prick test (SPT) to dust mite, dog, or 

### 10
cat allergen compared with concurrent blood Pb levels ( Figure 5

- 51 and Table 5

- 25). The greater risk 

### 11
associated with prenatal blood Pb measures was substantiated by the weak correlation observed between 

### 12

um bilical cord and age 5

- year blood Pb levels (r = 0.29). Larger risks were estimated for Pb than for other 

### 13
risk factors, including blood levels of mercury, polycyclic aromatic hydrocarbon DNA adducts, and 

### 14
residential levels of dust mite or pet allergen. 

### 15

Int erestingly, in a study of 216 adults without occupational Pb exposures, Pizent et al. ( 2008 ) found 

### 16
that among women, the association between blood Pb level and total IgE was statistically significant, 

### 17
whereas the association with positive SPT to common inhaled allergens was not. An increase in IgE 

### 18

mediates the acute inflamma tory response to allergens. Among men, not only was the opposite observed, 

### 19
but the association between blood Pb level and positive SPT was negative (OR: 0.92 [95% CI: 0.86, 0.98] 

### 20
in stepwise regression models that considered age, smoking intensity, and alc ohol consumption). 

### 21
Interpretation of the findings is difficult because only statistically significant effect estimates were 

### 22
reported; thus it is not possible to ascertain whether there were suggestions of association in women or 

### 23
whether there were discrepa nt findings for the related outcomes of IgE and positive SPT. 

### 24

5.6.4.3. Other Respiratory Effects

Increased bronchial responsiveness (BR) is a characteristic feature of asthma and other respiratory 

### 25
diseases and can result from activation of innate immune responses a nd increased airway inflammation. 

### 26
Compared with findings for IgE or asthma, evidence of association between blood Pb levels and BR is 

### 27

weak ( J. Y. Min et al., 2008 ; Pizent et al., 2008 ) . In a study of 525 middle

- aged adults in Seoul, Korea, 

### 28

Min et al. ( 2008 ) found an association between blood Pb levels and BR. Study subjects had a mean (SD) 

### 29

blood Pb level of 2.90 (1.59) μ g/dL. A 1 μ g/dL increase in blood Pb level was associated with an increase 

### 30
in BR index (log [% decline in forced expiratory volume in 1 second (FEV 1 ) /log of final methacholine 

### 31
concentration in mg/dL]) of 0.018 (95% CI: 0.004, 0.03), adjusting for age, sex , height, smoking

, lung 

### 32
function, and asthma diagnosis ( J. Y. Min et al., 2008 )

. In contrast to Min et al. ( 2008 )

, Pizent et al. ( 2008 ) 

### 33May 2011 5

- 263 DRAFT

– DO NOT CITE OR QUOTE observed a neg ative association between blood Pb level and nonspecific BR in men (2.4% decrease [95% 

### 1

CI:

- 4.2,

- 0.52%] in percent change FEV 1 post

- histamine challenge per 1 μ g/dL increase in blood Pb 

### 2
level). Although this counterintuitive finding was not discussed by in vestigators, it was consistent with 

### 3
the negative association found between blood Pb level and SPT among men in this study. 

### 4
In less rigorous analyses that compared occupational groups with relatively low Pb exposures, 

### 5

associations of blood Pb levels with lu ng function were less clearly indicated ( A. Y. M. Jones et al., 2008 ; 

### 6
A. Y. M. Jones et al., 2006 ) . In a comparison of male drivers of buses with (mean blood Pb level: 5.0 

### 7

μ g/dL) and without (mean blood Pb level: 3.7 μ g/dL) air conditioning in Hong Kong, China, dr ivers of 

### 8

non

- air conditioned buses had lower exposures to PM 10 , lower blood Pb levels but lower forced vital 

### 9
capacity and similar FEV 1 ( A. Y. M. Jones et al., 2006 ) . In this study, the authors attributed the slightly 

### 10
higher blood Pb levels among air conditioned bus drivers to the poor efficiency in the filters and higher 

### 11

PM 10 levels measure d on those buses versus the non

- air conditioned buses. In a comparison of roadside 

### 12
vendors and adjacent shopkeepers, blood Pb levels and various lung function parameters were similar 

### 13
between groups ( A. Y. M. Jones et al., 2008 ) . Neither study directly examined associations between blood 

### 14
Pb levels and lung function. 

### 15
In addition to blood Pb, several recent epidemiologic studies have used Pb measured in PM 10 and 

### 16

PM 2.5 air samples to represent Pb exposures. Some studies have analyzed the Pb component individually, 

### 17
whereas others have applied source apportionment techniques to analyze Pb as part of a group of 

### 18
correlated components. A majority of air

- Pb studies has found associations with asthma

- related morbidity 

### 19
in children and respiratory

- related hospitalizations and mortality in older adults (Table 5

- 26). Despite the 

### 20
concordance between the findings of air

- Pb and blood Pb studies, a common l imitation of air

- Pb studies is 

### 21

the variable size distribution of Pb

- bearing PM (Section 3.5.3) and its relationship with blood Pb levels. 

### 22
Additionally, in these air

- Pb studies, other PM components such as elemental carbon, copper, and zinc 

### 23

also were associ ated with respiratory effects. In the absence of detailed data on correlations among 

### 24
components or results adjusted for copollutants, it is difficult to exclude confounding by these other 

### 25

components. 

### 26May 2011 5

- 264 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 26. Associations of air

- Pb with respiratory eff ects

Study Population Air

- Pb Data Statistical analysis Outcome Effect Estimate (95% CI) a
Gent et al. ( 2009 ) CT, MA 149 asthmatic children, ages 4

- 12 yr 1

- yr follow

- up 2000

- 2003 2

- day avg Pb

- PM 2.5 Mean: 5.1 ng/m 3 Generalized estimating equations with one

- day lag autoregressive structure, adjusted for season, day of week, and date Wheeze Shortness of breath Fast

- acting inhaler

use OR per 5 ng/m 3 increase b 1.07 (p = 0.13) 1.12 (p = 0.01) 1.04 (p = 0.10) Hong et al. ( 2007 ) Dukjeok Island, Korea 43 children, grades 3

- 6 6

- wk follow up March

May 2004 Lag 1 Pb

- PM 10 Mean (SD): 51 ng/m 3 (31) Linear mixed effects models with random effect for subject adjusted for age, sex, height, weight, asthma history, passive smoking exposure at home, GST genotype, temperature, relative humidity, air pressure, day of week Morning PEF Daily average PEF Change per 1 log increase b

- 6.83 L/min (p <0.01)

- 6.37 L/min (p <0.01)

Ostro et al. ( 2007 ) 6 California counties Adults, ages ≥ 65 yr 2000

- 2003 Lag 3 Pb

- PM 2.5 Mean (IQR): 4 (4) ng/m 3 County

- specific poisson regression adjusted for day of week, smoothing splines for temperture and humidity (3 df), smoothing spline for time (4 df). County

- specific estimates combined using random effects model Respiratory mortality All

- year Summer

- only RR per 4 ng/m 3 increase 1.011 (0.99, 1.033) Statistically significant, quantitative results not reported

Bell et al. ( 2009 ) 106 U.S. coun ties Adults, ages ≥ 65 yr 1999

- 2005 Lag 0 Pb

- PM 2.5 Mean (SD): 4.89 ng/m 3 (31) Bayesian hierarchical regression to combine county

- specific estimates adjusted for day of week, seasonality, smooth function of time, daily temperature, previous 3

- day’s temperature and dew point temperature Respiratory hospital admissions Results were reported in a figure. RR per IQR increase was negative with wide 95% CI

Andersen et al. ( 2007 ) Copenhagen, Denmark Adults, ages ≥ 65 yr Children, ages 5

- 18 yr 1999

- 2004 Lag 3 “vehicle” PM 10 factor c comprising Pb, copper, iron, and other trace metals Generalized additive Poisson regression adjusted for smoothed splines for temperature and dew point temperature (4

- 5 df), smoothed spline for calendar time (3

- 5 df), influenza, day of week, public holidays, school holidays, and pollen (only for asthma mode ls) Respiratory hospital admissions (adults ages ≥ 65 yr) Asthma (children ages 5

- 18 yr) RR per 0.6 μ g/m 3 increase 0.95 (0.89, 1.02)

1.20 (0.98, 1.47)

Sarnat et al. ( 2008 ) 20 Atlanta, GA

- area counties 1998

- 2002 All ages “Woodsmoke” PM 2.5 factor c comprising Pb (minor contribution), potassium, organic carbon, ammonium Mean: 1.6 mg/m 3 (cool season), 0.8 mg/m 3 (warm season) Generalized linear Poisson regression adjusted for day of week, holidays, hospital, cubic splines for time (monthly knots), temperature, and mean dew point temperature (knots at 25 th and 75 th percentiles) Respiratory hospital admissions Increment NR 0.999 (0.993, 1.004)

OR = odds ratio, PEF = peak expiratory flow, RR = relative risk, NR = not reported

. a Effect estimates are reported as given in the study and are not standardized because of variability in exposure metrics among studie s

. b Investigators did not provide sufficient information to calculate 95% CIs

. c Souce apportionment techniques were applied to grou p correlated components into common source categories.



### 5.6.4.4. Autoimmunity

The 2006 AQCD described animal studies in which Pb exposure induced the generation of 

### 1

autoantibodies ( Bunn et al., 2000 ; El

- Fawal et al., 1999 ; Hudson et al., 2003 ; Waterman et al., 1994 )

. 

### 2
Whereas some evidence linked this risk of autoimmunity to a shift towards Th2 responses, other evidence 

### 3
pointed to a shift towards Th1 responses. While recent studies did not examine Pb

- induced production of 

### 4
autoantibodies, som e provided indirect evidence by indicating that the changes induced by Pb had broader 

### 5
implications for increasing risk of autoimmunity. For example, Kasten

- Jolly et al. ( 2010 ) examined the 

### 6May 2011 5

- 265 DRAFT

– DO NOT CITE OR QUOTE impact of developmental Pb exposure of mice on changes in gene expression in the spleen. They found 

### 1
that Pb upregulated digestive and catabolizing enzymes that could lead to the generation of self

- peptides, 

### 2
which in conjunction with other Pb

- induced immunomodulator y effects, had the potential to induce the 

### 3
generation of autoantibodies. In Carey et al. ( 2006 ) , the activation of neoantigen

- specific T cells in PbCl 2 - 

### 4
exposed mice also indicated the potential for autoantibody generation. Evidence of Pb

- associated 

### 5

au toimmune responses in humans is limited to a study of male Pb battery workers with blood Pb levels 

### 6
ranging from 10 to 40 μ g/dL ( El

- Fawal et al., 1999 ) . In this study, the Pb

- exposed workers h ad higher 

### 7
levels of IgM and IgG autoantibodies to neural proteins compared with unexposed controls (blood Pb 

### 8
levels not reported) ( El

- Fawal et al., 1999 )

. 

### 9

5.6.4.5. Specialized Cells in Other Tissues

R esident macrophages in tissues represent a significant target for Pb

- induced immun e effects, and 

### 10

a lteration in the function of these cells can contribute to organ/tissue dysfunction, cell death, tissue 

### 11
pathology and tissue

- specific autoimmune reactions . Am ong the specialized populations are microglia and 

### 12
astrocytes in the brain, Kupffer cells in the liver, alveolar macrophages in the lung, keratinocytes and 

### 13
Langerhans cells in the skin, osteoclasts in the bone, and preadipocytes in adipose tissue. The effec ts of 

### 14
Pb on these cells are important as they may contribute to disease in nonlymphoid tissues ( Figure 5

- 52). 

### 15
Because these specialized cells are not always recognized as macrophages, the resulting diseases and 

### 16
conditions are not always recognized as being linked with Pb

- induced immune dysfunction. 

### 17May 2011 5

- 266 DRAFT

– DO NOT CITE OR QUOTE Figure 5

- 52

. Specialized macrophages in nonlymphoid tissue may serve as a significant link between Pb and disease in multiple organ systems

.

Fan et al. ( 2009 ) reported that Kupffer cells undergo significant changes in phenotypic expression 

### 1
(e.g., CD68 and ferritin light chain), organization, and functional activity connected to Pb

- induced 

### 2
apoptosis in the liver. In the central nervous system, subacute exposure of Wistar rats to Pb (15 mg/kg of 

### 3
Pb acetate, i.p.) during early postnatal maturation was observed to produce chronic glial activation with 

### 4
coexisting features of inflammation and neurodegeneration ( Struzynska et al., 2007 ) . Among the 

### 5
cytokines detected in the brains of these Pb

- treated rats were IL

- 1β, TNF

- α and IL

- 6. In bone, resident 

### 6
tissue macrophages regulate osteoblast function ( M. K. Chang et al., 2008 ) , and osteoblasts are known 

### 7
targets of Pb. This can contribute to later life diseases such as arthritis [reviewed in Zoeger et al. ( 2006 ) ]. 

### 8

Pb

- induced elevation of TGF

- β production is also involved in chondrogenesis in bone. 

### 9
Resident immune cells in reproductive organs are also potentia l targets of Pb. Pace et al. ( 2005 ) 

### 10
reported that Pb exposure in mice contributed to poor reproductive performance that was concomitant 

### 11
with altered homeostasis of the testicular macrophage population in that organ. The authors proposed that 

### 12
increased oxidative damage and apoptosis among these macrophages and reduced potential to maintain 

### 13
organ homeostasis contributed to the observed pattern of male sterility. 

### 14 

# 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 macrophagesMay 2011 5

- 267 DRAFT

– DO NOT CITE OR QUOTE 

### 5.6.4.6. Tumors

While toxicological evidence supports high doses of Pb directly promoting tumor formation or 

### 1
inducing mutagenesis and genotoxicity ( Section 5. 10), evidence for involvement of the immune system is 

### 2

limited. Kerkvliet and Baecher

- Steppan ( 1982 ) observe d that male C57Bl/6 mice exposed to 130 and 

### 3
1300 ppm of Pb acetate in drinking water had enhanced moloney sarcoma virus

- induced tumor growth 

### 4
compared with control animals. These findings indicated that Pb

- induced immuno modulation affecting 

### 5
tumors likely re sults from a combination of suppressed Th1 responses and increased inflammation 

### 6
leading to excessive release of ROS into tissues. The promotion of cancer is a relatively common 

### 7

outcome in chemical

- induced immuntoxicology, particularly when early life expos ures are involved 

### 8
( Dietert )

. 

### 9



## 5.6.5. Mechanisms o f Lead

- Induced Immunomodulation

5.6.5.1. Inflammation

Misregulated inflammation represents one of the major immune

- related effects of Pb and a major 

### 10
mode of action for Pb effects in multiple organ systems (Section 5.2.5). It is important to note that 

### 11
inflammation can manifest in any tissue where immune cell m obilization and tissue insult occurs (as with 

### 12
an infection). Enhanced inflammation and tissue damage occurs through the modulation of inflammat ory 

### 13
cell function and production of proinflammatory cytokines and metabolites . Among the problems 

### 14
presented by th is immunomodulation are the overproduction of ROS and an apparent depletion of 

### 15
antioxidant protective enzymes and factors (e.g., selenium). Chetty et al. ( 2005 ) reported that Pb

- induced 

### 16
inflammatory damage involves the depletion of glutat hione. While several processes have been proposed 

### 17
to explain the mechanisms of Pb

- induced oxidative damage, the exact combination of processes involved 

### 18
remains to be determined (Section 5.2.4). 

### 19

Traditional immune

- mediated inflammation can be seen with ast hma, respiratory infections, and 

### 20
BR in association with Pb exposure. Nonetheless, inflammation also is a general response to tissue injury 

### 21
whether mediated by infection, toxic insult, or other stresses. Thus, Pb

- induced misregulation of 

### 22

inflammation could exacerbate disease and damage in almost any organ given the distribution of immune 

### 23
cells as both permanent residents and infiltrating cell populations. As described in Section 5.2.5, 

### 24
misregulation of inflammation represents a potential mode of action for P b induced effects on the liver, 

### 25
kidney, and vasculature. 

### 26
In epidemiologic studies, whereas Pb

- associated changes in proinflammatory cell function ( Section 

### 27

5.6.2 ) and cytokine production ( Section 5.6.5.4 ) have been demonstrated, less certain are the effect s of Pb 

### 28
exposure on nonspecific indicators of inflammation that may be related to multisystemic effects as have 

### 29
been demonstrated in toxicological and in vitro studies. Using 1999

- 2004 NHANES data for adults 40 

### 30May 2011 5

- 268 DRAFT

– DO NOT CITE OR QUOTE years of age or old er , Songdej et al. ( 2010 ) examined the relationship between blood Pb levels (mean: 

### 1
1.89 μg/dL) and the inflammation markers

, C

- reactive protein (CRP), fibrinogen, and white blood cell 

### 2
(WBC) count

. Adjusting for age, sex, race/ethnicit y, education, income, body mass index (BMI), physical 

### 3
activity, smoking status, diabetes status, inflammatory disease status, and cardiovascular disease status, 

### 4

t he researchers found that men appeared to be more susceptible than women to Pb

- associated 

### 5

infl ammation. Among women, most odds ratios for associations between quintiles of blood Pb level and 

### 6
tertiles of CRP, fibrinogen, and WBC count were less than 1.0 whereas corresponding odds ratios in men 

### 7
tended to be greater than 1.0. Additionally, compared wi th men with blood Pb levels less than 1.16 μg/dL, 

### 8

men with blood Pb levels of 1.16

- 1.63 μg/dL, 2.17

- 3.09 μg/dL, and ≥ 3.09 μg/dL had statistically 

### 9
significant increases in CRP (ORs: 2.22, 2.12, 2.85, respectively). For all inflammation markers, although 

### 10

th e highest quintile of blood Pb level was associated with the largest odds ratio, a blood Pb dose



### 11
dependent association was not observed

. Consistent with these findings, among men in Incheon, Korea 

### 12
without occupational Pb exposures, Kim et al. ( 2007 ) reported positive associations of blood Pb level 

### 13

with WBC as well as with IL

- 6. These findings of associations between blood Pb levels and inflammatory 

### 14

mediat ors are consistent with Pb effects on promoting a Th2 phenotype. Th2 cells produce IL

- 6 which is 

### 15
the primary stimulus for expression of CRP and fibrinogen ( Fuller & Zhang, 2001 ; Hage & Szalai, 2007 )

. 

### 16
In a genome

- wide association study that included 37 autistic and 15 nonautistic children (ages 2

- 5 

### 17
years; blood Pb level r ange: 0.37 to 5.2 μg/dL) in California, in models that included age, sex, and autism 

### 18
diagnosis, blood Pb level was associated with the expression of several genes related to immune function 

### 19
and inflammation, including human leukocyte antigen genes (HLA

- DRB ) and MHC Class II

- associated 

### 20
invariant chain CD74 (involved in antigen presentation) ( Y. Tian et al., 2011 ) . Although blood Pb levels 

### 21

were similar between autistic and nonautistic children and correlations were observ ed in both groups, they 

### 22
were in opposite directions (positive among autistic and negative among nonautistic). These results are 

### 23
consistent with findings that Pb increases MHC molecule surface expression in mouse and human HLA 

### 24
antigen presenting cells ( Guo et al., 1996a ; M. J. McCabe & Lawrence, 1991 ) and also suggest that Pb



### 25

associated ch anges in the expression of immune genes may be modified by underlying autism. 

### 26

5.6.5.2. Increased Prostaglandin E 2 

### and Decreased Nitric Oxide

Consistent with the findings presented in the 2006 Pb AQCD ( U.S. EPA, 2006 )

, recent studies 

### 27

continue to show that Pb exposure alters the levels of signaling mol ecules. These signaling molecules are 

### 28
involved in mediating inflammation and host resistance ( Figure 5

- 47). For example, Pb exposure 

### 29

increases arachidonic acid metab olis m, elevating the production of prostaglandin E 2 ( PG E 2 ) ( Chetty et al., 

### 30

2005 )

. Additionally, production of nitric oxide (NO) by macrophages is decreased at low

- moderate 

### 31
exposure levels ( Farrer et al., 2008 ; Pineda

- Zavaleta et al., 2004 ) . Decreases in NO can impact not only 

### 32
innate host defenses, but also, acquired immunity. This is proposed to occur via the Pb

- induced release of 

### 33May 2011 5

- 269 DRAFT

– DO NOT CITE OR QUOTE myeloid cell (CD11b+)

- mediated suppression of CD4+ T cell prolife ration ( Farrer et al., 2008 ) . The result 

### 1
is that Pb exposure leads to an increased production of alloreactive T and B cells. Farrer et al. ( 2008 ) 

### 2
found that Pb decreases inducible nitric oxide synthease function in myeloid cells without decreasing its 

### 3
abundance. The resulting loss of NO production also leads to a reduction in the production of the 

### 4
microbicidal metabolite peroxynitrite, thus weakeni ng host defenses against bacteria. When this is 

### 5
combined with the observation that Pb can alter antigen processing ( Farrer et al., 2005 ) and, hence, the 

### 6
quality and magnitude of the acquired immune response signal against pathogenic challenge, multiple 

### 7
arms of the host defense against infectious challenge can be compromised. The loss of NO production i n 

### 8
innate immune cells such as macrophages would be expected to affect other physiological systems (e.g., 

### 9
neurological, cardiovascular, endocrine) that require NO signaling cascades. 

### 10
Relative to studies in animal and in vitro models, fewer epidemiologic st udies have examined the 

### 11
effects of Pb on signaling molecules; however the limited data support associations of blood Pb level with 

### 12
suppressed NO production ( Barbosa et al., 2006 ; Mishra et al., 2006 ; Pineda

- Zavaleta et al., 2004 ; 

### 13
Valentino et al., 2007 ) and increased ROS production ( Pineda

- Zavaleta et al., 2004 )

. Among children, i n 

### 14

Pineda

- Zavaleta et al. ( 2004 ) , with increasing residential proximity to the Pb smelter, mean blood Pb 

### 15
levels increased (7.02 to 20.6 to 30.38 μ g/dL) as did superoxide anion release from macrophages (directly 

### 16
activated by IFN

- γ/LPS) isolated from children. NO release from macrophages ( indirectly activated by 

### 17
phytohemagglutinin, PHA) decreased with increasing blood Pb level. After adjusting for age and sex, a 1 

### 18

μ g/dL increase in blood Pb level was associated with a decrease in NO of 0.00089 (95% CI:

- 0.0017,

- 

### 19
0.00005) nmol/ μ g protein and an increase in superoxide anion of 0.00389 (95% CI: 0.00031, 0.00748) 

### 20

μ mol/mg protein. Because PHA activates macrophages indirectly through the activation of lymphocytes 

### 21

and IFN

- γ directly activates macrophages, these results suggest that Pb suppressed T cell

- mediated 

### 22
macrophage activation and stimulated cytokine

- induced macrophage activation. Results also 

### 23
demonstrated a larger magnitude of association between blood Pb levels and superoxide anion release in 

### 24
males. Although not described in detail, the asso ciation between blood Pb level and NO was reported to 

### 25
be not negative in girls. Barbosa et al. ( 2006 ) also observed negative associations between blood Pb level 

### 26
and plasma NO (quantitati ve results not provided) in a group of adults in Sao Paolo, Brazil residing near a 

### 27
battery plant, although it was not possible to identify immune cells as the specific sources of NO. 

### 28
In contrast to studies of populations without occupational Pb exposures, studies of occupationally



### 29
exposed groups provided less clear indication of association of blood Pb level with NO and ROS. Among 

### 30
30 male Pb recycling plant workers and 27 unexposed controls, despite large differences in blood Pb 

### 31
levels, levels of ROS relea sed from neutrophils (indicators of respiratory burst) were similar between 

### 32

workers and controls ( Mishra et al., 2006 ) . NO production of neutrophils after stimulation with zymosa n



### 33
A was lower in controls, but the difference was not statistically significant. In a study of male foundry 

### 34
workers (mean blood Pb level: 21.7 μg/dL), pottery workers (mean blood Pb level: 9.7 μg/dL), and 

### 35
unexposed workers (mean blood Pb level: 3.9 μg/dL), Valentino et al. ( 2007 ) also found lower pl asma NO 

### 36May 2011 5

- 270 DRAFT

– DO NOT CITE OR QUOTE levels in controls compared with Pb

- exposed workers. The means (ranges) of plasma NO levels in 

### 1
controls, pottery workers, and foundry workers were 23.73 μM (11.21 to 55.71 μM), 28.44 μM (15.23 to 

### 2
57.65 μM), and 25.30 μM (15.03 to 61.98 μM), respect ively. Further, although quantitative results were 

### 3
not reported, blood Pb level was reported to be not correlated with NO. 

### 4

5.6.5.3. Cellular D eath ( A poptosis, N ecrosis)

In a study in mice, Bishayi and Sengupta ( 2006 ) found that Pb exposure elevated DNA 

### 5
fragmentation in splenic macrophages. Using mouse resident peritoneal macrophages, Gargioni et al. 

### 6
( 2006 ) found that inorganic Pb induced both necrosis and apoptosis in vitro. While the exa ct pathways 

### 7
involved were not determined, the authors concluded that activation of the Bax pro

- apoptotic protein was 

### 8
not the key effect of Pb on inducing macrophage apoptosis. In an in vivo study in mice, Xu et al. ( 2008 ) 

### 9

found that a 4

- week administration of Pb acetate (50

- 100 mg/kg, oral) significantly elevated both ROS 

### 10
and malondialdehyde (an indicator of ROS

- induced peroxidation) levels in peripheral blood lymphocytes. 

### 11

The Pb

- induced DNA damage, determined by the comet assay, was accompanied by elevations in p53 and 

### 12
Bax expression with no change in Bcl

- 2 expression (creating a Bax/Bcl

- 2 imbalance). The authors 

### 13
propose that this is a likely route to Pb

- induced apoptosis a nd tumorigenesis. 

### 14

5.6.5.4. Cytokine P roduction

Toxicological studies have indicated that P b affects immune cytokine production via action on T 

### 15
cells, macrophages, and dendritic cells ( Section 5.6.2 ). The combination of these three pathways of 

### 16
cytokine changes induced by Pb creates a hyperinflammatory state among innate immune cells, and 

### 17
acquired immunity is skewed toward Th2 responses. As illustrated in Figure 5

- 47, downstream effects 

### 18

include altere d IgE production, ROS production, and inflammation. Cheng et al. ( 2006 ) found that Pb 

### 19

exp osure affected TNF

- α production in macrophages by affecting signaling pathways. This can result in 

### 20
local tissue damage during the course of immune responses affecting such targets as the liver. In a study 

### 21
involving macrophage

- mediated liver injury in mice (A/J), substimulatory levels of Pb (10 μM) 

### 22
coadministered with LPS stimulated the phosphorylation of p42/44 mitogen

- activated protein kinase 

### 23
(MAPK) and TNF

- α expression ( Y.

- J. Cheng et al., 2006 ) . Blocking protein kinase C or MAPK reduced 

### 24

TNF

- α production of macrophages in vitro, which in turn, protected against Pb + LPS

- induced liver 

### 25
injury in vivo. These findings are consistent with those of Gao et al. ( 2007 ) , which showed that treatment 

### 26
of mouse dendritic cells with Pb pr oduced an increased phosphorylation of the Erk/MAPK signaling 

### 27

molecule. 

### 28
The most consistent immunomodulatory effect of Pb is the skewing of immune responses away 

### 29
from Th1 and toward Th2. Pb was observed to skew toward Th2 cytokine production in both dendr itic 

### 30
cells (5.6.2.6) and T cells. Lynes et al. ( 2006 ) observed that Pb suppressed the production of Th1 cytokine 

### 31May 2011 5

- 271 DRAFT

– DO NOT CITE OR QUOTE IFN

- γ. Gao et al. ( 2007 ) observed that Pb exposure elevated production of Th2 cytokines such as IL

- 4, 

### 1

IL

- 5 and IL

- 6. This shift to a Th2 phenotype also was demonstrated in cultures of human blood 

### 2
monocytes activated with Salmonella enteritidis or with monoclonal antibodies of CD3, CD28, and CD40 

### 3
and exposed to inorganic Pb. Pb exposure suppressed expression of Th1 cytokines, IFN

- γ, IL

- 1β, and 

### 4

TNF

- α, and increased secretion of Th2 cytokines, I L

- 5, IL

- 6, and IL

- 10 ( Hemdan et al., 2005 )

. 

### 5

In a rec ent study conducted across a lifetime (developmental through adulthood) using a broad 

### 6
range of dietary Pb concentrations in Swiss mice (females and males), Iavicoli et al. ( 2006 ) suggested a 

### 7
nonlinear hierarchical cytokine response. At the lowest dietary Pb concentration (0.8 μ g/dL), IL

- 2 and 

### 8

IFN

- γ were elevated over the controls, indicating an enhanced Th1 response. However, as dietary Pb 

### 9
exposure increased (resulting in blood Pb levels 12

- 61 μ g/dL), a Th2 phenotype was observed with 

### 10
suppressed IFN

- γ and IL

- 2 and elevated IL

- 4 production. These findings support the notion that the 

### 11
immune system is remarkably sensitive to low Pb exposures and that compensatory mechanisms may be 

### 12
stimulated at low Pb exposures. Other studies have found variable Pb

- induced changes in IL

- 2, with no 

### 13

c hange or elevated production, depending upon the protocol used. Recently, Gao et al. ( 2007 ) found that 

### 14

Pb

- treated dendritic cells promoted a slight but statisti cally significant increase in IL

- 2 production among 

### 15
lymphocytes. A recent study on bone (in vivo and in vitro) chondrogenesis found Pb

- induced increases in 

### 16
production of TGF

- β ( Zuscik et al., 2007 )

. 

### 17
Consistent with toxicological studies, epidemiologic st udies also find higher blood Pb levels in 

### 18
humans to be associated with a shift towards production of Th2 cytokines relative to Th1 cytokines. One 

### 19
consequence of an increase in the Th2 cytokine IL

- 4 is the activation of B cells to induce B cell class 

### 20

switch ing to IgE. In particular, Lutz et al. ( 1999 ) provided evidence for Pb affecting this pathway by 

### 21
finding that children with blood Pb levels greater than 10 μ g/dL had both higher levels of IL

- 4 and IgE 

### 22
(Section 5.6.3). Among adults in Incheon, Korea without occupational Pb exposures with blood Pb levels 

### 23
ranging from 0.337 to 10.47 μ g/dL, Kim et al. ( 2007 ) found associations of blood Pb level with serum 

### 24

levels of TNF

- α and IL

- 6 that were larger among men with blood Pb levels ≥ 2.51 μ g/dL (median). In 

### 25
models that adjusted for age, sex, BMI, and smoking status, a 1 μ g/dL increase i n blood Pb level was 

### 26
associated with a 23% increase (95% CI: 4, 55%) in log of TNF

- α and a 26% increase in log of IL

- 6 (95% 

### 27
CI: 0, 55%). The associations between levels of blood Pb and plasma TNF

- α were greater among men 

### 28

who were GSTM1 null or had the TNF

- α GG genotype. For the association between blood Pb level and 

### 29
plasma IL

- 6, the effect estimate was slightly elevated in TNF

- α GG genotype but not elevated in the 

### 30
GSTM1 positive group. The effects of Pb on several physiological systems have been hypothesize d to be 

### 31
mediated by the generation of ROS ( Daggett et al., 1998 ) . Thus, the null variant of GSTM1, which is 

### 32
associated with reduced metabolism of ROS, may confer increased susceptibility to Pb

- associated 

### 33

immune effects. The results for the TNF

- α polymorphism were difficult to interpret. The GG genotype is 

### 34
associated with lower expression of TNF

- α, and the literature is mixed with respect to which variant 

### 35

increases risk of i nflammation

- related conditions. 

### 36May 2011 5

- 272 DRAFT

– DO NOT CITE OR QUOTE Results from studies of occupationally

- exposed adults also suggested that Pb exposure may be 

### 1
associated with decreases in Th1 cytokines and increases in Th2 cytokines; however, analysis were 

### 2
mostly limited to comparisons of mean cytokine levels among different blood Pb groups or Pb

- exposure 

### 3

groups ( Di Lorenzo et al., 2007 ; Valent ino et al., 2007 ; Yucesoy et al., 1997a )

. The exception was the 

### 4
study of male foundry workers, pottery workers, and unexposed workers by Valentino et al. ( 2007 )

. 

### 5
Multiple regression analyses were performed with age, BMI, smoking, and alcohol consumption included 

### 6
as covariates. Although effect estimates were not reported, statistically sig nificant associations were 

### 7

observed between blood Pb level and IL

- 10 and TNF

- α. Levels of IL

- 2, IL

- 6, and IL

- 10 showed an 

### 8
increasing trend from the lowest to highest blood Pb group. In contrast with most other studies, both 

### 9
exposed worker groups had lower IL

- 4 levels compared with controls. In a similar analysis, DiLorenzo et 

### 10

al. ( 2007 ) separate d exposed workers into intermediate (9.1

- 29.4 μ g/dL) and high (29.4

- 81.1 μ g/dL) 

### 11
blood Pb level groups, with unexposed workers comprising the low exposure group (blood Pb levels 1

- 11 

### 12

μ g/dL). Excluded from this study were exposed workers from the highest end of the distribution of blood 

### 13

Pb levels in DiLorenzo et al. ( 2006 ) . Mean TNF

- α levels showed a monotonic increase from the low to 

### 14
high blood Pb level group. A synergistic effect was observed between blood Pb levels and smoking. 

### 15
Among current sm okers, a 12

- to 16

- fold difference in TNF

- α levels was observed among blood Pb 

### 16
groups, whereas a less than twofold difference was observed among nonsmokers. In Yucesoy et al. 

### 17
( 1997a ) , levels of the Th1 cytokines, IL

- 1β an d IFN

- γ, were lower in workers than in controls; however, 

### 18
differences were not observed in levels of the Th2 cytokines, IL

- 2 and TNF

- α. 

### 19



## 5.6.6. Immune Effects of Lead within Mixtures

One of the most striking observations regarding Pb effects on the immune system s ince the 2006 Pb 

### 20
AQCD concern s the effects of metal mixtures . Recent studies indicate that immune effects may be 

### 21
observed with lower levels of Pb exposure when they occur in conjunction with other metals. Information 

### 22
on interactions among metal mixtures ma y improve risk assessment methods because most human 

### 23
exposures to chemicals involve mixtures at low environmental levels. In a study of mice exposed to Pb 

### 24
acetate (10 mg/kg by weight), As (0.5 mg/kg by weight), or both, Bishayi and Senguta ( 2006 ) reported a 

### 25
greater than additive effect of coadministered Pb and As on macrophages in producing a decrease in 

### 26
bacterial resistance, myeloperoxidase (MPO) release, and NO production. Investigators assessed the Pb



### 27

As interaction u sing the multivariate ANOVA for the experimental results of MPO release and 

### 28
constructing an isobologram by running an ordinary least squares regression between effects (% MPO 

### 29
release) and dose levels of metals (single and multimetal) in log

- linear form. 

### 30

Institoris et al. ( 2006 ) reached a similar conclusion after observing that lymph node weight 

### 31
decreased with exposure to 20 mg/kg Pb plus a second metal (Cd or Hg) but not with 20 mg/kg of Pb 

### 32
alone. Another study conducted in rats ( Jadhav et al., 2007 ) fou nd that mixtures of Pb, Hg, and other 

### 33May 2011 5

- 273 DRAFT

– DO NOT CITE OR QUOTE metals at 10 to 100 times the concentrations of the individual components typically measured in drinking 

### 1
water in India suppressed lymphocyte counts and antibody titers and increased neutrophil counts. In 

### 2

contrast with these aforementioned studies, Fortier et al. ( 2008 ) found that PbCl 2 - exposed (7.5

- 20.7 

### 3

μ g/dL) human leukocytes did not have alterations in lymphocyte proliferation, monocytic phago cytic 

### 4
activity, or NK cell activity. Although the combination of 20.7 μ g/dL PbCl 2 plus 12.0 μ g/dL 

### 5
methylmercuric chloride (MeHgCl) decreased lymphocyte proliferation, these effects were attributed to 

### 6
MeHgCl, which had a stronger suppressive effect individu ally. The majority of evidence indicating 

### 7
synergism between Pb and metals such as As, Cd, and Hg suggests that a threshold for producing Pb



### 8
induced immune effects may be lower if additional metals are present. 

### 9
In a study designed to mimic exposure to part icles associated with urban traffic, Wei et al. exposed 

### 10

human e ndothelial cells in culture (EA.hy926) to urban fine particles (PM 2.5 enriched with Pb and other 

### 11
metals). Investigators found that the particle mixture increased ROS production and mitochondrial



### 12
mediated apoptosis; however, they did not test metals individ ually and could not attribute findings to Pb 

### 13
individually or interactions between Pb and other metals within the mixture. Similarly, in two studies of 

### 14
mice exposed to Pb and Cd in drinking water (1 ppm of each metal in drinking water for 28 days), Pb and 

### 15

C d altered antibody titres ( Massadeh et al., 2007 ) . The main aims of these studies were to demonstrate 

### 16
that the effects of Pb and Cd could be reversed with administration of Nigella sativa L (black cumin) or 

### 17
garlic extract. However, investigators did no t test each metal individually to assess interactions between 

### 18

metals. 

### 19
Epidemiologic studies have not widely examined interactions between Pb and other metals. 

### 20
However, consistent with Bishayi and Sengupta ( 2006 ) , Pined a

- Zavaleta et al. ( 2004 ) (Section 5.6.5.2) 

### 21
reported interactions between Pb and As. In addition to Pb, As contamination of drinking water was a 

### 22
concern in the study area; however, urinary As levels were higher in children who had lower blood Pb 

### 23
levels. In multiple regression analyses, urinary As was negatively associated with NO (similar to Pb), and 

### 24
a statistically significant negative interaction was observed between Pb and As, indicating tha t high 

### 25
internal doses of both metals were associated with a larger decrease in NO than that associated with either 

### 26
metal alone. Urinary As was negatively associated with superoxide anion (opposite direction of Pb), and 

### 27
the Pb by As interaction was positive . Thus, although higher urinary As was associated with a lower 

### 28
superoxide anion level, higher internal doses both Pb and As were associated with a larger increase in 

### 29
superoxide anion than that associated with blood Pb level alone. 

### 30



## 5.6.7. Summary and Causal Determination

The collective body of evidence integrated across epidemiologic and toxicological studies 

### 31
consistently demonstrates that the immune system is a major target of Pb. Rather than resulting in overt 

### 32
cytotoxicity to lymphoid tissues, Pb exposure h as been associated predominantly with more subtle 

### 33May 2011 5

- 274 DRAFT

– DO NOT CITE OR QUOTE changes in a spectrum of immune mediators and functions. The importance of Pb as an immunomodulator 

### 1
is particularly evident when one considers: (1) the relative sensitivity of the immune system to Pb



### 2

induce d modulation; (2) the lifetime health ramifications of exposure to Pb in early development, 

### 3
particularly given the recognized sensitivity of the developing immune system to environmentally



### 4
induced programming; and (3) the consequent broad spectrum of disea ses and illnesses in multiple 

### 5
physiological systems potentially related to Pb

- associated immune dysfunction. The majority of results 

### 6
from animal studies indicates that immune changes are observable at blood Pb levels of <8.0 μ g/dL, with 

### 7

new evidence in mic e demonstrating that nonmonotonic cytokine changes occur at blood Pb levels of 2

- 3 

### 8

μ g/dL. Consistent with the animal studies, in the newly expanded body of epidemiologic studies in 

### 9
children and adults without occupational Pb exposures, changes in immune pa rameters are commonly 

### 10
demonstrated in association with mean blood Pb levels in the range of <2 μ g/dL to 10 μ g/dL. 

### 11
The strength of evidence for Pb

- associated immune effects is derived not only from the consistency 

### 12

of associations but also from the coherence of findings between toxicological and epidemiologic studies 

### 13
and coherence of findings among the spectrum of immune changes operating within particular pathways. 

### 14
A large body of toxicological evidence demonstrates Pb

- induced suppression of T cell prolifera tion, and 

### 15
epidemiologic evidence in children consistently links elevated blood Pb levels (as low as 2.2

- 3.4 μ g/dL) 

### 16
with decreases in T cell abundance. These changes can affect cell

- to

- cell interactions that mediate 

### 17
acquired immunity required in subsequent memory responses to antigen exposures. Despite the 

### 18
consistency of evidence for some T

- cell subtypes (e.g., CD3+, CD4+), it is unclear what effect the 

### 19
observed magnitudes of decrease may have in attenuating acquired immunity. 

### 20
The prominent effect of Pb exposure on T cells, in terms of coherence with effects on other 

### 21
immune endpoints and implications for developing immune

- based diseases, is the skewing of immune 

### 22
function away from a Th1 phenotype towards a Th2 phenotype. In toxi cological studies, this shift is well



### 23
established by suppressed production of Th1 cytokines (e.g., IFN

- γ) and increased production of Th2 

### 24
cytokines (e.g., IL

- 4). A recent toxicological study builds on this extant evidence by indicating that Pb 

### 25

may promote Th2 responses by acting directly on dendritic cells, the major effector in antigen response. 

### 26
Findings from recent epidemiologic studies strengthen the evidence with observations of similarly 

### 27
skewed cytokine profiles in association with blood Pb levels in h umans in the range of 2.5

- 10 μ g/dL. 

### 28
Further, toxicological and epidemiologic studies link the Pb

- associated predominance of Th2 cytokines 

### 29
with downstream effects on humoral immunity by demonstrating Pb

- associated changes in B cell 

### 30
abundance and changes in circulating antibody levels. An increase in IL

- 4 from activated Th2 cells 

### 31
induces differentiation of B cells into antibody

- producing cells, thereby amplifying B cell expansion to 

### 32
secrete IgE, IgA, and IgG. IgE is the primary mediator for type 1 hypersensit ivity resulting in various 

### 33
allergic conditions and asthma. In support of this well

- established mechanism, toxicological studies 

### 34

describe Pb

- induced (blood Pb levels 10

- 30 μ g/dL) changes in IgA, IgG, and IgM. Additionally, 

### 35
epidemiologic studies in children consistently demonstrate that blood Pb levels are positively associated 

### 36May 2011 5

- 275 DRAFT

– DO NOT CITE OR QUOTE with B cell abundance (blood Pb levels in the range of 5

- 10 μ g/dL) and increases in IgE (blood Pb levels 

### 1

as low as 2.8

- 3.4 μ g/dL). Observations of Pb

- associated increases in Th2 respon ses and circulating IgE 

### 2
levels provide biological plausibility for epidemiologic observations in children of associations of blood 

### 3
Pb levels (in the range of 1.16

- 10 μ g/dL) with asthma and allergic conditions. Such epidemiologic data 

### 4
are limited, and addit ional studies with more rigorous methodology (e.g., longitudinal design to establish 

### 5
temporality, improved assessment of Pb exposures, adjustment for potential confounders such as 

### 6
smoking, SES, and exposures to other metals) are needed to substantiate the findings. 

### 7
Suppression of Th1 function by Pb places individuals at greater risk of certain infectious diseases 

### 8
and cancer. Compared with the relationships between Pb

- suppressed Th1

- dependent antitumor processes 

### 9
and effects on tumor formation, Pb effects on decreasing host resistance are relatively well

- established. 

### 10
Pb exposure of animals (resulting in blood Pb levels <2

- 5 μ g/dL) suppresses the DTH response, and a 

### 11
recent in vitro study indicates such effects may be mediated by dendritic cells. Further eviden ce of Pb



### 12
associated suppressed Th1 activity is provided by toxicological and epidemiologic observations that Pb 

### 13
exposure and blood Pb levels, respectively, are associated impaired phagocytic and chemotactic activity 

### 14
of macrophages and neutrophils (blood Pb levels >25 μ g/dL in humans). Th1

- dependent IFN

- γ normally 

### 15
enhances the killing capacity of macrophages. Epidemiologic studies additionally demonstrate that higher 

### 16
blood Pb levels are associated with lower neutrophil respiratory burst (superoxide anion rel ease), 

### 17
indicative of diminished degradation of phagocytosed particles. Toxicological and epidemiologic 

### 18
evidence for suppressed Th1 activity and effects on macrophage and neutrophil functional activities 

### 19
provide biological plausibility for observations in a nimals and humans for associations between blood Pb 

### 20
levels (in the range of 3

- 10 μ g/dL in humans) and increased risk of infection. 

### 21
Toxicological studies demonstrate that Pb induces macrophages into a hyperinflammatory state as 

### 22
characterized by enhanced pro duction of ROS, suppressed production of NO, enhanced production of 

### 23

TNF

- α, and excessive metabolism of arachidonic acid into immunosuppressive metabolites (e.g., PGE 2 ). 

### 24
Although examined in fewer studies, epidemiologic studies find higher blood Pb levels ( in the range of 6

- 

### 25

20 μ g/dL) to be associated with higher serum levels of ROS, TNF

- α and lower serum levels of NO. 

### 26
These proinflammatory effects of Pb exposure on macrophages provide support for associations of Pb 

### 27
with elevated risk of disease in multiple physiological systems. The strongest evidence comprises Pb



### 28
induced changes in specialized macrophages such as alveolar macrophages, testicular macrophages, and 

### 29
microglia, whose altered homeostasis or function may contribute to documented associations of bl ood Pb 

### 30
levels with asthma and bronchial reactivity, poor reproductive performance, and neurodegeneration, 

### 31
respectively. Although limited mostly to toxicological studies, Pb has been shown to induce the 

### 32
generation of autoantibodies, suggesting that Pb expos ure may increase the risk of developing 

### 33
autoimmune conditions. These findings are supported by Pb

- induced inflammation and tissue damage and 

### 34

Pb

- induced T cell activation in response to new antigens. 

### 35May 2011 5

- 276 DRAFT

– DO NOT CITE OR QUOTE In summary, recent toxicological and epidemiologic studie s support the strong body of evidence 

### 1
presented in the 2006 Pb AQCD that Pb exposure is associated with changes in immune cell abundance 

### 2
and function that subsequently lead to a broad spectrum of changes in both cell

- mediated and humoral 

### 3
immunity to promot e a Th2 phenotype and hyperinflammatory state. Toxicological and epidemiologic 

### 4
findings of Pb

- associated decreases in T cells, inhibition of Th1

- type responses, and impaired phagocytic 

### 5
activity of macrophages and neutrophils provide biological plausibility for evidence linking Pb exposure 

### 6
with increased risk of bacterial and viral infection. Additionally, toxicological and epidemiologic evidence 

### 7

of a Pb

- associated promotion of the Th2 phenotype, increased B cell abundance, increased synthesis of 

### 8
IgE, and in creased inflammation support observations in children for associations of blood Pb levels with 

### 9
asthma and allergic conditions. Although not widely examined in humans, toxicological findings indicate 

### 10

that Pb

- induced immunomodulation may have broader implica tions for autoimmunity and mediating Pb 

### 11
effects in other physiological systems such as the nervous, cardiovascular, and reproductive systems. 

### 12
Animal studies and to a limited extent, epidemiologic studies, demonstrate increased susceptibility from 

### 13
prenatal exposures and enhanced responses with co

- exposures to other metals. The consistency of 

### 14
findings among toxicological and epidemiologic studies and the coherence of findings between these 

### 15
disciplines and across the continuum of related immune responses are s ufficient to conclude that there is a 

### 16

causal re lationship between Pb exposure s and immune system effects. 

### 17



# 5.7. Effects on Heme Synthesis and Red Blood Cell Function

5.7.1. Summary of Findings from 2006 Pb AQCD

The 2006 Pb AQCD reported that Pb affects developing RBCs (red blood cells [RBC]) as noted by 

### 18
anemia observed with blood Pb >40 μg/dL. Pb

- induced anemia is thought to occur due to decreased RBC 

### 19
life span and effects on hemoglobin (Hb) synthesis. The exact mechanism for these effects was not 

### 20
known, although Pbindu ced changes on iron uptake or inhibition of enzymes in the heme synthetic 

### 21
pathway may be responsible. 

### 22
The 2006 Pb AQCD indicated that Pb crosses RBC membranes through passive (i.e., energy



### 23
independent) carrier

- mediated mechanisms including a vanadate

- sens itive Ca 2+ pump. Once Pb enters the 

### 24
cells, it is predominantly found in protein

- bound form, with Hb and aminolevulinic acid dehydratase 

### 25
(ALAD) both identified as targets. Pb poisoning decreases RBC survival, as well as alters RBC mobility 

### 26
and morphology, although th e precise mechanisms by which it does so are not known. Pb exposure has 

### 27
been found to significantly decrease several hematological parameters including Hb, hematocrit (Hct), 

### 28
mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpusc ular 

### 29May 2011 5

- 277 DRAFT

– DO NOT CITE OR QUOTE hemoglobin concentration (MCHC). Pb has also been observed to exert multiple effects on RBC 

### 1
membranes, including altered microviscosity and fluidity, decreased sialic acid content, decreased 

### 2
lamellar organization, decreased lipid resistance to oxidati on (possibly mediated by perturbations in RBC 

### 3
membrane lipid profiles), and increased permeability. These alterations to RBC membranes potentially 

### 4
lead to RBC fragility, abnormal cellular function, RBC destruction, and ultimately anemic conditions. Pb 

### 5

expo sure also results in increased activation of RBC scramblase, an enzyme responsible for the 

### 6
expression of phosphotidylserine (PS) on RBC membranes. This expression of PS decreases the life span 

### 7
of RBCs via phagocytosis by macrophages. Pb exposure has been o bserved to alter the phosphorylation 

### 8
profiles of membrane proteins, which may influence the activity of membrane enzymes and the 

### 9
functioning of receptors and channels located on the membrane. 

### 10
The 2006 Pb AQCD reported that Pb affects heme synthesis throug h the inhibition of multiple key 

### 11
enzymes, most notably ALAD, the enzyme that catalyzes the second, rate

- limiting step in heme 

### 12
biosynthesis (See Figure 5

- 53 for a schematic representation of the heme biosynthetic pathway). The 

### 13
2006 Pb AQCD further reported that decreased RBC ALAD activity is the most sensitive measure of Pb 

### 14
exposure with a concentration

- response change in the ratio of activated/nonactivated ALAD activity that 

### 15
is not dependent on the method of Pb administration. The inhibition of the ALAD enz yme was observed 

### 16
in RBCs from multiple species, including birds, Cynomolgous monkeys, and humans. Pb was also 

### 17
observed to inhibit other enzymes responsible for heme biosynthesis, including ferrochelatase, 

### 18
porphobilinogen (PBG) deaminase, and coproporphyrin ogen oxidase. Pb also potentially alters heme 

### 19
biosynthesis through inhibition of transferrin (TF) endocytosis and iron transport. 

### 20
Pb has been found to alter RBC energy metabolism through inhibition of enzymes involved in 

### 21
anaerobic glycolysis and the pento se phosphate pathway. Pb was also found to inhibit pyrimidine 5’

- 

### 22
nucleotidase (P5N) activity and the 2006 Pb AQCD indicated that this might be another possible 

### 23
biomarker of Pb exposure. Inhibition of P5N results in an intracellular increase in pyrimidine n ucleotides 

### 24
leading to hemolysis. The 2006 Pb AQCD indicated that perturbations in RBC energy metabolism may be 

### 25
related to significant decreases in levels of nucleotide pools, including nicotinamide adenine nucleotide 

### 26
(NAD), possibly due to decreased NAD sy nthase activity, and nicotinamide adenine nucleotide phosphate 

### 27
(NADP) accompanying significant increases in purine degradation products. 

### 28
Pb was found to alter the activity of membrane

- bound ion pumps. Potassium (K + ) permeability was 

### 29

found to be increased by Pb due to altered sensitivity of the membrane calcium (Ca 2+ )

- binding site that 

### 30

caused selective efflux of K + ions from the RBC membrane. Inhibition of RBC sodium (Na + )

- K + 

### 31
adenosine triphosphate synthase (ATPase), acetylcholinesterase (ACh), and NADH deh ydrogenase was 

### 32
also observed. In human RBCs, Na +

- K + ATPase activity was more sensitive to Pb exposure than Ca 2+ or 

### 33
magnesium (Mg 2+ ) ATPases. 

### 34May 2011 5

- 278 DRAFT

– DO NOT CITE OR QUOTE The 2006 Pb AQCD document identified oxidative stress as an important potential mechanism of 

### 1
action resulting from Pb exposure. Increased lipid peroxidation and inhibition of antioxidant enzymes 

### 2
(e.g., superoxide dismutase [SOD], catalase [CAT]) was observed following exposure to Pb. 

### 3



## 5.7.2. Effects on Red Blood Cell F unctions

As stated in the 2006 Pb AQCD, Pb poisoning is as sociated with anemia resulting from shortened 

### 4
RBC life span and Pb effects on Hb synthesis. As of 2006, the mechanism for this was not clear, but it 

### 5
was determined not to be due to iron deficiency, which can be found to occur independently of Pb 

### 6

exposure. However, Zimmerman et al. ( 2006 ) found that blood Pb levels were statistically significantly 

### 7

reduced in non

- or mildly anemic, iron

- deficient children in India fed an iron

- fortified diet for 30 weeks 

### 8
(p <0.02); blood Pb levels were not reduced in the children not receiving the iron

- fortified diet. Although 

### 9
a number of studies find decreases in RBCs and/or Hct levels associated with Pb, it is not known whether 

### 10
this is due to reduced cell survival or a decrease in RBC cell production. Howe ver, decreased RBC 

### 11
survival and hematopoiesis can be expected to occur simultaneously, and any effect on RBC numbers is 

### 12
likely a combination of the two modes of action. 

### 13

5.7.2.1. Lead Uptake, Binding, and Transport into Red Blood Cells

The 2006 Pb AQCD reports that Pb uptake into human RBCs occurs via passive anion transport 

### 14
mechanisms. Although Pb can passively cross the membrane in both directions, little of the Pb leaves the 

### 15
cell after entry. Simons ( 1993b ) found that in vitro uptake of 203 Pb ( 1

- 10 μM) occurred via an anion 

### 16
exchanger while the efflux was via a vanadate

- sensitive pathway. A fter entry into the RBC, radioactive Pb 

### 17
was found to partition with Hb at a ratio estimated to be about 6000:1 bound to unbound ( Simons, 1986 )

. 

### 18
However, Bergdahl et al. ( 1997 ) suggested that ALAD was the primary Pb binding protein and not Hb. 

### 19
The 2006 Pb AQCD also reports that the majority (approximately 98%) of Pb accumulates in RBC 

### 20
cytoplasm bound to protein and only about 2% is found in the membrane. This is related to the high ratio 

### 21
of Pb in RBCs compared to plasma Pb. Further Information on Pb binding and transport in blood can be 

### 22
found in the kinetics section of Chapter 4 (Section 4.2). 

### 23
Although no studies were indentified that examined transport of Pb into RBCs, Lind et al. ( 2009 ) 

### 24
recently observed that several zinc (Zn) ionophores (8

- hy droxyquinoline derivatives and Zn and Na 

### 25
pyrithione) were able to effectively transport Pb out of RBCs into the extracellular space. 

### 26

5.7.2.2. Red Blood Cell Survival, Mobility, and Membrane Integrity

Although it is known that Pb exposure shortens the RBC life span and alters RBC mobility, as of 

### 27
the 2006 Pb AQCD the mechanism of this was not well understood. While the mechanism is still not fully 

### 28
understood, there has been some indication for a role in free Ca 2+ . There are also newer studies that 

### 29May 2011 5

- 279 DRAFT

– DO NOT CITE OR QUOTE examine the relatio nship between Pb and RBC survival, mobility, and membrane integrity. Pb

- exposed 

### 1
workers in a recycled automobile battery factory in Mexico had 7.5 times as much blood Pb as was found 

### 2
in unexposed workers (74.4 ± 21.9 μg/dL in 15 exposed workers versus. 9.9 ± 2 μg/dL in 15 unexposed 

### 3
workers, p <0.01) ( Quintanar

- Escorza et al., 2007 ) . Intracellular RBC free calcium levels [Ca 2+ ] i were 

### 4
significantly higher in the Pb

- exposed workers than in the unexposed workers (79 ± 13 versus. 30 ± 9 nM, 

### 5

p < 0.05). The level of [Ca 2+ ] i was highly correlated (R 2 = 0.754) with blood Pb levels in exposed workers; 

### 6
[Ca 2+ ] i was also elevated in the unexposed workers who had low blood Pb levels ranging from 7.9 μg/dL 

### 7
to 11.9 μg/dL. The observed elevation in [Ca 2+ ] i was related to an increased uptake and a decreased efflux 

### 8

due to reduced Ca 2+

- Mg 2+

- ATPase activity. In the RBCs of Pb

- exposed workers, the activity of Ca 2+

- 

### 9
Mg 2+

- ATPase was 28 ± 8 nmol Pi/mg protein/min versus. 40 ± 9 in unexposed workers (p <0.05). These 

### 10

c hanges were associated with increased fragility of the RBCs and dramatic morphological alterations, 

### 11
including the increased presence of ecinocytes (cells without normal biconcave shape) and crenocytes 

### 12
(speculated cells) in Pb

- exposed workers. Similar dose

- dependent effects were observed when RBCs 

### 13
from healthy subjects were incubated with Pb at concentrations from 0.2 to 6.0 μM ( Quintanar

- Escorza et 

### 14
al., 2010 ) . Abam et al. ( 2008 ) also obse rved a decrease in the activity of RBC membrane bound Ca 2+

- 

### 15
Mg 2+

- ATPase in workers exposed to Pb in a number of occupations. While the authors did not observe an 

### 16

increase in [Ca 2+ ] i or Mg in RBCs from exposed workers, the RBC membrane [Ca 2+ ] i and Mg 2+ 

### 17

conce ntration were increased. Abam et al. ( 2008 ) did not report on morphological changes in erythrocytes 

### 18
from exposed workers. Ciubar et al. ( 2007 ) found that RBC morphology was disrupted with 50% or more 

### 19
of RBCs exposed to Pb concentrations of 0 .5 μM or higher for 24 hours at 37°C having lost the typical 

### 20
discocytic morphology and displaying moderate to severe echinocytosis. Exposure of RBCs to higher 

### 21
concentrations of Pb nitrate resulted in cell shrinkage. Ademuyiwa et al. ( 2009 ) observed that the 

### 22
cholesterol content of RBC plasma membranes, but not the phospholipid content, was statistically 

### 23
significantly higher in rats exposed to 200 ppm Pb acetate (blood Pb = 40.63 ± 9. 21 μg/dL) through 

### 24
drinking water compared to controls. Further, the cholesterol/phospholipid ratio was increased in the rats 

### 25
with increased cholesterol, indicating that RBC membrane fluidity was decreased. 

### 26
A number of studies have investigated the effect of occupational exposure to Pb on various 

### 27
hematological parameters. ( Ukaejiofo et al., 2009 ) studied hematological effects of Pb in 81 male subjects 

### 28
exposed to Pb at three different manufacturing companies in Nigeria. Two control gro ups were used for 

### 29
comparison (30 individuals from the same industries not involved in handling Pb and 20 individuals from 

### 30
the same locality but not involved in Pb handling). The exposed individuals had an average blood Pb level 

### 31
of 7.00 μg/dL compared to 3 μg/dL in controls drawn from industries not involved in Pb handling (control 

### 32
group I) and 2 μg/dL in controls drawn from the general population (control group II) (p <0.05). Exposed 

### 33
subjects had significantly reduced Hb and packed cell volume (PCV) levels and increased percentage of 

### 34
reticulocytes. Although the differences were significant between the exposed and control subjects, the 

### 35
study authors state that the results in the exposed subjects were at the lower range of normal for 

### 36May 2011 5

- 280 DRAFT

– DO NOT CITE OR QUOTE Nigerians. The percent cel l lysis did not differ between controls and exposed workers; however, when 

### 1
workers and controls were stratified by age, there was a significant increase in cell lysis in workers under 

### 2
age 30 compared to similarly aged control group II (p <0.01). Patil et a l. ( 2006 ) examined hematology 

### 3
effects in jewelry workers in India occupationally exposed to Pb. Blood Pb was significantly higher in the 

### 4
jewelry workers (48.56 ± 7.39 μg/dL) compared to individuals not occupationally exposed (12.52 ± 4.08 

### 5
μg/dL). There was no significant difference in the Hb, MCV, or MCH; however, jewelry workers had a 

### 6
signifi cant decrease in PCV and total RBCs, accompanied by a significant increase in MCHC. In addition, 

### 7
the exposed workers had a significant increase in total leukocyte levels. In battery manufacturing workers 

### 8
in India, significant decreases in Hb, PCV, MCV, MCH , MCHC, and total RBCs were observed ( Patil, 

### 9
Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 2006 ) with similar blood Pb level s (53.63 ± 16.98 

### 10
μg/dL). Industrial workers in Pakistan occupationally exposed to Pb (blood Pb = 29.1 (range 9.0

- 61.1) 

### 11
μg/dL) had a significant increase (3.5

- fold higher median) in blood Pb levels compared to age and gender 

### 12
matched controls (blood Pb = 8.3 (range 1.0

- 21.7) μg/dL) ( D. A. Khan et al., 2008 ) . The industrial 

### 13
workers had a significantly lower Hb, but not a significant difference in the number of RBCs. There were 

### 14
no significant differences in the number of white blood cells (WBCs) or in platelet counts. Karita et al. 

### 15
( 2005 ) examined the relationship between hematological parameters in Pb

- exposed workers (blood Pb = 

### 16
26.9 μg/dL) in a variety of occupatio nal settings. Blood Pb was found to be negatively correlated with 

### 17
Hb, Hct, and total RBC count (p <0.01). Fonte et al. ( 2007 ) described a case

- report in which a 47

- year old 

### 18
male exposed to Pb fumes and vapors at a recycling plant (blood Pb = 148 μg/dL) experienced 

### 19
normocytic, norm ochromic anemia, along with reticulocytosis and RBC basophilic stipplings. Following 

### 20
chelation therapy, the hematological symptoms improved. Taken together, these studies provide consistent 

### 21
evidence that occupational exposure to Pb reduces the number of RB Cs in circulation. Although this 

### 22
decrease in RBCs may be explained by both decreased cell survival and/or disruption of hematopoiesis, 

### 23
the observation of increased reticulocytes in Ukaejiofo et al. ( 2009 ) seems to represent compensation for 

### 24

decreased RBC surviv al due to Pb exposure. 

### 25
Studies in children were more equivocal than those in adults. Riddell et al. ( 2007 ) found that 21% 

### 26
of children 6 months to 5 years of age living in rural Philippines had blood Pb levels greater than 10 

### 27
μg/dL (mean = 6.9 μg/dL). Hb levels were inversely related to blood Pb, with a decrease of 3% blood Pb 

### 28
associated with every 1 g/dL increase in Hb. However, Huo et al. ( 2007 ) found that children living near 

### 29

an area where electronic waste wa s recycled in China had significantly higher blood Pb levels than 

### 30
children in the neighboring town with no waste recycling (15.3 versus 9.94 μg/dL), but no difference in 

### 31
the Hb levels in the children in the two towns was detected (127.55 g/L in children wi th the higher blood 

### 32
Pb levels versus 123.46 g/L). Ahamed et al. ( 2006 ) studied male urban adolescents in India. The 39 

### 33
adolescents were separated in to groups according to their blood Pb level (group 1: <10 μg/dL, group 2: 

### 34
>10 μg/dL). Although the groups were similar in their age, height, weight, and body mass index, group 2 

### 35
had a significantly lower PCV compared to group 1. The equivocal findings in s tudies investigating 

### 36May 2011 5

- 281 DRAFT

– DO NOT CITE OR QUOTE hematological effects in children may be due to the comparatively shorter time period and magnitude of 

### 1
exposure versus. those seen in occupational studies. 

### 2

Baranowska

- Bosiacka et al. ( 2009 ) examined the effects of Pb on RBC hemolysis both in vitro 

### 3
measuring lysate in human RBCs incubated with Pb at concentrations ranging from 100 nM to 100 μM 

### 4

for 5

- 30 minutes, and in vivo using a rat RBC lysate from rats exposed to Pb acetate (0.1 %) in drinking 

### 5
water for 9 months. Rats exposed to Pb in the in vivo portion of the study achieved a blood Pb 

### 6
concentration of 7.1 μg/dL. Both studies demonstrated that Pb exposure resulted in increased hemolysis, 

### 7
observed as a significant increase in extracellular Hb. Pb

- induced hemolysis in these experiments may be 

### 8
due to inhibition of RBC phosphoribosyltransfer ases ( Section 5

. 7 .5.1 ). The in vitro studies indicated a 

### 9

concentration

- dependent increase in the amount of hemolysis, with a significant (threefold) increase even 

### 10
at the lowest concentration tested (i.e., 100 nM). Lee et al. ( 2005 ) observed that rats orally admini stered 

### 11
Pb (25 mg/kg) once a week for 4 weeks had an average plasma Pb level of 6.5 μg/dL (9.6

- fold higher than 

### 12
controls, p <0.05), and had significant decreases in Hct, Hb, and RBCs (p <0.05) ( M. K. Lee et al., 2005 )

. 

### 13

Male mice administered 5 0 mg/kg Pb nitrate in distilled water via gavage for 40 days had significantly 

### 14
reduced total RBC counts, total leukocyte counts, Hb, lymphocytes, and monocytes ( Sharma et al., 2010 )

. 

### 15
Rats exposed to 2 g/L Pb acetate in drinking water for 30 days had significantly decreased RBCs, Hb, 

### 16
PCV, MCH, and MCHC compared to controls (p <0.05) ( Simsek et al., 2009 ) . Microcytic and basophilic 

### 17
erythrocytic granulations were commonly seen in Pb

- exposed animals. An indication that the decrease in 

### 18
RBC count was related to decreased survival, and not a disruption of hematopoiesis, was the observation 

### 19
of significantly increased reticulocyte density and total count compared to controls (p <0.05). M ice 

### 20
exposed to 1 g/L Pb acetate in drinking water for 90 days , but not those exposed for 15 or 45 days, had 

### 21
significantly decreased RBC counts and Hct compared to controls (p <0.05) ( C. C. Marques et al., 2006 )

. 

### 22
Spleen weights were also observed to be increased relative to body weight in animals exposed to Pb for 

### 23
45 days. Male rats administered Pb acetate in th e drinking water for 4 weeks at concentrations ranging 

### 24
from 100 to 1,000 ppm had a dose

- dependent increase in blood Pb (range: 6.57 to 22.39 μg/dL), but there 

### 25
were no significant changes in any of the hematological parameters (complete blood cell count 

### 26

per formed) measured at the end of treatment ( M. Y. Lee et al., 2006 )

. Slight, nonsignificant, increases in 

### 27
PS expression on RBC membranes were also observed. In vitro experiments with rat and human blood 

### 28
did not demonstrate a significant increase i n hemolysis after 4 hours of treatment with Pb acetate at 

### 29
concentrations up to 10 μM. 

### 30

Khaĭrullina et al. ( 2008 ) observed that the surface profiles of RBC membrane shadows incubated 

### 31

with 0.5

- 10 μM/l Pb acetate for three hours were much smoother than untreated RBC membranes when 

### 32
examined by atomic force m icroscopy. The authors postulate that the observed smoothing in treated RBC 

### 33
membranes may be due to clusterization of band 3 protein. Band 3 (anion exchanger 1 [AE1]), is a 

### 34
chloride/bicarbonate (Cl - 1 /HCO 3 - ) exchanger and is the most abundant protein in RBC membranes. AE1 is 

### 35
integral in carbon dioxide (CO 2 ) transport and linkage of the cellular membrane to the underlying 

### 36May 2011 5

- 282 DRAFT

– DO NOT CITE OR QUOTE cytoskeleton ( Akel et al., 2007 ; Su et al., 2007 )

. The observed smoothing of the RBC membrane may due 

### 1
to Pb interfering wit h how the membrane attaches to the cytoskeletal structure of the RBC through 

### 2
perturbation of AE1 ’ s normal activity. 

### 3

Eryptosis

Eryptosis is the suicidal death of RBCs. It is characterized by cell shrinkage, membrane blebbing, 

### 4
and cell membrane phospholipid scrambling associated with PS exposure on the cell membrane that leads 

### 5
to cell destruction via macrophages ( Föller et al., 2008 ; Lang et al., 2008 ) . As previously reported in the 

### 6
2006 Pb AQCD, Kempe et al. ( 2005 ) found that exposing human RBCs to Pb at concentrations ranging 

### 7
from 0.3 to 3 μM caused increased activation of K + channels that lead to cell shrinkage and scramblase 

### 8
activation. The activation of scramblase increased the exposure to PS on the cell membran e, which causes 

### 9
an increase in destruction of the RBCs by macrophages. 

### 10

Shin et al. ( 2007 ) found that in vitro exposure of human RBCs to 1

- 5 μM Pb acetate increased PS 

### 11
expression in a time

- and concentration

- dependent manner. The maximum increase in expression of PS 

### 12
was 26.8 ± 3.15% (compared to deionized water) after incubation with 5 μM Pb for four hours. The 

### 13
expression of PS in RBCs is considered to be regulated through a C a 2+ dependent mechanism and, 

### 14
correspondingly, [Ca 2+ ] i was observed to increase with exposure to Pb (0.24 ± 0.21 μM in controls to 6.88 

### 15
± 1.13 μM in RBCs treated with 5 μ M Pb for one hour). Consistent with this finding, Shin et al ( 2007 ) 

### 16

also observed that scramblase activity, which is important for induction of PS exposure and is activated 

### 17
by [Ca 2+ ] i , was increased in Pb

- exposed RBCs. Flippase, which translates PS exposure to inner 

### 18
membranes, is inhibited by high levels of [Ca 2+ ] i and was observed to exhibi t reduced activity following 

### 19
Pb exposure. The inhibition of flippase is additionally influenced by the depletion of cellular adenosine 

### 20
triphosphate (ATP). ATP levels were decreased in a dose

- dependent manner following exposure to Pb. To 

### 21

confirm these findi ngs in vivo, Shin et al. ( 2007 ) exposed male rats i.p. to 25, 50, or 100 mg/kg Pb 

### 22

aceta te. Expression of PS was observed to increase in a concentration

- dependent manner at concentrations 

### 23 ≥ 50 mg/kg, confirming the in vitro results. No hemolysis or microvesicle formation was observed in the 

### 24
in vitro and in vivo experiments. Ciubar et al. ( 2007 ) also found that exposure to Pb nitrate (0.5

- 2 μM) 

### 25
resulted an increase in PS exposure and cell shrinkage, which they stated were indicator s of cell 

### 26 apoptosis. As reported above, Khaĭrullina et al. ( 2008 ) observed RBC membrane smoothing that may be 

### 27
due to alterations in AE1 activity. Disruptions in AE1 activity may also result in enhanced PS exposure 

### 28
and premature cell death. Akel et al. ( 2007 ) observed that in AE1 - /

- mice, PS exposure was much greater 

### 29
than in wild type mice. Decreased RBCs and increased reticulocytes were also observed, an indication of 

### 30
high cell turnover. 

### 31May 2011 5

- 283 DRAFT

– DO NOT CITE OR QUOTE 

### 5.7.2.3. Red Blo od Cell H ematopoiesis

Erythropoietin is a glycoprotein hormone excreted by the kidney to promote the development of 

### 1

RBCs in the bone marrow. Sakata et al. ( 2007 ) examined the relationship between blood Pb level and 

### 2
serum erythropoietin levels in Pb

- exposed tricycle taxi drivers (n = 27) working at Kathmandu who were 

### 3

not anemic. The average blood Pb level in the taxi drivers was 6.4 μg/dL compared to 2.4 μg/dL in 

### 4
nondrivers. Drivers had a significantly lower level of serum erythropoietin (12.7 versus 18.8 mU/mL) 

### 5
compared to the nondrivers and there was a statistically sig nificant inverse relationship between the level 

### 6
of serum erythropoietin and blood Pb (r =

- 0.68, p <0.001). No other hematological effects were 

### 7

observed. The Sakata et al. ( 2007 ) study demonstrated that serum erythropoietin levels are affected by Pb 

### 8
even at levels low enough not to cause anemia. While this is generally considered a measure of kidney 

### 9
toxicity, it can also be considered an indicator that Pb could possibly affect the level of RBCs through 

### 10
decreased levels of serum erythropoietin. 

### 11

Celik et al. ( 2005 ) observed that exposure of female rats to 140, 25 0, or 500 mg/kg Pb acetate once 

### 12
per week for 10 weeks resulted in statistically significantly decreased numbers of polychromatic RBCs 

### 13
(PCE) and increased numbers of micronucleated PCEs, compared to controls (p <0.001). Alghazal et al. 

### 14
( 2008 ) exposed male an d female rats to 100 mg/L Pb acetate daily for 125 days and observed statistically 

### 15
significant increases in micronucleated PCEs in female rats (p = 0.02) but no significant reduction in the 

### 16
ratio of PCEs to normochromic RBCs (NCE). In male rats, a signific ant increase in micronucleated PCEs 

### 17
was observed (p <0.001) along with a decrease in the PCE/NCE ratio (p = 0.02). While the results from 

### 18
Alghazal et al. ( 2008 ) indicate that Pb is cytotoxic in male rats only, but is genotoxic in both sexes, Celik 

### 19

et al. ( 2005 ) indicates Pb is cytotoxic in female rats as well. Mice exposed to 1 g/L Pb acetate in drinking 

### 20
water for 90 days had statistically significant increases in micronucleated PCEs; a small, but not 

### 21
statistically significant decrease in the PCE/N CE ratio was also observed ( C. C. Marques et al., 2006 )

. 

### 22
Cyto

- and genotoxicity in RBC precursor cells is a strong indication of altered hematopoiesis in bone 

### 23

marrow. 

### 24

5.7.2.4. Membrane Proteins

There have been few studies examini ng the effects of Pb on membrane proteins since the 2006 Pb 

### 25
AQCD. According to the 2006 Pb AQCD report, Pb has been found to affect RBC membrane 

### 26
polypeptides in exposed workers ( Fukumoto et al., 1983 ) ; ( Apostoli et al., 1988 ) . Fukumoto et al. ( 1983 ) 

### 27
found decreased levels of polypeptides in band 3, which Apostoli et al. ( 1988 ) suggested may represent an 

### 28
anion channel protein, and increases in the level of polypeptides in bands 2, 4, 6, and 7. Fukumoto et al. 

### 29
( 1983 ) suggested that the changes in the RBC membrane polypeptides may cause changes in me mbrane 

### 30
permeability. Apostoli et al. ( 1988 ) found that the changes in membrane polypeptides occurred at blood 

### 31
Pb levels greater than 50 μg/dL. Exposure to Pb acetate at concentrations above 100 nM for 60 minute 

### 32May 2011 5

- 28 4 DRAFT

– DO NOT CITE OR QUOTE has also been found to increase the phosphorylation of proteins in human RBC m embranes in vitro 

### 1
( Belloni

- Olivi et al., 1996 ) . Phosphorylation did not occur in cells depleted of protein kinase C (PKC), 

### 2
indicating a PKC

- dependent mechanism. 

### 3

Huel et al. ( 2008 ) found that newborn hair and cord blood Pb levels (1.22 ± 1.41 μg/g and 3.54 ± 

### 4
1.72 μg/dL) were negatively associated with Ca

- ATPase activity in plasma membranes of RBCs isolated 

### 5
from cord blood; newborn hair Pb levels were more strongly associated with cord Ca pump activity than 

### 6
cord blood Pb (p <0.0001 versus. p <0.05). Mate rnal Pb levels were not correlated with Ca pump activity 

### 7

in maternal or cord blood. Pb

- induced disruptions in Ca homeostasis in RBCs can lead to cytotoxicity and 

### 8
necrosis, and these effects may be representative of cellular dysfunction in other organ syste ms. 

### 9



## 5.7.3. Effects on Red Blood Cell H eme M etabolism

Pb has been found to inhibit several enzymes involved in heme synthesis, namely ALAD 

### 10
(cytoplasmic enzyme catalyzing the second, rate

- limiting, step of the heme biosynthesis pathway), 

### 11
coporphyrinogen oxidase (c atalyses the sixth step in heme biosynthesis converting coporphyrinogen III 

### 12
into protoporphyrinogen IX), and ferrochelatase (catalyses the terminal step in heme synthesis converting 

### 13
protoporphyrin IX into heme) (Figure 5

- 53). The observation of decreased H b (measured as total Hb, 

### 14
MCH, or MCHC) in occupationally exposed adults ( Karita et al., 2005 ; D. A. Khan et al., 2008 ; Patil, 

### 15
Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 2006 ; Ukaejiofo et al., 2009 ) , children ( Ri ddell et al., 

### 16

2007 ) , and experimental animal models ( M. K. Lee et al., 2005 ; C. C. Marques et al., 2006 ; Sharma e t al., 

### 17

2010 ; Simsek et al., 2009 ) is a direct measure of decreased heme synthesis due to Pb intoxication. 

### 18May 2011 5

- 285 DRAFT

– DO NOT CITE OR QUOTE Note: Steps in the pathway potentially affected by Pb are indicated with curved arrows pointing to the affected enzyme, and effects are represe nted by ↑ and ↓ arrows. Figure 5

- 53. Schematic representation of the enzymatic steps involved in the heme synthetic pathway.



### 5.7.3.1. Red Blood Cell 5

- Aminolevulinic Acid Dehydratase

Decreases in RBC 5

- aminolevulinic acid dehydratase (ALAD) levels are strongly associated with 

### 1
Pb exposure in humans to such an extent that RBC ALAD activity has been used to assess Pb toxicity. 

### 2
Several epidemiologic studies published since the 2006 Pb AQCD evaluated the relationship between Pb 

### 3
exposure, bl ood Pb levels and ALAD activity. 

### 4

Patil et al. ( 2006 ) examined jewelry workers (blood Pb = 48.46 ± 7.39 μg/dL) in India 

### 5
occupationally exposed to Pb. In the study both the activated and nonactivated ALAD activities were 

### 6
measured. The study authors state that this is because decreases in ALAD activity reached a plateau and 

### 7

anemia can result in increased ALAD levels. Therefore, they considered the ratio of 

### 8
activated/nonactivated ALAD to be a good indicator of Pb toxicity. As described in the 2006 Pb AQCD, 

### 9

Scheuhammer ( 1987 ) studied the usefulness of this ratio in avian RBCs and found it to be a sensitive, 

### 10

dose

- dependent measure of Pb exposure regardless of th e route of exposure. They found that the 

### 11
activated/nonactivated ALAD ratio could be used to determine the oral exposure due to the highly 

### 12
positive correlation between Pb exposure in the 5

– 100 ppm range and ALAD activity ratio. Patil et al. 

### 13
( 2006 ) observed a statistically significant decrease in nonactivated ALAD and an increase in the ratio of 

### 14
activated to nonactivated ALAD, compared to nonexposed controls (p <0.05 and 0.001, respectively) . 

### 15May 2011 5

- 286 DRAFT

– DO NOT CITE OR QUOTE The study authors state that this indicates the inhibition of heme synthesis. Urinary excretion of both ALA 

### 1
and PBG was statistically significantly increas ed in jewelry workers, a further indication of RBC ALAD 

### 2
inhibition. Similar results were seen in battery manufacturing workers in India ( Patil, Bhagwat, Patil, 

### 3
Dongre, Ambekar, Jailkhani, et al., 2006 ) . Pb

- exposed workers (blood Pb = 74.4 ± 21.9 μg/dL) in a 

### 4
recycled automobile battery factory in Mexico had a significant decrease (~90%) in RBC ALAD activity 

### 5
compared to unexposed worke rs (9.9 ± 2 μg/dL) ( Quintanar

- Escorza et al., 2007 ) . Painters in India with 

### 6
an average blood Pb level of 21.92 μg/dL had a significant decrease in ALAD levels compared to controls 

### 7
with an average blood Pb level of 3.06 μg/dL (p <0.01) ( Mohammad et al., 2008 ) . Stoleski et al. ( 2008 ) 

### 8
observed that workers in a Pb smelter in Macedonia (blood Pb = 16.4 ± 8.5 μg/dL) had significantly 

### 9
decreased ALAD activity (p <0.001) and increased ALA levels (p <0.0 005) compared to workers with no 

### 10
exposure to (blood Pb = 7.0 ± 5.4 μg/dL). Lastly, Ademuyiwa et al. ( 2005 ) observed that workers in 

### 11
mechanic workshops (blood Pb = 27.0 ± 1.1

- 48.9 ± 19.1 μg/dL μg/dL) had significant decreases in 

### 12
ALAD activity compared to controls (15.8 ± 2.8 μg/dL, p <0.001). Petrol station workers had the highest 

### 13
degree of ALAD inhibition relative to controls (77%), whereas welders only had a 36% decrease in 

### 14
ALAD activity compared to controls. A case report by Fonte et al. ( 2007 ) described a worker 

### 15
occupation ally exposed to Pb vapors (blood Pb = 148 μg/dL) with ALAD levels substantially decreased 

### 16
compared to normal levels (3 versus >25 U/L). Zinc protoporphyrin (ZPP) levels were also described to 

### 17
be greatly increased. Following chelation therapy, the patient’s clinical picture improved. 

### 18
Wang et al. ( 2010 ) found that even with low to moderate blood Pb levels, there was a 

### 19

concentration

- dependent decrease in ALAD activity in both children and adults (blood Pb = 7.1 and 6.4 

### 20
μg/dL, respectively) in rural southwest China. Further, Wang et al. ( 2010 ) observed that the relationship 

### 21
between blood Pb and ALAD activity was nonlinear and exponential, with more significant decreases in 

### 22
ALAD activity occurring with blood Pb levels grea ter than 10 μg/dL. No correlation was observed 

### 23
between urinary ALA levels and blood Pb. Ahamed et al. ( Ahamed et al., 2006 ) st udied male urban 

### 24
adolescents in India. The 39 adolescents were separated into groups according to their blood Pb levels 

### 25
(group 1: <10 μg/dL, group 2: >10 μg/dL). Although the groups were similar in their age, height, weight, 

### 26
and body mass index, group 2 ha d a significantly lower ALAD activity than group 1 (p <0.001). When all 

### 27
39 adolescents were examined together, an inverse relationship was found between blood Pb and ALAD 

### 28
activity. Ahamed et al. ( 2005 ) also observed decreased ALAD activity in Indian children (aged 4

- 12) with 

### 29

a mean blood Pb level of 11.39 ± 1.39 μg/dL compared to children with mean blood Pb levels of 3.93 ± 

### 30
0.61 μg/dL. Similar decreases were also observed in children 3

- 6 ye ars of age with >10 μg/dL, compared 

### 31
to children <10 μg/dL ( Y. P. Jin et al., 2006 )

. 

### 32
Rats administered 500 ppm Pb acetate in drinking water for 15 or 30 days had decreased blood 

### 33
ALAD activity that was related to duration of exposure and blood Pb ( Rendón

- Ramirez et al., 2007 )

. 

### 34

Administration of Pb (25 mg/kg) to rats once a week for 4 weeks achieved a blood Pb level of 6.5 μg/dL, 

### 35May 2011 5

- 287 DRAFT

– DO NOT CITE OR QUOTE which were associated with significant decreases (approximately 50% lower than control levels) in RBC 

### 1
ALAD activity ( M. K. Lee et al., 2005 )

. 

### 2



## 5.7.4. Other Heme Met abolism Enzymes

The 2006 Pb AQCD report indicates Pb affects RBC PBG synthase ( Farant & Wigfield, 1987 ) ; 

### 3
( Farant & Wigfield, 1990 ; Simons, 1995 ) , PBG deaminase ( Tomokuni & Ichiba, 1990 ) , and TF 

### 4
endocytosis and iron transport across me mbranes ( Z. M. Qian & Morgan, 1990 ) , all of which are directly 

### 5
or indirectly involved in heme synthesis. Although there are no new studies that measure the effect Pb has 

### 6
on other heme metabolism enzymes’ activities, a number of studies investigated the effect blood Pb had 

### 7
on concentrations of various intermediate products in the heme biosynthetic pathway. 

### 8
Pb intoxication is known to inhibit the function of ferrochelatase, the enzyme that catalyzes the las t 

### 9
step in the heme biosynthetic pathway. Under normal conditions, ferrochelatase incorporates ferrous iron 

### 10
(Fe 2+ ) into protoporphyrin IX, converting it into a heme molecule. However, Pb inhibits this insertion of 

### 11

Fe 2+ into the protoporphyrin ring and inste ad, Zn is inserted into the right creating ZPP. A number of 

### 12
recent studies have shown that blood Pb is statistically significantly associated with increased RBC ZPP 

### 13

levels in humans ( Ademuyiwa, Ugbaja, Ojo, et al., 2005 ; Counter et al., 2007 ; Mohammad et al., 2008 ; 

### 14
Patil, Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 2006 ) and animals ( Rendón

- Ramirez et al., 

### 15

2007 ) . Patil et al. ( 2006 ) observed a small, but not statistically significant increase in RBC ZPP levels 

### 16
among silver jewelry workers exposed to Pb, compared to nonexposed controls. Interestingly, Q. Wang et 

### 17

al. ( 2010 ) found that in children and adults living in a rural area of Southwest China, ZPP levels were 

### 18
decreased at the low blood levels of Pb and were only increased with higher blood Pb levels. The authors 

### 19
suggest that this may be representative of ALAD activitie s at low Pb levels, which contributes to lower 

### 20

ZPP levels. Scinicariello et al. ( 2007 ) performed a meta

- analysis and observed that Pb

- exposed 

### 21
individuals that carried the ALAD2 allele had slightly, but not statistically significant, lower 

### 22
concentrations of blood ZPP levels compared to carriers of the ALAD1 allele. 

### 23



## 5.7.5. Effects on Other Hematological Parameters

5.7.5.1. Energy Metabolism

RBCs use high energy purine nucleotides (i.e., ATP and guanine triphosphate [GTP]) to support 

### 24
basic metabolic functions. In mature RBCs, these nucleotides are synthesized via salvage reactions via 

### 25

either an adenin e pathway, which requires adenine phosphoribosyltransferase (APRT), or an adenosine 

### 26
pathway, which requires adenosine kinase. The 2006 Pb AQCD reports that Pb significantly reduces the 

### 27
nucleotide pool including NAD and NADP, as well as increases purine deg radation products resulting in 

### 28
altered RBC energetics. Since the 2006 report, there have been few studies examining Pb effects on 

### 29May 2011 5

- 288 DRAFT

– DO NOT CITE OR QUOTE energy metabolism. Baranowska

- Bosiacka et al. ( 2009 ) examined the effects of Pb on RBC APRT and 

### 1
hypoxanthine

- guanine phosphoribosyltransferase (HPRT) due to in vitro and in vivo ex posures. For the in 

### 2
vitro exposure, APRT and HPRT were measured in lysate of human RBCs after exposure to Pb at 

### 3
concentration range from 100 nM to 100 μM for 5

- 30 minutes of exposure. In vivo tests measured APRT 

### 4
and HPRT in rat RBC lysate from rats exposed to Pb acetate (0.1 %) in drinking water for 9 months. Both 

### 5
the in vivo and vitro studies found a significant decrease in both HPRT and APRT levels. The levels were 

### 6
significantly decreased in vitro after only 5 minutes of exposure to the 100 nM concentrati on, but the 

### 7

decrease was also dose

- dependent. However, the study authors considered the inhibition moderate 

### 8
(30

- 35%) even with the highest levels used in vitro. Shin et al. ( 2007 ) found a dose

- dependent decrease in 

### 9
intracellular ATP in human RBCs in vitro with significant decreases found even with the lowest 

### 10
concentration (i.e., 1 μM). 

### 11

5.7.5.2. Other Enzymes

The 2006 Pb AQCD reports that K + permeability was increased by Pb due to altered sensitivity of 

### 12

the membrane Ca 2+

- binding site that caused selective efflux of K + ions from the RBC membrane. 

### 13
However, inhibition of the RBC Na +

- K + ATPase is more sens itive to Pb exposure than the inhibition of 

### 14

Ca 2+

- Mg 2+ ATPase. Only two studies since the 2006 report were found that examined the effects of Pb 

### 15
exposure on other enzymes. Ekinci et al. ( 2007 ) tested the effects of Pb on two carbonic anhydrase 

### 16
isozymes (I and II) isolated from human RBCs. Carbonic anhydrases are metalloprotein that use Zn to 

### 17
catalyze the equilibrium betwee n carbon dioxide and bicarbonate in the cells of higher invertebrates. 

### 18
Although they found Pb nitrate inhibited both carbonic anhydrase isozymes in a concentration

- dependent 

### 19
manner, the concentrations used (i.e., 2 × 10 - 4 to 1 × 10 - 3 M) were above those th at would be 

### 20
physiologically relevant. Inhibition of isozyme I was noncompetitive, while the inhibition for isozyme II 

### 21
was uncompetitive. Bitto et al. ( 2006 ) examined the mechanisms of action of Pb

- induced inhibition of 

### 22
P5N, an enzyme important in the pyrimidine salvage pathway that requires manganese for normal 

### 23
activity. Pb was observed to bind directly to the enzyme’s active site in a different position than the 

### 24
manganese, thus possibly resulting in improper protein folding and inhibition of activity. 

### 25



## 5.7.6. Red Blood Cell Oxidative Stress

It has been suggested that the Pb

- associated decreases in ALAD activit y result in increased 

### 26
oxidative stress, owing to the buildup of ALA. ALA can act as an electron donor in the formation of 

### 27
reactive oxygen species (ROS) ( Ahamed & Siddiqui, 2007 ; Nemsadze et al., 2009 ) . Many studies have 

### 28
found an association between the level of blood Pb and lipid peroxidati on, antioxidant levels, or 

### 29
indicators of ROS production. 

### 30May 2011 5

- 289 DRAFT

– DO NOT CITE OR QUOTE 

### 5.7.6.1. Oxidative Stress, Lipid Peroxidation, and Antioxidant Enzymes

Malondialdehyde (MDA) is an end product of lipid peroxidation and is commonly used as an 

### 1
indicator of lipid peroxidation. Patil et al. ( 2006 ) found significantly higher blood Pb in the jewelry 

### 2
workers (48.56 ± 7.39 μg/dL) comp ared to individuals not occupationally exposed (12.52 ± 4.08 μg/dL) 

### 3
to Pb. These workers had significantly higher plasma MDA, along with significantly lower levels of RBC 

### 4
SOD, catalyses, and plasma ceruloplasm, all of which are indicators of oxidative stre ss in the RBCs. Patil 

### 5

et al. ( 2006 ) found similar effects in a group of battery manufacturing workers in India. Levels of MDA 

### 6
were significantly positively correlated with blood Pb in workers occ upationally exposed to Pb, but no 

### 7
correlation was observed in controls. These effects were also demonstrated in vitro by Ciubar et al. 

### 8
( 2007 ) , but only with concentrations of 2 μM (highest concentration tested). In this study, RBCs from 

### 9
nine volunteers were incubated with Pb at concentrations ranging from 0.1

- 2 μM for 24 hours. Evidence 

### 10
of lipid peroxidation was also observed in auto repair app rentices in Turkey with blood Pb levels as low 

### 11
as 7.9 μg/dL (compared to 2.6 μg/dL in controls) ( Ergurhan

- Ilhan et al., 2008 ) , including increases in 

### 12 glutathione peroxidase (GPx) and MDA, as well as decreases in α

- tocopherol and β

- carotene. Decreases 

### 13
were observed in SOD and CAT, but the results did not achieve statistical signi ficance. Decreased 

### 14
glutathione reductase (GR) activity was observed in human RBCs incubated with 5

- 18 μM Pb in vitro 

### 15
( Coban et al., 2007 ) . Industrial workers in Pakistan occupationally exposed to Pb had a significant 

### 16
increase in blood Pb levels (mean: 29.1 μg/dL, range: 9.0 to 61.1 μg/dL) compared to age and gender 

### 17
matched controls (mean: 8.3 μg/dL, range: 1.0 to 21.7 μg/dL) ( D. A. Khan et al., 2008 ) . The industrial 

### 18
workers also had increased oxidative stress as measured by increased levels of serum MDA and 

### 19

C

- reactive protein (CRP). In painters in India with an average b lood Pb level of 21.92 μg/dL (compared 

### 20
to 3.06 μg/dL in controls), there was a significant decrease in SOD, glutathione (GSH), and CAT 

### 21
accompanied by a significant increase in oxidized GSH (i.e., GSSG) and thiobarbituric acid reactive 

### 22
species (TBARS, expre ssed in terms of MDA) measured in plasma and RBC lysate ( Mohammad et al., 

### 23

2008 ) . Quintanar

- Escorza et al. ( 2 007 ) found elevated RBC lipid peroxidation measured as increased 

### 24

MDA levels in Pb

- exposed workers in a recycled automobile battery factory in Mexico. There was a 

### 25
correlation in the MDA levels and blood Pb even in the unexposed workers who had low (i.e., <12 μg/dL) 

### 26
blood Pb levels, although the observed correlation in exposed workers was greater. Similar effects were 

### 27 seen when RBCs from healthy volunteers with no Pb exposure were incubated with 0.4 μM Pb for 24 

### 28

hours ( Quintanar

- Escorza et al., 2010 ) . MDA concentrations, SOD and GPx activities were observed to be 

### 29

elevated in normotensive ex posed workers compared to controls ( S. Kasperczyk et al., 2009 ) . The 

### 30
concentration of MDA was statistically significantly greater in workers with hypertension compared to 

### 31

both controls and Pb

- exposed normotensive workers, whereas the act ivity of GR in hypertensive workers 

### 32
decreased to levels comparable to those seen in the control group. Exposure related increases in lipid 

### 33
peroxidation were also observed in occupationally exposed workers in Poland ( S. Kasperczyk et al., 

### 34May 2011 5

- 290 DRAFT

– DO NOT CITE OR QUOTE 2005 ) . The concentrations of MDA and 7

- ketocholesterol (oxidized cholesterol) were significantly greater 

### 1
in exposed hypertensive workers compared to normotensive wor kers. 

### 2

Ahamed et al. ( 2005 ) investigated the relationship between blood Pb levels and antioxidant enzyme 

### 3
levels and lipid peroxidation in children aged 4

- 12 years in Lucknow, India. A tota l of 62 children, with a 

### 4
mean blood Pb level of 7.47 ± 3.06 μg/dL, were included in the study; children were separated into three 

### 5
groups based on their blood Pb levels: group I, 3.93 ± 0.61 μg/dl; group II, 7.11 ± 1.25 μg/dL; and group 

### 6
III, 11.39 ± 1.39 μg /dL. Lipid peroxidation, measured as blood MDA, was statistically significant greater 

### 7
in group III, compared to group II and I, whereas GSH was decreased in group III relative to groups II 

### 8
and I. Catalase activity was the only measure of oxidative stress t hat was statistically significantly 

### 9
elevated in group II compared to group I. Additionally, blood Pb levels were found to be statistically 

### 10
significantly positively correlated with MDA and CAT and negatively correlated with GSH. Ahamed et al. 

### 11
( 2006 ) additionally studied male urban adolescents in India. The 39 adolescents were separated into 

### 12
groups according to their blood Pb level (group 1: <10 μg/dL, group 2: >10 μg/dL). Although the groups 

### 13
were similar in their age, height, weight, and body mass index, group 2 had significantly higher levels of 

### 14
CAT and MDA compared to group 1. There were no significant differences in blood GSH levels. 

### 15
Examining all th e study subjects together, there was a correlation between the blood Pb level and blood 

### 16
MDA and RBC CAT levels, as well as an inverse relationship between ALAD activity and MDA and CAT 

### 17
levels. In a similar study, Ahamed et al. ( 200 8 ) examined oxidative stress in Indian children with 

### 18
neurological disorders. There was a significantly higher blood Pb level in the study population compared 

### 19
to the control population (18.60 versus 10.37 μg/dL). In addition, the following indicators of oxidative 

### 20
stress were observed in the study population: increased blood MDA, RBC SOD, and CAT levels and 

### 21
decreased blood GSH levels. GPx levels were similar between the two groups. Typical indicators of Pb 

### 22
exposure (active/nonactive ALAD ratio and) were fo und to be correlated with lipid peroxidation and 

### 23
oxidative stress. Children aged 3

- 6 years old living near a steel refinery in China with blood Pb levels ≥ 

### 24
10 μg/dL also demonstrated a significant increase in plasma MDA compared to the children with blood 

### 25
Pb levels<10 μg/dL. However, levels of RBC SOD, GSH, and GPx were not different from controls ( Y. P. 

### 26
Jin et al., 2006 )

. 

### 27
Administration of Pb (25 mg/kg) to rats once a week for 4 weeks, which was related to a blood Pb 

### 28

level of about 6.5 μg/dL, caused a significant increase in RBC MDA levels ( M. K. Lee et al., 2005 ) . Other 

### 29
indications of oxidative stress included significant increases in RBC SOD and CAT levels accompanied 

### 30
by significant decreases in GSH and GPx. Exposure of rats to 750 mg/kg Pb acetate in drinking water for 

### 31
11 weeks resulted in decreased concentrations of plasma vitamin C, vitamin E, nonprotein thiol, and 

### 32

RBC

- reduced glutathione, while simultaneously increasing the activity of SOD and GPx ( Kharoubi, 

### 33
Slimani, Krouf, et al., 2008 ) . CAT activity was also slightly elevated in Pb

- exposed rats, but the increase 

### 34
failed to reach statistical signifi cance. SOD activity was significantly decreased in rats injected with 15 

### 35
mg/kg Pb i.p. for seven days, but not rats exposed to 5 mg/kg Pb ( Berrahal et al., 2007 ) . GPx activity and 

### 36May 2011 5

- 291 DRAFT

– DO NOT CITE OR QUOTE MDA concentrations were slightly elevated in the exposure groups, but failed to reach statistical 

### 1
significance. 

### 2

5.7.6.2. Antioxidant Defense

In addition to the studies li sted above that examine lipid peroxidation and oxidative stress, there 

### 3
have been studies that indicate that the use of antioxidants and free radical reactions were protective 

### 4
against Pb

- induced RBC oxidative stress. Rats treated with 500 ppm Pb acetate in drinking water for 15 

### 5
or 30 days had an increase in free RBC protoporphyrin and TBARS that was related to length of exposure 

### 6

and blood Pb ( Rendón

- Ramirez et al., 2007 ) . Vitamin E administration after exposure to Pb significantly 

### 7
reduced the TBARS levels and increased ALAD activity, compared to exposure to Pb alone. Co

- exposure 

### 8
to vitamin E and Pb simultaneously and exposure to vitamin E before Pb exposure also prevented Pb



### 9
induced oxidative stress. In vitro studies by Casado et al. ( 2006 ) , found that hemolysis and RBC 

### 10
membrane damage was mediated via oxidative stress. The in vitro studies demonstrated a concentration



### 11

and time

- dependent formation in lipid peroxide that was inhibited with a number of antioxidants, 

### 12
including desferrioxamine (iron chelator), trolox (chain breaking antioxidant), and mannitol and Na 

### 13
formate ( • OH scavengers). Results suggested the role of singlet oxygen in Pb

- mediated membrane 

### 14
damage and hemolysis of exposed RB Cs. In rats exposed to 2000 ppm Pb in drinking water for 5 weeks, 

### 15
MDA levels were significantly increased, whereas vitamin E concentrations were significantly decreased 

### 16
( Caylak et al., 2008 ) . In the case of MDA, co

- exposure to Pb and a number of sulfur

- containing 

### 17
antioxidants (e.g., L

- methionine, N

- acetylcysteine, and L

- homocysteine) reduced concentrations to a level 

### 18
not statistically significantly different from controls, but statistically smaller than concentrations observed 

### 19
with Pb alone. Exposure to L

- methionine an N

- acetylcysteine also reduced Pb

- induced depletion of 

### 20

vitamin E. 

### 21



## 5.7.7. Summary and Causal Dete rmination

There is consistent toxicological and epidemiologic evidence that exposure to Pb induces adverse 

### 22
effects on hematological endpoints, including altered heme synthesis, decreased RBC survival and 

### 23
function, and increased RBC oxidative stress

. Pb pre ferentially partitions into RBC s following exposure, 

### 24

with RBC concentrations approximately 100

- fold greater than those observed in the plasma ( C. Jin et al., 

### 25

2008 ; Timchalk et al., 2006 )

. 

### 26
Multiple occupational epidemiologic studies have observed that Pb affects several hematological 

### 27
parameters such as Hb , PCV, MCV, MCH, and MCHC ( Karita et al., 2005 ; D. A. Khan et al., 2008 ; Patil, 

### 28
Bhagwat, Patil, Dongre, Ambekar, & Das, 2006 ; Patil, Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 

### 29

2006 ; Ukaejiofo et al., 2009 )

. Although the majority of occup ationally exposed adults had blood Pb levels 

### 30
in excess of 20 μg/dL, decreases in Hb and PCV were observed in adults with blood Pb levels of 7 μg/dL

. 

### 31May 2011 5

- 292 DRAFT

– DO NOT CITE OR QUOTE In addition, Pb exposure was shown to reduce Ca 2+

- and Ca 2+

- Mg 2+

- ATPase activity in RBC membranes, 

### 1

which le ads to an increase in RBC [Ca 2+ ] i , increased membrane fragility, and abnormal morphological 

### 2
changes ( Ciubar et al., 2007 ; Huel et al., 2008 ; Quintanar

- Escorza et al., 2010 ; Quintana r

- Escorza et al., 

### 3

2007 ) . Heul observed a reduction of RBC Ca 2+

- Mg 2+

- ATPase activity at a cord blood Pb level of 3.54 

### 4
μg/dL. Studies in children are more equivocal than those investigating occup ationally exposed adults; this 

### 5
may due to the comparatively shorter duration of and magnitude of exposure experienced by children

. 

### 6
Toxicological studies have also observed decreases in Hct and Hb and increases in hemolysis and 

### 7
reticulocyte density in rats and mice with blood Pb levels as low as 6.6

- 7.1 μg/dL ( Baranowska

- Bosiacka 

### 8
et al., 2009 ; M. K. Lee et al., 2005 ; Sharma et al., 2 010 ; Simsek et al., 2009 )

. Pb exposure has also been 

### 9

observed to increase PS expression on RBC membranes, leading to cell shrinkage, eyrptosis, and 

### 10
destruction of the RBCs by macrophages ( Ciubar et al., 2007 ; Shin et al., 2007 )

. Suggestive evidence of 

### 11
disrupted hematopoiesis evidenced by decreased serum erythropoietin was observed in occupationally 

### 12
exposed adults with a blood Pb level of 6.4 μg/dL; toxicological studies in rats also indicate that Pb is 

### 13
cytotoxic to RBC progenitor cells

. Taken together, these studies provide consistent evidence that exposure 

### 14
to Pb adversely effects RBC function and survival, and leads to the reduct ion of RBC s in circulation

. 

### 15
Although this decrease in RBC s may be explained by both decreased cell survival and/or disruption of 

### 16
hematopoiesis, the observation of increased reticulocytes seems to represent compensation for decreased 

### 17

RBC survival due to Pb exposure

. 

### 18
Recently, numerous epidemiologic studies have confirmed that decreases in RBC ALAD levels and 

### 19
activity are strongly associated with Pb exposure in adults and children at blood Pb levels as low as 6.4 

### 20

and 7.1 μg/dL , respectively ( Ademuyiwa, Ugbaja, Ojo, et al., 2005 ; Mohammad et al., 2008 ; Patil, 

### 21
Bhagwat, Patil, Dongre, Ambekar, & Das, 2006 ; Patil, Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 

### 22

2006 ; Quintanar

- Escorza et al., 2007 )

. Decreases in blood ALAD activity were also seen in rats with 

### 23

blood Pb levels of 6.5 μg/dL ( M. K. L ee et al., 2005 )

. In addition to ALAD, recent studies have shown 

### 24
that Pb exposure inhibits the activity of ferrochelatase, leading to increased RBC ZPP in humans 

### 25
( Ademuyiwa, Ugbaja, Ojo, et al., 2005 ; Counter et al., 2007 ; Mohammad et al., 2008 ; Patil, Bhagwat, 

### 26
Patil, Dongre, Ambekar, Jailkhani, et al., 2006 ) and animals ( Rendón

- Ramirez et al., 2007 )

. Pb has also 

### 27
been shown to inhibit the activities of other enzymes in RBCs, including those involved in nucleotide 

### 28
scavenging, en ergy metabolism, and acid

- base homeostasis ( Baranowska

- Bosiacka et al., 2009 ; Ekinci et 

### 29
al., 2007 )

. 

### 30
Lastly, Pb exposure induces lipid peroxidation and oxidative stress in RBCs

. Epidemiologic studies 

### 31

have observe d increases in MDA in occupationally

- exposed adults with blood Pb levels as low as 7.9 

### 32
μg/dL ( Ergurhan

- Ilhan et al., 2008 ; D. A. Khan et al., 2008 ; Mohammad et al., 2008 ; Patil, Bhagwat, 

### 33
Patil, Dongre, Ambekar, & Das, 2006 ; Patil, Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 2006 ; 

### 34
Quintanar

- Escorza et al., 2007 )

. Other measures of oxidative stress observed included lowered activities 

### 35
of SOD, GR , and CAT, and increased CRP. Indices of R BC oxidative stress were also seen in adolescents 

### 36May 2011 5

- 293 DRAFT

– DO NOT CITE OR QUOTE and children exposed to Pb ( Ahamed et al., 2008 ; Ahamed et al., 2006 ; Y. P. Jin et al., 2006 )

. In vitro and 

### 1
vivo studies have also demonstrated that prior , con current, or subsequent treatment with various 

### 2
antioxidants has been shown to at least partially ameliorate Pb

- induced oxidative stress in RBCs ( Casadoa 

### 3
et al., 2006 ; Cecil et al., 2008 ; Rendón

- Ramirez et al., 2007 )

. 

### 4
Similar to the epidemiologic and toxicological studies that demonstrate an association between Pb 

### 5
exposure and hematological effects in humans and lab oratory animals, the ecological literature has 

### 6
consistently reported on hematological responses in aquatic and terrestrial invertebrates and vertebrates 

### 7
( Section 7.4.5 ). The most consistently observed effect in metal impacted environments is decreased RBC 

### 8
ALAD activity. This effect has been observed across a wide range of taxa, including bivalves, fish, 

### 9
amphibians, birds, and mammals. More limited evidence exists regarding deleterious effects of Pb on 

### 10
serum enzyme levels and white blood cell counts in birds and mammals. 

### 11
In conclusion, the recent epidemiologic and toxicological literature provides strong evidence that 

### 12
exposure to Pb is associated with numerous deleterious effects on the hematological system, including 

### 13
altered heme synthesis mediated through d ecreased ALAD and ferrochelatase activities, decreased RBC 

### 14
survival and function, decreased hematopoiesis, and increased oxidative stress and lipid peroxidation. The 

### 15
consistency of findings in the epidemiologic and toxicological literature and coherence ac ross the 

### 16
disciplines is sufficient to conclude that there is a causal relationship between Pb exposures and effects 

### 17
on heme synthesis and red blood cell function. 

### 18



# 5.8. Reproductive Effects and Birth Outcomes

The effect of Pb on reproductive outcomes has been of interest for years, starting in cohorts of 

### 19
occupationally exposed individuals. More recently, researchers have begun to focus on reproductive 

### 20
effects in people with environmentally relevant Pb exposure. In the toxicological and epidemiologic 

### 21
literature, r esearch on reproductive effects of Pb include female and male reproductive function (hormone 

### 22
levels, fertility, puberty, and effects on reproductive organs and estrus), birth defects, spontaneous 

### 23
abortions, infant mortality, preterm birth, low birth weight /fetal growth, and other developmental effects. 

### 24
In epidemiologic studies, various biological measures of Pb are used including Pb measured in blood and 

### 25
bone; toxicological studies only report exposure using blood Pb. Bone Pb is indicative of cumulative Pb 

### 26

exposure . Blood Pb can represent more recent exposure, although it can also represent remobilized Pb 

### 27
occurring during times of bone remodeling

. More detailed discussion of these measures and Pb transfer 

### 28
via umbilical cord blood Pb, across the placenta, and via lactation is given in Section 4.3.5 on Pb 

### 29

Toxicokinetics . A few studies of pregnancy

- induced hypertension and eclampsia have been conducted and 

### 30
are reported on in the section on hype rtension (Section 5.4.2.1). Briefly, t he relatively small number of 

### 31May 2011 5

- 294 DRAFT

– DO NOT CITE OR QUOTE studies found consistently positive associations between recent Pb exposure and pregnancy

- induced 

### 1
hypertension

. 

### 2
Overall, the recent reproductive literature continues to support associatio ns reported in earlier 

### 3
AQCDs between Pb exposure and adverse outcomes on various parameters of sperm (function, motility, 

### 4
count, integrity, histology). The toxicological and epidemiologic literature also support the finding that Pb 

### 5
exposure is consistently associated with delayed onset of puberty in both males and females. The new 

### 6
information from epidemiologic and toxicological studies and conclusions from previous AQCDs are 

### 7

summarized below. 

### 8



## 5.8.1. Effects on Female Reproductive Function

The epidemiologic studies presented on Pb and female reproductive function in the 2006 AQCD 

### 9
( U.S. EPA, 2006 ) provided little evidence on the possible associations between Pb exposure and female 

### 10
reproduction and fertility. However, the 1986 and 2006 Pb AQCDs ( U.S. EPA, 1986

, 2006 ) report ed 

### 11
toxicological findings that Pb exposure i s associated with effects on female repr oductive function that can 

### 12
be classified as alterations in female sexual maturation, effects on fertility and menstrual cycle, endocrine 

### 13
disruption, and changes in morphology or histology o f female reproductive organs including the placenta. 

### 14

Since the 2006 AQCD, many epidemiologic studies have been published regarding Pb levels in women 

### 15
and their effects on reproduction. In addition, r ecent toxicological studies add further knowledge of Pb



### 16

related effects on the female reproductive system

. 

### 17

5.8.1.1. Effects on Femal e Sex Endocrine System and Estrus Cycle

Multiple studies have examined the association between Pb and its effects on hormones and the 

### 18
estrus cyclce. Epidemiologic studies support the toxicological findings, which have the majority of the 

### 19

evidence. 

### 20

An epidemiologic study using the NHANES III database and including women aged 35

- 60 years 

### 21
old examined the relationship between blood Pb levels (mean 2.8 μg/dL) and serum follicle stimulating 

### 22
hormone (FSH) and luteinizing hormone (LH) ( E. F. Krieg, Jr., 2007 ) . Deviation from normal FSH and 

### 23
LH levels may indicate endocrine disruption related to ovary functioning. Researchers determined that as 

### 24

blood Pb levels increased, serum FSH and LH increased a mong both post

- menopausal women and 

### 25

women with both ovaries removed

. There was also an increasing trend for pre

- menopausal women who 

### 26
were not menstruating or pregnant, although the association was not statistically significant for LH

. 

### 27

I ncreasing blood Pb l evels were associated with decreasing levels of serum FSH among women taking 

### 28
birth control pills

. The inverse association was also present for LH, but it was not statistically significant

. 

### 29

No associations between blood Pb and FSH or LH were apparent for wo men who were menstruating at 

### 30
the time of the exam or were pregnant

. Further analysis found that the lowest level of blood Pb for which 

### 31May 2011 5

- 295 DRAFT

– DO NOT CITE OR QUOTE a statistically significant association could be observed between blood Pb and FSH was 1.7 μg/dL among 

### 1

women with their o varies removed . A limitation of the study is that FSH and LH were measured without 

### 2
attention to day of a woman’s menstrual cycle and LH and FSH are known to vary throughout the cycle. 

### 3
Another epidemiologic study was performed in Kaohsiung City, Taiwan amon g two groups of women 

### 4
aged 23

- 44 : those who were seeking help at a fertility clinic after one year of trying to conceive

, and those 

### 5
who had previously delivered an infant and were identified from medical records of a postpartum care 

### 6

unit ( S. H. Chang et al., 2006 ) . The mean blood Pb in this study was 3.12 μg/dL (SD 0.19 μg/dL ). The 

### 7
study reported a positive association between in creased blood Pb levels and serum estradiol 

### 8
concentrations, which reflects ovary activity. 

### 9
The effect of Pb exposure on the female endocrine system has been demonstrated in toxicological 

### 10

studies in the 1986 and 2006 Pb AQCD ( U.S. EPA, 1986

, 2006 ) . However, the mechanism by which Pb 

### 11
affects the endocrine system has not been fully elu cidated. Several recent articles continue to demonstrate 

### 12
that Pb alters the concentration of circulating hormones in female experimental animals. As mentioned 

### 13
previously, Pine et al. ( 2006 ) observed that maternal Pb exposure causes a decrease in basal LH levels in 

### 14

pre

- pubertal female Fisher 344 rat pups when compared to non

- Pb exposed pups during gestation and 

### 15

lactation. Dumitrescu et a l. ( 2008 ) observed alteration of hormone levels in female Wister rats after 

### 16
ingesting Pb acetate (50, 100, 150 ppb) in drinking water for six months. The authors reported decreases 

### 17
in FSH, estradiol, and progesteron e levels with increases in LH and testosterone levels. Nampoothiri and 

### 18
Gupta ( 2008 ) administered Pb acetate a t a concentration that did not affect reproduc tive performance, 

### 19

im plantation or pregnancy outcome ( 0.05 mg/kg body weight) to Charles Foster female rats 5 days before 

### 20
mating an d during the gestational period. They observed a decrease in steroidogenic enzymes, 3β

- HSD 

### 21 and 17β

- HSD, activity in reproducti ve organs, as well as a decrease in steroid hormones (progesterone 

### 22
and estradiol), suggesting that chronic exposure to low levels of Pb may affect reproductive function of 

### 23
mothers and their offspring. 

### 24

Kolesarova et al. ( 2010 ) conducted an in vitro study to examine the secretory activit y of porcine 

### 25
ovarian granulose cells after Pb administration

. The results of the study showed that Pb acetate 

### 26
concentrations of 0.046 mg/mL and 0.063 mg/mL statistically significantly inhibited IGF

- 1 release, but 

### 27
concentrations of 0.25 mg/mL and 0.5 mg/mL did not influence IGF

- 1 release. Progesterone release was 

### 28
not affected by Pb treatment ; however, Pb caused a reduction in LH and FSH binding in granulose cells 

### 29
and increased apoptosis as evidenced by increased expression of caspase

- 3 and cyclin B1, suggest ing a 

### 30

Pb

- induced alteration in the pathways of proliferation and apoptosis of porcine ovarian granulose cells. 

### 31
Decreased gonadotropin binding was also observed in rats after Pb exposure ( Nampoothiri & Gupt a, 

### 32

2006 )

. 

### 33

No recent toxicological studies were found that addressed P b

- induced effects on the estrus cycle. 

### 34
Overall, toxicological studies report alterations in hormone levels related to Pb concentration. This 

### 35
was also observed in epidemiologic studies. Although these changes are observed, there are discrepancies 

### 36May 2011 5

- 296 DRAFT

– DO NOT CITE OR QUOTE about the direction of the hormone changes related to Pb. One explanation is that the direction of effect 

### 1
could vary based on current hormonal and reproductive status. 

### 2

5.8.1.2. Effects on Fertility

Previ ous studies indicated that Pb exposure does not produce total sterility, but it can disrupt female 

### 3
fertility ( U.S. EPA, 2006 )

. Recent epidemiologic studies and studies in experimental animals support this 

### 4
finding . The epidemiologic studies are summarized in Table 5

- 2 7. 

### 5

Table 5

- 27. Summary of recent epidemiologic studies of effects on fertility for females

Reference Study
, Location
, and Years Outcome Study population Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Al

- Saleh et al. ( 2008 ) 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 levels >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) Chang et al. ( 2006 ) Kaohsiung City, Taiwan 1999, 2000

- 2001 Fertility 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) Fertility ≤2.5 μg/dl: 1.00 (Ref) >2.5 μg/dl: 2.94 (1.18, 7.34)

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

Epidemiologic studies examined women having difficulty conceiving by performing studies 

### 6
among patients of fertility clinics or undergoing in vitro fertilization (IVF)

. The first of these was 

### 7
performed in Kaohsiung City, Taiwan among women aged 23

- 44 ( S. H. Chang et al., 2006 )

. A difference 

### 8

in blood Pb was reported between women who were seeking help at a fertility clinic after one year of 

### 9
trying to conceive and women who had previously delivered an infant and were identified from medical 

### 10May 2011 5

- 297 DRAFT

– DO NOT CITE OR QUOTE records of a postpartum care unit at a medical center. Hi gher odds of infertility were observed when 

### 1
comparing women with blood Pb levels >2.5 μg/dL to those with blood Pb levels ≤ 2.5 μg/dL . Another 

### 2
study examining fertility reported on women in Saudi Arabia aged 1 9

- 50 years who were undergoing IVF 

### 3

treatment ( Al

- Saleh et al., 2008 )

. Women were categorized as having achieved a pregnancy versus not 

### 4
achieved a pregnancy and achieved fertilization versus not achieving fertilization

. The majority of women 

### 5
had follicular Pb levels that were below the level of detection, whereas less than 2% of women had blood 

### 6

Pb levels below the limit of detection

. In addition, less than 2% of women had blood Pb levels that were 

### 7
above 10 μg/dL

. Follicular Pb levels were not correlated with the blood Pb. No association was observed 

### 8
between blood or follicular Pb and pregnancy outcomes in either crude or adjusted models

. An association 

### 9

was not detected between follicular Pb and fertilization but an inverse association was detected between 

### 10

blood Pb and fertilization. Finally, a study that included nine women undergoing IVF treatment in Rhode 

### 11

Island ( Silberstein et al., 2006 ) found that median follicular Pb levels in women who achieved pregnancy 

### 12

wer e lower than the follicular Pb levels among non

- pregnant women . One limitation present in these 

### 13
studies is that the participants, especially in the later two studies, are women who are seeking help for 

### 14
fertility problems. The studies are not a sample of th e general population and therefore cannot be 

### 15
generalized to all women of childbearing age. 

### 16
Several studies observed a decrease in litter size when females were exposed to Pb before mating 

### 17
or during pregnancy ( Dumitrescu, Alexandra, et al., 2008 ; Iavicoli, Carelli, Stanek, Castellino, Li, et al., 

### 18

2006 ; Teijon et al., 2006 ) . Pups in Teijon et al .’ s study receiving 400 ppm Pb acetate in drinking water had 

### 19

blood Pb of 97 μ g Pb/ d L blood at 1 wk post

- weaning and 18.2 μ g Pb/ d L blood at 2 wk post

- weaning. 

### 20
Dumitrescu et al. observed a modification in sex ratio of pups born to dams exposed to Pb before mating 

### 21
and during pregnancy. As the dose of Pb increased, the number of fema les per litter also increased (i.e.

, 1 

### 22

male to 0.8 female in non

- Pb exposed group; 1 male to 0.66 female in 50 ppb Pb acetate group; 1 male to 

### 23
2.25 females in 100 ppb group; and 1 male to 2.5 females in 150 ppb group). These results are not 

### 24

consistent with earlier results of Ronis et al. ( 1998 ) , who did not observe differences in sex ratio if liters 

### 25
from females exposed only during pregnancy . Thus, Pb exposure in animal studies during or before 

### 26
pregnancy have shown effects on litter size and mixed effects on sex ratio. 

### 27

Nandi et al. ( 2010 ) demonstrated a dose

- dependent decline in viability rate, maturation, 

### 28

fertiliz ation, and cleavage rates of buffalo oocytes cult ured in medium containing 1

- 10 μ g/mL Pb acetate. 

### 29
Karaca and Şimşek ( 2007 ) observed an increase in the number of mast cells in ovary tissue after Pb 

### 30
exposure (2 g/L in drinking water) suggesting that Pb may stimulate an inflammatory response in the 

### 31
ovaries which may contr ibute to Pb

- induced female infertility. 

### 32
In contrast, Nampoothiri and Gupta ( 2008 ) did not observe any statistically significant change in 

### 33
fertility rate or litter size in female rats subcutaneously administered 0.05 mg/kg body weight daily before 

### 34
mating and during pregnancy with a resulting blood Pb of 2.49 μ g/mL. Although reproductive 

### 35May 2011 5

- 298 DRAFT

– DO NOT CITE OR QUOTE performance was not affected in this study, the authors did report an alt eration in implantation enzymes. 

### 1
Cathepsin

- D activity decreased and alkaline phosphatase activity increased after Pb exposure. 

### 2
Epidemiologic and toxicological studies on the effect of Pb on fertility outcomes have generated 

### 3
inconsistent results. However, t here is some indication that increased Pb exposure may decrease fertility. 

### 4

5.8.1.3. Effects on Puberty

Recent toxicological studies of rodents have examined the effects of Pb on pubertal and 

### 5
reproductive organ development and on biomarker development. There have al so been recent 

### 6
epidemiologic studies examining Pb levels and onset of puberty, which are summarized in Table 5

- 28 and 

### 7

in the text below. 

### 8

Table 5

- 28. Summary of recent epidemiologic studies of effects on puberty for females

Reference Study Location and Years Outcome Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

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 cente red 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 o dds of menarche attainment by 72% Gollenberg et al. ( 2010 ) U.S.A. 1988

- 1994 Luteinizing hormone (LH)

an d 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 pubertal inhi bi n B cutoff (>35pg/mL) <1 μg/dl: 1.00 (Ref) 1

- 4.9 μg/dl: 0.38 (0.12, 1.15) ≥ 5μg/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) ≥ 5μg/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 attenuatedMay 2011 5

- 299 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location and Years Outcome Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Naicker et al. ( 2010 ) Johann esburg/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 1990) 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 b reast development at age 13 <5 μg/dl: 1.00 (Ref) ≥ 5μg/dl: 2.34 (1.45, 3.79) Delay in p ubic hair development at age 13 <5 μg/dl: 1.00 (Ref) ≥ 5μg/dl: 1.81 (1.15, 2.84) Delay in a ttainment of menarche at age 13 <5 μg/dl: 1.00 (Ref) ≥ 5μg/dl: 2.01 (1.38, 2.94)
Selevan et al. ( 2003 ) U.S.A. 1988

- 1994 Tanner stag ing 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> 10μg/dL: NHWhites: 0.3 % NHBlacks: 1.6 % Mexican

Americans: 2.3 % OR (95% CI) Breast development NH Whites: 1 μg/dl: 1.00 (Ref) 3μg/dl: 0.82 (0 .47, 1.42) NH Blacks: 1 μg/dl: 1.00 (Ref) 3μg/dl: 0.64 (0.42, 0.97) Mexican Americans: 1 μg/dl: 1.00 (Ref) 3μg/dl: 0.76 (0.63 , 0.91)

Pubic hair development NH Whites: 1 μg/dl: 1.00 (Ref) 3μg/dl: 0.75 (0.37, 1.51) NH Blacks: 1 μg/dl: 1.00 (Ref) 3μg/dl: 0.62 (0.41, 0.96) Mexican Americans: 1 μg/dl: 1.00 (Ref) 3μg/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) 3μg/dl: 0.74 (0.55, 1.002) NH Blacks: 1 μg/dl: 1.00 (Ref) 3μg/dl: 0.78 (0.63, 0.98) Mexican Americans: 1 μg/dl: 1.00 (Ref) 3μg/dl: 0.90 (0.73, 1.11)
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 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% ≥ 10μg/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% ≥ 10μg/dl: Stage 2: 77.8% Stage 3: 22.2% Chi

- square p

- value>0.05

*Quantitative results for hormones not provided
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, assum e results are per 1 μg/dL) Breast stage: 1.01 (0.79, 1.30) Pubic hair stage: 1.25 (0.83, 1.88)May 2011 5

- 300 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location and Years Outcome Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Wu et al. ( 2003 ) U.S.A. 1988

- 1994 Tanner staging and age at menarche Girls a ges 8

- 16 from the NHANES III study Blood Pb 2.5 (2.2)

Weighted proportion of the sample with 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)

M enarche 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) Several epidemiologic studies investigated the association between blood Pb and indicators of 

### 1
puberty onset. A study performed in NYC among 9 year old girls reported no association between Pb 

### 2
levels and pubertal development ( Wolff et al., 2008 )

. However, a study among girls aged 10

- 13 (median 

### 3
age 12) reported decrea sed levels of FSH and LH levels in the group with blood Pb of at least 10 μg/dL 

### 4
compared to the group with blood Pb less than 10 μg/dL ( Tomoum et al., 2010 )

. In addition, there wer e 

### 5
some indications of lower Tanner stages of breast development associated with Pb levels of at least 10 

### 6
μg/dL, but this relationship was not present for stages of pubic hair development. A study of girls aged 

### 7

10

- 16.9 years of age in the Akwesasne Mohawk Nation reported a non

- linear positive association between 

### 8

blood Pb and age at menarche ( Denham et al., 2005 )

. No association was observed below blood Pb of 

### 9
0.49 μg/dL . A study conducted in South Africa reported a positive association between blood Pb levels 

### 10
and age at menarche and pube rtal development ( Naicker et al., 2010 ) . Blood Pb lev els were associated 

### 11
with delayed pubertal development and later age at menarche. This study illustrates not only the 

### 12
association between Pb and pubertal development, but that delays can occur at low Pb levels. Multiple 

### 13
studies have been performed examining blood Pb levels and puberty using the NHANES III database 

### 14
( Gollenberg et al., 2010 ; Selevan et al., 2003 ; T. Wu et al., 2003 )

. One study included girls aged 8

- 16 and 

### 15
reported an positive association for delayed attainment of menarche and pubic hair development , but not 

### 16
for breast development ( T. Wu et al., 2003 ) . The associations were observed even at low levels of blood 

### 17

Pb. An other NHANES III study included girls 8

- 18 years of age and reported the results stratified by race 

### 18
( Selevan et al., 2003 ) . Blood Pb levels were inversely associated with Tanner stage of breast and pubic 

### 19
hair development and age at menarche among African Americans and with breast and pubic hair 

### 20
development among Mexican Americans

. The associations were in the same directions for whites, but 

### 21
none of the associations reached statistical significance

. A third study using the NHANES III database 

### 22
examined the association between Pb and reproductive hormones among girls 6

- 11 years old ( Gollenberg 

### 23
et al., 2010 ) . Blood Pb levels were inversely associated with inhibin B , a prot ein that inhibits FSH 

### 24May 2011 5

- 301 DRAFT

– DO NOT CITE OR QUOTE production, but no association was observed for LH . The inverse association between blood Pb and 

### 1
inhibin B was greater among girls with iron deficiency compared to those with high Pb but sufficient iron 

### 2

levels. Inhibin B and LH were c hosen for this study because these hormones are, “ believed to be relevant 

### 3
for younger girls... near the onset of puberty and...serve as markers for hypothalamic

- pituitary

- gonadal 

### 4
functioning. ” 

### 5

6 Puberty; Neonate/adult; Mouse; Female ( Iavicoli, Carelli, Stanek, Castellino, Li, et al., 2006 )

Neurotransmitter; Adult; Mouse; Both ( Leasure et al., 2008 )

Physical development; Adult; Mouse; Male ( Leasure et al., 2008 )

Eye; Adult; Rat; Both ( Fox et al., 2008 )

Redox

- oxidative stress; Adult; Rat; Male ( Nava

- Hernandez et al., 2009 )

Sperm; Adult; Rabbit; Male ( Moorman et al., 1998 )

Neurobehavioral; Adult; Mouse; Male ( Leasure et al., 2008 )

Hematological parameters; Adult; Rat; Both ( Teijon et al., 2006 )

Histology; Adult; Rat; Both ( Teijon et al., 2006 )

Biomarkers; Adult; Rat; Both ( Teijon et al., 2006 )

Physical development; Adult; Rat; Both ( Teijon et al., 2006 )

Blood Pb Level (μg/dL) Figure 5

- 54. Toxicological Exposure

- Response Array for Reproductive Effects of Pb.May 2011 5

- 302 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 29. Toxicological Exposure

- Response Array Summaries for Reproductive Effects of Pb presented in Figure 5

- 54

R eference Blood Pb level with E ffect (μg/dl) A l tered Outcome

Iavicoli I. et al. ( 2006 ) 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 Physical 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

- Offspring 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 the onset 

### 1
of female puberty in rodents. Recent studies confirm these findings and show that puberty onset is one of 

### 2
the more sensitive markers of Pb exposure as is demonstrated in the exposure response array (Table 5

- 29 

### 3
and Figure 5

- 54) . Dumitres cu et al. ( 2008 ) exposed adult Wister female rats to varying doses of Pb acetate 

### 4
(50

- 150 ppb) in drinking water for 3 months before mating and during pregnancy

. Vaginal opening , an 

### 5
indicator of sexual maturation, was statistically sign ificantly delayed in pups from all Pb treated groups 

### 6
when compared to pups from non

- treated dams. The age at vaginal opening in female pups from the Pb 

### 7
treated groups increased, in a dose

- dependent manner, from 39 days to 43

- 47 days. The authors also 

### 8

obser ved a correlation between body weight and age at vaginal opening meaning that as body weight 

### 9
decreased the age at vaginal opening increased. This effect also exhibited a dose

- dependent relationship. 

### 10
In another recent study, I avicoli et al. ( 2006 ) reported a statistically significant delay in several 

### 11
biomarkers of sexual maturity in offspring (F 1 generation) born to dams that ingested 3.5

- 40 ppm in their 

### 12
daily diet. Maternal ingestion of Pb at the various doses resulted in female pup blood Pb levels of 3

. 5

- 13 

### 13
μg/dL. For all diet groups, there was a delay in age at vaginal opening, age of first estrus, age of vagin al 

### 14
plug formation, and age of first parturition. A novel finding in the Iavicoli study was that very low dose 

### 15
Pb (blood Pb of 0.7 μg/dL , food concentration of 0.02 ppm) induced statistically significant acceleration 

### 16

of markers of sexual maturation in femal e offspring versus background Pb level animals (blood Pb of 2 

### 17
μg/dL ) animals. There were statistically significant increases in time of vaginal opening (30% increased), 

### 18
first estrous, first vaginal plug formation, and first parturition at the very low Pb e xposure versus 2 μg/dL 

### 19
animals. Thus, the timing of puberty is delayed in a dose

- dependent fashion with very low dose Pb having 

### 20May 2011 5

- 303 DRAFT

– DO NOT CITE OR QUOTE a statistically significant earlier onset of puberty than the background Pb animals. Also, the animals 

### 1
exposed to the higher dos e of Pb (blood Pb up to 13 μg/dL ) had statistically significant delays in onset of 

### 2
puberty when compared to the other dose groups. 

### 3
In addition, Pb

- induced shifts in sexual maturity w ere observed in the subsequent generation (F2 

### 4
generation) across that dos e range

. These animals continued to be exposed to same concentrations of Pb 

### 5
over multiple generations through the diet. Data results of the F2 generation closely resembled those of 

### 6

the F 1 generation , as both generations received Pb exposure . The authors concluded that a modest 

### 7

elevation in blood Pb level (13 μg/dL) over background (2

- 3 μg/dL) can result in a profound delay in the 

### 8
onset of puberty (15

- 20%) . In the F 2 generation, reduction in blood Pb (0.7 μg/dL) below background (2

- 

### 9
3 μg/dL) w as associated with an earlier onset of sexual maturity (30% increase) above background

. 

### 10

In the 2006 Pb AQCD ( U.S. EPA, 2006 ) , it was reported that a statistically significant reduction in 

### 11
the circulating levels of insulin

- like growth factor 1 (IGF

- 1), LH, and estradiol (E2) was associated wi th 

### 12

Pb

- induced delayed puberty in Fisher 344 pups. Subsequently, Pine et al. ( 2006 ) evaluated whether IGF

- 1 

### 13
replacement could reverse the effects of Pb on female puberty. The authors reported that offspring 

### 14
exposed to Pb during gestation and lactation (12 mg/mL; mean maternal blood Pb level 40 μg/dL) 

### 15
exhibited a marked increase in LH and luteinizing hormone releasing hormone (LHRH) secretion after 

### 16

IGF

- 1 a dministration (200 ng 3 /μL) resulting in restored timing of vaginal opening such that they were the 

### 17
same as control. It should be noted that, IGF

- 1 replacement in Pb

- exposed animals did not cause 

### 18
advanced puberty over non

- Pb

- exposed controls. The results of this study provide support to the theory 

### 19

that Pb

- induced delayed onset of puberty may be due to disruption of pulsatile release of sex hormones 

### 20
( U.S. EPA, 2006 ) and not necessarily due to a direct toxic effect on the hypothalamic

- pituitary

- gonadal 

### 21

axis ( Salawu et al., 2009 ) , and IGF

- 1 may play a prominent role in the process. 

### 22
In sum, epidemiologic studies consistently show a positive association between blood Pb and 

### 23
delayed pubertal development in girls. This association is apparent even at low blood Pb levels. New 

### 24
evidence from the toxicology literature continues to support Pb

- induced delays in the onset of puberty. 

### 25
Further, the biological plausibility of delayed puberty is expanded with the toxicological literature that 

### 26
shows this pathway to be IGF

- 1

- dependent. 

### 27

5.8.1.4. Summary of Effects on Female Reproductive Function

In s ummary, Pb exposure affects female reproductive function as demonstrated by both 

### 28
epidemiologic and toxicological studies. At low Pb levels, associations are observed with delayed puberty. 

### 29
Some evidence is also available regarding Pb levels and altered horm one levels as well as decreased 

### 30
fertility, although studies reported inconsistent findings for the later. 

### 31May 2011 5

- 304 DRAFT

– DO NOT CITE OR QUOTE 

## 5.8.2. Effects on Male Reproductive Function

The 2006 Pb AQCD ( U.S. EPA, 2006 ) reported on male Pb exposure/levels and reproductive 

### 1
functions as measured by sperm count/motility/morphology, time to pregnancy, reproductive history, and 

### 2

chromosomal aberrations

. Despite limitations in many of the studies, most of the studies found slight 

### 3
associations between high Pb levels (i.e. ≥ 45 μg/dL) and reduced male fecundity or fertility ( U.S. EPA, 

### 4

2006 ) . E vidence provided in the 1986 Pb AQ CD ( U.S. EPA, 1986 ) also demonstrated that Pb exposure 

### 5
affects male reproductive function in humans and experimental animals. Recently published research has 

### 6
continued to support an association between Pb and reproductive function in males . These studies are 

### 7

described in the sections below. 

### 8

5.8.2.1. Effects on Sperm/Semen Production, Quality, and Function

Multiple epidemiologic and toxicological studies have examined the relationship between Pb and 

### 9
sperm and semen production, quality, and function. These studies are summarized in the text below. In 

### 10
addition, recent epidemiologic studies are included in Table 5

- 30. 

### 11

Table 5

- 30. Summary of recent epidemiologic studies of effects on sperm and semen

Reference Study Location and Years Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Hsu et al. ( 2009 ) Taiwan NS Men working at a battery pla nt 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 groups 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 immaturity, 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)May 2011 5

- 305 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location and Years Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Kasperczyk et al. ( 2008 ) Poland NS Healthy, non

- smoking, fertile men that worked at the Zn and Pb Metalworks Blood Pb; seminal fluid Pb
Categoriz ed 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 expos ure workers: 2.02 (0.23) Low expos ure workers: 2.06 (0.40) Controls : 1.73 (0.16) Mean (SE) S perm volume (mL) Controls: 2.94 (0.32) Low exposure : 2.89 (0.22) High exposure: 2.98 (0.22) (p

- value for ANOVA: 0.993)

S perm 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 (%) Controls: 63.3 (2.7) Low expos ure: 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 expos ure: 3.1 (0.8) (p

- value for ANO VA: 0.188)

p

- value for correlation between blood Pb and sperm cell motility after 1 h: 0.011
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 1 st quartile: 1.00 (ref) 2 nd quartile: 0.88 (0.32, 2.44) 3 rd quartile: 2.58 (0.86, 7.73) 4 th quartile: 1.16 (0.37, 3.60)

Motility 1 st quartile: 1.00 (ref) 2 nd quartile:1.04 (0.43, 2.53) 3 rd quartile: 1.95 (0.70, 5.46) 4 th quartile: 1.66 (0.64, 4.29)

Morphology 1 st quartile: 1.00 (ref) 2 nd quartile: 0.83 (0.37, 1.87) 3 rd quartile: 1.41 (0.54, 3.67) 4 th quartile: 1.18 (0.50, 2.79)

Models with adjustment for multiple metals Concentration 1 st quartile: 1.00 (ref) 2 nd quartile: 0.89 (1.57, 2.89) 3 rd quartile: 3.94 (1.15, 13.6) 4 th quartile: 2.48 (0.59, 10.4)May 2011 5

- 306 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location and Years Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Naha and Chowdhury ( 2006 ) Kolkata, India NS Men aged 31

- 45 that were non

- occupationally exposed co ntrols 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

- value s for difference across the three groups for mean values of semen profiles

were <0.01 for: sperm count, sperm protein, sperm D NA 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 fructose

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: liquefaction 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 chol esterol

Slivkova et al. ( 2009 ) NS Men aged 2 2

- 48 undergoing semen analysis 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): broken flagellum, separated flagellum, separated flagellum, small heads, retention of cytoplasmic drop, other pa thological spermatozoa, large heads, acrosomal changes, and knob twisted flagellum
Telisman et al. ( 2007 ) Croatia 2002

- 2005 Men aged 19

- 55,
never occupationally exposed to metals and going to a clinic for infe rtility examination or for semen donation to be used for artificial insemination Blood Pb Median: 4.92 (range 1.13

- 14.91) Standardized r egression coefficients for log blood 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, sperm concentration, slow sperm, short sperm, thin sperm, amorph sperm

International epidemiologic studies of men occupationally exposed to Pb have reported on 

### 1

associations between Pb levels and seme n/sperm count and quality. Most of these studies included 

### 2
individuals occupationally exposed to Pb and have reported blood Pb levels over 40 μg/dL

. For example, 

### 3
two studies performed in India ( Naha & Chowdhury, 2006 ; Naha & Manna, 2007 ) reported that men in 

### 4
the highest exposure group (men working in battery or paint manufacturing plants for 10

- 15 years for 8 

### 5
hours/day) had mean blood Pb levels of 77.22 μg/dL in one study ( Naha & Chowdhury, 2006 ) and 68.26 

### 6
in the other study ( Naha & Manna, 2007 )

. Control groups in these studies (those without occupational Pb 

### 7May 2011 5

- 307 DRAFT

– DO NOT CITE OR QUOTE exposure) had blood Pb lev els below 15 μg/dL. Increase s in levels of Pb in semen were also noted across 

### 1

exposure groups

. Both studies report decreases in sperm count and in sperm velocity and motility with 

### 2
increasing Pb exposure

. Higher Pb exposure was a lso associated with increase d hyploidy of sperm DNA 

### 3
and morphologic abnormalities ( Naha & Chowdhury, 2006 ; Naha & Manna, 2007 )

. Decreased viability 

### 4
and increased lipid peroxidat ion were detected ( Naha & Chowdhury, 2006 )

. A study performed in Taiwan 

### 5
reported that men with greater blood Pb levels had increased sperm head abnormalities, increased sperm 

### 6

DNA denaturatio n , and increased sensitivity to d enaturation compared to men with lower blood Pb levels 

### 7
( P. C. Hsu et al., 2009 )

. No difference was detected betwee n three Pb exposure groups and semen volume, 

### 8
sperm count, motility, velocity, and reactive oxygen species production

. A similar study in Poland 

### 9
included employees exposed t o Pb and compared them with a group of male office workers ( A. 

### 10
Kasperczyk et al., 2008 )

. Pb levels measured in seminal fluid were slightly higher among those in the 

### 11
exposed groups although they were not statistically different from the levels in the control group

. No 

### 12
difference was observed for semen volume, sperm count, or sperm morphology among the groups

. Sperm 

### 13
motility was lower in the highest exposure group compared to both the control and moderate exposure 

### 14

groups

. Lipid peroxidation, which induces tissue damage in sperm via reactive oxygen species, was 

### 15
greater in the highest exposure gr oup compared to the controls

. 

### 16
One study performed in Croatia recruited men who had never been occupationally exposed to 

### 17

metals ( Telisman et al., 2007 )

. Increased blood Pb was associated with increased percent of pathologic 

### 18
sperm, wide sperm, and round sperm

. There was also a slight increase in immature sperm although it was 

### 19
not statistically significant

. Similar results were seen when biomarkers for Pb (erythrocyte protoporphyrin 

### 20

and δ

- aminol evulinic acid dehydratase [ALAD] ) were used instead. 

### 21
Two studies examined Pb levels and semen quality of men at infertility clinics ( Meeker et al., 2008 ; 

### 22
Slivkova et al., 2009 )

. Meeker et al. ( 2008 ) detected no associations between increases in blood Pb and 

### 23
semen concentration, morphology, or motility (although a slight positive trend was observed between 

### 24
increasing Pb levels and motility in unadjusted models)

. In models that include multiple metals, blood Pb 

### 25
was associated with being below the WHO ’ s limit of sperm concentration levels (less than 20 million 

### 26
sperm/mL), although the 95% CI wa s wide for the 4th quartile of Pb levels and included the null

. 

### 27

Slivko v a et al

. ( 2009 ) reported a negativ e correlation between semen Pb and pathological changes in 

### 28
sperm (specifically, flagellum ball), but no correl ations were observed for other alterations in the sperm. 

### 29

An abundance of evidence in the toxicological literature demonstrates that Pb exposure is 

### 30
detrimental to the quality and overall health of testicular germ cells. Earlier studies showed that chronic 

### 31

P b exposure (15 weeks) in adult male rabbits induced statistically significant effects on semen quality and 

### 32
testicular pathology at blood Pb of 16

- 24 μg/dL ( Moorman et al., 1998 )

. Recent studies confirm earlier 

### 33
studies that Pb alters sperm parameters such as sperm count, viab ility, motility, and morphology. Oliveira 

### 34

et al. ( 2009 ) observed a negative correlation between Pb dose and intact acrosomes. Rubio et al ( 2006 )

, 

### 35

Biswas and Ghosh ( 2006 ) , and Salawu et al. ( 2009 ) observed a decrease in absolute testicular weight after 

### 36May 2011 5

- 308 DRAFT

– DO NOT CITE OR QUOTE Pb exposure. Rubio et al ( 2006 ) and Biswas and Ghosh ( 2006 ) also observed a Pb

- induced decrease in 

### 1
seminal vesicle and ventral prostate weights and Rubio et al. ( 2006 ) reported that Pb acetate, in a dose



### 2
dependent fashion (8

- 24 mg/kg body weight), reduced the length of certain stages of the spermatogenic 

### 3
cycle of rat seminif erous tubules and thus affected spermatogenesis. Reshma Anjum et al. reported 

### 4

decreased testicu lar and epididymal weights, sperm count, and viable sperm of male rats exposed to Pb 

### 5
acetate (273 mg/L or 819 mg/L in drinking water)

. Pb induced morphological abnormalities in sperm in a 

### 6

dose

- dependent manner ( Allouche et al., 2009 ; Massanyi et al., 2007 ; Oliveira et al., 2009 ; Salawu et al., 

### 7

2009 ; Shan et al., 2009 ; Tapisso et al., 2009 ; C. H. Wang et al., 2006 )

. S perm abnormalities reported after 

### 8
Pb exposures are amorphous sperm head, abnormal tail, and abnormal neck. Dong et al. ( 2009 ) reported 

### 9
decreased epididymis and body weights in mice after exposure to 0.6% Pb acetate in drinking water. 

### 10
However, the majority of studies did not observe a statistically significant difference in body weight or 

### 11
reproductive organ weights after Pb exposure at the doses used in the studies. Not all the above studies 

### 12
observed changes in every parameter. This may be due to the use of different strains or species , chemical 

### 13
form of the Pb compound administered, dosage schedule, duration of exposure, and age of animals at the 

### 14
time of the study ( Oliveira et al., 2009 )

. 

### 15
Data from recent studies suggest that a compo nent of Pb

- induced toxicity is the generation of 

### 16
reactive oxygen species (ROS) which can then affect antioxidant defense systems of cells ( Pandya et al., 

### 17

2010 ) . Salawu et al. ( 2009 ) observed a statistically significant increase in malondialdehyde (MDA, 

### 18
oxidative stress marker) and a significant decrease in the activity of antioxidant enzymes superoxide 

### 19
dismutase (SOD) and catalse (CAT) in plasma and testes of adult male Sprague Dawl ey rats after 

### 20
administration of 1% Pb acetate in drinking water for 8 weeks. Supplementation with tomato paste (used 

### 21
as a source of antioxidants) reduced ROS production and prevented the increase in MDA formation and 

### 22
decrease in SOD and CAT activity. Furth ermore, co

- treatment of Pb with substances that are known to 

### 23
have antioxidant properties (i.e. tomato paste, Maca (Lepidium meyenii), and ascorbic acid) prevented the 

### 24
reduction in sperm count, sperm motility, and sperm viability ( Madhavi et al., 2007 ; Rubio et al., 2006 ; 

### 25

Salawu et al., 2009 ; Shan et al., 2009 ; C. H. Wang et al., 2006 )

. 

### 26
Recent studies also demonstrate that Pb may be directly tox ic to mature spermatozoa ( Hernandez



### 27
Ochoa et al., 2006 ; Tapisso et al., 2009 ) as well as primary spermatocytes ( Nava

- Hernandez et al., 2009 ; 

### 28
Rafique et al., 2009 ) . Nava

- Hernandez et al. had two Pb exposure groups via drinking water

. In their 

### 29
study, all Pb

- treated animals had blood Pb levels statistically significantly higher than controls (L 1 = 

### 30
19.54 μg/dL and L2 = 21.90 μ g/dL); no statistically significant difference in blood Pb levels existed 

### 31
between the two Pb exposure groups because the L2 group drank less water than the L1 group. Piao et al. 

### 32
( 2007 ) report Pb exposure caused DNA damage to sperm; the Pb exposure group had blood Pb of 67 μ g/l. 

### 33

Piao et al. ( 2007 ) looked at the effect of Zn supplementation on Pb

- induced sperm aberrations and found 

### 34
that the proportion of abnormal sperm was statistically significantly higher in t he Pb group and the Pb+Zn 

### 35

group than in controls. However, the proportion of abnormal sperm in Pb+Zn group was statistically 

### 36May 2011 5

- 309 DRAFT

– DO NOT CITE OR QUOTE significantly lower than in Pb alone group

. Hernandez

- Ochoa et al. ( 2006 ) report that Pb reaches the 

### 1
sperm nucleus in the epididymis of mice chronically exposed to Pb (mean blood Pb 75.6 μg/dL) by 

### 2
binding to nuclear sulfhydryl groups from the DNA

- protamine complex, increasing sperm chro matin 

### 3
condensation, and thereby interfering with the sperm maturation process without altering sperm quality 

### 4
parameters. Tapisso et al. ( 2009 ) observed a statistically significant increase in the number of micronuclei 

### 5
and frequency of sister chromatid exchange with increasing treatment duration in adult male mice 

### 6
administered 21.5 mg/kg body weight Pb acetate by intraperitoneal injection. Nava

- Hernandez ( 2009 ) 

### 7
reported a dose

- dependent increase in DNA damage in rat primary spermatocytes after a 13

- week 

### 8
exposure period to Pb acetate in drinking water (mean blood Pb levels between 19.5 and 21.9 μg/dL). 

### 9
Rafique et al. ( 2009 ) reported degenerative changes from pyknosis to apoptosis in primary spermatocytes. 

### 10

Pb

- induced apoptosis i n germ cells within the seminiferous tubules is another suggested 

### 11
mechanism by which Pb exerts its toxic effects on sperm production and function ( C. H. Wang et al., 

### 12

2006 ) . Dong et al. ( 2009 ) reported a dose

- related increase in apoptosis in spermatogonia and 

### 13
spermatocytes of Kunming mice after exposure to 0.15

- 0.6% Pb acetate in drinking water. Pb

- induced 

### 14
testicular germ cell apoptosis w as associated with up

- regulation of genes involved in the signal pathway 

### 15
of MAPK and death receptor signaling pathway of FAS. For instance, up

- regulation of K

- ras and Fas 

### 16
expressions was concomitant with activation of c

- fos and active caspase

- 3 proteins. W ang et al. ( 2006 ) 

### 17

observed a dose

- dependent increase in the expression of apoptotic markers TGFβ1 and caspase

- 3 in 

### 18
spermatogenic cells, Sertoli cells, and L eydig cells. Shan et al. ( 2009 ) also reported a statistically 

### 19
significant increase in mRNA expression and protein levels of Fas, Fas

- L and caspase

- 3 after Pb 

### 20
exposure. Supplementation with ascorbic acid inhibited or reduced the Pb

- induced apoptosis in germ cells 

### 21
and protected testicular structure and function ( Shan et al., 2009 ; C. H. Wang et al., 2006 ) suggesting 

### 22
ROS generation is a major contributing factor in decreased male fertility observed after chronic Pb 

### 23

exposure. 

### 24
Similar to the results summarized in previous AQCDs, recent epidemiologic and toxicological 

### 25

studies report negative effects of high levels of Pb on sperm and semen. Future studies will aid in 

### 26
determining whether this association is observed at lower Pb levels. 

### 27

5.8.2.2. H ormone Levels

The 2006 Pb AQCD ( U.S. EPA, 2006 ) provided evidence that Pb acts as an endocrine disruptor in 

### 28
males at various po ints along the hypothalamic

- pituit a r y

- gonadal axis. The 2006 document also reported 

### 29
inconsistencies in the reported effects of Pb exposure on circulating testosterone levels. Recent 

### 30
epidemiologic and toxicological studies are reported below. Epidemiologic stud ies are summarized in 

### 31

Table 5

- 31. 

### 32May 2011 5

- 310 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 31. Summary of recent epidemiologic studies of effects on hormones for males

Reference Study Location and Years Outcome Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Meeker et al. ( 200 8 ) Michigan NS FSH, LH, Inhibin B, testosterone, SHBG, FAI, testosterone/LH 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) Regression coefficients (95% CI) FSH 1 st quartile: 1.00 (ref) 2 nd quartile: 0.13 (

- 0.10, 0.37) 3 rd quartile: 0.10 (

- 0.15, 0.35) 4 th quartile: 0.07 (

- 0.18, 0.31)

LH 1 st quartile: 1.00 (ref) 2 nd quartile:0.004 (

- 0.20, 0.21) 3 rd quartile: 0.13 (

- 0.09, 0.35) 4 th quartile: 0.88 (

- 0.14, 0.29)

Inhibin B 1 st quartile: 1.00 (ref) 2 nd quartile:

- 6.45 (

- 27.2, 14.3) 3 rd quartile:

- 4.62 (

- 26.6, 17.4) 4 th quartile:

- 7.79 (

- 29.0, 13.4)

Testosterone 1 st quartile: 1.00 (ref) 2 nd quartile: 28.6 (

- 6.82, 64.1) 3 rd quartile : 15.8 (

- 21.8, 53.3) 4 th quartile: 39.9 (3.32, 76.4)

SHBG 1 st quartile: 1.00 (ref) 2 nd quartile:

- 0.01 (

- 0.16, 0.15) 3 rd quartile: 0.04 (

- 0.12, 0.21) 4 th quartile: 0.07 (

- 0.10, 0.23)

FAI 1 st quartile: 1.00 (ref) 2 nd quartile: 0.8 (

- 0.04, 0.20) 3 rd quartile: 0.03 (

- 0.10, 0.17) 4 th quartile: 0.08 (

- 0.05, 0.21)

Testosterone/LH 1 st quartile: 1.00 (ref) 2 nd quartile: 0.07 (

- 0.16, 0.30) 3 rd quartile:

- 0.05 (

- 0.29, 0.19) 4 th quartile: 0.07 (

- 0.17, 0.31)May 2011 5

- 311 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location and Years Outcome Study P opulation Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

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 differ ence >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 >0.05
Telisman et al. ( 2007 ) Croatia 2002

- 2005 FSH, LH, t estosterone, estradiol, prolactin Men aged 19

- 55, never occupationally exposed to metals and going to a clinic for inf ertility 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 <0.003) 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)

Hormone levels were measured in a few recent epidemiologic studies. In a study of men non



### 1
occupationally exposed to Pb in Croatia, increased blood Pb was associated with increasing serum 

### 2

testosterone and estradio l but decreasing serum prolactin ( Telisman et al., 2007 )

. In addition, the 

### 3

interaction term of blood Pb and blood ca d mium levels demonstrated a synergistic effect o n increasing 

### 4

serum testosterone levels . No association was observed bet ween blood Pb and FSH or LH. A nother study 

### 5
of men with high blood Pb levels reported no difference in serum FSH, LH, and testosterone among the 

### 6
three groups ( Naha & Manna, 2007 )

. Among men recruited from infertility clinics in Michigan, median 

### 7

blood Pb levels were much lower than those observed in the other studies of Pb and hormone levels 

### 8

among men ( Meeker et al., 2010 )

. No association was detected between blood Pb and levels of FSH, LH, 

### 9
InhibinB, sex hormone

- binding globulin (SHBG), free androgen index (FAI) or a measure of Leydig cell 

### 10
function (T/LH)

. A positive association between the highest quartile of blood Pb and testosterone was 

### 11
present, but this association did not persist when other metals were included in the model

. 

### 12
In a recent toxicological study , Rubio et al. ( 2006 ) observed a decrease in testosterone levels in Pb 

### 13

acetate

- treated rats in a dose

- related fashion (8

- 24 mg/kg bod y weight), and this decrease correlated with 

### 14
reduced lengths of spermatogenic cycle stages VII

- VIII (spermiation) and IX

- XI (onset of 

### 15
spermatogenesis). Pandya et al. ( 2010 ) reported altered hepatic sterodogenic enzyme activity. Biswas and 

### 16

Ghosh ( 2006 ) reported a Pb

- induced decrease in serum testosterone and gonadotropins (FSH, LH) with 

### 17
inhibition of spermatogenesis, however, there was a statistically significant increase in adrenal 

### 18May 2011 5

- 312 DRAFT

– DO NOT CITE OR QUOTE steroidogen ic enzyme, Δ5

- 3β

- HSD activity and serum corticosterone levels indicating disruption of the 

### 1
adrenocortical process. Dose

- dependent decreases in serum testosterone were reported in Pb

- exposed 

### 2

male rats ( Reshma Anjum et al. )

. In contrast, Salawu et al. ( 2009 ) did not observe a decrease in serum 

### 3
testosterone between control animals and animals administered 1% Pb acetate in drinking water for 8 

### 4

weeks. Allouche et al. ( 2009 ) not only did not observe any statistically significant changes in serum FSH 

### 5
or LH, but reported an increase in serum testosterone levels after 0.05

- 0.3% Pb acetate treatment in 

### 6
drinking wat er (only statistically significant in animals administered 0.05% Pb acetate). The results of 

### 7
these recent studies further support the theory that compensatory mechanisms i n the hypothalamic



### 8
pituit a r y

- gonadal axis may allow for the adaptation of exposed ani mals to the toxic endocrine effects of 

### 9

Pb ( Rubi o et al., 2006 ; U.S. EPA, 2006 )

. 

### 10
Overall, r ecent epidemiologic and toxicological studies report mixed outcomes regarding hormone 

### 11
aberrations associated with Pb exposur e . These results are similar to those from the 2006 Pb AQCD o n 

### 12
the effects of Pb exposure on circulating testosterone levels. 

### 13

5.8.2.3. F ertility

Epidemiologic studies have been performed comparing Pb and infertility in men. A study 

### 14
conducted in Turkey reported blood and seminal plasma Pb levels were different in fertile and infertile 

### 15
men [mean blood Pb 23.16 μg/dL (SD 5.59 μg/dL) for fertile men an d 36.82 μg/dL (SD 12.30 μg/dL) for 

### 16

infertile men (p

- values for ANOVA <0.001) ] ( Kiziler et al., 2007 )

. Another study examined occupational 

### 17
Pb exposure (determined by self

- report of occupational exposure) and detected no difference in reported 

### 18

exposure for infertile versus fertile men [OR 0.95 (95% CI: 0.6, 1.6)] ( Gracia et al., 2005 ) . Blood Pb was 

### 19
not measured but approximately 5.0% of infertile men and 5.3% fertile men reporte d occupational 

### 20
exposure to Pb . As with the fertility studies among women, a limitation present in these studies is that the 

### 21
cases included are men who are seeking help at fertility clinics; the studies are not a sample of the general 

### 22
population regarding f ertility. 

### 23

5.8.2.4. P uberty

Research has also been published examining the association between blood Pb and onset of puberty 

### 24

in males. These studies are summarized in Table 5

- 32. 

### 25May 2011 5

- 313 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 32. Summary of recent epidemiologic studies of effects on puberty for males

.

R eference Study Location Outcome Study P opulation Exposure Measurement 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 (IQR 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)
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 for Pb contamination and one with no Pb source) Blood Pb NS for boys only (combined with girls in the study the mean was 9.46 (3.08)) Testicular size <10 μg/dl: Stage 1: 0% Stage 2: 44.4% Stage 3: 55.6% ≥ 10μg/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% ≥ 10μg/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% ≥ 10μg/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) ≥ 10μg/dl: 1.84 (SD 1.04)

*Quantitative results for LH and FSH not providedMay 2011 5

- 314 DRAFT

– DO NOT CITE OR QUOTE Williams et al. ( 2010 ) 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 μ g/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)

Studies were performed among a cohort of Russian boys enrolled between ages 8

- 9 ( Hauser et al., 

### 1

2008 ; P. L. Williams et al., 2010 )

. Both the cross

- sectional study ( Hauser et al., 2008 ) and the prospective 

### 2
study with annual follow

- ups ( P. L. Williams et al., 2010 ) demonstrated an association between increased 

### 3

blood Pb levels and later onset of puberty

. In addition, in a st udy of boys and girls in Egypt boys with 

### 4
higher blood Pb had delayed pubertal development compared to those with lower levels (median age in 

### 5
the high blood Pb group was 12.5 y ea rs compared to 13.0 y ea rs in the low blood Pb group) ( Tomoum et 

### 6
al., 2010 )

. In addition, compared to the low Pb group, those boys with higher blood Pb had lower 

### 7
testosterone, FSH, and LH levels. 

### 8
Thus, recent studies have demonstrated a negative effect of Pb on pubertal development among 

### 9
boys that exists even at low blood Pb levels. N o recent toxicological studies were found that addressed the 

### 10

effect of Pb on male sex ual development and maturation; however, t he 2006 Pb AQCD ( U.S. EPA, 2006 ) 

### 11
supported earlier findings that Pb exposure may result in delayed onset of male puberty and altered 

### 12
reproductive function later in life in experimental animals. 

### 13

5.8.2.5. Effects on Morphology and Histology of Male Sex Organs

Recent toxicological studies further support historical findings that showed an 14 association between Pb exposure and histological changes in the testes as well as germ cells. 15 Histological changes of testes in Pb

- treated animals included seminife rous tubule atrophy, 16 Sertoli cell and Leydig cell shrinkage with pyknotic nuclei ( Shan et al., 2009 ; C. H. Wang et 17 al., 2006 ) , dilatation of blood capillaries in the interstitium, undulation of basal membrane, 18 and occurrence of empty s paces in seminiferous epithelium ( Massanyi et al., 2007 )

. 19

5.8.2.6. Summary of Effects on Male Reproductive Function

Associations between Pb exposure and male reproductive function vary by out come. The strongest 

### 20
evidence of an association is the relationship observed between high levels of Pb and negative effects on 

### 21
sperm and semen in both recent epidemiologic and toxicological studies and previous AQCDs. Recent 

### 22
toxicological studies also repor ted an association between Pb exposure and histological changes in the 

### 23

testes and germ cells . In addition, recent epidemiologic studies found Pb exposure to be associated with 

### 24May 2011 5

- 315 DRAFT

– DO NOT CITE OR QUOTE delayed pubertal development at low blood Pb levels. This is supported by earlie r toxicological studies. 

### 1

Similar to the 2006 Pb AQCD, r ecent epidemiologic and toxicological studies report ed inconsistent 

### 2

results regarding hormone aberrations associated with Pb exposure . Mixed findings were also apparent 

### 3
among epidemiologic studies of fertility among men. 

### 4



## 5.8.3. Effects on Ovaries, Embryo Development, Placental function, and Spontaneous Abortions

The 2006 Pb AQCD ( U.S. EPA, 2006 ) included studies of Pb exposure among men and women and 

### 5
their associations with spontaneous abortions . T he 2006 AQCD concluded that overall th ere wa s little 

### 6
evidence to support an association between Pb levels among women and spontaneous abortion ( U.S. EPA, 

### 7

2006 )

. Most of the studies examined in the 2006 AQCD assigned exposure based on living near a smelter 

### 8
or working in occupations that often result in Pb exposure and the results of these studies were 

### 9

inconsistent

. Little evidence was available in the 2006 AQCD to suggest an association with paternal Pb 

### 10

levels ( U.S. EPA, 2006 ) , and no recent studies have been performed to examine paternal Pb levels and 

### 11
spontaneous abortion

. Since the 2006 AQCD, multiple epidemiologic studies have been published that 

### 12

examine Pb levels in women and the ir possible association with spontaneous abortion . Additionally, 

### 13
toxicological studies have studied the effects of Pb on fetal loss and the contribution of the ovaries and 

### 14
placenta to fetal loss. 

### 15

Table 5

- 33. Summary of recent epidemiologic studies of effects on spontaneous abortions

.

Reference Study Location Outcome Study population Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Gundacker et al. ( 2010 ) Vienna, Austria 2005 Previous miscarriage W omen recruited during the second trimester of pregnancy W hole 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)
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 ≤14 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) Contro ls: Blood Pb: 6.5 (4.9) Plasma Pb: 0.013 (0.013) Categorized Plasma Blood Pb ratio: 1 st tertile: 1.00 (Ref) 2 nd tertile: 1.16 (p

- value 0.61) 3 rd 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

- v alue 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)May 2011 5

- 316 DRAFT

– DO NOT CITE OR QUOTE Yin et al. ( 2008 ) Shanxi Province, 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 after miscarriage for

case s 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

Table 5

- 33, above, provides a summary of the recent epidemiologic studies examining the 

### 1
association between Pb levels and past and current spontaneous abortion. Yin et al. ( 2008 ) performed a 

### 2
study in the Shanxi Province of China to examine if plasma Pb levels were associated with anembryonic 

### 3
pregnancies (spontaneous abortions during the first trimester, which account for 15% of all spontaneous 

### 4
abortions)

. Women were enrolled at 8

- 12 weeks of gestation . W omen who delivered a term pregnancy had 

### 5
mean plasma Pb levels that were lower than those of women wh o had an anembryonic pregnancy. Of 

### 6
note, among cases Pb was inversely correlated with folate and vitamin B12, but this correlatio n was not 

### 7

observed among those who delivered at term; no models examining Pb levels were controlled for nutrient 

### 8

status . A study in Turkey reported on groups of women who either had a spontaneous abortion before the 

### 9

20 th week of gestation or who had a viab le pregnancy ( Faikoglu et al., 2006 ) . N o difference was detected 

### 10

bet ween the blood Pb levels of the two groups ( Pb levels not reported here due to calculation errors 

### 11
discovered in the paper; errors do not appear to affect conclusions). A study in Mexico City examined a 

### 12
group of pregnant women (maximum gestational period at enrollment was 14 weeks) who had previously 

### 13
been pregnant and either given birth or had a spontaneous abortion ( Lamadrid

- Figueroa et al., 2007 )

. 

### 14
Women in the highest tertile of plasma /blood Pb ratio had higher rates of previous spontaneous abortions 

### 15

than women in the lowest tertile

. The authors state that the plasma/whole blood ratio represents the 

### 16
availability of Pb capable of crossing the placental barrier for a given blood concentra tion

. No association 

### 17
was observed when examining the relationship between Pb and spontaneous abortions using whole blood, 

### 18
plasma, or bone Pb alone. Similarly, a study of placental Pb levels among pregnant women in Austria 

### 19
observed higher placenta Pb levels among women who had miscarried a previous pregnancy compared to 

### 20
women who had not miscarried a previous pregnancy although the number of women included in the 

### 21
study was small (only 8 women reported previously having a miscarriage) ( Gundacker et al., 2010 )

. 

### 22
Isolated embryo cultures are often used to understand the mechanisms responsible for aberrant 

### 23
embryo development as it may contribute to teratogenesis, fetal loss or negative postnatal pup outcomes. 

### 24

Nandi et al. ( 2010 ) demonstrated a dose

- dependent decline in embryo development of fertilized buffalo 

### 25
oocytes cultured in medium containing 0.05

- 10 μ g/mL Pb acetate as evidenced by reduced 

### 26
morula/blastocyst yield and inc reased four

- to eight

- cell arrest, embryo degeneration, and asynchronous 

### 27

division. This study provides evidence of the negative effect of Pb on embryo development and 

### 28

contributes mechanistic understand to Pb

- dependent pregnancy loss. 

### 29May 2011 5

- 317 DRAFT

– DO NOT CITE OR QUOTE A possible explanation for reduced fertility and impaired female reproductive success as a result of 

### 1
Pb exposure is changes in morphology or histology in female sex organs and the placenta ( Dumitrescu et 

### 2
al., 2007 ; U.S. EPA, 2006 ) . Wang et al. ( 2009 ) observed that elevated maternal blood Pbs (0.6

- 1.74 μM) 

### 3
compared to control (0.04 μM) were associated with decreased fetal body weight, pup body length, and 

### 4
placental weight in Wistar rats. The authors reported that placentae from P b

- exposed groups showed 

### 5

dose

- dependent increasing pathology of cytoarchitecture and cytoplasmic organelles. The authors also 

### 6
reported a positive expression of NF

- κB, a transcription factor that controls the expression of genes 

### 7
involved in immune responses, apoptosis, and cell cycle, in the cytotrophoblasts, decidual cells, and small 

### 8
vascular endothelial cells in rat placenta under a low Pb level exposure condition which correlated to 

### 9

blood levels. 

### 10

Pb

- exposed (273 mg/L or 819 mg/L in drinking water , 0.05 or 0.15% Pb Acetate, respectively ) 

### 11

male rats from Reshma Anjum et al. that had dose

- dependent decreases in serum testosterone , decreased 

### 12
male reproductive organ weight and decreased sperm were mated to untreated females. These u ntreated 

### 13

dams had dose

- depen dent decreased implantation rate and higher pre

- and post

- implantation loss

, 

### 14
indicating paternally mediated fetal loss

. 

### 15
As observed in sperm cells, Pb stimulates changes in antioxidant enzyme activity in rat ovaries 

### 16
indicating that a contributing factor in Pb

- induced ovarian toxicity may be oxidat ive stress. Nampoothiri 

### 17

et al. ( 2007 ) observed a reduction in SOD activity and an increase in CAT activity along with a decrease 

### 18
in glutathione content and an increase in lipid peroxidation in rat granulosa cells after 15 days of Pb 

### 19
treatment (0.05 mg/kg body weight). 

### 20
Previous studies demonstrated that Pb accumulates in the ovaries and causes histological changes, 

### 21
thus contributing to Pb

- induced effects on female fertility ( U.S. EPA, 2006 ) . In support of historical 

### 22
studies, rec ent studies demonstrate histological changes in ovarian cells of pigs ( Kolesarova et al., 2010 ) 

### 23

and rats ( Nampoothiri et al., 2007 ; Nampoothiri & Gupta, 2006 ) . Kolesarova et al. ( 2010 ) observed a 

### 24
reduction of the monolayer of granulosa cells after Pb addition (0.5 mg/mL). Nampoothiri and Gupta 

### 25
( 2006 ) reported that Pb exposure caused a decrease in cholesterol and total phospholipid con tent in the 

### 26
membranes of granulosa cells which resulted in increased membrane fluidity. A possible explanation for 

### 27
reduced fertility and impaired female reproductive success as a result of Pb exposure is changes in 

### 28
morphology or histology in female sex org ans and the placenta ( Dumitrescu et al., 2007 ; U.S. EPA, 

### 29

2006 )

. 

### 30
Overall, the recent studies support the conclusions of the last Pb AQCD that there is insufficient 

### 31
evidence among epidemiologic studies to suggest an association between Pb and spont aneous abortions. 

### 32
In addition, studies of spontaneous abortions are difficult to conduct. The majority of spontaneous 

### 33
abortions are during the first trimester, which makes them difficult to capture. Women may miscarry 

### 34
before being enrolled in a study and m any 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 lead to 

### 36May 2011 5

- 318 DRAFT

– DO NOT CITE OR QUOTE increased risk of early spontaneous abortions. Toxicological data provide mechanistic understa nding of 

### 1
the contribution of Pb to spontaneous abortions. These laboratory data show that Pb exposure impaired 

### 2
placental function, induced oxidative stress and histological changes in the ovaries, and affected embryo 

### 3
development. The toxicological and epid emiologic data up to the current date provide mixed effects on 

### 4
the role of Pb in spontaneous abortions. 

### 5



## 5.8.4. Infant Mortality and Embryogenesis

The 2006 AQCD ( U.S. EPA, 2006 ) concluded that Pb exposure can increase fetal mortality and 

### 6
produce sublethal effects (disrupt growth and development) in offspring of Pb exposed dams at 

### 7
concentrations that do not result in clinical toxicity to the dams. There is substantial evidence to show that 

### 8
there is no apparent maternal

- fetal barrier to Pb and it ca n easily cross the placenta and accumulate in 

### 9

fetal t issue during gestation ( Pillai et al., 2009 ; Uzbekov et al., 2007 ; Y.

- Y. Wang et al., 2009 )

. No recent 

### 10
epidemiologic studies have reported on the relationship between Pb levels and infant mortality. 

### 11



## 5.8.5. Birth Defects

The 2006 Pb AQCD ( U.S. EPA, 2006 ) reported the possibility of small associations between high 

### 12
Pb exposure and birth defects , but many of the studies used occupation al histories instead of actu al 

### 13

measures of blood Pb levels. Among the studies included in the 2006 AQCD, two studies reported 

### 14
possible associations between parental exposure to Pb and neural tube defects ( Bound et al., 1997 ; Irgens 

### 15
et al., 1998 ) . R ecent studies also examined Pb levels and neural tube defects (Table 5

- 34). No other recent 

### 16
studies of Pb levels/exposure and birth defects were identified in the literature . N o r ecent toxicological 

### 17
studies were found that investigated Pb

- induced changes in morphology, teratology effects, or skeletal 

### 18
malformations of developing fetuses as a result of maternal Pb exposure; however, in the 2006 AQCD 

### 19
toxicological studies demonstrated associations between exposure to high doses of Pb and increased 

### 20
incidences of teratogenic effect in experimenta l animals

. 

### 21

Table 5

- 34. Summary of recent epidemiologic studies of effects on neural tube defects

.

Reference Study Location Study P opulation Ex posure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Brender et al. ( 2006 ) Texas 1995

- 2000 Infants of Mexican

American women Maternal blood Pb taken 5

- 6 wks post

- partum Cases: 2.4 (1.9) Controls: 2.5 (1.6) Blood Pb<6.0 μg/dL: 1.0 (Ref) B lood Pb≥6.0 μg/dL: 1.5 (95% CI : 0.6, 4.3)May 2011 5

- 319 DRAFT

– DO NOT CITE OR QUOTE 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

Among the recent epidemiologic studies (described in Table 5

- 34), a study of women in Turkey 

### 1

detected no difference between the blood Pb of mothers or the umbilical cord blood Pb of the newborns 

### 2
for healthy infants compared with infants with neural tube defects ( cases of spina bifida occulta were 

### 3
excluded, but other forms of spina bifida were included ) ( Zeyrek et al., 2009 )

. Brender et al. ( 2006 ) 

### 4
performed a study of Mexican

- American women living in Texas . These measurements were taken 5

- 6 

### 5
weeks postpartum , which is a limitation of this study because the levels may be different than during the 

### 6
developmental period of gestation. The OR co mparing those with at least 6 μg/dL Pb to those with less 

### 7
than 6 μg/dL Pb was 1.5 (95% CI : 0.6, 4.3)

. This increased after adjusting for breast feeding , although 

### 8
this variable was not a confounder due to its inability to be associated with neural tube defe cts. For these 

### 9

women neither occupational exposure to Pb or proximity of reside nce to a facility with Pb air e missions at 

### 10

the time of conception were associated with increased odds of neural tube defects. 

### 11
Overall, in contrast to studies from the 2006 AQCD, recent studies of Pb and neural tube defects 

### 12

observed no associations. 

### 13



## 5.8.6. Preterm Birth

Research on preterm birth included in the 2006 Pb AQCD ( U.S. EPA, 2006 ) reported inconsistent 

### 14
findings regarding the relationship between Pb and gestationa l age . R ecent studies have examined this 

### 15
potential association and again mixed results were reported (Table 5

- 35). Of these studies, the ones that 

### 16
categorized births as preterm or term all defined preterm birth as less than 37 weeks of gestation. One 

### 17

limit ation to note for these studies is that if Pb affects spontaneous abortion and length of gestation via a 

### 18
similar pathway, then the studies that only collect data at delivery and not at earlier stages of pregnancy 

### 19

would be biased towards the null. 

### 20May 2011 5

- 320 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 35. Summary of recent epidemiologic studies of effects on preterm birth.

Reference Study Location Outcome Study P opulation Exposure Measurement Mean Pb (SD) in g/dL Adjusted Effect estimates

Berkowitz et al. ( 2006 ) Idaho 1970

- 1981 Preterm birth (<37wk) Singleton infants with 28

- 45 wk gestation 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 ai r Pb concentrations for 6 mo ), and “ post

- fire ” NS OR (90% CI) (unexposed 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)
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 M aximum 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) ≥ 40μg/dl: 4.7 (1.1, 19.9)
<10 μg/dl: 1.00 (Ref) ≥ 10μg/dl: 3.2 (1.2, 7.4 )

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 C o unty 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

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 difference: <0.05

OR (95% CI) 1.41 (1.08, 1.84) (unit not given, assume per 1 μg/dL)

One study of preterm birth included women living in two different residential areas over three 

### 1
different time periods ( Berkowitz et al., 2006 )

. One residential area was consistently unexposed but the 

### 2
other had a period of high Pb emissions due to damage at a l ocal factory

. The three time periods examined 

### 3
preterm birth rates before, during, and after the time of high exposure

. No association was observed 

### 4
between women living in the high exposure area compared to those in the low exposure area during any 

### 5

of the t ime periods, but the number of preterm infants born during the period of high exposure was small

. 

### 6
In another study, measurements of umbilical cord blood were taken after birth at a hospital in Nagpur, 

### 7May 2011 5

- 321 DRAFT

– DO NOT CITE OR QUOTE India ( Patel & Prabhu, 2009 )

. A sample of women had their blood Pb measured and among this sample, 

### 1

maternal blood Pb was correlated with the umbilical cord Pb levels. Mean g estational age varied between 

### 2 infants with >5 μg/dL Pb and infants with ≤ 5 μg/dL Pb. In a linear regression model, gestational age was 

### 3
found to decrease with increasing umbilical cord Pb levels. A study of women in Tennessee consisted 

### 4
primarily of Africa n American women living in an urban setting ( E. A. Jones et al., 2010 )

. Mean levels of 

### 5

umbilical cord blood Pb were slightly higher among infants born preterm but the difference was not 

### 6
statistically significant . I n a study taking place in California, women with information on blood Pb levels 

### 7
based on their participation in a surveillance program (reason for participation in th e surveillance program 

### 8

was unknown but the authors speculate it was likely because of potential Pb exposure) were matched with 

### 9

the birth certificates of their infants ( Jelliffe

- Pawlowski et al., 2006 )

. Almost 70% of women had 

### 10

maximum blood Pb measurements <10 μg/dL with the majority being <5 μg/dL . P reterm birth was 

### 11

associated with increased blood Pb when comparing women with maximum blood Pb levels ≥ 10 μg/dL 

### 12

to women with blood Pb levels < 10 μg/dL in adjusted analyses. In analyses of maximum Pb levels refined 

### 13
into further categories, when compared to maximum blood Pb levels ≤ 5 μg/dL the positive association 

### 14

between maxi mum blood Pb measurement and preterm birth was present at 20 μg/dL and higher. Finally, 

### 15
a study in Iran reported higher maternal blood Pb for preterm births than term births ( Vigeh et al., 2011 )

. 

### 16
A positive associatio n between maternal blood Pb levels and preterm birth was observed. 

### 17
In sum, as in the 2006 Pb AQCD, recent epidemiologic studies report inconsistent findings for the 

### 18
relationship between Pb and preterm birth. 

### 19



## 5.8.7. Low Birth Weight /Fetal Growth

The 2006 Pb AQCD reported inconsistent study results examining the associations between Pb and 

### 20
birth weight/fetal growth and concluded that there could be a small effect of Pb exposure on birth weight 

### 21
and fetal growth ( U.S. EPA, 2006 )

. Since then, multiple studies on the relationship between Pb exposure 

### 22

and birth weight and fetal growth have been published using various measures of exposure, such as air 

### 23
levels, umbilical cord blood, and maternal blood and bone . These studies are summarized in Table 5

- 36 

### 24
below. Additionally, there have been a few recent toxico logical studies evaluating the effect of Pb 

### 25
exposure during gestation on birth weight. 

### 26May 2011 5

- 322 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 36. Summary of recent epidemiologic studies of effects on low birth weight and fetal growth.

Reference Study Location Outcome Study population Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Berkowitz et al. ( 2006 ) Idaho 1970

- 1 981 L ow birth weight (<2

, 500 g and ≥37 wk) S mall for gestational age ( birth weight ≤5th percentile of sex

- and gestational wk weights for singletons in Idaho) Singleton infants with 28

- 45 wk gestation Three time periods of two locations (unexposed and expo sed/near smelter): pre

- fire, “high

- exposure period ” (when a fire happened at the smelter and resulted in damages leading to high ai r Pb concentrations for 6 mo ), and “ post

- fire ” Not specified Term Low birth weight : OR (90% CI) (unexposed location is refere nt 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)
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) Umbilical 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

- value <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) 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) Controls: 13.5 (2.7) Umbilical cord blood Pb: Cases: 10.7 (1 .7) Controls: 1 1.3 (1.9) P

- values for t

- tes ts: 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 N ormal birth weight : 0.3 P

- values for correlations: Umbilical cord blood Pb and birth weight : Low birth weight : 0.84 normal birth weight : 0.26 Janjua et al. ( 2009 ) Karachi, Pakistan 2005 Low birth weight (≤ 2

, 500g) Infants of

r andomly 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) ≥ 10μg/dl: 0.82 (0.57, 1.17)May 2011 5

- 323 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location Outcome Study population Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Jelliffe

- Pawlowski et al. ( 2006 ) 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 n on

- 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 and matched to birth records M aximum 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) ≥ 40μg/dl:

-- <10 μg/dl: 1.00 (Ref) ≥ 10μg/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) ≥ 40μg/dl:

-- <10 μg/dl: 1.00 (Ref) ≥ 10μg/dl: 4.2 (1.3, 13.9)
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 C ounty area for at least 5

mo during pregnancy 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: >0.10

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 and 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, infant 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
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)

- Llanos and Ronco ( 2009 ) Santiago, Chile NS Fetal growth restriction (1,000

- 2,500g) *note normal birth 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 M ann

- Whitney U

- test <0.01

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/m 3 (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 decrea se in birth weight Increase of Pb from 0 to 0.13 (maximum) relates to a 124g decrease in birth weightMay 2011 5

- 324 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location Outcome Study population Exposure Measurement Mean Pb (SD) in μg/dL Adjusted Effect Estimates

Zentner et al. ( 2006 ) Santo Amaro, Brazil 2002 Birth weight and length Single ton 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)

Women residing in two different towns in Yugoslavia (one with a Pb smelter and one without a 

### 1
Pb smelter) were recruited during their first prenatal visit ( Lamb e t al., 2008 ) (study based on previous 

### 2
work by Factor

- Litvak et al. ( 1991 ) ). The blood Pb levels were greater in the town with a Pb smelter . No 

### 3

association was reported between maternal blood Pb and height or BMI at birth for the infants of these 

### 4

women

. Despite the differences in maternal blood Pb between the two towns , no difference s in the 

### 5

associations were detected

. Another stu dy using maternal blood Pb was conducted in California ( Jelliffe



### 6
Pawlowski et al., 2006 ) . W omen ’ s blood Pb measu rements during pregnancy were matched with the 

### 7
corresponding birth certificates

. The adjusted OR for low birth weight that compared women with blood 

### 8

Pb levels ≥ 10 μg/dL to women with levels <10 μg/dL was elevated . However, it was difficult to draw 

### 9

conclus ions about the relationship between blood Pb and birth weight due to small nu mbers (n = 9 for low 

### 10
birth weight) and the subsequently large 95% CI

. An association was detected for high blood Pb and 

### 11
having an infant who was small of his/her gestational age ( SGA ; defined as birth weight <10th percentile 

### 12
of normal weight for population

- based singleton race and gender specific infants of the same gestational 

### 13

age )

. A study of term births in Iran reported no difference in blood Pb of women giving birth to a normal 

### 14
weight infant and women giving birth to an infant with low birth weight ( Iranpour et al., 2007 ) . Finally, a 

### 15
study in Vienna, Austria reported an inverse association between maternal blood Pb levels and birth 

### 16
weight but no associations for birth length or head circumference ( Gundacker et al., 2010 )

. 

### 17
One study examining the association between Pb levels and birth weight u sed tibia bone 

### 18
measurements from the mothers living in Mexico City ( Kordas et al., 2009 )

. Pb tibia levels were inversely 

### 19

associated with birth weight but not with birth length

. This association between Pb and birth weight was 

### 20
not modified by maternal folat e consumption or maternal or infant MTHFR genotype, although the 

### 21

association between tibia Pb levels and birth weight was increased among women with certain genotypes 

### 22
(statistical tests not reported). 

### 23
Multiple studies examined the relationship using Pb me asured from the placenta or umbilical cord

. 

### 24
First, the study by Iranpour et al. ( 2007 ) discussed above investigated the association between umbilical 

### 25

cord blood Pb levels in addition to their examination of whole blood

. They again report no difference in 

### 26

levels between term infants of normal and low birth weight. Researchers in Chile collected the placentas 

### 27

from term births and compared the Pb levels for those born with normal birth weight s to those with low 

### 28May 2011 5

- 325 DRAFT

– DO NOT CITE OR QUOTE birth weight s ( Llanos & Ronco, 2009 ) . Pb levels were greater in the placentas of infants with low birth 

### 1
weights. In addition, the authors note that 3 low birth weight infants had extremely high Pb levels in the 

### 2
placentas (>1.5 μg/g ) and were excluded from these analyses

. A study in Brazil examined Pb levels in 

### 3

umbilical cord blood from term births of women residing within 5 km of a Pb smelter ( Zentner et al., 

### 4

2006 )

. The Pb level was found to be inversely correlated with length and weight of the infants

. A third 

### 5
study recruited women in Pakistan ( Janjua et al., 2009 )

. Umbilical cord blood Pb levels were not 

### 6

associated with low birth weight. A study comparing geometric mean umbilical cord blood Pb level s 

### 7
reported no difference in the levels for n ormal and low birth weight infants among infants born to women 

### 8
living primaril y in urban areas of Memphis, TN ( E. A. Jones et al., 2010 )

. Finally, a study in Vienna 

### 9
measured Pb in the placenta ( Gundacker et al., 2010 ) . A positive correlation was observed between 

### 10
placenta Pb and birth length and weight, howev er, in the same study, maternal blood Pb was inversely 

### 11
related to birth weight. 

### 12
Two studies examined air exposures and reported inverse associations between air Pb 

### 13
concentrations and birth weight. Williams et al. ( 2007 ) examined Pb concentrations in the air during the 

### 14

first trimester

. The purpose of thei r study was to demonstrate the use of hierarchical line a r models and 

### 15
they used the example of air pollution and birth weight in Tennessee. The model results showed an 

### 16

association between ambient Pb concentration and birth weight , with an estimated decrease in birth 

### 17
weight of 38 grams for every 0.04 μg/m 3 (i.e.

, one standard deviation) increase in Pb concentration

. The 

### 18
other study of air Pb levels was conducted in Idaho and included two areas over three time periods

. One 

### 19
study area was affected by damage to a local factory that lead to high Pb emissions during one of the time 

### 20
periods under study ( Berkowitz et al., 2006 )

. No levels of Pb are provided

. Mean birth weight for term 

### 21

births was decreased among women living in high exposure areas during the period of high exposure 

### 22
compared to those living in unexposed areas

. The diffe rence in birth weight of term births remained, but 

### 23
was reduced, between the two areas during the time period after the exposure ended

. During the period of 

### 24
exposure, the odds of low birth weight among term births was increased among those living in the 

### 25

exp osed area compared to those in the unexposed area but the odds were not different between the two 

### 26
study areas during the time periods before or after the high level of exposure

. An increase in SGA infants 

### 27
(defined as infants with weights less than or equal to the lowest fifth percentile of birth weight for their 

### 28
sex and age) was also associated with living in the exposed area during the time period of exposure

. The 

### 29
odds of SGA infants decreased during the time period after the exposure but the odds were sti ll elevated 

### 30
compared to those residing in the unexposed area. 

### 31
Evidence from previous toxicological studies has shown an association between gestational Pb 

### 32
exposure and reduced birth weight and impaired postnatal growth ( U.S. EPA, 2006 ) . More recent studies 

### 33
have reported conflicting resu lts. Wang et al. ( 2009 ) demonstrated a statistically significant decrease in 

### 34
fetal body weight and body length of Wistar rats after maternal exposure to 0.025% Pb acetate during 

### 35
gestation days 1

- 10 , 11

- 20, or 1

- 20. The greatest decrease in fetal body weight and length was observed in 

### 36May 2011 5

- 326 DRAFT

– DO NOT CITE OR QUOTE the group exposed to Pb during gestation days 1

- 20 followed by the group exposed to Pb during gestation 

### 1
days 11

- 20. Teijón et al. ( 2006 ) observed that when pregnant dams were administered 200 ppm or 400 

### 2
ppm Pb acetate in drinking water, female pups had a decreased birth weight when compared to male pups 

### 3
of the same litter (only stati stically significant at the 400 ppm dose). This effect did not persist in the 

### 4
postnatal growth of the rats. The results of these studies indicate that as Pb exposure increases, the body 

### 5
weight of exposed offspring decreases. Massó

- González and Antonia

- Garc ía ( 2009 ) also observed a n 8

- 

### 6
20% decrease in body weight of pups from rat dams given 300 mg/L Pb acetate in drinking water (mean 

### 7

blood Pb level 2 2.8 μg/100mL), but no changes in body length were reported

. In contrast, Leasure et al. 

### 8
( 2008 ) reported a statistically significant inverse relationship between Pb exposure and body weight for 

### 9
male mice exposed to low

- and high

- levels of Pb during gestation. Male mice e xposed to the low and high 

### 10
Pb concentrations during gestation were 26% and 13% heavier than controls at 1 year of age, respectively. 

### 11
In this study, dams were administered 27 ppm (low), 55 ppm (moderate), and 109 ppm (high) Pb in 

### 12
drinking water beginning 2 weeks before mating and continuing until PND 10. Resulting blood Pb levels 

### 13
ranged from 10 μg/dL or less in the low

- exposure offspring to 42 μg/dL in the high

- exposure offspring at 

### 14

PND 10. The authors also reported that when dams received low or moderate leve ls of Pb in drinking 

### 15
water from birth to weaning neither male nor female offspring exposed to Pb postnatally exhibited a 

### 16
difference in body weight when compared to control offspring. 

### 17
In summary, a ssociations were observed between Pb and low birth weight in a study of maternal 

### 18
bone Pb and studies of Pb air exposures and birth weight. However, the associations were less consistent 

### 19
when using maternal blood Pb or umbilical cord and placenta Pb as the expo sure measurement. P revious 

### 20
toxicological studies observe d an association between gestational Pb exposure and redu ced birth weight 

### 21
with moderate to high dose Pb. More recent findings using low dose Pb exposure reported increased 

### 22
offspring body weight after developmental Pb exposure. 

### 23



## 5.8.8. Toxicological Studies of Developmental Effects

5.8.8.1. Developmental Effects on Blood and Liver

The 1986 and 2006 AQCD reported studies that suggest Pb may alter hematopoietic and hepati c 

### 24
function during development. Some r ecent studies provide evidence that support these findings; however 

### 25
recent results are not consistent among the studies. 

### 26

Massó et al. ( 2007 ) reported a decrease in liver weights of pups born to dams that consumed 300 

### 27
mg/L Pb in drinking water during gestation and lactation. They also report an increase in the number of 

### 28
erythrocytes; however their size was diminished by 62%. Pb produced microcitic anemia as evidenced by 

### 29
decreased hemoglobin content and hematocrit values with out changes in mean corpuscular hemoglobin 

### 30
(MCH) concentration. No changes were observed in alkaline phosphatase (ALP) activity, CAT activity, or 

### 31May 2011 5

- 327 DRAFT

– DO NOT CITE OR QUOTE thiobarbituric acid reactive substances (TBARS) production in pups at postnatal 0, but increased 

### 1
statistically significantly by PND 21 indicating reactive oxygen generation. No change in acid phosphatase 

### 2
(ACP) activity was observed in the livers of pups at PND 0 or 21. 

### 3

Massó

- González and Antonia

- García ( 2009 ) reported normochromic and microcytic anemia and a 

### 4 significant decrease in hematocrit values and blood δ

- aminolevulinic acid dehydratase (ALAD) activity 

### 5
(90% reduction) in pups from dams administered 300 mg/L Pb acetate in drinking water during gestation. 

### 6
The authors also reported that erythrocyte osmotic fragility was four times greater in Pb

- exposed pups 

### 7
than in control pups. Massó

- González and Antonia

- García reported increases in TBARS and CAT activity 

### 8
in the liver after Pb exposure. Intoxication with Pb also resulted in decreased liver protein concentrations 

### 9
and manganese

- dependent SOD activity. Abnormalities in liver function were further exemplified by 

### 10

increases in liver concentrations of ALP and ACP

. 

### 11
Teijón et al. ( 2006 ) observed that gestational exposure to Pb caused a decrease in erythrocytes, 

### 12
hemoglobin, and MCH at weaning; however

, by 1 and 3 months postweani ng, these parameters had 

### 13
returned to normal values. The authors observed a slight increase in serum ALP, alanine aminotransferase 

### 14
(ALT), and aspartate aminotransferase (AST) levels after Pb exposure in the absence of liver histological 

### 15
changes. 

### 16

Pb

- induced effects on SOD activity in the liver of fetuses after Pb intoxication was supported in a 

### 17
study by Uzbekov et al. ( 2007 ) . The authors rep orted an initial increase in SOD activity in livers of pups 

### 18
exposed to 0.3 mg/L and 3.0 mg/L Pb nitrate during gestation for 1 mo nth (mean daily consumption 27 

### 19

μ g/kg). In contrast, long

- term exposure (5 months) to the same concentrations of Pb nitrate conc entration 

### 20
during gestation resulted in decreased hepatic SOD activity. 

### 21
Effects on hepatic Phase I and Phase II enzymes after early developmental exposure of offspring to 

### 22
Pb during gestation and lactation was evaluated by Pillai et al. ( 2009 ) . In the study, pregnant Charles 

### 23

Foster rats were administered 0.05 mg/kg body weight Pb subcutaneously throughout gestation until 

### 24

PND 21. Pups were evaluated on PND 56. Results of the study show that Phase I xenobiotic

- metabolizing 

### 25
enzymes (NADPH

- and NADH cytochrome c reductase) and Phase II xenobiotic

- and steroid



### 26

metabo lizing enzymes (δ

- glutamyl transpeptidase, UDPGT, glutathione

- s

- transferase, and 17β



### 27
hydroxysteroid oxidoreductase) were reduced in both male and female pups by PND 56. Only inhibition in 

### 28
glutathione

- s

- transferase and 17β

- hydroxysteroid oxidoreductase activ ities demonstrated a sex

- specific 

### 29
pattern (glutathione

- s

- transferase inhibition in males; 17β

- hydroxysteroid oxidoreductase inhibition 

### 30
greater in females). Pb

- induced histological changes observed include massive fatty degeneration in 

### 31
hepatocytes, large va cuoles in cytoplasm, appearance of pycnotic nuclei, and infiltration of lymphocytes 

### 32
in the liver. Antioxidant enzymes (SOD, CAT, glutathione peroxidase, and glutathione reductase) were 

### 33
also reduced after Pb intoxication. Alterations in biochemical paramete rs included decreased DNA, RNA, 

### 34

and cholesterol content. 

### 35May 2011 5

- 328 DRAFT

– DO NOT CITE OR QUOTE 

### 5.8.8.2. Developmental Effects on Skin

The 2006 Pb AQCD ( U.S. EPA, 2006 ) reported one study that demonstrated Pb

- induced 

### 1
abnormalities in skin development. No current studies were identified that addressed Pb

- induced skin 

### 2

alterations. 

### 3

5.8.8.3. Developmen tal Effects on the Retina

The 2006 AQCD concluded that Pb exposure during early postnatal development ( blood Pb ~20 

### 4

μg /dL) impaired retinal development in female Long

- Evans hooded rats. A more recent study ( Fox et al., 

### 5

2008 ) exposed female Long

- Evans hooded rats to low (27 ppm), moderate (55 ppm), and high (109 ppm) 

### 6
levels of Pb acetate in drinking water beginning 2 weeks before mating, throughout gestation, and until 

### 7

PND 10. blood Pb levels in pups on postnatal days 0

- 10 exposed to Pb during gestation were 10

- 12 μg/dL 

### 8
(l ow), 21

- 24 μg/dL (moderate), and 40

- 46 μg/dL (high). Results of the study demonstrated supernormal 

### 9
persistent rod photoreceptor

- mediated (scotopic) electroretinograms (ERG) in adult rats similar to ERG 

### 10
findings in male and female children after gestational exposure to low

- and moderate

- levels of Pb

. Low



### 11

and moderate

- levels of Pb increased neurogenesis of rod photoreceptors and rod bipolar cells without 

### 12
affecting Müller glial cells and statistically significantly increased the number of rods in central and 

### 13
peripheral retina. High

- level Pb exposure (109 ppm) statistically significantly decreased the number of 

### 14
rods in central and peripheral retina Pb

- exposure induced dose

- dependent decreases in adult rat retinal 

### 15
dopamine synthesis and utilization/release. 

### 16

5.8.8.4. Deve lopment al Effects on Teeth

Pb has been associated with multiple health effects including dental caries, however, there is very 

### 17
limited information available on the temporal and spatial incorporation of Pb in dental tissue ( Arora et al., 

### 18

2005 ) . Arora et al. ( 2005 ) demonstrated that W istar rat pups exposed to Pb during gestation and lactation 

### 19
(40 mg/L of Pb nitrate in drinking water of pregnant dams) had higher concentrations of Pb on the surface 

### 20
of enamel and in the dentine immediately adjacent to the pulp. The authors concluded that additional 

### 21
research is needed on the intracellular uptake of Pb during tooth development to fully understand the 

### 22
spatial distribution of Pb in teeth. 

### 23



## 5.8.9. Summar y and Causal Determination

Many epidemiologic and toxicological studies of the effects of Pb on rep roductive outcomes have 

### 24
been performed since the 2006 AQCD

. These studies covered outcomes such as female and male 

### 25
reproductive function, birth defects spontaneous abortions, infant mortality, preterm birth, low birth 

### 26
weight , and developmental effects. There is an abundance of evidence in the literature demonstrating that 

### 27May 2011 5

- 329 DRAFT

– DO NOT CITE OR QUOTE Pb induces reproductive and developmental effects in laboratory animals exposed to Pb during gestation 

### 1

and/or lactation

. Many of the Pb

- induced effects occur in a dose

- dependent manner and have been 

### 2
observed at maternal blood Pb levels that do not result in clinical toxicity in the dams . Additionally, 

### 3
epidemiologic studies have demonstrated strong evidence of an association between Pb and delayed 

### 4
puberty as well as decrements to sperm/se men quality and function. 

### 5
Many of the animal toxicology studies included in the 2006 AQCD explored the effect of Pb on 

### 6
reproduction and development at blood Pb levels greater than 40 μg /dL, a dose where maternal toxicity 

### 7
can develop during pregnancy

. Data from the 2006 AQCD on male fertility showed perturbed semen 

### 8
quality. Recent studies have shown the e ffects of Pb exposure during early development to include 

### 9
disruption of endocrine function; delay in the onset of puberty and alteration in reproductive fun ction later 

### 10
in life; and changes in morphology or histo logy in sex organs and placenta. Additionally, epidemiologic 

### 11
studies of reproductive factors among males and females investigated whether Pb levels were associated 

### 12

with hormone levels, fertility, and o nset of puberty

. Epidemiologic studies showed associations between 

### 13

blood Pb and hormone levels for females

. Studies of Pb and fertility are limited and inconsistent for 

### 14

females and males

. Strong and consistent associations were observed between Pb levels i n males in 

### 15
occupational settings with blood Pb levels as low as 20

- 45 μg/dL and sperm count and quality

. Multiple 

### 16
studies of Pb and puberty have shown associations between blood Pb levels and delayed pubertal 

### 17
development for girls and boys

. These associati ons are consistently observed in multiple epidemiologic 

### 18
studies and demonstrate effects on pubertal development at blood Pb levels <10ug/dL

. 

### 19

Pb

- mediated changes in levels or function of repr oductive and growth hormones have been 

### 20
demonstrated in past and m ore recent toxicological studies; however the findings are inconsistent. More 

### 21
data are needed to determine whether Pb exerts its toxic effects on the reproductive system by affecting 

### 22
the responsiveness of the hypothalamic

- pituitary

- gonad axis or by suppres sing circulating hormone levels

. 

### 23

More recent toxicological studies suggest that oxidative stress is a major contributor to the toxic effects of 

### 24
Pb on male and female reproductive systems. The effects of ROS may involve interference with cellular 

### 25
defense sy stems leading to increased lipid peroxidation and free radical attack on lipids, proteins, and 

### 26
DNA. Several recent studies showed an association between increased generation of ROS and germ cell 

### 27
injury as evidenced by destruction of germ cell structure and function. Co

- administration of Pb with 

### 28
various antioxidant compounds either eliminated Pb

- induced injury or greatly attenuated its effects. In 

### 29
addition, many studies that observed increased oxidative stress also observed increased apoptosis which is 

### 30

likel y a critical underlying mechanism in Pb

- induced germ cell DNA damage and dysfunction. 

### 31
Overall, results of pregnancy outcomes were similar to those of the 2006 AQCD; inconsistent 

### 32
evidence of a relationship with Pb was available for preterm birth and little evidence was available to 

### 33
study the associations with spontaneous abortions

. The previous AQCD included a few studies that 

### 34
reported possible associations between Pb and neural tube defects, but the recent epidemiologic studies 

### 35

found no association

. Possib le associations were observed between Pb and low birth weight when 

### 36May 2011 5

- 330 DRAFT

– DO NOT CITE OR QUOTE epidemiologic studies used measures of maternal bone Pb or air exposures, but the associations were less 

### 1
consistent when using maternal blood Pb or umbilical cord and placenta Pb

. Effects o f Pb exposure 

### 2
during early development on toxicological studies included reduction in litter size, implantation, birth 

### 3
weight and postnatal growth

. 

### 4
Toxicological studies demonstrated that the e ffects of Pb exposure during early development 

### 5

in clude im pairme nt of retinal development and alterations in the developing hematopoietic and hepatic 

### 6

systems . Negative developmental outcomes were also noted including effects on the skin and teeth. 

### 7
Similar to toxicological and epidemiologic studies that observed Pb to be associated with delayed 

### 8
puberty, delays of dynamic changes in the HPT axis are seen in the ecological literature, i.e., delayed 

### 9
metamorphosis in Pb exposed frogs

. Additionally, Pb exposure has been shown to have detrimental 

### 10
effects on sperm, albeit ofte n at higher blood Pb levels in epidemiology studies but in lower doses in the 

### 11
toxicology literature

. Again, these findings agree with the ecological literature where Pb

- dependent sperm 

### 12
effects are seen in rotifers, earthworms, and trout. 

### 13
In conclusion, th e recent toxicological and epidemiologic literature provides strong evidence that 

### 14

Pb exposure is related to delayed onset of puberty in both males and females. Additionally, Pb exposure 

### 15

has been shown to ha ve detrimental effects on sperm ( at high blood Pb levels in epidemiologic studies 

### 16

and in low doses in the toxicological literature ). The data on preterm birth, low birth weight, spontaneous 

### 17
abortions, birth defects, hormonal influences, and fecundity are a bit more mixed and less consistent 

### 18

between the to xicological and epidemiologic literature. The collective body of evidence integrated across 

### 19
epidemiologic and toxicological studies with a focus on the strong relationship observed with negative 

### 20
effects on sperm and delayed pubertal onset is sufficient to conclude that there is a causal relationship 

### 21
between Pb exposures and reproductive effects and birth outcomes. 

### 22



# 5.9. Effects on Other Organ Systems

5.9.1. Effects on the Hepatic System

Hepatotoxic effects of Pb in various animal and human models include alterations in hepatic 

### 23
metabolism, hepatic cell proliferation, changes in cholesterol metabolism, as well as oxidative stress



### 24
related injury. Animal studies have also shown that exposure to Pb causes a decrease in Phase I along with 

### 25

a simultaneous increase in Phase II enzymes following exposure to various forms of Pb. Induction of 

### 26
oxidative stress is well supported by an increase in lipid peroxidation along with a decrease in glutathione 

### 27
( GSH ) levels and catalase ( CAT )

, superoxide dismutase ( SOD ) and glutathione peroxidase (GPx) 

### 28

activities 

### 29May 2011 5

- 331 DRAFT

– DO NOT CITE OR QUOTE 

### 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 some of the frequent toxicological eff ects in the liver following 

### 1
exposure to Pb included increased hepatic cell proliferation, cholesterol synthesis, DNA synthesis and 

### 2
glucose

- 6

- phosphotase dehydrogenase (G6DP) activity resulting in Pb

- induced hyperplasia. The AQCD 

### 3
concluded that cytochrome ( CYP) P450 levels decreased following single exposures to Pb, primarily Pb 

### 4
nitrate. Inhibition of induced and constitutive expression of microsomal CYP 1A1 and 1A2 was observed 

### 5
among various P450 isozymes. Inhibition of Phase I enzymes was accompanied by an increase in Phase II 

### 6
enzymes following exposure to Pb nitrate and other Pb compounds, suggesting that Pb is capable of 

### 7
causing a biochemical phenotype similar to hepatic nodules. Studies relating to Pb

- induced hepatic 

### 8
hyperplasia suggested alterations in the gluconeogenic mechanism, DNA hypomethylation along with 

### 9
changes in proto

- oncogene expression as well as cholesterol synthesis. Cholesterol metabolism changes 

### 10
following exposure to Pb were reportedly mediated as a result of induction of several enzymes related to 

### 11 cholesterol metabolism as well as a decrease in the cholesterol catabolizing enzyme, 7 α

- hydroxylase. 

### 12 Tumor necrosis factor α (TNF

- α) was reported to be one of the major mitogenic signals that mediated Pb 

### 13

nitrate

- induced hepatic hyperplasia in s tudies using inhibitors to block TNF

- α activity. Other Pb

- related 

### 14
effects presented in the 2006 Pb AQCD include liver cell apoptosis mediated by Kupffer cell derived 

### 15
signals and Pb

- induced oxidative stress in vitro cell cultures. More recent Pb exposure ex periments 

### 16
suggested that alterations in liver heme metabolism may involve changes in 5

- aminolevulinic acid 

### 17
dehydrogenase (ALAD) activity, porphyrin metabolism, Transferrin (TF) gene expression and changes in 

### 18

iron metabolism. 

### 19
In humans, the 2006 Pb AQCD st ated that nonspecific liver injury generally observed as increases 

### 20
in liver enzymes in the serum was reported in occupational studies. In addition, similar to effects noted in 

### 21
animal studies, cytochrome P450 activity was also suppressed in humans following exposure to Pb under 

### 22
various conditions. The 2006 Pb AQCD concluded that hepatic effects occurred only at high Pb exposure 

### 23

levels. 

### 24

5.9.1.2. New Epidemiologic Studies

A few studies examined liver biochemical parameters effects on antioxidant status and oxidative 

### 25

st ress resulting from occupational exposures. Patil et al. ( 2007 ) examined the effect of occupational Pb 

### 26
exposure to liver and kidney function in silver jewelry workers (SJW), battery manufacturing workers 

### 27
(BMW) and spray painters (SP) in western Maharashtra, India. Blood Pb was statistically significantly 

### 28

increased in all three groups: 53.63 ± 16.98 (BMW)

, 48.56 ± 7.39 (SJW) , and 22.32 ± 8.87 μg/dL (SP ), 

### 29
compared to controls (12.52 ± 4.08). Liver function enzymes including serum glutamic oxaloacetic 

### 30

transaminase (SGOT)/AST, and serum glutamic pyruvic transaminase (SGPT) /ALT levels were only 

### 31May 2011 5

- 332 DRAFT

– DO NOT CITE OR QUOTE increased in the SP group compared to the concurrent control group but not in the SJW and BMW groups. 

### 1
Total serum protein levels were decreased in all three groups, while serum albumin levels were decreased 

### 2
in SJW and SP groups and in creased in the BMW group. Serum globulin levels were decreased and the 

### 3
albumin/globulin levels were increased in the BMW and SJW groups compared to controls. In addition, 

### 4
bilirubin levels were increased only in the BMW group. In a study examining the impac t of Pb exposure 

### 5
in an occupational setting, Khan et al. ( 2008 ) reported that workers in Pakistan occupationally exposed to 

### 6
Pb (blood Pb = 29.1 [range 9.0 to 61.1] μg/dL) had a signific ant increase (3.5

- fold higher median) in 

### 7
blood levels compared to age and gender matched controls (blood Pb = 8.3 [range 1.0 to 21.7] μg/dL). 

### 8
Oxidative stress markers such as MDA, and g amma

- glutamyl transpeptidase (GGT) were significantly 

### 9

increased in work ers as were ALT levels. Serum albumin and total protein levels were significantly 

### 10
decreased in the examined workers, compared to controls. Based on these results, study authors 

### 11
concluded that Pb exposure causes oxidative stress and changes in liver enzymes that may lead to hepatic 

### 12
toxicity in exposed workers. Can et al. ( 2008 ) al so reported changes in liver function enzymes among 

### 13
battery workers and muffler repair workers exposed to Pb in an occupational setting. Blood Pb was 

### 14
elevated in both worker groups ( 36.83 ± 8.13 and 26.99 ± 9.42 μg/dL for battery workers and muffler 

### 15
repair workers, respectively) versus. controls ( 14.81 ± 3.01 μg/dL). The study authors reported that total 

### 16
protein, globulin, and lactate dehydrogenase (LDH) levels were within or very close to the normal range, 

### 17
but were statistically significantly higher in bot h worker groups compared to controls. Additionally, 

### 18
increases in cholesterol and ALP were increased only in battery workers and muffler repair workers, 

### 19
respectively. Though an increase in LDH levels among the workers was observed, the study authors stated 

### 20
that this increase was not related to liver injury. Total protein, globulin, ALP, and LDH were also observed 

### 21
to be significantly correlated to blood Pb levels in workers. Though liver enzyme function changes were 

### 22
nominal, the study authors concluded that i n an occupational setting, exposure to Pb may lead to liver 

### 23
injury. While Can et al. ( 2008 ) did consider the impact of smoking in their analysis, it is not clear whether 

### 24

Patil et al. ( 2007 ) and Khan et al. ( 2008 ) considered the impact on these factors in their analysis. I n a 

### 25
single case study report of a 40

- year old Iranian male accustomed to using opium as a pain reliever, 

### 26
Verheij et al. ( 2009 ) repo rted that a liver biopsy taken following elevated liver function enzymes exhibited 

### 27
bile intracytoplasmic pigmentation in the hepatocytes. The study authors reported that the blood Pb levels 

### 28
were highly elevated in the patient (86.0 μg/dL), and attributed e xposure to Pb from Pb

- contaminated 

### 29
opium consumption. The liver parenchyma also revealed disrupted architecture along with regenerated 

### 30
nodules. Pathomorphological changes, rarely seen in humans, were also reported in the form of active 

### 31
hepatitis along with microvesicular and macrovesicular steatosis, hemosiderosis, and cholestatis as well as 

### 32
lymphocytic cholangitis. The study authors stated that following chelation therapy, liver enzymes returned 

### 33
to normal suggesting reversal of the histological findings. H owever, the reversibility was not confirmed 

### 34
with another liver biopsy. A case report by Fonte et al. ( 2007 ) described a worker occupationally exposed 

### 35
to Pb vapors (blood Pb = 148 μg/dL) with hypersideremia, mixed bilirubinemia, and elevated levels of 

### 36May 2011 5

- 333 DRAFT

– DO NOT CITE OR QUOTE ALT and AST. Following chelation therapy, the patient’s clinical symptoms resolved, indicating the 

### 1
reversibility of the Pb

- induced effects on the liver. 

### 2

5.9.1.3. New Toxicological Studies

Hepatic Metabolism

As stated in the AQCD 2006, acute exposures to Pb nitrate and other Pb compounds causes a 

### 3
decrease in Phase I enzymes accompanied by a simultaneous increase in Phase II e nzymes. The 

### 4
conclusions presented in the AQCD 2006 were also reviewed and corroborated by Mudipalli ( 2007 )

. 

### 5
Changes in biochemical parameters, suggestive of liver damage, in male Wistar rats t reated with 

### 6
500 ppm Pb acetate in drinking water over a 10 month period included decreases in serum protein and 

### 7
albumin levels as well as an increase in aspartate aminotransferase (AST), alanine aminotransferase 

### 8
(ALT), serum alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (GGT) levels ( S. et al., 

### 9

2009 ) . In treated animals, the blood Pb levels steadily increased throughout the study period, reaching a 

### 10

maximum of approximately 110 μg/dL. The study authors reported that similar biochemical changes were 

### 11

not observed in animals treated with Pb acetate as well a mineral rich diet and concluded that nutritional 

### 12
management is important in managing Pb

- related poisoning. S warup et al. ( 2007 ) investigated serum 

### 13
biochemical changes in cows living in Pb

- contaminated environments. Serum levels of ALT, AST, 

### 14
alkaline phosphatase, total protein, albumin, globulin, and A/G ratio were statistically significantly altered 

### 15
in cows living near Pb

- Zn smelters (blood Pb = 86 ± 6) compared to control cows (blood P b = 7 ± 1 

### 16
μg/dL). Significant positive correlations were found between blood Pb and ALT and AST, whereas a 

### 17
negative correlation was observed between blood Pb and total lipids, protein, and albumin. Upadhyay et 

### 18

al. ( 2009 ) investigated the effects of Pb exposure on biochemical alterat ions in Sprague

- Dawley rats 

### 19
exposed to 35 mg/kg via i.p. injection for 3 days. The activities of ALT, AST, serum ALP, and acid 

### 20
phosphatase were all significantly increased over control in exposed animals, whereas alkaline 

### 21
phosphatase activity was decreased in exposed animals. Concomitant treatment with zinc and varying 

### 22

levels of ascorbic acid were observed to ameliorate the toxic effects of Pb. 

### 23

Pillai et al. ( 2009 ) investigated gestational and lactational exposure to Pb on hepatic phase I and II 

### 24
enzymes in male and female rats. Pregnant rats were exposed to 50 μg/kg Pb acetate via subcutaneous 

### 25
injection daily throughout gestation, and continuing until PND21. The female and male pups were then 

### 26
allowed to reach sexual maturity (PND55

- 56) to assess continuing exposure to bioaccumulated Pb. The 

### 27
activities of hepa tic phase I enzymes NADPH

- and NADH

- cytochrome c reductase were statistically 

### 28
significantly reduced in Pb

- exposed male and female rats on PND56, compared to controls. In rats treated 

### 29
with 25 μg/kg Pb and Cd, the effect on phase I enzymes was increased. Pb exposure additionally 

### 30
decreased the activities of phase II enzymes uridine diphosphate

- glucoronyl transferase and GST in males 

### 31May 2011 5

- 334 DRAFT

– DO NOT CITE OR QUOTE and females, but no effect was observed on GGT or 17β

- hydroxysteroid oxidoreductase. Additionally, no 

### 1

effect was observed in Pb

- e xposed rats regarding serum glutamate pyruvate dehydrogenase or ALP 

### 2
activities in males or females. Histological observations in both male and female rats demonstrated fatty 

### 3
degeneration, vacuolization, and pycnotic nuclei, indicating general hepatotoxicit y following Pb 

### 4
exposure. In a similar study, Teijon et al. ( 2006 ) exposed Wistar rats to 200 or 400 ppm throughout 

### 5
gestation, lactation, and 3 months postweaning, or o nly 1 month postweaning. In the animals exposed 

### 6
continuously throughout gestation and lactation, the concentrations of Pb in the liver were elevated in the 

### 7

200

- and 400

- ppm groups 1 and 3 months postweaning. Liver concentrations of Pb were greater in the 

### 8

2 00 ppm animals compared to the 400 ppm animals at one month postweaning, but were similar between 

### 9
the 2 dosing regimens at 3 months postweaning. ALP activity was increased at 2 weeks postweaning in 

### 10
animals continuously exposed to Pb throughout gestation an d lactation, whereas ALT activity was 

### 11
decreased only at 2 and 3 months postweaning. In animals exposed only for 1 month postweaning, serum 

### 12
ALP activity was significantly increased, although in a non

- dose dependent manner. ALT and AST 

### 13

activities were not af fected. 

### 14
Cheng et al. ( 2006 ) studied the mechanism of Pb effects on bacterial lipopolysacc haride (LPS)

- 

### 15

induced TNF

- α expression. A/J mice were injected via i.p with 100 μmol/kg Pb, with or without 5 mg/kg 

### 16
LPS. Pb alone did not affect liver function (measured as AST or ALT activity) or the level of TNF

- α in the 

### 17
serum. In comparison, exposing the mice to low doses of Pb and LPS together caused a statistically 

### 18
significant increase in TNF

- α induction as well as enhanced liver injury, suggesting that Pb potentiated 

### 19

LPS

- induced 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 increased TNF

- α 

### 21
expression in mouse whole blood cells, peritoneal macrophages, and RAW264.7 cells (a macrophage cell 

### 22
line) and concluded that monocytes/macro phages (rather than hepatocytes) were primarily responsible for 

### 23

Pb increased LPS

- induced TNF

- α levels via the protein kinase C (PKC)/MAPK pathway. 

### 24

Lipid Metabolism

In a lipid metabolism study, Ademuyiwa et al. ( 2009 ) reported that male albino Sprague Dawley 

### 25
rats exposed to 200, 300 and 400 ppm Pb in drinking water had blood Pb levels of 40.63 ± 9.21, 61.44 ± 

### 26
4.63, and 39.00 ± 7.90 μg/dL, respectively. Animals exposed to 200 ppm had liver Pb concentrations of 

### 27
10.04 ± 1.14 μg/g, compared to 3.24 ± 1.19 and 2.41 ± 0.31 in animals exposed to 300 or 400 ppm Pb. 

### 28
Animals exposed to Pb exhibited increased hepatic cholesterogenesis at all doses tested compared to 

### 29
controls. Additionally, a decrease in triglyceride was observed at 300 and 400 ppm, a decrease in 

### 30
phospholipid levels was observed at 400 ppm. The authors also reported a positive correlation between 

### 31
tissue cholesterol and phospholipids compared to Pb accumulation in liver across all doses. I n contrast, 

### 32
the association between tissue triglyceride levels and Pb accumulation was negative. In a related study, 

### 33May 2011 5

- 335 DRAFT

– DO NOT CITE OR QUOTE Khotimchenko and Kolenchenko ( 2007 ) reported that adult male albino rats treated with 100 mg/kg Pb 

### 1
acetate for as little as 14 days exhibited disorders in lipid metabolism that were supported by increased 

### 2
levels of total cholesterol and triglyceride levels in the liver tissue. Pillai et al. ( 2009 ) observed decreases 

### 3

in total liver cholestero l in PND56 male and female rats that had been exposed to 50 μg/kg Pb acetate 

### 4
continuously throughout gestation and lactation. These results suggest that induction of cholesterogenesis 

### 5
and phospholipidosis in the liver may cause subtle effects at the cellul ar level that may lead to 

### 6
hepatotoxicity. Kojima and Degawa ( 2006 ) examined the gender

- related differences in the hepatic sterol 

### 7
regulatory element binding protein

- 2 (SREB P

- 2) and 3

- hydroxy

- 3

- methylglutaryl

- CoA reductase 

### 8
(HMGR) gene expressions in male and female Sprague Dawley rats injected with 100 μmol/kg body 

### 9
weight of Pb nitrate intravenously. The SREBP

- 2 expression, which is a transcription factor for the 

### 10
HMGR gene, w as significantly increased in males and females with the increase occurring earlier in male 

### 11
rats (6

- 12 hours, compared to 24

- 36 hours in females). In contrast, expression of the HMGR gene, a rate 

### 12
limiting enzyme in cholesterol biosynthesis, was significant ly increased in both males and females at 

### 13
earlier time frames (3

- 48 hours in males; 12

- 48 hours in females) compared to the SREBP

- 2 gene 

### 14
expression. Significant increases in total liver cholesterol were also observed in males and females at 3

- 48 

### 15

and 24

- 48 hours, respectively. These results suggest that the SREBP

- 2 and HMGR gene expressions and 

### 16
increase in total cholesterol levels in the liver occur earlier in males compared to females and also suggest 

### 17
that the HMGR gene expression and increase in total chol esterol levels in the liver occur before an 

### 18

increase in the SREBP

- 2 gene expression in either sex. 

### 19

Hepatic Oxidative Stress

A number of studies pertaining to hepatic oxidative stress as a result of exposure to various Pb 

### 20
compounds were identified. Adegbes an and Adenuga ( 2007 ) reported that protein undernourished male 

### 21
Wistar rats injected with 100 μmol/kg Pb nitrate exhibited increased lipid peroxidation, increased CAT 

### 22
activity, decreased SOD activity, and increased GSH levels, compared to undernourished rats not exposed 

### 23
to Pb. Increased lipid peroxidation and decreased CAT and SOD activity were also observed when 

### 24
comparing undernourished Pb

- exposed rats to wellnourished control rats. Study authors concl uded that 

### 25
malnutrition exacerbated Pb exposure effects on liver lipid peroxidation and the involvement of free 

### 26
radicals in Pb toxicity. Male Foster rats exposed to 0.025 mg/kg Pb via i.p. injection also exhibited 

### 27
statistically significant increases in lipi d peroxidation levels and decreases in SOD, CAT, and glucose

- 6

- 

### 28
phosphatase dehydrogenase (G6PD) levels in liver mitochondrial and postmitochondrial fraction ( Pandya 

### 29
et al., 2010 ) . Nonstatistically significant decreases were also observed in GSH levels and GPx and GR 

### 30
activities in exposed animals. Yu et al. ( 2008 ) reported similar dose

- dependent increases in lipid peroxide 

### 31
levels and decreases in GSH levels and CAT, SOD and GPx activities in castrated boars that received a 

### 32
supplemental diet with 0, 5, 10, or 20 mg/kg Pb. The level of hepatic CuZn SOD mRNA was also reduced 

### 33May 2011 5

- 336 DRAFT

– DO NOT CITE OR QUOTE in treated animals. The study authors suggested that this decrease in SOD mRNA expression and activity 

### 1
of antioxidant enzymes may lead to a reduction free radical scavenging capability, along with increased 

### 2
lipid peroxidation, pot entially causing serious damage to hepatic function and structure. Khotimchenko 

### 3

and Kolinchenko ( 2007 ) also reported an increase in lipid peroxidation and development of hepatitis in 

### 4
male albino rat liver parenchyma following treatment with 100 mg/kg Pb acetate for as little as 14 days. 

### 5
Lipid peroxidation was confirmed by increases in malondialdehyde (MDA) levels along with decreases in 

### 6
GSH and thiol groups indicating inj ury in the liver antioxidant system. In another experiment, Jurczuk et 

### 7

al. ( 2007 ) reported that male Wistar rats treated with 500 mg/L Pb in dri nking water exhibited decreases 

### 8
in liver vitamin E and GSH levels along with an increase in lipid peroxidation. The study authors 

### 9
hypothesized that vitamin E is involved in the mechanism of peroxidative action of Pb in the liver, and 

### 10
concluded that the sug gested protective role of vitamin E in the potential toxicity by Pb may be related to 

### 11
scavenging of free radicals that are generated either directly or indirectly by Pb. In a study examining the 

### 12
effects of Pb exposure to fetuses, Massó et al. ( 2007 ) exposed pregnant Wistar rats with 300 mg/L Pb in 

### 13
drinking water starting at day 1 of pregnancy to parturition or until weaning to determine the effects of Pb 

### 14
exposure i n the fetal liver. Pups exhibited liver damage that was supported by an increase in thiobarbituric 

### 15

acid

- reactive species (TBARS) production and increased CAT activity compared to controls. In addition, 

### 16
increased ALP and acid phosphatase activity was also o bserved. Uzbekov et al. ( 2007 ) exposed female 

### 17
Wistar rats to 0.3 and 3.0 mg/L Pb nitrate for 1 and 5 months prior to, and continuing duri ng pregnancy, 

### 18
and measured fetal hepatic SOD activity on GD20. In the fetuses from dams exposed for 1 month prior to 

### 19
pregnancy, an dose

- dependent increase in liver SOD activity was observed, whereas SOD activity was 

### 20
decreased in the fetuses from dams expos ed for 5 months prior to pregnancy. The increase in SOD 

### 21
activity in the livers of fetuses from dams exposed to 0.3 or 3.0 mg/L Pb nitrate for one month suggests 

### 22
that activation of SOD in response to increased free radical production, while the decrease in SOD 

### 23
production in fetal livers from dams exposed to the same concentrations for 5 months suggests that longer 

### 24
durations of Pb exposure impairs the antioxidant defense mechanism. In a study examining the role of 

### 25
low molecular weight thiols on peroxidative m echanism, Jurczuk et al. ( 2006 ) stated that male Wistar rats 

### 26
treated with 500 mg/L Pb acetate in drinking water exhibited a decrease in blood ALAD as well as 

### 27
decreases in GSH and nonprotein sulfhydryl (NPSH) levels in the liver. Metalothionine levels were also 

### 28
reported to be higher in t he liver following exposure to Pb. Levels of hepatic lipid peroxidation were 

### 29
observed to be significantly increased in rats exposed to 35 mg/kg Pb via i.p. injection, whereas hepatic 

### 30
GSH was significantly decreased ( Upadhyay et al., 2009 ) . No effects on GSH or MDA levels were 

### 31
observed in PND56 male and female rats following continuous exposure to 50 μg/kg Pb acetate 

### 32
throughout gestation and lactation ( Pillai et al., 2009 )

. 

### 33
The studies presented above all confirm the possible oxidative stress impacts following exposure to 

### 34

various doses of Pb administered in various forms and the potential for hepatotoxicity as a result of 

### 35

oxidative stress. 

### 36May 2011 5

- 337 DRAFT

– DO NOT CITE OR QUOTE 

## 5.9.2. Effects on the Gastrointestinal System

Gastrointestinal effects of Pb exposure primarily include abdominal pain, constipation, and internal 

### 1
paralysis i n humans. In animals, degeneration of the intestinal epithelial mucosa and a decrease in 

### 2
duodenal motility has been reported. 

### 3

5.9.2.1. Summary of Key Findings on the Effects on the Gastrointestinal System from the 2006 Lead AQCD

The 2006 Pb AQCD states that a numbe r of factors influence the gastrointestinal absorption of Pb 

### 4
including the chemical and physical form of Pb, the age at Pb intake, as well as various nutritional factors. 

### 5
Potential malabsorption of Pb as a result of degeneration of the intestinal epithelia l mucosa has been 

### 6
observed in rats exposed to Pb. Casein micelles incidences were reported as a result of Pb present in 

### 7
bovine and rat milk and in infant milk formula. Pb ingestion through water was more toxic compared to 

### 8
Pb ingestion via milk. Pb ingested in milk was reported to be taken up by the ileal tissue, whereas Pb 

### 9
administered intragastrically as a soluble salt was primarily accumulated in the duodenum irrespective of 

### 10
vehicle used for administration. Decreases in duodenal motility and the amplitude of contractility in the 

### 11
intestinal tract were observed in rats following Pb exposure. Nutritional studies examining different 

### 12
dietary levels of Pb, calcium, and vitamin D indicated competition in absorption between Pb and calcium. 

### 13
Diet supplement with vit amin D Pb to an increase in intestinal absorption of Pb and calcium. In instances 

### 14
where severe calcium deficiency was noted, ingestion of Pb caused a clear decrease in 1,25

- dihydroxy 

### 15
vitamin D (1,25

- (OH)2D3) levels. Overall, the 2006 Pb AQCD states that st udies in rat intestine have 

### 16
shown that the largest amount of absorption occurs in the duodenum with the mechanisms of absorption 

### 17
involving active transport and diffusion via the intestinal epithelial cells. Absorption has been reported to 

### 18
occur, through bo th saturable and nonsaturable pathways based on results from various animal studies. 

### 19
The AQCD also states that the 1986 Pb AQCD reported that common gastrointestinal effects in humans 

### 20
following exposure to Pb include early symptoms of abdominal pain, const ipation, and internal paralysis 

### 21
with these symptoms generally observed at blood Pb range of 30

– 80 μg/dL

. 

### 22

5.9.2.2. New Epidemiologic Studies

The 2006 Pb AQCD reported that in humans, gastrointestinal effects generally include abdominal 

### 23
pain, constipation, and intern al paralysis. In a case study, Cabb et al. ( 2008 ) reported similar symptoms in 

### 24

a 3

- year

- old child diagnosed with elevated blood Pb (19 μg/dL). The child was reported to be 

### 25
complaining of no nlocalized abdominal pain along with vomiting, nausea, constipation, lack of appetite, 

### 26
joint pains, fatigue, irritability as well as headaches. Based on detailed nutritional information obtained 

### 27
from the mother, the study authors inferred that the child wa s regularly ingesting candy contaminated 

### 28
with Pb. Following this the child was treated by a folk healer with “greta” an orange powder that 

### 29May 2011 5

- 338 DRAFT

– DO NOT CITE OR QUOTE worsened the abdominal symptoms. When analyzed, it was determined that “greta” contained 14,000 

### 1
mg/kg Pb monoxide. Wh en the child was taken off “greta” his GI symptoms began to resolve. Based on 

### 2
this study report, it may be concluded that Pb is capable of causing severe GI trauma and the GI effects 

### 3
may be reversible once exposure to Pb is ceased. In a similar case study, Fonte et al. ( 2007 ) repo rted that 

### 4

a 47

- year

- old male exposed to Pb fumes and vapors at a recycling plant experienced similar symptoms 

### 5
reported by Cabb et al. ( 2008 ) in the 3

- year

- old child. The male patients’ Pb l evels were elevated with a 

### 6
blood Pb level of 148 μg/dL. Once exposure to Pb had ceased and the patient was treated with EDTA, his 

### 7
condition improved and the blood Pb dropped to 16 μg/dL. Kuruvilla et al. ( 2006 ) also reported 

### 8
gastrointestinal effects including stomach pain and gastritis along with other Pb

- related clinical 

### 9
manifestations in battery workers and painters (blood Pb = 42.40 ± 25.53 and 8.04 ± 5.04 μg/dL, 

### 10
respectively) occupationally exposed to Pb in India. 

### 11

5.9.2.3. New Toxicological Studies

Two studies pertaining to gastro intestinal effects of Pb exposure were identified. Santos et al. 

### 12
( 2006 ) examined the impact of Pb exposure on nonadrenergic noncholinergic (NANC) relaxations in rat 

### 13
gastric fundus. Male Wistar rats treated with 0.008% Pb acetate via drinking water for 15, 30, and 120 

### 14
days exhibited significant difference in NANC relaxations in the gastric fundus following electrical field 

### 15
stimulus (EFS). Whil e frequency

- dependent relaxations were observed in all groups, including the control 

### 16
group, the relaxations were significantly inhibited in rats treated with Pb acetate at all three durations. 

### 17
When gastric fundus strips from rats were incubated with L

- nitr oarginine (L

- NOARG), a nitric oxide 

### 18
synthase (NOS) inhibiter, no additional inhibition in relaxations was observed. In contrast, incubation 

### 19
with sodium nitroprusside and 8

- Br

- GMPc (a Cyclic guanosine monophosphate [cGMP] analog), 

### 20

exhibited at dose

- dependen t relaxation in strips in the control group and group exposed to Pb acetate for 

### 21
120 days. Study authors concluded that chronic exposure to Pb causes inhibition in NANC relaxation 

### 22
probably due to the modulated release of NO from the NANC nerves or due to in teraction with the 

### 23
intracellular transducer mechanism in the rat gastric fundus. 

### 24
In another study examining Pb

- induced oxidative stress in the gastric mucosa, Olaleye et al. ( 2007 ) 

### 25
treated Albino Wistar rats with 100 or 5,000 mg/L of Pb acetate for 15 weeks. Exposure to Pb acetate 

### 26
caused a significant increase in gastric mucosal damage caused by pretreatment with acidified ethanol. 

### 27
Study authors reported that though the basal gastric acid secretory rate was not altered, stomach res ponse 

### 28
to histamine was significantly higher in animals treated with Pb acetate compared to the controls. 

### 29
Additionally, there was a significant increase in gastric lipid peroxidation in both the 100 and 5,000 mg/L 

### 30
dose levels. In contrast, CAT, and SOD acti vities and nitrite levels were significantly decreased in the 

### 31
gastric mucosa. Study authors concluded that exposure to Pb may increase the formation of gastric ulcers 

### 32
as a result of changes in the oxidative metabolism in the stomach. 

### 33May 2011 5

- 339 DRAFT

– DO NOT CITE OR QUOTE 

## 5.9.3. Effects on the Endocri ne System

Endocrine processes that are most commonly impacted by Pb exposure include changes in the 

### 1
thyroid, such as changes in the thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine 

### 2
(T4). In addition changes in the male sex hormone le vels have also been reported following exposure to 

### 3

Pb. 

### 4

5.9.3.1. Summary of Key Findings of the Effects on the E ndocrine System from
the 2006 Lead AQCD

Endocrine processes that may be impacted by Pb include thyroid hormone levels, changes in male 

### 5
sex hormone levels, as well as changes in the production of 1,25

- (OH)2D3 levels. However, these effects 

### 6
were reported to be observed only at blood Pb levels exceeding 30

– 40 μg/dL. Summary of key findings 

### 7
pertaining to reproductive hormones in females is presented in the sect ion on reproductive effects and 

### 8
birth outcomes (Section 5.8.1). 

### 9

5.9.3.2. New Epidemiologic Studies

Thyroid hormone levels were reported to be impacted following exposure to various environmental 

### 10
contaminates, including Pb, by Abdelouahab et al. ( 2008 ) , Croes et al. ( 2009 ) , and Dundar et al. ( 2006 )

. 

### 11

Abdelouahab et al. ( 2008 ) performed a cros s

- sectional study in a Canadian population exposed to various 

### 12
environmental contaminants, including Pb, following consumption of freshwater fish. The median blood 

### 13
Pb level of men included in this study was 3.1 μg/dL, whereas for women the median was 1.7 μg /dL. It is 

### 14
important to note that the median blood Pb level for women is lower than the limit of detection for Pb in 

### 15
the blood (2.1 μg/dL), effectively meaning that greater than 50% of women in the study had 

### 16

nondetectable levels of Pb in their blood. The s tudy authors conducted a stratified analysis and concluded 

### 17
that TSH levels were negatively correlated with blood Pb in women that consumed fish contaminated 

### 18
with Pb and other environmental pollutants. No impacts in T3 and T4 levels were reported in women. 

### 19
TSH, T3 and T4 levels were not observed to be correlated with blood Pb in males. However, study 

### 20
authors stated that occupational exposure to Pb in men can affect pituitary thyroid axis homeostasis and 

### 21
the relation between low level Pb exposure thyroid horm one homeostasis in men and women needs to be 

### 22
investigated further. Dundar et al. ( 2006 ) examined the effects of bl ood Pb on thyroid function in 42 male 

### 23
adolescent auto repair workers exposed long term to Pb. A control group comprised of 55 healthy subjects 

### 24
was also used for comparison purpose. Mean blood Pb levels were reported to be higher in the auto repair 

### 25

workers compared to the control subjects (7.3 ± 2.92 versus. 2.08 ± 1.24 μg/dL). Free T4 (FT4) levels 

### 26
were significantly lower in the study group compared to the control group, which had no abnormal FT4 

### 27
levels reported. In contrast, free T3 (FT3) and TSH levels we re comparable between the study and control 

### 28
group. Blood Pb level was reported to be negatively correlated with FT4 levels. Based on the study 

### 29May 2011 5

- 340 DRAFT

– DO NOT CITE OR QUOTE outcome, the study authors reported that long

- term exposures that result in the studied blood Pb levels 

### 1
may lead to lower FT4 levels without impact on T3 and TSH levels in adolescents. The study authors 

### 2
stated that this effect is likely secondary to the toxic effects of Pb on the pituitary

- thyroid axis and to the 

### 3
earlier findings of primary hypothyroidism as a result of impaired production of peripheral thyroid 

### 4
hormones. Similar findings were reported by Croes et al. ( 2009 ) in a study conducted in Belgium. Croes 

### 5

et al. ( 2009 ) examined the hormone levels and degree of sexual maturity in 1679 adoles cents residing in 

### 6
nine study areas with varying exposures to multiple industrial pollutants including Pb. The median blood 

### 7
Pb of the participants from the nine different regions ranged from 1.6 to 2.8 μg/dL. The study authors 

### 8
reported that, after correctio n for confounding, significant interregional differences were observed in the 

### 9
levels of sex hormones including total and free testosterone, estradiol, aromatase, and luteinizing hormone 

### 10
as well as FT3 hormone levels. When individual neighborhoods were anal yzed within the larger study 

### 11
areas, altered levels of testosterone, aromatase, and FT3 levels were also observed. Altered sexual 

### 12
maturation was also observed among boys and girls of individual study areas, compared to the sexual 

### 13

maturation of the entire co hort. Though varying levels of sex hormones and rates of sexual maturation 

### 14
was observed, the study authors reported that these changes are not wholly due to exposure to various 

### 15
pollutants, including Pb that were measured in the study and stated that other pollutants and 

### 16
environmental factors may also contribute to the effects noted. 

### 17
Gump et al. ( 2008 ) examined cortisol response to acute stress in children (aged 9.5 years) whose 

### 18
prenatal and postnatal blood Pb levels had been determined prior to the study at birth (from cord blood) 

### 19
and at age 2.62 ± 1.2 years, respecti vely. For prenatal blood Pb, the children were broken into the 

### 20 following quartiles: ≤ 1, 1.1

- 1.4, 1.5

- 1.9, and 2.0

- 6.3 μg/dL. For postnatal blood Pb, the quartiles were 

### 21

1.5

- 2.8, 2.9

- 4.1, 4.2

- 5.4, and 5.5

- 13.1 μg/dL. T he study authors reported that blood Pb was not associated 

### 22
with initial salivary cortisol levels . However following an acute stressor , which was comprised of a gallon 

### 23
of one part ice to one part water into which a child was asked to submerge his or her dominant arm for a 

### 24
minute, increasing pren atal and postnatal blood Pb level s were statistically significantly associated with 

### 25
increases in salivary cortisol responses . Children in the 2nd, 3rd and 4th prenatal blood Pb quartiles and in 

### 26

the 4 th postnatal quartile appeared to have increased salivary cortisol responses compared to children in 

### 27

the 1 st quartile. When blood Pb was treated as a continuous variable, regression analysis showed that both 

### 28
prenatal and postnatal blood Pb levels were significantly correlated to salivary cortisol reactivity. Bas ed 

### 29
on these results, the study authors reported that relatively low prenatal and postnatal blood Pb levels, 

### 30
notably those well below 10 μg/dL, can alter children’s adrenocortical responses following acute stress 

### 31
and the health impact and behavioral aspects of this Pb

- induced HPA deregulation in children needs to be 

### 32

further examined. 

### 33
In another study on the impact of Pb in children, Kemp et al. ( 2007 ) examined the blood levels in 

### 34
142 young, urban African

- American and Hispanic children in winter and summer to determine the 

### 35

seasonal increase in blood Pb and its association with vitamin D, age and race

. Ther e was a 

### 36May 2011 5

- 341 DRAFT

– DO NOT CITE OR QUOTE winter/summer (W/S) increase in blood Pb levels in children aged between 1 and 3 years (winter blood Pb 

### 1
= 4.94 ± 0.45 (SE) μg/dL, summer blood Pb = 6.54 ± 0.82 (SE) μg/dL), with a smaller W/S increase 

### 2
observed in children aged between 4 and 8 years (winter blood Pb = 3.68 ± 0.31 (SE) μg/dL, summer 

### 3

blood Pb = 4.16 ± 0.36 (SE) μg/dL)

. Additionally, the winter and summer blood Pb levels were highly 

### 4

correlated with one another

. The percentage of African

- American children with blood Pb levels ≥ 10 

### 5
μg/dL increased from 12.2% in winter to 22.5% in summer

. Summer and winter concentrations of 1,25

- 

### 6
(OH)2D3 were observed to differ in children aged 4

- 8 years and the correlation between the serum 1,25

- 

### 7
(OH)2D3

. No difference in 1,25

- (OH)2D3 was observed in childre n 1

- 3 years old

. There was a significant 

### 8

correlation between seasonal differences in blood Pb and serum 1,25

- (OH)2D3 in all children and 

### 9

African

- American children between 4 and 8 years

. Based on these results, the study authors concluded 

### 10
that higher summer time increase in serum 1,25

- (OH)2D3 levels in children between 4 and 8 years is most 

### 11
likely due to increased sunlight

- induced vitamin D synthesis and may be a contributing factor to seasonal 

### 12
changes in blood Pb levels. 

### 13

5.9.3.3. New Toxicological Studies

A single s tudy examining the impact of Pb exposure on the endocrine system was identified. In a 

### 14
study examining the effects of Pb and cadmium in adult cows reared in a polluted environment in India, 

### 15
Swarup et al. ( 2007 ) stated that the mean plasma T3 and T4 levels were significantly higher in cows near 

### 16
Pb and zinc smelters (blood Pb = 86 ± 6 μg/dL) and near closed Pb and operational zinc smelters (blood 

### 17
Pb = 51 ± 9 μg/dL) when compared to cows in unpolluted areas (blood Pb = 7 ± 1 μg/dL). Regression 

### 18
analyses from 269 cows examined in the study showed a significant positive correlation betwee n blood 

### 19
Pb and plasma T3 and T4 levels, whereas the correlation between blood Pb and plasma cortisol was 

### 20
nonsignificant. Mean plasma estradiol level was significantly higher in cows near closed Pb and 

### 21
operational zinc smelter industries compared to the con trol group. Based on these results, the study 

### 22
authors concluded that endocrine profile in animals can be impacted following exposure to Pb in polluted 

### 23

environments. 

### 24

Biswas and Ghosh ( 2006 ) investigated the effect of Pb exposure on adrenal and male gonadal 

### 25
functions in Wistar rats exposed to 8.0 mg/kg Pb acetate via i.p. injection for 21 days. Exposure to Pb was 

### 26
observed to significantly increase adrenal steroidogenic enzyme activity and serum corticosterone levels. 

### 27
Accessory sex organ (prostate and seminal vesicle) weights were decreased in P b

- exposed animals, 

### 28
whereas adrenal weights were increased. Spermatogenesis was decreased and the percent of spermatid 

### 29
degeneration was increased in animals exposed to Pb. Lastly, serum concentrations of testosterone, FSH, 

### 30
and LH, were decreased in Pb

- expos ed animals. Supplementation with testosterone during the last 14 

### 31
days of exposure to Pb was observed to ameliorate these effects. 

### 32May 2011 5

- 342 DRAFT

– DO NOT CITE OR QUOTE 

## 5.9.4. Effects on Bone and Teeth

Primary effects on bone as a result of Pb exposure include an increase in osteoporosis, increased 

### 1

f requencies of falls and fractures, changes in bone cell function as a result of replacement of bone calcium 

### 2
with Pb and depression in early bone growth. Similar to bone, calcium in the teeth is easily substituted by 

### 3
Pb following Pb exposure. Exposure to hi gh levels of Pb may result in the formation of “Pb line” and Pb 

### 4
can also cause a decrease in cell proliferation, procollagen type I production, intracellular protein, and 

### 5
osteocalcin in human dental pulp cell cultures. Accumulation of Pb was also associate d with tooth loss 

### 6
and higher incidence of periodontitis. 

### 7

5.9.4.1. Summary of Key Findings of the Effects on Bone and Teeth from the 2006 Lead AQCD

The 2006 Pb AQCD reported many effects on bone and some in teeth following exposure to Pb. 

### 8
Calcium in bone was easily substituted by Pb and taken up by the bone causing changes in bone cell 

### 9
function. Exposure to Pb during gestation and immediate postnatal period was reported to significantly 

### 10
depress early bone growth with the effects showing a dose

- dependent trend, though this effect was not 

### 11
manifested below certain exposure thresholds. Bone effects following short

- term exposure to Pb were not 

### 12
reported in mature animals; however, long

- term exposures to Pb along with poor nutrition had an adverse 

### 13
effect on bone growth as we ll bone density. Systemic effects of Pb exposure include disruption in bone 

### 14
mineralization during growth, alteration in bone cell differentiation and function due to alterations in 

### 15
plasma levels of growth hormones and calcitropic hormones such as 1,2

- [OH]2 D3 and impact on calcium 

### 16
binding proteins and increases in calcium and phosphorus concentrations in the blood stream. Bone cell 

### 17
cultures exposed to Pb were shown to impact vitamin D

- stimulated production of osteocalcin 

### 18
accompanied by inhibition of secretio n of bone

- related proteins such as osteonectin and collagen. In 

### 19
addition, Pb exposure also caused suppression in bone cell proliferation most likely due to interference 

### 20
from factors such as growth hormone (GH), epidermal growth factor (EGF), transforming g rowth factor



### 21
beta 1 (TGF

- β1), and parathyroid hormone

- related protein (PTHrP). 

### 22
Like the bone, Pb can easily substitute for calcium in the teeth and is taken and incorporated into 

### 23
developing teeth in experimental animals. Since teeth do not undergo remodeli ng like the bone during 

### 24
growth, most of the Pb in the teeth remains in a state of permanent storage. High dose exposure of Pb to 

### 25

animals has lead to the formation of a “Pb line” that is visible in both the enamel and dentin and is 

### 26

localized in areas of rec ently formed tooth structure. Areas of mineralization are easily evident in the 

### 27
enamel and the dentin within these “Pb lines.” Pb has also been shown to decrease cell proliferation, 

### 28
procollagen type I production, intracellular protein, and osteocalcin in h uman dental pulp cell cultures. 

### 29
Adult rats exposed to Pb have exhibited an inhibition of the posteruptive enamel proteinases, delayed 

### 30
teeth eruption times, as well as decrease in microhardness of surface enamel. Pb was reported to be 

### 31May 2011 5

- 343 DRAFT

– DO NOT CITE OR QUOTE widely dispersed and i ncorporated into developing apatite crystal during enamel formation process; 

### 1
however, post formation, Pb was reported to be capable of entering and concentrating in enamel areas that 

### 2
were calcium deficient. The AQCD also reported that a number of epidemiol ogic and animal studies have 

### 3
both separately suggested that Pb is a caries

- promoting element. 

### 4

5.9.4.2. New Toxicological and Epidemiologic Studie s

As reported in the 2006 Pb AQCD, Pb is capable of causing significant toxicological effects in 

### 5

bones of humans and an imals following short

- term and long

- term exposure. The association between Pb 

### 6
exposure and osteoporosis was examined in three different epidemiologic studies. Campbell and Auigner 

### 7
( 2007 ) examined subjects ≥ 50 years of age using the Third National Health and Nutrition Examination 

### 8
Survey (NHANES III) for association between Pb exposure and osteoporosis. The study authors used the 

### 9
bone mineral density (BMD ) in the hip as the primary variable to examine groups comprised of non



### 10
Hispanic white men (mean blood Pb = 4.9, range: 0.7 to 48.1 μg/dl), non

- Hispanic white women (mean 

### 11
blood Pb = 3.6, range: 0.7 to 28.7 μg/dL), African

- American men (mean blood Pb = 7.7, range: 0.7 to 

### 12
52.9 μg/dL) and African

- American women (mean blood Pb = 4.5, range: 0.7 to 23.3 μg/dL). The results 

### 13
indicated that the adjusted mean total hip BMD in the non

- Hispanic white males who had the lowest 

### 14
blood Pb levels was statistically significa ntly higher than the males with higher blood Pb levels. Similar 

### 15
associations, although not statistically significant, were reported among white females. Due to the small 

### 16
sample size, similar results were not observed among African

- American men and women. N o association 

### 17
was observed between blood Pb and osteoporotic fractures in any gender/race. Since the NHANES data 

### 18
were comprised of a cross

- sectional design, no inferences could be made regarding the temporal sequence 

### 19
of the observed association. The study authors concluded that further inquiry was needed to study the 

### 20
possible causal association between Pb exposure and osteoporosis. In a similar study, Sun et al. ( 2008 ) 

### 21
examined the association between Pb exposure and osteoporosis in 192 (155 males; 37 females) Chinese 

### 22
individuals occupationally

- exposed to Pb (blood Pb = 20.22 and 15.5 μg/dL, respectively). BMD was 

### 23
reported to be statistically significantly lower in expo sed females compared to exposed males. When all 

### 24
participants (including 36 male and 21 female unexposed controls) were divided into groups according to 

### 25
blood Pb and urinary Pb levels, the study authors reported that there were significant decreases in BMD in 

### 26 groups that had high urinary Pb levels (≥ 5 μg/g creatinine) compared to groups with low urinary Pb in 

### 27
both genders. In contrast a significant difference was only observed between blood Pb and BMD in males 

### 28
with blood Pb >30 μg/dL. Prevalence of osteopor osis was reported to increase significantly with 

### 29
increasing blood Pb in a linear manner. Khalil et al. ( 2008 ) reported similar associations between Pb 

### 30
exposure and osteoporosis in older women. The study authors conducted a prospective s tudy using 533 

### 31
women aged 65

- 78 years with a mean blood Pb of 5 .3 ± 2.3 μg/dL to determine the association between 

### 32
blood Pb and recurring fractures. The BMD was 7% lower in the total hip (p <0.02) and 5% lower in the 

### 33May 2011 5

- 344 DRAFT

– DO NOT CITE OR QUOTE femoral neck (p <0.03) in the highest b lood Pb group (≥ 8 μg/dL) compared to the lowest blood Pb group 

### 1 (≤ 3 μg/dL). The trend across all dose groups was also observed to be statistically significant for hip and 

### 2
femoral neck BMD. In addition, hip, femoral neck, and calcaneus bone loss was observ ed to be greater in 

### 3
the medium (blood Pb = 4

- 7 μg/dL) and high Pb groups compared to the low Pb group, but the observed 

### 4
trend was only significant for calcaneus bone loss. Multivariate analysis indicated that women with high 

### 5

blood Pb levels had an increase d risk of nonspine fracture and women with medium or high blood Pb 

### 6
levels had a higher risk of falls compared to the low blood Pb level group. Based on these results, the 

### 7
study authors concluded that blood Pb is associated with an increased risk of falls a nd fractures leading to 

### 8
negative health consequences including osteoporosis

- related fractures. 

### 9
The effect of Pb exposure on bone development in younger children has been studied before and an 

### 10
association has been found between Pb exposure and bone develop ment. Ignasiak et al. ( 2006 ) conducted 

### 11
a study on school children aged 7

- 15 years (463 males, 436 females) living close to copper smelters and 

### 12
refineries in Poland to assess the impact of Pb exposure on their growth status. The mean blood Pb for all 

### 13
participants was 7.7 ± 3.5 μg/dL (range: 2.0 to 33.9). Blood Pb levels were similar between males and 

### 14
females except at age 14.8 years, when females had lower blood Pb than males. Study results indicated 

### 15
that there was a statistically significant and linear relationship between blood Pb and reduced weight, 

### 16
height, and trunk, leg and arm lengths. Regression analysis revealed that, per every 10 μg/dL inc rease in 

### 17
blood Pb levels, height decreased 2.1 and 2.9 cm for males and females, respectively. This decrease in 

### 18
height was more influenced by decreases in leg length than trunk length. Based on these results, the study 

### 19

authors concluded that linear skeleta l growth was reduced with increases in blood Pb levels and these 

### 20
effects were seen even at levels below 10 μg/dL. These results also indicated that there was attenuation in 

### 21
osteoblast activity as a result of Pb exposure. 

### 22
To understand the significance of bone as a target tissue of Pb toxicity, Jang et al. ( 2008 ) studied 

### 23
the effect of Pb on calcium release activated calcium influx (CRACI) using primary cultures of human 

### 24

osteoblast

- like cells (OLC). When cells were incubated with 1 or 3 mM Pb, a dose

- dependent impact on 

### 25
the CRACI observed, as was a dose

- dependent increase in the influx of Pb into human OLC. These 

### 26
results suggest that Pb interferes with CARCI in human OLCs by initiating th e CRACI (i.e. the 

### 27
measurable influx of calcium upon re

- addition of calcium is partially inhibited by Pb) and the influx of Pb 

### 28
was enhanced after CRACI had been induced. Since skeletal growth in Pb

- exposed children is stunted, 

### 29

Zuscik et al. ( 2007 ) conducted a study using murin e limb bud mesenchymal cells (MSCs) to test the 

### 30
hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and also assessed the effects of 

### 31
Pb on various signaling pathways. Exposure to 1 μM Pb caused increased basal and TGF

- β/BMP 

### 32

induction of chondrogenesis in MSCs which was supported by nodule formation and upregulation of Sox



### 33
9, type 2 collagen, and aggrecan which are all key markers of chondrogenesis. The study authors also 

### 34
observed enhanced chondrogenesis during ectopic bone formation in mi ce that had been pre

- exposed to 

### 35
Pb in drinking water (55 or 233 ppm, corresponding to 14 or 40 μg/dL blood Pb). MSCs exposed to Pb 

### 36May 2011 5

- 345 DRAFT

– DO NOT CITE OR QUOTE exhibited an increase in TGF

- β, but BMP

- 2 signaling was inhibited. Pb was also reported to induce NF κB 

### 1

and inhibit AP

- 1 signa ling. Based on these results, the study authors concluded that chondrogenesis 

### 2
following exposure to Pb most likely involved modulation and integration of multiple signaling pathways 

### 3
including TGF

- β, BMP, AP

- 1, and NF κB

. 

### 4
Effects of Pb exposure on teeth were examined in three epidemiologic studies. Since individuals 

### 5
previously exposed to Pb may be impacted due to the endogenous release of Pb stored in their skeletal 

### 6
compartments, Arora et al. ( 2009 ) examined the association between bone Pb concentrations and loss of 

### 7
natural teeth in 333 male participants of the Normative Aging Study. Tooth loss in men was categorized as 

### 8
0, 1

- 8 or ≥ 9. Individuals with ≥ 9 teeth missing had significantly higher bone Pb concentrations 

### 9
compared to those with no tooth loss; no significant difference in blood Pb levels was observed between 

### 10
the categories of teeth loss. Following adjustment for different variables (age, education, smoking status, 

### 11
pack

- years of smoking, and diabetes), men with the highest tibia Pb concentrations (>23μg/g) had higher 

### 12 odds of tooth loss (OR: 3.03 [95% CI: 1.60,5.75]) compared to men with tibia Pb ≤ 1 5 μg/g. Men with the 

### 13
highest patellar Pb (>36 μg/g) also had higher odds of tooth loss (OR: 2.41 [95% CI: 1.30,4.49]) 

### 14 compared to men with patellar Pb ≤ 22.0 μg/g. Odds of tooth loss were not statistically associated with 

### 15

blood Pb levels. Based on these re sults, the study authors concluded that long

- term cumulative exposure 

### 16
to Pb is associated with increased odds of tooth loss. In a study examining the effects of Pb exposure on 

### 17
periodontitis in the United States (US), Saraiva et al. ( 2007 ) used data from NHANESIII by analyzing 

### 18
data for 2,500 men and 2,399 women aged between 30 and 56 years. The analysis took into account 

### 19
various covariates including age, NHANESIII phase, cotinine levels, poverty ration, race/ethnicity, 

### 20
education, BMD, diabetes, calcium intake, dental visits, and menopause in women. After adjusting for 

### 21
these covariates and comparing individuals with a blood Pb level of >7 μg/dL to tho se with a blood Pb 

### 22
level of <3 μg/dL, the prevalence ratios of periodontitis was 1.70 (95% CI: 1.02,2.85) for men and 3.80 

### 23
(95% CI: 1.66,8.73) for women. Based on these results, the study authors concluded that there was a 

### 24
positive and statistical associat ion between periodontitis and Pb levels for both men and women. In a 

### 25
similar study, Yetkin et al. ( 2007 ) recruited 60 male subjects (30 apprentices with Pb exposure, 30 

### 26
controls), to examine the impact of occupational exposure to Pb on periodontal status and association 

### 27
between periodontitis and blood Pb or oxidative stress. The results of their analysis indicated that blood 

### 28
Pb was significantly higher in apprentices exposed to Pb compared to controls (7.38 ± 4.41 versus. 2.27 ± 

### 29
1.49 μg/dL). No clinical periodontal or oxidative stress parameters were significantly difference between 

### 30
apprentices and controls. While the correlation between blood Pb and periodontal parameters was not 

### 31
reported, significant correlations between plaque index and CAT, probing depth and SOD, clinical 

### 32
attachment level and SOD, and clinical attachment l evel and malondialdehyde in Pb

- exposed apprentices 

### 33
was observed. These results demonstrate that there is significant association between clinical periodontal 

### 34
parameters and oxidative stress/damage indices in Pb

- exposed apprentices. Following multiple regre ssion 

### 35May 2011 5

- 346 DRAFT

– DO NOT CITE OR QUOTE analysis, a statistically significant association between gingival index and working status, family income 

### 1
and either probing depth or clinical attachment level was noted. 

### 2



## 5.9.5. Effects on Ocular Health

Ocular effects most commonly observed following expo sure to Pb include formation of cataract, 

### 3
impaired vision, edema and retinal stippling. 

### 4

5.9.5.1. Summary of Key Findings of the Effects o n Ocular Health from the 2006 Lead AQCD

The 2006 Pb AQCD stated that various changes in the visual system were observed with Pb 

### 5
poisoning including retinal stippling and edema, cataract, ocular muscle paralysis and impaired vision. 

### 6
The AQCD reported that retinal responses were observed in children of mothers with a blood Pb range of 

### 7

10.5 to 32.5 μg/dL during pregnancy, while cataracts were noted in middle

- aged male with tibia bone Pb 

### 8

levels of 31

- 126 μg/g. 

### 9

5.9.5.2. New Toxicology and Epidemiology Studies

New animal studies pertaining to the ocular effects of Pb have investigated endpoints such as 

### 10
retinal progenitor cell proliferation and neurogenesis, and have observed effects at exposures that resulted 

### 11
in blood Pb levels as low as <10 μg/dL (Section 5.3.4 .3). One human study pertaining to ocular effects of 

### 12

Pb was identified. Mosad et al. ( 2010 ) studied the association between subcapsula r cataract and Pb, 

### 13
cadmium, vitamin C, vitamin E, and beta carotene blood levels in middle

- aged male smokers compared to 

### 14
nonsmokers. Blood Pb was statistically significantly elevated in light (14.5 ± 0.41 μg/dL), moderate (14.5 

### 15
± 0.41 μg/dL), and heavy smo kers (18.7 ± 1.24 μg/dL) compared to nonsmokers (12.2 ± 0.21 μg/dL). Pb 

### 16
concentrations were also observed to be statistically higher in the cataracts of smokers versus. 

### 17
nonsmokers. Similar associations were also observed for cadmium blood and lens levels, while vitamins 

### 18
C, E, and beta carotene levels were significantly decreased in smokers. Based on these results, the study 

### 19
authors concluded that the Pb and cadmium present in high concentration in smokers were associated 

### 20

with cataracts due to oxidative stre ss which was indicated by reduced levels of antioxidants such as 

### 21
vitamins C, and E and beta carotene. 

### 22



## 5.9.6. Effects on the Respiratory System

The collective body of toxicological and epidemiologic studies demonstrates Pb

- induced effects on 

### 23
multiple immunologica l pathways, including a shift from a Th1 to a Th2 phenotype, increased IgE 

### 24
antibody production, and increased inflammatory responses (Sections 5.2.5.1. and 5.6). These are well 

### 25May 2011 5

- 347 DRAFT

– DO NOT CITE OR QUOTE recognized pathways that contribute to increased susceptibility to infections a nd also to the development 

### 1
of respiratory diseases such as asthma. Recent investigation of the respiratory effects of Pb exposure has 

### 2
been limited; however, cross

- sectional studies have indicated an association of increasing blood Pb level 

### 3
with increased p revalence of respiratory tract illnesses (Section 5.6.4.1) and asthma in children (Section 

### 4
5.6.4.2). As described in Section 5.2.4, Pb has been shown to induce the generation of ROS. ROS are 

### 5
implicated in mediating increases in bronchial responsiveness and activating neural reflexes leading to 

### 6
decrements in lung function. Studies investigating these airway responses also are limited in number and 

### 7
collectively do not provide strong evidence of an association with blood Pb (Section 5.6.4.3). 

### 8
Collectively, pan el and time

- series studies demonstrate associations between Pb measured in PM 2.5 or 

### 9

PM 10 air samples and decreases in lung function and increases in respiratory symptoms, hospitalizations, 

### 10
and mortality (Section 5.6.4.3). However, common limitations of the se air

- Pb studies are the variable size 

### 11

distribution of Pb

- bearing PM (Section 3.5.3) and its relationship with blood Pb levels as well as the lack 

### 12
of adjustment for other correlated PM chemical components. 

### 13



## 5.9.7. Summary

There is evidence from epidemiologic and toxicological studies that exposure to Pb results in 

### 14
altered liver function and hepatic toxicity

. Biochemical changes indicative of liver injury, including 

### 15
decreases in serum protein and albumin levels and increased AST, ALT, ALP, and GGT activities, have 

### 16

been observed in humans ( Can et al., 2008 ; D. A. Khan et al., 2008 ; Patil et al., 2007 ) and animals ( Y.

- J. 

### 17
Cheng et al., 2006 ; S. et al., 2009 )

. Increased hepatic cholesterogenesis, altered triglyceride and 

### 18
phospholipid levels, and disorders in lipid metabolism supported by increased levels of total cholesterol 

### 19
and triglycerides has been reported in the animal literature ( Ademuyiwa et al., 2009 ; Khotimchenko & 

### 20
Kolenchenko, 2007 )

. These results suggest that induction of cholesterogenesis and phospholipids in the 

### 21
liver may cause subtle effects at the cellular level, leading to hepatic injury

. Multiple studies in humans 

### 22
and animals have observed hepatic oxidative stress, generally indicated by an increase in lipid 

### 23
peroxidation along with a decrease in GSH levels and CAT, SOD, and GPx activities following exposure 

### 24

to Pb ( Adegbesan & Adenuga, 2007 ; D. A. Khan et al., 2008 ; Khotimchenko & Kolenchenko, 2007 ; D. Y. 

### 25
Yu et al., 2008 )

. Indices of oxidative stress were additionally observed in the livers of fetuses exposed 

### 26
throughout gestation ( Uzbekov et al., 2007 )

. 

### 27
Relatively few human studies have been conducted on the gastrointestinal toxicity of Pb since the 

### 28
completion of the 2006 Pb AQCD. Two ca ses studies reporting on GI symptoms in a child and adult 

### 29
report that elevated blood Pb was associated with nonlocalized abdominal pain, vomiting, nausea, 

### 30
constipation, lack of appetite, fatigue, and headaches ( Cabb et al., 2008 ; Fonte et al., 2007 )

. The adult ’ s 

### 31

blood Pb level was reported as 148 μg/dL

. Both subjects ’ symptoms were reported to diminish following 

### 32
cessation of exposure or che lation therapy

. Similar GI symptoms (stomach pain and gastritis) were 

### 33May 2011 5

- 348 DRAFT

– DO NOT CITE OR QUOTE observed in battery works and painters exposed to Pb in India with blood Pb levels ranging from 0.4

- 

### 1
116.6 μg/dL ( Kuruvilla et al., 2006 )

. Toxicological evidence for Pb

- induced GI health effects in rats 

### 2
includes inhibition of NANC relaxations in the gastric fundus and the observation of oxidative stress 

### 3
(lipid peroxidation, decreased SOD and CAT) in the gastric mucosa ( Olaleye et al., 2007 ; M. R. V. Santos 

### 4
et al., 2006 )

. The observation of oxidative stress was accompanied gastric mucosal damage. 

### 5
The endocrine processes most impacted by exposure to Pb include chan ges in thyroid function, as 

### 6
well as alteration in sex and stress hormone profiles

. TSH was negatively associated with blood Pb in 

### 7

women that ate contaminated fish (median blood Pb = 1.7 μg/dL) but not men, and FT4, but not FT3, was 

### 8

decreased in adolescent male auto repair workers ( Abdelouahab et al., 2008 ; Dundar et al., 2006 )

. 

### 9
Alterations in the levels of sex hormones, including total and free testosterone, estradiol, aromatase, and 

### 10
luteinizing hormone, were observed in Belgian adolescents environmentally exposed to Pb ( Croes et al., 

### 11

2009 )

. Toxicological evidence for similar effects was observed in adults cows reared in a contaminated 

### 12
environment and exposed to Pb

. A positive correlation was reported between blood Pb and plasma T3 and 

### 13

T4 levels ( Swarup et al., 2007 )

. Mean plasma estradiol levels were also significantly higher in Pb

- exposed 

### 14

cows

. In children challenged with an acute stressor, increasing blood Pb was associated with significant 

### 15
increases in salivary cortisol responses, even at blood Pb levels less than 10 μg/dL ( Gump et al., 2008 )

. 

### 16
Numerous epidemiologic studies investigated the as sociation between Pb exposure and 

### 17
osteoporosis in adults

. High blood Pb has been observed to be associated with decreased BMD in non



### 18
Hispanic white males ( Campbell & Auinger, 2007 ) , whereas urinary Pb, but not blood Pb , was seen to be 

### 19
associated with decreased BMD in Chinese individuals occupationally exposed to Pb ( Y. Sun, Sun, Zhou, 

### 20
Zhu, Zhang, et al., 2008 )

. In elderly women, blood Pb levels were associated with an increased risk of 

### 21
falls and fractures, including osteoporosis

- related falls ( Khalil et al., 2008 )

. Linear skeletal growth in 

### 22
children exposed to Pb was reduced with increased blood Pb ; these eff ects were seen at blood levels <10 

### 23
μg/dL ( Ignasiak et al., 2006 )

. In vitro studies indicate that Pb interferes with C ARCI in human OLCs and 

### 24
that Pb perturbs multiple signaling pathways during murine limb bud growth, potentially resulted in 

### 25
altered skeletal development ( Jang et al., 2008 ; Zuscik et al., 2007 )

. Epidemiologic studies investigating 

### 26
Pb exposure and tooth loss report that long

- term , cumulative exposure to Pb is associated with increased 

### 27
odds of tooth loss, periodontitis in men and women, an d that periodontitis is associated with oxidative 

### 28
stress/damage in individuals exposed in an occupational setting ( Arora et al., 2009 ; Saraiva et al., 2007 ; 

### 29

Yetkin

- Ay et al., 2007 )

. 

### 30
New toxicology studies have reported ocular effects (i.e., retinal progenitor cell proliferation) at 

### 31
blood Pb levels as low as <10 μg/dL ( Section 5. 3

. 4

. 3 ), and one human study reports and association 

### 32
between heavy smoking, increased blood Pb levels , and ca taracts ( Mosad et al., 2010 )

. Investigation of 

### 33
the respiratory effects of Pb exposure has been limited; however, c ross

- sectional studies have indicated an 

### 34
association of increasing blood Pb with increased prevalence of r espiratory tract illnesses ( Section 

### 35May 2011 5

- 349 DRAFT

– DO NOT CITE OR QUOTE 5. 6

. 4

. 1) and asthma in children (S ection 5. 6

. 4 .2)

. Additionally, Pb

- induced production of RO S is 

### 1
implicated in inc reased BR and d ecrements in lung function ( Section 5. 6

. 4

. 3 ). 

### 2
In summary, recent toxicological and epidemiologic evidence regarding the effects of Pb 

### 3
exposure on the liver, GI tract, endocrine system, bone and teeth, eyes, and respiratory tract largely 

### 4

are confirmatory of those effects noted in the 2006 Pb AQCD. However, recent evidence of these 

### 5
effects is relatively limited in number, and therefore no causal determinations are made regarding 

### 6

Pb

- induced effects in these organ systems. 

### 7



# 5.10. Cancer

The previous ep idemiologic studies included in the 2006 Pb AQCD ( U.S. EPA, 2006 ) “ provide[d] 

### 8
only very limited evidence suggestive of Pb exposure associations with carcinogenic or genotoxic effects 

### 9

in humans ” and the studies were summarized as follows: 

### 10

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 consistently with chromosomal aberrations. The International Agency for Research on Cancer (IARC) has recently classified inorganic Pb 

### 11
compounds as probable human carcinogens (Group 2A of IARC classifications) based on stronger 

### 12
evidence in animal studies than human studies, and organic Pb compounds as not classifiable (Group 3 of 

### 13
IARC classifications) ( IARC, 2006 ; Rousseau et al., 2005 )

. Additionally, the National Toxicology 

### 14
Program has listed Pb and Pb compounds as “ reasonably anticipated to be human carcinogens ” ( NTP, 

### 15

2004 )

. 

### 16
In the following sections, recent epidemiologic and toxicological studies published since the 2006 

### 17
Pb AQCD regarding Pb and cancer mortality and incidence are examined

. In addition, recent studies of Pb 

### 18
and DNA and cellular damage, as well as epigenetics studies, are summarized

. In epidemiologic studies, 

### 19
various biological measures of Pb are used including Pb measured in blood and bone

. Bone Pb is 

### 20
indicative of cumulative Pb exposure . Blood Pb can represent more recent exposure, although it can also 

### 21
represent remobilized Pb occurring during times of bone remodeling . Toxicological studies only repor t 

### 22
exposure by blood Pb. More detailed discussion of these measures is given in Section 4. 3.5

. 

### 23



## 5.10.1. Cancer Incidence and Mortality

Recent studies have included epidemiologic evaluations of the associations between Pb and both 

### 24
specific cancers, such as lung cance r and brain cancer, and overall cancer . Table 5

- 37 provides an 

### 25
overview of the study characteristics and results for the epidemiologic studies that reported effect 

### 26
estimates. This section also presents toxicological evidence on the carcinogenicity of Pb. 

### 27May 2011 5

- 350 DRAFT

– DO NOT CITE OR QUOTE Table 5

- 37. Summary of epidemiologic studies of cancer incidence and mortality

Reference Study Location Cancer Outcome Study P opulation Exposure Measurement Mean Pb (SD) Adjusted Effect Estimates

( M enke et al., 2006 ) U.S.A. Overall

cancer mortality NHANES III cohort with Blood Pb measures in 1988

- 1994

- at least 12 years of follow

- up

- blood Pb <10 μg/dL Blood Pb 2.58 μg/dL (geometric mean)

Tertile 1: <1.93 μg/dL Tertile 2: 1.94

- 3.62 μg/dL Terti le 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 ) U.S.A. Overall

cancer mortality NHANES III cohort

– at least 40 years of age Blood Pb 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 Normative Aging Study

- included men only

- mean follow

- up period for this study: 8.9 yr Blood Pb, patella Pb 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: <22 μg/g Tertil e 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. ( 2009 ) Baltimore, MD, and Monongahel a Valley, PA Overall

cancer mortality Subgroup of the Study of Osteoporotic Fractures cohort

- included white women aged 65

- 87 12 yr (+/

- 3 yr) follow

- up 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)

Lundstrom et al. ( 2006 ) Sweden Lung cancer (incidence and mortality) 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: cases 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 c umulative 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 r estricted to smokers onlyMay 2011 5

- 351 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location Cancer Outcome Study P opulation Exposure Measurement 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 conduc ted 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 a ged 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) Lung 1.3 (95% CI: 0.5, 3.1) 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)

Ino rganic 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) Lung 1.1 (95% CI: 0.7, 1.7) 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) Lung 0.8 (95% CI: 0.6, 1.1) 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 types of cancers were similar except no association was present between organic Pb and rectal

cancerMay 2011 5

- 352 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location Cancer Outcome Study P opulation Exposure Measurement Mean Pb (SD) Adjusted Effect Estimates

van Wijngaarden and Dosemeci ( 2006 ) U.S.A. Brain cancer mortality 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 exposure 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 Tumo r Study

– included individuals > =18 yr diagnosed with brain cancer less than 8 wk before hospitalization; frequency

matched controls were individuals admitted to the same hospitals for

non

- neoplastic conditions Interviews of lifetime work history and expos ure 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 incidence

Bhatti et al. ( 2009 ) Phoenix, AZ, Boston, MA, and Pittsburgh, PA Brain cancer incidence NCI Brain Tumor Study

– included non

Hispanic whites

18 yr diagnosed with brain cancer less than 8 wk before hospitalization; frequency

matched controls were individuals admitted to the same hospitals for

non

- neoplastic conditions Interviews of lifetime work history and exposu re 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

Absalon and Slesak ( 2010 ) Silesia province, Poland O verall

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 co e fficients between

- 0.3 and 0.2)
Obhodas et al. ( 2007 ) Island of Krk, Croa tia 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 (n umerical results not provided)May 2011 5

- 353 DRAFT

– DO NOT CITE OR QUOTE Reference Study Location Cancer Outcome Study P opulation Exposure Measurement 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 exposure 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 ≥15yr



### 5.10.1.1. O verall Cancer Mortality

Recent studies have been performed examining the association between biologically measured Pb 

### 1
levels and cancer mortality

. The Third National Health and Nutrition Examination Survey (NHANES III) 

### 2
included a nationally representative sample of U.S. adults who had blood Pb measurements taken and 

### 3
were followed up for 12 years

. Mean blood Pb levels were 2.58 μg/dL (individuals with blood Pb levels 

### 4
greater than 10 μg/dL were excluded from the study)

. No association was observed between blood Pb and 

### 5
cancer mortality ( HR of highest tertile compared to lo west tertile: 1.10 [95% CI: 0.82, 1.47]) ( Menke et 

### 6
al., 2006 )

. Another analysis of the NHANES III population, restricted to individuals 40 years and older at 

### 7

the time of blood Pb collection, included individuals with all blood Pb levels (including those greater than 

### 8
10 μg/dL) ( Schober et al., 2006 ) . Overall, 68% of the study population had blood Pb levels less than 5 

### 9
μg/dL and 6% had blood Pb levels greater than 10 μg/dL . Among individuals who died of cancer during 

### 10
the study period, 52% had blood Pb levels less than 5 μg/dL and 12% had blood Pb levels greater than 10 

### 11
μg/dL

. In this study, median foll ow

- up time was 8.6 years and a positive association was observed 

### 12

between blood Pb and cancer mortality . The RRs were 1.69 (95% CI: 1.14, 2.52) for individuals with 

### 13

blood Pb levels of at least 10 μg/dL and 1.44 (95% CI: 1.12, 1.86) for blood Pb levels of 5

- 9 μg/dL 

### 14
compared to individuals with blood Pb levels less than 5 μg/dL

. When categorized by age groups, point 

### 15
estimates comparing blood Pb levels of 5

- 9 versus less than 5 μg/dL were similar across all age groups 

### 16
but only statistically significant among 75

- 84 year olds

. A positive association for blood Pb levels of 10 

### 17
μg/dL and greater was present among those 40

- 74 years old and 85 years and older

. A study o f m en from 

### 18
the greater Boston area enrolled in the Department of Veterans Affairs N ormative Aging Study reported 

### 19

mean blood Pb levels of 5.6 μg/dL (SD 3.4 μg/dL) but this measure was poorly correlated with measured 

### 20

bone Pb ( Weisskopf et al., 2009 )

. No association was detected between either measure of Pb and cancer 

### 21May 2011 5

- 354 DRAFT

– DO NOT CITE OR QUOTE mortality in adjusted analyses

. As part of the Study of Osteoporotic Fractures s tudy, White women aged 

### 1

65

- 87 were included in a sub

- study of blood Pb levels and cancer mortality and were followed for 

### 2
approximately 12 years ( Khalil, Wilson, et al., 2009 )

. Mean blood Pb levels were 5.3 μg/d L (SD 2.3 

### 3
μg/dL) and no association was detected between blood Pb and cancer mortality in the study population

. 

### 4

5.10.1.2. L ung Cancer

A study was conducted among smelter workers to examine the relationship between Pb and lung 

### 5
cancer (incidence and mortality combine d) ( Lundstrom et al., 2006 )

. Pb exposure was measured with 

### 6

three different variables (peak blood Pb values, number of years Pb samples were obtained, and 

### 7
cumulative blood Pb index) but none showed an association with lung cancer

. Median follow

- up in the 

### 8
study was about 30 years and the peak blood Pb values during employment were 49.7 μg/dL for lung 

### 9
cancer cases and 55.9 μg/dL for controls

. A study in the UK of tin smelters reported no association 

### 10
between Pb exposure and lung cancer mortality in unweighted analyses, but when the analyses were 

### 11
weigh ted by age and time since exposure, positive associations were apparent ( S. R. Jones et al., 2007 )

. 

### 12
Pb exposure was calculated in this study by combining h istorical air sampling data and personnel record 

### 13
cards, which specified work histories. The median Pb exposure was estimated to be approximately 2 mg



### 14
year/m 3 and the smelters were exposed to other metals as well, such as arsenic and antimon y. A 

### 15
population

- based case

- control study performed among men in Montreal, Canada assessed Pb exposure via 

### 16
interviews regarding job histories and calculated the likely Pb exposures associated with the job activities 

### 17
( Rousseau et al., 2007 )

. No association was apparent between organic Pb, inorganic Pb, or Pb from 

### 18
gasoline emissions and lung cancer. 

### 19

Other studies of Pb and lung cancer were performed by comparing the lung tissue of individuals 

### 20
with lung cancer to those without lung cancer. The controls for these studies were individuals with 

### 21
metastases in the lung from other primary cancers ( De Palma et al., 2008 ) and individuals with non



### 22

cance rous lung diseases ( De Palma et al., 2008 ; Kuo et al., 2006 ) . One study reported increased Pb 

### 23

concentrations were observed in the cancerous and non

- cancerous lung tissue of individuals with non



### 24
small cell lung cancer compared to control gro ups (although the authors report these results may be 

### 25
confounded by smoking) ( De Palma et al., 2008 )

, but n o statistical difference in Pb levels was reported 

### 26
for lung tissue of individuals with lung cancer compared to control s in the other study ( Kuo et al., 2006 )

. 

### 27

5.10.1.3. B rain Cancer

A few studies of brain cancer examined the association between cancer and Pb using exposures 

### 28
determined via exposure databases and patient interviews about past jobs and known exposures. The 

### 29
National Longitudinal Mortality Study, a study that include d a national sample of the U.S. population, 

### 30
estimated Pb exposure based on current/most recent employment among individuals ( van Wijngaarden & 

### 31May 2011 5

- 355 DRAFT

– DO NOT CITE OR QUOTE Dos emeci, 2006 )

. Although not all estimates are statistically significant, a pattern of increased 

### 1
associations between Pb exposure and brain cancer mortality was observed in the study population

. In a 

### 2

case

- control study of brain tumors, glioma was reported to have no association with any Pb exposure 

### 3
metric; however, positive associations were observed between high cumulative Pb exposure and 

### 4
meningioma among individuals with ALAD2 genotypes ( Rajaraman et al., 2006 )

. The association was not 

### 5
present among individuals with ALAD1 genotypes

. A thi rd study of the association between Pb exposure 

### 6
and brain tumors reported none or slight overall associations with types of brain tumors; however, 

### 7
positive associations were observed among individuals with certain single nucleotide polymorphisms 

### 8
(SNPs) ( Bhatti et al., 2009 )

. After control for multiple comparisons, individuals with GPX1 variants had 

### 9
positive associations between cumulative Pb exposure and glioblastoma multiforme and meningioma

, 

### 10

whereas individuals wi thout RAC2 variants showed a positive association between Pb and glioblastoma 

### 11

multiforme and individuals without XDH variants displayed a positive association between Pb and 

### 12
meningioma

. 

### 13

5.10.1.4. B reast Cancer

A few studies examined Pb levels and breast tumors amon g individuals with and without breast 

### 14
tumor and/or cancer present. One study of newly diagnosed breast cancer patients and controls examined 

### 15
Pb levels in blood and hair samples and reported higher levels in both for cancer cases, although the 

### 16

difference in the hair samples was not statistically significant ( Alatise & Schrauzer, 2010 )

. Siddiqui et al. 

### 17
( 2006 ) observed higher blood Pb levels in women with benign and malignant tumors compared to 

### 18

controls

. Additionally, although blood Pb levels were higher among those with malignant breast tumors 

### 19
compared to those with benign tumors, both had similar levels of Pb detected in breast tissues

. Another 

### 20
study of Pb levels present in breast tissue also reported no statistical difference in Pb levels ( Pasha, Malik, 

### 21
et al., 2008b )

. However, one study of breast tissue did observe a statistically significant difference 

### 22

between Pb levels in the breast tissue of cancer cases and controls ( Ionescu et al., 2007 )

. Finally, a study 

### 23
of Pb levels in urine reported a positive association between urine Pb and breast cancer but this 

### 24
association became null when women taking nonsteroidal aromatase inhibitors but not taking 

### 25
bisphostphonates (a combination responsible for bone loss) were excluded from the analysis ( McElroy et 

### 26
al., 2008 )

. 

### 27
Overall, these studies demonstrate the possibility that women with breast cancer may have 

### 28
increased Pb levels in blood measurement, whereas the results for actual breast tissue are mixed. 

### 29
However, these studies are limited by their study design. The sample s are taken after cancer is already 

### 30
present in the cases, leading to issues of temporality for the Pb levels. Additionally, the sample sizes are 

### 31
often small and the studies may be underpowered. 

### 32May 2011 5

- 356 DRAFT

– DO NOT CITE OR QUOTE 

### 5.10.1.5. O ther Cancers

Studies of multiple cancers or cancers not list ed above have also been performed. An ecologic 

### 1
analysis compared levels of Pb in the air from 1990 to 2005 with incidence rates of cancer (cancer sites 

### 2
not specified) among children during this time period ( Absalon & Slesak, 2010 )

. The highest Pb levels 

### 3
were measured in 1990 when over 50% of the study area exceeded the limit of 1 μg/m 2

· year

. No 

### 4

correlation was observed both overall and in sex

- specific analyses . A similar study examined corr elations 

### 5
between Pb concentrations in soil, water, vegetation, and hair samples with incidence of neoplasms 

### 6
( Obhodas, 2007 ) . No correlations were reported. 

### 7
A study performed among men evaluated multiple cancer outcomes and determined exposures to 

### 8
organic Pb, inorganic Pb, and Pb in gasoline emissions via interviews regarding job hist ories and then 

### 9
subsequent exposure approximations by chemists and hygenists ( Rousseau et al., 2007 )

. Organic Pb 

### 10
exposure was positively associated with stomach cancer

. A positive association was also observed for 

### 11
rectal cancer when population

- based controls were used but was null when the control population was 

### 12

limited to individuals with other types of cancers

. No association was detected for cancers of the 

### 13
esophageous, colon, pancreas, prostate, bladder, kidney, melanoma, or non

- Hodgkin ’ s lymphoma

. None 

### 14
of the cancers were associated with exposure to inorganic Pb

. When occupational exposure to Pb in 

### 15
gasoline was c ategorized as unexposed, nonsubstantial level, and substantial level, a positive association 

### 16
with stomach cancer was observed when cancer controls were used (association not present when 

### 17
population controls were employed as the control group)

. A nother case

- control study using participant 

### 18
interviews and a job exposure matrix, including only men , reported no association between Pb exposure 

### 19
and esophageal squamous cell carcinomas, but an association was present between high Pb exposure and 

### 20

adenocarcinmoa of th e esophag us ( Santibanez et al., 2008 )

. 

### 21
Several studies compared Pb levels in blood, tissue, and urine of individuals who have cancer with 

### 22

individuals who are cancer

- free. Compared to control groups, increased Pb levels were observed in the 

### 23

blood and bladder tissue of individuals with bladde r cancer ( Golabek et al., 2009 ) , the kidney ti ssue of 

### 24
individuals with renal cell carcinoma (with highest levels among those with the highest stage tumors) 

### 25
( Calvo et al., 2009 ) , the tissue (but not serum) of individuals with laryngeal cancer ( Olszewski et al., 

### 26

2006 ) , the blood of individuals with gastric cancer ( Khorasani et al., 2008 )

, the plasma and hair of 

### 27
individuals with gastrointestinal cancer ( Pasha et al., 2010 )

, the blood and hair of individuals with non



### 28
specified types of cancer ( Pasha et al., 2007 ; Pasha, Malik, & Shah, 2008 ) , and the hair of individuals 

### 29
with benign tumors ( Pasha, Malik, et al., 2008a )

. No statistical difference in Pb levels was reported for 

### 30
colon tissue of individuals with colorectal polyps ( Alimonti et al., 2008 ) or urine of individuals with 

### 31

bladder cancer ( C. N. Lin et al., 2009 ) compared to control groups . A study exa mining Pb levels in kidney 

### 32
tissue reported the highest levels of Pb in normal kidney tissue samples that were adjacent to neoplastic 

### 33
tumors. The Pb levels reported in the kidney tissue of neoplastic tumors were elevated compared to those 

### 34May 2011 5

- 357 DRAFT

– DO NOT CITE OR QUOTE detected in corpse s without neoplastic tumors of the kidney ( Cerulli et al., 2006 )

. All of these comparison 

### 1
studies are limited by the inability to examine tempor ality ; the presence of Pb may be due to changes that 

### 2
result from having cancer, not changes that result in cancer

. Many of these studies attempted to control for 

### 3
this by including only cases who have not undergone certain treatments . Additionally, studies are limited 

### 4
by their small sample size and the selection of the control populations. Control populations are supposed 

### 5
to represent the general population from which the cases are drawn; some of the control subjects in these 

### 6

studies are individuals with dis eases/conditions warranting tissue resections, which are not prevalent in 

### 7
the general population. 

### 8

5.10.1.6. Toxicological Models of Carcinogenicity

Carcinogenicity in Animal Models

Inorganic Pb has been shown to act as a carcinogen in animal toxicology models. Most commonly 

### 9
the kidneys ( Azar et al., 1973 ; Kasprzak et al., 1985 ; Koller et al., 1985 ; Van Esch & Kroes, 1969 ) are 

### 10
targeted but the testes, brain, adrenals, prostate, pituitary, and mammary gland have also been affected 

### 11
( IARC, 2006 )

. More recently it has been shown that early life transplacental and lactational exposure of 

### 12
laboratory rodents to inorganic Pb induces carcinogenicity in adulthood ( Waalkes et al., 1995 )

. Chronic, 

### 13
lifetime exposure to Pb is also associated with carci nogenicity in laboratory rodents ( Koller et al., 1985 )

. 

### 14

One recent study considered Pb

- dependent carcinogenesis in laboratory animals. Tokar et al. ( 2010 ) 

### 15
considered tumorigenesis in rodents. Homozygous metallothionein I/II knockout mice and their 

### 16
corresponding wild type controls (groups of ten mice each) were exposed by drinking water to 2,000 or 

### 17
4,000 pp m Pb(Ac) 2 and compared to untreated controls. Study animals were exposed in utero, through 

### 18
birth and lactation, and then directly to drinking water until 8 weeks old. The metallothionein I/II 

### 19
knockout mice had increased testicular teratomas and renal and urinary bl adder preneoplasia. Pb exposed 

### 20

wild

- type mice were not statistically significantly different than controls

. The data suggest 

### 21
metallothionein can protect against Pb

- induced tumorigenesis. Concerns with the study are that the doses 

### 22

are at levels of Pb humans would not likely be exposed to and there is no metallothionein null condition 

### 23
in humans though there is variability in the expression of metallothionein. The data do not address 

### 24
whether this variability has any impact on Pb carcinogenesis. 

### 25

Neoplastic T ran sformation S tudies, Human Cell Cultures

T hree studies considered Pb

- dependent carcinogenesis in human cells

. Xie et al. ( 2007 ) treated 

### 26
BEP2D cells (human papilloma virus

- immortalized human bronchial cells) with 0, 1, 5, or 10 μg / cm2 

### 27

PbCrO 4 for 120 h. PbCrO 4 induced foci formation in a concen tration

- dependent manner. Xie et al. ( 2008 ) 

### 28
treated BJhTERT cells (hTERT

- immortalized human skin fibroblasts) and ATLD

- 2 cells (hTERT

- 

### 29May 2011 5

- 358 DRAFT

– DO NOT CITE OR QUOTE immortalized human skin fibroblasts deficient in Mre11) with 0, 0.1, 0.5, and 1 μ g/cm 2 PbCrO 4 for 120 h. 

### 1

PbCrO 4 induc ed foci formation in a concentration

- dependent manner in the Mre11 deficient cells. Xie et 

### 2
al., ( 2008 ) treated BJhTERT cells (hTERT

- immortalized human skin fibroblasts) and ATLD

- 2 cells 

### 3
(hTERT

- immortalized human skin fibroblasts deficient in Mre11) with 0, 0.1, 0.5, and 1 μ g/cm 2 PbCrO 4 

### 4
for 24 or 120 h. Mre11 was required to prevent PbCrO 4 - induced neoplasti c transformation. 

### 5

Immune Modulation of Tumorogenesis by Pb

As described in the prior AQCD for Pb ( U.S. EPA, 2006 ) , Pb

- induced immunotoxicity affecting 

### 6
tumors results from a combination of suppressed Th1 responses and misregulated inflammation

. The 

### 7
intersection of these two general Pb

- induced alterations that elevate t he risk of cancer. First, Pb

- induced 

### 8
misregulation of inflammation involving innate immune cells results in chronic insult to tissues

. Decades 

### 9
of excessive lipid and DNA oxidation production by overproduction of ROS and weakened a nti

- oxidant 

### 10
defenses increase the likelihood of mutagenesis, cellular instability, and tumor cell formation

. In support 

### 11
of this, Xu et al. ( J. Xu et al., 200 8 ) found evidence that supports the association with Pb exposure and 

### 12
DNA damage and concluded that it is a route to increased Pb

- induced tumorigenesis

. The second 

### 13
component of increased risk of cancer involves Pb

- induced suppression of Th1

- dependent ant i

- tumor 

### 14
immunity as acquired immunity shifts statistically significantly toward Th2 responses

. With cytotoxic T 

### 15
lymphocytes and other cell

- mediated defenses dramatically lessened, the capacity to resist cancer may be 

### 16
compromised. 

### 17



## 5.10.2. Cancer Biomarkers

A study of men aged 21

- 40 years never occupationally exposed to metals examined prostate 

### 18
specific antigen ( PSA) , a biomarker for prostate cancer . This study reported a positive association 

### 19

between blood Pb and PSA levels in adjusted analyses ( Pizent et al., 2009 ) . The m edian blood Pb level 

### 20

w as 2.6 μg/dL (range 1.0

- 10.8 μg/dL)

. The authors note that the study population was young and at lower 

### 21
risk of prostate cancer than older men. 

### 22



## 5.10.3. DNA and Cellular Damage

Multiple studies have been performed examining the relationship between Pb and DNA and 

### 23
cellular damage. Details of the recent epidemiologic and toxicological studies follow. 

### 24

5.10.3.1. Epidemiologic Evidence for DNA and Cellular Damage

Multiple studies examined the relationship between Pb and sister chromatid exchange (SCE). A 

### 25
study of male policeman reported mean blood Pb levels for the study population of 43.5 μg/dL 

### 26May 2011 5

- 359 DRAFT

– DO NOT CITE OR QUOTE ( Wiwanitkit et al., 2008 )

. When categorized as having high or low blood Pb levels (cut

- off at 49.7 μg/dL), 

### 1
the higher blood Pb group was observed to have higher mean SCE

. Another study of adult males 

### 2
compared the SCE of storage battery manufacturing workers (mean blood Pb levels of 40.14 μg/dL) and 

### 3
office workers (mean blood Pb levels of 9.77 μg/dL) ( Duydu et al., 2005 )

. The exposed workers had 

### 4
higher SCE levels and also a greater number of cells with the SCEs per cell higher than the 95 th 

### 5
percentile of the population. Finally, a study of children aged 5

- 14 years old ( mean blood Pb levels of 

### 6
7.69 μg/dL [ SD 4.29 μg/dL]) reported no correlation with SCE ( Mielzyńska et al., 2006 )

. However, the 

### 7
study did report a positive association between blood Pb and micronuclei levels

. 

### 8
Other studies of DNA damage have reported mixed results. A study of children ages 6

- 11 years old 

### 9
and environmentally

- exposed to Pb reported no association between blood Pb and DNA basal damage or 

### 10
repair ability after a peroxide challenge ( Méndez

- Gómez et al., 2008 )

. Another study included adult 

### 11
participants aged 50

- 65 years and reported an association between blood Pb and carcinoembryonic 

### 12
antigen (CEA) but not with DNA

- strand breaks or oxidative DNA dama ge ( De Coster et al., 2008 )

. A 

### 13
study conducted among workers exposed to Pb (mean blood Pb levels of 30.3 μg/dL) and une xposed 

### 14
controls (mean blood Pb levels of 3.2 μg/dL) reported positive associations between the exposed group 

### 15
and cytogenetic damage (measured by micronuclei frequency), chromosomal aberrations, and DNA 

### 16
damage (although this was not statistically significan t in linear regression models controlling for age) 

### 17
( Grover et al., 2010 ) . A study of painters in India, where Pb

- concentrations in paint are high, reported 

### 18

mean blood Pb levels of 21.56 μg/dL (SD 6.43 μg/dL ) among painters who reported painting houses for 

### 19

8

- 9 hours/day for 5

- 10 years ( M. I. Khan et al., 2010 ) ; the mean blood Pb levels were 2.84 μg/dL (SD 

### 20

0.96 μg/dL ) for healthy workers who had not been occupationally exposed to Pb. Cytogenetic damage 

### 21
was increased among th e painters compared to the healthy controls. Another study compared the blood Pb 

### 22
of metal workers and office workers and reported greater blood Pb levels (both current and 2 year 

### 23
average) among the metal workers (blood Pb levels at least 20 μg/dL for metal workers compared to 

### 24

blood Pb levels less than 10 μg/dL for the office workers) ( Olewińska et al., 2010 ) . Overall, the workers 

### 25
had increased DNA strand breaks versus the office workers (this held true at various blood Pb levels). 

### 26
Finally, a study of Pb battery workers with symptoms of Pb toxicity and a group of controls were 

### 27

examined ( Shaik & Jamil, 2009 )

. Higher chromosomal aberrations, micronuclei frequency, and DNA 

### 28
damage were reported for the battery workers as compared to the controls

. 

### 29

5.10.3.2. Toxicological Evidence for DNA and Cellular Damage

Sister Chromat id Exchanges

One study, Tapisso et al. ( 2009 ) , considered sister chromatid exchanges ( SCE ) in rodents. Algerian 

### 30
mice (groups of six mice each) were exposed by intraperitoneal injection to 5 or 10 doses of 0.46 mg/kg 

### 31May 2011 5

- 360 DRAFT

– DO NOT CITE OR QUOTE Pb(Ac) 2 and compared to untreated controls. The SCE in bone marrow were elevated after Pb exposure 

### 1
alone, which increased with time. Co

- exposure with cadmium or zinc further increased SCE levels

. 

### 2
Two studies considered SCE in cultured human cells. In one study, Ustundag and Duydu ( 2007 )

, 

### 3
considered the ability of N

- acetylcysteine and melatonin to reduce led nitrate

- induced SCE in a si ngle 

### 4
human donor. Cells were treated with 0, 1, 5, 10, or 50 μ M Pb(NO 3 ) 2 . SCE statistically significantly 

### 5
increased at every Pb concentration in a concentration dependent manner. Both 1 and 2 mM N



### 6
acetylcysteine and melatonin were able to statistically significantly reduce SCE levels

. Exposure times 

### 7
were not provided. The full interpretation of these data is limited by the limited number of donors and the 

### 8
absence of an exposure time for the SCE assay. In the other study, Turkez et al. considered the ability of 

### 9
boron comp ounds to prevent Pb chloride

- induced SCE in human lymphocytes. Cells were obtained from 

### 10

4 non

- sm oking donors. Both 3 and 5 ppm Pb chloride induced a statistically significant increase in SCE 

### 11

levels over controls. Boron was able to statistically significant ly diminish these levels. Exposure times 

### 12
were not provided. The full interpretation of these data is limited by the limited number of donors and the 

### 13
absence of an exposure time for the SCE assay. 

### 14

Micronuclei Formation

Two studies considered MN in rodents. Alghazal et al. ( 2008 ) , considered the ability of Pb(Ac) 2 

### 15
trihydrate to induce MN in bone marrow of Wistar rats. Animals were given a daily dose of 100 mg/l in 

### 16
their drinking water for 125 days. The mean number of MN in male and female rats was statisticall y 

### 17
significantly higher than unexposed controls. The second study, Tapisso et al., ( 2009 ) , considered Pb 

### 18
alone, Pb plus zinc and Pb plus cadmium

- induced MN i n rodents. Algerian mice were exposed by 

### 19
intraperitoneal injection to 5 or 10 doses of 0.46 mg/kg Pb(Ac) 2 and compared to untreated controls. The 

### 20

MN in bone marrow were elevated after Pb exposure, which increased with time. Co

- exposure with 

### 21

cadmium or zinc did not further increase MN levels. 

### 22

Three studies considered MN in cultured human cells. In one study, Ustundag and Duydu ( 2007 ) 

### 23
considered the ability of N

- acetylcysteine and melatonin to reduce led nitrate

- induced MN in a single 

### 24
human donor. Cells were treated with 0, 1, 5, 10, or 50 μ M Pb(NO 3 ) 2 . MN statistically significantly 

### 25
increased at the two highest Pb concentrations in a concentration dependent manner. Both 1 and 2 mM N



### 26
acetylcysteine and melatonin were not able to statistically significantly reduce MN levels. Exposure times 

### 27
were not provided. The full interpretation of these data is limited by the lim ited number of donors and the 

### 28
absence of an exposure time for the MN assay. The second study, Turkez et al., considered the ability of 

### 29
boron compounds to prevent Pb chloride

- induced MN in human lymphocytes. Cells were obtained from 4 

### 30

non

- smoking donors. Both 3 and 5 ppm Pb chloride induced a statistically significant increase in MN 

### 31

levels over controls. Boron induced a statistically significantly attenuation of these levels. Exposure times 

### 32
were not provided. The full interpretation of these data is limited by the limited nu mber of donors and the 

### 33May 2011 5

- 361 DRAFT

– DO NOT CITE OR QUOTE absence of an exposure time for the MN assay. T he third study, Gastaldo et al. ( 2007 ) , evaluated the 

### 1
ability of Pb to induce MN. Human endothelial HMEC cell line was treated with 1

– 1000 μ M Pb(NO 3 ) 2 

### 2

for 24 h

. MN increased in a statistically significant, concentration

- dependent manner. 

### 3

HPRT Mutations

Two studies evaluated HPR T mutations in human cell cultures. Li et al., ( 2008 ) , evaluated Pb(Ac) 2 - 

### 4

induced HPRT in the non

- small

- cell lung carcinoma tumor cell line, CL3 and normal human diploid 

### 5
fibroblasts (specific tissue source not provided). All cells were exposed to 0, 100, 300 or 500 μ M Pb(Ac) 2 

### 6

for 24 h ours in serum

- free medium ± a 1

- h our pretreatment with a MKK1/2 inhibitor or a PKC

- alpha 

### 7

inhibitor. P b alone did not induce HPRT mutations. Inhibiting the ERK pathway via either inhibitor 

### 8
statistically significantly increased Pb

- induced mutagenesis. A second study, Wang et al. ( 2008 )

, 

### 9
investigated Pb(Ac) 2 - induced HPRT mutations in CL3 cell s. All cells were exposed to 0, 100, 300 or 500 

### 10

μ M Pb(Ac) 2 for 24 h ours in serum

- free medium ± a 1

- h our pretreatment with a PKC

- alpha inhibitor or 

### 11
siRNA fpr PKC

- alpha. Pb alone did not induce HPRT mutations. Inhibiting the PKC

- alpha via either 

### 12

inhibitor st atistically significantly increased Pb

- induced mutagenesis. 

### 13
One study considered HPRT in anim al cell culture. McNeill et al. ( 2007 ) considered Pb(Ac) 2 

### 14
induced HPRT mutations in Chinese hamster ovary AA8 cells an d AA8 cells overexpressing human 

### 15
Ape1. Cells were treated with 5 μ M Pb(Ac) 2 for 6 h ours . No increases in HPRT mutations were observed 

### 16

after Pb exposure in either cell line. 

### 17

Chromosomal Aberrations

Only one study ( El

- Ashmawy et al., 2006 ) considered Pb in laboratory rode nts. The study focused 

### 18
on dietary exposure to Pb(Ac) 2 administered as a single dose of 0.5% w/w. Male Swiss albino mice, 30 

### 19
per group, were studied. In addition, the authors considered the protective effects of turmeric and myrrh 

### 20
powder. The study reported statistically significant levels of chromosomal aberrations in the Pb treatment 

### 21
alone group, particularly with respect to fragments, deletions, ring chromosomes, gaps, and end

- to

- end 

### 22
associations. The turmeric and myrrh powders were protective. Concerns with the study include the use of 

### 23
only a single dose of Pb(Ac) 2 along with the high levels of unusual aberrations such as ring chromosomes 

### 24

and end

- to

- end associations

. Typically, these aberrations are rare after metal exposure, but were the most 

### 25

c ommon in this study raising questions about the quality of the metaphase preparations. One additional 

### 26
concern was that only 50 metaphases per dose were analyzed instead of the more common 100 

### 27
metaphases per dose. The authors did not explain why their spect rum of aberrations was so different or 

### 28
why they only used one dose or analyzed fewer metaphases per dose. 

### 29
Seven studies considered the ability of Pb to induce chromosomal aberrations in cultured human 

### 30
cells. One study ( Pasha Shaik et al., 2006 ) considered the ability of Pb(NO 3 ) 2 to induce chromosomal 

### 31May 2011 5

- 362 DRAFT

– DO NOT CITE OR QUOTE aberrations in primary human peripheral blood lymphocytes obtaine d from healthy, non

- smoking donors

. 

### 1
Cells were treated with 0, 1.2 or 2 mM Pb(NO 3 ) 2 for 2 h

. No increase in chromosomal aberrations was 

### 2
reported. Some aneuploidy was observed. Concerns with the study are that only a 2 hour exposure was 

### 3
used, which may not be long enough for DNA damage to be expressed as a chromosomal aberration. It 

### 4
also appears from the data presentation that only three subjects were used; one for a control, one for the 

### 5
low dose and one for the high dose. Experiments were not repeated, thus given the small number of 

### 6
subjects, this study may not have had sufficient power to detect any effects. Holmes et al. ( 2006 ) , treated 

### 7

WHTBF

- 6 cells (hTERT

- immortalized human lung cells) with 0, 0.1, 0.5, or 1 μ g/cm 2 PbCrO 4 for 24

- 120 

### 8
hours or with 0, 0.1, 0.5, 1, 5 or 10 μ g/ cm 2 PbO for 24 or 120 hours

. PbCrO 4 induced statistically 

### 9
significant, concentration

- dependent increases in centrosome abnormalities and aneuploidy. Wise et al. 

### 10
( 2006 ) treated BEP2D cells with 0, 0.5, 1, 5, or 10 μ g/cm 2 PbCrO 4 for 24 h ours

. PbCrO 4 induced 

### 11
statistically significant concentration

- dependent increases in chromosomal aberrations. Holmes et al. 

### 12
( 2006 ) , treated WHTBF

- 6 cells with 0, 0.1, 0.5, or 1 μ g/cm 2 PbCrO 4 for 24

- 72 hours

. PbCrO 4 induced 

### 13
statistically significant, concentration

- dependent increases in chromosoma l aberrations

. The effects were 

### 14

attributed to the chromate anion. Wise et al. ( 2006 ) , treated WHTBF

- 6 cells with 0, 0.1, 0.5, or 1 μ g/cm 2 

### 15

PbCrO 4 f or 24

- 120 hours

. PbCrO 4 induced statistically significant, concentration

- dependent increases in 

### 16
spindl e assembly checkpoint disruption, effects of mitosis and aneuploidy. By contrast, chromate

- free 

### 17

PbO did not induce centrosome amplification. The effects were attributed to the chromate anion. Xie et al. 

### 18
( 2007 ) treated BEP2D cells with 0, 1, 5, or 10 μ g/cm 2 PbCrO 4 for 24 h ours

. PbCrO 4 induced statistically 

### 19
significant, concentration

- dependent increases in chromosomal aberrations and aneuploidy. 

### 20

Wise et al. ( 2010 ) treated WHTBF

- 6 cells with 0, 0.1, 0.5, or 1 μ g/cm 2 PbCrO 4 for 24 h ours in a 

### 21
study comparing 4 chromate compounds. PbCrO 4 induced statistically significant, concentration



### 22
dependent increases in chromosomal aberrations, but was the least potent chromate based on administered 

### 23

concentration

. 

### 24
Five studies considered the ability of PbCrO 4 to induce chromosome aberrations in roden t cell 

### 25

cultures. All focused on PbCrO 4 . Duzevik et al. ( 2006 ) treated Chinese hamster ovary (CHO) cells with 0, 

### 26
0.1, 0.5, or 1 μ g/cm 2 PbCrO 4 for 24 h. Specific CHO lines used included AA8 (wildtype) EM9 (XRCC1

- 

### 27
deficient), and H9T3 (EM9 complemented with human XRCC1 gene). PbCrO 4 induced statistically 

### 28
significant, concentration

- dependent increases in chromosomal aberrations that were statistica lly 

### 29
significantly increased by XRCC1 deficiency. Nestmann and Zhang ( 2007 ) treated Chinese hamster ovary 

### 30
cells (clone WB(L)) with 0, 0.1, 0.5, 1, 5, or 10 μ g/cm 2 PbCrO 4 (as pigment yellow) for 18 h. No 

### 31

increases in chromosomal aberrations were observed. Savery et al. ( 2007 ) treated CHO cells with 0, 0.1, 

### 32
0.5, 1, or 5 μ g/cm 2 PbCrO 4 for 24 h. Specific CHO lines used included AA8 (wildtype) KO40 (Fancg



### 33
deficient), and 40 BP6 (Fancg complemented). PbCrO 4 induced statistically significant, concentration



### 34
dependent increases in chromosomal aberrations that were increased by Fancg deficiency. Camrye et al., 

### 35
( 2007 ) treated CHO cells with 0, 0.1, 0.5, 1, 5, or 10 μ g/cm 2 PbCrO 4 for 24 hours. Specific CHO lines 

### 36May 2011 5

- 363 DRAFT

– DO NOT CITE OR QUOTE used included CHO

- K1 (parental), xrs

- 6 (Ku80 deficient), and 2E (xrs

- 6 complemented with Ku80 gene). 

### 1

PbCrO 4 induced statistically significant, concentration

- dependent increases in chromosomal aberrations 

### 2
that were not affected by Ku80 de ficiency. Stackpole et al. ( 2007 ) treated CHO and C hinese hamster lung 

### 3
(CHL) cells with 0, 0.1, 0.5, or 1 μ g/ cm 2 PbCrO 4 for 24 hours. Specific CHO lines used included AA8 

### 4
(wildtype), irs1SF (XRCC3

- deficient), and 1SFwt8 (XRCC3 complemented). CHL lines used included 

### 5
V79 (wildtype), irs3 (Rad51C deficient) a nd irs3#6 (Rad51C complemented) . PbCrO 4 induced 

### 6
statistically significant, concentration

- dependent increases in chromosomal aberrations that were 

### 7
statistically significantly increased by both XRC3 and Rad51C deficiency. 

### 8
Three studies considered the ability of PbCrO 4 to induce chromosome aberrations in marine 

### 9

mammal cell cultures. All focused on PbCrO 4 . Li Chen et al. ( 2009 ) treated primary North Atlantic right 

### 10
whale lung and skin fibroblasts with 0, 0.5, 1.0, 2.0, and 4.0 μ g/cm 2 PbCrO 4 for 24 hours . Wise et al

. 

### 11
( 2009 ) treated primary Steller sea lion lung fibroblasts with 0, 0.1, 0.5, 1 and 5 μ g/cm 2 PbCrO 4 for 24 

### 12

h ours

. Wise et al. ( 2011 ) treated primary sperm whale skin fibroblasts with 0, 0.5, 1, 3, 5, and 10 μ g/cm 2 

### 13

PbCrO 4 for 24 h ours . In all three studies, PbCrO 4 induced statistically significant, concentration



### 14
dependent increases in chromosomal aberrati ons. 

### 15

COMET Assay

Three studies considered the ability of Pb to induce DNA single strand breaks measured by the 

### 16

comet assays in laboratory animals. Xu et al. ( 2008 ) conside red the ability of Pb(Ac) 2 to induce DNA 

### 17
damage measured by the comet assay in lymphocytes in male ICR mice. Animals (5 per group) were 

### 18
given Pb(Ac) 2 by gavage at doses of 0, 10, 50, or 100 mg/kg body weight every other day for 4 weeks. Pb 

### 19
exposure statist ically significantly increased both tail length and tail moment in a dose

- dependent manner. 

### 20

Nava

- Hernandez et al. ( 2009 ) considered the ability of Pb(Ac) 2 to induce DNA damage measured by the 

### 21
comet assay in primary spermatocyte DNA of male Wistar rats. Animals (3 per group) were treated for 13 

### 22
weeks with 0, 250 or 500 mg/l Pb in their drinking water. There was statistically significantly less DNA 

### 23
damage in the controls compared to the two treatment groups. Narayana and Al

- Bader ( 2011 ) considered 

### 24
the ability of Pb(NO3) 2 to induce DNA damage measured by the comet assay in liver tissue of adult male 

### 25
Wistar rats. Animals (8 per group) were treated for 60 days with doses of 0, 0.5 or 1% Pb(NO 3 )2 in their 

### 26
drinking water. There were no statistical differences between treated groups and controls. 

### 27

Two studie s considered the ability of Pb to induce DNA strand breaks measured by the comet assay 

### 28
in cultured human cells. Only, one study ( Pasha Shaik et al., 2006 ) considered the ability of Pb to induce 

### 29
DNA single strand breaks using the comet assay in primary human peripheral blood lymphocytes 

### 30
obtained from healthy, non

- smoking donors

. Cells were treated with 0, 2.1, 2.4, 2 .7, 3.0, 3.3 Pb(NO 3 ) 2 for 

### 31

2 hours. Increased comet tail length with increased dose was reported. 

### 32May 2011 5

- 364 DRAFT

– DO NOT CITE OR QUOTE Concerns with the study are that apparently no untreated control was used. It also appears from the 

### 1
data presentation that only five subjects were used; one f or each dose. Experiments were not repeated, 

### 2
thus given the small number of subjects and the absence of a negative control, this study may only be 

### 3
detecting background levels. Xie et al. ( 2008 ) treated BJhTERT cells (hTERT

- immortalized human skin 

### 4

fibr oblasts) and ATLD

- 2 cells (hTERT

- immortalized human skin fibroblasts deficient in Mre11) with 0, 

### 5
0.1, 0.5, and 1 μ g/cm 2 PbCrO 4 for 24 hours. PbCrO 4 induced a concentration

- dependent increase in DNA 

### 6
double strand breaks measured by the comet assay. 

### 7

Two stud ies considered Pb

- induced DNA single strand breaks in rodent cell cultures using the 

### 8
comet assay. Xu e t al. ( 2006 ) , treated PC12 cells with 0, 0.1, 1 or 10 μ M Pb(Ac) 2 . Both tail length and tail 

### 9
moment statistically significantly increased in a concentration

- dependent manner. Kermani et al. ( 2008 ) 

### 10
exposed mouse bone marrow

- mesenchymal stem cells to 60 μ M Pb(Ac) 2 for 48 hours. There was an 

### 11
increase in several comet assay measurements including tail length. 

### 12

Other Assays

Three studies considered the ability of Pb to induce DNA double strand breaks measured by 

### 13
measuring gamma

- H2A.X foci formation in c ultured human cells. Xie et al. ( 2008 ) treated BJhTERT 

### 14
cells (hTERT

- immortalized human skin fibroblasts) and ATLD

- 2 cells (hTERT

- immortalized human skin 

### 15
fibroblasts deficient in Mre11) with 0, 0.1, 0.5, and 1 μ g/cm 2 PbCrO 4 for 24 hours. PbCrO 4 induced a 

### 16

conce ntration

- dependent increase in DNA double strand breaks measure d by gamma

- H2A.X foci 

### 17

formation. Gastaldo et al. ( 2007 ) evaluated the ability of Pb to induce DNA double strand breaks measure 

### 18
with gamma

- H2A.X foci formation and by pulse

- field gel electrophoresis in cultured human cells. Human 

### 19

endothelial HMEC cell line was treated with 1 to 1

, 000 μ M Pb(NO 3 ) 2 for 24 hours. DNA double strand 

### 20

breaks increased in a concentration

- dependent manner. Wise et al. ( 2010 ) treated WHTBF

- 6 cells with 0, 

### 21
0.1, 0.5, or 1 μ g/cm 2 PbCrO 4 for 24 hours in a study comparing 4 chromate compounds. PbCrO 4 induced 

### 22
statistically significant, concentration

- dependent increases in DNA double strand breaks measured by 

### 23

g amma

- H2A.X foci formation, at a similar level to the other 3 compounds

. 

### 24

Four studies considered Pb and DNA repair. All were done in cultured cells. Li et al., ( 2008 )

, 

### 25

evaluated Pb(Ac) 2 - induced effects on nucleotide excision repair efficiency in CL3 cells. All cells were 

### 26
exposed to 0, 100, 300 or 500 μ M Pb(Ac) 2 for 24 h ours in serum

- free medium. Pb increased nucleotide 

### 27
excision repa ir efficiency. Gastaldo et al. ( 2007 ) evaluated the ability of Pb to affect DNA repair in 

### 28

cu ltured human cells. Human endothelial HMEC cell line was treated with 100 μ M Pb(NO 3 ) 2 for 24 

### 29

hours. Pb inhibited non

- homologous end joining (NHEJ) repair, over activates MRE11

- dependent repair 

### 30

and increased Rad51

- related repair. Xie et al. ( 2008 ) trea ted BJhTERT cells (hTERT

- immortalized human 

### 31
skin fibroblasts) and ATLD

- 2 cells (hTERT

- immortalized human skin fibroblasts deficient in Mre11) with 

### 32
0, 0.1, 0.5, and 1 μ g/cm 2 PbCrO 4 for 24 or 120 hours. Mre11 was required to prevent PbCrO 4 - induced 

### 33May 2011 5

- 365 DRAFT

– DO NOT CITE OR QUOTE DNA double strand breaks. McNeill et al

, ( 2007 ) considered Pb(Ac) 2 effects on Ape1. Chinese hamster 

### 1
ovary cells (AA8) were treated with 0, 0.5, 5, 50, or 500 μ M Pb(Ac) 2 and then whole cell extracts were 

### 2

used to determine AP site incision activity. The data show that Pb reduced AP endonuclease function

. 

### 3
Finally, t wo studies considered Pb

- induced cellular proliferation in laboratory animals. Fortoul et al. 

### 4
( 2005 ) exposed adult male CD1 mice (24 animals per group) to 0.01 M Pb(Ac) 2 , 0.006 M ca

. dmium 

### 5
chloride or a mixture of the two chemicals for 1 h twice a week for 4 weeks by inhalation. The lungs were 

### 6
then examined by electron microscopy for changes. Pb i nduced cellular proliferation in the lungs. 

### 7

Kermani et al. ( 2008 ) exposed mouse bone marrow

- mesenchymal stem cells to 0

- 100 μ M Pb(Ac) 2 for 48 

### 8

hours. As measured by the MTT assay, Pb decrease cell proliferation at all concentrations tested. 

### 9

5.10.3.3. Mechanism s of A ction

The carcinogenic mechanism of action of Pb is poorly underst ood. Three well

- accepted general 

### 10
paradigms of carcinogenesis include multi stage carcinogenesis (including initiation, promotion, and 

### 11
progression) , genomic instability, and epigenetic modification. Of the aforementioned paradigms, i t is 

### 12

u nclear which of the se best fit Pb. For example, multistage carcinogenesis involves a series of cellular and 

### 13
molecular changes that result from the progressive accumulation of mutations that induce alterations in 

### 14

cancer

- related genes. Pb does not appear to follow this paradig m and the literature suggest s it is weakly 

### 15
mutagenic . Pb does appear to have some ability to induce chromosomal mutation and DNA damage, i.e. 

### 16
clastogenicity. However, the ability of Pb to alter gene expression (epigenetic effects) and to interact with 

### 17

prot eins may be a means by which Pb induces its carcinogenicity. It is known that Pb can replace zinc in 

### 18

zinc

- binding (zinc

- finger) proteins, which include hormone receptors, cell

- cycle regulatory proteins, the 

### 19
Ah receptor, estrogen receptor, p53, DNA repair p roteins, protamines, and histones. These zinc

- finger 

### 20
proteins all bind to specific recognition elements in DNA. Thus, Pb may act at a post

- translational stage 

### 21
to alter protein structure of Zn

- finger proteins, which can in turn alter gene expression, DNA re pair and 

### 22

other cellular functions. To recapitulate, cancer develops from one or a combination of multiple 

### 23
mechanisms including modification of DNA via epigenetics or enzyme dysfunction and genetic instability 

### 24
or mutation(s). These modifications then provid e the cancer cells with a selective growth advantage. In 

### 25
this schematic, Pb appears to contribute to epigenetic changes, and chromosomal aberrations. 

### 26
The genomic instability paradigm requires a cascade of genome

- wide changes caused by 

### 27
interfering with DNA repair, kinetochore assembly, cellular checkpoints, centrosome duplication, 

### 28
microtubule dynamics or a number of cell maintenance processes. There are some data that suggest Pb 

### 29
may interfere with some of these processes, but the data are few as these areas are rarely studied for Pb

. 

### 30
Furthermore, the bulk of the literature in this area involves PbCrO 4 and it i s unclear if the effects are due 

### 31

to Pb or chromate. Epigenetic modifications lead to cancer by altering cellular functions without altering 

### 32
the genetic material

. The most commonly studied epigenetic change is methylation alterations

. Data show 

### 33May 2011 5

- 366 DRAFT

– DO NOT CITE OR QUOTE Pb can induce epigenetic changes, but studies are still missing to clearly tie these effects to Pb

- induced 

### 1
carcinogenesis and genotoxicity. Thus, the mechanism is d ifficult to define but, if Pb is a human 

### 2
carcinogen, the mechanism likely involves either genomic instability or epigenetic modification 

### 3
paradigms or some combination of the two, but it not likely to occur by a multistage carcinogenesis 

### 4
paradigm. More work is needed to determine the mechanism

. 

### 5
No recent studies of the prot ective role of calcium or zinc in Pb

- dependent carcinogenesis or 

### 6
genotoxicity were found. There were some data suggesting that metallothionein protects rodents from Pb



### 7

induced cancers. Th ere were some data suggesting that boron, melatonin, N

- acetylcysteine, turmeric and 

### 8
myrrh protecting cells against Pb

- induced genotoxicity. There were some data suggesting that Pb mimic s 

### 9
or antagonizes selenium in rodents . These data are discussed in more detail elsewhere in the cancer 

### 10

section

. 

### 11

5.10.3.4. Effects of Lead within Mixtures

Three studies considered the impact of mixtures with Pb . All considered genotoxicity. Mendez



### 12
Gomez et al., ( 2008 ) , considered 65 children from Mexico exposed to bo th arsenic and Pb . DNA damage 

### 13
and decreased DNA repair were seen using the comet assay and other assays , but did not correlate with 

### 14

arsenic or Pb levels. Tapisso et al., ( 2009 ) , considered Pb alone, Pb plus zinc and Pb plus cadmium



### 15
induced MN in rodents. Algerian mice (groups of six mice each) were exposed by intraperitoneal to 5 or 

### 16
10 doses of 0.46 mg/kg Pb(Ac) 2 compared to untreated controls. The MN in bone marrow were elevated 

### 17

after Pb alone exposure, which increased with time. Co

- exposure with cadmium or zinc increased SCE 

### 18
levels, but did not further increase MN levels. Glahn et al., ( 2008 ) performed a gene array stu dy in 

### 19
primary normal human bronchial epithelial cells from 4 donors treated with 550 μ g/l Pb chloride, 15 μ g/l 

### 20
cadmium sulphate, 25 μ g/l cobalt chloride or all three combined for 72 hours. There was a clear 

### 21
interaction of all three metals impacting RNA exp ression. 

### 22
One new publication details the interaction of Pb and selenium in virus

- dependent mammary tumor 

### 23

formation. No recent studies of the protective role of calcium in Pb

- dependent carcinogenesis or 

### 24
genotoxicity were found. There were some data suggesti ng that boron, melatonin, N

- acetylcysteine, 

### 25
turmeric and myrrh protect cells against Pb

- induced genotoxicity (Sections 5.10.3.5, 5.10.3.7 and 

### 26

5.10.3.10

. ). 

### 27
One study considered Pb and selenium interactions in carcinogenesis in laboratory animals. 

### 28

Schrauzer ( 2008 ) considered the impact of selenium on carcinogensis by studying 4 groups of weanling 

### 29
virgin female C3H/St mice infected with murine mammary tumor virus (groups of 20

- 30 mice)

. One set 

### 30
of two groups were fed a diet containing 0.15 ppm selenium and then were exposed via drinking water to 

### 31
acetic acid (control group) or 0.5 ppm Pb(Ac) 2 (treated group)

. The second set of two groups were fed a 

### 32
diet containing 0.65 ppm selenium and then similarly exposed to acetic acid or 0.5 ppm Pb(Ac) 2 . The 

### 33May 2011 5

- 367 DRAFT

– DO NOT CITE OR QUOTE effects of selenium and Pb on the tumors caused by the virus were studied

. The study is primarily focused 

### 1
on the general effects of a low selenium diet. The data suggest that selenium is anticarcinogenic as in the 

### 2
control groups the a nimals exposed to the higher selenium levels had fewer mammary tumors and these 

### 3
tumors had a delayed onset. Pb exposure with low selenium caused the same delayed onset as the higher 

### 4
dose of selenium and also caused some reduction in the tumor frequency. Pb exposure with higher 

### 5
selenium increased the tumor frequency and the onset of the tumors. Pb also induced weight loss at 14 

### 6
months in both exposed groups

. The data suggest that there may be interactions of Pb and selenium, but 

### 7
they suggest that Pb mimics o r antagonizes selenium. They do not suggest that selenium is protective of 

### 8

Pb

- induced toxicity or carcinogenesis. 

### 9



## 5.10.4. Epigenetics

E pigenetic studies have been conducted to examine the associations between Pb levels and global 

### 10
DNA methylation markers [Alu and long interspersed nuclear element

- 1 (LINE

- 1)] ( Pilsner et al., 2009 ; 

### 11
R. O. Wright et al., 2010 )

. Wright et al. ( 2010 ) utilized a sample of participants from the Normative Aging 

### 12
Study with mean Pb levels of 20.5 g/g (SD 14.8 g/g) for tibia measures, 27.4 g/g (SD 19.7 g/g) for patella 

### 13
measures, and 4.1 μg/dL (SD 2.4 μg/dL) for blood measures

. In both crude and adjusted analyses, patella 

### 14
Pb levels were inversely associated with LINE

- 1 methylation but not with Alu

. When examining the 

### 15
relationship between pate lla Pb and LINE

- 1 more closely, a non

- linear trend was observed with leveling 

### 16
off at higher Pb levels

. No associations were observed for tibia or blood Pb and either LINE

- 1 or Alu

. The 

### 17
second study included maternal

- infant pairs from the Early Life Exposur es in Mexico to Environmental 

### 18
Toxicants (ELEMENT) study and measured LINE

- 1 and Alu methylation in umbilical cord blood samples 

### 19
( Pilsner et al., 2009 )

. In unadjusted models , maternal tibia Pb levels [mean 10.5 μg/g (SD 8.4 μg/g)] were 

### 20
inversely associated with Alu methylation and maternal patella Pb levels [12.9 μg/g (SD 14.3 μg/g)] were 

### 21
inversely associated with LINE

- 1 methylation

. The associations persisted in adjusted mod els although the 

### 22
association between patella Pb and LINE

- 1 was only apparent when the adjusted models also included 

### 23

umbilical cord blood Pb levels

. No association was detected between umbilical cord Pb levels and the 

### 24
DNA methylation markers . Overall, the s tudies consistently demonstrate an association between patella 

### 25

Pb levels and LINE

- 1 methylation. 

### 26
Toxicological studies have been performed examining Pb

- dependent epigenetic changes and gene 

### 27
expression, DNA repair, and mitogenesis. Glahn et al., ( 2008 ) performed a gene array study in primary 

### 28
normal human bronchial epithelial cells from 4 donors after in vitro treat ment of the cells with 550 μ g/l 

### 29

Pb chloride, 15 μ g/l cadmium sulphate, 25 μ g/l cobalt chloride or all thr ee combined for 72 hours. The 

### 30
authors describe a pattern of RNA expression changes indicating “ ...coordinated stress

- response and cell



### 31
survival signaling, deregulation of cell proliferation, increased steroid metabolism, and increased 

### 32
expression of xen obi otic metabolizing enzymes”. These are all known targets of possible epigentic 

### 33May 2011 5

- 368 DRAFT

– DO NOT CITE OR QUOTE changes, but f ull interpretations of the data as epigenetic changes are complicated by the absence of a 

### 1
measure to determine if these changes were a result of genotoxic effects. 

### 2



## 5.10.5. Summary and Causal Determination

In summary, the toxicology literature on the genotoxic, mutagenic, and carcinogenic potential of Pb 

### 3
have strong evidence of effects in laboratory animals. In laboratory studies, high

- dose Pb has been 

### 4

demonstrated to be an animal carcinogen. There are data to suggest Pb is a human carcinogen among 

### 5
toxicological studies , but they are not definitive. The three toxicological studies of neoplastic 

### 6
transformation in cultured cells both were positive, but both focused on PbCrO 4 an d attributed the 

### 7
positive response to the CrO4 and not the Pb. Mechanistic understanding of the carcinogenicity of Pb is 

### 8
expanding with work on the antioxidant selenium and metallothionein, a protein that binds Pb and 

### 9
reduces its bioavailability. In separate studies, selenium and metallothionein are protective aga inst the 

### 10
effect of Pb on carcinogenicity. Pb is clastogenic and mutagenic in some but not all models. 

### 11
Clastogenicity and mutagenicity may be possible mechanisms contributing to cancer but are not 

### 12
absolutely associated with the induction of cancer. Because Pb has a higher atomic weight than zinc, Pb 

### 13
replaces zinc at many zinc binding or zinc finger proteins. This substitution has the potential to induce 

### 14
indirect effects that can contribute to carcinogenicity via interactions at hormone receptors, at cell

- cyc le 

### 15
regulatory proteins, with tumor suppressor genes like p53, with DNA repair enzymes, with histones, etc. 

### 16
These indirect effects may act at a post

- translational level to adversely alter protein structure and DNA 

### 17
repair. Also, epigenetic changes associated with Pb exposure are beginning to appear in the literature. 

### 18
These modifications may further alter DNA repair or change the expression of a tumor suppressor gene or 

### 19
oncogene in an adverse fashion. Thus, the animal toxicology literature provides a strong ba se for 

### 20
understanding the potential contribution of Pb exposure to cancer in laboratory animals. 

### 21

M ultiple epidemiologic studies have been performed examining cancer incidence and mortality, 

### 22
determined with biological measures and exposure databases . M ixed results have been reported for cancer 

### 23
mortality studies; one strong epidemiologic study demonstrated a positive association between blood Pb 

### 24
and cancer mortality, but the other studies reported null results. Although the previous Pb AQCD reported 

### 25

some stud ies demonstrating an association between Pb exposure and lung cancer, current studies mostly 

### 26
include studies of occupational exposure and observed no associations. Most studies of Pb and brain 

### 27
cancer were null among the overall study population, but positi ve associations were observed among 

### 28
individuals with certain genotypes. A limited amount of research on other types of cancer has been 

### 29
performed. The previous AQCD reported evidence that suggested an association between Pb exposure 

### 30
and stomach cancer, but recent studies of this association are lacking. One study examining Pb and 

### 31
stomach cancer has been performed since the last AQCD and the results of the study are mixed. 

### 32May 2011 5

- 369 DRAFT

– DO NOT CITE OR QUOTE Among epidemiologic studies, positive associations were observed between high Pb level s and 

### 1
SCE among adults but not children. Other epidemiologic studies of DNA damage reported inconsistent 

### 2

results. Consistent with previous toxicological findings

, Pb does appear to have genotoxic activity 

### 3
inducing SCE, MN and DNA strand breaks, but continu es to not produce chromosomal aberrations except 

### 4

for PbCrO 4 ; this again is likely due to the chromate . Pb does not appear to be very mutagenic as the HPRT 

### 5
assays were typically negative unless a cell signaling pathway was disturbed. 

### 6
Epigenetic effects, pa rticularly with respect to methylation and effects on DNA repair were also 

### 7
consistently seen. Epigenetic studies examining Pb and LINE

- 1 and Alu consistently demonstrated an 

### 8
inverse association between patella Pb and LINE

- 1 methylation. Toxicological s tudi es do show that Pb 

### 9
can activate or interfere with a number of signaling and repair pathways, though it is unclear whether 

### 10
these are in response to epigenetics or to genotoxicity. Thus, an underlying mechanism is still uncertain, 

### 11
but likely involves either genomic instability or epigenetic modifications or both. 

### 12
Overall, there is some epidemiologic evidence supporting associations between Pb and cancer. 

### 13
Strong evidence from toxicological studies demonstrates an association between Pb and cancer, 

### 14
genotoxicity /clastogenicity or epigenetic modification. The collective body of evidence integrated across 

### 15
epidemiologic and toxicological studies is sufficient to conclude that there is a likely causal relationship 

### 16
between Pb exposures and cancer
. 

### 17



# 5.11. Overall Summary

The evidence reviewed in this chapter describes the recent findings regarding the health effects of 

### 18

Pb. Table 5

- 38 provides an overview of the causal determinations for each of the health categories 

### 19

evaluated. 

### 20

Table 5

- 38. Summary of causal determinations for P b.

Health Category Causal Determination
Neurological Effects Causal relationship
Cardiovascular Effects Causal relationship

Renal Effects Causal relationship Immune System Effects Causal relationship

Effects on Heme Synthesis and Red Blood Cell Function Causal relationship Reproductive Effects and Birth Outcomes Causal relationship

Cancer Likely causal relationshipMay 2011 5

- 370 DRAFT

– DO NOT CITE OR QUOTE 

# Chapter 5. References

Abam, E., Okediran, B. S., Odukoya, O. O., Adamson, I., & Ademuyiwa, O. (2008). Reversal of ionoregulatory disruptions in occupational lead exposure by vitamin C. Environmental Toxicology and Pharmacology, 26 (3), 297

- 304. http://dx.doi.org/10.1016/j.etap .2008.05.008

Abdallah, G. M., El

- Sayed, E. S., & Abo

- Salem, O. M. (2010). Effect of lead toxicity on coenzyme Q levels in rat tissues. Food and Chemical Toxicology, 48 (6), 1753

- 1756. http://dx.doi.o rg/10.1016/j.fct.2010.04.006

Abdelouahab, N., Mergler, D., Takser, L., Vanier, C., St

- Jean, M., Baldwin, M., . . . Chan, H. M. (2008). Gender differences in the effects of organochlorines, mercury, and lead on thyroid hormone levels in lakeside communities of Quebec (Canada). Environmental Research, 107 (3), 380

- 392. http://dx.doi.org/10.1016/j.envres.2008.01.006

Abe, A., Hiraoka, M., & Shayman, J. A. (2007). A role for lysosomal phospholipase A2 i n drug induced phospholipidosis. Drug Metabolism Letters, 1 (1), 49

- 53. http://dx.doi.org/10.2174/187231207779814292

Aboul

- Ela, E. I. (2002). The protective effect of calcium against genotoxicity of lead acetate administration on bone marrow and spermatocyte cells of mice in vivo. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 516 , 1

- 9. http://dx.doi.org/10.1016/S1383

- 5718(01)00332

- 1

Absalon, D., & Slesak, B. (2010). The effects of changes in cadmium and lead air pollution on cancer incidence in children. Science of the Total Environment, 408 (20), 4420

- 4428. http://dx.doi.org/10.1016/j.scitotenv.2010.06.030

Adegbesan, B. O., & Adenuga, G. A. (2007). Effect of lead exposure on liver lipid peroxidative and antioxidant defense system s of protein

- undernourished rats. Biological Trace Element Research, 116 (2), 2 19

- 225. http://dx.doi.org/10.1007/BF02685932

Ademuyiwa, O., Agarwal, R., Chandra, R., & Behari, J. R. (2009). Lead

- induced phospholipidosis and cholesterogenesis in rat
tissues. Chemico

- Biological Interact ions, 179 (2

- 3), 314

- 320. http://dx.doi.org/10.1016/j.cbi.2008.10.057

Ademuyiwa, O., Ugbaja, R. N., Idumebor, F., & Adebawo, O. (2005). Plasma lipid profiles and risk of cardiovascular disease in

occ upational lead exposure in Abeokuta, Nigeria. Lipids in Health and Disease, 4 , 19. http://dx.doi.org/10.1186/1476

- 511X

- 4

- 19

Ademuyiwa, O., Ugbaja, R. N., Ojo, D. A., Owoigbe, A. O., & Adeokun, S. E. (2 005). Reversal of aminolevulinic acid dehydratase (ALAD) inhibition and reduction of erythrocyte protoporphyrin levels by Vitamin C in occupational lead exposure in Abeokuta, Nigeria. Environmental Toxicology and Pharmacology, 20 (3), 404

- 411. http://dx.doi.org/10.1016/j.etap.2005.04.002

Adonaylo, V. N., & Oteiza, P. I. (1999). Pb2+ promotes lipid oxidation and alterations in membrane physical properties. Toxicology, 132 (1), 19

- 32. http://dx.doi.org/10.1016/S0300

- 483X(98)00134

- 6

Agarwal, S., Roy, S., Ray, A., Mazumder, S., & Bhattacharya, S. (2009). Arsenic trioxide and lead acetate induce apoptosis in adult rat hepatic stem cell s. Cell biology and toxicology, 25 (4), 403

- 413. http://dx.doi.org/10.1007/s10565

- 008

- 9094

- 6

Aguilar, R. P., Genta, S., & Sanchez, S. (2008). Renal gangliosides are involved in lead intoxication. Jou rnal of Applied Toxicology, 28 (2), 122

- 131. http://dx.doi.org/10.1002/jat.1256

Ahamed, M., Fareed, M., Kumar, A., Siddiqui, W. A., & Siddiqui, M. K. J. (2008). Oxidative stress and neurological disorders in
relation to blood lead levels in children. Redox Report, 13 (3), 117

- 122. http://dx.doi.org/10.1179/135100008X259213May 2011 5

- 371 DRAFT

– DO NOT CITE OR QUOTE Ahamed, M., Mehrotra, P. K., Kumar, P., & Siddiqui, M. K. J. (2009). Placental lead

- induced oxidative stress and preterm delivery. Environmental Toxicology and Pharmacology, 27 (1), 70

- 74. http://dx.doi.org/10.1016/j.etap.2008.08.013

Ahamed, M., & Siddiqui, M. K. (2007). Low level lead exposure and oxidative stress: Current opinions. Clinica Chimica Acta,
383 (1

- 2), 57

- 64. http://dx.doi.org/10.1016/j.cca.2007.04.024

Ahamed, M., Verma, S., Kumar, A., & Siddiqui, M. K. (2005). Environmental exposure to lead and its correlation with
biochemical indices in children. Science of the Total Environment, 346 , 48

- 55. http://dx.doi.org/10.1016/j.scitotenv.2004.12.019

Ahamed, M., Verma, S., Kumar, A., & Siddiqui, M. K. (2006). Delta

- aminolevulinic acid dehydratase inhibition and oxidative stress in relation to blood lead among urban adolescents. Human and Experimental Toxicology, 25 (9), 547

- 553. http://dx.doi.org/10.1191/0960327106het657oa

Akel, A., Wagner, C. A., Kovacikova, J., Kasinathan, R. S., Kiedaisch, V., Koka, S., . . . Lang, F. (2007). Enhanced suicidal death of erythrocytes from gene

- targeted mice lacking the Cl

- /HCO3

- exchanger AE1. American Journal of Physiology: Cell Physiology, 292 (5), C1759

- C1767. http://dx.doi.org/10.1152/ajpcell.00158.2006

Akesson, A., Lundh, T., Vahter, M., Bjellerup, P., Lidfe ldt, J., Nerbrand, C., . . . Skerfving, S. (2005). Tubular and glomerular kidney effects in Swedish women with low environmental cadmium exposure. Environmental Health Perspectives, 113

, 1627

- 1631. http://dx.doi.org/10.1289/ehp.8033

Al

- Saleh, I., Coskun, S., Mashhour, A., Shinwari, N., El

- Doush, I., Billedo, G., . . . El Din Mohamed, G. (2008). Exposure to heavy metals (lead, cadmium and mercury) and its effect on the outcome of in

- vitro fertilizatio n treatment. International Journal of Hygiene and Environmental Health, 211 (5

- 6), 560

- 579. http://dx.doi.org/10.1016/j.ijheh.2007.09.005

Al

- Saleh, I., Nester, M., DeVol, E., Shinwari, N., Munchari , L., & Al

- Shahria, S. (2001). Relationships between blood lead concentrations, intelligence, and academic achievement of Saudi Arabian schoolgirls. International Journal of Hygiene and Environmental Health, 204 (2

- 3), 165

- 174. http://dx.doi.org/10.1078/1438

- 4639

- 00091

Alatise, O. I., & Schrauzer, G. N. (2010). Lead exposure: A contributing cause of the current breast cancer epidemic in Niger ian

women. Biological Trace Element Research, 136 (2), 127

- 139. http://dx.doi.org/10.1007/s12011

- 010

- 8608

- 2

Alghazal, M. A., Lenártová, V., Holovská, K., Sobeková, A., Falis, M., & Legáth, J. (2008). Activities of antioxidant and detoxifying enzymes in rats aft er lead exposure. Acta Veterinaria, 77 (3), 347

- 354. http://dx.doi.org/10.2754/avb200877030347

Alghazal, M. A., Sutiakova, I., Kovalkovicova, N., Legath, J., Falis, M., Pistl, J., . . . Vaczi, P. (2008 ). Induction of micronuclei in rat bone marrow after chronic exposure to lead acetate trihydrate. Toxicology and Industrial Health, 24 (9), 587

- 593. http://dx.doi.org/10.1177/0748233708100089

Alimonti , A., Bocca, B., Lamazza, A., Forte, G., Rahimi, S., Mattei, D., . . . Pino, A. (2008). A study on metals content in patients with colorectal polyps. Journal of Toxicology and Environmental Health, Part A: Current Issues, 71 (5), 342

- 347. http://dx.doi.org/10.1080/15287390701839133

Alinovi, R., Scotti, E., Andreoli, R., De Palma, G., Goldoni, M., Apostoli, P., & Mutti, A. (2005). [Neuroendocrine and renal effects of inorganic lead]. Giornale Italiano di Medicina del Lavoro ed Ergonomia, 27 (Suppl 1), 33

- 38. http://www.ncbi.nlm.nih.gov/pubmed/15915652

Allouche, L., Hamadouche, M., & Touabti, A. (2009). Chronic effects of low lead levels on sperm quality, gonadotropins and
testosterone in albino rats. Experimental and Toxicologic Pathology, 61 (5), 503

- 510. http://dx.doi.org/10. 1016/j.etp.2008.12.003May 2011 5

- 372 DRAFT

– DO NOT CITE OR QUOTE Alomran, A. H., & Shleamoon, M. N. (1988). The influence of chronic lead exposure on lymphocyte proliferative response and immunoglobulin levels in storage battery workers. Journal of Biological Sciences, 19 , 575

- 585.

Alvarez, J., G arcia

- Sancho, J., & Herreros, B. (1986). Inhibition of Ca2+

- dependent K+ channels by lead in one

- step inside

- out vesicles from human red cell membranes. Biochimica et Biophysica Acta, 857 , 291

- 294. http://dx.doi.org/10.1016/0005

- 2736(86)90359

- 7

Amici, A., Emanuelli, M., Magni, G., Raffaelli, N., & Ruggieri, S. (1997). Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase activities specific for pyrimidine nucleotides. FEBS Let ters, 419 (2

- 3), 263

- 267. http://dx.doi.org/10.1016/S0014

- 5793(97)01464

- 6

Andersen, Z. J., Wahlin, P., Raaschou

- Nielsen, O., Scheike, T., & Loft, S. (2007). Ambient particle source apportionment and daily hospital admissions among children and elderly in Copenhagen. Journal of Exposure Science and Environmental Epidemiology, 17 (7), 625

- 636. http://www.ncbi.nlm.nih.gov/pubmed/17495872

Andreae, M. O. (1983). Biotransformation of arsenic in the marine environment. In W. H. Lederer & R. J. Fensterheim (Eds.), Arsenic: Industrial, biomedical and environmental perspectives: Proceedings of the Arsenic Symposium (pp. 378

- 391). New York, NY: Van Nostrand Reinhold.

Anetor, J. I., & Adeniyi, F. A. A. (1998). Decreased immune status in Nigerian workers occupationally exposed to lead. African Journal of Medicine and Medical Sciences, 28 (3

- 4), 169

- 172

. http://www.ncbi.nlm.nih.gov/pubmed/10497641

Annesi

- Maesano, I., Pollitt, R., King, G., Bousquet, J., Hellier, G., Sahuquillo, J., & Huel, G. (2003). In utero exposure to lead
and cord blood total IgE: Is there a connection? Allergy, 58 (7), 589

- 594. http://dx.doi.org/10.1034/j.1398

- 9995.2003.00111.x

Antonowicz, J., Andrzejak, R., & Smolik, R. (1990). Influence of heavy metal mixtures o n erythrocyte metabolism. International Archives of Occupational and Environmental Health, 62 , 195

- 198. http://dx.doi.org/10.1007/BF00379431

Antonyuk, S. V., Strange, R. W., Marklund, S. L., & Hasnain, S. S. (2009). The structure of human extracellular copper

- zinc superoxide dismutase at 1.7 A resolution: Insights into heparin and collagen binding. Journal of Molecular Biology,
338 (2), 310

- 326. http: //dx.doi.org/10.1016/j.jmb.2009.03.026

Anwar, W. A., & Kamal, A. A. M. (1988). Cytogenetic effects in a group of traffic policemen in Cairo. Mutation Research Letters, 208 (3

- 4), 225

- 231. http://d x.doi.org/10.1016/0165

- 7992(88)90065

- 6

Apostoli, P., Huard, C., Chaumontet, C., Martel, P., Allesio, L., & Mazzoleni, G. (2000). Effects of four inorganic lead compounds on the proliferation and junctional coupling of cultured REL liver cells. American Jou rnal of Industrial Medicine, 38 (3), 340

- 348. http://www.ncbi.nlm.nih.gov/pubmed/10940974

Apostoli, P., Romeo, L., De Matteis, M. C., Menegazzi, M., Faggionato, G., & Vettore, L. (1988). Effects of l ead on red blood cell membrane proteins. International Archives of Occupational and Environmental Health, 61 (1

- 2), 71

- 75. http://dx.doi.org/10.1007/BF00381610

Aquilonius, S. M., & Hartvig, P. (1986). A Swe dish county with unexpectedly high utilization of anti

- parkinsonian drugs. Acta Neurologica Scandinavica, 74 (5), 379

- 382. http://dx.doi.org/10.1111/j.1600

- 0404.1986.tb03529.x

Ariza, M. E., Bijur, G. N., & Williams, M. V. (1998). Lead and mercury mutagenesis: Role of H2O2, superoxide dismutase, and
xanthine oxidase. Environmental and Molecular Mutagenesis, 31 , 352

- 361. http://www.ncbi.nlm.nih.gov/pubmed/9654245

Ariza, M. E., & Williams, M. V. (1996). Mutagenesis of AS52 cells by low concentrations of lead(II) and mercury(II). Environmental and Molecular Mutagenesis, 27 , 30

- 33. http://www.ncbi.nlm.nih.gov/pubmed/8625945May 2011 5

- 373 DRAFT

– DO NOT CITE OR QUOTE Ariza, M. E., & Williams, M. V. (1999). Lead and mercury mutagenesis: Type of mutation dependent upon metal concentration. Journal of Biochemical and Molecular Toxicology, 13 (2), 107

- 112. http://dx.doi.org/10.1002/(SICI)1099

- 0461(1999)13:2<107::AID

- JBT6>3.0.CO;2

- 0

Arnold, W. J., & Kelley, W. N. (1978). Adenine phosphoribosyltransferase. Methods in Enzymology, 51 , 568

- 574. http://dx.doi.org/10.1016/S0076

- 6879(78)51079

- 3

Arora, M., Chan, S. W., Ryan, C. G., Kennedy, B. J., & Walker, D. M. (2005). Spatial distribution of lead in enamel and coron al
dent ine of wistar rats. Biological Trace Element Research, 105 (1

- 3), 159

- 170. http://dx.doi.org/10.1385/BTER:105:1

- 3:159

Arora, M., Weuve, J., Weisskopf, M. G., Sparrow, D., Nie, H. L., Garcia, R. I., & Hu, H. (2009). Cumulative lead exposure and tooth loss in men: The normative aging study. Environmental Health Perspectives, 117 (10), 1531

- 1534. http://dx.doi.org/10.1289/ehp.0900739

Aruoma, O. I., Halliw ell, B., Laughton, M. J., Quinlan, G. J., & Gutteridge, J. M. (1989). The mechanism of initiation of lipid peroxidation. Evidence against a requirement for an iron(II)

- iron(III) complex. Biochemical Journal, 258 (2), 617

- 620. http://www.ncbi.nlm.nih.gov/pubmed/2706005

Aslani, M. R., Najarnezhad, V., & Mohri, M. (2010). Individual and combined effect of meso

- 2,3

- dimercaptosuccinic acid and
allicin on blood and tissue lead content in mice. Planta Medica, 76 (3), 241

- 244. http://dx.doi.org/10.1055/s

- 0029

- 1186141

Astrin, K. H., Bishop, D. F., Wetmur, J. G., Kaul, B., Davidow, B., & Desnick, R. J. (1987). Delta

- aminolevulinic acid dehydratase isozymes and lead toxicity. Annals of the New York Academy of Sciences, 514 , 23

- 29. http://dx.doi.org/10.1111/j.1749

- 6632.1987.tb48757.x

Attri, J., Dhawan, V., Mahmood, S., Pandhi, P., Parwana, H. K., & Nath, R. (2003). Effect of vitamin C supplementation on oxidative DNA damage in an experimental model of lead

- induced hypertension. Annals of Nutrition and Metabolism,
47 (6), 294

- 301. http://dx.doi.org/10 .1159/000072402

Audesirk, G., & Audesirk, T. (1991). Effects of inorganic lead on voltage

- sensitive calcium channels in N1E

- 115 neuroblastoma
cells. NeuroToxicology, 12 (3), 519

- 528. http://www.ncbi.nlm.nih.gov/pubmed/1660583

Audesirk, G., & Audesirk, T. (1993). The effects of inorganic lead on voltage

- sensitive calcium channels differ among cell types and among channel subtypes. NeuroToxicology, 142 (2

- 3), 259

- 266. http://www.ncbi.nlm.nih.gov/pubmed/8247399

Azar, A., Trochimowicz, H. J., & Maxfield, M. E. (1973). Review of lead studies in animals carried out at Haskell Laboratory: Two year feeding study and response to hemorrhage study. In D. Barth, A. Berlin, R. Engel, P. Recht & J. Smeets (Eds.), Environmental health aspects of lead: Proceedings of an international symposium (pp. 199

- 210). Amsterdam, The Netherlands: Commission of the European Communities.

Badavi, M., Mehrgerdi, F. Z., Sarkaki, A., Naseri, M. K., & Dianat, M. (2008). Effect of grape seed extract on lead induced hypertension and heart rate in rat. Pakistan Journal of Biological Sciences, 11 (6), 882

- 887. http://www.ncbi.nlm.nih.gov/pubmed/18814650

Bagchi, D., & Preuss, H. G. (2005). Effects of acute and chronic oral exposure of lead on blood pressure and bone mineral density in rats. Journal of Inorganic Biochemistry, 99 , 1155

- 1164. http://dx.doi.org/10.1016/j.jinorgbio.2005.02.011

Baghurst, P. A., McMichael, A. J., Wigg, N. R., Vimpani, G. V., Robertson, E. F., Roberts, R. J., & Tong, S.

- L. (1992). Environmental exposure to lead and children's intelligence at the age of seven years: The Port Pirie cohort study. New England Journal of Medicine, 327 (18), 1279

- 1284. http://www.ncbi.nlm.nih.gov/pubmed/1383818May 2011 5

- 374 DRAFT

– DO NOT CITE OR QUOTE Baker, E. L., Feldman, R. G., White, R. A., Harley, J. P., Niles, C. A., Dinse, G. E., & Berkey, C. S. (1984). Occupational lead neurotoxicity: A behavioural and electrophysiological evaluation: Study design and year one results. Occupational and Environmental Medicine, 41 (3), 352

- 361. http://dx.doi.org/10.1136/oem.41.3.352

Baker, E. L., Landrigan, P. J., Barbour, A. G., Cox, D. H., Folland, D. S., Ligo, R. N., & Throckmorton, J. (1979). Occupatio nal lead poisoning in the United States: Clinical and biochemical findings related to blood lead levels. Occupational and Environmental Medicine, 36 (4), 314

- 322. http://dx.doi.org/10.1136/oem.36.4.314

Baker, E. L., White, R. F., Pothier, L. J., Berkey, C. S., Dinse, G. E., Travers, P. H., . . . Feldman, R. G. (1985). Occupational lead neurotoxicity: Improvement in behavioural effects after reduction of exposure. British Journal of Industrial Medicine,
42 (8), 507

- 516. http://dx.doi.org/10.1136/oem.42.8.507

Ball, G. V., & Sorensen, L. B. (1969). Pathogenesis of hyperuricemia in saturnine gout. New England Journal of Medicine, 280

, 1199

- 1202. http://dx.doi.org/10.1056/nejm196905292802203

Bandeen

- Roche, K., Glass, T. A., Bolla, K. I., Todd, A. C., & Schwartz, B. S. (2009). Cumulative lead dose and cognitive
function in older adults. Epidemiology, 20 (6), 831

- 839. http://dx.doi.org/10.1097/EDE.0b013e3181b5f100

Baranowska

- Bosiacka, I., Dąbrowska

- Bouta, B., & Strużyńska, L. (2011). Regional changes in purines and selected purinergic receptors in immature rat brain ex posed to lead. Toxicology, 279 (1

- 3), 100

- 107. http://dx.doi.org/10.1016/j.tox.2010.09.016

Baranowska

- Bosiacka, I., Dziedziejko, V., Safranow, K., Gutowska, I., Marchlewicz, M., Dołegowska, B., . . . Chlubek, D. (2009). Inhibition of erythrocyte phosphoribosyltransferases (APRT and HPRT) by Pb2+: A potential mechanism of lead toxicity. Toxicolog y, 259 (1

- 2), 77

- 83. http://dx.doi.org/10.1016/j.tox.2009.02.005

Baranowska

- Bosiacka, I., & Hlynczak, A. J. (2003). The effect of lead ions on the energy metabolism of human erythrocytes in
vitro. Co mparative Biochemistry and Physiology

- Part C: Toxicology and Pharmacology, 134 (3), 403

- 416. http://dx.doi.org/10.1016/S1532

- 0456(03)00008

- 5

Baranowska

- Bosiacka, I., & Hlynczak, A. J. (2004). E ffect of lead ions on rat erythrocyte purine content. Biological Trace Element Research, 100 , 259

- 273. http://dx.doi.org/10.1385/BTER:100:3:259

Barbeito, A. G., Martinez

- Palma, L., Vargas, M. R., Pehar , M., Mañay, N., Beckman, J. S., . . . Cassina, P. (2010). Lead exposure stimulates VEGF expression in the spinal cord and extends survival in a mouse model of ALS. Neurobiology of Disease, 37 (3), 574

- 580. http://dx.doi.org/10.1016/j.nbd.2009.11.007

Barbosa, F., Sandrim, V. C., Uzuelli, J. A., Gerlach, R. F., & Tanus

- Santos, J. E. (2006). eNOS genotype

- dependent correlation between whole blood lead and plasma nitric oxide products concentrations. Nit ric Oxide, 14 (1), 58

- 64. http://dx.doi.org/10.1016/j.niox.2005.09.007

Baronas, E. T., Lee, J. W., Alden, C., & Hsieh, F. Y. (2007). Biomarkers to monitor drug

- induced phospholipidosis. Toxicology

a nd Applied Pharmacology, 218 (1), 72

- 78. http://dx.doi.org/10.1016/j.taap.2006.10.015

Basha, M. R., Murali, M., Siddiqi, H. K., Ghosal, K., Siddiqi, O. K., Lashuel, H. A., . . . Zawia, N. H. (2005). Lead (Pb) exposure and its effect on APP proteolysis and A beta aggregation. FASEB Journal, 19 (12), 2083

- 2084. http://dx.doi.org/10.1096/fj.05

- 4375fje

Basha, M. R., Wei, W., Brydie, M., Razmiafshari, M ., & Zawia, N. H. (2003). Lead

- induced developmental perturbations in hippocampal Sp1 DNA

- binding are prevented by zinc supplementation: In vivo evidence for Pb and Zn competition. International Journal of Developmental Neuroscience, 21 (1), 1

- 12. http://dx.doi.org/10.1016/S0736

- 5748(02)00137

- 5

Batuman, V. (1993). Lead nephropathy, gout, and hypertension. American Journal of the Medical Sciences, 305 (4), 241

- 247. http://www.ncbi.nlm.nih.gov/pubmed/8475950May 2011 5

- 375 DRAFT

– DO NOT CITE OR QUOTE Bayer, S. A. (1989). Cellular aspects of brain development. NeuroToxicology, 10 , 307

- 320.

Bear, M. F., & Malenka, R. C. (1994). Synaptic plasticity: LTP and LTD. Current Opinion in Neurobio logy, 4 (3), 389

- 399. http://dx.doi.org/10.1016/0959

- 4388(94)90101

- 5

Beaudin, S. A., Stangle, D. E., Smith, D. R., Levitsky, D. A., & Strupp, B. J. (2007). Succimer chelation normalizes reactivity to
reward omission and errors in lead

- exposed rats. Neurotoxicology and Teratology, 29 (2), 188

- 202. http://dx.doi.org/10.1016/j.nett. 2006.11.004

Bechara, E. J. H., Medeiros, M. H. G., Monteiro, H. P., Hermes

- Lima, M., Pereira, B., Demasi, M., . . . Mascio, P. D. (1993). A free radical hypothesis of lead poisoning and inborn porphyrias associated with 5

- aminolevulinic acid overload. Quim ica Nova, 16 (4), 385

- 392.

Behl, M., Zhang, Y., & Zheng, W. (2009). Involvement of insulin

- degrading enzyme in the clearance of beta

- amyloid at the
blood

- CSF barrier: Consequences of lead exposure. Cerebrospinal Fluid Research, 6 (1), 11. http://dx.doi.org/10.1186/1743

- 8454

- 6

- 11

Bell, M., Ebisu, K., Peng, R., Samet, J., & Dominici, F. (2009). Hospital admissions and chemical composition of fine particl e air pollution. American Journal of Respiratory and Critical Care Medicine, 179 , 1115

- 1120. http://dx.doi.org/10.1164/rccm.200808

- 1240OC

Bellinger, D., Hu, H., Titlebaum, L., & Needleman, H. L. (1994). Attentional correlates of dentin and bone lead levels in
adolescents. Archives of Environmental Health, 49 (2), 98

- 105. http://dx.doi.org/10.1080/00039896.1994.9937461

Bellinger, D., Leviton, A., & Sloman, J. (1990). Antecedents and correlate s of improved cognitive performance in children exposed in utero to low levels of lead. Environmental Health Perspectives, 89 , 5

- 11.

Bellinger, D., Leviton, A., Waternaux, C., Needleman, H., & Rabinowitz, M. (1987). Longitudinal analyses of prenatal and

p ostnatal lead exposure and early cognitive development. New England Journal of Medicine, 316 , 1037

- 1043. http://dx.doi.org/10.1056/NEJM198704233161701

Bellinger, D., & Needleman, H. L. (2003). Intellectual impairment and blood lead levels [letter]. New England Journal of Medicine, 349 , 500. http://dx.doi.org/10.1056/NEJM200307313490515

Bellinger, D., Needleman, H. L., Bromfield, R., & Mintz, M. (1984). A followup study of the academic attainment and classroom
behavior of children with elevated dentine lead levels. Biological Trace Element Research, 6 , 207

- 223.

Bellinger, D., & Rappaport, L. (2002). Developmenta l assessment and interventions. In B. Harvey (Ed.), Managing elevated blood lead levels among young children: recommendations from the Advisory Committee on Childhood Lead Poisoning
Prevention (pp. 79

- 95). Atlanta, GA: Centers for Disease Control.

Bellinge r, D., Sloman, J., Leviton, A., Rabinowitz, M., Needleman, H. L., & Waternaux, C. (1991). Low

- level lead exposure and children's cognitive function in the preschool years. Pediatrics, 87 (2), 219

- 227. http://www.ncbi.nlm.nih.gov/pubmed/1987535

Bellinger, D., & Stiles, K. M. (1993). Epidemiologic approaches to assessing the developmental toxicity of lead. NeuroToxicology, 14 (2

- 3), 151

- 160. http://www.ncbi.nlm.nih.gov/pubmed/7504226

Bellinger, D., Stiles, K. M., & Needleman, H. L. (1992). Low

- level lead exposure, intelligence and academic achievement: A long

- term follow

- up study. Pediatrics, 90 (6), 855

- 861. http://www.ncbi.nlm.nih.gov/pubmed/1437425

Belloni

- Olivi, L., Annadata, M., Goldstein, G. W., & Bressler, J. P. (1996). Phosphorylation of membrane proteins in erythrocytes
treated with lead. Biochemistry, 315 , 401

- 406.May 2011 5

- 376 DRAFT

– DO NOT CITE OR QUOTE Bener, A., Ob ineche, E., Gillett, M., Pasha, M. A. H., & Bishawi, B. (2001). Association between blood levels of lead, blood pressure and risk of diabetes and heart disease in workers. International Archives of Occupational and Environmental Health, 74 (5), 375

- 378. http://dx.doi.org/10.1007/s004200100231

Bennet, C., Bettaiya, R., Rajanna, S., Baker, L., Yallapragada, P. R., Brice, J. J., . . . Bokara, K. K. (2007). Region speci fic increase in the antioxidant enzymes and lipid peroxidation products in the brain of rats exposed to lead. Free Radical Research, 41 (3), 267

- 273. http://dx.doi.org/10.1080/10715760600889855

Bergdahl, I. A., Grubb, A., Schutz, A., Desni ck, R. J., Wetmur, J. G., Sassa, S., & Skerfving, S. (1997). Lead binding to delta

aminolevulinic acid dehydratase (ALAD) in human erythrocytes. Basic and Clinical Pharmacology and Toxicology,
81 (4), 153

- 158. http://dx.doi.org/10.1111/j.1600

- 0773.1997.tb02061.x

Bergdahl, I. A., Schutz, A., Gerhardsson, L., Jensen, A., & Skerfving, S. (1997). Lead concentrations in human plasma, urine and
whole blood. Scandinavian Journal of Work, Environment and Health, 23 , 359

- 363.

Bergdahl, I. A., Sheveleva, M., Schutz, A., Artamonova, V. G., & Skerfving, S. (1998). Plasma and blood lead in humans: Capacity

- limited binding to delta

- aminolevulinic acid dehydratase and other lead

- binding components. Toxicolo gical Sciences, 46 (2), 247

- 253. http://dx.doi.org/10.1093/toxsci/46.2.247

Bergeret, A., Pouget, E., Tedone, R., Meygert, T., Cadot, R., & Descotes, J. (1990). Neutrophil functions in lead

- exposed
work ers. Human and Experimental Toxicology, 9 , 231

- 233. http://dx.doi.org/10.1177/096032719000900405

Berkowitz, Z., Price

- Green, P., Bove, F. J., & Kaye, W. E. (2006). Lead exposure and birth outcomes in five communities in Shoshone County, Idaho. International Journal of Hygiene and Environmental Health, 209 (2), 123

- 132. http://dx.doi.org/10.1016/j.ijheh.2005.11.001

Berrahal, A. A., Lasram, M. , El Elj, N., Kerkeni, A., Gharbi, N., & El

- Fazâa, S. (2011). Effect of age

- dependent exposure to lead on hepatotoxicity and nephrotoxicity in male rats. Environmental Toxicology, 26 (1), 68

- 78. http://dx.do i.org/10.1002/tox.20530

Berrahal, A. A., Nehdi, A., Hajjaji, N., Gharbi, N., & El

- Fazâa, S. (2007). Antioxidant enzymes activities and bilirubin level in
adult rat treated with lead. Academie des Sciences. Comptes Rendus. Biologies, 330 (8), 581

- 588. http://dx.doi.org/10.1016/j.crvi.2007.05.007

Berry, W. D. J., Moriarty, C. M., & Lau, Y. S. (2002). Lead attenuation of episodic growth hormone secretion in male rats. International Journal of Toxicology, 21 (2), 93

- 98. http://www.ncbi.nlm.nih.gov/pubmed/12022635

Bhatti, P., Stewart, P. A., Hutchinson, A., Rothman, N., Linet, M. S., Inskip, P. D., & Rajaraman, P. (2009). Lead exposure, pol ymorphisms in genes related to oxidative stress, and risk of adult brain tumors. Cancer Epidemiology Biomarkers and Prevention, 18 (6), 1841

- 1848. http://dx.doi.org/10.1158/1055

- 9965.EPI

- 09

- 0197

Bielarczyk, H., Tian, X., & Suszkiw, J. B. (1996). Cholinergic denervation

- like changes in rat hippocampus following developmental lead exposure. Brain Research, 708 (1

- 2), 108

- 115. http://dx.doi. org/10.1016/0006

- 8993(95)01315

- 6

Bilban, M. (1998). Influence of the work environment in a Pb

- Zn mine on the incidence of cytogenetic damage in miners. American Journal of Industrial Medicine, 34 , 455

- 463. http://www.ncbi.nlm.nih.gov/pubmed/9787849

Bishayi, B., & Sengupta, M. (2006). Synergism in immunotoxicological effects due to repeated combined administration of
arsenic and lead in mice. International Immunopharmacology, 6 (3), 454

- 464. http://dx.doi.org/10.1016/j.intimp.2005.09.011

Biswas, N. M., & Ghosh, P. K. (2006). Protection of adrenal and male gonadal functions by androgen in lead

- treated rats. Kathmandu University Medical Journal, 4 (2), 218

- 221. http://www.ncbi.nlm.nih.gov/pubmed/1860390May 2011 5

- 377 DRAFT

– DO NOT CITE OR QUOTE Bitto, E., Bingman, C. A., Wesenberg, G. E., McCoy, J. G., & Phillips, G. N., Jr. (2006). Structure of pyrimidine 5'

- nucleotidase type 1: Ins ight into mechanism of action and inhibition during lead poisoning. Journal of Biological Chemistry,
281 (29), 20521

- 20529. http://dx.doi.org/10.1074/jbc.M602000200

Blackman, S. S., Jr. (1936). Intranuc lear inclusion bodies in the kidney and liver caused by lead poisoning. Johns Hopkins Medical Journal, 58 , 384

- 402.

Bleecker, M. L., Ford, D. P., Celio, M. A., Vaughan, C. G., & Lindgren, K. N. (2007). Impact of cognitive reserve on the relationship of le ad exposure and neurobehavioral performance. Neurology, 69 (5), 470

- 476. http://dx.doi.org/10.1212/01.wnl.0000266628.43760.8c

Bleecker, M. L., Ford, D. P., Vaughan, C. G., Walsh, K. S., & Li ndgren, K. N. (2007). The association of lead exposure and motor performance mediated by cerebral white matter change. NeuroToxicology, 28 (2), 318

- 323. http://dx.doi.org/10.1016/j.neuro.2006.04.00 8

Boerrigter, G., & Burnett, J. C., Jr. (2009). Soluble guanylate cyclase: Not a dull enzyme. Circulation, 119 (21), 2752

- 2754. http://dx.doi.org/10.1161/CIRCULATIONAHA.109.860288

Bogden, J. D., Kemp, F. W., Han, S., Murphy, M., Fraiman, M., Czerniach, D., . . . Gertner, S. B. (1995). Dietary calcium and lead interact to modify maternal blood pressure, erythropoiesis, and fetal and neonatal growth in rats during pregnancy
and lactat ion. Journal of Nutrition, 125 , 990

- 1002.

Boillee, S., Yamanaka, K., Lobsiger, C. S., Copeland, N. G., Jenkins, N. A., Kassiotis, G., . . . Cleveland, D. W. (2006). On set and progression in inherited ALS determined by motor neurons and microglia. Science, 312 (5778), 1389

- 1392. http://dx.doi.org/10.1126/science.1123511

Bokara, K. K., Blaylock, I., Denise, S. B., Bettaiya, R., Rajanna, S., & Yallapragada, P. R. (2009). Influence of lead acetat e on gluta thione and its related enzymes in different regions of rat brain. Journal of Applied Toxicology, 29 (5), 452

- 458. http://dx.doi.org/10.1002/jat.1423

Bolin, C. M., Basha, R., Cox, D., Zawia, N. H., Maloney, B. , Lahiri, D. K., & Cardozo

- Pelaez, F. (2006). Exposure to lead (Pb) and the developmental origin of oxidative DNA damage in the aging brain. FASEB Journal, 20 (6), 788

- 790. http://dx.doi.org/10.1096/fj.0 5

- 5091fje

Bonacker, D., Stoiber, T., Bohm, K. J., Prots, I., Wang, M., Unger, E., . . . Degen, G. H. (2005). Genotoxicity of inorganic lead
salts and disturbance of microtubule function. Environmental and Molecular Mutagenesis, 45 (4), 346

- 353. http://dx.doi.org/10.1002/em.20100

Bonucchi, D., Mondaini, G., Ravera, F., Minisci, E., Albertazzi, V., Arletti, S., . . . Cappelli, G. (2007). ["Terzo fuoco," lead poisoning and chronic renal failure]. Giornale Italian o di Nefrologia, Suppl 38 , 76

- 79. http://www.ncbi.nlm.nih.gov/pubmed/17922453

Borgstahl, G. E., Parge, H. E., Hickey, M. J., Beyer, W. F., Jr., Hallewell, R. A., & Tainer, J. A. (1992). The structur e of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4

- helix bundles. Cell, 71 (1),
107

- 118. http://dx.doi.org/10.1016/0092

- 8674(92)90270

- M

Bouchard, M. F., Bellinger, D. C., Weuve, J., Matthews

- Bellinger, J., Gilman, S. E., Wright, R. O., . . . Weisskopf, M. G. (2009). Blood lead levels and major depressive disorder, panic disorder, and generalized anxiety disorder in US young adults. Archives of Gene ral Psychiatry, 66 (12), 1313

- 1319. http://dx.doi.org/10.1001/archgenpsychiatry.2009.164

Boudene, C., Despaux

- Pages, N., Comoy, E., & Bohuon, C. (1984). Immunological and enzymatic studies o f erythrocytic delta

aminolevulinate dehydratase [Comparison of results obtained in normal and lead

- exposed subjects]. International Archives of Occupational and Environmental Health, 55 (1), 87

- 96. http:// dx.doi.org/10.1007/BF00378071May 2011 5

- 378 DRAFT

– DO NOT CITE OR QUOTE Bound, J. P., Harvey, P. W., Francis, B. J., Awwad, F., & Gatrell, A. C. (1997). Involvement of deprivation and environmental lead
in neural tube defects: A matched case

- control study. Archives of Disease in Childhood, 76 (2), 107

- 112. http://dx.doi.org/10.1136/adc.76.2.107

Bouton, C. M., Frelin, L. P., Forde, C. E., Arnold Godwin, H., & Pevsner, J. (2001). Synaptotagmin I is a molecular target fo r lead. Journal of Neurochemis try, 76 (6), 1724

- 1735. http://dx.doi.org/10.1046/j.1471

- 4159.2001.00168.x

Bradberry, S., & Vale, A. (2009). A comparison of sodium calcium edetate (edetate calcium disodium) and succimer (DMSA) in the treatment of inorganic lead poisoning. Clinical Toxicology, 47 (9), 841

- 858. http://dx.d oi.org/10.3109/15563650903321064

Bragadin, M., Marton, D., & Manente, S. (2007). Trialkyllead compounds induce the opening of the MTP pore in rat liver
mitochondria. Journal of Inorganic Biochemistry, 101 (5), 876

- 878. http://dx.doi.org/10.1016/j.jinorgbio.2007.01.016

Bragadin, M., Marton, D., Murgia, M., Rizzoli, V., Scutari, G., & Deana, R. (1998). Interactions of trialkyllead compounds wi th
rat liver mitochondria. Journal of Inorganic Biochem istry, 69 (4), 259

- 262. http://dx.doi.org/10.1016/S0162

- 0134(97)10036

- 8

Bratton, G. R., Hiney, J. K., & Dees, W. L. (1994). Lead (Pb) alters the norepinephrine

- induced secretion of luteinizing ho rmone releasing hormone from the medium eminence of adult male rats in vitro. Life Sciences, 55 (8), 563

- 571. http://dx.doi.org/10.1016/0024

- 3205(94)00482

- X

Braun, J. M., Froehlich, T. E., Daniels , J. L., Dietrich, K. N., Hornung, R., Auinger, P., & Lanphear, B. P. (2008). Association of
environmental toxicants and conduct disorder in U.S. children: NHANES 2001

- 2004. Environmental Health Perspectives, 116 , 956

- 962. http://dx.doi.org/10.1289/ehp.11177

Braun, J. M., Kahn, R. S., Froehlich, T., Auinger, P., & Lanphear, B. P. (2006). Exposures to environmental toxicants and attention deficit hyperactivity disorder in U.S. children. Environmental Health P erspectives, 114 , 1904

- 1909. http://dx.doi.org/10.1289/ehp.9478

Bravo, Y., Quiroz, Y., Ferrebuz, A., Vaziri, N. D., & Rodriguez

- Iturbe, B. (2007). Mycophenolate mofetil administration reduces
renal inflammat ion, oxidative stress, and arterial pressure in rats with lead

- induced hypertension. American Journal of Physiology: Renal Physiology, 293 (2), F616

- F623. http://dx.doi.org/10.1152/ajprenal.00507.2 006

Brender, J. D., Suarez, L., Felkner, M., Gilani, Z., Stinchcomb, D., Moody, K., . . . Hendricks, K. (2006). Maternal exposure to arsenic, cadmium, lead, and mercury and neural tube defects in offspring. Environmental Research, 101 (1), 132

- 139. http://dx.doi.org/10.1016/j.envres.2005.08.003

Brubaker, C. J., Dietrich, K. N., Lanphear, B. P., & Cecil, K. M. (2010). The influence of age of lead exposure on adult gray
matter volume. NeuroToxicology , 31 (3), 259

- 266. http://dx.doi.org/10.1016/j.neuro.2010.03.004

Brubaker, C. J., Schmithorst, V. J., Haynes, E. N., Dietrich, K. N., Egelhoff, J. C., Lindquist, D. M., . . . Cecil, K. M. (2 009).
A ltered myelination and axonal integrity in adults with childhood lead exposure: A diffusion tensor imaging study. NeuroToxicology, 30 (6), 867

- 875. http://dx.doi.org/10.1016/j.neuro.2009.07.007

Budtz

- Jorgensen, E., Keiding, N., & Grandjean, P. (2001). Benchmark dose calculation from epidemiological data. Biometrics,
57 (3), 698

- 706. http://dx.doi.org/10.1111/j.0006

- 341X.2001.00698.x

Bunn, T. L., Ladics, G. S., Holsapple, M. P., & Dietert, R. R. (2001). Developmental immunotoxicology assessment in the rat: Age, gender and strain comparisons after exposure to Pb. Toxicology Mechanisms and Methods, 11 (1), 41

- 58. http://dx.doi.org/10.1080/105172301300055151May 2011 5

- 379 DRAFT

– DO NOT CITE OR QUOTE Bunn, T. L., Marsh, J. A., & Dietert, R. R. (2000). Gender differences in developmental immunotoxicity to lead in a chicken: Analysis following a single early low

- level exposure in ovo. Journal of Toxicology and Environmental Health, Part A: Current Issues, 61 (8), 677

- 693. http://dx.doi.org/10.1080/00984100050195152

Bunn, T. L., Parsons, P. J., Kao, E., & Dietert, R. R. (2 001a). Exposure to lead during critical windows of embryonic development: Differential immunotoxic outcome based on stage of exposure and gender. Toxicological Sciences, 64 (1),
57

- 66. http://dx.doi.org /10.1093/toxsci/64.1.57

Bunn, T. L., Parsons, P. J., Kao, E., & Dietert, R. R. (2001b). Gender

- based profiles of developmental immunotoxicity to lead in the rat: Assessment in juveniles and adults. Journal of Toxicology and Environmental Health, Part A: Cu rrent Issues,
64 (3), 223

- 240. http://dx.doi.org/10.1080/15287390152543708

Burger, J., & Gochfeld, M. (2005). Effects of lead on learning in herring gulls: An avian wildlife model for neurobehavioral
deficits. NeuroToxicology, 26 (4), 615

- 624. http://dx.doi.org/10.1016/j.neuro.2005.01.005

Burns, J. M., Baghurst, P. A., Sawyer, M. G., McMichael, A. J., & Tong, S.

- L. (1999). Lifetime low

- level exposure to environmental lead and children's emotional and behavioral development at ages 11

- 13 years: The Port Pirie cohort study. American Journal of Epidemiology, 149 (8), 740

- 749. http://www.ncbi.nlm.nih.gov/pubmed/10206624

Bussolaro, D., Neto, F. F., Gargioni, R., Fernandes, L. C., Randi, M. A. F., Pelletier, E., & Oliveira Ribeiro, C. A. (2008). The immune response of peritoneal macrophages due to exposur e to inorganic lead in the house mouse Mus musculus. Toxicology In Vitro, 22 (1), 254

- 260. http://dx.doi.org/10.1016/j.tiv.2007.09.003

Cabb, E. E., Gorospe, E. C., Rothweiler, A. M., & Gerstenberger, S. L. (2008). Toxic remedy: A case of a 3

- year

- old child with lead colic treated with lead monoxide (Greta). Clinical Pediatrics, 47 (1), 77

- 79. http://dx.doi.org/10.1177/0009922807304385

Caffo, B., Chen, S., Stewart, W., Bolla, K., Yousem, D., Davatzikos, C., & Schwartz, B. S. (2008). Are brain volumes based on magnetic resonance imaging mediators of the associations of cumulative lead dose with cognitive function? American Journal of Epidemiology, 1 67 (4), 429

- 437. http://dx.doi.org/10.1093/aje/kwm326

Cai, M. Y., & Arenaz, P. (1998). Antimutagenic effect of crown ethers on heavy metal

- induced sister chromatid exchanges. Mutagenesis, 13 (1), 27

- 32. http://dx.doi.org/10.1093/mutage/13.1.27

Calderon

- Salinas, J. V., Quintanar

- Escorcia, M. A., Gonzalez

- Martinez, M. T., & Hernandez

- Luna, C. E. (1999). Lead and calcium transport in human erythrocyte. Hum an and Experimental Toxicology, 18 (5), 327

- 332. http://dx.doi.org/10.1191/096032799678840138

Calderon

- Salinas, J. V., Quintanar

- Escorza, M. A., Hernandez

- Luna, C. E., & Gonzalez

- Martinez, M. T. (19 99). Effect of lead on the calcium transport in human erythrocyte. Human and Experimental Toxicology, 18 (3), 146

- 153. http://dx.doi.org/10.1177/096032719901800303

Calvo, F. B., Santos, D., Rodrigue s, C. J., Krug, F. J., Marumo, J. T., Schor, N., & Bellini, M. H. (2009). Variation in the
distribution of trace elements in renal cell carcinoma. Biological Trace Element Research, 130 (2), 107

- 113. http://dx.doi.org/10.1007/s12011

- 009

- 8325

- x

Camoratto, A. M., White, L. M., Lau, Y.

- S., Ware, G. O., Berry, W. D., & Moriarty, C. M. (1993). Effect of exposure to low level lead on growth and growth hormone release in rats. Toxicology, 83 (1

- 3), 101

- 114. http://dx.doi.org/10.1016/0300

- 483X(93)90095

- A

Campbell, J. R., & Auinger, P. (2007). The association between blood lead levels and osteoporosis among adults: Results from
the Third National Healt h and Nutrition Examination Survey (NHANES III). Environmental Health Perspectives,
115 (7), 1018

- 1022. http://dx.doi.org/10.1289/ehp.9716May 2011 5

- 380 DRAFT

– DO NOT CITE OR QUOTE Camyre, E., Wise, S. S., Milligan, P., Gordon, N., Goodale, B., Stack pole, M., . . . Wise, J. P. (2007). Ku80 deficiency does not affect particulate chromate

- induced chromosome damage and cytotoxicity in chinese hamster ovary cells. Toxicological Sciences, 97 (2), 348

- 354. http://dx.doi.org/10.1093/toxsci/kfm045

Can, S., Bagci, C., Ozaslan, M., Bozkurt, A. I., Cengiz, B., Cakmak, E. A., . . . Tarakcioglu, M. (2008). Occupational lead exposure effect on liver functions and biochemical parameters. Acta Physiologica Hungarica , 95 (4), 395

- 403. http://dx.doi.org/10.1556/APhysiol.95.2008.4.6

Canfield, R. L., Henderson, C. R., Jr., Cory

- Slechta, D. A., Cox, C., Jusko, T. A., & Lanphear, B. P. (2003). Intellectual impairm ent in children with blood lead concentrations below 10 micrograms per deciliter. New England Journal of Medicine, 348 , 1517

- 1526. http://dx.doi.org/10.1056/NEJMoa022848

Canfield, R. L., Kreher, D. A., C ornwell, C., & Henderson, C. R., Jr. (2003). Low

- level lead exposure, executive functioning, and learning in early childhood. Neuropsychology, Development, and Cognition. Section C: Child Neuropsychology, 9 (1),
35

- 53. http://dx.doi.org/10.1076/chin.9.1.35.14496

Cantarow, A., & Trumper, M. (1944). Lead poisoning . Hagerstown, MD: Williams & Wilkins Co.

Carafoli, E. (2005). The Theodor Bucher Lecture

- Calcium

- a universal carrier of biological signals. Delivered on 3 July 2003 at the Special FEBS Meeting in Brussels. The F E B S Journal, 272 (5), 1073

- 1089. http://dx.doi.org/10.1111/j.1742

- 4658.2005.04546.x

Cardenas, A., Roels, H., Bernard, A. M., Barbon, R., Buchet, J. P., Lauwerys, R. R., . . . Price, R. G. (1993). Markers of ea rly renal changes induced by industrial pollutants: II Applica tion to workers exposed to lead. British Journal of Industrial Medicine, 50 (1), 28

- 36. http://dx.doi.org/10.1136/oem.50.1.28

Carey, J. B., Allshire, A., & Van Pelt, F. N. (2006). Immune modulation by cadm ium and lead in the acute reporter antigen

popliteal lymph node assay. Toxicological Sciences, 91 (1), 113

- 122. http://dx.doi.org/10.1093/toxsci/kfj142

Carmignani, M., Boscolo, P., Poma, A., & Volpe, A. R. (1999). Kininergic system and arterial hypertension following chronic exposure to inorganic lead. International Immunopharmacology, 44 (1

- 2), 105

- 110. http://dx.doi.org/10.1016/S0162

- 3109(99)0 0115

- 0

Carmignani, M., Volpe, A. R., Boscolo, P., Qiao, N., MDi, G., Grilli, A., & Felaco, M. (2000). Catcholamine and nitric oxide systems as targets of chronic lead exposure in inducing selective functional impairment. Life Sciences, 68 (4), 401

- 415. http://dx.doi.org/10.1016/S0024

- 3205(00)00954

- 1

Carreras, H. A., Wannaz, E. D., & Pignata, M. L. (2009). Assessment of human health risk related to metals by the use of biomonitors in the province o f Cordoba, Argentina. Environmental Pollution, 157 (1), 117

- 122. http://dx.doi.org/10.1016/j.envpol.2008.07.018

Casadoa, M. F., Cecchinia, A. L., Simãoa, A. N. C., Oliveiraa, R. D., & Cecchini, R. (2006). Free radical

- mediated pre

- hemolytic injury in human red blood cells subjected to lead acetate as evaluated by chemiluminescence. Food and Chemical Toxicology, 45 (6), 945

- 952. http://dx.doi. org/10.1016/j.fct.2006.12.001

Caylak, E., Aytekin, M., & Halifeoglu, I. (2008). Antioxidant effects of methionine, alpha

- lipoic acid, N

- acetylcysteine and homocysteine on lead

- induced oxidative stress to erythrocytes in rats. Experimental and Toxicologic P athology, 60 (4

- 5), 289

- 294. http://dx.doi.org/10.1016/j.etp.2007.11.004

CDC. (Centers for Disease Control and Prevention). (2000). Linked birth and infant death data set: 1998 birth cohort data. Hya ttsville, MD: Author.May 2011 5

- 381 DRAFT

– DO NOT CITE OR QUOTE Cecil, K. M., Brubaker, C. J., Adler, C. M., Dietrich, K. N., Altaye, M., Egelhoff, J. C., . . . Lanphear, B. P. (2008). Decr eased brain volume in adults with childhood lead exposure. PLoS Medicine, 5 (5), e112. http://dx.doi.org/10.1371/journal.pmed.0050112

Cecil, K. M., Yuan, W., Holland, S., Wessel, S., Dietrich, K., Ris, D., & Lanphear, B. (2005). The influence of childhood lead exposure on language function in young adul ts: An fMRI study . International Society for Magnetic Resonance Imaging 12th Scientific Meeting and Exhibition. Miami, FL.

Celik, A., Ogenler, O., & Comelekoglu, U. (2005). The evaluation of micronucleus frequency by acridine orange fluorescent staining i n peripheral blood of rats treated with lead acetate. Mutagenesis, 20 (6), 411

- 415. http://dx.doi.org/10.1093/mutage/gei055

Cerulli, N., Campanella, L., Grossi, R., Politi, L., Scandurra, R., Soda, G., . . . Caroli, S. (2006). Determination of Cd, C u, Pb and Zn in neoplastic kidneys and in renal tissue of fetuses, newborns and corpses. Journal of Trace Elements in Medicine
and Biology, 20 (3), 171

- 179. http://dx.doi.org/10.1016/j.jtemb.2006.03.002

Cezard, C., Demarquilly, C., Boniface, M., & Haguenor, J. M. (1992). Influence of the degree of exposure to lead on relations between alcohol consumption and the biological indices of lead exposure: epidemiological study in a lead acid battery factory. Occupational and Environmental Medicine, 49 (9), 645

- 647. http://dx.doi.org/ 10.1136/oem.49.9.645

Chai, S., & Webb, R. C. (1988). Effects of lead on vascular reactivity. Environmental Health Perspectives, 78 , 85

- 89. http://www.ncbi.nlm.nih.gov/pubmed/3060355

Chakraborty, I., Sharma, A., & Talukder, G. (1987). Antagonistic and synergistic effects of lead and selenium in Rattus norvegicus. Toxicology Letters, 37 (1), 21

- 26. http://dx.doi.org/10.1016/0378

- 4274(87)90162

- 7

Chandramouli, K., Steer, C. D., Ellis, M., & Emond, A. M. (2009). Effects of early childhood lead exposure on academic performance and behaviour of school age children. Archives of Disease in Childhood, 94 (11), 844

- 848. http://dx.doi.org/10.1136/adc.2008.149955

Chang, H.

- R., Chen, S.

- S., Tsao, D.

- A., Cheng, J.

- T., Ho, C.

- K., & Yu, H.

- S. (1997). Change of cardiac "beta"

- adrenoceptors in
lead

- exposed rats. Toxicology, 123 (1

- 2), 27

- 32. http://dx.doi.org/10.1016/S0300

- 483X(97)00100

- 5

Chang, H.

- R., Tsao, D.

- A., Yu, H.

- S., & Ho, C.

- K. (2005). The change of "beta"

- adrenergic system after cessation of lead

exposure. Toxicology, 207 (1), 73

- 80. http://dx.doi.org/10.1016/j.tox.2004.08.018

Chang, M. K., Raggatt, L. J., Alexander, K. A., Kuliwaba, J. S., Fazzalari, N. L., Schroder, K., . . . Pettit, A. R. (2008). Osteal tissue macrophages are inter calated throughout human and mouse bone lining tissues and regulate osteoblast function
in vitro and in vivo. Journal of Immunology, 181 (2), 1232

- 1244. http://www.ncbi.nlm.nih.gov/pubmed/18606677

Ch ang, S. H., Cheng, B. H., Lee, S. L., Chuang, H. Y., Yang, C. Y., Sung, F. C., & Wu, T. N. (2006). Low blood lead concentration in association with infertility in women. Environmental Research, 101 (3), 380

- 386. http://dx.doi.org/10.1016/j.envres.2005.10.004

Chang, W. C., Chang, C. C., Wang, Y. S., Weng, W. T., Yoshioka, T., & Juo, S. H. (2011). Involvement of the epidermal growth factor receptor in Pb(2+)

- induced activation of cPLA(2)/COX

- 2 genes and PGE(2) production in vascular smooth
muscle cells. Toxicology, 279 (1

- 3), 45

- 53. http://dx.doi.org/10.1016/j.tox.2010.09.004

Chao, S. L., Moss, J. M., & Harry, G. J. (2007). Lead

- induced alterations of apoptosis and neurotrophic factor mRNA in the developing rat cortex, hippocampus, and cerebellum. Journal of Biochemical and Molecular Toxicology, 21 (5), 265

- 272. http://dx.doi.org/10.1002/jbt.20191May 2011 5

- 382 DRAFT

– DO NOT CITE OR QUOTE Chaurasia, S. S., Gupta, P., Maiti, P. K., & Kar, A. (1998). Possible involvement of lipid peroxidation in the inhibition of type I iodothyronine 5'

- monodeiodinase activity by lead in chicken liver. Journal of App lied Toxicology, 18 (4), 299

- 300. http://www.ncbi.nlm.nih.gov/pubmed/9719431

Chaurasia, S. S., & Kar, A. (1997). Protective effects of vitamin E against lead

- induced deterioration of membrane associat ed type

- I iodothyronine 5'

- monodeiodinase (5'D

- I) activity in male mice. Toxicology, 124 (3), 203

- 209. http://dx.doi.org/10.1016/S0300

- 483X(97)00155

- 8

Chen, A., Cai, B., Dietrich, K. N., Radcliff e, J., & Rogan, W. J. (2007). Lead exposure, IQ, and behavior in urban 5

- to 7

- year

olds: Does lead affect behavior only by lowering IQ? Pediatrics, 119 (3), e650

- e658. http://dx.doi.org/10.1542/peds.20 06

- 1973

Chen, A., Dietrich, K. N., Ware, J. H., Radcliffe, J., & Rogan, W. J. (2005). IQ and blood lead from 2 to 7 years of age: Are the effects in older children the residual of high blood lead concentrations in 2

- year

- olds? Environmental Health Perspect ives, 113 (5), 597

- 601. http://dx.doi.org/10.1289/ehp.7625

Chen, B., Lamberts, L. V., Behets, G. J., Zhao, T. T., Zhou, M. X., Liu, G., . . . D'Haese, P. C. (2009). Selenium, lead, and
cadmium levels in renal failure patients in China. Biological Trace Element Research, 131 (1), 1

- 12. http://dx.doi.org/10.1007/s12011

- 009

- 8340

- y

Chen, H. I., Chiu, Y. W., Hsu, Y. K., Li, W. F., Chen, Y. C., & Chuang, H. Y. (2010). The association of metallothionein

- 4 gene polymorphism and renal function in long

- term lead

- exposed workers. Biological Trace Element Research, 137 , 55

- 62. http://dx.doi.org/10.1007/s12011

- 009

- 8564

- x

Chen, S., Golemboski, K. A., Piepenbrink, M., & Dietert, R. R. (2004). Developmental immunotoxicity of lead in the rat:
Influence of maternal diet. Journal of Toxicology and Environmental Health, Part A: Current Issues, 67 (6), 495

- 511. http://dx.doi.org/10.1080/15287390490276520

Chen, S., Golemboski, K. A., Sanders, F. S., & Dietert, R. R. (1999). Persistent effect of in utero meso

- 2,3

- dimercaptosuccinic acid (DMSA) on immune function and lead

- induced immunotoxicity. Toxicology, 132 (1), 67

- 79. http://dx.doi.org/10.1016/S0300

- 483X(98)00139

- 5

Chen, X.

- K., Yang, Q., Smith, G., Krewski, D., Walker, M., & Wen, S. W. (2006). Environmen tal lead level and pregnancy

induced hypertension. Environmental Research, 100 (3), 424

- 430. http://dx.doi.org/10.1016/j.envres.2005.07.006

Chen, Y., Zhao, J. X., Liu, J. W., Cui, J., Li, L., & Tian, W. (2008). Lack of association of delta

- aminolevulinic acid dehydratase genotype with blood lead levels in environmentally exposed children of Uygur and Han populations. Acta Paediatrica,
97 (12), 1717

- 1720. http://dx.doi.org/10.1111/j.1651

- 2227.2008.01003.x

Cheng, Y.

- J., Yang, B.

- C., Hsieh, W.

- C., Huang, B.

- M., & Liu, M.

- Y. (2002). Enhancement of TNF

- alpha expression does not trigger apoptosis upon exposure of glial cells to lead and li popolysaccharide. Toxicology, 178 (3), 183

- 191. http://dx.doi.org/10.1016/S0300

- 483X(02)00225

- 1

Cheng, Y.

- J., Yang, B. C., & Liu, M. Y. (2006). Lead increases lipopolysaccharide

- induced liver inj ury through tumor necrosis
factor

- alpha overexpression by monocytes/macrophages: Role of protein kinase C and p42/44 mitogen

- activated protein kinase. Environmental Health Perspectives, 114 (4), 507

- 513. http ://dx.doi.org/10.1289/ehp.8550

Cheng, Y., Schwartz, J., Sparrow, D., Aro, A., Weiss, S. T., & Hu, H. (2001). Bone lead and blood lead levels in relation to baseline blood pressure and the prospective development of hypertension: The Normative Aging Study. American Journal of Epidemiology, 153 (2), 164

- 171. http://dx.doi.org/10.1093/aje/153.2.164

Cheng, Y., Schwartz, J., Vokonas, P. S., Weiss, S. T., Aro, A., & Hu, H. (1998). Electrocardiographic conductio n disturbances in
association with low

- level lead exposure (the Normative Aging Study). The American Journal of Cardiology, 82 (5),
594

- 599. http://dx.doi.org/10.1016/S0002

- 9149(98)00402

- 0May 2011 5

- 383 DRAFT

– DO NOT CITE OR QUOTE Chetty , C. S., Reddy, G. R., Murthy, K. S., Johnson, J., Sajwan, K., & Desaiah, D. (2001). Perinatal lead exposure alters the expression of neuronal nitric oxide synthase in rat brain. International Journal of Toxicology, 20 (3), 113

- 120. http://dx.doi.org/10.1080/109158101317097692

Chetty, C. S., Vemuri, M. C., Campbell, K., & Suresh, C. (2005). Lead

- induced cell death of human neuroblastoma cells involves GSH deprivation. Cellular and Molecular Biology L etters, 10 (3), 413

- 423. http://www.ncbi.nlm.nih.gov/pubmed/16217553

Chia, S.

- E., Zhou, H., Tham, M. T., Yap, E., Dong, N. V., Tu, N. H., & Chia, K. S. (2005). Possible influence of "delta"

- aminolevulinic acid dehydratase polymorphism and susceptibility to renal toxicity of lead: A study of a Vietnamese population. Environmental Health Perspectives, 113 , 1313

- 1317. http://dx.doi.org/10.1289/ehp.7904

Chia, S.

- E., Zhou, H. J., Theng, T. M., & Yap, E. (2007). Possibilities of newer ALAD polymorphism influencing human susceptibility to effects of inorganic lead on the neurobehavioral functions. Neur oToxicology, 28 (2), 312

- 317. http://dx.doi.org/10.1016/j.neuro.2006.04.003

Chia, S.

- E., Zhou, H. J., Yap, E., Tham, M. T., Dong, N.

- V., Hong Tu, N. T., & Chia, K.

- S. (2006). Association of renal f unction
and delta

- aminolevulinic acid dehydratase polymorphism among Vietnamese and Singapore workers exposed to inorganic lead. Occupational and Environmental Medicine, 63 (3), 180

- 186. http://dx.doi. org/10.1136/oem.2005.021154

Chiodo, L. M., Jacobson, S. W., & Jacobson, J. L. (2004). Neurodevelopmental effects of postnatal lead exposure at very low
levels. Neurotoxicology and Teratology, 26 , 359

- 371.

Cho, S. C., Kim, B. N., Hong, Y. C., Shin, M. S., Yoo, H. J., Kim, J. W., . . . Kim, H. W. (2010). Effect of environmental ex posure to lead and tobacco smoke on inattentive and hyperactive symptoms and neurocognitive performance in children. Journal of Chi ld Psychology and Psychiatry, 51 (9), 1050

- 1057. http://www.ncbi.nlm.nih.gov/pubmed/20406335

Chobanian, A. V., Bakris, G. L., Black, H. R., Cushman, W. C., Green, L. A., Izzo, J. L., Jr., . . . Rocce lla, E. J. (2003). Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension, 42 , 1206

- 1252. http://dx.doi.org/ 10.1161/01.HYP.0000107251.49515.c2

Choie, D. D., & Richter, G. W. (1972). Lead poisoning: Rapid formation of intranuclear inclusions. Science, 177 (4055), 1194

- 1195. http://dx.doi.org/10.1126/sci ence.177.4055.1194

Choie, D. D., Richter, G. W., & Young, L. B. (1975). Biogenesis of intranuclear lead

- protein inclusions in mouse kidney. Pathology

- Research and Practice, 155 (2), 197

- 203. http://w ww.ncbi.nlm.nih.gov/pubmed/168862

Chuang, H.

- Y., Kuo, C.

- H., Chiu, Y.

- W., Ho, C.

- K., Chen, C.

- J., & Wu, T.

- N. (2007). A case

- control study on the relationship of hearing function and blood concentrations of lead, manganese, arsenic, and selenium. Science o f the Total Environment,
387 (1

- 3), 79

- 85. http://dx.doi.org/10.1016/j.scitotenv.2007.07.032

Chuang, H.

- Y., Schwartz, J., Gonzales

- Cossio, T., Lugo, M. C., Palazuelos, E., Aro, A., . . . Hernan dez

- Avila, M. (2001). Interrelations of lead levels in bone, venous blood, and umbilical cord blood with exogenous lead exposure through maternal plasma lead in peripartum women. Environmental Health Perspectives, 109 , 527

- 532. http://dx.doi.org/10.1289/ehp.01109527

Church, H. J., Day, J. P., Braithwaite, R. A., & Brown, S. S. (1993a). Binding of lead to a metallothionein

- like protein in human erythrocytes. Journal of Inorganic Biochemistry, 49 (1), 55

- 68. http://dx.doi.org/10.1016/0162

- 0134(93)80048

- E

Church, H. J., Day, J. P., Braithwaite, R. A., & Brown, S. S. (1993b). The speciation of lead in erythrocytes in relation to lead toxicity: Case st udies of two lead

- exposed workers. NeuroToxicology, 14 (2

- 3), 359

- 364. http://www.ncbi.nlm.nih.gov/pubmed/8247409May 2011 5

- 384 DRAFT

– DO NOT CITE OR QUOTE Ciubar, R., Ciofrangeanu, C., Mitran, V., Cimpean, A., Stanescu, A., & Iordachescu, D. (2007). The effect of lead ions on human erythrocytes in vitro. Revista de Chimie, 58 (9), 895

- 897.

Cline, H. T., Witte, S., & Jones, K. W. (1996). Low lead levels stunt neuronal growth in a reversible manner. Proceedings of the National Academy of Scienc es, 93 (18), 9915

- 9920. http://www.ncbi.nlm.nih.gov/pubmed/8790431

Coban, T. A., Senturk, M., Ciftci, M., & Kufrevioglu, O. I. (2007). Effects of some metal ions on human erythrocyte glutathio ne reduc tase: an in vitro study. Protein and peptide letters, 14 (10), 1027

- 1030. http://dx.doi.org/10.2174/092986607782541060

Cocco, P., Fadda, D., Atzeri, S., Avataneo, G., Meloni, M., & Flore, C. (2007). Causes of death among lead smelters in relation to the glucose

- 6

- phosphate dehydrogenase polymorphism. Occupational and Environmental Medicine, 64 (6), 414

- 416. http://dx.doi.org/10. 1136/oem.2006.028779

Cockcroft, D. W., & Gault, M. H. (1976). Prediction of creatinine clearance from serum creatinine. Nephron, 16 (1), 31

- 41. http://www.ncbi.nlm.nih.gov/pubmed/1244564

Cohen, B. S., Li, W., Xiong, J. Q., & Lippmann, M. (2000). Detecting H+ in ultrafine ambient aerosol using iron nano

- film detectors and scanning probe microscopy. Applied Occupational and Environmental Hygiene, 15 (1), 80

- 89. http://dx.doi.org/10.1080/104732200301881

Cohen, N., Modai, D., Golik, A., Weissgarten, J., Peller, S., Katz, A., . . . Shaked, U. (1989). Increased concanavalin A

- induced suppressor cell activity in humans with occupational lead exposure. Envi ronmental Research, 48 (1), 1

- 6. http://dx.doi.org/10.1016/S0013

- 9351(89)80079

- 9

Columbano, A., Endoh, T., Denda, A., Noguchi, O., Nakae, D., Hasegawa, K., . . . Konishi, Y. (1996). Effects of ce ll proliferation and cell death (apoptosis and necrosis) on the early stages of rat hepatocarcinogenesis. Carcinogenesis, 17 (3), 395

- 400. http://dx.doi.org/10.1093/carcin/17.3.395

Columbano, A., Ledda

- Columbano, G. M., Coni, P., & Pani, P. (1987). Failure of mitogen

- induced cell proliferation to achieve initiation of rat liver carcinogenesis. Carcinogenesis, 8 (2), 345

- 347. http://dx.doi.org/10.1093 /carcin/8.2.345

Columbano, A., Ledda

- Columbano, G. M., Ennas, M. G., Curto, M., Chelo, A., & Pani, P. (1990). Cell proliferation and promotion of rat liver carcinogenesis: Different effect of hepatic regeneration and mitogen induced hyperplasia on the
deve lopment of enzyme

- altered foci. Carcinogenesis, 11 (5), 771

- 776. http://dx.doi.org/10.1093/carcin/11.5.771

Comper, W. D., & Russo, L. M. (2009). The glomerular filter: An imperfect barrier is required for perfect renal function. Current Opinion in Nephrology and Hypertension, 18 (4), 336

- 342. http://dx.doi.org/10.1097/Mnh.0b013e32832cb96a

Coni, P., Pichiri

- Coni, G., Ledda

- Columbano, G. M., Semp le, E., Rajalakshmi, S., Rao, P. M., . . . Columbano, A. (1992). Stimulation of DNA synthesis by rat plasma following in vivo treatment with three liver mitogens. Cancer Letters,
61 (3), 233

- 238. http://dx.doi.org/10.1016/0304

- 3835(92)90293

- 5

Conterato, G. M. M., Augusti, P. R., Somacal, S., Einsfeld, L., Sobieski, R., Torres, J. R. V., & Emanuelli, T. (2007). Effec t of lead acetate on cytosolic thioredoxin reductase activity and oxidative stress p arameters in rat kidneys. Basic and Clinical Pharmacology and Toxicology, 101 (2), 96

- 100. http://dx.doi.org/10.1111/j.1742

- 7843.2007.00084.x

Coon, S., Stark, A., Peterson, E., Gloi, A., Kortsha, G., Pounds, J., . . . Gorell, J. (2006). Whole

- body lifetime occupational lead exposure and risk of Parkinson's disease. Environmental Health Perspectives, 114 (12), 1872

- 1876. http://dx.doi.org/10.1289/ehp.9102

Cooper, G. P., & Manalis, R. S. (1984). Interactions of lead and cadmium on acetylcholine release at the frog neuromuscular junction. Toxicology and Applied Pharmacology, 74 (3), 411

- 416. http://dx.doi.org/10.1016/0041

- 008X(84)90294

- 1May 2011 5

- 385 DRAFT

– DO NOT CITE OR QUOTE Coratelli, P., Giannattasio, M., Lomonte, C., Marzolla, R., Rana, F., & L'Abbate, N. (1988). Enzymuria to detect tubular injury in workers exposed to lead: A 12

- month follow

- up. In C. Bianchi, V. Bocci, F. A. Carone & R. Rabkin (Eds.), Kidney and proteins in health and disease: Fifth international symposium in health and dise ase; July 1987; Montecatini Terme, Italy (pp. 207

- 211). Basel, Switzerland

Montecatini Terme, Italy: S. Karger.

Coria, C., Cabello, A., Tassara, E., Lopez, E., Rosales, H., Perez, M., . . . Kirsten, L. (2009). Long term consequences amon g children exposed to lead poisoning. Revista Medica de Chile, 137 (8), 1037

- 1044. http://www.ncbi.nlm.nih.gov/pubmed/19915767

Coria, F., Berciano, M. T., Berciano, J., & Lafarga, M. (1984). Axon membrane remodeling in the lead

- induced demyelinating neuropathy of the rat. Brain Research, 291 (2), 369

- 372. http://dx.doi.org/10.1016/0006

- 8993(84)91271

- x

Corongiu, F. P., & Milia, A. (1982). Rise of hepatic glutath ione concentration induced in rats by chronic lead nitrate treatment. Its
role in aflatoxin B1 intoxication. Research Communications in Chemical Pathology and Pharmacology, 38 (1), 97

- 112. http://www. ncbi.nlm.nih.gov/pubmed/6815742

Cory

- Slechta, D. A., Pokora, M. J., & Widzowski, D. V. (1992). Postnatal lead exposure induces supersensitivity to the stimulus properties of D2D3 agonist. Brain Research, 598 (1

- 2), 162

- 172. http://dx.doi.org/10.1016/0006

- 8993(92)90180

- H

Cory

- Slechta, D. A., Stern, S., Weston, D., Allen, J. L., & Liu, S. (2010). Enhanced learning deficits in female rats following lifetime pb exposure combined with prenatal stress. T oxicological Sciences, 117 (2), 427

- 438. http://dx.doi.org/10.1093/toxsci/kfq221

Cory

- Slechta, D. A., Virgolini, M. B., Rossi

- George, A., Thiruchelvam, M., Lisek, R., & Weston, D. (2008). Lifetime

conseq uences of combined maternal lead and stress. Basic and Clinical Pharmacology and Toxicology, 102 (2), 218

- 227. http://dx.doi.org/10.1111/j.1742

- 7843.2007.00189.x

Cory

- Slechta, D. A., Virgolini , M. B., Rossi

- George, A., Weston, D., & Thiruchelvam, M. (2009). Experimental manipulations
blunt time

- induced changes in brain monoamine levels and completely reverse stress, but not Pb +/

- stress

- related modifications to these trajectories. Behavioural Brain Research, 205 (1), 76

- 87. http://dx.doi.org/10.1016/j.bbr.2009.06.040

Cory

- Slechta, D. A., Virgolini, M. B., Thiruchelvam, M., Weston, D. D., & Bauter, M. R. (2004). Maternal stress modulates t he effects of developmental lead exposure. Environmental Health Perspectives, 112 , 717

- 730. http://dx.doi.org/10.1289/ehp.6481

Costa, L. G., & Fox, D. A. (1983). A selective decrease of cholinergic muscarinic receptors in the visual cortex of adult rats following developmental lead exposure. Brain Research, 276 , 259

- 266. http://dx.doi. org/10.1016/0006

- 8993(83)90733

- 3

Costa, M., Zhitkovich, A., Gargas, M., Paustenbach, D., Finley, B., Kuykendall, J., . . . Bogdanffy, M. (1996). Interlaborato ry validation of a new assay for DNA

- protein crosslinks. Mutation Research: Genetic Toxicology, 36 9 (1

- 2), 13

- 21. http://dx.doi.org/10.1016/S0165

- 1218(96)90043

- 9

Counter, S. A., Buchanan, L. H., Ortega, F., Rifai, N., & Shannon, M. W. (2007). Comparative analysis of zinc protoporphyrin
and bl ood lead levels in lead

- exposed Andean children. Clinical Biochemistry, 40 (11), 787

- 792. http://dx.doi.org/10.1016/j.clinbiochem.2007.03.003

Courtois, E., Marques, M., Barrientos, A., Casado , S., & Lopez

- Farre, A. (2003). Lead

- induced downregulation of soluble guanylage cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxygenase

- 2. Journal of the American Society of Nephrology: JASN, 14 (6), 1464

- 1470. http://dx.doi.org/10.1097/01.ASN.0000064947.1499May 2011 5

- 386 DRAFT

– DO NOT CITE OR QUOTE Coyle, J. T., & Tsai, G. (2004). The NMDA receptor glycine modulatory site: A therapeutic target for improving cognition and reducing negative symptom s in schizophrenia. Psychopharmacology, 174 (1), 32

- 38. http://dx.doi.org/10.1007/s00213

- 003

- 1709

- 2

Croes, K., Baeyens, W., Bruckers, L., Den Hond, E., Koppen, G., Nelen, V., . . . Van Larebeke, N. ( 2009). Hormone levels and sexual development in Flemish adolescents residing in areas differing in pollution pressure. International Journal of Hygiene and Environmental Health, 212 (6), 612

- 625. h ttp://dx.doi.org/10.1016/j.ijheh.2009.05.002

Crofton, K. M., Taylor, D. H., Bull, R. J., Sivulka, D. J., & Lutkenhoff, S. D. (1980). Developmental delays in exploration a nd locomotor activity in male rats exposed to low level lead. Life Sciences, 26 (10), 8 23

- 831. http://dx.doi.org/10.1016/0024

- 3205(80)90289

- 1

Crooks, D. R., Ghosh, M. C., Haller, R. G., Tong, W. H., & Rouault, T. A. (2010). Posttranslational stability of the heme biosynthetic enzym e ferrochelatase is dependent on iron availability and intact iron

- sulfur cluster assembly machinery. Blood, 115 (4), 860

- 869. http://dx.doi.org/10.1182/blood

- 2009

- 09

- 243105

Daggett, D. A., Oberle y, T. D., Nelson, S. A., Wright, L. S., Kornguth, S. E., & Siegel, F. L. (1998). Effects of lead on rat kidney and liver: GST expression and oxidative stress. Toxicology, 128 (3), 191

- 206. http:/ /dx.doi.org/10.1016/S0300

- 483X(98)00080

- 8

Danadevi, K., Rozati, R., Saleha Banu, B., Hanumanth, R. P., & Grover, P. (2003). DNA damage in workers exposed to lead using comet assay. Toxicology, 187 (2

- 3), 183

- 193. http://dx.doi.org/10.1016/S0300

- 483X(03)00054

- 4

Davenport, A., Murcutt, G., & Whiting, S. (2009). Cross

- sectional audit of blood lead levels in regular outpatient haemodialysis patients dialysing in north London. Nephrology, 14 (5), 476

- 481. http://dx.doi.org/10.1111/j.1440

- 1797.2009.01087.x

David, O. J., Clark, J., & Voeller, K. (1972). Lead and hyperactivity. Lancet, 300 (7783), 900

- 903. http://dx.doi.org/10.1016/S0140

- 6736(72)92534

- 2

Davidson, R., & MacKinnon, J. G. (1981). Several tests for model specification in the presence of alternative hypotheses. Econometrica, 49 (3), 781

- 793.

Davis, J. M. (1982). Ethological approaches to behavioral toxicology. In C. L. Mitchell (Ed.), Nervous system toxicology (pp. 29

- 44). New York, NY: Raven Press.

De, M., Ghosh, S., Palit, S., Ghosh, A., & Talukder. (1995). Clastogenic effects in human samples following prolong ed exposure in metal industry. Bulletin of Environmental Contamination and Toxicology, 54 , 357

- 362. http://dx.doi.org/10.1007/BF00195105

de Restrepo, H. G., Sicard, D., & Torres, M. M. (2000). DNA damage and repair in cells of lead exposed people. American Journal of Industrial Medicine, 38 (3), 330

- 334.

De Burbure, C., Buchet, J. P., Leroyer, A., Nisse, C., Haguenoer, J. M., Mutti, A., . . . Bernard, A. (2006). Renal and neurologic effects of cadmium, lead, mercury, and arsenic in children: Evidence of early effects and multiple interactions at environmental exposure levels. Environmental Health Perspectives, 114 (4), 584

- 590. http://dx.doi.org/10.1289/ehp.8202

De Coster, S., Koppen, G., Bracke, M., Schroijen, C., Den Hond, E., Nelen, V., . . . van Larebeke, N. (2008). Pollutant effects on genotoxic parameters and tumor

- associated protein levels in adults: A cross sectional st udy. Environmental Health: A Global Access Science Source, 7 , 26. http://dx.doi.org/10.1186/1476

- 069X

- 7

- 26

De Keyser, J., Mostert, J. P., & Koch, M. W. (2008). Dysfunctional astrocytes as key players i n the pathogenesis of central nervous system disorders. Journal of the Neurological Sciences, 267 (1

- 2), 3

- 16. http://dx.doi.org/10.1016/j.jns.2007.08.044May 2011 5

- 387 DRAFT

– DO NOT CITE OR QUOTE De Palma, G., Goldoni, M., Catalani, S., Car bognani, P., Poli, D., Mozzoni, P., . . . Apostoli, P. (2008). Metallic elements in pulmonary biopsies from lung cancer and control subjects. L' Ateneo Parmense. Sezione I: Acta Bio

- medica, 79 (Suppl 1), 43

- 51. http://www.ncbi.nlm.nih.gov/pubmed/18924309

de Souza Lisboa, S. F., Goncalves, G., Komatsu, F., Queiroz, C. A. S., Almeida, A. A., & Moreira, E. G. (2005). Developmental lead exposure induces depressive

- like behavior in female rats. Drug and Ch emical Toxicology, 28 (1), 67

- 77. http://dx.doi.org/10.1081/dct

- 200039696

Dearth, R. K., Hiney, J. K., Srivastava, V., Burdick, S. B., Bratton, G. R., & Dees, W. L. (2002). Effects of lead (Pb) expos ure during gestation and lactation on female pubertal development in the rat. Reproductive Toxicology, 16 (4), 343

- 352. http://dx.doi.org/10.1016/S0890

- 6238(02)00037

- 0

Degawa, M., Arai, H., Miura, S., & Hashimoto, Y. (1993). Preferential inhibitions of hepatic P450IA2 expression and induction by lead nitrate in the rat. Carcinogenesis, 14 (6), 1091

- 1094. http://dx.d oi.org/10.1093/carcin/14.6.1091

Dehpour, A. R., Essalat, M., Ala, S., Ghazi

- Khansari, M., & Ghafourifar, P. (1999). Increase by NO synthase inhibitor of lead

induced release of N

- acetyl

- "beta"

- D

- glucosaminidase from perfused rat kidney. Toxicology, 132 (2

- 3 ), 119

- 125. http://dx.doi.org/10.1016/S0300

- 483X(98)00143

- 7

Deng, H., Callender, R., Schramm, V. L., & Grubmeyer, C. (2010). Pyrophosphate activation in hypoxanthine

-- guanine phosphoribosyltrans ferase with transition state analogue. Biochemistry, 49 (12), 2705

- 2714. http://dx.doi.org/10.1021/bi100012u

Deng, W., & Poretz, R. D. (2002). Protein kinase C activation is required for the lead

- induced inh ibition of proliferation and differentiation of cultured oligodendroglial progenitor cells. Brain Research, 929 (1), 87

- 95. http://dx.doi.org/10.1016/S0006

- 8993(01)03385

- 6

Denham, M., Schell, L. M., Deane, G., Gallo, M. V., Ravenscroft, J., & DeCaprio, A. P. (2005). Relationship of lead, mercury, mirex, dichlorodiphenyldichloroethylene, hexachlorobenzene, and polychlorinated biphenyls to timing of menarche among Akwesasne Mohawk girls. Pediatrics, 115 (2), e127

- e134. http://dx.doi.org/10.1542/peds.2004

- 1161

Dentener, M. A., Greve, J. W., Maessen, J. G., & Buurman, W. A. (1989). Role of tumour necrosis factor in the enhanced sensitivity of mice t o endotoxin after exposure to lead. Immunopharmacology and Immunotoxicology, 11 (2

- 3), 321

- 334. http://dx.doi.org/10.3109/08923978909005373

Devarajan, P. (2007). Emerging biomarkers of acute kidney i njury. Contributions to Nephrology, 156 , 203

- 212. http://dx.doi.org/10.1159/000102085

Deveci, E. (2006). Ultrastructural effects of lead acetate on brain of rats. Toxicology and Industrial Health, 22 (10), 4 19

- 422. http://dx.doi.org/10.1177/0748233706074171

Devi, C. B., Reddy, G. H., Prasanthi, R. P. J., Chetty, C. S., & Reddy, G. R. (2005). Developmental lead exposure alters
mitochondrial monoamine oxi dase and synaptosomal catecholamine levels in rat brain. International Journal of Developmental Neuroscience, 23 (4), 375

- 381. http://dx.doi.org/10.1016/j.ijdevneu.2004.11.003

Devi, K. D., Banu, B. S., Grover, P., & Jamil, K. (2000). Genotoxic effect of lead nitrate on mice using SCGE (Comet assay). Toxicology, 145 (2

- 3), 195

- 201. http://dx.doi.org/10.1016/S0300

- 483X(00)00154

- 2

Dhir, H., Ghosh, S., Sharma, A., & Talukder, G. (1992). Interaction between 2 group IV metals

- lead and zirconium

- in bone
marrow cells of mus

- musculus in vivo. Biology of Metals, 5 (2), 81

- 86. http://dx.doi. org/10.1007/BF01062218

Dhir, H., Roy, A. K., & Sharma, A. (1993). Relative efficiency of Phyllanthus emblica fruit extract and ascorbic acid in modifying lead and aluminium

- induced sister

- chromatid exchanges in mouse bone marrow. Environmental and
Molecula r Mutagenesis, 21 (3), 229

- 236. http://dx.doi.org/10.1002/em.2850210305May 2011 5

- 388 DRAFT

– DO NOT CITE OR QUOTE Dhir, H., Roy, A. K., Sharma, A., & Talukder, G. (1990). Modification of clastogenicity of lead and aluminium in mouse bone

marrow cells by dietary ingestion of Phyllanthus emblica fruit extract. Mutation Research: Genetic Toxicology, 241 (3),
305

- 312. http://dx.doi.org/10.1016/0165

- 1218(90)90029

- 2

Dhir, H., Sharma, A., & Tal ukder, G. (1992). Modifying effect of iron on lead

- induced clastogenicity in mouse bone marrow
cells. Biological Trace Element Research, 34 , 279

- 286.

Di Lorenzo, L., Silvestroni, A., Martino, M. G., Gagliardi, T., Corfiati, M., & Soleo, L. (2006). Evaluat ion of peripheral blood neutrophil leucocytes in lead

- exposed workers. International Archives of Occupational and Environmental Health,
79 (6), 491

- 498. http://dx.doi.org/10.1007/s00420

- 005

- 0073

- 4

Di Lorenzo, L., Vacca, A., Corfiati, M., Lovreglio, P., & Soleo, L. (2007). Evaluation of tumor necrosis factor

- alpha and granulocyte colony stimulating factor serum levels in lead

- exposed smoker workers. International Journal of Immunopathology and Pharmaco logy, 20 (2), 239

- 247. http://www.ncbi.nlm.nih.gov/pubmed/17624258

Dietert, R. R. (2011). Role of developmental immunotoxicity and immune dysfunction in chronic disease and cancer. Reproductive Toxic ology, 31 (3), 319

- 326. http://dx.doi.org/10.1016/j.reprotox.2010.09.006

Dietert, R. R., & McCabe, M., Jr. (2007). Lead immunotoxicity Immunotoxicology and Immunopharmacology (pp. 207

- 223).
Boca Raton, FL: CRC Press.

Dietert, R. R., & Piepenbrink, M. S. (2006). Perinatal immunotoxicity: Why adult exposure assessment fails to predict risk. Environmental Health Perspectives, 114 (4), 477

- 483. http://d x.doi.org/10.1289/ehp.8566

Dietrich, K. N., Berger, O. G., & Succop, P. A. (1993). Lead exposure and the motor developmental status of urban six

- year

- old children in the Cincinnati prospective study. Pediatrics, 91 (2), 301

- 307. http://www.ncbi.nlm.nih.gov/pubmed/7678702

Dietrich, K. N., Berger, O. G., Succop, P. A., Hammond, P. B., & Bornschein, R. L. (1993). The developmental consequences of low to moderate prenatal and postnatal lead exposure: Intellectual attainment in the Cincinnati Lead Study Cohort following school entry. Neurotoxicology and Teratology, 15 (1), 37

- 44. http://dx.doi.org/10.1016/0892

- 0362(93)90043

- N

Dietrich, K. N., Krafft, K. M., Bier, M., Succop, P. A., Berger, O., & Bornschein, R. L. (1986). Early effects of fetal lead exposure: Neurobehavioral findings at 6 months. International Journal of Biosocial and Medical Research, 8 (2), 151

- 168.

Dietrich, K. N., Krafft, K. M., Bornschein, R. L., Hammond, P. B., Berger, O., S uccop, P. A., & Bier, M. (1987). Low

- level fetal lead exposure effect on neurobehavioral development in early infancy. Pediatrics, 80 (5), 721

- 730. http://www.ncbi.nlm.nih.gov/pubmed/2444921

Dietrich, K. N., Ris, M. D., Succop, P. A., Berger, O. G., & Bornschein, R. L. (2001). Early exposure to lead and juvenile delinquency. Neurotoxicology and Teratology, 23 (6), 511

- 518. http://dx.doi.org/1 0.1016/S0892

- 0362(01)00184

- 2

Dietrich, K. N., Succop, P. A., Berger, O. G., Hammond, P. B., & Bornschein, R. L. (1991). Lead exposure and the cognitive development of urban preschool children: The Cincinnati lead study cohort at age 4 years. Neurotoxicolog y and Teratology, 13 (2), 203

- 211. http://dx.doi.org/10.1016/0892

- 0362(91)90012

- L

Dietrich, K. N., Succop, P. A., Berger, O. G., & Keith, R. W. (1992). Lead exposure and the central auditory proce ssing abilities and cognitive development of urban children: The Cincinnati lead study cohort at age 5 years. Neurotoxicology and Teratology, 14 (1), 51

- 56. http://dx.doi.org/10.1016/0892

- 0362(92) 90028

- 9

Ding, Y., Gonick, H. C., & Vaziri, N. D. (2000). Lead promotes hydroxyl radical generation and lipid peroxidation in cultured
aortic endothelial cells. American Journal of Hypertension, 13 (5), 552

- 555. http://dx.doi.org/10.1016/S0895

- 7061(99)00226

- 5May 2011 5

- 389 DRAFT

– DO NOT CITE OR QUOTE Ding, Y., Gonick, H. C., Vaziri, N. D., Liang, K., & Wei, L. (2001). Lead

- induced hypertension. III. Increased hydroxyl radical production. American Journal of Hypertension, 14 , 169

- 173. http://dx.doi.org/10.1016/S0895

- 7061(00)01248

- 6

Diouf, A., Garcon, G., Diop, Y., Ndiaye, B., Thiaw, C., Fall, M., . . . Shirali, P. (2006). Environmental lead exposure and i ts relationship to traffic density among Senegalese children: A cross

- sectional study. Human and Experimental Toxicology, 25 (11), 637

- 644. http://dx.doi.org/10.1177/0960327106074591

Dogru, M. K., Dogru, A. K., Gul, M., Esrefo glu, M., Yurekli, M., Erdogan, S., & Ates, B. (2008). The effect of adrenomedullin on rats exposed to lead. Journal of Applied Toxicology, 28 (2), 140

- 146. http://dx.doi.org/10.1002/jat.1259

Dogu, O., Louis, E. D., Tamer, L., Unal, O., Yilmaz, A., & Kaleagasi, H. (2007). Elevated blood lead concentrations in essential
tremor: A case

- control study in Mersin, Turkey. Environmental Health Perspectives, 115 (11), 1564

- 1568. http://dx.doi.org/10.1289/ehp.10352

Dolinoy, D. C., & Jirtle, R. L. (2008). Environmental epigenomics in human health and disease. Environmental and Molecular Mutagenesis, 49 (1), 4

- 8. http://dx.doi.org/10.1002/em.20366

Donaldson, W. E., & Knowles, S. O. (1993). Is lead toxicosis a reflection of altered fatty acid composition of membranes? Comparative Biochemistry and Physiology

- Part C: Toxicology and Pharmacology, 104 (3), 377

- 379. http://dx.doi.org/10.1016/0742

- 8413(93)90003

- 4

Dong, S. Y., Liang, D. P., An, N., Jia, L., Shan, Y. J., Chen, C., . . . Fu, S. B. (2009). The role of MAPK and FAS death rec eptor pathways in testicul ar germ cell apoptosis induced by lead. Acta Biochimica et Biophysica Sinica, 41 (9), 800

- 807. http://dx.doi.org/10.1093/abbs/gmp069

Dorman, R. V., & Freeman, E. J. (2002). Lead

- dependent effects on arachi donic acid accumulation and the proliferation of
vascular smooth muscle. Journal of Biochemical and Molecular Toxicology, 16 (5), 245

- 253. http://dx.doi.org/10.1002/jbt.10045

Dorsey, C. D., Lee, B. K., Bolla , K. I., Weaver, V. M., Lee, S. S., Lee, G. S., . . . Schwartz, B. S. (2006). Comparison of patella
lead with blood lead and tibia lead and their associations with neurobehavioral test scores. Journal of Occupational and Environmental Medicine, 48 (5), 489

- 496. http://dx.doi.org/10.1097/01.jom.0000199678.86629.3b

Dowd, T. L., & Gupta, R. K. (1991). 19F

- NMR study of the effect of lead on intracellular free calcium in human platelets.
Biochimic a et Biophysica Acta, 1092 , 341

- 346. http://dx.doi.org/10.1016/S0167

- 4889(97)90010

- 0

Dowd, T. L., Rosen, J. F., Gundberg, C. M., & Gupta, R. K. (1994). The displacement of calcium from osteocalc in at
submicromolar concentrations of free lead. Biochimica et Biophysica Acta, 1226 (2), 131

- 137. http://dx.doi.org/10.1016/0925

- 4439(94)90020

- 5

Dowd, T. L., Rosen, J. F., & Gupta, R. K. (1990). 31P NMR and saturation transfer studies of the effect of PB2+ on cultured
osteoblastic bone cells. Journal of Biological Chemistry, 265 , 20833

- 20838.

Dowd, T. L., Rosen, J. F., Mints, L., & Gundberg, C. M. (2001). The effect of Pb2+ on the structure and h ydroxyapatite binding properties of osteocalcin. Biochimica et Biophysica Acta, 1535 (2), 153

- 163. http://dx.doi.org/10.1016/S0925

- 4439(00)00094

- 6

Dumitrescu, E., Alexandra, T., & Snejana, P. (20 08). Lead acetate impact on some markers of female reproductive system performances (litter size, sex ratio) and physical development (vaginal opening) in rats. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj

- Napoca

- Veterinar y Medicine, 65 (2), 283

- 287.

Dumitrescu, E., Trif, A., & Argherie, D. (2007). Consequences of chronic exposure of female rats to lead acetate on ovarian a nd
uterine histoarhitectonic. Lucrari Stiintifice, Universitatea de Stiinte Agronomice si Medicina Vet erinara Bucuresti, Seria C: Medicina Veterinara, LII , 198

- 205.May 2011 5

- 390 DRAFT

– DO NOT CITE OR QUOTE Dumitrescu, E., Trif, A., & Florin, M. (2008). The consequences of female rats chronic exposure to lead acetate in the biomar kers emphasizing the hormonal disrupting potential of the reproduct ive function for in vivo evaluation. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj

- Napoca

- Veterinary Medicine, 65 (1), 382

- 387.

Dundar, B., Oktem, F., Arslan, M. K., Delibas, N., Baykal, B., Arslan, C., . . . Ilhan, I. E. ( 2006). The effect of long

- term low

dose lead exposure on thyroid function in adolescents. Environmental Research, 101 (1), 140

- 145. http://dx.doi.org/10.1016/j.envres.2005.10.002

Dursun, N., Arifoglu, C., Suer, C., & Keskinol, L. (2005). Blood pressure relationship to nitric oxide, lipid peroxidation, r enal function, and renal blood flow in rats exposed to low lead levels. Biological Trace Element Research, 104 (2), 141

- 150. http://dx.doi.org/10.1385/BTER:104:2:141

DuVal, G., & Fowler, B. A. (1989). Preliminary purification and characterization studies of a low molecular weight, high affi nity cytosolic lead

- binding protein in rat brain. Biochemical and Biophysical Research Communications, 159 (1), 177

- 184. http://dx.doi.org/10.1016/0006

- 291X(89)92420

- 0

Duydu, Y., Dur, A., & Suzen, H. S. (2005). Evaluation of increased pro portion of cells with unusually high sister chromatid exchange counts as a cytogenetic biomarker for lead exposure. Biological Trace Element Research, 104 (2), 121

- 129. http://dx.doi.org/10.1385/BTER:10 4:2:121

Duydu, Y., Suzen, H. S., Aydin, A., Cander, O., Uysal, H., Isimer, A., & Vural, N. (2001). Correlation between lead exposure indicators and sister chromatid exchange (SCE) frequencies in lymphocytes from inorganic lead exposed workers. Archives of Environmental Contamination and Toxicology, 41 (2), 241

- 246. http://dx.doi.org/10.1007/s002440010244

Dyatlov, V. A., & Lawrence, D. A. (2002). Neonatal lead exposure potentiates sickness behavior induced by Listeria monocytogenes infection of mice. Brain, Behavior, and Immunity, 16 (4), 477

- 492. http://dx.doi.org/10.1006/brbi.2001.0641

Dyatlov, V. A., Platoshin, A. V., Lawrence, D. A., & Carpenter, D. O. (1998). Lead potentiates cytokine

- and glutamate

- mediated increases in permeability of the blood

- brain barrier. NeuroToxicology, 19 (2), 283

- 292. http://www.ncbi.nlm.nih.gov/pubmed/9553965

Eder, K. , Reichlmayr

- Lais, A. M., & Kirchgessner, M. (1990). Activity of Na

- K

- ATPase and Ca

- Mg

- ATPase in red blood cell
membranes of lead

- depleted rats. Journal of Trace Elements in Medicine and Biology, 4 (1), 21

- 24. http://www.ncbi.nlm.nih.gov/pubmed/1967007

Egle, P. M., & Shelton, K. R. (1986). Chronic lead intoxication causes a brain

- specific nuclear protein to accumulate in the nuclei of cells lining kidney tubules. Journal of Biological Chemistry, 261 (5) , 2294

- 2298. http://www.ncbi.nlm.nih.gov/pubmed/3003107

Ehrlich, R., Robins, T., Jordaan, E., Miller, S., Mbuli, S., Selby, P., . . . Landrigan, P. (1998). Lead absorption and renal dysfunction in a South African battery factory. Occupational and Environmental Medicine, 55 , 453

- 460. http://dx.doi.org/10.1136/oem.55.7.453

Ekinci, D., Beydemir, S., & Küfrevioğlu, O. I. (2007). In vitro inhibitory effe cts of some heavy metals on human erythrocyte carbonic anhydrases. Journal of Enzyme Inhibition and Medicinal Chemistry, 22 (6), 745

- 750. http://dx.doi.org/10.1080/14756360601176048

El

- Ashmawy, I. M. , Ashry, K. M., El

- Nahas, A. F., & Salama, O. M. (2006). Protection by turmeric and myrrh against liver oxidative damage and genotoxicity induced by lead acetate in mice. Basic and Clinical Pharmacology and Toxicology,
98 (1), 32

- 37. http://dx.doi.org/10.1111/j.1742

- 7843.2006.pto_228.xMay 2011 5

- 391 DRAFT

– DO NOT CITE OR QUOTE El

- Fawal, H. A. N., Waterman, S. J., De Feo, A., & Shamy, M. Y. (1999). Neuroimmunotoxicology: Humoral assessment of neurotoxicity and aut oimmune mechanisms. Environmental Health Perspectives, 107 (Suppl 5), 767

- 775. http://www.ncbi.nlm.nih.gov/pubmed/10502543

El

- Nekeety, A. A., El

- Kady, A. A., Soliman, M. S., Hassan, N. S., & Abdel

- Wa hhab, M. A. (2009). Protective effect of Aquilegia vulgaris (L.) against lead acetate

- induced oxidative stress in rats. Food and Chemical Toxicology, 47 (9), 2209

- 2215. http://dx.doi.org/10.1016/j.fc t.2009.06.019

El

- Neweshy, M. S., & El

- Sayed, Y. S. (2011). Influence of vitamin C supplementation on lead

- induced histopathological
alterations in male rats. Experimental and Toxicologic Pathology, 63 (3), 221

- 227. http://dx.doi.org/10.1016/j.etp.2009.12.003

El

- Sokkary, G. H., Abdel

- Rahman, G. H., & Kamel, E. S. (2005). Melatonin protects against lead

- induced hepatic and renal toxicity in male rats. Toxicology, 213 (1

- 2), 25

- 33. http://dx.doi.org/10.1016/j.tox.2005.05.003

Elinder, C. G., Friberg, L., Lind, B., Nilsson, B., Svartengren, M., & Övermark, I. (1986). Decreased blood lead levels in residents of Stockholm for the period 1980

- 1984. Scand inavian Journal of Work, Environment and Health, 12 (2), 114

- 120. http://www.ncbi.nlm.nih.gov/pubmed/3726492

Elmarsafawy, S. F., Jain, N. B., Schwartz, J., Sparrow, D., Nie, H. L., & Hu, H. (2006). Di etary calcium as a potential modifier of the relationship of lead burden to blood pressure. Epidemiology, 17 (5), 531

- 537. http://dx.doi.org/10.1097/01.ede.0000231285.86968.2b

Emmerson, B. T . (1965). The renal excretion of urate in chronic lead nephropathy. Internal Medicine Journal, 14 (4), 295

- 303. http://www.ncbi.nlm.nih.gov/pubmed/5861252

Emmerson, B. T., & Ravenscroft, P. J. (1975). Abnormal renal urate homeostasis in systemic disorders. Nephron, 14 (1), 62

- 80. http://dx.doi.org/10.1159/000180436

Ercal, N., Treeratphan, P., Hammond, T. C., Matthews, R. H., Grannemann, N. H., & Spitz, D . R. (1996). In vivo indices of
oxidative stress in lead

- exposed C57BL/6 mice are reduced by treatment with meso

- 2,3

- dimercaptosuccinic acid or N

acetylcysteine. Free Radical Biology and Medicine, 21 (2), 157

- 161. http://dx.doi.org/10.1016/0891

- 5849(96)00020

- 2

Ergurhan

- Ilhan, I., Cadir, B., Koyuncu

- Arslan, M., Gultepe, F. M., & Ozkan, G. (2008). Level of oxidative stress and damage in erythrocytes in apprentices indirectly exposed to lead. Pediatri cs International, 50 (1), 45

- 50. http://dx.doi.org/10.1111/j.1442

- 200X.2007.02442.x

Ernhart, C. B., Wolf, A. W., Kennard, M. J., Erhard, P., Filipovich, H. F., & Sokol, R. J. (1986). Intrauterine exposure to low
levels of lead: the status of the neonate. Archives of Environmental and Occupational Health, 41 , 287

- 291.

Evans, M., Fored, C. M., Nise, G., Bellocco, R., Nyren, O., & Elinder, C. G. (2010). Occupational lead exposure and severe CKD: A population

- based case

- control and prospective observational cohort study in Sweden. American Journal of Kidney Diseases, 55 (3), 497

- 506. http://dx.doi.org/10.1053/j .ajkd.2009.11.012

Ewers, U., Stiller

- Winkler, R., & Idel, H. (1982). Serum immunoglobulin, complement C3, and salivary IgA levels in lead
workers. Environmental Research, 29 (2), 351

- 357. http://d x.doi.org/10.1016/0013

- 9351(82)90036

- 6

Factor

- Litvak, P., Graziano, J. H., Kline, J. K., Popovac, D., Mehmeti, A., Ahmedi, G., . . . Stein, Z. A. (1991). A prospective study of birthweight and length of gestation in a population surrounding a lead smelter in Kosovo, Yugoslavia. International Journal of Epidemiology, 20 (3), 722

- 728. http://dx.doi.org/10.1093/ije/20.3.722

Factor

- Litvak, P., Wasserman, G., Kline, J. K., & Graziano, J. (1999). The Yugoslavia prospective study of environmental lead

exposure. Environmental Health Perspectives, 107 , 9

- 15. http://dx.doi.org/10.1289/ehp.991079May 2011 5

- 392 DRAFT

– DO NOT CITE OR QUOTE Fadrowski, J. J., Navas

- Acien, A., Tellez

- Plaza, M., Guallar, E., Weaver , V. M., & Furth, S. L. (2010). Blood lead level and kidney function in US adolescents: The Third National Health and Nutrition Examination Survey. Archives of Internal Medicine, 170 (1), 75

- 82. http://dx.doi.org/10.1001/archinternmed.2009.417

Fahmy, M. A. (1999). Lead acetate genotoxicity in mice. Cytologia, 64 , 357

- 365.

Faikoglu, R., Savan, K., Utku, C., Takar, N., & Zebitay, A. G. (2006). Significance of maternal plasma lead level in early

pr egnancy loss. Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 41 (3), 501

- 506. http://dx.doi.org/10.1080/10934520500428435

Faith, R. E., Luster, M. I., & Kimmel, C. A. (1979). Effect of chronic developmental lead exposure on cell

- mediated immune
functions. Clinical and Experimental Immunology, 35 (3), 413

- 420. http://www.ncbi.nlm.nih.g ov/pubmed/455779

Fan, G., Feng, C., Li, Y., Wang, C. H., Yan, J., Li, W., . . . Bi, Y. Y. (2009). Selection of nutrients for prevention or ame lioration of
lead

- induced learning and memory impairment in rats. Annals of Occupational Hygiene, 53 (4), 341

- 351. http://dx.doi.org/10.1093/annhyg/mep019

Fan, G., Feng, C., Wu, F., Ye, W., Lin, F., Wang, C., . . . Bi, Y. (2010). Methionine choline reverses lead

- induced cognitive and N

methyl

- d

- aspartate receptor su bunit 1 deficits. Toxicology, 272 (1

- 3), 23

- 31. http://dx.doi.org/10.1016/j.tox.2010.03.018

Fan, Y., Yamada, T., Shimizu, T., Nanashima, N., Akita, M., Suto, K., & Tsuchida, S. (2009). Ferritin expression in rat hepatocytes and Kupffer cells after lead nitrate treatment. Toxicologic Pathology, 37 (2), 209

- 217. http://dx.doi.org/10.1177/0192623308328544

Fang, F. , Kwee, L. C., Allen, K. D., Umbach, D. M., Ye, W., Watson, M., . . . Kamel, F. (2010). Association between blood lead and the risk of amyotrophic lateral sclerosis. American Journal of Epidemiology, 171 (10), 1126

- 1133. http://dx.doi.org/10.1093/aje/kwq06

Farant, J.

- P., & Wigfield, D. C. (1987). Interaction of divalent metal ions with normal and lead

- inhibited human erythrocytic porphobilinogen synthase in vitro. Toxicology and Applied Pharmacology, 89 (1), 9

- 18. http://dx.doi.org/10.1016/0041

- 008X(87)90171

- 2

Farant, J.

- P., & Wigfield, D. C. (1990). The effects of copper, zinc, mercury, and cadmium on rabbit erythrocytic porphobilinogen synthase in v ivo. Journal of Analytical Toxicology, 14 (4), 222

- 226. http://www.ncbi.nlm.nih.gov/pubmed/2395343

Farmand, F., Ehdale, A., Roberts, C. K., & Sindhu, R. K. (2005). Lead

- induced dysregulation of supero xide dismutases, catalase, glutathione peroxidase, and guanylate cyclase. Environmental Research, 98 (1), 33

- 39. http://dx.doi.org/10.1016/j.envres.2004.05.016

Farrer, D. G., Hueber, S., Laiosa, M . D., Eckles, K. G., & McCabe, M. J., Jr. (2008). Reduction of myeloid suppressor cell derived nitric oxide provides a mechanistic basis of lead enhancement of alloreactive CD4(+) T cell proliferation. Toxicology and Applied Pharmacology, 229 (2), 135

- 145. http://dx.doi.org/10.1016/j.taap.2007.12.011

Farrer, D. G., Hueber, S. M., & McCabe, M. J. J. R. (2005). Lead enhances CD4+ T cell proliferation indirectly by targeting antigen presenting cells and modulating antigen

- specific interactions. Toxicology and Applied Pharmacology, 207 (2),
125

- 137. http://dx.doi.org/10.1016/j.taap.2004.12.017

Favalli, L., Chiari, M. C., Piccinini, F., & Rozza, A. (1977). Experimental investigations on the contraction induced by lead in
arterial smooth muscle. Acta Pharmacologica et Toxicologica, 41 (Suppl 2), 412

- 420. http://dx.doi.org/10.1111/j.1600

- 0773.1977.tb03362.x

Fazli

- Tabaei, S., Fahim, M., & Khoshbaten, A. (2006). Acute lead exposure and contraction of rat isolated aorta induced by D1

- dopaminergic and alpha

- adrenergic drugs. Archives of Iranian Medicine, 9 (2), 119

- 123.May 2011 5

- 393 DRAFT

– DO NOT CITE OR QUOTE Fehlau, R., Grygorczyk , R., Fuhrmann, G. F., & Schwarz, W. (1989). Modulation of the Ca2+

- or Pb2+

- activated K+

- selective channels in human red cells. II. Parallelisms to modulation of the activity of a membrane

- bound oxidoreductase. Biochimica et Biophysica Acta, 978 (1), 37

- 42

. http://dx.doi.org/10.1016/0005

- 2736(89)90495

- 1

Feinberg, A. P. (2007). Phenotypic plasticity and the epigenetics of human disease. Nature, 447 (7143), 433

- 440. http://dx.doi.org/10.1038/nature05919

Fels, L. M., Wunsch, M., Baranowski, J., Norska

- Borowka, I., Price, R. G., Taylor, S. A., . . . Stolte, H. (1998). Adverse effects of chronic low level lead exposure on kidney function: A risk group study in children. Nephrology, Dialysis, Transplantation, 13 (9), 2248

- 2256. http://dx.doi.org/10.1093/ndt/13.9.2248

Ferguson, C., Kern, M., & Audesirk, G. (2000). Nanomolar concentrations of inorganic lead increase Ca2+ efflux and decrease
intracellular free Ca2+ ion concentrations in cultured rat hippocampal neurons by a calmodulin

- dependent mechanism. NeuroToxicology, 21 (3), 365

- 378. http://www.ncbi.nlm.nih.gov/pubmed/10894126

Ferguson, M. A., Vaidya, V. S., & Bonventre, J. V. (2008). Biomarkers of nephrotoxic acute kidney injury. Toxicology, 245 (3),
182

- 193. http://dx.doi.org/10.1016/j.tox.2007.12.024

Fergusson, D. M., Horwood, L. J., & Lynskey , M. T. (1993). Early dentine lead levels and subsequent cognitive and behavioural development. Journal of Child Psychology and Psychiatry, 34 (2), 215

- 227. http://dx.doi.org/10.1111/j.1469

- 7610.1993.tb00980.x

Finkelstein, Y., Markowitz, M. E., & Rosen, J. F. (1998). Low

- level lead

- induced neurotoxicity in children: An update on central nervous system effects. Brain Research, 27 (2), 168

- 176. http://dx.doi.org/10.1016/S0165

- 0173(98)00011

- 3

Fischbein, A., Tsang, P., J

- CJ, L., Roboz, J. P., Jiang, J. D., & Bekesi, J. G. (1993). Phenotypic aberrations of the CD3+ and CD4+ cells and functional impairments of lymphocytes at low

- level occu pational exposure to lead. 66 (2), 163

- 168. http://dx.doi.org/10.1006/clin.1993.1020

Flegal, A. R., & Smith, D. R. (1992). Lead levels in preindustrial humans. New England Journal of Medicine, 326 (19), 1293

- 1294.

Flohe, S. B., Bruggemann, J., Herder, C., Goebel, C., & Kolb, H. (2002). Enhanced proinflammatory response to endotoxin after priming of macrophages with lead ions. Journal of the Lepidopterists Society, 71 (3), 417

- 424. http://www.ncbi.nlm.nih.gov/pubmed/11867679

Flora, S. J. S., Saxena, G., Gautam, P., Kaur, P., & Gill, K. D. (2007). Response of lead

- induced oxidative stress and alterations in biogenic amines in different rat brain regio ns to combined administration of DMSA and MiADMSA. Chemico

Biological Interactions, 170 (3), 209

- 220. http://dx.doi.org/10.1016/j.cbi.2007.08.003

Flora, S. J. S., Singh, S., & Tandon, S. K. (1989). T hiamine and zinc in prevention or therapy of lead intoxication. Journal of International Medical Research, 17 (1), 68

- 75. http://www.ncbi.nlm.nih.gov/pubmed/2707476

Foglieni, C., Fulgenzi, A., Ticozzi , P., Pellegatta, F., Sciorati, C., Belloni, D., . . . Ferrero, M. E. (2006). Protective effect of EDTA preadministration on renal ischemia. BMC Nephrology, 7 , 5. http://dx.doi.org/10.1186/1471

- 2369

- 7

- 5

Föller, M., Huber, S. M., & Lang, F. (2008). Erythrocyte programmed cell death. IUBMB Life, 60 (10), 661

- 668. http://dx.doi.org/10.1002/iub.106

Fonte, R., Agosti, A., Scafa, F., & Candura, S. M. (2007). Anaem ia and abdominal pain due to occupational lead poisoning. Haematologica, 92 (2), e13

- e14. http://dx.doi.org/10.3324/haematol.10951

Forst, L. S., Freels, S., & Persky, V. (1997). Occupational lead exposu re and hearing loss. Journal of Occupational and Environmental Medicine, 39 (7), 658

- 660. http://www.ncbi.nlm.nih.gov/pubmed/9253727May 2011 5

- 394 DRAFT

– DO NOT CITE OR QUOTE Fortier, M., Omara, F., Bernier, J., Brousseau, P., & Fournier, M. (2008). Effects of physiological concentrations of heavy metals both individually and in mixtures on the viability and function of peripheral blood human leukocytes in vitro. Journal of Toxicology and Environmental Health, Part A: Current Iss ues, 71 (19), 1327

- 1337. http://dx.doi.org/10.1080/15287390802240918

Fortoul, T. I., Saldivar, L., Espejel

- Mayab, G., Bazarro, P., Mussali

- Galante, P., Avila

- Casado, M. D., . . . Avila

- Costa, M. R. ( 2005). Inhalation of cadmium, lead or its mixture effects on the bronchiolar structure and its relation with metal tissue
concentrations. Environmental Toxicology and Pharmacology, 19 (2), 329

- 334. http://dx.doi.org/10.1016/j.etap.2004.08.007

Fortune, T., & Lurie, D. I. (2009). Chronic low

- level lead exposure affects the monoaminergic system in the mouse superior olivary complex. Journal of Comparative Neurology, 513 (5), 542

- 558. http://dx.doi.org/10.1002/cne.21978

Fowler, B. A., & DuVal, G. (1991). Effects of lead on the kidney: Roles of high

- affinity lead

- binding proteins. Environmental Health Perspectives, 91 (1), 77

- 80. http://dx.doi.org/10.1289/ehp.919177

Fowler, B. A., Kahng, M. W., Smith, D. R., Conner, E. A., & Laughlin, N. K. (1993). Implications of lead binding proteins for risk assessment of lead exposure. Journal of Exposure Science and Envir onmental Epidemiology, 3 (4), 441

- 448. http://www.ncbi.nlm.nih.gov/pubmed/8173344

Fowler, B. A., Kimmel, C. A., Woods, J. S., McConnell, E. E., & Grant, L. D. (1980). Chronic low

- level lead toxicity i n the rat: III An integrated assessment of long

- term toxicity with special reference to the kidney. Toxicology and Applied Pharmacology, 56 (1), 59

- 77. http://dx.doi.org/10.1016/0041

- 008X(80)90131

- 3

Fox, D. A., Campbell, M. L., & Blocker, Y. S. (1997). Functional alterations and apoptotic cell death in the retina following developmental or adult lead exposure. NeuroToxicology, 18 (3), 645

- 664. http://www.ncbi.nlm.nih.gov/pubmed/9339814

Fox, D. A., & Chu, L. W.

- F. (1988). Rods are selectively altered by lead: II. Ultrastructure and quantitative histology. Experimental Eye Research, 46 (4), 613

- 625. http://dx.doi.org/10.1016/S0014

- 4835(88)80017

- 4

Fox, D. A., & Farber, D. B. (1988). Rods are selectively altered by lead: I. Electrophysiology and biochemistry. Experimental Eye Research, 46 (4), 5 97

- 611. http://dx.doi.org/10.1016/S0014

- 4835(88)80016

- 2

Fox, D. A., Kala, S. V., Hamilton, W. R., Johnson, J. E., & O'Callaghan, J. A. (2008). Low

- level human equivalent gestational lead exposur e produces supernormal scotopic electroretinograms, increased retinal neurogenesis, and decreased retinal dopamine utilization in rats. Environmental Health Perspectives, 116 (5), 618

- 625. http://dx.doi.org/ 10.1289/ehp.11268

Fox, D. A., Katz, L. M., & Farber, D. B. (1991). Low level developmental lead exposure decreases the sensitivity, amplitude a nd temporal resolution of rods. NeuroToxicology, 12 (4), 641

- 654. http://www.ncbi.nlm.nih.gov/pubmed/1665551

Fox, D. A., Opanashuk, L., Zharkovsky, A., & Weiss, B. (2010). Gene

- chemical interactions in the developing mammalian nervous system: Effects on proliferation, neurogenesis and differentiation. NeuroToxic ology, 31 (5), 589

- 597. http://dx.doi.org/10.1016/j.neuro.2010.03.007

Fox, D. A., & Sillman, A. J. (1979). Heavy metals affect rod, but not cone, photoreceptors. Science, 206 (4414), 78

- 80. http://dx.doi.org/10.1126/science.314667

Fracasso, M. E., Perbellini, L., Solda, S., Talamini, G., & Franceschetti, P. (2002). Lead induced DNA strand breaks in lymphocytes of exposed workers: Role of reactive oxygen species and protein kinase C. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 515 (1

- 2), 159

- 169. http://dx.doi.org/10.1016/S1383

- 5718(02)00012

- 8May 2011 5

- 395 DRAFT

– DO NOT CITE OR QUOTE Francis, D. D., Dior io, J., Plotsky, P. M., & Meaney, M. J. (2002). Environmental enrichment reverses the effects of maternal separation on stress reactivity. Journal of Neuroscience, 22 (18), 7840

- 7843. http://www.ncbi .nlm.nih.gov/pubmed/12223535

Fried, L. F. (2009). Creatinine and cystatin C: What are the values? Kidney International, 75 (6), 578

- 580. http://dx.doi.org/10.1038/Ki.2008.688

Froehlich, T. E., Lanphear, B. P., Auinger, P., Hornung, R., Epstein, J. N., Braun, J., & Kahn, R. S. (2009). Association of tobacco and lead exposures with attention

- deficit/hyperactivity disorder. Pediatrics, 124 (6), E1054

- E1063. http://dx.doi.org/10.1542/peds.2009

- 0738

Froehlich, T. E., Lanphear, B. P., Dietrich, K. N., Cory

- Slechta, D. A., Wang, N., & Kahn, R. S. (2007). Interactive effects of a DRD4 polymorphism, lead and sex on executive functions in children. Biological Psychiatry, 62 , 243

- 249. http://dx.doi.org/10.1016/j.biopsych.2006.09.039

Fujita, H., Orii, Y., & Sano, S. (1981). Evidence of increased synthesis of "delta"

- aminolevulinic acid dehydratase in experimental lead

- poisoned rats. Biochimica et Biophysica Acta, 678 (1), 39

- 50. http://dx.doi.org/10.1016/0304

- 4165(81)90045

- 3

Fujita, H., Sato, K., & Sano, S. (1982). Increase in the amount of er ythrocyte delta

- aminolevulinic acid dehydratase in workers with moderate lead exposure. International Archives of Occupational and Environmental Health, 50 (3), 287

- 297. http://dx.doi.org/10.1007/BF00378090

Fujiwara, Y., Kaji, T., Sakurai, S., Sakamoto, M., & Kozuka, H. (1997). Inhibitory effect of lead on the repair of wounded monolayers of cultured vascular endothelial cells. Toxicology, 117 (2

- 3), 193

- 198. http://dx.doi.org/10.1016/S0300

- 483X(96)03575

- 5

Fukumoto, K., Karai, I., & Horiguchi, S. (1983). Effect of lead on erythrocyte membranes. Occupational and Environmental Medicine, 40 , 220

- 223. http://dx.doi.org/10.1136/oem.40.2.220

Fuller, G. M., & Zhang, Z. (2001). Transcriptional control mechanism of fibrinogen gene expression. Annals of the New York Academy of Sciences, 936 , 469

- 479. http://dx.doi.org/10.1111/j.1749

- 6632.2001.tb03534.x

Gao, D., Kasten

- Jolly, J., & Lawrence, D. A. (2006). The paradoxical effects of lead in interferon

- gamma knockout BALB/c
mice. Toxicological Sciences, 89 (2), 444

- 453. http://dx.doi.org/10.1093/toxsci/kfj043

Gao, D., & Lawrence, D. A. (2010). Dendritic cells in immunotoxicity testing. Methods in Molecular Biology, 598 , 259

- 281. http:/ /dx.doi.org/10.1007/978

- 1

- 60761

- 401

- 2_19

Gao, D., Mondal, T. K., & Lawrence, D. A. (2007). Lead effects on development and function of bone marrow

- derived dendritic cells promote Th2 immune responses. Toxicology and Applied Pharmacology, 222 (1), 69

- 79. http://dx.doi.org/10.1016/j.taap.2007.04.001

Gao, S. J., Jin, Y. L., Unverzagt, F. W., Ma, F., Hall, K. S., Murrell, J. R., . . . Hendrie, H. C. (2008). Trace element lev els and cognitive function in rural elderly Chinese. Journals of Gerontology. Series A: Biological Sciences and Medical Sciences, 63 (6), 635

- 641. http://www.ncbi.nlm.nih.gov/pubmed/18559640

Garavan, H., Morgan, R. E., Levitsky, D. A., Hermer

- Vazquez, L., & Strupp, B. J. (2000). Enduring effects of early lead exposure: Evidence for a specific deficit in associative ability. Neurotoxicology and Teratology, 22 , 151

- 164.

Garcon, G., Leleu, B., Marez, T., Zerimech, F., Jean

- Marie, H. D., Daniel, F. B., & Shirali, P. (2007). Biomonitoring of the adverse effects induced by the chronic exposure to lead and cadmium on kidney function: Usefulness of alpha

glutathione S

- transfera se. Science of the Total Environment, 377 (2

- 3), 165

- 172. http://dx.doi.org/10.1016/j.scitotenv.2007.02.002May 2011 5

- 396 DRAFT

– DO NOT CITE OR QUOTE Gargioni, R., Neto, F. F., Buchi, D. F., Randi, M. A. F., Franco, C. R. C., Paludo, K . S., . . . Ribeiro, C. A. O. (2006). Cell death and DNA damage in peritoneal macrophages of mice (Mus musculus) exposed to inorganic lead. Cell Biology International, 30 (7), 615

- 623. http://dx.d oi.org/10.1016/j.cellbi.2006.03.010

Garza, A., Vega, R., & Soto, E. (2006). Cellular mechanisms of lead neurotoxicity. Medical Science Monitor, 12 (3), RA57

- RA65. http://www.ncbi.nlm.nih.gov/pubmed/1 6501435

Gastaldo, J., Viau, M., Bencokova, Z., Joubert, A., Charvet, A. M., Balosso, J., & Foray, N. (2007). Lead contamination resul ts in late and slowly repairable DNA double

- strand breaks and impacts upon the ATM

- dependent signaling pathways. Toxicology Letters, 173 (3), 201

- 214. http://dx.doi.org/10.1016/j.toxlet.2007.08.003

Gedeon, Y., Ramesh, G. T., Wellman, P. J., & Jadhav, A. L. (2001). Changes in mesocorticolimbic dopamine and D1/D2 recept or levels after low level lead exposure: A time course study. Toxicology Letters, 123 (2

- 3), 217

- 226. http://dx.doi.org/10.1016/S0378

- 4274(01)00408

- 8

Gent, J. F., Koutrakis, P., Belanger, K., Tri che, E., Holford, T. R., Bracken, M. B., & Leaderer, B. P. (2009). Symptoms and
medication use in children with asthma and traffic

- related sources of fine particle pollution. Environmental Health Perspectives, 117 (7), 1168

- 1174. http://dx.doi.org/10.1289/ehp.0800335

Ghering, A. B., Jenkins, L. M. M., Schenck, B. L., Deo, S., Mayer, R. A., Pikaart, M. J., . . . Godwin, H. A. (2005). Spectro scopic
and functional determination of the interaction of Pb2+ with GATA proteins. Journal of the American Chemical Society, 127 (11), 3751

- 3759. http://dx.doi.org/10.1021/ja0464544

Ghosh

- Narang, J., Jones, T. M., Menke, A., Todd, A. C., Muntner, P., & Batuman, V. (2007). Pa rathyroid hormone status does not influence blood and bone lead levels in dialysis patients. American Journal of the Medical Sciences, 334 (6), 415

- 420. http://dx.doi.org/10.1097/MAJ.0b013e318068b 237

Giddabasappa, A., Hamilton, W. R., Chaney, S., Xiao, W., Johnson, J. E., Mukherjee, S., & Fox, D. A. (2011). Low

- level gestational lead exposure increases retinal progenitor cell proliferation and rod photoreceptor and bipolar cell neurogenesis in mice

. Environmental Health Perspectives, 119 (1), 71

- 77. http://dx.doi.org/10.1289/ehp.1002524

Gilbert, M. E., Kelly, M. E., Samsam, T. E., & Goodman, J. H. (2005). Chronic developmental lead exposure reduces neurogenesis in adult rat hippocampus but does not impair spatial learning. Toxicological Sciences, 86 , 365

- 374.

Gill, S. K., O'Brien, L., & Koren, G. (2009). The safety of histamine 2 (H2) blockers in pregnancy: A meta

- analysis. Digestive
Diseases and Sc iences, 54 (9), 1835

- 1838. http://dx.doi.org/10.1007/s10620

- 008

- 0587

- 1

Giridhar, J., & Isom, G. E. (1990). Interaction of lead acetate with atrial natriuretic factor in rats. Life Sciences, 46 (8), 569

- 576. http://dx.doi.org/10.1016/0024

- 3205(90)90124

- A

Gittleman, R., & Eskenazi, B. (1983). Lead and hyperactivity revisited: An investigation of non

- disadvantaged children. Archives of Ge neral Psychiatry, 40 (8), 827

- 833. http://dx.doi.org/10.1001/archpsyc.1983.01790070017002

Glahn, F., Schmidt

- Heck, W., Zellmer, S., Guthke, R., Wiese, J., Golka, K., . . . Foth, H. (2008). Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors. Archives of Toxi cology,
82 (8), 513

- 524. http://dx.doi.org/10.1007/s00204

- 008

- 0331

- 9

Glass, T. A., Bandeen

- Roche, K., McAtee, M., Bolla, K., Todd, A. C., & Schwartz, B. S. (2009). Neighborhood psychosocial
hazards a nd the association of cumulative lead dose with cognitive function in older adults. American Journal of Epidemiology, 169 (6), 683

- 692. http://dx.doi.org/10.1093/aje/kwn390May 2011 5

- 397 DRAFT

– DO NOT CITE OR QUOTE Glenn, B. S., Bandeen

- Roche, K., Lee, B. K., Weaver, V. M., Todd, A. C., & Schwartz, B. S. (2006). Changes in systolic blood pressure associated with lead in blood and bone. Epidemiology, 17 (5), 538

- 544. http://dx.doi.org/ 10.1097/01.ede.0000231284.19078.4b

Goering, P. L., & Fowler, B. A. (1984). Regulation of lead inhibition of delta

- aminolevulinic acid dehydratase by a low molecular weight, high affinity renal lead

- binding protein. Journal of Pharmacology and Experimental Therapeutics, 231 (1), 66

- 71. http://www.ncbi.nlm.nih.gov/pubmed/6092606

Goering, P. L., & Fowler, B. A. (1985). Mechanism of renal lead

- binding protein reversal of delta

- aminolevulinic acid dehydrata se inhibition by lead. Journal of Pharmacology and Experimental Therapeutics, 234 (2), 365

- 371. http://www.ncbi.nlm.nih.gov/pubmed/4020676

Goering, P. L., & Fowler, B. A. (1987a). Kidney zinc

- thionein regulation of delta

- aminolevulinic acid dehydratase inhibition by
lead. Archives of Biochemistry and Biophysics, 253 (1), 48

- 55. http://dx.doi.org/10.1016/0003

- 9861(87)90635

- 7

Goering, P. L., & F owler, B. A. (1987b). Regulatory roles of high

- affinity metal

- binding proteins in mediating lead effects on
delta

- aminolevulinic acid dehydratase. Annals of the New York Academy of Sciences, 514 , 235

- 247. http://dx.doi.org/10.1111/j.1749

- 6632.1987.tb48778.x

Goering, P. L., Mistry, P., & Fowler, B. A. (1986). A low molecular weight lead

- binding protein in brain attenuates lead inhibition
of delta

- aminolevulinic acid dehydratase: Comparison wi th a renal lead

- binding protein. Journal of Pharmacology and Experimental Therapeutics, 237 , 220

- 225. http://www.ncbi.nlm.nih.gov/pubmed/3958965

Golabek, T., Darewicz, B., Borawska, M., Markiewicz, R., Socha, K., & Kudelski, J. (2009). Lead concentration in the bladder tissue and blood of patients with bladder cancer. Scandinavian Journal of Urology and Nephrology, 43 (6), 467

- 470. http://dx.doi.org/10.3109/00365590903198991

Goldstein, G. W. (1993). Evidence that lead acts as a calcium substitute in second messenger metabolism. NeuroToxicology, 14 (2

- 3), 97

- 101. http://www.ncbi.nlm.nih.gov/pubmed/8247416

Gollenberg, A. L., Hediger, M. L., Lee, P. A., Himes, J. H., & Buck Louis, G. M. (2010). Association between lead and cadmium and reproductive hormones in peripubertal U.S. girls. Environm ental Health Perspectives, 118 (12), 1782

- 1787. http://dx.doi.org/10.1289/ehp.1001943

Gomaa, A., Hu, H., Bellinger, D., Schwartz, J., Tsaih, S.

- W., Gonzalez

- Cossio, T., . . . Hernandez

- Avila, M. (2002). Ma ternal bone lead as an independent risk factor for fetal neurotoxicity: A prospective study. Pediatrics, 110 , 110

- 118.

Gong, Z., & Evans, H. L. (1997). Effect of chelation with meso

- dimercaptosuccinic acid (DMSA) before and after the appearance
of lead

- in duced neurotoxicity in the rat. Toxicology and Applied Pharmacology, 144 (2), 205

- 214. http://dx.doi.org/10.1006/taap.1997.8118

Gonick, H. C., Cohen, A. H., Ren, Q., Saldanha, L. F., Khalil

- Manesh, F., Anzalone, J., & Sun, Y. Y. (1996). Effect of 2,3

- dimercaptosuccinic acid on nephrosclerosis in the Dahl rat I role of reactive oxygen species. Kidney International,
50 (5), 1572

- 1581. http://dx.doi.org/10.1038/ki.1996.473

Gonick, H. C., Ding, Y., Bondy, S. C., Ni, Z., & Vaziri, N. D. (1997). Lead

- induced hypertension: Interplay of nitric oxide and reactive oxygen species. Hypertension , 30 (6), 1487

- 1492. http://www.ncbi.nlm.nih.gov/pubmed/9403571

Gonick, H. C., Ding, Y., & Vaziri, N. D. (1998). Effect of low lead exposure on eicosanoid excretion in rats. Prostaglandins and
Other L ipid Mediators, 55 (2

- 3), 77

- 82. http://dx.doi.org/10.1016/S0090

- 6980(98)00010

- 0

Gonick, H. C., Khalil

- Manesh, F., & Raghavan, S. R. V. (1985). Characterization of human erythrocyte lead

- binding protein. In T. D. Lekkas (Ed.), Heavy metals in the environment: Athens, September 1985 (pp. 313

- 316). Edinburgh, UK: CEP
Consultants.May 2011 5

- 398 DRAFT

– DO NOT CITE OR QUOTE Gorell, J. M., Johnson, C. C., Rybicki, B. A., Peterson, E. L., Kortsha, G. X., Brown, G. G., & Richardson, R. J. (1997). Occupational exposures to metals as risk factors for Parkinson's disease. Neurology, 48 (3), 650

- 658. http://www.ncbi.nlm.nih.gov/pubmed/9065542

Goswami, K., Gachhui, R., & Bandopadhyay, A. (2005). H epatorenal dysfunctions in lead pollution. Journal of Environmental Science and Engineering, 47 (1), 75

- 80. http://www.ncbi.nlm.nih.gov/pubmed/16669340

Governa, M., Valentino, M., & Visona, I. (1987) . In vitro impairment of human granulocyte functions by lead. Archives of Toxicology, 59 (6), 421

- 425. http://dx.doi.org/10.1007/BF00316208

Goyer, R. A. (1968). The renal tubule in lead poisoning. I. Mitochondrial swelling and aminoaciduria. Laboratory Investigation,
19 (1), 71

- 77. http://www.ncbi.nlm.nih.gov/pubmed/5671355

Goyer, R. A., Cherian, M. G., & Delaquerriere

- Richardson, L. (1978). Renal effects of repeated administration of calcium disodium ethylenediaminetetraacetate during excessive exposure to lead in rats. Journal of Environmental Pathology, Toxicology and Oncology, 1 (4), 40 3

- 410. http://www.ncbi.nlm.nih.gov/pubmed/102714

Goyer, R. A., Krall, A., & Kimball, J. P. (1968). The renal tubule in lead poisoning. II. In vitro studies of mitochondrial s tructure and function. Lab oratory Investigation, 19 (1), 78

- 83. http://www.ncbi.nlm.nih.gov/pubmed/4970361

Goyer, R. A., Leonard, D. L., Moore, J. F., Rhyne, B., & Krigman, M. R. (1970). Lead dosage and the role of the intranu clear inclusion body: An experimental study. Archives of Environmental and Occupational Health, 20 (6), 705

- 711. http://www.ncbi.nlm.nih.gov/pubmed/5443344

Goyer, R. A., May, P., Cates, M. M., & Krigm an, M. R. (1970). Lead and protein content of isolated intranuclear inclusion bodies from kidneys of lead

- poisoned rats. Laboratory Investigation, 22 (3), 245

- 251. http://www.ncbi.nlm.nih.gov/pubmed/5 436518

Goyer, R. A., & Rhyne, B. C. (1973). Pathological effects of lead. International Review of Experimental Pathology, 12 , 1

- 77. http://www.ncbi.nlm.nih.gov/pubmed/4349348

Grabowska, M., & Guminsk a, M. (1996). The effect of lead on lactate formation, ATP level and membrane ATPase activities in human erythrocytes in vitro. International Journal of Occupational Medicine and Environmental Health, 9 (3), 265

- 274. http://www.ncbi.nlm.nih.gov/pubmed/8972169

Gracia, C. R., Sammel, M. D., Coutifaris, C., Guzick, D. S., & Barnhart, K. T. (2005). Occupational exposures and male infertility. American Journal of Epidemiology, 162 (8), 729

- 733. http://dx.doi.org/10.1093/aje/kwi269

Gramigni, E., Tadini

- Buoninsegni, F., Bartolommei, G., Santini, G., Chelazzi, G., & Moncelli, M. R. (2009). Inhibitory effect of Pb2+ on the transport cycle of the Na+,K+

- ATPase. Ch emical Research in Toxicology, 22 (10), 1699

- 1704. http://dx.doi.org/10.1021/tx9001786

Grant, L. D., Kimmel, C. A., West, G. L., Martinez

- Vargas, C. M., & Howard, J. L. (1980). Chronic low

- level lead toxicit y in the rat: II Effects on postnatal physical and behavioral development. Toxicology and Applied Pharmacology, 56 , 42

- 58. http://dx.doi.org/10.1016/0041

- 008X(80)90130

- 1

Greene, T., & Ernhart, C. B. (1993). Dentine lead and intelligence prior to school entry: A statistical sensitivity analysis. Journal of Clinical Epidemiology, 46 (4), 323

- 339. http://dx.doi.org/10.1016/0895

- 4 356(93)90147

- S

Grizzo, L. T., & Cordellini, S. (2008). Perinatal lead exposure affects nitric oxide and cyclooxygenase pathways in aorta of
weaned rats. Toxicological Sciences, 103 (1), 207

- 214. http://d x.doi.org/10.1093/toxsci/kfn018May 2011 5

- 399 DRAFT

– DO NOT CITE OR QUOTE Grlickova

- Duzevik, E., Wise, S. S., Munroe, R. C., Thompson, W. D., & Wise, J. P., Sr. (2006). XRCC1 protects against particulate chromate

- induced chromosome damage and cytotoxicity in Chinese hamster ovary cells. Toxicologi cal Sciences, 92 (2), 409

- 415. http://dx.doi.org/10.1093/toxsci/kfl021

Grover, P., Rekhadevi, P. V., Danadevi, K., Vuyyuri, S. B., Mahboob, M., & Rahman, M. F. (2010). Genotoxicity evaluation in
workers occupationally exposed to lead. International Journal of Hygiene and Environmental Health, 213 (2), 99

- 106. http://dx.doi.org/10.1016/j.ijheh.2010.01.005

Guilarte, T. R., & McGlothan, J. L. (1998). Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure. Brain Research, 790 (1

- 2), 98

- 107. http://dx.doi.org/10.1016/S0006

- 8993(98)00054

- 7

Guilarte, T. R., Toscano, C. D., McGlothan, J. L., & Weaver, S. A. (2003). Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure. Annals of Neurology, 53 (1), 50

- 56. http://dx.doi.org/10.1002/ana.10399

Gulson, B., Mahaffey, K. R., Jameson, C. W., Patison, N., Law, A. J., Mizon, K. J., . . . Pederson, D. (1999). Impact of diet on lead in blood and urine in female adults and relevance to mobilization of lead from bone stores. Environmental Health Perspectives, 107 (4), 257

- 263. http://dx.doi.org/10.1289/ehp.99107257

Gulson, B., Mizon, K. J., Korsch, M. J., Palmer, J. M., & Donnelly, J. B. (2003). Mobilization of lead f rom human bone tissue during pregnancy and lactation: A summary of long

- term research. Science of the Total Environment, 303 (1

- 2), 79

- 104. http://dx.doi.org/10.1016/S0048

- 9697(02)00355

- 8

Gump, B . B., Reihman, J., Stewart, P., Lonky, E., Darvill, T., & Matthews, K. A. (2007). Blood lead (Pb) levels: A potential environmental mechanism explaining the relation between socioeconomic status and cardiovascular reactivity in
children. Health Psychology, 26 (3), 296

- 304. http://dx.doi.org/10.1037/0278

- 6133.26.3.296

Gump, B. B., Stewart, P., Reihman, J., Lonky, E., Darvill, T., Matthews, K. A., & Parsons, P. J. (2005). Prenatal and early
childhood b lood lead levels and cardiovascular functioning in 9 1/2 year old children. Neurotoxicology and Teratology,
27 , 655

- 665. http://dx.doi.org/10.1016/j.ntt.2005.04.002

Gump, B. B., Stewart, P., Reihman, J., Lonky, E., Darvill, T., Parsons, P. J., & Granger, D. A. (2008). Low

- level prenatal and postnatal blood lead exposure and adrenocortical responses to acute stress in children. Environmental Health Perspectives, 116 (2) , 249

- 255. http://dx.doi.org/10.1289/ehp.10391

Gundacker, C., Fröhlich, S., Graf

- Rohrmeister, K., Eibenberger, B., Jessenig, V., Gicic, D., . . . Husslein, P. (2010). Perinatal lead and mercury exposure in Austria. Science of the Total Environment, 408 (23), 5744

- 5749. http://dx.doi.org/10.1016/j.scitotenv.2010.07.079

Guo, T. L., Mudzinski, S. P., & Lawrence, D. A. (1996a). The heavy metal lead m odulates the expression of both TNF

- "alpha"
and TNF

- "alpha" receptors in lipopolysaccharide

- activated human peripheral blood mononuclear cells. Journal of Leukocyte Biology, 59 (6), 932

- 939. http://ww w.ncbi.nlm.nih.gov/pubmed/8691080

Guo, T. L., Mudzinski, S. P., & Lawrence, D. A. (1996b). Regulation of HLA

- DR and invariant chain expression by human peripheral blood mononuclear cells with lead, interferon

- "gamma", or interleukin

- 4. Cellular Immunology, 171 (1), 1

- 9. http://dx.doi.org/10.1006/cimm.1996.0166

Gupta, P., Husain, M. M., Shankar, R., Seth, P. K., & Maheshwari, R. K. (2002). Lead exposure enhances virus multiplication a nd pathogenesis in mi ce. Veterinary and Human Toxicology, 44 (4), 205

- 210. http://www.ncbi.nlm.nih.gov/pubmed/12136965

Gustafson, A., Hedner, P., Schutz, A., & Skerfving, S. (1989). Occupational lead exposure and pituita ry function. International Archives of Occupational and Environmental Health, 61 (4), 277

- 281. http://dx.doi.org/10.1007/BF00381426May 2011 5

- 400 DRAFT

– DO NOT CITE OR QUOTE Gustavsson, P., Plato, N., Hallqvist, J., Hogstedt, C., Lewne, M., Reuterw all, C., & Scheele, P. (2001). A population

- based case

referent study of myocardial infarction and occupational exposure to motor exhaust, other combustion products, organic solvents, lead, and dynamite stockholm heart epidemiology program (SHEEP) study gr oup. Epidemiology,
12 (2), 222

- 228. http://www.ncbi.nlm.nih.gov/pubmed/11246584

Habermann, E., Crowell, K., & Janicki, P. (1983). Lead and other metals can substitute for Ca2+ in calmodulin. Archives of Toxicology, 54 , 61

- 70. http://dx.doi.org/10.1007/BF00277816

Hage, F. G., & Szalai, A. J. (2007). C

- reactive protein gene polymorphisms, C

- reactive protein blood levels, and cardiovascular
disease risk. Journal of the American College of Cardiology, 50 (12), 1115

- 1122. http://dx.doi.org/10.1016/j.jacc.2007.06.012

Hamidinia, S. A., Erdahl, W. L., Chapman, C. J., Steinbaugh, G. E., Taylor, R. W., & Pfeiffer, D. R. (2006). Monensin improves
the effectiveness of meso

- dimercaptosuccinate when used to treat lead intoxication in rats. Environmental Health Perspectives, 114 (4), 484

- 493. http://dx.doi.org/10. 1289/ehp.8279

Hanas, J. S., Rodgers, J. S., Bantle, J. A., & Cheng, Y. G. (1999). Lead inhibition of DNA

- binding mechanism of Cys(2)His(2) zinc finger proteins. Molecular Pharmacology, 56 (5), 982

- 988. http://www.ncbi.nlm.nih.gov/pubmed/10531404

Hara, N., Yamada, K., Terashima, M., Osago, H., Shimoyama, M., & Tsuchiya, M. (2003). Molecular identification of human glutamine

- and ammonia

- dependent NAD synthetases: Carbon

- nitrogen hydrolase domain confers glutamine dependency. Journal of Biological Chemistry, 278 (13), 10914

- 10921. http://dx.doi.org/10.1074/jbc.M209203200

Harry, G. J., Schmitt, T. J. , Gong, A., Brown, H., Zawia, N., & Evans, H. L. (1996). Lead

- induced alterations of glial fibrillary acidic protein (GFAP) in the developing rat brain. Toxicology and Applied Pharmacology, 139 (1), 84

- 93. http://dx.doi.org/10.1006/taap.1996.0145

Hartwig, A., Schlepegrell, R., & Beyersmann, D. (1990). Indirect mechanism of lead

- induced genotoxicity in cultured
mammalian cells. Mutation Research: Genetic Toxicology, 241 (1), 75

- 82. http://dx.doi.org/10.1016/0165

- 1218(90)90110

- N

Hashemzadeh

- Gargari, H., & Guilarte, T. R. (1999). Divalent cations modulate N

- methyl

- D

- aspartate receptor function at the glycine site. Journal of Pharmacology and Experim ental Therapeutics, 290 (3), 1356

- 1362. http://www.ncbi.nlm.nih.gov/pubmed/10454514

Hashmi, N. S., Kachru, D. N., Khandelwal, S., & Tandon, S. K. (1989). Interrelationship between iron deficiency and lead intoxication (part 2). Biological Trace Element Research, 22 (3), 299

- 307. http://dx.doi.org/10.1007/BF02916618

Hauser, R., Sergeyev, O., Korrick, S., Lee, M. M., Revich, B., Gitin, E., . . . Williams , P. L. (2008). Association of blood lead levels with onset of puberty in Russian boys. Environmental Health Perspectives, 116 (7), 976

- 980. http://dx.doi.org/10.1289/ehp.10516

He, L. H., Poblenz, A. T., Med rano, C. J., & Fox, D. A. (2000). Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore. Journal of Biological Chemistry, 275 (16), 12175

- 12184. http://dx.doi.org/10.1074/jbc.275.16.12175

He, S., Dong, W., Deng, Q., Weng, S., & Sun, W. (2003). Seeing more clearly: Recent advances in understanding retinal circuitry. Science, 302 (5644), 408

- 411. http://dx.doi.org/10.1126/science.1085457

He, T., Hirsch, H. V. B., Ruden, D. M., & Lnenicka, G. A. (2009). Chronic lead exposure alters presynaptic calcium regulation and synaptic facilitation in Drosophila larvae. NeuroToxicology, 30 (5), 777

- 784. http://dx.doi.org/10.1016/j.neuro.2009.08.007May 2011 5

- 401 DRAFT

– DO NOT CITE OR QUOTE Hechtenberg, S., & Beyersmann, D. (1991). Inhibition of sarcoplasmic reticulum Ca2+

- ATPase activity by cadmium, lead and

mercury. Enzyme, 45 (3), 109

- 115. http://www.ncbi.nlm.nih.gov/pubmed/1840035

Heiman, A. S., & Tonner, L. E. (1995). The acute effect of lead acetate on glucocorticoid regulation of tyrosine aminotransferase in hepatoma cells. Toxicology, 100 (1

- 3), 57

- 68. http://dx.doi.org/10.1016/0300

- 483X(95)03061

- J

Hellstrom, L., Elinder, C.

- G., Dahlberg, B., Lundberg, M., Jarup, L., Persson, B., & Axelson, O. (2001). Cadmium exposu re and
end

- stage renal disease. American Journal of Kidney Diseases, 38 (5), 1001

- 1008. http://dx.doi.org/10.1053/ajkd.2001.28589

Hemdan, N. Y. A., Emmrich, F., Adham, K., Wichmann, G., Lehmann, I., El

- Massry, A., . . . Sack, U. (2005). Dose

- dependent modulation of the in vitro cytokine production of human immune competent cells by lead salts. Toxicological Sciences,
86 (1), 75

- 83. http://dx.doi.org/1 0.1093/toxsci/kfi177

Hengstler, J. G., Bolm

- Audorff, U., Faldum, A., Janssen, K., Reifenrath, M., Gotte, W., . . . Oesch, F. (2003). Occupational exposure to heavy metals: DNA damage induction and DNA repair inhibition prove co

- exposures to cadmium, cobalt and lead as more dangerous than hitherto expected. Carcinogenesis, 24 (1), 63

- 73. http://dx.doi.org/10.1093/carcin/24.1.63

Heo, Y., Lee, B.

- K., Ahn, K.

- D., & Lawrence, D. A. (2004). Serum IgE elevation correlates with blood lead levels in battery manufacturing workers. Human and Experimental Toxicology, 23 (5), 209

- 213. http://dx.doi.org/10.1191/0960327104ht442oa

Heo, Y., Mondal, T. K., Gao, D. H. , Kasten

- Jolly, J., Kishikawa, H., & Lawrence, D. A. (2007). Posttranscriptional inhibition of
interferon

- gamma production by lead. Toxicological Sciences, 96 (1), 92

- 100. http://dx.doi.org/10.1093/toxsc i/kfl182

Heo, Y., Parsons, P. J., & Lawrence, D. A. (1996). Lead differentially modifies cytokine production in vitro and in vivo. Toxicology and Applied Pharmacology, 138 (1), 149

- 157. http://dx.doi.org/10.1006/taap.1996.0108

Hermes

- Lima, M., Pereira, B., & Bechara, E. J. H. (1991). Are free radicals involved in lead poisoning? Xenobiotica, 21 (8),
1085

- 1090. http://dx.doi.org/10.3 109/00498259109039548

Hernandez

- Avila, M., Smith, D., Meneses, F., Sanin, L. H., & Hu, H. (1998). The influence of bone and blood lead on plasma lead levels in environmentally exposed adults. Environmental Health Perspectives, 106 , 473

- 477.

Hernandez

- Ocho a, I., Sanchez

- Gutierrez, M., Solis

- Heredia, M. J., & Quintanilla

- Vega, B. (2006). Spermatozoa nucleus takes up lead during the epididymal maturation altering chromatin condensation. Reproductive Toxicology, 21 (2), 171

- 178. http://dx.doi.org/10.1016/j.reprotox.2005.07.015

Hernandez

- Serrato, M. I., Fortoul, T. I., Rojas

- Martinez, R., Mendoza

- Alvarado, L. R., Canales

- Trevino, L., Bochichio

Riccardelli, T., . . . Olaiz

- Fernandez, G. (2006). Lead bl ood concentrations and renal function evaluation: Study in an exposed Mexican population. Environmental Research, 100 (2), 227

- 231. http://dx.doi.org/10.1016/j.envres.2005.03.004

Herold, C. (2010). NMDA and D2

- like receptors modulate cognitive flexibility in a color discrimination reversal task in pigeons. Behavioral Neuroscience, 124 (3), 381

- 390. http://dx.doi.org/10.1037/a0019504

H eydari, A., Norouzzadeh, A., Khoshbaten, A., Asgari, A., Ghasemi, A., Najafi, S., & Badalzadeh, R. (2006). Effects of short

term and subchronic lead poisoning on nitric oxide metabolites and vascular responsiveness in rat. Toxicology Letters,
166 (1), 88

- 94

. http://dx.doi.org/10.1016/j.toxlet.2006.05.014

Hilson, J. A., & Strupp, B. J. (1997). Analyses of response patterns clarify lead effects in olfactory reversal and extradimensional
shift tasks: Assessment of inhibitory control, associative ability, and memory. Behavioral Neuroscience, 111 (3), 532

- 542. http://www.ncbi.nlm.nih.gov/pubmed/9189268May 2011 5

- 402 DRAFT

– DO NOT CITE OR QUOTE Holmes, A. L., Wise, S. S., Sandwick, S. J., Li ngle, W. L., Negron, V. C., Thompson, W. D., & Wise, J. P., Sr. (2006). Chronic exposure to lead chromate causes centrosome abnormalities and aneuploidy in human lung cells. Cancer Research,
66 (8), 4041

- 4048. http://dx.doi.org/10.1158/0008

- 5472.can

- 05

- 3312

Holmes, A. L., Wise, S. S., Sandwick, S. J., & Wise, J. P. (2006). The clastogenic effects of chronic exposure to particulate and soluble Cr(VI) in human lung cells. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 610 (1

- 2),
8

- 13. http://dx.doi.org/10.1016/j.mrgentox.2006.06.006

Holtzman, D., Olson, J. E., DeVries, C., & Bensch, K. (1987). Lead toxicity in primary cultured cerebral astrocytes and cerebellar granular neurons. Toxicology and Applied Pharmacology, 89 (2), 211

- 225. http://dx.doi.org/10.1016/0041

- 008X(87)90042

- 1

Hong, Y. C., Hwang, S. S., Kim, J. H., Lee, K. H., Lee, H. J., Yu, S. D., & Kim, D. S. (2007). Metals in particulate pollutants affect peak expiratory flow of schoolchildren. Environmental Health Perspectives, 115 (3), 430

- 434. http://dx.do i.org/10.1289/ehp.9531

Hornung, R. W., Lanphear, B. P., & Dietrich, K. N. (2009). Age of greatest susceptibility to childhood lead exposure: A new statistical approach. Environmental Health Perspectives, 117 (8), 1309

- 1312. http://dx.doi.org/10.1289/ehp.0800426

Hotz, P., Buchet, J. P., Bernard, A., Lison, D., & Lauwerys, R. (1999). Renal effects of low

- level environmental cadmium exposure: 5

- year follow

- up of a subcohort from the Cadmibel study. Lancet, 354 (9189), 1508

- 1513. http://dx.doi.org/10.1016/S0140

- 6736(99)91145

- 5

Howard, J. K. (1974). Human erythrocyte glutathione reductase and glucose 6

- phosphate dehydrogenase activities in normal subjec ts and in persons exposed to lead. Clinical Science and Molecular Medicine, 47 (5), 515

- 520. http://www.ncbi.nlm.nih.gov/pubmed/4434692

Hsiao, C. Y., Wu, H. D., Lai, J. S., & Kuo, H. W. (2001). A longitudinal study of the effects of long

- term exposure to lead among lead battery factory workers in Taiwan (1989

- 1999). Science of the Total Environment, 279 (1

- 3), 151

- 158. http://dx.doi.org/10.1016/S0048

- 9697(01)00762

- 8

Hsieh, T. J., Chen, Y. C., Li, C. W., Liu, G. C., Chiu, Y. W., & Chuang, H. Y. (2009). A proton magnetic resonance spectroscopy study of the chronic lead effect on the basal ganglion and frontal and occipi tal lobes in middle

- age adults. Environmental Health Perspectives, 117 (6), 941

- 945. http://dx.doi.org/10.1289/ehp.0800187

Hsu, C. W., Lin, J. L., Lin

- Tan, D. T., Yen, T. H., Huang, W. H., Ho, T. C., . . . Huang, L. M. (2009). Association of environmental cadmium exposure with inflammation and malnutrition in maintenance haemodialysis patients. Nephrology, Dialysis, Transplantation, 24 (4), 1282

- 1288. http:/ /dx.doi.org/10.1093/ndt/gfn602

Hsu, J. M. (1981). Lead toxicity as related to glutathione metabolism. Journal of Nutrition, 111 (1), 26

- 33. http://www.ncbi.nlm.nih.gov/pubmed/7452372

Hsu, P. C., Chang , H. Y., Guo, Y. L., Liu, Y. C., & Shih, T. S. (2009). Effect of smoking on blood lead levels in workers and role of reactive oxygen species in lead

- induced sperm chromatin DNA damage. Fertility and Sterility, 91 (4), 1096

- 1103. http://dx.doi.org/10.1016/j.fertnstert.2008.01.005

Hsu, P. C., Hsu, C. C., Liu, M. Y., Chen, L. Y., & Guo, Y. L. (1998). Lead

- induced changes in spermatozoa function and
metabolism. Journal of Toxicology and Environment al Health, Part A: Current Issues, 55 (1), 45

- 64. http://dx.doi.org/10.1080/009841098158610

Hsu, P. C., Liu, M. Y., Hsu, C. C., Chen, L. Y., & Guo, Y. L. (1998). Effects of vitamin E and/or C on reacti ve oxygen species

related lead toxicity in the rat sperm. Toxicology, 128 (3), 169

- 179. http://dx.doi.org/10.1016/S0300

- 483X(98)00068

- 7May 2011 5

- 403 DRAFT

– DO NOT CITE OR QUOTE Hu, H. (1991). A 50

- year follow

- up of childhood plumbism: H ypertension, renal function, and hemoglobin levels among
survivors. American Journal of Diseases of Children, 145 (6), 681

- 687. http://www.ncbi.nlm.nih.gov/pubmed/2035497

Hu, H., Aro, A., Payton, M., Korrick, S., & Sparrow. (1996). The relationship of bone and blood lead to hypertension: The Normative Aging Study. JAMA: Journal of the American Medical Association, 275 (15), 1171

- 1176. http ://dx.doi.org/10.1001/jama.1996.03530390037031

Hu, H., & Hernandez

- Avila, M. (2002). Lead, bones, women, and pregnancy: The poison within? American Journal of Epidemiology, 156 (12), 1088

- 1091. http://dx.do i.org/10.1093/aje/kwf164

Hu, H., Tellez

- Rojo, M. M., Bellinger, D., Smith, D., Ettinger, A. S., Lamadrid

- Figueroa, H., . . . Hernandez

- Avila, M. (2006). Fetal lead exposure at each stage of pregnancy as a predictor of infant mental development. Environmental Health Perspectives, 114 , 1730

- 1735. http://dx.doi.org/10.1289/ehp.9067

Hu, Q. S., Fu, H. J., Ren, T. L., Wang, S. Y., Zhou, W., Song, H., . . . Dong, S. Z. (2008). Maternal low

- level lead expo sure reduces the expression of PSA

- NCAM and the activity of sialyltransferase in the hippocampi of neonatal rat pups. NeuroToxicology, 29 (4), 675

- 681. http://dx.doi.org/10.1016/j.neuro.2008.04.002

Huang, B. M., Lai, H. Y., & Liu, M. Y. (2002). Concentration dependency in lead

- inhibited steroidogenesis in MA

- 10 mouse Leydig tumor cells. Journal of Toxicology and Environmental Health, Part A: Current Issues, 65 (7), 557

- 567. http://dx.doi.org/10.1080/15287390252808000

Huang, B. M., & Liu, M. Y. (2004). Inhibitory actions of lead on steroidogenesis in MA

- 10 mouse Leydig tumor cells. Systems Biology in Reproductive Medicine , 50 (1), 5

- 9. http://dx.doi.org/10.1080/01485010490250434

Huang, M., Krepkiy, D., Hu, W., & Petering, D. H. (2004). Zn

- , Cd

- , and Pb

- transcription factor IIIA: Properties, DNA binding, and compariso n with TFIIIA

- finger 3 metal complexes. Journal of Inorganic Biochemistry, 98 (5), 775

- 785. http://dx.doi.org/10.1016/j.jinorgbio.2004.01.014

Huang, X. P., Feng, Z. Y., Zhai, W. L., & Xu, J. H. (1988). Chromosomal aberrations and sister chromatid exchanges in workers exposed to lead. Biomedical and Environmental Sciences, 1 (4), 382

- 387. http://www.ncbi.nlm.nih.gov/pubmed/3268122

Hudson, C. A., Cao, L., Kasten

- Jolly, J., Kirkwood, J. N., & Lawrence, D. A. (2003). Susceptibility of lupus

- prone NZM mouse strains to lead exacerbation of systemic lupus erythematosus symptoms. Journal of Toxicology and Environmental Health, Part A: Current Issues , 66 (10), 895

- 918. http://dx.doi.org/10.1080/15287390306456

Huel, G., Sahuquillo, J., Debotte, G., Oury, J. F., & Takser, L. (2008). Hair mercury negatively correlates with calcium pump activity in hum an term newborns and their mothers at delivery. Environmental Health Perspectives, 116 (2), 263

- 267. http://dx.doi.org/10.1289/ehp.10381

Huo, X., Peng, L., Xu, X. J., Zheng, L. K., Qiu, B., Qi, Z. L., . . . Piao, Z. X. (2007). Elevated blood lead levels of children in Guiyu, an electronic waste recycling town in China. Environmental Health Perspectives, 115 (7), 1113

- 1117. http://dx.doi.org/10.1289/ehp.9697

Huse man, C. A., Moriarty, C. M., & Angle, C. R. (1987). Childhood lead toxicity and impaired release of thyrotropin

- stimulating
hormone. Environmental Research, 42 (2), 524

- 533. http://dx.doi.org/10. 1016/S0013

- 9351(87)80219

- 0

Huseman, C. A., Varma, M. M., & Angle, C. R. (1992). Neuroendocrine effects of toxic and low blood lead levels in children. Pediatrics, 90 (2), 186

- 189. http://www.ncbi.nlm.nih.gov/pubmed/8361827

Hwang, K.

- Y., Lee, B.

- K., Bressler, J. P., Bolla, K. I., Stewart, W. F., & Schwartz, B. S. (2002). Protein kinase C activity and the
relations between blood lead and neurobehavioral function in lead workers. Envir onmental Health Perspectives, 110 (2),
133

- 138. http://dx.doi.org/10.1289/ehp.02110133May 2011 5

- 404 DRAFT

– DO NOT CITE OR QUOTE Hwang, Y.

- H., Chiang, H.

- Y., Yen

- Jean, M.

- C., & Wang, J.

- D. (2009). The association between low levels of lead in blo od and occupational noise

- induced hearing loss in steel workers. Science of the Total Environment, 408 (1), 43

- 49. http://dx.doi.org/10.1016/j.scitotenv.2009.09.016

Hwua, Y. S., & Yang, J. L. ( 1998). Effect of 3

- aminotriazole on anchorage independence and mutagenicity in cadmium

- and lead

treated diploid human fibroblasts. Carcinogenesis, 19 (5), 881

- 888. http://dx.doi.org/10.1093/carcin/19. 5.881

IARC. (International Agency for Research on Cancer). (2006). Inorganic and organic lead compounds . Lyon, France: Author.
Retrieved from http://monographs.iarc.fr/ENG/Monographs/v ol87/index.php

.

Iavicoli, I., Carelli, G., Stanek, E. J., Castellino, N., Li, Z., & Calabrese, E. J. (2006). Low doses of dietary lead are associated with a profound reduction in the time to the onset of puberty in female mice. Reproductive Toxicology, 22 ( 4), 586

- 590. http://dx.doi.org/10.1016/j.reprotox.2006.03.016

Iavicoli, I., Carelli, G., Stanek, E. J., III, Castellino, N., & Calabrese, E. J. (2006). Below background levels of blood lead imp act cytokine levels in male and female mice. Toxicology and Applied Pharmacology, 210 (1

- 2), 94

- 99. http://dx.doi.org/10.1016/j.taap.2005.09.016

Iavicoli, I., Sgambato, A., Carelli, G., Ardito, R., Cittadini, A., & Castellino, N. (2001). Lead

- related effects on rat fibroblasts. Molecular and Cellular Biochemistry, 222 (1

- 2), 35

- 40. http://dx.doi.org/10.1023/A:1017930401906

Ignasiak, Z., Slawinska , T., Rozek, K., Little, B. B., & Malina, R. M. (2006). Lead and growth status of schoolchildren living in the copper basin of south

- western Poland: Differential effects on bone growth. Annals of Human Biology, 33 (4), 401

- 414. http://dx.doi.org/10.1080/03014460600730752

Ikebuchi, H., Teshima, R., Suzuki, K., Terao, T., & Yamane, Y. (1986). Simultaneous induction of Pb

- metallothionein

- like protein
and Zn

- thionein in the liver of rats given lead acetat e. Biochemistry, 233 (2), 541

- 546. http://www.ncbi.nlm.nih.gov/pubmed/3954751

Ingelsson, E., Gona, P., Larson, M. G., Lloyd

- Jones, D. M., Kannel, W. B., Vasan, R. S., & Levy, D. (2008). Altered blood pressure progression in the community and its relation to clinical events. Archives of Internal Medicine, 168 (13), 1450

- 1457. http://dx.doi.org/10.1001/archinte.168.13.1450

Inglis, J. A., Henderson, D. A., & Emmerson, B. T. (1978). The pathology and pathogenesis of chronic lead nephropathy occurring in Queensland. Journal of Pathology, 124 (2), 65

- 76. http://dx.doi.org/10 .1002/path.1711240202

Institoris, L., Kovacs, D., Kecskemeti

- Kovacs, I., Lukacs, A., Szabo, A., Lengyel, Z., . . . Desi, I. (2006). Immunotoxicological investigation of subacute combined exposure with low doses of PB, HG and CD in rats. Acta Biologica Hung arica,
57 (4), 433

- 439. http://dx.doi.org/10.1556/ABiol.57.2006.4.5

Ionescu, J. G., Novotny, J., Stejskal, V., Latsch, A., Blaurock

- Busch, E., & Eisenmann

- Klein, M. (2007). Breast tumours strongly

ac cumulate transition metals. Maedica

- a Journal of Clinical Medicine, 2 (1), 5

- 9.

Iranpour, R., Besharati, A. A., Nasseri, F., Hashemipour, M., Balali

- Mood, M., & Kelishadi, R. (2007). Comparison of blood lead
levels of mothers and cord blood in intrauteri ne growth retarded neonates and normal term neonates. Saudi Medical Journal, 28 (6), 877

- 880. http://www.ncbi.nlm.nih.gov/pubmed/17530103

Irgens, A., Kruger, K., Skorve, A. H., & Irgens, L. M. (1998) . Reproductive outcome in offspring of parents occupationally exposed to lead in Norway. American Journal of Industrial Medicine, 34 , 431

- 437.

Iwata, T., Yano, E., Karita, K., Dakeishi, M., & Murata, K. (2005). Critical dose of lead affecting postural bal ance in workers. American Journal of Industrial Medicine, 48 , 319

- 325.May 2011 5

- 405 DRAFT

– DO NOT CITE OR QUOTE Jaako

- Movits, K., Zharkovsky, T., Romantchik, O., Jurgenson, M., Merisalu, E., Heidmets, L.

- T., & Zharkovsky, A. (2005). Developmental lead exposure impairs contextual fear conditionin g and reduces adult hippocampal neurogenesis in the
rat brain. International Journal of Developmental Neuroscience, 23 (7), 627

- 635. http://dx.doi.org/10.1016/j.ijdevneu.2005.07.005

Jacobsen, C. , Hartvigsen, K., Thomsen, M. K., Hansen, L. F., Lund, P., Skibsted, L. H., . . . Meyer, A. S. (2001). Lipid oxidation in fish oil enriched mayonnaise: Calcium disodium ethylenediaminetetraacetate, but not gallic acid, strongly inhibited
oxidative deterior ation. Journal of Agricultural and Food Chemistry, 49 (2), 1009

- 1019. http://dx.doi.org/10.1021/jf000729r

Jadhav, S. H., Sarkar, S. N., Ram, G. C., & Tripathi, H. C. (2007). Immunosuppressive effect of subchronic exposure to a mixture of eight heavy metals, found as groundwater contaminants in different areas of india, through drinking water in male

rats. Archives of Environmental Contamination and Tox icology, 53 (3), 450

- 458. http://dx.doi.org/10.1007/s00244

- 006

- 0177

- 1

Jagetia, G. C., & Aruna, R. (1998). Effect of various concentrations of lead nitrate on the induction of micronuclei in mouse bon e

marrow. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 415 (1

- 2), 131

- 137. http://dx.doi.org/10.1016/S1383

- 5718(98)00052

- 7

Jain, N. B., Potula, V., Schwartz, J., Vokonas, P. S., Sparrow, D., Wright, R. O., . . . Hu, H. (2007). Lead levels and ischemic heart disease in a prospective study of middle

- aged and elderly men: The VA Normative Aging Study. Environmental Health Perspectives, 115 (6), 871

- 875. http://dx.doi.org/10.1289/ehp.9629

Jamieson, J. A., Shuhyta, J. N., & Taylor, C. G. (2007). Lead does not affect transcription of intestinal zinc

- binding proteins in growing rats. Experimental Biology and Medicine, 232 (6), 744

- 753. http://www.ncbi.nlm.nih.gov/pubmed/17526766

Jamieson, J. A., Stringer, D. M., Zahradka, P., & Taylor, C. G. (2008). Dietary zinc attenuates renal lead deposition but
metallothionein is not directly involved. BioMetals, 21 (1), 29

- 40. http://dx.doi.org/10.1007/s10534

- 007

- 9090

- y

Jang, H. O., Kim, J. S., Kwon, W. C., Kim, J. K., Ko, M. S., Kim, D. H., . . . Yun, I. (2008). The effect of lead on calcium release activated calcium influx in primary cultures of human osteoblast

- like cells. Archives of Pharmacal Research, 31 (2),
188

- 194. http://dx.doi.org/10.1007/s12272

- 001

- 1140

- 3

J anjua, N. Z., Delzell, E., Larson, R. R., Meleth, S., Kristensen, S., Kabagambe, E., & Sathiakumar, N. (2009). Determinants of low birth weight in urban Pakistan. Public Health Nutrition, 12 (6), 789

- 798. http://dx.doi.org/10.1017/S1368980008002942

Jayakumar, T., Sridhar, M. P., Bharathprasad, T. R., Ilayaraja, M., Govindasamy, S., & Balasubramanian, M. P. (2009). Experimental studies of Achyranthes aspera (L) preventing nephrotoxicity induced by lead in albino rats. Journal of Health Science, 55 (5), 701

- 708. http://dx.doi.org/10.1248/jhs.55.701

Jedrychowski, W., Perera, F., Jankowski, J., Mrozek

- Budzyn, D., Mroz, E., Flak, E., . . . Lisowska

- Miszczyk, I. (2009a). Gender specific differences in neurodevelopmental effects of prenatal exposure to very low

- lead levels: The prospective cohort study in three

- year olds. Early Human Development, 85 (8), 503

- 510. http://dx.doi.org/10.1016/j.earlhumdev.2009.04.006

Jedrychowski, W., Perera, F., Maugeri, U., Rembiasz, M., Flak, E., Mroz, E., . . . Zembala, M. (2011). Intrauterine exposure to lead may enhance sensitization to common inhalant allergens i n early childhood: A prospective prebirth cohort study. Environmental Research, 111 (1), 119

- 124. http://dx.doi.org/10.1016/j.envres.2010.11.002

Jedrychowski, W., Perera, F. P., Jankowski, J., Mro zek

- Budzyn, D., Mroz, E., Flak, E., . . . Lisowska

- Miszczyk, I. (2009b). Very low prenatal exposure to lead and mental development of children in infancy and early childhood: Krakow prospective cohort study. Neuroepidemiology, 32 (4), 270

- 278. http://dx.doi.org/10.1159/000203075May 2011 5

- 406 DRAFT

– DO NOT CITE OR QUOTE Jehan, Z. S., & Motlag, D. B. (1995). Metal induced changes in the erythrocyte membrane of rats. Toxicology Letters, 78 (2), 127

- 133. http://dx.doi.org/10.1016/0378

- 4274(94)03245

- 3

Jelliffe

- Pawlowski, L. L., Miles, S. Q., Courtney, J. G., Materna, B., & Charlton, V. (2006). Effect of magnitude and timing of maternal pregnancy blood lead (Pb) levels on birth outcomes. Journal o f Perinatology, 26 (3), 154

- 162. http://dx.doi.org/10.1038/sj.jp.7211453

Jenq, C. C., Hsu, C. W., Huang, W. H., Chen, K. H., Lin, J. L., & Lin

- Tan, D. T. (2009). Serum ferritin levels predict all

- cause

a nd infection

- cause 1

- year mortality in diabetic patients on maintenance hemodialysis. American Journal of the Medical Sciences, 337 (3), 188

- 194. http://dx.doi.org/10.1097/MAJ.0b013e31818d8bbe

Jett, D. A., Kuhlmann, A. C., Farmer, S. J., & Guilarte, T. R. (1997). Age

- dependent effects of developmental lead exposure on performance in the Morris water maze. Pharmacology, Biochemistry and Behavior, 57 (1

- 2), 271

- 279. http://dx.doi.org/10.1016/S0091

- 3057(96)00350

- 4

Jett, D. A., Kuhlmann, A. C., & Guilarte, T. R. (1997). Intrahippocampal administration of lead (Pb) impairs performance of rats
in the Morris water maze. Pharmacology, Biochemis try and Behavior, 57 (1

- 2), 263

- 269. http://dx.doi.org/10.1016/S0091

- 3057(96)00349

- 8

Jiang, Y.

- M., Long, L.

- L., Zhu, X.

- Y., Zheng, H., Fu, X., Ou, S.

- Y., . . . Zheng, W. (2008). Evidence for alte red hippocampal volume and brain metabolites in workers occupationally exposed to lead: A study by magnetic resonance imaging and 1H magnetic resonance spectroscopy. Toxicology Letters, 181 (2), 118

- 125. http://dx.doi.org/10.1016/j.toxlet.2008.07.009

Jin, C., Li, Y., Li, Y. L., Zou, Y., Zhang, G. L., Normura, M., & Zhu, G. Y. (2008). Blood lead: Its effect on trace element levels and iron structure in hemoglobin. Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms, 266 (16), 3607

- 3613. http://dx.doi.org/10.1016/j.nimb.2008.05.087

Jin, Y. P., Liao, Y. J., Lu, C. W., Li, G. X., Yu, F., Zhi, X. P., . . . Yang, J. (2006). Health effects in children aged 3

- 6 years induced by environmental lead exposure. Ecotoxicology and Environmental Safety, 63 (2), 313

- 317. http://dx.doi.org/10.1016/j .ecoenv.2005.05.011

Johnson, B. L., Elbatawi, M., Xintaras, C., Baker, E. L., Jr., Hanninen, H., & Seppalainen, A. M. (Eds.). (1987). Prevention of neurotoxic illness in working populations . New York: John Willey & Sons.

Johnson, R. J., Kang, D.

- H., Feig, D., Kivlighn, S., Kanellis, J., Watanabe, S., . . . Mazzali, M. (2003). Is there a pathogenetic role for uric acid in hypertension and cardiovascular and renal disease? Hypertension, 41 , 1183

- 1190. http://dx.doi.org/10.1161/01.HYP.0000069700.62727.C5

Jones, A. Y. M., Lam, P. K., & Gohel, M. D. (2008). Respiratory health of road

- side vendors in a large industrialized city. Environmental Science and Pollution Research, 15 (2), 150

- 154. http://dx.doi.org/10.1065/espr2006.12.368

Jones, A. Y. M., Lam, P. K. W., & Dean, E. (2006). Respiratory health of bus drivers in Hong Kong. International Archives of Occupational and Environmental Health, 79 (5), 414

- 418. http://dx.doi.org/10.1007/s00420

- 005

- 0061

- 8

Jones, E. A., Wright, J. M., Rice, G., Buckley, B. T., Magsumbol, M. S., Barr, D. B., & Williams, B. L. (2010). Metal exposures
in an inner

- city neonatal population. Environment International, 36 (7), 649

- 654. http://dx.doi.org/10.1016/j.envint.2010.04.007

Jones, S. R., Atkin, P., Holroyd, C., Lutman, E., Batlle, J. V. I., Wakeford, R ., & Walker, P. (2007). Lung cancer mortality at a UK
tin smelter. Occupational Medicine, 57 (4), 238

- 245. http://dx.doi.org/10.1093/occmed/kql153

Joseph, C. L. M., Havstad, S., Ownby, D. R., Peterson, E. L., Maliarik, M., McCabe, M. J., . . . Johnson, C. C. (2005). Blood lead
levels and risk of asthma. Environmental Health Perspectives, 113 (7), 900

- 904. http://dx.doi.org/10.1289/ehp.7453May 2011 5

- 407 DRAFT

– DO NOT CITE OR QUOTE Jurczuk, M., Brzoska, M. M., & Moniuszko

- Jakoniuk, J. (2007). Hepatic and renal concentrations of vitamins E and C in lead

and ethanol

- exposed rats. An assessment of their involvement in the mechanisms of peroxidative damage. Food and Chemical Toxicology, 45 (8), 1478

- 1486. http://dx.doi.org/10.1016/j.fct.2007.02.007

Jurczuk, M., Moniuszko

- Jakoniuk, J., & Brzóska, M. M. (2006). Involvement of some low

- molecular thiols in the peroxidative mechanisms of lead and ethanol action on rat liver and kidney. Toxicology, 219 (1

- 3), 11

- 21. http://dx.doi.org/10. 1016/j.tox.2005.10.022

Kaji, T., Suzuki, M., Yamamoto, C., Mishima, A., Sakamoto, M., & Kozuka, H. (1995). Severe damage of cultured vascular endothelial cell monolayer after simultaneous exposure to cadmium and lead. Archives of Environmental
Contaminatio n and Toxicology, 28 , 168

- 172. http://dx.doi.org/10.1007/BF00217612

Kamel, F., Umbach, D. M., Hu, H., Munsat, T. L., Shefner, J. M., Taylor, J. A., & Sandler, D. P. (2005). Lead exposure as a risk
factor f or amyotrophic lateral sclerosis. Neurodegenerative Diseases, 2 (3

- 4), 195

- 201. http://dx.doi.org/10.1159/000089625

Kamel, F., Umbach, D. M., Munsat, T. L., Shefner, J. M., Hu, H., & Sandler, D. P. (2002). L ead exposure and amyotrophic lateral
sclerosis. Epidemiology, 13 , 311

- 319. http://www.ncbi.nlm.nih.gov/pubmed/11964933

Kamel, F., Umbach, D. M., Stallone, L., Richards, M., Hu, H., & Sandler, D. P. (2008). Association of lead exposure with survival in amyotrophic lateral sclerosis. Environmental Health Perspectives, 116 (7), 943

- 947. http://dx.doi.org/10.1289/ehp.11193

Kanduc, D., Rossiello, M. R., Are sta, A., Cavazza, C., Quagliariello, E., & Farber, E. (1991). Transitory DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate. Archives of Biochemistry and Biophysics, 286

, 212

- 216. http://dx.doi.org/10.1016/0003

- 9861(91)90030

- M

Kannel, W. B. (2000a). Elevated systolic blood pressure as a cardiovascular risk factor. The American Journal of Cardiology, 85

, 251

- 255. http://dx.doi.org/10.1016/S0002

- 9149(99)00635

- 9

Kannel, W. B. (2000b). Risk stratification in hypertension: New insights from the Framingham Study. American Journal of Hypertension, 13 , 3S

- 10S. http://dx.doi.org/10.1016/S0895

- 7061(99)00252

- 6

Karaca, T., & Simsek, N. (2007). Effects of spirulina on the number of ovary mast cells in lead

- induced toxicity in rats. Phytotherapy Research, 21 (1), 44

- 46. http://dx.doi.org/10.1002/ptr.2015

Karita, K., Yano, E., Dakeishi, M., Iwata, T., & Murata, K. (2005). Benchmark dose of lead inducing anemia at the workplace. Risk Analysis, 25 (4), 957

- 962. http://dx.doi.org/10.1111/j.1539

- 6924.2005.00652.x

Karmaus, W., Brooks, K. R., Nebe, T., Witten, J., Obi

- Osius, N., & Kruse, H. (2005). Immune function biomarkers in children exposed to lead and organochlorine compounds: A cross

- sectional study. Environmental Health: A Global Access Science Source, 4 (5), 1

- 10. http://dx.doi.org/10.1186/1476

- 069X

- 4

- 5

Kasperczyk, A., Kasperczyk, S., Horak, S., Ostalowska, A., Grucka

- Mamczar, E., Romuk, E., . . . Birkner, E. (2008). Assessment of semen function and lipid peroxidation among lead exposed men. Toxicology and Applied Pharmacology, 228 (3),
378

- 384. http://dx. doi.org/10.1016/j.taap.2007.12.024

Kasperczyk, S., Birkner, E., Kasperczyk, A., & Kasperczyk, J. (2005). Lipids, lipid peroxidation and 7

- ketocholesterol in workers exposed to lead. Human and Experimental Toxicology, 24 (6), 287

- 295. http://dx.doi.org/10.1191/0960327105ht528oa

Kasperczyk, S., Kasperczyk, J., Ostałowska, A., Zalejska

- Fiolka, J., Wielkoszyński, T., Swietochowska, E., & Birkner, E. (2009). The role of the antioxidant enzymes in erythrocy tes in the development of arterial hypertension among humans exposed
to lead. Biological Trace Element Research, 130 (2), 95

- 106. http://dx.doi.org/10.1007/s12011

- 009

- 8323

- zMay 2011 5

- 408 DRAFT

– DO NOT CITE OR QUOTE Kasprzak, K. S., Hoover, K. L., & Poirier, L. A. (1985). Effects of dietary calcium acetate on lead subacetate carcinogenicity in kidneys of male Sprague

- Dawley rats. Carcinogenesis, 6 (2), 279

- 282. http://dx.doi.org/10.1093/c arcin/6.2.279

Kasten

- Jolly, J., Heo, Y., & Lawrence, D. A. (2010). Impact of developmental lead exposure on splenic factors. Toxicology and Applied Pharmacology, 247 (2), 105

- 115. http://dx.doi.org/ 10.1016/j.taap.2010.06.003

Kazi, T. G., Jalbani, N., Kazi, N., Jamali, M. K., Arain, M. B., Afridi, H. I., . . . Pirzado, Z. (2008). Evaluation of toxic metals in blood and urine samples of chronic renal failure patients, before and after dialysis. Renal F ailure, 30 (7), 737

- 745. http://dx.doi.org/10.1080/08860220802212999

Ke, H. (2004). Implications of PDE4 structure on inhibitor selectivity across PDE families. International Journal of Impotence
Res earch, 16 , S24

- S27. http://dx.doi.org/10.1038/sj.ijir.3901211

Kemp, F. W., Neti, P., Howell, R. W., Wenger, P., Louria, D. B., & Bogden, J. D. (2007). Elevated blood lead concentrations and vitamin D deficiency in winter and summer in young urban children. Environmental Health Perspectives, 115 (4), 630

- 635. http://dx.doi.org/10.1289/ ehp.9389

Kempe, D. A., Lang, P. A., Eisele, K., Klarl, B. A., Wieder, T., Huber, S. M., . . . Lang, F. (2005). Stimulation of erythrocyte phosphatidylserine exposure by lead ions. American Journal of Physiology, 288 , C396

- C402. http://dx.doi.org/10.1152/ajpcell.00115.2004

Kempermann, G., Krebs, J., & Fabel, K. (2008). The contribution of failing adult hippocampal neurogenesis to psychiatric
disorders. Current Opinion in Psychiatry, 21 (3), 290

- 295. http://dx.doi.org/10.1097/YCO.0b013e3282fad375

Kerkvliet, N. I., & Baecher

- Steppan, L. (1982). Immunotoxicology studies on lead: Effects of exposure on tumor growth and
cell

- mediated immunity afte r syngeneic or allogeneic stimulation. International Immunopharmacology, 4 , 213

- 224. http://dx.doi.org/10.1016/0162

- 3109(82)90003

- 0

Kermani, S., Karbalaie, K., Madani, S. H., Jahangirnejad, A. A. , Eslaminejad, M. B., Nasr

- Esfahani, M. H., & Baharvand, H. (2008). Effect of lead on proliferation and neural differentiation of mouse bone marrow

- mesenchymal stem cells. Toxicology In Vitro, 22 (4), 995

- 1001. http://dx.doi.org/10.1016/j.tiv.2008.02.009

Kern, M., & Audesirk, G. (2000). Stimulatory and inhibitory effects of inorganic lead on calcineurin. Toxicology, 150 , 171

- 178. http://dx. doi.org/10.1016/S0300

- 483X(00)00258

- 4

Kern, M., Wisniewski, M., Cabell, L., & Audesirk, G. (2000). Inorganic lead and calcium interact positively in activation of
calmodulin. NeuroToxicology, 21 (3), 353

- 363. http://www.ncbi.nlm.nih.gov/pubmed/10894125

Khaĭrullina, A. Y., Ol’shanskaya, T. V., Filimonenko, D. S., Yasinski, V. M., Slobozhanina, E. I., & Kozlova, N. M. (2008). Study of optical and nanostructural metal

- induced changes in erythrocyte membr anes by scattering and atomic force microscopy. Optics and Spectroscopy, 105 (1), 154

- 160. http://dx.doi.org/10.1134/S0030400X08070242

Khalil

- Manesh, F., Gonick, H. C., Cohen, A., Bergamaschi, E., & Mutti, A. (1992). Experimental model of lead nephropathy II Effect of removal from lead exposure and chelation treatment with dimercaptosuccinic acid (DMSA). Environmental Research, 58 (1

- 2), 35

- 54. http://dx.doi.org/10.1016/S0013

- 9351(05)80203

- 8

Khalil

- Manesh, F., Gonick, H. C., & Cohen, A. H. (1993). Experimental model of lead nephropathy III Continuous low

- level lead
administration. Archives of Environmental and Occupational Health, 48 , 271

- 278. http://dx.doi.org/10.1080/00039896.1993.9940372

Khalil

- Manesh, F., Gonick, H. C., Cohen, A. H., Alinovi, R., Bergamaschi, E., Mutti, A., & Rosen, V. J. (1992). Experimental
model of l ead nephropathy I Continuous high

- dose lead administration. Kidney International, 41 , 1192

- 1203. http://dx.doi.org/10.1038/ki.1992.181May 2011 5

- 409 DRAFT

– DO NOT CITE OR QUOTE Khalil

- Manesh, F., Gonick, H. C., Weiler, E. W. J., Prins, B., Weber, M. A., Purdy, R., & Ren, Q. (1994). Effect of chelation treatment with dimercaptosuccinic acid (DMSA) on lead

- related blood pressure changes. Environmental Research, 65

, 86

- 99. http://dx.doi.org/10.1006/enrs.1994.1023

Khalil

- Manesh, F., Gonick, H. C., Weiler, E. W. J., Prins, B., Weber, M. A., & Purdy, R. E. (1993). Lead

- induced hypertension:
Possible role of endothelial factors. Amer ican Journal of Hypertension, 6 , 723

- 729. http://www.ncbi.nlm.nih.gov/pubmed/8110424

Khalil, N. (2010). [Correspondence re: Blood lead mortality study: Khalil et al., 2009].

Khalil, N., Cauley, J. A. , Wilson, J. W., Talbott, E. O., Morrow, L., Hochberg, M. C., . . . Cummings, S. R. (2008). Relationship of blood lead levels to incident nonspine fractures and falls in older women: The study of osteoporotic fractures. Journal of Bone and Mineral Research , 23 (9), 1417

- 1425. http://dx.doi.org/10.1359/jbmr.080404

Khalil, N., Morrow, L. A., Needleman, H., Talbott, E. O., Wilson, J. W., & Cauley, J. A. (2009). Association of cumulative lead and neurocognitive function in an occupational cohort. Neuropsychology, 23 (1), 10

- 19. http://dx.doi.org/10.1037/a0013757

Khalil, N., Wilson, J. W., Talbott, E. O., Morrow, L. A., Hochberg, M. C., Hillier, T. A., . . . Cauley, J. A. (2009). Association of blood lead concentrations with mortality in older women: a prospective cohort study. Environmental Health: A Global Access Science Source, 8 , 15. http://dx.doi.org/10.1186 /1476

- 069x

- 8

- 15

Khan, D. A., Qayyum, S., Saleem, S., & Khan, F. A. (2008). Lead

- induced oxidative stress adversely affects health of the occupational workers. Toxicology and Industrial Health, 24 (9), 611

- 618. http://dx.doi.org/10.1177/0748233708098127

Khan, M. I., Ahmad, I., Mahdi, A. A., Akhtar, M. J., Islam, N., Ashquin, M., & Venkatesh, T. (2010). Elevated blood lead levels and cytogenetic markers in buccal epithelial cells of painters in India: Ge notoxicity in painters exposed to lead containing paints. Environmental Science and Pollution Research, 17 (7), 1347

- 1354. http://dx.doi.org/10.1007/s11356

- 010

- 0319

- x

Kharoubi, O., Slimani, M., Aoues , A., & Seddik, L. (2008). Prophylactic effects of Wormwood on lipid peroxidation in an animal
model of lead intoxication. Indian Journal of Nephrology, 18 (2), 51

- 57. http://dx.doi.org/10.4103/0971

- 40 65.42333

Kharoubi, O., Slimani, M., Krouf, D., Seddik, L., & Aoues, A. (2008). Role of wormwood (Artemisia absinthium) extract on oxidative stress in ameliorating lead induced haematotoxicity. African Journal of Traditional, Complementary and
Alternative M edicines, 5 (3), 263

- 270. http://www.ncbi.nlm.nih.gov/pubmed/20161947

Khorasani, G., Shokrzade, M., Salehifar, E., Asadi, M., Shabankhani, B., & Rezaeinejad, S. (2008). The comparison of lead and
zin c plasma levels in gastric cancer patients with healthy volunteers. Research Journal of Biological Sciences, 3 (6),
631

- 634. http://dx.doi.org/10.3923/rjbsci.2008.631.634

Khotimchenko, M. Y., & Kolenchenko, E. A. (2007). Efficiency of low

- esterified pectin in toxic damage to the liver inflicted by
lead treatment. Bulletin of Experimental Biology and Medicine, 144 (1), 60

- 62. http://dx.doi. org/10.1007/s10517

- 007

- 0254

- 0

Kibayashi, K., Nakao, K. I., Taki, T., & Koyama, H. (2010). Changes in the brain after intracerebral implantation of metal lead in
a rat model. Journal of Neurotrauma, 27 , 1925

- 1934. http://dx.doi.org/10.1089/neu.2010.1379

Kim, D., & Lawrence, D. A. (2000). Immunotoxic effects of inorganic lead on host resistance of mice with different circling behavior preferences. Brain, Behavior, and Immunity, 14 , 305

- 317. http://dx.doi.org/10.1006/brbi.2000.0609

Kim, H.

- S., Lee, S.

- S., Lee, G.

- S., Hwangbo, Y., Ahn, K.

- D., & Lee, B.

- K. (2004). The protective effect of delta

- aminolevulinic acid dehydratase 1

- 2 and 2

- 2 isozymes against b lood lead with higher hematologic parameters. Environmental Health Perspectives, 112 (5), 538

- 541. http://dx.doi.org/10.1289/ehp.6464May 2011 5

- 410 DRAFT

– DO NOT CITE OR QUOTE Kim, H. K., Yoon, E. K., Jang, J., Hwang, M., Kim, J., Ha, J. H., . . . Pa rk, K. L. (2009). Assessment of heavy metal exposure via
the intake of oriental medicines in Korea. Journal of Toxicology and Environmental Health, Part A: Current Issues,
72 (21

- 22), 1336

- 1342. http ://dx.doi.org/10.1080/15287390903212485

Kim, J. H., Lee, K. H., Yoo, D. H., Kang, D., Cho, S. H., & Hong, Y. C. (2007). GSTM1 and TNF

- alpha gene polymorphisms and relations between blood lead and inflammatory markers in a non

- occupational population. Mutat ion Research: Genetic Toxicology and Environmental Mutagenesis, 629 (1), 32

- 39. http://dx.doi.org/10.1016/j.mrgentox.2007.01.004

Kim, R., Rotnitsky, A., Sparrow, D., Weiss, S. T., Wager, C., & H u, H. (1996). A longitudinal study of low

- level lead exposure and impairment of renal function The Normative Aging Study. JAMA: Journal of the American Medical Association,
275 , 1177

- 1181. ht tp://dx.doi.org/10.1001/jama.1996.03530390043032

Kim, Y., Kim, B. N., Hong, Y. C., Shin, M. S., Yoo, H. J., Kim, J. W., . . . Cho, S. C. (2009). Co

- exposure to environmental lead and manganese affects the intelligence of school

- aged children. NeuroToxicolo gy, 30 (4), 564

- 571. http://dx.doi.org/10.1016/j.neuro.2009.03.012

Kimber, I., Stonard, M. D., Gidlow, D. A., & Niewola, Z. (1986). Influence of chronic low

- level exposure to lead on plasma immunog lobulin concentration and cellular immune function in man. International Archives of Occupational and Environmental Health, 57 (2), 117

- 125. http://dx.doi.org/10.1007/BF00381379

Király, E., & Jones, D. G. ( 1982). Dendritic spine changes in rat hippocampal pyramidal cells after postnatal lead treatment: A golgi study. Experimental Neurology, 77 (1), 236

- 239. http://dx.doi.org/10.1016/0014

- 4886(82)90158

- 3

Kirberger, M., & Yang, J. J. (2008). Structural differences between Pb2+

- and Ca2+

- binding sites in proteins: Implications with respect to toxicity. Journal of Inorganic Biochemistry, 102 (10), 1901

- 1909. http://dx.doi.org/10.1016/j.jinorgbio.2008.06.014

Kishi, R., Ikeda, T., Miyake, H., Uchino, E., Tsuzuki, T., & Inoue, K. (1983). Effects of low lead exposure on neuro

- behavioral
function in the rat

. Archives of Environmental and Occupational Health, 38 , 25

- 33. http://www.ncbi.nlm.nih.gov/pubmed/6830315

Kishimoto, T., Oguri, T., Ueda, D., & Tada, M. (1995). Effect of lead on tube formation by c ultured human vascular endothelial
cells. Archives of Toxicology, 69 , 718

- 721. http://www.ncbi.nlm.nih.gov/pubmed/8572931

Kiziler, A. R., Aydemir, B., Onaran, I., Alici, B., Ozkara, H., Gulyasar, T., & Akyolcu, M. C. (2007). High levels of cadmium and lead in seminal fluid and blood of smoking men are associated with high oxidative stress and damage in infertile subjects. Biological Trace Element Research, 120 (1

- 3), 82

- 91. http://dx.doi.org/10.1007/s12011

- 007

- 8020

- 8

Klann, E., & Shelton, K. R. (1989). The effect of lead on the metabolism of a nuclear matrix protein which becomes prominent in
lead

- induced intranuclear inclusion bodies. Journal of Biological Chemistry, 264 , 16969

- 16972. http://www.ncbi.nlm.nih.gov/pubmed/2571613

Knowles, S. O., & Donaldson, W. E. (1990). Dietary modification of lead toxicity: Effects on fatty acid and eicosan oid
metabolism in chicks. Comparative Biochemistry and Physiology

- Part C: Toxicology and Pharmacology, 95 (1), 99

- 104. http://dx.doi.org/10.1016/0742

- 8413(90)90088

- Q

Knowles, S. O., & Donaldson, W. E. (1997). Lead disrupts eicosanoid metabolism, macrophage function, and disease resistance
in birds. Biological Trace Element Research, 60 , 13

- 26. http://dx.doi.org/10.1007/BF02783306

Kojima, M., & De gawa, M. (2006). Gender

- related difference in altered gene expression of a sterol regulatory element binding protein, SREBP

- 2, by lead nitrate in rats: Correlation with development of hypercholesterolemia. Journal of Applied Toxicology, 26 (4), 381

- 384. http://dx.doi.org/10.1002/jat.1138May 2011 5

- 411 DRAFT

– DO NOT CITE OR QUOTE Kojima, M., Sekikawa, K., Nemoto, K., & Degawa, M. (2005). Tumor necrosis factor

- alpha

- independent downregulation of hepatic cholesterol 7 alpha

- hydroxylase gene in mice treat ed with lead nitrate. Toxicological Sciences, 87 (2), 537

- 542. http://dx.doi.org/10.1093/toxsci/kfi267

Koka, S., Huber, S. M., Boini, K. M., Lang, C., Foller, M., & Lang, F. (2007). Lead decreases parasi temia and enhances survival of Plasmodium berghei

- infected mice. Biochemical and Biophysical Research Communications, 363 (3), 484

- 489. http://dx.doi.org/10.1016/j.bbrc.2007.08.173

Kolesarova, A., Roychoudhury, S., Slivkova, J., Sirotkin, A., Capcarova, M., & Massanyi, P. (2010). In vitro study on the effects of lead and mercury on porcine ovarian granulosa cells. Journal of Environmental Science and Health, Part A: Toxic/Hazardous S ubstances and Environmental Engineering, 45 (3), 320

- 331. http://dx.doi.org/10.1080/10934520903467907

Koller, L. D., Kerkvliet, N. I., & Exon, J. H. (1985). Neoplasia induced in male rats fed lead ac etate, ethyl urea, and sodium
nitrite. Toxicologic Pathology, 13 , 50

- 57. http://dx.doi.org/10.1177/019262338501300107

Korashy, H. M., & Ei

- Kadi, A. O. S. (2008). The role of redox

- sensitive transcr iption factors NF

- kappa B and AP

- 1 in the modulation of the Cyp1a1 gene by mercury, lead, and copper. Free Radical Biology and Medicine, 44 (5), 795

- 806. http://dx.doi.org/10.1016/j.freerad biomed.2007.11.003

Korashy, H. M., & El

- Kadi, A. O. S. (2004). Differential effects of mercury, lead and copper on the constitutive and inducible expression of aryl hydrocarbon receptor (AHR)

- regulated genes in cultured hepatoma Hepa 1c1c7 cells. Toxicolog y, 201 , 153

- 172. http://dx.doi.org/10.1016/j.tox.2004.04.011

Korashy, H. M., & El

- Kadi, A. O. S. (2008). NF

- kappa B and AP

- 1 are key signaling pathways in the modulation of NAD(P)H: Quinone oxidored uctase 1 gene by mercury, lead, and copper. Journal of Biochemical and Molecular Toxicology, 22 (4),
274

- 283. http://dx.doi.org/10.1002/jbt.20238

Kordas, K., Canfield, R. L., Lopez, P., Rosado, J. L., Vargas , G. G., Cebrian, M. E., . . . Stoltzfus, R. J. (2006). Deficits in cognitive function and achievement in Mexican first

- graders with low blood lead concentrations. Environmental Research, 100 (3), 371

- 386. http://dx.doi.org/10.1016/j.envres.2005.07.007

Kordas, K., Ettinger, A. S., Lamadrid

- Figueroa, H., Tellez

- Rojo, M. M., Hernandez

- Avila, M., Hu, H., & Wright, R. O. (2009). Methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C and G1793A genot ypes, and the relationship between maternal folate intake, tibia lead and infant size at birth. British Journal of Nutrition, 102 (6), 907

- 914. http://dx.doi.org/10.1017/s0007114509318280

Kottgen, A. , Selvin, E., Stevens, L. A., Levey, A. S., Van Lente, F., & Coresh, J. (2008). Serum cystatin C in the United States: The Third National Health and Nutrition Examination Survey (NHANES III). American Journal of Kidney Diseases,
51 (3), 385

- 394. http://dx.doi.org/10.1053/j.ajkd.2007.11.019

Krieg, E. F., Jr, Chrislip, D. W., Crespo, C. J., Brightwell, W. S., Ehrenberg, R. L., & Otto, D. A. (2005). The relationship
between blood lead levels and neurobe havioral test performance in NHANES III and related occupational studies. Public Health Reports, 120 , 240

- 251. http://www.ncbi.nlm.nih.gov/pubmed/16134563

Krieg, E. F., Jr. (2007). The relationships between blood lead levels and serum follicle stimulating hormone and luteinizing hormone in the third national health and nutrition examination survey. Environmental Research, 104 (3), 374

- 382.
h ttp://dx.doi.org/10.1016/j.envres.2006.09.009

Krieg, E. F., Jr., & Butler, M. A. (2009). Blood lead, serum homocysteine, and neurobehavioral test performance in the third National Health and Nutrition Examination Survey. NeuroToxicology, 30 (2), 281

- 289. http://dx.doi.org/10.1016/j.neuro.2008.12.014May 2011 5

- 412 DRAFT

– DO NOT CITE OR QUOTE Krieg, E. F., Jr., Butler, M. A., Chang, M. H., Liu, T. B., Yesupriya, A., Lindegren, M. L., & Dowling, N. (2009). Lead and cognitive function in ALAD g enotypes in the Third National Health and Nutrition Examination Survey. Neurotoxicology and Teratology, 31 (6), 364

- 371. http://dx.doi.org/10.1016/j.ntt.2009.08.003

Krieg, E. F., Jr., Butler, M. A., M

- h, C., Liu, T., Yesupriya, A., Dowling, N., & Lindegren, M. L. (2010). Lead and cognitive function in VDR genotypes in the Third National Health and Nutrition Examination Survey. Neurotoxicology and Teratology, 32 (2), 262

- 272. http://dx.doi.org/10.1016/j.ntt.2009.12.004

Kristal

- Boneh, E., Coller, D., Froom, P., Harari, G., & Ribak, J. (1999). The association between occupational lead exposure and serum cholesterol and lipoprotein levels. American J ournal of Public Health, 89 , 1083

- 1087. http://www.ncbi.nlm.nih.gov/pubmed/10394320

Kuhlmann, A. C., McGlothan, J. L., & Guilarte, T. R. (1997). Developmental lead exposure causes spatial learning d eficits in
adult rats. Neuroscience Letters, 233 , 101

- 104. http://dx.doi.org/10.1016/S0304

- 3940(97)00633

- 2

Kuo, C. Y., Wong, R. H., Lin, J. Y., Lai, J. C., & Lee, H. (2006). Accumulation of chro mium and nickel metals in lung tumors from lung cancer patients in Taiwan. Journal of Toxicology and Environmental Health, Part A: Current Issues, 69 (14),
1337

- 1344. http://dx.doi.org/10.1080/152873 90500360398

Kuruvilla, A., Pillay, V. V., Adhikari, P., Venkatesh, T., Chakrapani, M., Rao, H. T. J., . . . Rai, M. (2006). Clinical manifestations of lead workers of Mangalore, India. Toxicology and Industrial Health, 22 (9), 405

- 413. http://dx.doi.org/10.1177/0748233706074174

Laden, F., Neas, L. M., Dockery, D. W., & Schwartz, J. (2000). Association of fine particulate matter from different sources with daily mortality in six US cities. Environmental Health Perspectives, 108 , 941

- 947. http://www.ncbi.nlm.nih.gov/pubmed/11049813

Lai, C.

- C., Lin, H. H., Chen, C. W., Chen, S.

- H., & Chiu, T. H. (2002). Excitatory action of lead on rat sympathetic p reganglionic
neurons in vitro and in vivo. Life Sciences, 71 , 1035

- 1045. http://dx.doi.org/10.1016/S0024

- 3205(02)01789

- 7

Lai, L. H., Chou, S. Y., Wu, F. Y., Chen, J. J. H., & Kuo, H. W. (2008). Renal dysfunction and hyperuricemia with low blood lead levels and ethnicity in community

- based study. Science of the Total Environment, 401 (1

- 3), 39

- 43. http://dx.doi.org/10.1016/j.scitotenv. 2008.04.004

Lal, B., Murthy, R. C., Anand, M., Chandra, S. V., Kumar, R., Tripathi, O., & Srimal, R. C. (1991). Cardiotoxicity and hypertension in rats after oral lead exposure. Drug and Chemical Toxicology, 14 (3), 305

- 318. http://dx.doi.org/10.3109/01480549109002192

Lamadrid

- Figueroa, H., Téllez

- Rojo, M. M., Hernández

- Avila, M., Trejo

- Valdivia, B., Solano

- González, M., Mercado

- Garcia, A., . . . Wright, R. O. (2007). Association between the plasma/whole blood lead ratio and history of spontaneous abortion:
A nested cro ss

- sectional study. BMC Pregnancy and Childbirth, 7 , 22. http://dx.doi.org/10.1186/1471

- 2393

- 7

- 22

Lamb, M. R., Janevic, T., Liu, X., Cooper, T., Kline, J., & Factor

- Litvak, P. (2008). Environmental lea d exposure, maternal thyroid function, and childhood growth. Environmental Research, 106 (2), 195

- 202. http://dx.doi.org/10.1016/j.envres.2007.09.012

Landrigan, P. J., Suk, W. A., & Amler, R. W. ( 1999). Chemical wastes, children's health, and the Superfund Basic Research Program. Environmental Health Perspectives, 107 , 423

- 427. http://www.ncbi.nlm.nih.gov/pubmed/10339440

Lang, F., Gulbins, E ., Lerche, H., Huber, S. M., Kempe, D. S., & Foller, M. (2008). Eryptosis, a window to systemic disease. Cellular Physiology and Biochemistry, 22 (5

- 6), 373

- 380. http://www.ncbi.nlm.nih.gov/pubmed/19 088418May 2011 5

- 413 DRAFT

– DO NOT CITE OR QUOTE Lanphear, B. P., Dietrich, K., Auinger, P., & Cox, C. (2000). Cognitive deficits associated with blood lead concentrations <10 microg/dL in US children and adolescents. Public Health Reports, 115 , 521

- 529. http://www.ncbi.nlm.nih.gov/pubmed/11354334

Lanphear, B. P., Hornung, R., Khoury, J., Yolton, K., Baghurst, P., Bellinger, D. C., . . . Roberts, R. (2005). Low

- level environmental lead exposure and children's intellectual function: An intern ational pooled analysis. Environmental Health Perspectives, 113 , 894

- 899. http://www.ncbi.nlm.nih.gov/pubmed/16002379

Lara

- Tejero, M., & Pamer, E. G. (2004). T cell responses to Listeria monocytogen es. Current Opinion in Microbiology, 7 (1), 45

- 50. http://dx.doi.org/10.1016/j.mib.2003.12.002

Laschi

- Loquerie, A., Decotes, J., Tachon, P., & Evreux, J. C. (1984). Influence of lead acetate on hyper sensitivity: Experimental study. Journal of Immunopharmacology, 6 (1

- 2), 87

- 93. http://dx.doi.org/10.3109/08923978409026461

Lasley, S. M., & Gilbert, M. E. (2002). Rat hippocampal glutamate and GABA release exhibit biphasic effects as a function of chronic lead exposure level. Toxicological Sciences, 66 , 139

- 147. http://dx.doi.org/10.1093/toxsci/66.1.139

Lau, Y. S., Camoratto, A. M., White, L. M. , & Moriarty, C. M. (1991). Effect of lead on TRH and GRF binding in rat anterior pituitary membranes. Toxicology, 68 , 169

- 179. http://dx.doi.org/10.1016/0300

- 483X(91)90019

- W

Laviola, G., Hannan, A. J., Marci, S., Solinas, M., & Jaber, M. (2008). Effects of enriched environment on animal models of neurodegenerative diseases and psychiatric disorders. Neurobiology of Disease, 31 , 159

- 168. http://dx.doi.org/10.1016/j.nbd.2008.05.001

Laviola, G., Rea, M., Moreley

- Fletcher, S., Di Carlo, S., Bacosi, A., De Simone, R., . . . Pacifici, R. (2004). Beneficial effects of enriched environment on adolescent rats from stressed pregna ncies. European Journal of Neuroscience, 20 , 1655

- 1664. http://dx.doi.org/10.1111/j.1460

- 9568.2004.03597.x

Lawton, L. J., & Donaldson, W. E. (1991). Lead

- induced tissue fatty acid alterations and lipid peroxidation. Biological Trace Element Research, 28 (2), 83

- 97. http://dx.doi.org/10.1007/BF02863075

Leasure, J. L., Giddabasappa, A., Chaney, S., Johnson, J. E., Pothakos, K., Lau, Y. S., & Fox, D. A. (2008). Low

- level human equivalent gestational lead exposure produces sex

- specific motor and coordination abnormalities and late

- onset obesity in year

- old mice. Environmental Health Perspectives, 116 (3), 355

- 361. http://dx.doi.org/10.1289/ehp.10862

Ledda

- Columbano, G. M., Coni, O., Curto, M., Giacomini, O., Faa, G., Sarma, D. S. R., & Columbano, A. (1992). Mitogen

induced liver hyperplasia does not substitute for compensatory regeneration during promo tion of chemical hepatocarcinogenesis. Carcinogenesis, 13 , 379

- 383. http://dx.doi.org/10.1093/carcin/13.3.379

LeDoux, J. (2003). The synaptic self: How our brains become who we are . New York, NY: Peng uin Group.

Lee, B.

- K., Lee, G.

- S., Stewart, W. F., Ahn, K.

- D., Simon, D., Kelsey, K. T., . . . Schwartz, B. S. (2001). Associations of blood pressure and hypertension with lead dose measures and polymorphisms in the vitamin D receptor and delta

aminolevuli nic acid dehydratase genes. Environmental Health Perspectives, 109 , 383

- 389. http://www.ncbi.nlm.nih.gov/pubmed/11335187

Lee, D. H., Blomhoff, R., & Jacobs, D. R., Jr. (2004). Is serum gamma glutamy ltransferase a marker of oxidative stress? Free Radical Research, 38 (6), 535

- 539. http://dx.doi.org/10.1080/10715760410001694026

Lee, D. H., Lim, J. S., Song, K., Boo, Y., & Jacobs, D. R. (2006). Graded associations of blood lead and urinary cadmium
concentrations with oxidative

- stress

- related markers in the US population: Results from the Third National Health and Nutrition Examination Survey. Environmental Health Perspectives, 114 (3), 350

- 354. http://dx.doi.org/10.1289/ehp.8518May 2011 5

- 414 DRAFT

– DO NOT CITE OR QUOTE Lee, J.

- E., Chen, S., Golemboski, K. A., Parsons, P. J., & Dietert, R. R. (2001). Developmental windows of differential lead

induced immunotoxicity in chickens. Toxicology, 156 , 161

- 170. http://dx.doi.org/10.1016/S0300

- 483X(00)00350

- 4

Lee, J. J., & Battles, A. H. (1994). Lead toxicity via arachidonate signal transduction to growth responses in the splenic macrophag e. Environmental Research, 67 , 209

- 219. http://dx.doi.org/10.1006/enrs.1994.1075

Lee, M. K., Cho, S. Y., Kim, D. J., Jang, J. Y., Shin, K. H., Park, S. A., . . . Kim, M. J. (2005). Du

- zhong (Eucommia ulmoides Oliv.) cortex water extract alters heme biosynthesis and erythrocyte antioxidant defense system in lead

- administered

rats. Journ al of Medicinal Food, 8 (1), 86

- 92. http://dx.doi.org/10.1089/jmf.2005.8.86

Lee, M. Y., Shin, J. H., Han, H. S., & Chung, J. H. (2006). In vivo effects of lead on erythrocytes following chronic exposure through drinking water. Archives of Pharmacal Research, 29 (12), 1158

- 1163. http://www.ncbi.nlm.nih.gov/pubmed/17225467

Lee, T.

- H., Tseng, M.

- C., Chen, C.

- J., & Lin, J.

- L. (2009). Association of high body lead store with severe intracranial carotid
atherosclerosis. NeuroToxicology, 30 (6), 876

- 880. http://dx.doi.org/10.1016/j.neuro.2009.07.004

Legare, M. E., Barhoumi, R., Burghardt, R. C., & T iffany

- Castiglioni, E. (1993). Low

- level lead exposure in cultured astroglia: Identification of cellular targets with vital fluorescent probes. NeuroToxicology, 14 (2

- 3), 267

- 272. http://www.ncbi.nlm. nih.gov/pubmed/8247400

Leret, M. L., Garcia

- Uceda, F., & Antonio, M. T. (2002). Effects of maternal lead administration on monoaminergic, GABAergic and glutamatergic systems. Brain Research Bulletin, 58 (5), 469

- 473. http://dx.doi.org/10.1016/S0361

- 9230(02)00819

- 5

Leseney, A. M., Dème, D., Legué, O., Ohayon, R., Chanson, P., Sales, J. P., . . . Virion, A. (1999). Biochemical characterization of a Ca2+/NAD(P)H

- dependent H2O2 generator in human thyr oid tissue. Biochimie, 81 (4), 373

- 380. http://dx.doi.org/10.1016/S0300

- 9084(99)80084

- 4

Levey, A. S., Bosc, J. P., Lewis, J. B., Greene, T., Rogers, N., & Roth, D. (1999). A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Annals of Internal Medicine, 130 (6), 461

- 470. http://www.ncbi.nlm.ni h.gov/pubmed/10075613

Levey, A. S., Greene, T., Kusek, J. W., & Beck, G. (2000). A simplified equation to predict glomerular filtration rate from serum
creatinine. Journal of the American Society of Nephrology: JASN, 11 , A0828.

Levey, A. S., Stevens, L. A ., Schmid, C. H., Zhang, Y. P., Castro, A. F., Feldman, H. I., . . . Coresh, J. (2009). A new equation to estimate glomerular filtration rate. Annals of Internal Medicine, 150 (9), 604

- 612. http://ww w.ncbi.nlm.nih.gov/pubmed/19414839

Leviton, A., Bellinger, D., Allred, E. N., Rabinowitz, M., Needleman, H., & Schoenbaum, S. (1993). Pre

- and postnatal low

- level lead exposure and children's dysfunction in school. Environmental Research, 60 , 30

- 43. http://dx.doi.org/10.1006/enrs.1993.1003

Lewis, M., & Pitts, D. (2004). Inorganic lead exposure in the rat activates striatal cFOS expression at lower blood levels and inhibits amphetamine

- induced cFOS expre ssion at higher blood levels. Journal of Pharmacology and Experimental Therapeutics, 310 (2), 815

- 820. http://dx.doi.org/10.1124/jpet.103.063941

Li, J. P., Wang, C. Y., Tang, Y. A., Lin, Y. W., & Yang, J. L. (2008). Role of extracellular signal

- regulated kinase (ERK) signaling in nucleotide excision repair and genotoxicity in response to As(III) and Pb(II). Pure and Applied Chemistry, 80 (12),
2735

- 2750. http://dx.doi.org/10.1351/pac200880122735

Li, N., Yu, Z. L., Wang, L., Zheng, Y. T., Jia, J. X., Wang, Q., . . . Li, W. J. (2009). Early

- life lead exposure affects the activity of
TNF

- alpha and expression of SNARE complex in hippocampus of mous e pups. Biological Trace Element Research,
132 (1

- 3), 227

- 238. http://dx.doi.org/10.1007/s12011

- 009

- 8551

- 2May 2011 5

- 415 DRAFT

– DO NOT CITE OR QUOTE Li, N., Yu, Z. L., Wang, L., Zheng, Y. T., Jia, J. X., Wang, Q., . . . Li, W. J. (2010). Inc reased tau phosphorylation and beta amyloid in the hipocampus of mouse pups by early life lead exposure. Acta Biologica Hungarica, 61 (2), 123

- 134. http://dx.doi.org/10.1556/ABiol.61.2010.2.1

Li, S., Zhao, Z., Zhou, X., & Liu, S. (2008). The effect of lead on intracellular Ca<sup>2+</sup> in mouse lymphocytes. Toxicology In Vitro, 22 (8), 1815

- 1819. http://dx.doi.org/10.1016/j.tiv.2008.08.005

Li Chen, T., Wise, S. S., Kraus, S., Shaffiey, F., Levine, K. M., Thompson, W. D., . . . Wise, J. P., Sr. (2009). Particulate hexavalent chromium is cytotoxic and genotoxic to the North Atlantic right whale (Eubalaena glacialis) lung and skin
fibroblasts. En vironmental and Molecular Mutagenesis, 50 (5), 387

- 393. http://dx.doi.org/10.1002/em.20471

Lidsky, T. I., & Schneider, J. S. (2003). Lead neurotoxicity in children: basic mechanisms and clinical correlates. B rain, 126 , 5

- 19. http://dx.doi.org/10.1093/brain/awg014

Lidsky, T. I., & Schneider, J. S. (2004). Lead and public health: Review of recent findings, re

- evaluation of clinical risks. Journal of Environmen tal Monitoring, 6 (4), 36N

- 42N. http://dx.doi.org/10.1039/B403259b

Lilienthal, H., Kohler, K., Turfeld, M., & Winneke, G. (1994). Persistent increases in scotopic b

- wave amplitudes after lead exposure in monk eys. Experimental Eye Research, 59 , 203

- 209. http://dx.doi.org/10.1006/exer.1994.1098

Lilienthal, H., Lenaerts, C., Winneke, G., & Hennekes, R. (1988). Alteration of the visual evoked potential and the electroretinogram in lead

- treated monkeys. Neurotoxicology and Teratology, 10 (5), 417

- 422. http://dx.doi.org/10.1016/0892

- 0362(88)90002

- 5

Lilis, R., Fischbein, A., Eisinger, J., Blumberg, W. E., Diamond, S., Anderson, H. A., . . . Selikoff, I. J. (1977). Prevalence of lead disease among secondary lead smelter workers and biological indicators of lead exposure. Environmental Research, 14

, 255

- 285. http://dx.doi.org/10.1016/0013

- 9351(77)90037

- 8

Lin

- Tan, D. T., Lin, J. L., Wang, L. H., Wang, L. M., Huang, L. M., Liu, L., . . . Huang, Y. L. (2007). Fasting glucose levels in predicting 1

- year all

- cause mortality in patients who do not have diabetes and are on maintenance hemodialysis. Journal of the American Society of Nephrology: JASN, 18 (8), 2385

- 2391. http://dx.doi.org/10.1681/Asn.2006121409

Lin

- Tan, D. T., Lin, J. L., Yen, T. H., Chen, K. H., & Huang, Y. L. (2007). Long

- term outcome of repeat ed lead chelation therapy in progressive non

- diabetic chronic kidney diseases. Nephrology, Dialysis, Transplantation, 22 (10), 2924

- 2931. http://dx.doi.org/10.1093/ndt/gfm342

Lin, C. N., Wang, L. H., & Shen , K. H. (2009). Determining urinary trace elements (Cu, Zn, Pb, As, and Se) in patients with
bladder cancer. Journal of Clinical Laboratory Analysis, 23 (3), 192

- 195. http://dx.doi.org/10.1002/jcla.20318

Li n, J.

- L., Lin

- Tan, D.

- T., Chen, K.

- H., Hsu, C.

- W., Yen, T.

- H., Huang, W.

- H., & Huang, Y.

- L. (2010). Blood lead levels
association with 18

- month all

- cause mortality in patients with chronic peritoneal dialysis. Nephrology, Dialysis, Transplantation, 25 (5), 1627

- 1633. http://dx.doi.org/10.1093/ndt/gfp663

Lin, J.

- L., Lin

- Tan, D.

- T., Hsu, K.

- H., & Yu, C.

- C. (2003). Environmental lead exposure and progression of chronic renal diseases in patients without diabete s. New England Journal of Medicine, 348 , 277

- 286. http://dx.doi.org/10.1097/01.ASN.0000118529.01681

Lin, J.

- L., Lin

- Tan, D.

- T., Li, Y.

- J., Chen, K.

- H., & Huang, Y.

- L. (2006). Low

- level environ mental exposure to lead and progressive chronic kidney diseases. The American Journal of Medicine, 119 (8), 1

- 9. http://dx.doi.org/10.1016/j.amjmed.2006.01.005

Lin, J.

- L., Lin

- Tan, D.

- T., Yen, T.

- H., Hsu, C.

- W., Jenq, C.

- C., Chen, K.

- H., . . . Huang, Y.

- L. (2008). Blood lead levels, malnutrition, inflammation, and mortality in patients with diabetes treated by long

- term hemodialysis. American Journal of Kidney Diseases, 51 (1), 107

- 115. http://dx.doi.org/10.1053/j.ajkd.2007.10.002May 2011 5

- 416 DRAFT

– DO NOT CITE OR QUOTE Lin, J.

- L., Lin

- Tan, D.

- T., Yu, C.

- C., Li, Y.

- J., Huang, Y.

- Y., & Li, K.

- L. (2006). Environmental exposure to lead and progressive diabetic nephropathy in patients with type II diabetes. Kidney International, 69 (11), 2049

- 2056. http://dx.doi.org/10.1038/sj.ki.5001505

Lin, J.

- L., Yu, C.

- C., Lin

- Tan, D.

- T., & Ho, H.

- H. (2001). Lead chelation therapy and urate excret ion in patients with chronic renal diseases and gout. Kidney International, 60 , 266

- 271. http://dx.doi.org/10.1046/j.1523

- 1755.2001.00795.x

Lin, R. H., Lee, C. H., Chen, W. K., & Lin

- Shiau, S . Y. (1994). Studies on cytotoxic and genotoxic effects of cadmium nitrate and lead nitrate in Chinese hamster ovary cells. Environmental and Molecular Mutagenesis, 23 , 143

- 149. http://dx.doi.org/10.100 2/em.2850230212

Lin, T. A., & Tai

- yi, J. (2007). Benchmark dose approach for renal dysfunction in workers exposed to lead. Environmental Toxicology, 22 (3), 229

- 233. http://dx.doi.org/10.1002/tox.20260

Lind, S. E., Park, J. S., & Drexler, J. W. (2009). Pyrithione and 8

- hydroxyquinolines transport lead across erythrocyte
membranes. Translational Research: Journal of Laboratory and Clinical Medicine, 154 (3), 153

- 159. http://dx.doi.org/10.1016/j.trsl.2009.06.002

Lison, D., Raguzzi, F., & Lauwerys, R. (1990). Comparison of the effects of auranofin, heavy metals and retinoids on protein kinase C in vitro and on a protein kinase C mediated response in macrophages. Basic and Clinical Pharmacology and Toxicology, 67 , 239

- 242. http://dx.doi.org/10.1111/j.1600

- 0773.1990.tb00820.x

Liu, H. T., Niu, R. Y., Wang, J. M., He, Y., & Wang, J. D. (2008). Changes caused by fluoride and lead in energy metabolic enzyme activities in the repro ductive system of male offspring rats. Fluoride, 41 (3), 184

- 191.

Liu, J., Kershaw, W. C., & Klaassen, C. D. (1991). The protective effect of metallothionein on the toxicity of various metals in rat primary hepatocyte culture. Toxicology and Applied Pharma cology, 107 (1), 27

- 34. http://dx.doi.org/10.1016/0041

- 008X(91)90327

- B

Liu, L., Wong, T., Pozza, M., Lingenhoehl, K., Wang, Y., Sheng, M., & Auberson, Y. (2004). Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science, 304 (5673), 1021

- 1024. http://dx.doi.org/10.1126/science.1096615

Liu, M. Y., Hsieh, W. C., & Yang, B. C. (2000). In vitro aberrant g ene expression as the indicator of lead

- induced neurotoxicity in
U

- 373MG cells. Toxicology, 147 , 59

- 64. http://dx.doi.org/10.1016/S0300

- 483X(00)00186

- 4

Llanos, M. N., & Ronco, A. M. (2009). Feta l growth restriction is related to placental levels of cadmium, lead and arsenic but not
with antioxidant activities. Reproductive Toxicology, 27 (1), 88

- 92. http://dx.doi.org/10.1016/j.reprotox .2008.11.057

Lohmann, C., & Bonhoeffer, T. (2008). A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia. Neuron, 59 (2), 253

- 260. http://dx.doi.org/10.1016 /j.neuron.2008.05.025

Lolin, Y., & O'Gorman, P. (1988). An intra

- erythrocyctic low molecular weight lead

- binding protein in acute and chronic lead exposure and its possible protective role in lead toxicity. Annals of Clinical Biochemistry, 25 (6), 688

- 697. http://www.ncbi.nlm.nih.gov/pubmed/3254111

Long, G. J., Pounds, J. G., & Rosen, J. F. (1992). Lead intoxication alters basal and parathyroid hormone

- regulated cellular
calcium homeostasis in rat oste osarcoma (ROS 17/28) cells. Calcified Tissue International, 50 (5), 451

- 458. http://dx.doi.org/10.1007/BF00296777

Long, G. J., & Rosen, J. F. (1992). Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism and collagen synthesis in clonal rat osteoblastic (ROS 17/28) cells. Toxicology and Applied Pharmacology, 114 (1), 63

- 70. http://dx.doi.org/10.1016/0041

- 008X(92)90097

- CMay 2011 5

- 417 DRAFT

– DO NOT CITE OR QUOTE Long, G. J., Rosen, J. F., & Schanne, F. A. X. (1994). Lead activation of protein kinase C from rat brain: Determination of free calcium, lead, and zinc by 19F NMR. Journ al of Biological Chemistry, 269 , 834

- 837. http://www.ncbi.nlm.nih.gov/pubmed/8288636

Lopez, C. M., Pineiro, A. E., Nunez, N., Avagnina, A. M., Villaamil, E. C., & Roses, O. E. (2000). Thyroid hormone changes in males exposed to lead in the Buenos Aires area (Argentina). Pharmacological Research, 42 , 599

- 602. http://dx.doi.org/10.1006/phrs.2000.0734

Louis, E. D., Applegate, L., Graziano, J. H., Par ides, M., Slavkovich, V., & Bhat, H. K. (2005). Interaction between blood lead
concentration and delta

- amino

- levulinic acid dehydratase gene polymorphisms increases the odds of essential tremor. Movement Disorders, 20 (9), 1170

- 1177. http://dx.doi.org/10.1002/mds.20565

Louis, E. D., Jurewicz, E. C., Applegate, L., Factor

- Litvak, P., Parides, M., Andrews, L., . . . Todd, A. (2003). Association
between essential tremor and blood lead concentration. Environmenta l Health Perspectives, 111 , 1707

- 1711. http://www.ncbi.nlm.nih.gov/pubmed/14594619

Lu, H., Guizzetti, M., & Costa, L. G. (2002). Inorganic lead activates the mitogen

- activated protein kinase kinase

- mitogen

activated protein kinase

- p90RSK signaling pathway in human astrocytoma cells via a protein kinase C

- dependent
mechanism. Journal of Pharmacology and Experimental Therapeutics, 300 , 818

- 823. http://www.ncbi.nlm.nih.gov/pubmed/11861786

Lundstrom, N. G., Englyst, V., Gerhardsson, L., Jin, T., & Nordberg, G. (2006). Lung cancer development in primary smelter
workers: A nested case

- referent study. Journal of Occupational and Environmental Medicine , 48 (4), 376

- 380. http://dx.doi.org/10.1097/01.jom.0000201556.95982.95

Lurie, D. I., Brooks, D. M., & Gray, L. C. (2006). The effect of lead on the avian auditory brainstem. NeuroToxicology , 27 (1),
108

- 117. http://dx.doi.org/10.1016/j.neuro.2005.07.005

Lustberg, M., & Silbergeld, E. (2002). Blood lead levels and mortality. Archives of Internal Medicine, 162 (21), 2443

- 2449. http://www.ncbi.nlm.nih.gov/pubmed/12437403

Lutz, P. M., Wilson, T. J., Ireland, A. L., Gorman, J. S., Gale, N. L., Johnson, J. C., & Hewett, J. E. (1999). Elevated immunoglobulin E (IgE) levels in children with exposure to environmental lead. Toxicology, 134 , 63

- 78. http://dx.doi.org/10.1016/S0300

- 483X(99)00036

- 0

Lynes, M. A., Fontenot, A. P., Lawrence, D. A., Rosenspire, A. J., & Pollard, K. M. (2006). Gene expression influences on metal
immunomodulation. Toxicology and Applied Pharmacology, 210 (1

- 2), 9

- 16. http://dx.doi.org/10.1016/j.taap.2005.04.021

Ma, T., Chen, H., & H o, I. (1999). Effects of chronic lead (Pb) exposure on neurobehavioral function and dopaminergic neurotransmitter receptors in rats. Toxicology Letters, 105 (2), 111

- 121. http://dx.doi.org/10.101 6/S0378

- 4274(98)00388

- 9

MacDonald, J. F., Jackson, M. F., & Beazely, M. A. (2006). Hippocampal long

- term synaptic plasticity and signal amplification of NMDA receptors. Critical Reviews in Neurobiology, 18 (1

- 2), 71

- 84.

Madhavi, D., Devi, K. R., Rao, K. K. , & Reddy, P. P. (2007). Modulating effect of Phyllanthus fruit extract against lead genotoxicity in germ cells of mice. Journal of Environmental Biology, 28 (1), 115

- 117. http://www.ncbi.nlm.nih.gov /pubmed/17717996

Maeda, K., Sugino, H., Hirose, T., Kitagawa, H., Nagai, T., Mizoguchi, H., . . . Yamada, K. (2007). Clozapine prevents a decrease in neurogenesis in mice repeatedly treated with phencyclidine. Journal of Pharmacological Sciences, 103 (3), 2 99

- 308. http://dx.doi.org/10.1254/jphs.FP0061424May 2011 5

- 418 DRAFT

– DO NOT CITE OR QUOTE Maes, M. (2011). Depression is an inflammatory disease, but cell

- mediated immune activation is the key component of depression. Progress in Neuro

- Psycho pharmacology and Biological Psychiatry, 35 (3), 664

- 675. http://dx.doi.org/10.1016/j.pnpbp.2010.06.014

Magalhaes, C., Socodato, R., & Paes

- de

- Carvalho, R. (2006). Nitric oxide regulates the prolife ration of chick embryo retina cells by a cyclic GMP

- independent mechanism. International Journal of Developmental Neuroscience, 24 (1), 53

- 60. http://dx.doi.org/10.1016/j.ijdevneu.2005.10.004

Ma gyar, J. S., Weng, T. C., Stern, C. M., Dye, D. F., Rous, B. W., Payne, J. C., . . . Godwin, H. A. (2005). Reexamination of lead(II) coordination preferences in sulfur

- rich sites: Implications for a critical mechanism of lead poisoning. Journal of
the Amer ican Chemical Society, 127 (26), 9495

- 9505. http://dx.doi.org/10.1021/ja0424530

Mahaffey, K. R., Capar, S. G., Gladen, B. C., & Fowler, B. A. (1981). Concurrent exposure to lead, cadmium, and arsenic. Effects on toxicity and tissue metal concentrations in the rat. Translational Research: Journal of Laboratory and Clinical Medicine, 98 (4), 463

- 481. http://www.ncbi.nlm.nih.gov/pubmed/7288264

Mahaffey, K. R., & Fowler, B. A. (1977). Effects of concurrent administration of lead, cadmium, and arsenic in the rat. Environmental Health Perspectives, 19 , 165

- 171. http://www.ncbi.nlm.nih.gov/pubmed/198203

Maitani, T., Watahiki, A., & Suzuki, K. T. (1986). Induction of metallothionein after lead administration by three injection routes
in mice. Toxicology and Applied Pharmacology, 8 3 , 211

- 217. http://dx.doi.org/10.1016/0041

- 008X(86)90298

- X

Maizlish, N. A., Parra, G., & Feo, O. (1995). Neurobehavioural evaluation of Venezuelan workers exposed to inorganic lead. Occupational and Environmental Medicine, 52 , 408

- 414. http://www.ncbi.nlm.nih.gov/pubmed/7627319

Malvezzi, C. K., Moreira, E. G., Vassilieff, I., Vassilieff, V. S., & Cordellini, S. (2001). Effect of L

- arginine, dimercaptosuccinic acid (DMSA) and the association of L

- arginine and DMSA on tissue lead mobilization and blood pressure level in plumbism. Brazilian Journal of Medical and Biological Research, 34 , 1341

- 1346. http://dx.doi.org/10.1590/S0100

- 879X2001001000016

Marchlewicz, M., Baranowska

- Bosiacka, I., Kolasa, A., Kondarewicz, A., Chlubek, D., & Wiszniewska, B. (2009). Disturbances of energetic metabolism in rat epididymal epithelial cells as a co nsequence of chronic lead intoxication. BioMetals,
22 (6), 877

- 887. http://dx.doi.org/10.1007/s10534

- 009

- 9238

- z

Marchwinska

- Wyrwal, E., Dziubanek, G., Skrzypek, M., & Hajok, I. (2010). Study of the h ealth effects of long

- term exposure to cadmium and lead in a region of Poland. International Journal of Environmental Health Research, 20 (2), 81

- 86. http://dx.doi.org/10.1080/09603120903394656

Marcu s, D. K., Fulton, J. J., & Clarke, E. J. (2010). Lead and conduct problems: A meta

- analysis. Journal of Clinical Child and Adolescent Psychology, 39 (2), 234

- 241. http://dx.doi.org/10.1080/1537441100 3591455

Markovac, J., & Goldstein, G. W. (1988a). Lead activates protein kinase C in immature rat brain microvessels. Toxicology and Applied Pharmacology, 95 , 14

- 23. http://dx.doi.org/10.1016/004 1

- 008X(88)90242

- 6

Markovac, J., & Goldstein, G. W. (1988b). Picomolar concentrations of lead stimulate brain protein kinase C. Nature, 334 , 71

- 73. http://dx.doi.org/10.1038/334071a0

Marques, C. C., Nunes, A. C., Pinheiro, T., Lopes, P. A., Santos, M. C., Viegas

- Crespo, A. M., . . . Mathias, M. L. (2006). An
assessment of time

- dependent effects of lead exposure in algerian mice (Mus spretus) using different methodological approaches. Biological Trace Element R esearch, 109 (1), 75

- 90. http://dx.doi.org/10.1385/BTER:109:1:075

Marques, M., Millas, I., Jimenez, A., Garcia

- Colis, E., Rodriguez

- Feo, J. A., Velasco, S., . . . Lopez

- Farre, A. (2001). Alteration of

t he soluble guanylate cyclase system in the vascular wall of lead

- induced hypertension in rats. Journal of the American Society of Nephrology: JASN, 12 , 2594

- 2600. http://www.ncbi.nlm.nih.gov/pubmed/ 11729227May 2011 5

- 419 DRAFT

– DO NOT CITE OR QUOTE Martin, D., Glass, T. A., Bandeen

- Roche, K., Todd, A. C., Shi, W. P., & Schwartz, B. S. (2006). Association of blood lead and tibia lead with blood pressure and hypertension in a community sample of older adults. American Journal of Epidemiology, 163 (5), 467

- 478. http://dx.doi.org/10.1093/aje/kwj060

Mas

- Oliva, J. (1989). Effect of lead on the erythrocyte (Ca2+,Mg2+)

- ATPase activity Calmodulin involvement. Molecular and Cellular Biochemistry, 89 , 87

- 93. http://dx.doi.org/10.1007/BF00228283

Massadeh, A. M., Al

- Safi, S. A., Momani, I. F., Alomary, A. A., Jaradat, Q. M., & AlKofahi, A. S. (2007). Garlic (Allium sativum L.) as a potential antidote fo r cadmium and lead intoxication: Cadmium and lead distribution and analysis in different mice organs. Biological Trace Element Research, 120 (1

- 3), 227

- 234. http://dx.doi.org/10.1007/s12011

- 007

- 8017

- 3

Massanyi, P., Lukac, N., Makarevich, A. V., Chrenek, P., Forgacs, Z., Zakrzewski, M., . . . Flesarova, S. (2007). Lead

- induced alterations in rat kidneys and testes in vivo. Journal of Environmental Science and Health, Part A: Toxic/Hazardous
Substances and Environmental Engineering, 42 (5), 671

- 676. http://dx.doi.org/10.1080/10934520701244474

Massó

- González, E. L., & Antonio

- García, M. T. (2009). Natural antioxidants protect against lead

- induced da mage during pregnancy and lactation in rat's pups. Ecotoxicology and Environmental Safety, 72 (8), 2137

- 2142. http://dx.doi.org/10.1016/j.ecoenv.2009.03.013

Massó, E. L., Corredor, L., & Antonio, M. T. (2007). Oxidative damage in liver after perinatal intoxication with lead and/or
cadmium. Journal of Trace Elements in Medicine and Biology, 21 (3), 210

- 216. http://dx.doi.org/10.1016/j.jtemb. 2007.03.002

Mauel, J., Ransijn, A., & Buchmuller

- Rouiller, Y. (1989). Lead inhibits intracellular killing of Leishmania parasites and extracellular cytolysis of target cells by macrophages exposed to macrophage activating factor. Journal of the Lepidopteri sts Society, 45 , 401

- 409. http://www.ncbi.nlm.nih.gov/pubmed/2496192

McCabe, M. J., Jr., Singh, K. P., & Reiners, J. J., Jr. (1999). Lead intoxication impairs the generation of a delayed type hyperse nsitivity response. Toxicology, 139 , 255

- 264. http://dx.doi.org/10.1016/S0300

- 483X(99)00147

- X

McCabe, M. J., & Lawrence, D. A. (1991). Lead, a major environmental pollutant, is immunomodulatory by its differential
effects on CD4+ T cell subsets. Toxicology and Applied Pharmacology, 111 , 13

- 23. http://dx.doi.org/10.1016/0041

- 008X(91)90129

- 3

McElroy, J. A., Shafer, M. M., Gangnon, R. E., Crouch, L. A., & Newcomb, P. A. (2008). Urinary lead exposure and breast cancer risk in a population

- based case

- control study. Cancer Epidemiology Biomarkers and Prevention, 17 (9), 2311

- 2317. ht tp://dx.doi.org/10.1158/1055

- 9965.epi

- 08

- 0263

McGrath, J., Saha, S., Chant, D., & Welham, J. (2008). Schizophrenia: A concise overview of incidence, prevalence, and mortality. Epidemiologic Reviews, 30 (1), 67

- 76. http://dx.doi.org/10.1093/epirev/mxn001

McLachlin, J. R., Goyer, R. A., & Cherian, M. G. (1980). Formation of lead

- induced inclusion bodies in primary rat kidney epithelial cell cultures: effect of actinomycin D and cycloheximide. Toxicology and Applied Pharmacology, 56 , 418

- 431. http://dx.doi.org/10.1016/0041

- 008X(80)90076

- 9

McNeill, D. R., Wong, H. K., Narayana, A., & Wilson, D. M. (2007). Lead promotes abasic site accumulation and co

mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1. Molecular Carcinogenesis, 46 (2),
91

- 99. http://dx.doi.org /10.1002/mc.20196

Meeker, J. D., Rossano, M. G., Protas, B., Diamond, M. P., Puscheck, E., Daly, D., . . . Wirth, J. J. (2008). Cadmium, lead, and other metals in relation to semen quality: Human evidence for molybdenum as a male reproductive toxicant.
Env ironmental Health Perspectives, 116 (11), 1473

- 1479. http://dx.doi.org/10.1289/ehp.11490May 2011 5

- 420 DRAFT

– DO NOT CITE OR QUOTE Meeker, J. D., Rossano, M. G., Protas, B., Padmanahban, V., Diamond, M. P., Puscheck, E., . . . Wirth, J. J. (2010).
E nvironmental exposure to metals and male reproductive hormones: Circulating testosterone is inversely associated with blood molybdenum. Fertility and Sterility, 93 (1), 130

- 140. http://dx.doi. org/10.1016/j.fertnstert.2008.09.044

Méndez

- Gómez, J., García

- Vargas, G. G., López

- Carrillo, L., Calderón

- Aranda, E. S., Gómez, A., Vera, E., . . . Rojas, E. (2008). Genotoxic effects of environmental exposure to arsenic and lead on children in region Lagunera, Mexico. Annals of the
New York Academy of Sciences, 1140 , 358

- 367. http://dx.doi.org/10.1196/annals.1454.027

Menegazzi, M., Carcereri

- De Prati, A., Suzuki, H., Shinozuka, H., Pibiri, M., Piga, R., . . . Ledda

- Columbano, G. M. (1997 ). Liver cell proliferation induced by nafenopin and cyproterone acetate is not associated with increases in activation of transcription factors NF

- "kappa"B and AP

- 1 or with expression of tumor necrosis factor "alpha". Hepatology, 25 , 585

- 592. http://www.ncbi.nlm.nih.gov/pubmed/9049203

Meng, X.

- M., Zhu, D.

- M., Ruan, D.

- Y., She, J.

- Q., & Luo, L. (2005). Effects of chronic lead exposure on H MRS of hippocampus and frontal lobes in children. Neurology, 6 4 , 1644

- 1647. http://www.ncbi.nlm.nih.gov/pubmed/15883337

Menke, A., Muntner, P., Batuman, V., Silbergeld, E. K., & Guallar, E. (2006). Blood lead below 0.48 micromol/L (10 microg/dL) and mortality among US adults. Circulation, 114 (13), 1388

- 1394. http://dx.doi.org/10.1161/circulationaha.106.628321

Messerschmidt, A., Huber, R., & Poulos, T. (Eds.). (2001). Handbook of metalloproteins

. Chichester, UK: Wiley.

Mielzyńska, D., Siwińska, E., Kapka, L., Szyfter, K., Knudsen, L. E., & Merlo, D. F. (2006). The influence of environmental exposure to complex mixtures including PAHs and lead on genotoxic effects in children living in Upper Silesia , Poland. Mutagenesis, 21 (5), 295

- 304. http://dx.doi.org/10.1093/mutage/gel037

Miller, T. E., Golemboski, K. A., Ha, R. S., Bunn, T., Sanders, F. S., & Dietert, R. R. (1998). Developmental exposure to l ead causes persistent immunotoxicity in Fischer 344 rats. Toxicological Sciences, 42 , 129

- 135. http://dx.doi.org/10.1006/toxs.1998.2424

Min, J. Y., Min, K. B., Kim, R., Cho, S. I., & Paek, D. (2008). B lood lead levels and increased bronchial responsiveness. Biological Trace Element Research, 123 (1

- 3), 41

- 46. http://dx.doi.org/10.1007/s12011

- 008

- 8099

- 6

Min, M. Y. O., Singer, L. T., Kirchner, H. L., Minnes, S., Short, E., Hussain, Z., & Nelson, S. (2009). Cognitive development and
low

- level lead exposure in poly

- drug exposed children. Neurotoxicology and Teratology, 31 (4), 225

- 231. http://dx.doi.org/10.1016/j.ntt.2009.03.002

Minozzo, R., Deimling, L. I., Gigante, L. P., & Santos

- Mello, R. (2004). Micronuclei in peripheral blood lymphocytes of workers exposed to lead. DNA Repair, 565 , 53

- 60. http://dx.doi.org/10.1016/j.mrgentox.2004.09.003

Minozzo, R., Deimling, L. I., & Santos

- Mello, R. (2010). Cytokinesis

- blocked micronucleus cytome and comet assays in peripheral blood lymphocytes of wor kers exposed to lead considering folate and vitamin B12 status. Mutation Research, 697 (1

- 2), 24

- 32. http://dx.doi.org/10.1016/j.mrgentox.2010.01.009

Miranda, M. L., Kim, D., Galeano, M., Paul, C. J., Hull, A. P., & Morgan, S. P. (2007). The relationship between early childhood blood lead levels and performance on end of grade tests. Environmental Health Perspectives, 115 (8), 1242

- 1247.
ht tp://www.ncbi.nlm.nih.gov/pubmed/17687454

Miranda, M. L., Kim, D., Reiter, J., Overstreet Galeano, M. A., & Maxson, P. (2009). Environmental contributors to the achievement gap. NeuroToxicology, 30 (6), 1019

- 1024. http://dx.doi.org/10.1016/j.neuro.2009.07.012

Mishra, K. P. (2009). Lead exposure and its impact on immune system: A review. Toxicology In Vitro, 23 (6), 969

- 972. http://dx.doi.org/1 0.1016/j.tiv.2009.06.014May 2011 5

- 421 DRAFT

– DO NOT CITE OR QUOTE Mishra, K. P., Chauhan, U. K., & Naik, S. (2006). Effect of lead exposure on serum immunoglobulins and reactive nitrogen and oxygen intermediate. Human and Experimental Toxicology, 25 (11), 661

- 665. http://dx.doi.org/10.1177/0960327106070453

Mishra, K. P., Rani, R., Yadav, V. S., & Naik, S. (2010). Effect of lead exposure on lymphocyte subsets and activation markers. Immunopharmacology and Immunotoxicology, 32 (3), 446

- 449. http://dx.doi.org/10.3109/08923970903503668

Mishra, K. P., Singh, V. K., Rani, R., Yadav, V. S., Chandran, V., Srivastava, S. P., & Seth, P. K. (2003). Effect of lead exposure on the immune response of some occupationally exposed individuals. Toxicology, 188 , 251

- 259. http://dx.doi.org/10.1016/S0300

- 483X(03)00091

- X

Mistry, P., Lucier, G. W., & Fowler, B. A. (1985). High

- affinity lead binding p roteins in rat kidney cytosol mediate cell

- free
nuclear translocation of lead. Journal of Pharmacology and Experimental Therapeutics, 232 , 462

- 469. http://www.ncbi.nlm.nih.gov/pubmed/3968645

Mistry, P., Mastri, C., & Fowler, B. A. (1986). Influence of metal ions on renal cytosolic lead

- binding proteins and nuclear uptake of lead in the kidney. Biochemical Pharmacology, 35 , 711

- 713. h ttp://dx.doi.org/10.1016/0006

- 2952(86)90371

- 0

Moffitt, T. E., Caspi, A., Rutter, M., & Silva, P. A. (2001). Sex differences in antisocial behavior: Conduct disorder, delinquency, and violence in the Dunedin Longitudinal Study . Cambridge, UK: University of Cambridge Press.

Mohammad, I. K., Mahdi, A. A., Raviraja, A., Najmul, I., Iqbal, A., & Thuppil, V. (2008). Oxidative stress in painters exposed to
low lead levels. Arhiv za Higijenu Rada i Toksikologiju, 59 (3), 161

- 169. http://dx.doi.org/10.2478/10004

- 1254

- 59

- 2008

- 1883

Molero, L., Carrasco, C., Marques, M., Vaziri, N. D., Mateos

- Caceres, P. J., Casado, S., . . . Lopez

- Farre, A. J. (2006).
Involvement of endothelium and endothelin

- 1 in lead

- indu ced smooth muscle cell dysfunction in rats. Kidney International, 69 (4), 685

- 690. http://dx.doi.org/10.1038/sj.ki.5000103

Monsell, S. (2003). Task switching. Trends in Cognitive Sciences, 7 (3), 134

- 140. http://dx.doi.org/10.1016/S1364

- 6613(03)00028

- 7

Monteiro, H. P., Abdalla, D. S., Augusto, O., & Bechara, E. J. (1989). Free radical generation during delta

- aminolevulinic acid
autoxidation: ind uction by hemoglobin and connections with porphyrinpathies. Archives of Biochemistry and Biophysics, 271 (1), 206

- 216. http://dx.doi.org/10.1016/0003

- 9861(89)90271

- 3

Monteiro, H. P., Abdalla, D. S ., Faljoni

- Alàrio, A., & Bechara, E. J. (1986). Generation of active oxygen species during coupled autoxidation of oxyhemoglobin and delta

- aminolevulinic acid. Biochimica et Biophysica Acta, 881 (1), 100

- 106. http://dx.doi.org/10.1016/0304

- 4165(86)90102

- 9

Monteiro, H. P., Bechara, E. J., & Abdalla, D. S. (1991). Free radicals involvement in neurological porphyrias and lead poisoning. Molecular and Cellular Biochemistry, 103 (1), 73

- 83. http://dx.doi.org/10.1007/BF00229595

Montenegro, M. F., Barbosa, F., Jr., Sandrim, V. C., Gerlach, R. F., & Tanus

- Santos, J. E. (2006). A polymorphism in the delta

aminolevulinic acid dehydratase gene modifies plasma/w hole blood lead ratio. Archives of Toxicology, 80 (7), 394

- 398. http://dx.doi.org/10.1007/s00204

- 005

- 0056

- y

Moore, J. F., & Goyer, R. A. (1974). Lead

- induced inclusion bodies: Composition and probable role in lead metabolism. Environmental Health Perspectives, 7 , 121

- 127. http://www.ncbi.nlm.nih.gov/pubmed/4364645

Moore, J. F., Goyer, R. A., & Wilson, M. (1973). Lead

- induced inclusion bodies: Solubility, amino acid content, and relationship to residual acidic nuclear proteins. Laboratory Investigation, 29 , 488

- 494. http://www.ncbi.nlm.nih.gov/pubmed/4356630May 2011 5

- 422 DRAFT

– DO NOT CITE OR QUOTE Moorhouse, S. R., Carden, S., Drewitt, P. N., Eley, B. P., Hargreaves, R. J., & Pelling, D. (1988). The effect of chronic low level lead exposure on blood

- brain barrier function in the developing rat. Biochemical Pharmacology, 37 , 4539

- 4547. http://dx.doi.org/10.1016/0006

- 2952(88)90670

- 3

Moorman, W. J., Skaggs, S. R., Clark, J. C., Turner, T. W., Sharpnack, D. D., Murrell, J. A., . . . Schrader, S. M. (19 98). Male reproductive effects of lead, including species extrapolation for the rabbit model. Reproductive Toxicology, 12 (3), 333

- 346. http://dx.doi.org/10.1016/S0890

- 6238(98)00010

- 0

Moreley

- Fle tcher, S., Rea, M., Maccari, S., & Laviola, G. (2003). Environmental enrichment during adolescence reverses the effects of prenatal stress on play behaviour and HPA axis reactivity in rats. European Journal of Neuroscience, 18

, 3367

- 3374. http://dx.doi.org/10.1111/j.1460

- 9568.2003.03070.x

Morita, Y., Sakai, T., Araki, S., Araki, T., & Masuyama, Y. (1997). Nicotinamide adenine dinucleotide synthetase activity in erythrocytes as a tool for the b iological monitoring of lead exposure. International Archives of Occupational and Environmental Health, 70 , 195

- 198. http://dx.doi.org/10.1007/s004200050206

Morris, S., van Aardt, W. J., & Ahern, M. D. (2005). The effect of lead on the metabolic and energetic status of the Yabby, Cherax destructor, during environmental hypoxia. Aquatic Toxicology, 75 (1), 16

- 31. http://dx.doi.org/10.1016/j.aqua tox.2005.07.001

Mosad, S. M., Ghanem, A. A., El

- Fallal, H. M., El

- Kannishy, A. M., El Baiomy, A. A., Al

- Diasty, A. M., & Arafa, L. F. (2010). Lens cadmium, lead, and serum vitamins C, E, and beta carotene in cataractous smoking patients. Current Eye
Resear ch, 35 (1), 23

- 30. http://dx.doi.org/10.3109/02713680903362880

Moser, R., Oberley, T. D., Daggett, D. A., Friedman, A. L., Johnson, J. A., & Siegel, F. L. (1995). Effects of lead administration
on de veloping rat kidney: I Glutathione S

- transferase isoenzymes. Toxicology and Applied Pharmacology, 131 (1), 85

- 93. http://dx.doi.org/10.1006/taap.1995.1050

Mudipalli, A. (2007). Lead hepatotoxicity & pot ential health effects. Indian Journal of Medical Research, 126 (6), 518

- 527.

Muller, S., Gillert, K.

- E., Krause, C., Gross, U., L'Age

- Stehr, J., & Diamantstein, T. (1977). Suppression of delayed type hypersensitivity of mice by lead. Cellular and Molecular Life Sciences (CMLS), 33 , 667

- 668. http://dx.doi.org/10.1007/BF01946564

Muntner, P., He, J., Vupputuri, S., Coresh, J., & Batuman, V. (2003). Blood lead and chronic kidney disease in the general United
St ates population: Results from NHANES III. Kidney International, 63 (3), 1044

- 1050. http://dx.doi.org/10.1046/j.1523

- 1755.2003.00812.x

Muntner, P., Menke, A., Batuman, V., Rabito, F. A., He, J., & Todd, A. C. (2007). Association of tibia lead and blood lead with
end

- stage renal disease: A pilot study of African

- Americans. Environmental Research, 104 (3), 396

- 401. http://dx.doi.org/10.1016/j.envres.2007.04.001

Muntner, P., Menke, A., DeSalvo, K. B., Rabito, F. A., & Batuman, V. (2005). Continued decline in blood lead levels among
adults in the United States

- The National Health an d Nutrition Examination Surveys. Archives of Internal Medicine,
165 (18), 2155

- 2161. http://www.ncbi.nlm.nih.gov/pubmed/16217007

Murakami, K., Feng, G., & Chen, S. G. (1993). Inhibition of brain prot ein kinase

- C subtypes by lead. Journal of Pharmacology and Experimental Therapeutics, 264 , 757

- 761. http://www.ncbi.nlm.nih.gov/pubmed/8437124

Naha, N., & Chowdhury, A. R. (2006). Inorganic lead expo sure in battery and paint factory: Effect on human sperm structure and functional activity. Journal of University of Occupational and Environmental Health, 28 (2), 157

- 171. http://www.ncbi.nlm.nih.go v/pubmed/16780224May 2011 5

- 423 DRAFT

– DO NOT CITE OR QUOTE Naha, N., & Manna, B. (2007). Mechanism of lead induced effects on human spermatozoa after occupational exposure. Kathmandu University Medical Journal, 5 (1), 85

- 94. http://www.ncbi .nlm.nih.gov/pubmed/18603992

Naicker, N., Norris, S. A., Mathee, A., Becker, P., & Richter, L. (2010). Lead exposure is associated with a delay in the onset of puberty in South African adolescent females: Findings from the birth to twenty cohort. Science o f the Total Environment, 408 (21), 4949

- 4954. http://dx.doi.org/10.1016/j.scitotenv.2010.07.037

Nakagawa, K. (1989). Hepatic glutathione metabolism in mice acutely treated with lead acetate. Ja panese Journal of Pharmacology, 51 (2), 173

- 179. http://dx.doi.org/10.1254/jjp.51.173

Nakagawa, K. (1991). Decreased glutathione S

- transferase activity in mice livers by acute treatment with lead, independe nt of alteration in glutathione content. Toxicology Letters, 56 (1

- 2), 13

- 17. http://dx.doi.org/10.1016/0378

- 4274(91)90085

- K

Nakajima, T., Deguchi, T., Kagawa, K., Hikita, H., Ueda, K., Katagishi, T., . . . Ashihara, T. (1995). Identification of apoptotic hepatocytes in situ in rat liver after lead nitrate administration. Journal of Gastroenterology, 30 (6), 725

- 730. http://dx.doi.org/10.1007/BF0234 9638

Nampoothiri, L. P., Agarwal, A., & Gupta, S. (2007). Effect of co

- exposure to lead and cadmium on antioxidant status in rat ovarian granulose cells. Archives of Toxicology, 81 (3), 145

- 150. http ://dx.doi.org/10.1007/s00204

- 006

- 0133

- x

Nampoothiri, L. P., & Gupta, S. (2006). Simultaneous effect of lead and cadmium on granulosa cells: A cellular model for ovarian toxicity. Reproductive Toxicology, 21 (2), 179

- 185. http://dx.doi.org/10.1016/j.reprotox.2005.07.010

Nampoothiri, L. P., & Gupta, S. (2008). Biochemical effects of gestational coexposure to lead and cadmium on reproductive performance, placenta, a nd ovary. Journal of Biochemical and Molecular Toxicology, 22 (5), 337

- 344. http://dx.doi.org/10.1002/jbt.20246

Nandi, S., Gupta, P. S., Selvaraju, S., Roy, S. C., & Ravindra, J. P. (2010). Effects of exposu re to heavy metals on viability, maturation, fertilization, and embryonic development of buffalo (Bubalus bubalis) oocytes in vitro. Archives of Environmental Contamination and Toxicology, 58 (1), 194

- 204. http://dx.doi.org/10.1007/s00244

- 009

- 9342

- 7

Narayana, K., & Al

- Bader, M. (2011). Ultrastructural and DNA damaging effects of lead nitrate in the liver. Experimental and Toxicologic Pathology, 63 (1

- 2), 43

- 51. http://dx.doi.org/10.1016/j.etp.2009.09.007

National Kidney Foundation. (2002). K/DOQI clinical practice guidelines for chronic kidney disease: Evaluation, classification, and stratification, Part 4: Definition and stages of chronic kidney disease. American Journal of Kidney Diseases, 39 (2),
S46

- S75. http://dx.doi.org/10.1053/ajkd.2002.30943

Nava

- Hernandez, M. P., Hauad

- Marroquin, L. A., Bassol

- Mayagoitia, S., Garcia

- Arenas, G., Mercado

- Hernandez, R., Echavarri

Guzman, M. A., & Cerda

- Flores, R. M. (2009). Lead

- , cadmium

- , and arsenic

- induced DNA damage in rat germinal
cells. DNA and Cell Biolo gy, 28 (5), 241

- 248. http://dx.doi.org/10.1089/dna.2009.0860

Navarro

- Moreno, L. G., Quintanar

- Escorza, M. A., Gonzalez, S., Mondragon, R., Cerbon

- Solorzano, J., Valdes, J., & Calderon

Salinas, J. V. (200 9). Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells. Toxicology In Vitro, 23 (7), 1298

- 1304. http://dx.doi.org/10.1016/j.tiv.200 9.07.020

Navas

- Acien, A., Schwartz, B. S., Rothenberg, S. J., Hu, H., Silbergeld, E. K., & Guallar, E. (2008). Bone lead levels and blood pressure endpoints: A meta

- analysis. Epidemiology, 19 (3), 496

- 504. http://dx.doi.org/10.1097/EDE.0b013e31816a2400

Navas

- Acien, A., Selvin, E., Sharrett, A. R., Calderon

- Aranda, E., Silbergeld, E., & Guallar, E. (2004). Lead, cadmium, smoking, and increased risk of peripheral arterial disease. Circulation, 109 , 3 196

- 3201. http://dx.doi.org/10.1161/01.CIR.0000130848.18636.B2May 2011 5

- 424 DRAFT

– DO NOT CITE OR QUOTE Navas

- Acien, A., Silbergeld, E. K., Sharrett, A. R., Calderon

- Aranda, E., Selvin, E., & Guallar, E. (2005). Metals in urine an d peripheral arterial disease. Environmental Health Perspectives, 113 , 164

- 169. http://dx.doi.org/10.1289/ehp.7329

Navas

- Acien, A., Tellez

- Plaza, M., Guallar, E., Muntner, P., Silbergeld, E., Jaar, B., & Wea ver, V. (2009). Blood cadmium and lead and chronic kidney disease in US adults: A joint analysis. American Journal of Epidemiology, 170 (9), 1156

- 1164. http://dx.doi.org/10.1093/aje/kwp248

Nawrot, T. S., Thijs, L., Den Hond, E. M., Roels, H. A., & Staessen, J. A. (2002). An epidemiological re

- appraisal of the association between blood pressure and blood lead: A meta

- analysis. Journal of Human Hypertension, 16 , 123

- 131. http://dx.doi.org/10.1038/sj/jhh/1001300

Nayak, B. N., Ray, M., & Persaud, T. V. N. (1989). Maternal and fetal chromosomal aberrations in mice following prenatal exposure to subembryotoxic doses of lead nitrate. Cells Tissu es Organs, 135 , 185

- 188. http://dx.doi.org/10.1159/000146751

Nayak, B. N., Ray, M., Persaud, T. V. N., & Nigli, M. (1989). Relationship of embryotoxicity to genotoxicity of lead nitrate in
mice. Experimenta l and Toxicologic Pathology, 36 , 65

- 73. http://www.ncbi.nlm.nih.gov/pubmed/2767206

Neal, A., Stansfield, K., Worley, P., Thompson, R., & Guilarte, T. (2010). Lead exposure during synaptogenesis alter s vesicular proteins and impairs vesicular release: Potential role of NMDA receptor

- dependent BDNF signaling. Toxicological Sciences, 116 (1), 249

- 263. http://dx.doi.org/10.1093/toxsci/kfq111

Neaton, J. D., Kuller, L., Stamler, J., & Wentworth, D. N. (1995). Impact of systolic and diastolic blood pressure on cardiovascular mortality. In J. H. Laragh & B. M. Brenner (Eds.), Hypertension: Pathophysiology, diagnosis, and
management (2nd ed., pp. 127

- 144). Ne w York, NY: Raven Press Ltd.

Needleman, H. L., & Gatsonis, C. A. (1990). Low

- level lead exposure and the IQ of children: A meta

- analysis of modern studies. JAMA: Journal of the American Medical Association, 263 (5), 673

- 678. http://dx.doi.org/10.1001/jama.1990.03440050067035

Needleman, H. L., Gunnoe, C., Leviton, A., Reed, R., Peresie, H., Maher, C., & Barrett, P. (1979). Deficits in psychologic and classroom performance of children with elevat ed dentine lead levels. New England Journal of Medicine, 300 (13), 689

- 695. http://www.ncbi.nlm.nih.gov/pubmed/763299

Needleman, H. L., McFarland, C., Ness, R. B., Fienberg, S. E., & Tobin, M. J. (2002 ). Bone lead levels in adjudicated delinquents: A case control study. Neurotoxicology and Teratology, 24 (6), 711

- 717. http://dx.doi.org/10.1016/S0892

- 0362(02)00269

- 6

Needleman, H. L., Riess, J. A., Tobin, M. J., Biesecker, G. E., & Greenhouse, J. B. (1996). Bone lead levels and delinquent
behavior. JAMA: Journal of the American Medical Association, 275 , 363

- 369. http://dx.doi.org/10 .1001/jama.1996.03530290033034

Needleman, H. L., Schell, A., Bellinger, D., Leviton, A., & Allred, E. N. (1990). The long

- term effects of exposure to low doses
of lead in childhood: An 11

- year follow

- up report. New England Journal of Medicine, 322 , 83

- 88. http://www.ncbi.nlm.nih.gov/pubmed/2294437

Nehez, M., Lorencz, R., & Desi, I. (2000). Simultaneous action of cypermethrin and two environmental pollutant metals, cadmium and lead, on bone marrow cell chromosomes of rats in subchronic administration. Ecotoxicology and Environmental Safety, 45 , 55

- 60. http://dx.doi.org/10.1006/eesa.1999.1831

Nemsadze, K., Sanikidze, T., Ratiani, L., Gabunia, L., & Sharashenidze, T. (2009). Mechanisms of lead

- induced poisoning. Georgian medical news (172

- 173), 92

- 96. http://www.ncbi.nlm.nih.gov/pubmed/19644200May 2011 5

- 425 DRAFT

– DO NOT CITE OR QUOTE Nenov, V. D., Taal, M. W., Sakharova, O. V., & Bre nner, B. M. (2000). Multi

- hit nature of chronic renal disease. Current Opinion in Nephrology and Hypertension, 9 , 85

- 97. http://www.ncbi.nlm.nih.gov/pubmed/10757212

Nestmann, E. R., & Zhang, B. W. ( 2007). Chromosome aberration test of Pigment Yellow 34 (lead chromate) in Chinese hamster ovary cells. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 633 (2), 126

- 132. http ://dx.doi.org/10.1016/j.mrgentox.2007.05.012

Neuberger, J. S., Hu, S. C., Drake, K. D., & Jim, R. (2009). Potential health impacts of heavy

- metal exposure at the Tar Creek Superfund site, Ottawa County, Oklahoma. Environmental Geochemistry and Health, 31 (1 ), 47

- 59. http://dx.doi.org/10.1007/s10653

- 008

- 9154

- 0

Newland, M. C., Yezhou, S., Logdberg, B., & Berlin, M. (1994). Prolonged behavioral effects of in utero exposure to lead or methyl mercury: Redu ced sensitivity to changes in reinforcement contingencies during behavioral transitions and in steady state. Toxicology and Applied Pharmacology, 126 (1), 6

- 15. http://dx.doi.org/10.1006/taap.1994.1084

Ni, Z., Hou, S., Barton, C. H., & Vaziri, N. D. (2004). Lead exposure raises superoxide and hydrogen peroxide in human
endothelial and vascular smooth muscle cells. Kidney International, 66 , 2329

- 2336.

Nicolescu, R., Petcu, C., Cordeanu, A., Fabritius, K. , Schlumpf, M., Krebs, R., . . . Winneke, G. (2010). Environmental exposure to lead, but not other neurotoxic metals, relates to core elements of ADHD in Romanian children: Performance and questionnaire data. Environmental Research, 110 (5), 476

- 483. http://dx.doi.org/10.1016/j.envres.2010.04.002

Nigg, J., Knottnerus, G., Martel, M., Nikolas, M., Cavanagh, K., Karmaus, W., & Rappley, M. (2008). Low blood lead levels associated with clinically diagn osed attention

- deficit/hyperactivity disorder and mediated by weak cognitive control. Biological Psychiatry, 63 , 325

- 331. http://dx.doi.org/10.1016/j.biopsych.2007.07.013

Nikolova, P., & Kavald zhieva, B. (1991). The effect of certain heavy metals (Mn and Pb) on parameters of erythrocyte energy
metabolism. Central European Journal of Public Health, 35 , 361

- 365. http://www.ncbi.nlm.nih.gov/p ubmed/1804866

Nordberg, M., Winblad, B., Fratiglioni, L., & Basun, H. (2000). Lead concentrations in elderly urban people related to blood pressure and mental performance: Results from a population

- based study. American Journal of Industrial Medicine,
38 (3 ), 290

- 294. http://dx.doi.org/10.1002/1097

- 0274(200009)38:3<290::AID

- AJIM7>3.0.CO;2

- T

Novak, J., & Banks, R. O. (1995). Lead and nickel alter the cardiorenal actions of endothelin in the rat. Proceedings of the Society for Experimental Biology and Medicine, 208 (2), 191

- 198. http://www.ncbi.nlm.ni h.gov/pubmed/7831352

Nowak, P., Szczerbak, G., Nitka, D., Kostrzewa, R. M., Sitkiewicz, T., & Brus, R. (2008). Effect of prenatal lead exposure on nigrostriatal neurotransmission and hydroxyl radical formation in rat neostriatum: Dopaminergic

- nitrergic int eraction. Toxicology, 246 (1), 83

- 89. http://dx.doi.org/10.1016/j.tox.2007.12.026

NRC. (National Research Council). (2000). Toxicological effects of methylmercury . Washington, DC: National Academy Pr ess.

NTP. (National Toxicology Program). (2004). Eleventh report on carcinogens: Lead (CAS no 7439

- 92

- 1) and lead compounds

. Research Triangle Park, NC: U.S. Department of Health and Human Services, National Toxicology Program. Retrieved
from http://ntp.niehs.nih.gov/ntp/roc/eleventh/profiles/s101lead.pdf

.

Nuyts, G. D., Van Vlem, E., Thys, J., De Leersnijder, D., D'Haese, P. C., Elseviers, M. M., & De Broe, M. E. (1995). New

occu pational risk factors for chronic renal failure. Lancet, 346 , 7

- 11. http://dx.doi.org/10.1016/S0140

- 6736(95)92648

- 8

Oberley, T. D., Friedman, A. L., Moser, R., & Siegel, F. L. (1995). Effects of lead administration on developing rat kidney: II Functional, morphologic, and immunohistochemical studies. Toxicology and Applied Pharmacology, 131 (1), 94

- 107. http://dx.doi.org/10.1006/taap.1995.1051May 2011 5

- 426 DRAFT

– DO NOT CITE OR QUOTE Oberto, M. N., Evans, H. L., & Guidotti, A. (1996). Lead (Pb+2) promotes apoptosis in newborn rat cerebellar neurons: Pathological implications. Journal of Pharmacology and Experimental Therapeutics, 279 (1), 435

- 442. http://www.ncbi.nlm.nih.gov/pubmed/8859023

Obhodas, J., Tucak

- Zorić, S., Kutle, A., Valković, V. (2007). Indications for synergetic and antagonistic effects between trace
elements in the environment to human health. Collegium Antropologi cum, 31 , 209

- 219. http://www.ncbi.nlm.nih.gov/pubmed/17598404

Oishi, H., Nakashima, M., Totoki, T., & Tomokuni, K. (1996). Chronic lead exposure may inhibit endothelium

- dependent hyperpolarizing fac tor in rats. Journal of Cardiovascular Pharmacology, 28 (4), 558

- 563. http://www.ncbi.nlm.nih.gov/pubmed/8891882

Olaleye, S. B., Adaramoye, O. A., Erigbali, P. P., & Adeniyi, O. S. (2007). Lead exposu re increases oxidative stress in the gastric
mucosa of HCl/ethanol

- exposed rats. World Journal of Gastroenterology, 13 (38), 5121

- 5126. http://www.ncbi.nlm.nih.gov/pubmed/17876879

Olewińska, E., Kasp erczyk, A., Kapka, L., Kozłowska, A., Pawlas, N., Dobrakowski, M., . . . Kasperczyk, S. (2010). Level of DNA damage in lead

- exposed workers. Annals of Agricultural and Environmental Medicine, 17 (2), 231

- 236. http://www.ncbi.nlm.nih.gov/pubmed/21186764

Oliveira, H., Spano, M., Santos, C., & Pereira, M. D. (2009). Lead chloride affects sperm motility and acrosome reaction in mice: Lead affects mice sperm mo tility and acrosome reaction. Cell biology and toxicology, 25 (4), 341

- 353. http://dx.doi.org/10.1007/s10565

- 008

- 9088

- 4

Olszewski, J., Latusinski, J., Kita, A., Pietkiewicz, P., Starostecka, B., & Ma jak, J. (2006). Comparative assessment of aluminum and lead concentrations in serum and tissue bioptates in patients with laryngeal papilloma or cancer. B

- ENT, 2 (2), 47

- 49. http://www.ncbi.nlm.nih.g ov/pubmed/16910286

Ong, C. N., & Lee, W. R. (1980a). Distribution of lead

- 203 in human peripheral blood in vitro. Occupational and Environmental Medicine, 37 , 78

- 84. http://www.ncbi.nlm.nih.gov/pubme d/7370196

Ong, C. N., & Lee, W. R. (1980b). High affinity of lead for fetal haemoglobin. Occupational and Environmental Medicine, 37 (3),
292

- 298. http://dx.doi.org/10.1136/oem.37.3.292

Opler, M. G. A., B rown, A. S., Graziano, J., Desai, M., Zheng, W., Schaefer, C., . . . Susser, E. S. (2004). Prenatal lead exposure,
delta

- aminolevulinic acid, and schizophrenia. Environmental Health Perspectives, 112 , 548

- 552. http://dx.doi.org/10.1289/ehp.10464

Opler, M. G. A., Buka, S. L., Groeger, J., McKeague, I., Wei, C., Factor

- Litvak, P., . . . Susser, E. S. (2008). Prenatal lead exposure, delta

- aminolevulinic acid, and schizophrenia: Further evidence. Environmental H ealth Perspectives, 116 (11),
1586

- 1590. http://dx.doi.org/10.1289/ehp.10464

Orisakwe, O. E., Nwachukwu, E., Osadolor, H. B., Afonne, O. J., & Okocha, C. E. (2007). Liver and kidney function tests amongst pa int factory workers in Nkpor, Nigeria. Toxicology and Industrial Health, 23 (3), 161

- 165. http://dx.doi.org/10.1177/0748233707081908

Oskarsson, A., & Fowler, B. A. (1985). Effects of lead inclusion bo dies on subcellular distribution of lead in rat kidney: the relationship to mitochondrial function. Experimental and Molecular Pathology, 43 , 397

- 408. http://dx.doi.org/10.1016/0014

- 4800(85)90076

- 0

Oskarsson, A., Squibb, K. S., & Fowler, B. A. (1982). Intracellular binding of lead in the kidney: The partial isolation and characterization of postmitochondrial lead binding components. Biochemical and Biophysical Research Communications, 104 (1), 290

- 298. http://dx.doi.org/10.1016/0006

- 291X(82)91973

- 8May 2011 5

- 427 DRAFT

– DO NOT CITE OR QUOTE Osman, K., Pawlas, K., Schutz, A., Gazdzik, M., Sokal, J. A., & Vahter, M. (1999). Lead exposure and hearing effects in children in Katowice, P oland. Environmental Research, 80 (1), 1

- 8. http://dx.doi.org/10.1006/enrs.1998.3886

Osterode, W., & Ulberth, F. (2000). Increased concentration of arachidonic acid in erythrocyte membranes in chronically lead

exposed men. Journal of Toxicology and Environmental Health, Part A: Current Issues, 59 (2), 87

- 95. http://dx.doi.org/10.1080/009841000156998

Ostro, B., Feng, W.

- Y., Broadwin, R., Green, S., & Lipsett, M. (2007). The effects of components of fine particulate air pollution on mortality in California: Results from CALFINE. Environmental Health Per spectives, 115 (1), 13

- 19. http://www.ncbi.nlm.nih.gov/pubmed/17366813

Oteiza, P. I., Kleinman, C. G., Demasi, M., & Bechara, E. J. H. (1995). 5

- Aminolevulinic acid induces iron release from ferritin

. Archives of Biochemistry and Biophysics, 316 (1), 607

- 611. http://dx.doi.org/10.1006/abbi.1995.1080

Otto, D. A., Baumann, S. B., Robinson, G. S., Schroeder, S. R., Kleinbaum, D. G., Barton, C. N., & M ushak, P. (1985). Auditory and visual evoked potentials in children with undue lead absorption. Toxicologist, 5 , 81.

Otto, D. A., & Fox, D. A. (1993). Auditory and visual dysfunction following lead exposure. NeuroToxicology, 14 (2

- 3), 191

- 207. http://www.ncbi.nlm.nih.gov/pubmed/8247393

Overmann, S. R. (1977). Behavioral effects of asymptomatic lead exposure during neonatal development in rats. Toxicology and Applied Pharmacology, 41 , 459

- 471. http://dx.doi.org/10.1016/S0041

- 008X(77)80002

- 1

Ozsoy, S. Y., Ozsoy, B., Ozyildiz, Z., & Aytekin, I. (In Press). (In Press). Protective effect of L

- carnitine on experimental lead toxicity in rats: A clinical, histopathological and immunohistochemical study. Biotechnic and Histochemistry

. http://dx.doi.org/10.3109/10520295.2010.529825

Pace, B. M., Lawrence, D. A., Behr, M. J., Parsons, P. J., & Dias, J. A. (2005). Neonatal lead exposure changes quality of sperm and number of macrophages in testes of BALB/c mice. Toxicology, 210 (2

- 3), 247

- 256. http://dx.doi.org/10.1016/j.tox.2005.02.004

Pace, T. W. W., & Miller, A. H. (2009). Cytokines and glucocorticoid receptor signaling: Relevance to major depression. Annals of the New York Ac ademy of Sciences, 1179 , 86

- 105. http://dx.doi.org/10.1111/j.1749

- 6632.2009.04984.x

Paglia, D. E., & Valentine, W. N. (1975). Characteristics of a pyrimidine

- specific 5'

- nucleotidase in human erythrocytes. Journal of Biological Chemistry, 250 (20), 7973

- 7979. http://www.ncbi.nlm.nih.gov/pubmed/240846

Palus, J., Rydzynski, K., Dziubaltowska, E., Wyszynska, K., Natarajan, A. T., & Nilsson, R

. (2003). Genotoxic effects of occupational exposure to lead and cadmium. Mutation Research: Genetic Toxicology and Environmental Mutagenesis,
540 (1), 19

- 28. http://dx.doi.org/10.1016/S1383

- 5718(03)00167

- 0

Pandya, C. D., Pillai, P. P., & Gupta, S. S. (2010). Lead and cadmium co

- exposure mediated toxic insults on hepatic steroid metabolism and antioxidant system of adult male rats. Biological Trace Element Resea rch, 134 (3), 307

- 317. http://dx.doi.org/10.1007/s12011

- 009

- 8479

- 6

Park, S. K., Elmarsafawy, S., Mukherjee, B., Spiro, A., III, Vokonas, P. S., Nie, H., . . . Hu, H. (2010). Cumulative lead exposure and age

- related hearing loss: The VA Normative Aging Study. Hearing Research, 269 (1

- 2), 48

- 55. http://dx.doi.org/10.1016/j.heares.2010.07.004

Park, S. K., Hu, H., Wright, R. O., Schwartz, J., Che ng, Y., Sparrow, D., . . . Weisskopf, M. G. (2009). Iron metabolism genes,
low

- level lead exposure, and QT interval. Environmental Health Perspectives, 117 (1), 80

- 85. http://dx.doi.org/10.1289/ehp.11559May 2011 5

- 428 DRAFT

– DO NOT CITE OR QUOTE Par k, S. K., Mukherjee, B., Xia, X., Sparrow, D., Weisskopf, M. G., Nie, H., & Hu, H. (2009). Bone lead level prediction models and their application to examine the relationship of lead exposure and hypertension in the third National Health and
Nutrition Exam ination survey. Journal of Occupational and Environmental Medicine, 51 (12), 1422

- 1436. http://dx.doi.org/10.1097/JOM.0b013e3181bf6c8d

Park, S. K., Schwartz, J., Weisskopf, M., Sparrow, D., Vokona s, P. S., Wright, R. O., . . . Hu, H. (2006). Low

- level lead exposure, metabolic syndrome, and heart rate variability: The VA Normative Aging Study. Environmental Health Perspectives,
114 (11), 1718

- 1724. http://www.ncbi.nlm.nih.gov/pubmed/17107858

Parkinson, D. K., Ryan, C., Bromet, E. J., & Connell, M. M. (1986). A psychiatric epidemiologic study of occupational lead

exposure. American Journal of Epidemiology, 123 (2), 261

- 269. http://www.ncbi.nlm.nih.gov/pubmed/3946375

Parr, D. R., & Harris, E. J. (1976). The effect of lead on the calcium

- handling capacity of rat heart mitochondria. Biochemistry,
158 (2), 289

- 294. http://www.ncbi.nlm.nih.gov/pubmed/985429

Pasha, Q., Malik, S. A., Iqbal, J., & Shah, M. H. (2007). Characterization and distribution of the selected metals in the scalp hair of cancer patients in comparison with normal donors. Bio logical Trace Element Research, 118 (3), 207

- 216. http://dx.doi.org/10.1007/s12011

- 007

- 0035

- 7

Pasha, Q., Malik, S. A., Iqbal, J., Shaheen, N., & Shah, M. H. (2008a). Comparative distribution of the s calp hair trace metal contents in the benign tumour patients and normal donors. Environmental Monitoring and Assessment, 147 (1

- 3), 377

- 388. http://dx.doi.org/10.1007/s10661

- 007

- 0127

- z

Pasha, Q., Mal ik, S. A., Iqbal, J., Shaheen, N., & Shah, M. H. (2008b). Comparative evaluation of trace metal distribution and correlation in human malignant and benign breast tissues. Biological Trace Element Research, 125 (1), 30

- 40. http://dx.doi.org/10.1007/s12011

- 008

- 8158

- z

Pasha, Q., Malik, S. A., & Shah, M. H. (2008). Statistical analysis of trace metals in the plasma of cancer patients versus controls. Journal of Hazardous Materials, 153 (3), 1215

- 1221. http://dx.doi.org/10.1016/j.jhazmat.2007.09.115

Pasha, Q., Malik, S. A., Shaheen, N., & Shah, M. H. (2010). Investigation of trace metals in the blood plasma and scalp hair of gastrointestinal can cer patients in comparison with controls. Clinica Chimica Acta, 411 (7

- 8), 531

- 539. http://dx.doi.org/10.1016/j.cca.2010.01.010

Pasha Shaik, A., Sankar, S., Reddy, S. C., Das, P. G., & Jamil, K. (200 6). Lead

- induced genotoxicity in lymphocytes from peripheral blood samples of humans: In vitro studies. Drug and Chemical Toxicology, 29 (1), 111

- 124. http://dx.doi.org/10.1080/01480540500408739

Past or

- Barriuso, R., Banegas, J. R., Damián, J., Appel, L. J., & Guallar, E. (2003). Systolic blood pressure, diastolic blood pressure, and pulse pressure: An evaluation of their joint effect on mortality. Annals of Internal Medicine, 139 (9), 731

- 739. http://www.ncbi.nlm.nih.gov/pubmed/14597457

Patel, A. B., & Prabhu, A. S. (2009). Determinants of lead level in umbilical cord blood. Indian Pediatrics, 46 (9), 791

- 793. http://www.ncbi.nlm.nih.gov/pubmed/19213980

Patil, A. J., Bhagwat, V. R., Patil, J. A., Dongre, N. N., Ambekar, J. G., & Das, K. K. (2006). Biochemical aspects of lead exposure in silver jewelry workers in western Maharashtra (India). Journal of Basic and Clinical Physiology and Pharmacology, 17 (4), 213

- 229. http://www.ncbi.nlm.nih.gov/pubmed/17338278

Patil, A. J., Bhagwat, V. R., Patil, J. A., Dongre, N. N., Ambekar, J. G., & Da s, K. K. (2007). Occupational lead exposure in battery manufacturing workers, silver jewelry workers, and spray painters in western Maharashtra (India): Effect on liver and kidney function. Journal of Basic and Clinical Physiology and Pharmacology, 18 (2), 87

- 100. http://www.ncbi.nlm.nih.gov/pubmed/17715565May 2011 5

- 429 DRAFT

– DO NOT CITE OR QUOTE Patil, A. J., Bhagwat, V. R., Patil, J. A., Dongre, N. N., Ambekar, J. G., Jailkhani, R., & Das, K. K. (2006). Effect of lead (Pb) exposure on the activity of superoxide dismutase and catalase in battery manufacturing workers (BMW) of Western Maharashtra (India) with reference to heme biosynthesis. International Journal of Environmental Research and Public Health, 3 (4), 329

- 337. http://www.ncbi.nlm.nih.gov/pubmed/17159274

Payton, M., Hu, H., Sparrow, D., & Weiss, S. T. (1994). Low

- level lead exposure and renal function in the Normative Aging Study. American Journal of Epidemiology, 140 (9), 821

- 829. http://www.ncbi.nlm.nih.gov/pubmed/7977292

Payton, M., Riggs, K. M., Spiro, A., III, Weiss, S. T., & Hu, H. (1998). Relations of bone and blood lead to cognitive function: The VA Normative Agin g Study. Neurotoxicology and Teratology, 20 , 19

- 27. http://www.ncbi.nlm.nih.gov/pubmed/9511166

Peng, S., Hajela, R. K., & Atchison, W. D. (2002). Characteristics of block by Pb2+ of function of human neuronal L

- , N

- , and R

type Ca2+ channels transiently expressed in human embryonic kidney 293 cells. Molecular Pharmacology, 62 (6),
1418

- 1430. http://dx.doi.org/10.1124/mol.62.6.1418

Pentyala, S., Ruggeri, J., Veerraju, A., Yu, Z., Bhatia, A., Desaiah, D., & Vig, P. (2010). Microsomal Ca2+ flux modulation as an indicator of heavy metal toxicity. Indian Journal of Experimental Biology, 48 (7), 737

- 743. http://www.ncbi.nlm.nih.gov/pubmed/20929057

Pérez

- Bravo, F., Ruz, M., Morán

- Jiménez, M. J., Olivares, M., Rebolledo, A., Codoceo, J., . . . Fontanellas, A. (2004). Association between aminolevulinate dehydrase genotypes and blood lead levels in children from a lead

- contaminated area in Antofagasta, Chile. Archives of Environmental Contamination and Toxicology, 47 (2), 276

- 280. http://dx.doi.org/10.1007/s00244

- 004

- 2215

- 1

Perlstein, T., Weuve, J., Schwartz, J., Sparrow, D., Wright, R., Litonjua, A., . . . Hu, H. (2007). Cumulative community

- level lead exposure and pulse pressure: The Normative Aging Study. Environmental Health Perspectives, 115 (12), 1696

- 1700. http://dx.doi.org/10.1289/ehp.10350

Pe ters, J. L., Kubzansky, L., McNeely, E., Schwartz, J., Spiro, A., III, Sparrow, D., . . . Hu, H. (2007). Stress as a potential modifier of the impact of lead levels on blood pressure: The Normative Aging Study. Environmental Health Perspectives, 115 (8), 11 54

- 1159. http://dx.doi.org/10.1289/ehp.10002

Petit, T. L., & LeBoutillier, J. C. (1979). Effects of lead exposure during development on neocortical dendritic and synaptic

structure. Experimental Neurology, 64 (3), 482

- 492. http://dx.doi.org/10.1016/0014

- 4886(79)90226

- 7

Petri, S., Calingasan, N. Y., Alsaied, O. A., Wille, E., Kiaei, M., Friedman, J. E., . . . Beal, M. F. (2007). The lipophilic metal
chelators DP

- 109 and DP

- 460 are neuroprotective in a transgenic mouse model of amyotrophic lateral sclerosis. Journal of Neurochemistry, 102 (3), 991

- 1000. http://dx.doi.org/10.1111/j.1471

- 415 9.2007.04604.x

Piao, F., Cheng, F., Chen, H., Li, G., Sun, X., Liu, S., . . . Yokoyama, K. (2007). Effects of zinc coadministration on lead
toxicities in rats. Industrial Health, 45 (4), 546

- 551. htt p://www.ncbi.nlm.nih.gov/pubmed/17878626

Piccinini, F., Favalli, L., & Chiari, M. C. (1977). Experimental investigations on the conctraction induced by lead in arterial
smooth muscle. Toxicology, 8 (1), 43

- 51. http://dx.doi.org/10.1016/0300

- 483X(77)90022

- 1

Pillai, P., Patel, R., Pandya, C., & Gupta, S. (2009). Sex

- specific effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic/steroid

- metabo lizing enzymes and antioxidant status. Journal of Biochemical and Molecular Toxicology, 23 (6), 419

- 431. http://dx.doi.org/10.1002/jbt.20305

Pilsner, J. R., Hu, H., Ettinger, A., Sánchez, B. N., Wright, R. O ., Cantonwine, D., . . . Hernández

- Avila, M. (2009). Influence of prenatal lead exposure on genomic methylation of cord blood DNA. Environmental Health Perspectives, 117 (9), 1466

- 1471. http://dx.doi.org/10.1289/ehp.0800497May 2011 5

- 430 DRAFT

– DO NOT CITE OR QUOTE Pilsner, J. R., Hu, H., Wright, R. O., Kordas, K., Ettinger, A. S., Sánchez, B. N., . . . Hernández

- Avila, M. (2010). Maternal MTHFR genotype and haplotype predict deficits in early cognitive development in a lead

- expo sed birth cohort in Mexico City. American Journal of Clinical Nutrition, 92 (1), 226

- 234. http://dx.doi.org/10.3945/ajcn.2009.28839

Pine, M. D., Hiney, J. K., Dearth, R. K., Bratton, G. R., & Dees, W. L. (2006). IGF

- 1 administration to prepubertal female rats can overcome delayed puberty caused by maternal Pb exposure. Reproductive Toxicology, 21 (1), 104

- 109. http://dx.doi.org/10.1016/j.repr otox.2005.07.003

Pineda

- Zavaleta, A. P., García

- Vargas, G., Borja

- Aburto, V. H., Acosta

- Saavedra, L. C., Aguilar, E. V., Gómez

- Muñoz, A., . . .
Calderón

- Aranda, E. S. (2004). Nitric oxide and superoxide anion production in monocytes from children exposed t o arsenic and lead in region Lagunera, Mexico. Toxicology and Applied Pharmacology, 198 (3), 283

- 290. http://dx.doi.org/10.1016/j.taap.2003.10.034

Pinkerton, L. E., Biagini, R. E., Ward, E. M., Hull , R. D., Deddens, J. A., Boeniger, M. F., . . . Luster, M. I. (1998). Immunologic findings among lead

- exposed workers. American Journal of Industrial Medicine, 33 (4), 400

- 408. http://dx.doi.org/10.1002/(SICI)1097

- 0274(199804)33:4<400::AID

- AJIM11>3.0.CO;2

- 2

Pinto, D., Ceballos, J. M., Garcia, G., Guzman, P., Del Razo, L. M., Vera, E., . . . Gonsebatt, M. E. (2000). Increased cytogenetic damage in outdoor painte rs. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 467 (2), 105

- 111. http://dx.doi.org/10.1016/S1383

- 5718(00)00024

- 3

Pizent, A., Colak, B., Kljakovic Gaspic, Z., & Telisman, S. (2009). Prostate

- specific antigen (PSA) in serum in relation to blood lead concentration and alcohol consumption in men. Arhiv za Higijenu Rada i Toksikologiju, 60 (1), 69

- 78. http://dx.doi .org/10.2478/10004

- 1254

- 60

- 2009

- 1901

Pizent, A., Macan, J., Jurasovic, J., Varnai, V. M., Milkovic

- Kraus, S., & Kanceljak

- Macan, B. (2008). Association of toxic and essential metals with atopy markers and ventilatory lung function in women and men. Science of the Total Environment,
390 (2

- 3), 369

- 376. http://dx.doi.org/10.1016/j.scitotenv.2007.10.049

Pocock, S. J., Smith, M., & Baghurst, P. (1994). Environmental lead and children's intelligence: A systematic review of the epidemiological evidence. British Medical Journal, 309 (6963), 1189

- 1197. http://www.ncbi.nlm.nih.gov/pubmed/7987149

Ponce

- Canchihuamán, J. C., Pérez

- Méndez, O., Hernández

- Muñoz, R., Torres

- Durán, P. V., & Juárez

- Oropeza, M. A. (2010). Protective effects of Spirulina maxima on hyperlipidemia and oxidative

- stress induced by lead acetate in the liver and kidney. Lipids in Health and Disease, 9 , 35. http://dx.doi.org/10.1186/1476

- 511X

- 9

- 35

Poreba, R., Gac, P., Poreba, M., Derkacz, A., Pilecki, W., Antonowicz

- Juchniewicz, J., & Andrzejak, R. (2010). [Relationship between chronic exposure to lead, cadmium and manganese, blood pressure values and incidence of arterial hypertension]. Medycyna Pracy, 61 (1), 5

- 14. http://www.ncbi.nlm.nih.gov/pubmed/20437884

Prentice, R. C., & Kopp, S. J. (1985). Cardiotoxicity of lead at various perfusate calcium concentrations: Functional and metabolic responses of the perfused rat heart. Toxicology and Applied Pharmacology, 81 (3 pt. 1), 491

- 501. http://dx.doi.org/10.1016/0041

- 008X(85)90420

- X

Prins, J. M., Park, S., & Lurie, D. I. (2010). Decreased expression of the voltage

- dependent anion channel in differentiated PC

- 12
and SH

- SY5Y cells following low

- level Pb exposure. Toxicologica l Sciences, 113 (1), 169

- 176. http://dx.doi.org/10.1093/toxsci/kfp249

Prospective Studies Collaboration. (2002). Age

- specific relevance of usual blood pressure to vascular mortality: A meta

- analysis
of i ndividual data for one million adults in 61 prospective studies. Lancet, 360 (9349), 1903

- 1913. http://dx.doi.org/10.1016/S0140

- 6736(02)11911

- 8May 2011 5

- 431 DRAFT

– DO NOT CITE OR QUOTE Prozialeck, W. C., Grunwald, G. B., Dey, P. M., Reu hl, K. R., & Parrish, A. R. (2002). Cadherins and NCAM as potential targets in metal toxicity. Toxicology and Applied Pharmacology, 182 (3), 255

- 265. http://dx.doi.org/10.1006/taap.2002.9422

Purdy, R. E ., Smith, J. R., Ding, Y., Oveisi, F., Vaziri, N. D., & Gonick, H. C. (1997). Lead

- induced hypertension is not associated with altered vascular reactivity in vitro. American Journal of Hypertension, 10 (9 Pt. 1), 997

- 1003. http://dx.doi.org/10.1016/S0895

- 7061(97)00108

- 8

Putnam, C. D., Arvai, A. S., Bourne, Y., & Tainer, J. A. (2000). Active and inhibited human catalase structures: Ligand and NADPH binding and catalytic mechanism. Journal of Molecu lar Biology, 296 (1), 295

- 309. http://dx.doi.org/10.1006/jmbi.1999.3458

Pyatt, D. W., Zheng, J.

- H., Stillman, W. S., & Irons, R. D. (1996). Inorganic lead activates NF

- kB in primary human CD4+ T lymphoc ytes. Biochemical and Biophysical Research Communications, 227 , 380

- 385. http://dx.doi.org/10.1006/bbrc.1996.1516

Qian, L., & Flood, P. M. (2008). Microglial cells and Parkinson's disease. Immunologic Research, 41 (3), 155

- 164. http://dx.doi.org/10.1007/s12026

- 008

- 8018

- 0

Qian, Y., Harris, E. D., Zheng, Y., & Tiffany

- Castiglioni, E. (2000). Lead targets GRP78, a molecular chaperone, in C6 rat glioma
cells. Toxicology and Applied Pharmacology, 163 (3), 260

- 266. http://dx.doi.org/10.1006/taap.1999.8878

Qian, Y., Zheng, Y., Ramos, K. S., & Tiffany

- Castiglioni, E. (2005). GRP78 compartmentalized redistribution in Pb

- treated glia:
Role of GRP78 in lead

- induced oxidative stress. NeuroToxicology, 26 (2), 267

- 275. http://dx.doi.org/10.1016/j.neuro.2004.09.002

Qian, Z. M., & Morgan, E. H. (1990). Effect of lead on the transport of transferrin

- free and transferrin

- bound iron into rabbit reticulocytes. Biochemical Pharmacology, 40 (5), 1049

- 1054. http://dx.doi.org/10.1016/0006

- 2952(90)90492

- 4

Qiao, Y.

- F., Jiang, Y.

- S., & Pang, D.

- Z. (2006). [Expression of renal nuclear factor

- kappaB, transforming growth factor

- beta and
fibronectin of rats e xposed to lead]. Zhonghua Laodong Weisheng Zhiyebing Zazhi, 24 (3), 139

- 142. http://www.ncbi.nlm.nih.gov/pubmed/16600131

Qu, W., Diwan, B. A., Liu, J., Goyer, R. A., Dawson, T., Horton, J. L., . . . Waalkes, M. P. (2002). The metallothionein

- null phenotype is associated with heightened sensitivity to lead toxicity and an inability to form inclusion bodies. The American Journal of Pathology, 160 (3), 1047

- 1056. http://dx.doi.org/10.1016/S0002

- 9440(10)64925

- 5

Queiroz, M. L. S., Almeida, M., Gallão, M. I., & Höehr, N. F. (1993). Defective neutrophil function in workers occupationally exposed to lead. Basic and Clinical Pharmacology and Toxicolog y, 72 (2), 73

- 77. http://dx.doi.org/10.1111/j.1600

- 0773.1993.tb00293.x

Queiroz, M. L. S., Costa, F. F., Bincoletto, C., Perlingeiro, R. C. R., Dantas, D. C. M., Cardoso, M. P., & Almeida, M. (1994). Engulfment and killing capabilities of neutrophils and phagocytic splenic function in persons occupationally exposed to
lead. International Journal of Immunopharmacology, 16 (3), 239

- 244. http://dx.doi.org/10.1016/0192

- 0561(94)90018

- 3

Queiroz, M. L. S., Perlingeiro, R. C. R., Bincoletto, C., Almeida, M., Cardoso, M. P., & Dantas, D. C. M. (1994). Immunoglobulin levels and cellular immune function in lead exposed workers. Immunopharmacology and Immunotoxicology, 16 (1), 115

- 128. http://dx.doi.org/10.3109/08923979409029904

Quinlan, G. J., Halliwell, B., Moorhouse, C. P., & Gutteridge, J. M. C. (1988). Action of lead(II) and aluminium(II I) ions on iron

stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions. Biochimica et Biophysica Acta, 962 (2), 196

- 200. http://dx.doi.org/10.1016/0005

- 2760(88 )90159

- 2May 2011 5

- 432 DRAFT

– DO NOT CITE OR QUOTE Quintanar

- Escorza, M. A., González

- Martínez, M. T., del Pilar, I.

- O. M., & Calderón

- Salinas, J. V. (2010). Oxidative damage increases intracellular free calcium [Ca2+]i concentration in human erythrocytes incubated with lead. Toxicology In Vitro, 24 (5), 1338

- 1346. http://dx.doi.org/10.1016/j.tiv.2010.05.002

Quintanar

- Escorza, M. A., González

- Martínez, M. T., Navarro, L., Maldonado, M., Arévalo, B., & Calderón

- Salinas, J. V. (2007).
Intracell ular free calcium concentration and calcium transport in human erythrocytes of lead

- exposed workers. Toxicology and Applied Pharmacology, 220 (1), 1

- 8. http://dx.doi.org/10.1016/j.taap.2006.10.016

Q uintanilla

- Vega, B., Smith, D. R., Kahng, M. W., Hernandez, J. M., Albores, A., & Fowler, B. A. (1995). Lead

- binding proteins in brain tissue of environmentally lead

- exposed humans. Chemico

- Biological Interactions, 98 (3), 193

- 209. http://dx.doi.org/10.1016/0009

- 2797(95)03646

- 6

Rabinowitz, M. B. (1988). Lead and pregnancy. Birth: Issues in Perinatal Care, 15 (4), 236

- 241. http://dx.doi.org/10.1111/j.1523

- 536X.1988.tb01117.x

Rabinowitz, M. B., Allred, E. N., Bellinger, D. C., Leviton, A., & Needleman, H. L. (1990). Lead and childhood propensity to infectious and allergic disorders: Is there an association? Bulletin of Enviro nmental Contamination and Toxicology, 44

, 657

- 660. http://dx.doi.org/10.1007/BF01701784

Rafalowska, U., Struzyńska, L., Dabrowska

- Bouta, B., & Lenkiewicz, A. (1996). Is lead toxicosis a reflection of alter ed energy metabolism in brain synaptosomes? Acta Neurobiologiae Experimentalis, 56 (2), 611

- 617. http://www.ncbi.nlm.nih.gov/pubmed/8768312

Rafique, M., Khan, N., Perveen, K., & Naqvi, A. (2009). The effects of lead and zinc on the quality of semen of albino rats. Journal of the College of Physicians and Surgeons

- Pakistan, 19 (8), 510

- 513. http://www.ncbi.nlm.nih.gov/pubmed/19651015

Raghavan, S . R. V., Culver, B. D., & Gonick, H. C. (1980). Erythrocyte lead

- binding protein after occupational exposure: I Relationship to lead toxicity. Environmental Research, 22 (1), 264

- 270. http://dx.do i.org/10.1016/0013

- 9351(80)90138

- 3

Raghavan, S. R. V., Culver, B. D., & Gonick, H. C. (1981). Erythrocyte lead

- binding protein after occupational exposure: II Influence on lead inhibition of membrane Na+, K+

- adenosinetriphosphatase. Journal of Toxicology and Environmental Health, 7 (3

- 4), 561

- 568. http://dx.doi.org/10.1080/15287398109530001

Raghavan, S. R. V., & Gonick, H. C. (1977). Isolation of low

- molecular

- weight lead

- binding protein from human erythrocytes. Proceedings of the Society for Experimental Biology and Medicine, 155 (2), 164

- 167. http://www.ncbi.nlm.nih.gov/pubmed/866346

Rajah, T., & Ahuja, Y. R. (1995). In vivo genotoxic effects o f smoking and occupational lead exposure in printing press workers. Toxicology Letters, 76 (1), 71

- 75. http://dx.doi.org/10.1016/0378

- 4274(94)03200

- 9

Rajah, T. T., & Ahuja, Y. R. (1996). In vivo g enotoxicity of alcohol consumption and lead exposure in printing press workers. Alcohol, 13 (1), 65

- 68. http://dx.doi.org/10.1016/0741

- 8329(95)02014

- 4

Rajan, P., Kelsey, K. T., Schwartz, J. D., Be llinger, D. C., Weuve, J., Sparrow, D., . . . Wright, R. O. (2007). Lead burden and psychiatric symptoms and the modifying influence of the delta

- aminolevulinic acid dehydratase (ALAD) polymorphism: The VA Normative Aging Study. American Journal of Epidemi ology, 166 (12), 1400

- 1408. http://dx.doi.org/10.1093/aje/kwm220

Rajan, P., Kelsey, K. T., Schwartz, J. D., Bellinger, D. C., Weuve, J., Spiro, A., III, . . . Wright, R. O. (2008). Interaction of the
delta

- aminolevulinic acid dehydratase polymorphism and lead burden on cognitive function: The VA Normative Aging Study. Journal of Occupational and Environmental Medicine, 50 (9), 1053

- 1061. http://dx.doi.org/10.1097/JOM.0b013e3181792463May 2011 5

- 433 DRAFT

– DO NOT CITE OR QUOTE Rajaraman, P., Stewart, P. A., Samet, J. M., Schwartz, B. S., Linet, M. S., Zahm, S. H., . . . Inskip, P. D. (2006). Lead, genetic susceptibility, and risk of adult brain tumors. Cancer Epidemiology Biomarkers and Prevention, 15 (12), 2514

- 2520. http://dx.doi.org/10.1158/1055

- 9965.EPI

- 06

- 0482

Ramesh, G. T., Manna, S. K., Aggarwal, B. B., & Jadhav, A. L. (1999). Lead activates nuclear transcription fac tor

- kB, activator protein

- 1, and amino

- terminal c

- Jun kinase in pheochromocytoma cells. Toxicology and Applied Pharmacology, 155 (3),
280

- 286. http://dx.doi.org/10.1006/taap.1999.8624

Ramesh, G. T., Ma nna, S. K., Aggarwal, B. B., & Jadhav, A. L. (2001). Lead exposure activates nuclear factor kappa B, activator protein

- 1, c

- Jun N

- terminal kinase and caspases in the rat brain. Toxicology Letters, 123 , 195

- 207. http://dx.doi.org/10.1016/S0378

- 4274(01)00395

- 2

Rana, S. V. S. (2008). Metals and apoptosis: Recent developments. Journal of Trace Elements in Medicine and Biology, 22 (4),
262

- 284. http://dx.doi.org/10.1016/j.jtemb.2008.08.002

Razmiafshari, M., Kao, J., d'Avignon, A., & Zawia, N. H. (2001). NMR identification of heavy metal

- binding sites in a synthetic zinc finger peptide: Toxicological implications for the interactions of xenobiotic metals with zinc finger proteins. Toxicology and Applied Pharmacology, 172 (1), 1

- 10. http://dx.doi.org/10.1006/taap.2001.9132

Razmiafshari, M., & Zawia, N. H. (2000). Utilization of a synthetic peptide as a tool to study the interaction of heavy metals with the zinc finger domain of prote ins critical for gene expression in the developing brain. Toxicology and Applied Pharmacology, 166 (1), 1

- 12. http://dx.doi.org/10.1006/taap.2000.8950

Reddy, G. R., & Zawia, N. H. (2000). Lead exposure alters Egr

- 1 DNA

- binding in the neonatal rat brain. International Journal of Developmental Neuroscience, 18 (8), 791

- 795. http://dx.doi.org/10.1016/S0736

- 5748(00)00048

- 4

Reddy, S. Y., Pullakhanda m, R., & Dinesh Kumar, B. (2010). Thiamine reduces tissue lead levels in rats: Mechanism of
interaction. BioMetals, 23 (2), 247

- 253. http://dx.doi.org/10.1007/s10534

- 009

- 9282

- 8

Regunathan, S., & Sund aresan, R. (1984). Pyruvate metabolism in the brain of young rats intoxicated with organic and inorganic
lead. Journal of Neurochemistry, 43 (5), 1346

- 1351. http://dx.doi.org/10.1111/j.1471

- 4159.1984.tb05393.x

Rehman, S., Chandra, O., & Abdulla, M. (1995). Evaluation of malondialdehyde as an index of lead damage in rat brain homogenates. BioMetals, 8 (4), 275

- 279. http://dx.doi.org/10.1007/BF0 0141599

Reif, A., Fritzen, S., Finger, M., Strobel, A., Lauer, M., Schmitt, A., & Lesch, K. (2006). Neural stem cell proliferation is decreased in schizophrenia, but not in depression. Molecular Psychiatry, 11 (5), 514

- 522.

Rendón

- Ramirez, A., Cerbón

- Solórzano, J., Maldonado

- Vega, M., Quintanar

- Escorza, M. A., & Calderón

- Salinas, J. V. (2007).
Vitamin

- E reduces the oxidative damage on δ

- aminolevulinic dehydratase induced by lead intoxication in rat erythrocytes. Toxicology In Vitro, 21 (6), 1121

- 1126. http://dx.doi.org/10.1016/j.tiv.2007.04.019

Reshma Anjum, M., Sainath, S. B., Suneetha, Y., & Sreenivasula Reddy, P. (In Press). Lead acetate induced reproductive and pater nal mediated developmental toxicity in rats. Ecotoxicology and Environmental Safety

. http://dx.doi.org/10.1016/j.ecoenv.2010.10.044

Reuhl, K. R., Rice, D. C., Gilbert, S. G., & Mallett, J. (1989) . Effects of chronic developmental lead exposure on monkey neuroanatomy: Visual system. Toxicology and Applied Pharmacology, 99 (3), 501

- 509. http://dx.doi.org/10.1016/0041

- 008X(89)90157

- 9

Revis, N. W., Zinsmeister, A. R., & Bull, R. (1981). Atherosclerosis and hypertension induction by lead and cadmium ions: An effect prevented by calcium ion. Proceedings of the National Academy of Sciences, 78 (10), 6494

- 6498. http://www.ncbi.nlm.nih.gov/pubmed/6947240May 2011 5

- 434 DRAFT

– DO NOT CITE OR QUOTE Reza, B., Ali, N., Azhdar, H., Alireza, A., & Ali, K. (2008). Effects of low

- level lead exposure on blood pressure and function of
the rat isolated heart. Indian Journal of Pharmacology, 40 (2), 69

- 72. http://dx.doi.org/10.4103/0253

- 7613.41041

Rhodes, D., Spiro, A., III, Aro, A., & Hu, H. (2003). Relationship of bone and blood lead levels to psychiatric symptoms: The Normative Aging Study. J ournal of Occupational and Environmental Medicine, 45 (11), 1144

- 1151. http://www.ncbi.nlm.nih.gov/pubmed/14610395

Rice, D. C. (1990). Lead

- induced behavioral impairment on a spatial discrimination r eversal task in monkeys exposed during different periods of development. Toxicology and Applied Pharmacology, 106 (2), 327

- 333. http://dx.doi.org/10.1016/0041

- 008X(90)90251

- O

Rice, D. C. (1992). E ffect of lead during different developmental periods in the monkey on concurrent discrimination performance. NeuroToxicology, 13 , 583

- 592. http://www.ncbi.nlm.nih.gov/pubmed/1475062

Rice, D. C. (1996 ). Behavioral effects of lead: Commonalities between experimental and epidemiologic data. Environmental Health Perspectives, 104 (Suppl 2), 337

- 351. http://www.ncbi.nlm.nih.gov/pubmed/9182041

Rice, D. C. (1997). Effects of lifetime lead exposure in monkeys on detection of pure tones. Fundamental and Applied Toxicology, 36 (2), 112

- 118. http://dx.doi.org/10.1006/faat.1996.2268

Rice, D. C., & Barone, S., Jr. (2000). Critical periods of vulnerability for the developing nervous system: Evidence from humans
and animal models. Environmental Health Perspectives, 108 (S3), 511

- 533. http://www .ncbi.nlm.nih.gov/pubmed/10852851

Richardt, G., Federolf, G., & Habermann, E. (1986). Affinity of heavy metal ions to intracellular Ca2+

- binding proteins. Biochemical Pharmacology, 35 (8), 1331

- 1335. http://dx.doi.org/10.1016/0006

- 2952(86)90278

- 9

Richetti, S., Rosemberg, D., Ventura

- Lima, J., Monserrat, J., Bogo, M., & Bonan, C. (2010). Acetylcholinesterase activity and antioxidant capacity of zebrafish brain is altered by heavy metal exposure. Neu roToxicology, 32 (1), 116

- 122. http://dx.doi.org/10.1016/j.neuro.2010.11.001

Riddell, T. J., Solon, O., Quimbo, S. A., Tan, C. M., Butrick, E., & Peabody, J. W. (2007). Elevated blood

- lead levels a mong children living in the rural Philippines. Bulletin of the World Health Organization, 85 (9), 674

- 680. http://www.ncbi.nlm.nih.gov/pubmed/18026623

Ris, M. D., Dietrich, K. N., Succop, P. A., Berg er, O. G., & Bornschein, R. L. (2004). Early exposure to lead and neuropsychological outcome in adolescence. Journal of the International Neuropsychological Society, 10 , 261

- 270. http://dx.doi.org/1 0.1017/S1355617704102154

Rizzi, E., Castro, M. M., Fernandes, K., Barbosa, F., Arisi, G. M., Garcia

- Cairasco, N., . . . Gerlach, R. F. (2009). Evidence of early involvement of matrix metalloproteinase

- 2 in lead

- induced hypertension. Archives of Toxicology, 83 (5), 439

- 449. http://dx.doi.org/10.1007/s00204

- 008

- 0363

- 1

Robins, J. M., Cullen, M. R., Connors, B. B., & Kayne, R. D. (1983). Depressed thyroid indexes associated with occupational exposure to i norganic lead. Archives of Internal Medicine, 143 (2), 220

- 224. http://dx.doi.org/10.1001/archinte.143.2.220

Rodriguez

- Iturbe, B., Sindhu, R. K., Quiroz, Y., & Vaziri, N. D. (2005). Chronic exposure to low doses of lead results in renal infiltration of immune cells, NF

- "kappa"B activation, and overexpression of tubulointerstitial angiotensin II. Antioxidants & Redox Signaling, 7 (9

- 10), 1269

- 1274. http://dx.doi.org/10.1089/ars.2005.7.1269

Rodriguez

- Iturbe, B., Vaziri, N. D., Herrera

- Acosta, J., & Johnson, R. J. (2004). Oxidative stress, renal infiltration of immune cells, and salt

- sensitive hypertension: All for one and one for all. American Journ al of Physiology, 286 (4), F606

- F616. http://dx.doi.org/10.1152/ajprenal.00269.2003May 2011 5

- 435 DRAFT

– DO NOT CITE OR QUOTE Roels, H., Lauwerys, R., Konings, J., Buchet, J.

- P., Bernard, A., Green, S., . . . Chettle, D. (1994). Renal func tion and hyperfiltration capacity in lead smelter workers with high bone lead. Occupational and Environmental Medicine, 51 (8),
505

- 512. http://www.ncbi.nlm.nih.gov/pubmed/7951773

Roncal, C., Mu, W., Reungjui, S., Kim, K. M., Henderson, G. N., Ouyang, X., . . . Johnson, R. J. (2007). Lead, at low levels, accelerates arteriolopathy and tubulointerstitial injury in chronic kidney disease. American Journal of Physiology: Renal Physiolo gy, 293 (4), F1391

- F1396. http://dx.doi.org/10.1152/ajprenal.00216.2007

Ronis, M. J. J., Badger, T. M., Shema, S. J., Roberson, P. K., & Shaikh, F. (1996). Reproductive toxicity and growth effects in rats exposed to lead at different periods during development. Toxicology and Applied Pharmacology, 136 (2), 361

- 371. http://dx.doi.org/10.1006/taap.1996.0044

Ronis, M. J. J., Gandy, J., & Badger, T. (1998). Endocrine mechanisms underlying reproductive toxicity in the developing rat chronically exposed to dietary lead. Journal of Toxicology and Environmental Health, Part A: Current Issues, 54 (2),
77

- 99. http://www.ncbi.nlm.nih.gov/pubmed/9652546

Rossi

- George, A., Virgolini, M., Weston, D., Thiruchelvam, M., & Cory

- Slechta, D. (2011). Interactions of lifetime lead exposure and stress: Behavioral, neurochemical and HPA axis effects. NeuroToxicology, 32 (1), 83

- 99. http://dx.doi.org/10.1016/j.neuro.2010.09.004

Rossi

- George, A., Virgolini, M. B., Weston, D., & Cory

- Slechta, D. A. (2009). Alterations in glucocorticoid negative feedback following maternal Pb, prenatal stress and the combination: A potential biological unifying mechanism for their corresponding disease profiles. Toxicology and Applied Pharmacology, 234 (1), 117

- 127. http://d x.doi.org/10.1016/j.taap.2008.10.003

Rothenberg, S. J., Poblano, A., & Garza

- Morales, S. (1994). Prenatal and perinatal low level lead exposure alters brainstem auditory evoked responses in infants. NeuroToxicology, 15 (3), 695

- 699. http://www.ncbi.nlm.nih.gov/pubmed/7854608

Rothenberg, S. J., Poblano, A., & Schnaas, L. (2000). Brainstem auditory evoked response at five years and prenatal and postnatal blood lead. Neurotoxicology and Teratology, 22 (4), 503

- 510. http://dx.doi.org/10.1016/S0892

- 0362(00)00079

- 9

Rothenberg, S. J., & Rothenberg, J. C. (2005). Testing the dose

- response specification in epidemiology: Public health and policy

consequ ences for lead. Environmental Health Perspectives, 113 (9), 1190

- 1195. http://dx.doi.org/10.1289/ehp.7691

Rothenberg, S. J., Schnaas, L., Cansino

- Ortiz, S., Perroni

- Hernandez, E., De La Torre, P., Neri

- Mendez , C., . . . Svendsgaard, D. (1989). Neurobehavioral deficits after low level lead exposure in neonates: The Mexico City pilot study. Neurotoxicology and Teratology, 11 (2), 85

- 93. http://dx.doi.or g/10.1016/0892

- 0362(89)90046

- 9

Rothenberg, S. J., Schnaas, L., Salgado

- Valladares, M., Casanueva, E., Geller, A. M., Hudnell, H. K., & Fox, D. A. (2002).
Increased ERG a

- and b

- wave amplitudes in 7

- to 10

- year

- old children resulting from prenatal lead expo sure. Investigative Ophthalmology and Visual Science, 43 (6), 2036

- 2044. http://www.ncbi.nlm.nih.gov/pubmed/12037016

Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G., & Hoe kstra, W. G. (1973). Selenium: Biochemical role as a component of glutathione peroxidase. Science, 179 (4073), 588

- 590. http://dx.doi.org/10.1126/science.179.4073.588

Rousseau, M. C., Parent, M. E., Nadon, L., Latrellie, B., & Siemiatycki, J. (2007). Occupational exposure to lead compounds and risk of cancer among men: A population

- based case

- control study. American Journal of Epidemiology, 166 (9), 1005

- 1014. http://dx.doi.org/10.1093/aje/kwm183

Rousseau, M. C., Straif, K., & Siemiatycki, J. (2005). IARC carcinogen update. Environmental Health Perspectives, 113 (9),
A580

- A581. http://dx.doi.org/10.1289/ehp.113

- a580May 2011 5

- 436 DRAFT

– DO NOT CITE OR QUOTE Roy, A., Bellinger, D., Hu, H., Schwartz, J., Ettinger, A. S., Wright, R. O., . . . Balakrishnan, K. (2009). Lead exposure and behavior among young children in Chennai, India. Environmental Health Perspectives , 117 (10), 1607

- 1611. http://dx.doi.org/10.1289/ehp.0900625

Roy, A. K., Dhir, H., & Sharma, A. (1992). Modification of metal

- induced micronuclei formation in mouse bone marrow erythrocytes by Phyllanthus fruit extract and ascorbic acid. Toxicology Letters, 62 , 9

- 17.

Roy, N. K., & Rossman, T. G. (1992). Mutagenesis and comutagenesis by lead compounds. Mutation Research, 298 (2), 97

- 103. http://www.ncb i.nlm.nih.gov/pubmed/1282217

Rubio, J., Riqueros, M. I., Gasco, M., Yucra, S., Miranda, S., & Gonzales, G. F. (2006). Lepidium meyenii (Maca) reversed the
lead acetate induced

- damage on reproductive function in male rats. Food and Chemical Toxicology, 44 (7), 1114

- 1122. http://dx.doi.org/10.1016/j.fct.2006.01.007

Rybicki, B. A., Johnson, C. C., Uman, J., & Gorell, J. M. (1993). Parkinson's disease mortality and the industrial use of heavy
metals in Michigan. Movement Disorders, 8 (1), 87

- 92. http://dx.doi.org/10.1002/mds.870080116

S., H. D., Geraldine, M., & T, V. (2009). Influence of minerals on lead

- induced alterations in liver function in rats e xposed to long

- term lead exposure. Journal of Hazardous Materials, 166 (2

- 3), 1410

- 1414. http://dx.doi.org/10.1016/j.jhazmat.2008.12.070

Sabbioni, E., & Marafante, E. (1976). Identification of le ad

- binding components in rat liver: In vivo study. Chemico

- Biological Interactions, 15 (1), 1

- 20. http://dx.doi.org/10.1016/0009

- 2797(76)90124

- 1

Saimi, Y., & Kung, C. (2002). Calmodulin as an ion channel subunit. Annual Review of Physiology, 64 , 289

- 311. http://dx.doi.org/10.1146/annurev.physiol.64.100301.111649

Sakai, T., & Morita, Y. (1996). delta

- Aminolevulinic acid in plas ma or whole blood as a sensitive indicator of lead effects, and its
relation to the other heme

- related parameters. International Archives of Occupational and Environmental Health, 68 (2),
126

- 132. http://dx.doi.org/10.1007/BF00381245

Sakata, S., Shimizu, S., Ogoshi, K., Hirai, K., Ohno, Y., Kishi, T., . . . Mori, I. (2007). Inverse relationship between serum erythropoietin and blood lead concentrations in Kathmandu tricycle taxi drivers. Internation al Archives of Occupational and Environmental Health, 80 (4), 342

- 345. http://dx.doi.org/10.1007/s00420

- 006

- 0125

- 4

Sakuma, H., Kusama, M., Yamaguchi, K., Matsuki, T., & Sugawara, S. (1984). The distr ibution of cigarette smoke components
between mainstream and sidestream smoke: II Bases. Beitraege zur Tabakforschung International, 12 (4), 199

- 209.

Salawu, E. O., Adeeyo, O. A., Falokun, O. P., Yusuf, U. A., Oyerinde, A., & Adeleke, A. A. (2009). Tomato (Lycopersicon esculentum) prevents lead

- induced testicular toxicity. Journal of Human Reproductive Sciences, 2 (1), 30

- 34. http://dx.doi.org/10.4103/0974

- 1208.51346

Saleh, M., Mathison, J., Wolinski, M ., Bensinger, S., Fitzgerald, P., Droin, N., . . . Nicholson, D. (2006). Enhanced bacterial clearance and sepsis resistance in caspase

- 12

- deficient mice. Nature, 440 (7087), 1064

- 1068. http://dx.doi.org/10 .1038/nature04656

Sanchez

- Fructuoso, A. I., Blanco, J., Cano, M., Ortega, L., Arroyo, M., Fernandez, C., . . . Barrientos, A. (2002). Experimental lead nephropathy: Treatment with calcium disodium ethylenediaminetetraacetate. American Journal of Kidney
Dis eases, 40 (1), 59

- 67. http://dx.doi.org/10.1053/ajkd.2002.33936

Sanchez

- Fructuoso, A. I., Cano, M., Arroyo, M., Fernandez, C., Prats, D., & Barrientos, A. (2002). Lead mobilization during calcium disodium ethylenediaminetetraacetate chelation therapy in treatment of chronic lead poisoning. American Journal of Kidney Diseases, 40 (1), 51

- 58. http://dx.doi.org/10.1053/ajkd.2002.33913May 2011 5

- 437 DRAFT

– DO NOT CITE OR QUOTE Sandhir, R., & Gill, K. D. (1995). Effect of lead on lipid peroxidation in liver of rats. Biological Trace Element Research, 48 (1),
91

- 97. http://dx.doi.org/10. 1007/BF02789081

Sandhir, R., Julka, D., & Gill, K. D. (1994). Lipoperoxidative damage on lead exposure in rat brain and its implications on membrane bound enzymes. Pharmacology and Toxicology, 74 (2), 66

- 71. http://dx.doi.org/10.1111/j.1600

- 0773.1994.tb01077.x

Santibanez, M., Vioque, J., Alguacil, J., Barber, X., de la Hera, G., & Kauppinen, T. (2008). Occupational exposures and risk of oesophageal cancer by histological type: a case

- control study in eastern Spain. Occupational and Environmental Medicine, 65 (11), 774

- 781. http://dx.doi.org/10.1136/oem.2007.037929

Santos, F. W., Rocha, J. B. T., & Nogueira, C. W. (2006). 2,3

- dimercaptoprop anol, 2,3

- dimercaptopropane

- 1

- sulfonic acid and

meso

- 2,3

- dimercaptosuccinic acid increase lead

- induced inhibition of delta

- aminolevulinate dehydratase in vitro and ex
vivo. Toxicology In Vitro, 20 (3), 317

- 323. http://dx.doi.org/10.1016/j.tiv.2005.08.006

Santos, M. R. V., Marchioro, M., & Antoniolli, A. R. (2006). Lead effects on non

- adrenergic non

- cholinergic relaxations in the rat gastric fundus. Toxicology In Vitro, 20 (1), 38

- 42. http://dx.doi.org/10.1016/j.tiv.2005.05.005

Saraiva, M. C. P., Taichman, R. S., Braun, T., Nriagu, J., Eklund, S. A., & Burt, B. A. (2007). Lead exposure and periodontitis in
US adults. Journal of Per iodontal Research, 42 (1), 45

- 52. http://dx.doi.org/10.1111/j.1600

- 0765.2006.00913.x

Sarasua, S. M., Mueller, P., Kathman, S., Campagna, D., Uddin, M. S., & White, M. C. (2003). Confirming the utility of four kidney biomarker tests in a longitudinal follow

- up study. Renal Failure, 25 (5), 797

- 817. http://www.ncbi.nlm.nih.gov/pubmed/14575288

Sarasua, S. M., Vogt, R. F., Henderson, L. O., J ones, P. A., & Lybarger, J. A. (2000). Serum immunoglobulins and lymphocyte subset distributions in children and adults living in communities assessed for lead and cadmium exposure. Journal of Toxicology and Environmental Health, Part A: Current Issues, 60 , 1

- 15. http://dx.doi.org/10.1080/009841000156556

Sarnat, J. A., Marmur, A., Klein, M., Kim, E., Russell, A. G., Sarnat, S. E., . . . Tolbert, P. E. (2008). Fine particle sources and cardiorespiratory morbidity: An application of chemical mass balance and factor analytical source

- apportionment
methods. Environmental Health Perspectives, 116 , 459

- 466. http://dx.doi.org/10.1289/ehp.10873

Savery, L. C., Gr lickova

- Duzevik, E., Wise, S. S., Thompson, W. D., Hinz, J. A., Thompson, L. H., & Wise, J. P., Sr. (2007). Role of the Fancg gene in protecting cells from particulate chromate

- induced chromosome instability. Mutation Research: Genetic Toxicology and Envir onmental Mutagenesis, 626 (1

- 2), 120

- 127. http://dx.doi.org/10.1016/j.mrgentox.2006.09.005

Saxena, G., & Flora, S. J. S. (2004). Lead

- induced oxidative stress and hematological alterations and t heir response to combined
administration of calcium disodium EDTA with a thiol chelator in rats. Journal of Biochemical and Molecular Toxicology, 18 (4), 221

- 233. http://dx.doi.org/10.1002/jbt.20027

Saxena, G., Pathak, U., & Flora, S. J. S. (2005). Beneficial role of monoesters of meso

- 2,3

- dimercaptosuccinic acid in the mobilization of lead and recovery of tissue oxidative injury in rats. Toxicology, 214 (1

- 2), 39

- 56. http://dx.doi.org/10.1016/j.tox.2005.05.026

Schanne, F. A. X., Dowd, T. L., Gupta, R. K., & Rosen, J. F. (1989). Lead increases free Ca2+ concentration in cultured
osteoblastic bone cells: simultaneous detection of intracellular free Pb2+ b y 19F NMR. Proceedings of the National Academy of Sciences, 86 , 5133

- 5135. http://www.ncbi.nlm.nih.gov/pubmed/2500664

Schanne, F. A. X., Long, G. J., & Rosen, J. F. (1997). Lead induced rise in intra cellular free calcium is mediated through activation of protein kinase C in osteoblastic bone cells. Biochimica et Biophysica Acta, 136 (3), 247

- 254. http://dx.doi.org/10.1016/S0925

- 4439(97)00006

- 9May 2011 5

- 438 DRAFT

– DO NOT CITE OR QUOTE Schell, L. M., Denham, M., Stark, A. D., Gomez, M., Ravenscroft, J., Parsons, P. J., . . . Samelson, R. (2003). Maternal blood lead concentration, diet during pregnancy, and anthropometry predict neonatal blood lead in a socioeconomically disadvantaged population. Environmental Health Perspectives, 111 (2), 195

- 200. http://dx.doi.org/10.1289/ehp.5592

Scheuhammer, A. M. (1987). Erythrocyte delta

- aminolevulinic acid dehydratase in birds: II The effects of lead exposure in vivo. Toxicology, 45 (2), 165

- 175. http://dx.doi.org/10.1016/0300

- 483X(87) 90102

- 8

Schirrmacher, K., Wiemann, M., Bingmann, D., & Busselberg, D. (1998). Effects of lead, mercury, and methyl mercury on gap junctions and [CA2+]i in bone cells. Calcified Tissue International, 63 , 134

- 139. http://dx.doi.org/10.1007/s002239900503

Schnaas, L., Rothenberg, S. J., Flores, M.

- F., Martinez, S., Hernandez, C., Osorio, E., . . . Perroni, E. (2006). Reduced intellectual development in children with prenatal lead exposure. Environmental Heal th Perspectives, 114 (5), 791

- 797. http://dx.doi.org/10.1289/ehp.8552

Schneider, J. S., Lee, M. H., Anderson, D. W., Zuck, L., & Lidsky, T. I. (2001). Enriched environment during development is protective aga inst lead

- induced neurotoxicity. Brain Research, 896 (1

- 2), 48

- 55. http://dx.doi.org/10.1016/S0006

- 8993(00)03249

- 2

Schober, S. E., Mirel, L. B., Graubard, B. I., Brody, D. J., & Flegal, K. M. (20 06). Blood lead levels and death from all causes, cardiovascular disease, and cancer: Results from the NHANES III Mortality Study. Environmental Health Perspectives,
114 (10), 1538

- 1541. http://dx.doi.org/10. 1289/ehp.9123

Schrauzer, G. N. (1987). Effects of selenium antagonists on cancer susceptibility: New aspects of chronic heavy metal toxicity. Journal of University of Occupational and Environmental Health, 9 Suppl , 208

- 215. http://www.ncbi.nlm.nih.gov/pubmed/3299603

Schrauzer, G. N. (2008). Effects of selenium and low levels of lead on mammary tumor development and growth in MMTV

infected female mice. Biological Trace Element Research, 125 (3), 268

- 275

. http://dx.doi.org/10.1007/s12011

- 008

- 8172

- 1

Schwartz, B. S., Chen, S., Caffo, B., Stewart, W. F., Bolla, K. I., Yousem, D., & Davatzikos, C. (2007). Relations of brain volumes with cognitive funct ion in males 45 years and older with past lead exposure. NeuroImage, 37 (2), 633

- 641. http://dx.doi.org/10.1016/j.neuroimage.2007.05.035

Schwartz, B. S., Lee, B.

- K., Bandeen

- Roche, K., Stewart , W., Bolla, K., Links, J., . . . Todd, A. (2005). Occupational lead exposure and longitudinal decline in neurobehavioral test scores. Epidemiology, 16 (1), 106

- 113. http://dx.doi.org/10.109 7/01.ede.0000147109.62324.51

Schwartz, B. S., Lee, B.

- K., Lee, G.

- S., Stewart, W. F., Simon, D., Kelsey, K., & Todd, A. C. (2000). Associations of blood lead, dimercaptosuccinic acid

- chelatable lead, and tibia lead with polymorphisms in the vitamin D recep tor and delta

aminolevulinic acid dehydratase genes. Environmental Health Perspectives, 108 (10), 949

- 954. http://dx.doi.org/10.2307/3435053

Schwartz, B. S., Lee, B.

- K., Stewart, W., Ahn, K.

- D., Kelsey, K., & Bresssler, J. (1997). Associations of subtypes of hemoglobin
with delta

- aminolevulinic acid dehydratase genotype and dimercaptosuccinic acid

- chelatable lead levels. Archives of Environmental and Occupational Health, 52 (2), 97

- 103. http://dx.doi.org/10.1080/00039899709602871

Schwartz, B. S., Lee, B.

- K., Stewart, W., Sithisarankul, P., Strickland, P. T., Ahn, K.

- D., & Kelsey, K. (1997). delta

Aminolevulinic acid dehydratase genotype modifies four hour urinary lead excretion after oral administration of dimercaptosuccinic acid. Occupational and Environmental Medicine, 54 (4), 241

- 246. http://www.ncbi.nlm.nih.gov/pubmed/9166129May 2011 5

- 439 DRAFT

– DO NOT CITE OR QUOTE Schwartz, B. S., Lee, B. K., Lee, G. S., Stewart, W. F., Lee, S. S., Hwang, K. Y., . . . Todd, A. C. (2001). Associations of blood lead, dimercaptosuccinic acid

- chelatable lead, and tibia lead with neurobehavioral test scores in South Korean lead
workers. American Journal of Epidemiology, 153 (5), 453

- 464. http://dx.doi.org/10.1093/aje/153.5.453

Schwartz, J. (1991). Lead, blood pressure, and cardiovascular disease in men and women. Environmental Health Perspectives,
91 , 71

- 75. http://www.ncbi.nlm.nih.gov/pubmed/1828226

Schwartz, J. (1994). Low

- level lead exposure and children's IQ: A met a

- analysis and search for a threshold. Environmental Research, 65 (1), 42

- 55. http://dx.doi.org/10.1006/enrs.1994.1020

Schwartz, J., & Otto, D. (1987). Blood lead, hearing thresholds, and neurobehaviora l development in children and youth. Archives of Environmental and Occupational Health, 42 (3), 153

- 160. http://dx.doi.org/10.1080/00039896.1987.9935814

Schwartz, J., & Otto, D. (1991). Lead and minor hearing impairment. Archives of Environmental and Occupational Health, 46 (5),
300

- 305. http://dx.doi.org/10.1080/00039896.1991.9934391

Scinicariello, F., Murray, H. E., Moffett, D. B., Aba din, H. G., Sexton, M. J., & Fowler, B. A. (2007). Lead and delta

aminolevulinic acid dehydratase polymorphism: Where does it lead? A meta

- analysis. Environmental Health Perspectives, 115 (1), 35

- 41. http://d x.doi.org/10.1289/ehp.9448

Scinicariello, F., Yesupriya, A., Chang, M. H., & Fowler, B. A. (2010). Modification by ALAD of the association between blood lead and blood pressure in the U.S. population: Results from the Third National Health and Nutrition Ex amination Survey. Environmental Health Perspectives, 118 (2), 259

- 264. http://dx.doi.org/10.1289/ehp.0900866

Seki, T., & Arai, Y. (1993). Highly polysialylated NCAM expression in the developing and adult r at spinal cord. Brain Research,
73 (1), 141

- 145. http://dx.doi.org/10.1016/0165

- 3806(93)90056

- G

Selevan, S. G., Rice, D. C., Hogan, K. A., Euling, S. Y., Pfahles

- Hutchens, A., & Bethel, J. (2003). Blood lead concentration and delayed puberty in girls. New England Journal of Medicine, 348 (16), 1527

- 1536. http://dx.doi.org/10.1056/NEJMoa020880

Selvin

- Testa, A., Lopez

- Costa, J. J. , Nessi

- de Avinon, A. C., & Saavedra, J. P. (1991). Astroglial alterations in rat hippocampus during chronic lead exposure. Glia, 4 (4), 384

- 392. http://dx.doi.org/10.1002/glia.440040406

Senger, M. R., Rico, E. P., Arizi, M. D. B., Frazzon, A. P. G., Dias, R. D., Bogo, M. R., & Bonan, C. D. (2006). Exposure to Hg2+ and Pb2+ changes NTPDase and ecto

- 5 '

- nucleotidase activities in central nervous system of zebrafish (Danio rerio). Toxicology, 226 (2

- 3), 229

- 237. http://dx.doi.org/10.1016/j.tox.2006.07.012

Shabani, A., & Rabbani, A. (2000). Lead nitrate induced apoptosis in alveolar macrophages from rat lung. Toxicology, 149 , 109

- 114. http://dx.doi.org/10.1016/S0300

- 483X(00)00232

- 8

Shadick, N. A., Kim, R., Weiss, S., Liang, M. H., Sparrow, D., & Hu, H. (2000). Effect of low level lead exposure on hyperuricemia and gout among middle aged and e lderly men: the normative aging study. Journal of Rheumatology, 27

, 1708

- 1712. http://www.ncbi.nlm.nih.gov/pubmed/10914856

Shaik, A. P., & Jamil, K. (2009). Individual susceptibility and genotoxicit y in workers exposed to hazardous materials like lead. Journal of Hazardous Materials, 168 (2

- 3), 918

- 924. http://dx.doi.org/10.1016/j.jhazmat.2009.02.129

Shakoor, A., Gupta, P. K., Singh, Y. P., & Kataria, M. (2000). Beneficial effects of aluminum on the progression of lead

- induced nephropathy in rats. Basic and Clinical Pharmacology and Toxicology, 87 (6), 258

- 260. http://dx.doi .org/10.1034/j.1600

- 0773.2000.pto870603.xMay 2011 5

- 440 DRAFT

– DO NOT CITE OR QUOTE Shan, G., Tang, T., & Zhang, X. B. (2009). The protective effect of ascorbic acid and thiamine supplementation against damage caused by lead in the testes of mice. Journal of Huazhong University of Science and Tech nology

- Medical Sciences,
29 (1), 68

- 72. http://dx.doi.org/10.1007/s11596

- 009

- 0114

- 4

Sharifi, A. M., Darabi, R., Akbarloo, N., Larijani, B., & Khoshbaten, A. (2004). Investigation of circulatory and tissue ACE activity during development of lead

- induced hypertension. Toxicology Letters, 153 (2), 233

- 238. http://dx.doi.org/10.1016/j.toxlet.2004.04.013

Sharifi, A. M., & Mousavi, S. H. (2008). Studying the effects of lead on DNA fragmentation and proapoptotic bax and antiapoptotic Bcl

- 2 protein expression in PC12 cells. Toxicology Mechanisms and Methods, 18 (1), 75

- 79. http://dx.doi.org/10 .1080/15376510701665814

Sharifi, A. M., Mousavi, S. H., & Jorjani, M. (2010). Effect of chronic lead exposure on pro

- apoptotic Bax and anti

- apoptotic
Bcl

- 2 protein expression in rat hippocampus in vivo. Cellular and Molecular Neurobiology, 30 (5), 769

- 774. http://dx.doi.org/10.1007/s10571

- 010

- 9504

- 1

Sharma, V., Sharma, A., & Kansal, L. (2010). The effect of oral administration of Allium sativum extracts on lead nitrate induced toxicity in male mice. F ood and Chemical Toxicology, 48 (3), 928

- 936. http://dx.doi.org/10.1016/j.fct.2010.01.002

Shelkovnikov, S. A., & Gonick, H. C. (2001). Influence of lead on rat thoracic aorta contraction and relaxati on. American Journal of Hypertension, 14 (9), 873

- 878. http://dx.doi.org/10.1016/S0895

- 7061(01)02149

- 5

Shelton, K. R., Cunningham, J. G., Klann, E., Merchant, R. E., Egle, P. M., & Bigbee, J. W. (1990). Low

- abundance 32

- kilodalton nuclear protein specifically enriched in the central nervous system. Journal of Neuroscience Research, 25

, 287

- 294. http://www.ncbi.nlm.nih.gov/pubmed/2325156

Shel ton, K. R., & Egle, P. M. (1982). The proteins of lead

- induced intranuclear inclusion bodies. Journal of Biological Chemistry,
257 (19), 11802

- 11807. http://www.ncbi.nlm.nih.gov/pubmed/7118911

Shen, X

. - M., Yan, C.

- H., Guo, D., Wu, S.

- M., Li, R.

- Q., Huang, H., . . . Tang, J.

- M. (1998). Low

- level prenatal lead exposure and neurobehavioral development of children in the first year of life: A prospective study in Shanghai. Environmental Research, 79 , 1

- 8. http://dx.doi.org/10.1006/enrs.1998.3851

Shih, R. A., Glass, T. A., Bandeen

- Roche, K., Carlson, M. C., Bolla, K. I., Todd, A. C., & Schwartz, B. S. (2006). Environmental lead exposure and cognitive fun ction in community

- dwelling older adults. Neurology, 67 (9), 1556

- 1562. http://dx.doi.org/10.1212/01.wnl.0000239836.26142.c5

Shin, J. H., Lim, K. M., Noh, J. Y., Bae, O. N., Chung, S. M., Le e, M. Y., & Chung, J. H. (2007). Lead

- induced procoagulant activation of erythrocytes through phosphatidylserine exposure may lead to thrombotic diseases. Chemical Research in Toxicology, 20 (1), 38

- 43. http ://dx.doi.org/10.1021/tx060114+

Shinkai, Y., Yamamoto, C., & Kaji, T. (2010). Lead induces the expression of ER chaperones GRP78 and GRP94 in vascular
endothelial cells via the JNK

- AP

- 1 pathway. Toxicological Sciences, 114 (2), 378

- 386. http://dx.doi.org/10.1093/toxsci/kfq008

Shinozuka, H., Ohmura, T., Katyal, S. L., Zedda, A. I., Ledda

- Columbano, G. M., & Columbano, A. (1996). Possible roles of nonparenchymal cells in hepatocyte prolife ration induced by lead nitrate and by tumor necrosis factor alpha. Hepatology, 23 (6), 1572

- 1577. http://dx.doi.org/10.1002/hep.510230638

Siddiqui, M. K. J., Jyoti, Singh, S., Mehrotra, P. K., Singh, K., & Sarangi, R. (2006). Comparison of some trace elements concentration in blood, tumor free breast and tumor tissues of women with benign and malignant breast lesions: An Indian study. Environment International, 32 (5), 630

- 637. http://dx.doi.org/10.1016/j.envint.2006.02.002May 2011 5

- 441 DRAFT

– DO NOT CITE OR QUOTE Silberstein, T., Saphier, O., Paz

- Tal, O., Trimarchi, J. R., Gonzalez, L., & Keefe, D. L. (2006). Lead concentrates in ovarian follicle compromises pr egnancy. Journal of Trace Elements in Medicine and Biology, 20 (3), 205

- 207. http://dx.doi.org/10.1016/j.jtemb.2006.05.001

Silkin, Y. A., Silkina, E. N., Sherstobitov, A. O., & Gusev, G. P. (2001). Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus. Membrane and Cell Biology, 14 (6), 773

- 782. http://www.ncbi.nlm.nih.gov/pubmed/11817573

Silva, P. A., Hughes, P., Williams, S., & Faed, J. M. (1988). Blood lead, intelligence, reading attainment, and behaviour in eleven year old children in Dunedin, New Zealand. Journal of Child Psychology and Psychiatry, 29 (1), 43

- 52. http://dx.doi.org/10.1111/j.1469

- 7610.1988.tb00687.x

Silveira, E. A., Lizardo, J. H., Souza, L. P., Stefanon, I., & Vassallo, D. V. (2010). Acute lead

- induced vasoconstriction in the vascular beds of isolated perfused rat tails is endoth elium

- dependent. Brazilian Journal of Medical and Biological Research, 43 (5), 492

- 499. http://dx.doi.org/10.1590/S0100

- 879X2010007500027

Simons, T. J. B. (1986). Passive transport and binding of lead by human red blood cells. Journal of Physiology, 378 , 267

- 286. http://www.ncbi.nlm.nih.gov/pubmed/3795106

Simons, T. J. B. (1993a). Lead

- calcium interactions in cellular lead toxicity. NeuroT oxicology, 14 (2

- 3), 77

- 85. http://www.ncbi.nlm.nih.gov/pubmed/8247414

Simons, T. J. B. (1993b). Lead transport and binding by human erythrocytes in vitro. Pflügers Archiv European Journal of Physiolo gy, 423 (3

- 4), 307

- 313. http://dx.doi.org/10.1007/BF00374410

Simons, T. J. B. (1995). The affinity of human erythrocyte porphobilinogen synthase for Zn2+ and Pb2+. FEBS Journal, 234 (1),
178

- 183. http://dx.doi.org/10.1111/j.1432

- 1033.1995.178_c.x

Simsek, N., Karadeniz, A., Kalkan, Y., Keles, O. N., & Unal, B. (2009). Spirulina platensis feeding inhibited the anemia

- and leucopenia

- induced lead and cadmium in rats. Journal of Hazardous Materials, 164 (2

- 3), 1304

- 1309. http://dx.doi.org/10.1016/j.jhazmat.2008.09.041

Singh, B., Chandran, V., Bandhu, H. K., Mittal, B. R., Bhattacharya, A., Jindal, S. K., & Varma, S. (2000). Impact of lead exposure on pituitary

- thyroid axis in humans. BioMetals, 13 (2), 187

- 192. http://dx.doi.org/10.1023/A:1009201426184

Singh, J., Pritchard, D. E., Carlisle, D. L., Mclean, J. A., Montaser, A., Orenstein, J. M., & Patierno, S. R. (1999). Internalization of carcinogenic lead chromate particles by cultured normal human lung epithelial cells: Formation of intracellular lead

i nclusion bodies and induction of apoptosis. Toxicology and Applied Pharmacology, 161 (3), 240

- 248. http://dx.doi.org/10.1006/taap.1999.8816

Singh, U. S., Saxena, D. K., Singh, C., Murthy, R. C., & Chand ra, S. V. (1991). Lead

- induced fetal nephrotoxicity in iron

deficient rats. Reproductive Toxicology, 5 (3), 211

- 217. http://dx.doi.org/10.1016/0890

- 6238(91)90053

- I

Sivaprasad, R., Nagaraj, M., & V aralakshmi, P. (2003). Combined efficacies of lipoic acid and meso

- 2,3

- dimercaptosuccinic acid
on lead

- induced erythrocyte membrane lipid peroxidation and antioxidant status in rats. Human and Experimental Toxicology, 22 (4), 183

- 192. http://dx.doi.org/10.1191/0960327103ht335oa

Skoczynska, A., & Stojek, E. (2005). The impact of subchronic lead poisoning on the vascular effect of nitric oxide in rats. Environmental Toxicology and Pharmacology, 19 (1), 9 9

- 106. http://dx.doi.org/10.1016/j.etap.2004.05.004

Slivkova, J., Popelkova, M., Massanyi, P., Toporcerova, S., Stawarz, R., Formicki, G., . . . Guzik, M. (2009). Concentration of
trace elements in human semen and relation to spermatozoa quality. Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 44 (4), 370

- 375. http://dx.doi.org/10. 1080/10934520802659729May 2011 5

- 442 DRAFT

– DO NOT CITE OR QUOTE Smejkalova, J. (1990). The chromosomal aberrations investigation in children permanently living in the lead polluted area. Sbornik Vedeckych Praci Lekarske Fakulty University Karlovy V Hradci Kralove, 33 (5), 539

- 564.

Smith, C. M., H u, H., Wang, X., & Kelsey, K. T. (1995). ALA

- D genotype is not associated with HT or HB levels among workers exposed to low levels of lead. La Medicina del Lavoro, 86 (3), 229

- 235. http://www.ncbi.nlm .nih.gov/pubmed/7565283

Smith, C. M., Wang, X., Hu, H., & Kelsey, K. T. (1995). A polymorphism in the delta

- aminolevulinic acid dehydratase gene may modify the pharmacokinetics and toxicity of lead. Environmental Health Perspectives, 103 (3), 248

- 253. http://www.ncbi.nlm.nih.gov/pubmed/7768225

Smith, D. R., Kahng, M. W., Quintanilla

- Vega, B., & Fowler, B. A. (1998). High

- affinity renal lead

- binding proteins in environmentally

- exposed humans. Chemico

- Bi ological Interactions, 115 (1), 39

- 52. http://dx.doi.org/10.1016/S0009

- 2797(98)00060

- X

Snyder, J. E., Filipov, N. M., Parsons, P. J., & Lawrence, D. A. (2000). The efficiency of maternal transfer of lead and its influence on plasma IgE and splenic cellularity of mice. Toxicological Sciences, 57 (1), 87

- 94. http://dx.doi.org/10.1093/toxsci/57.1.87

Snyder, R. D., & Lachmann, P. J. (1989). Thiol i nvolvement in the inhibition of DNA repair by metals in mammalian cells. Molecular Toxicology, 2 (2), 117

- 128. http://www.ncbi.nlm.nih.gov/pubmed/2702302

Sobekova, A., Holovska, K., Lenartova, V., Legath, J., & Javorsky, P. (2009). The alteration of glutathione peroxidase activity in rat organs after lead exposure. Acta Physiologica Hungarica, 96 (1), 37

- 44. http://dx.doi.org/10.1556/APhysiol.96.2009.1.4

Sokol, R. Z., & Berman, N. (1991). The effect of age of exposure on lead

- induced testicular toxicity. Toxicology, 69 (3), 269

- 278. http:// dx.doi.org/10.1016/0300

- 483X(91)90186

- 5

Solon, O., Riddell, T. J., Quimbo, S. A., Butrick, E., Aylward, G. P., Lou Bacate, M., & Peabody, J. W. (2008). Associations between cognitive function, blood lead concentration, and nutrition among children in the c entral Philippines. Journal of Pediatrics, 152 (2), 237

- 243. http://dx.doi.org/10.1016/j.jpeds.2007.09.008

Songdej, N., Winters, P. C., McCabe, M. J., Jr., & Wijngaarden, E. V. (2010). A population

- based assessment of blood lead levels
in relation to inflammation. Environmental Research, 110 (3), 272

- 277. http://dx.doi.org/10.1016/j.envres.2009.12.008

Spector, J. T., Navas

- Acien, A., Fadrowski, J., Guallar, E., Jaar, B., & Weaver, V. M. (2011). Associations of blood lead with estimated glomerular filtration rate using MDRD, CKD

- EPI and serum cystatin C

- based equations. Nephrology, Dialysis, Transplanta tion

. http://dx.doi.org/10.1093/ndt/gfq773

Srivastava, D., Hurwitz, R. L., & Fox, D. A. (1995). Lead

- and calcium

- mediated inhibition of bovine rod cGMP phosphodiesterase: Interactions with magnesium. Toxi cology and Applied Pharmacology, 134 (1), 43

- 52. http://dx.doi.org/10.1006/taap.1995.1167

Srivastava, V., Dearth, R. K., Hiney, J. K., Ramirez, L. M., Bratton, G. R., & Dees, W. (2004). The effects of l ow

- level Pb on steroidogenic acute regulatory protein (StAR) in the prepubertal rat ovary. Toxicological Sciences, 77 (1), 35

- 40. http://dx.doi.org/10.1093/toxsci/kfg249

Stacchiotti, A., Morandini, F., B ettoni, F., Schena, I., Lavazza, A., Grigolato, P. G., . . . Aleo, M. F. (2009). Stress proteins and oxidative, damage in a renal derived cell line exposed to inorganic mercury and lead. Toxicology, 264 (3), 215

- 224. http://dx.doi.org/10.1016/j.tox.2009.08.014May 2011 5

- 443 DRAFT

– DO NOT CITE OR QUOTE Stackpole, M. M., Wise, S. S., Goodale, B. C., Duzevik, E. G., Munroe, R. C., Thompson, W. D., . . . Wise, J. P. (2007). Homologous recombination repair protects against particulate chromate

- ind uced chromosome instability in Chinese
hamster cells. Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis, 625 (1

- 2), 145

- 154. http://dx.doi.org/10.1016/j.mrfmmm.2007.06.003

Sta essen, J. A., Lauwerys, R. R., Buchet, J.

- P., Bulpitt, C. J., Rondia, D., Van Renterghem, Y., & Amery, A. (1992). Impairment of renal function with increasing blood lead concentrations in the general population. New England Journal of Medicine,
327 (3), 151

- 156. http://dx.doi.org/10.1056/NEJM199207163270303

Staessen, J. A., Nawrot, T., Den Hond, E., Thijs, L., Fagard, R., Hoppenbrouwers, K., . . . Roels, H. A. (2001). Renal function, cytogenetic measurements, and sexual developments in adolescents in relation to environmental pollutants: A feasibility study o f biomarkers. Lancet, 357 (9269), 1660

- 1669. http://dx.doi.org/10.1016/S0140

- 6736(00)04822

- 4

Stangle, D. E., Smith, D. R., Beaudin, S. A., Strawderman, M. S., Levitsky, D. A., & Strupp, B. J. (20 07). Succimer chelation improves learning, attention, and arousal regulation in lead

- exposed rats but produces lasting cognitive impairment in the absence of lead exposure. Environmental Health Perspectives, 115 (2), 201

- 209. http://dx.doi.org/10.1289/ehp.9263

Stayner, L., Steenland, K., Dosemeci, M., & Hertz

- Picciotto, I. (2003). Attenuation of exposure

- response curves in occupational cohort studies at high exposure levels. Scandinavian Journal of Work, Envir onment and Health, 29 (4), 317

- 324. http://www.ncbi.nlm.nih.gov/pubmed/12934726

Steenland, N. K., Thun, M. J., Ferguson, C. W., & Port, F. K. (1990). Occupational and other exposures associated with male end

stage renal disease: A case/control study. American Journal of Public Health, 80 (2), 153

- 157. http://www.ncbi.nlm.nih.gov/pubmed/2153349

Sterling, G., O'Neill, K., McCafferty, M., & O'Neill, J. (1982). Effect of chronic lead ingestion by rats on glucose metabolism and acetylcholine synthesis in cerebral cortex slices. Journal of Neurochemistry, 39 (2), 592

- 596. http://dx.doi.or g/10.1111/j.1471

- 4159.1982.tb03989.x

Stevens, L. A., Coresh, J., Schmid, C. H., Feldman, H. I., Froissart, M., Kusek, J., . . . Levey, A. S. (2008). Estimating GFR using serum cystatin C alone and in combination with serum creatinine: A pooled analysis of 3,418 individuals with CKD. American Journal of Kidney Diseases, 51 (3), 395

- 406. http://dx.doi.org/10.1053/j.ajkd.2007.11.018

Stewart, P. W., Blaine, C., Cohen, M., Burright, R. G., & Donovick, P. J. (1996). Acute and longer term effects of meso

- 2,3 dimercaptosuccinic acid (DMSA) on the behavior of lead

- exposed and control mice. Physiology and Behavior, 59 (4

- 5),
849

- 855. http://dx.doi.org/ 10.1016/0031

- 9384(95)02185

- X

Stewart, W. F., Schwartz, B. S., Davatzikos, C., Shen, D., Liu, D., Wu, X., . . . Youssem, D. (2006). Past adult lead exposure is linked to neurodegeneration measured by brain MRI. Neurology, 66 (10), 1476

- 1484. http://dx.doi.org/10.1212/01.wnl.0000216138.69777.15

Stewart, W. F., Schwartz, B. S., Simon, D., Kelsey, K., & Todd, A. C. (2002). ApoE genotype, past adult lead exposure, and
neurobehavioral function. Env ironmental Health Perspectives, 110 (5), 501

- 505. http://dx.doi.org/10.1289/ehp.02110501

Stoleski, S., Karadzinska

- Bislimovska, J., Stikova, E., Risteska

- Kuc, S., Mijakoski, D., & Minov, J. (2008). Advers e effects in workers exposed to inorganic lead. Arhiv za Higijenu Rada i Toksikologiju, 59 (1), 19

- 29. http://dx.doi.org/10.2478/10004

- 1254

- 59

- 2008

- 1866

Strömberg, U., Schütz, A., & Skerfving, S. (1995). Substantial decrease of blood lead in Swedish children, 1978

- 94, associated with petrol lead. Occupational and Environmental Medicine, 52 , 764

- 769. http://dx.doi.org/10.1136/oem.5 2.11.764

Struzynska, L., Dabrowska

- Bouta, B., Koza, K., & Sulkowski, G. (2007). Inflammation

- like glial response in lead

- exposed
immature rat brain. Toxicological Sciences, 95 (1), 156

- 162. http://dx.doi .org/10.1093/toxsci/kfl134May 2011 5

- 444 DRAFT

– DO NOT CITE OR QUOTE Struzynska, L., Dabrowska

- Bouta, B., & Rafałowska, U. (1997). Acute lead toxicity and energy metabolism in rat brain synaptosomes. Acta Neurobiologiae Experimentalis, 57 (4), 275

- 281. http://www.ncbi.nlm.nih.gov/pubmed/9519545

Struzynska, L., Walski, M., Gadamski, R., Dabrowska

- Bouta, B., & Rafalowska, U. (1997). Lead

- induced abnormalities in
blood

- brain barrier permeability in experimental chronic toxicity. Molecular and Che mical Neuropathology, 31 (3),
207

- 224. http://dx.doi.org/10.1007/BF02815125

Su, Y., Ding, Y., Jiang, M., Hu, X., & Zhang, Z. (2007). Protein 4.2 Komatsu (D175Y) associated with the lack of interaction with ankyrin in human red blood cells. Blood Cells Molecules and Diseases, 38 (3), 221

- 228. http://dx.doi.org/10.1016/j.bcmd.2006.11.004

Sun, H., Wang, H. L., & Wang, S. (2007). D

- serine relieves chronic lead exposure

- impaired long

- term potentiation in the CA1 region of the rat hippocampus in vitro. Neuroscience Letters, 417 (2), 118

- 122. http://dx.doi.org/10.1016/j.neulet.2007.01.085

Sun, L., Hu , J., Zhao, Z., Li, L., & Cheng, H. (2003). Influence of exposure to environmental lead on serum immunoglobulin in preschool children. Environmental Research, 92 (2), 124

- 128. http://dx.doi.org/1 0.1016/S0013

- 9351(02)00090

- 7

Sun, L. R., & Suszkiw, J. B. (1995). Extracellular inhibition and intracellular enhancement of Ca2+ currents by Pb2+ in bovine
adrenal chromaffin cells. Journal of Neurophysiology, 74 (2), 574

- 581. http://www.ncbi.nlm.nih.gov/pubmed/7472365

Sun, Y., Sun, D. H., Zhou, Z. J., Zhu, G. Y., Lei, L. J., Zhang, H. Y., . . . Jin, T. Y. (2008). Estimation of benchmark dose for bone damage and renal dysfunction in a Chinese male popu lation occupationally exposed to lead. Annals of Occupational Hygiene, 52 (6), 527

- 533. http://dx.doi.org/10.1093/annhyg/men031

Sun, Y., Sun, D. H., Zhou, Z. J., Zhu, G. Y., Zhang, H. Y., Chang, X. L., . . . Jin, T. Y. (2008). Osteoporosis in a Chinese population due to occupational exposure to lead. American Journal of Industrial Medicine, 51 (6), 436

- 442. http://dx.doi.org/10.1002/ajim.20567

Sundstrom, R., Muntzing, K., Kalimo, H., & Sourander, P. (1985). Changes in the integrity of the blood

- brain barrier in suckling rats with low dose lead encephalopathy. Acta Neuropathologica, 68 (1), 1

- 9. http://dx.doi.org/10.1007/BF00688948

Surkan, P. J., Schnaas, L., Wright, R. J., Téllez

- Rojo, M. M., Lamadrid

- Figueroa, H., Hu, H., . . . Wright, R. O. (2008). Maternal
self

- esteem, exposure to lead, and child neurodevelopment. NeuroToxicology, 29 (2), 278

- 2 85. http://dx.doi.org/10.1016/j.neuro.2007.11.006

Surkan, P. J., Zhang, A., Trachtenberg, F., Daniel, D. B., McKinlay, S., & Bellinger, D. C. (2007). Neuropsychological function in
children with b lood lead levels <10 μg/dL. NeuroToxicology, 28 (6), 1170

- 1177. http://dx.doi.org/10.1016/j.neuro.2007.07.007

Suszkiw, J., Toth, G., Murawsky, M., & Cooper, G. P. (1984). Effects of Pb2+ and Cd2+ o n acetylcholine release and Ca2+ movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ. Brain Research,
323 (1), 31

- 46. http://dx.doi.org/10.1016/0006

- 8993( 84)90262

- 2

Suszkiw, J. B. (2004). Presynaptic disruption of transmitter release by lead. NeuroToxicology, 25 (4), 599

- 604. http://dx.doi.org/10.1016/j.neuro.2003.09.009

Suzuki, T., Morimura, S., Di ccianni, M. B., Yamada, R., Hochi, S.

- I., Hirabayashi, M., . . . Muramatsu, M. (1996). Activation of glutathione transferase P gene by lead requires glutathione transferase P enhancer I. Journal of Biological Chemistry,
271 (3), 1626

- 1632. http://dx.doi.org/10.1074/jbc.271.3.1626

Swarup, D., Naresh, R., Varshney, V. P., Balagangatharathilagar, M., Kumar, P., Nandi, D., & Patra, R. C. (2007). Changes in plasma hormones profile and liver function in cows n aturally exposed to lead and cadmium around different industrial

areas. Research in Veterinary Science, 82 (1), 16

- 21. http://dx.doi.org/10.1016/j.rvsc.2006.05.002May 2011 5

- 445 DRAFT

– DO NOT CITE OR QUOTE Taketani, S., Tanaka, A., & Tokuna ga, R. (1985). Reconstitution of heme

- synthesizing activity from ferric ion and porphyrins, and the effect of lead on the activity. Archives of Biochemistry and Biophysics, 242 , 291

- 296. http://d x.doi.org/10.1016/0003

- 9861(85)90504

- 1

Tanner, C. M., Chen, B., Wang, W., Peng, M., Liu, Z., Liang, X., . . . Schoenberg, B. S. (1989). Environmental factors and
Parkinson's disease: A case

- control study in China. Neurology, 39 (5), 660

- 664. http://www.ncbi.nlm.nih.gov/pubmed/2710356

Tapisso, J. T., Marques, C. C., Mathias, M. L., & Ramalhinho, M. G. (2009). Induction of micronuclei and sister chromatid exchange in bone

- marrow cells and abnormalities i n sperm of Algerian mice (Mus spretus) exposed to cadmium, lead
and zinc. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 678 (1), 59

- 64. http://dx.doi.org/10.1016/j.mrgento x.2009.07.001

Tavakoli

- Nezhad, M., Barron, A. J., & Pitts, D. K. (2001). Postnatal inorganic lead exposure decreases the number of spontaneously active midbrain dopamine neurons in the rat. NeuroToxicology, 22 , 259

- 269. http://dx.doi.org/10.1016/S0161

- 813X(01)00010

- 9

Technische Universität Braunschweig. (2011). BRENDA: Comprehensive enzyme information system, from http://www.brenda

enzymes.org/

Teijon, C., Blanco, M. D., Romero, C. S., Beneit, J. V., Villarino, A. L., Guerrero, S., & Olmo, R. (2010). Study of response of thymic and submaxillary lymph node lymphocytes to administration of lead by different routes. Biological Trace
Element Research , 135 (1

- 3), 74

- 85. http://dx.doi.org/10.1007/s12011

- 009

- 8495

- 6

Teijon, C., Olmo, R., Blanco, D., Romero, A., & Teijon, J. M. (2006). Low doses of lead: Effects on reproduction and development in rat s. Biological Trace Element Research, 111 (1

- 3), 151

- 165. http://dx.doi.org/10.1385/BTER:111:1:151

Telisman, S., Colak, B., Pizent, A., Jurasovic, J., & Cvitkovic, P. (2007). Reproductive toxicity of lo w

- level lead exposure in

men. Environmental Research, 105 (2), 256

- 266. http://dx.doi.org/10.1016/j.envres.2007.05.011

Tellez

- Rojo, M. M., Bellinger, D. C., Arroyo

- Quiroz, C., Lamadrid

- Figueroa, H ., Mercado

- Garcia, A., Schnaas

- Arrieta, L., . . . Hu, H. (2006). Longitudinal associations between blood lead concentrations lower than 10 microg/dL and neurobehavioral development in environmentally exposed children in Mexico City. Pediatrics, 118 , e323

- e 330. http://dx.doi.org/10.1542/peds.2005

- 3123

Tian, L., & Lawrence, D. A. (1995). Lead inhibits nitric oxide production in vitro by murine splenic macrophages. Toxicology and Applied Pharmacology, 132

, 156

- 163. http://dx.doi.org/10.1006/taap.1995.1096

Tian, L., & Lawrence, D. A. (1996). Metal

- induced modulation of nitric oxide production in vitro by murine macrophages: Lead, nickel, and cobalt utili ze different mechanisms. Toxicology and Applied Pharmacology, 141 (2), 540

- 547. http://dx.doi.org/10.1006/taap.1996.0320

Tian, Y., Green, P., Stamova, B., Hertz

- Picciotto, I., Pessah, I. N., Hansen, R., . . . Sharp, F. (2011). Correlations of gene expression with blood lead levels in children with autism compared to typically developing controls. Neurotoxicity Research, 19 (1), 1

- 13. http://dx.doi. org/10.1007/s12640

- 009

- 9126

- x

Tiffany

- Castiglioni, E., Sierra, E. M., Wu, J.

- N., & Rowles, T. K. (1989). Lead toxicity in neuroglia. NeuroToxicology, 10 (3),
417

- 443. http://www.ncbi.nlm.nih.gov/pubme d/2696897

Timchalk, C., Lin, Y., Weitz, K. K., Wu, H., Gies, R. A., Moore, D. A., & Yantasee, W. (2006). Disposition of lead (Pb) in saliva and blood of Sprague

- Dawley rats following a single or repeated oral exposure to Pb

- acetate. Toxicology, 222 (1

- 2), 8 6

- 94. http://dx.doi.org/10.1016/j.tox.2006.01.030May 2011 5

- 446 DRAFT

– DO NOT CITE OR QUOTE Tokar, E. J., Diwan, B. A., & Waalkes, M. P. (2010). Early life inorganic lead exposure induces testicular teratoma and renal and urinary bladder pr eneoplasia in adult metallothionein

- knockout mice but not in wild type mice. Toxicology, 276 (1), 5

- 10. http://dx.doi.org/10.1016/j.tox.2010.06.006

Tomokuni, K., & Ichiba, M. (1990). Effect of lead on the activity of erythrocyte porphobilinogen deaminase in vivo and in vitro. Toxicology Letters, 50 (2

- 3), 137

- 142. http://dx.doi.org/10.1016/0378

- 4274(90)9000 3

- 5

Tomoum, H. Y., Mostafa, G. A., Ismail, N. A., & Ahmed, S. M. (2010). Lead exposure and its association with pubertal development in school

- age Egyptian children: Pilot study. Pediatrics International, 52 (1), 89

- 93. http://dx.doi.org/10.1111/j.1442

- 200X.2009.02893.x

Tomsig, J. L., & Suszkiw, J. B. (1993). Intracellular mechanism of Pb(2+)

- induced norepinephrine release from bovine
chromaffin cells. American Journal of Physiology: Cell Physi ology, 265 (6), C1630

- C1636. http://www.ncbi.nlm.nih.gov/pubmed/8279523

Tomsig, J. L., & Suszkiw, J. B. (1995). Multisite interactions between Pb2+ and protein kinase C and its role in norepinephrine
release from bovine adrenal chromaffin cells. Journal of Neurochemistry, 64 (6), 2667

- 2673. http://dx.doi.org/10.1046/j.1471

- 4159.1995.64062667.x

Tonelli, M., Wiebe, N., Hemmelgarn, B., Kla renbach, S., Field, C., Manns, B., . . . Gill, J. (2009). Trace elements in hemodialysis patients: A systematic review and meta

- analysis. BMC Medicine, 7 , 25. http://dx.doi.org/10.1186/1741

- 7015

- 7

- 25

Tong, S., Baghurst, P., McMichael, A., Sawyer, M., & Mudge, J. (1996). Lifetime exposure to environmental lead and children's intelligence at 11

- 13 years: The Port Pirie cohort study. British Medical Journal, 312 (7046), 1569

- 1575. http://www.ncbi.nlm.nih.gov/pubmed/8664666

Tong, S., Baghurst, P. A., Sawyer, M. G., Burns, J., & McMichael, A. J. (1998). Declining blood lead levels and changes in cognitive function during childhood: the Port Pirie cohort study. JAMA: Journal of the American Medical Association,
280 (22), 1915

- 1919. http://dx.doi.org/10.1001/jama.280.22.1915

Tong, S., McMichael, A. J., & Baghurst, P. A. (2000). Interactions between en vironmental lead exposure and sociodemographic factors on cognitive development. Archives of Environmental and Occupational Health, 55 , 330

- 335. http://www.ncbi.nlm.nih.gov/pubmed/11063408

Trope, I. , Lopez

- Villegas, D., Cecil, K. M., & Lenkinski, R. E. (2001). Exposure to lead appears to selectively alter metabolism of cortical gray matter. Pediatrics, 107 (6), 1437

- 1443. http://dx.doi.org/10.154 2/peds.107.6.1437

Trope, I., Lopez

- Villegas, D., & Lenkinski, R. E. (1998). Magnetic resonance imaging and spectroscopy of regional brain
structure in a 10

- year

- old boy with elevated blood lead levels. Pediatrics, 101 (6), E7. http://dx.doi.org/10.1542/peds.101.6.e7

Tsaih, S.

- W., Korrick, S., Schwartz, J., Amarasiriwardena, C., Aro, A., Sparrow, D., & Hu, H. (2004). Lead, diabetes, hypertension, and renal function: The normative aging study. Environmental Health Perspectives, 112 (11), 1178

- 1182. http://dx.doi.org/10.1289/ehp.7024

Tsao, D.

- A., Yu, H.

- S., Cheng, J.

- T., Ho, C.

- K., & Chang, H.

- R. (2000). The change of beta

- adrenergic system in lead

- induced hyper tension. Toxicology and Applied Pharmacology, 164 (2), 127

- 133. http://dx.doi.org/10.1006/taap.1999.8871

Tsuang, M. (2000). Schizophrenia: Genes and environment. Biological Psychiatry, 47 (3), 210

- 220. http://dx.doi.org/10.1016/S0006

- 3223(99)00289

- 9

Tuppurainen, M., Wagar, G., Kurppa, K., Sakari, W., Wambugu, A., Froseth, B., . . . Nykyri, E. (1988). Thyroid function as assessed by routine labo ratory tests of workers with long

- term lead exposure. Scandinavian Journal of Work, Environment and Health, 14 (3), 175

- 180. http://www.ncbi.nlm.nih.gov/pubmed/3393853May 2011 5

- 447 DRAFT

– DO NOT CITE OR QUOTE Turkez, H., Geyikoglu, F., Tatar , A., Keles, M. S., & Kaplan, I. (In Press). The effects of some boron compounds against heavy metal toxicity in human blood. Experimental and Toxicologic Pathology

. http://dx.doi.org/10.1016/j.etp. 2010.06.011

U.S. EPA. (U.S. Environmental Protection Agency). (1986). Air quality criteria for lead . (Report No. EPA/600/8

- 83/028aF

- dF). Washington, DC: Author.

U.S. EPA. (U.S. Environmental Protection Agency). (2006). Air quality criteria for lead . (Repor t No. EPA/600/R

- 05/144aF

- bF). Research Triangle Park, NC: U.S. Environmental Protection Agency, National Center for Environmental Assessment.
Retrieved from http://cfpub.epa.gov/ncea/ CFM/recordisplay.cfm?deid=158823

.

U.S. Renal Data System. (2009). USRDS 2009 annual data report: Atlas of chronic kidney disease and end

- stage renal disease in
the United States . Bethesda, MD: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases. Retrieved from http://www.usrds.org/adr_2009.htm

.

Ueda, D., Kishimoto, T., Dekio, S., & Tada, M. (1997). Inhibitory effect of lead on tube formation by cultured human vascul ar endothelial cells. Human Cell, 10 (4), 283

- 291. http://www.ncbi.nlm.nih.gov/pubmed/9573489

Ujihara, H., Sasa, M., & Ban, T. (1995). Selective blockade of P

- type calcium channels by lead in cultured hippocampal neurons. Journal of Pharmacological Sciences, 67 (3), 267

- 269. http://dx.doi.org/10.1254/jjp.67.267

Ukaejiofo, E. O., Thomas, N., & Ike, S. O. (2009). Haematological assessment of occupational exposure to lead handlers in Enugu urban, Enugu State, Nigeria. Nigerian Journal of Clinical Practice, 12 (1), 58

- 64. http://www.ncbi.nlm.nih.gov/pubmed/19562924

Undeger, U., Basaran, N., Canpinar, H., & Kansu, E. (1996). Immune alterations in lead

- exposed workers. Toxicology, 109 , 167

- 172. http://dx.doi.org/10.1016/0300

- 483X(96)03333

- 1

Upadhyay, A. K., M athur, R., Bhadauria, M., & Nirala, S. K. (2009). Therapeutic influence of zinc and ascorbic acid against lead
induced biochemical alterations. Therapie, 64 (6), 383

- 388. http://dx.doi.org/10.2515/the rapie/2009055

Ustundag, A., & Duydu, Y. (2007). The influence of melatonin and N

- acetylcysteine in delta

- aminolevulinic acid and lead induced genotoxicity in lymphocytes in vitro. Biological Trace Element Research, 117 (1

- 3), 53

- 64. http://dx.doi.org/10.1007/BF02698083

Uzbekov, M. G., Bubnova, N. I., & Kulikova, G. V. (2007). Effect of prenatal lead exposure on superoxide dismutase activity in
the brain and liver of rat fetuses. Bulletin of Experimental Biol ogy and Medicine, 144 (6), 783

- 785. http://dx.doi.org/10.1007/s10517

- 007

- 0431

- 1

Vaglenov, A., Carbonell, E., & Marcos, R. (1998). Biomonitoring of workers exposed to lead genotoxic effects, its modul ation by polyvitamin treatment and evaluation of the induced radioresistance. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 418 (2

- 3), 79

- 92. http://dx.doi.org/10.1016/S138 3

- 5718(98)00111

- 9

Vaglenov, A., Creus, A., Laltchev, S., Petkova, V., Pavlova, S., & Marcos, R. (2001). Occupational exposure to lead and induction of genetic damage. Environmental Health Perspectives, 109 (3), 295

- 298. http://www.ncbi.nlm.nih.gov/pubmed/11333192

Valencia, I., Castillo, E. E., Chamorro, G., Bobadilla, R. A., & Castillo, C. (2001). Lead induces endothelium

- and Ca2+

- independent contraction in rat aortic rings. Pharmacology and Toxicolo gy, 89 (4), 177

- 182. http://dx.doi.org/10.1111/j.0901

- 9928.2001.890406.x

Valentino, M., Governa, M., Marchiseppe, I., & Visona, I. (1991). Effects of lead on polymorphonuclear leukocyte (PMN) functions in occupationally exposed workers. Archives of Toxicology, 65 (8), 685

- 688. http://dx.doi.org/10.1007/BF02098038May 2011 5

- 448 DRAFT

– DO NOT CITE OR QUOTE Valentino, M., Rapisarda, V., Santarelli, L., Bracci, M., Scorcelletti, M., Di Lor enzo, L., . . . Soleo, L. (2007). Effect of lead on the levels of some immunoregulatory cytokines in occupationally exposed workers. Human and Experimental Toxicology, 26 (7), 551

- 556. http://dx.doi.o rg/10.1177/0960327107073817

Valverde, M., Fortoul, T. I., Diaz

- Barriga, F., Majia, J., & Del Castillo, E. R. (2002). Genotoxicity induced in CD

- 1 mice by inhaled lead: Differential organ response. Mutagenesis, 17 (1), 55

- 61. http://dx.doi.org/10.1093/mutage/17.1.55

Van Esch, G. J., & Kroes, R. (1969). The induction of renal tumours by feeding basic lead acetate to mice and hamsters. British Journal of Cancer, 23 (4), 765

- 771. http://dx.doi.org/10.1038/bjc.1969.95

Van Larebeke, N., Koppen, G., Nelen, V., Schoeters, G., Van Loon H Albering H Riga L Vlietinck, R., & Kleinjans, J. (2004). Differences in HPRT mutant frequency among middle

- aged Flemish women in association with area of residence and
blood lead levels. Biomarkers, 9 (1), 71

- 84. http://dx.doi.org /10.1080/13547500310001652160

van Wijngaarden, E., Campbell, J. R., & Cory

- Slechta, D. A. (2009). Bone lead levels are associated with measures of memory impairment in older adults. NeuroToxicology, 30 (4), 572

- 580. http://dx.doi.org/10.1016/j.neuro.2009.05.007

van Wijngaarden, E., & Dosemeci, M. (2006). Brain cancer mortality and potential occupational exposure to lead: Findings from the National Longitudinal Mortality Study, 1979

- 1989. Internation al Journal of Cancer, 119 (5), 1136

- 1144. http://dx.doi.org/10.1002/ijc.21947

Vander, A. J. (1988). Chronic effects of lead on the renin

- angiotensin system. Environmental Health Perspectives, 78 , 77

- 83. http://www.ncbi.nlm.nih.gov/pubmed/3060354

Vargas

- Robles, H., Romo, E., Sanchez

- Mendoza, A., Rios, A. P., Soto, V., Avila

- Casado, C., . . . Escalante, B. (2007). Lead exposure effect on angiotensin II re nal vasoconstriction. Human and Experimental Toxicology, 26 (6), 499

- 507. http://dx.doi.org/10.1177/0960327106077597

Vassallo, D. V., Lebarch, E. C., Moreira, C. M., Wiggers, G. A., & Stefanon, I. (20 08). Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium. Brazilian Journal of Medical and Biological Research,
41 (9), 789

- 795. http://dx.do i.org/10.1590/S0100

- 879X2008000900008

Vaziri, N. D. (2008a). Causal link between oxidative stress, inflammation, and hypertension. Iranian Journal of Kidney Diseases,
2 (1), 1

- 10.

Vaziri, N. D. (2008b). Mechanisms of lead

- induced hypertension and cardiovas cular disease. American Journal of Physiology: Heart and Circulatory Physiology, 295 (2), H454

- H465. http://dx.doi.org/10.1152/ajpheart.00158.2008

Vaziri, N. D., & Ding, Y. (2001). Effect of lead o n nitric oxide synthase expression in coronary endothelial cells: Role of superoxide. Hypertension, 37 (2), 223

- 226. http://www.ncbi.nlm.nih.gov/pubmed/11230275

Vaziri, N. D., Ding, Y., & Ni, Z. (199 9). Nitric oxide synthase expression in the course of lead

- induced hypertension. Hypertension, 34 (4 Pt 1), 558

- 562. http://www.ncbi.nlm.nih.gov/pubmed/10523326

Vaziri, N. D., Ding, Y., Ni, Z., & Barton, C. H. (2005). Bradykinin down

- regulates, whereas arginine analogs up

- regulates,
endothelial nitric

- oxide synthase expression in coronary endothelial cells. Journal of Pharmacology and Experimental Therapeutics, 313 (1), 121

- 126. http://dx.doi.org/10.1124/jpet.104.076497

Vaziri, N. D., Ding, Y., Ni, Z., & Gonick, H. C. (1997). Altered nitric oxide metabolism and increased oxygen free radical activity
in lead

- induced hypertension: effect of lazar oid therapy. Kidney International, 52 , 1042

- 1046. http://dx.doi.org/10.1038/ki.1997.426May 2011 5

- 449 DRAFT

– DO NOT CITE OR QUOTE Vaziri, N. D., & Khan, M. (2007). Interplay of reactive oxygen species and nitric oxide in the pathogenesis of exper imental lead

induced hypertension. Clinical and Experimental Pharmacology and Physiology, 34 (9), 920

- 925. http://dx.doi.org/10.1111/j.1440

- 1681.2007.04644.x

Vaziri, N. D., Liang, K., & Ding, Y. (1999). Increased nitric oxide inactivation by reactive oxygen species in lead

- induced hypertension. Kidney International, 56 , 1492

- 1498. http://dx.doi.org/10.1046/j.1523

- 1755.1999.00670.x

Vaziri, N. D., Lin, C.

- Y., Farmand, F., & Sindhu, R. K. (2003). Superoxide dismutase, catalase, glutathione peroxidase and
NADPH oxidase in lead

- induced hypertension. Kidney International, 63 , 186

- 194. http://dx.doi.org/10.1046/j.1523

- 1755.2003.00711.x

Vaziri, N. D., & Wang, X. Q. (1999). cGMP

- mediated negative

- feedback regulation of endothelial nitric oxide synthase expression by nitric oxide. Hypertension, 34 (6), 1237

- 1241. http://www.ncbi.nlm.nih.gov/pubmed/10601124

Vaziri, N. D., Wang, X. Q., Oveisi, F., & Rad, B. (2000). Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats. Hypertension, 36 (1), 142

- 146. http://www.ncbi.nlm.nih.gov/pubmed/10904027

Verheij, J., Voortman, J., van Nieuwkerk, C. M., Jarbandhan, S. V., Mulder, C. J., & Bloemena, E. (2009). Hepatic morphopathologic finding s of lead poisoning in a drug addict: a case report. Journal of Gastrointestinal and Liver Diseases, 18 (2), 225

- 227. http://www.ncbi.nlm.nih.gov/pubmed/19565057

Verina, T., Rohde, C. A., & Guilarte, T. R. (2007). Environmental lead exposure during early life alters granule cell neurogenesis and morphology in the hippocampus of young adult rats. Neuroscience, 145 (3), 1037

- 1047. http:// dx.doi.org/10.1016/j.neuroscience.2006.12.040

Verma, S. K., Dua, R., & Gill, K. D. (2005). Impaired energy metabolism after co

- exposure to lead and ethanol. Basic and Clinical Pharmacology and Toxicology, 96 (6), 475

- 479. http://dx.doi.org/10.1111/j.1742

- 7843.2005.pto_96611.x

Vetter, S. W., & Leclerc, E. (2003). Novel aspects of calmodulin target recognition and activation. European Journal of Biochemistry, 270 (3), 404

- 414. http://dx.doi.org/10.1046/j.1432

- 1033.2003.03414.x

Vigeh, M., Yokoyama, K., Seyedaghamiri, Z., Shinohara, A., Matsukawa, T., Chiba, M., & Yunesian, M. (2011). Blood lead at currently acceptable levels may cause preterm labour. Occupational and Environmental Medicine, 68 , 231

- 234. http://dx.doi.org/10.1136/oem.2009.050419

Vinceti, M., Guidetti, D., Bergomi, M., Caselgrandi, E., Vivoli, R., O lmi, M., . . . Solime, F. (1997). Lead, cadmium, and selenium in the blood of patients with sporadic amyotrophic lateral sclerosis. Italian Journal of Neurological Sciences,
18 (2), 87

- 92. http://dx.doi.org /10.1007/BF01999568

Virgolini, M. B., Chen, K., Weston, D. D., Bauter, M. R., & Cory

- Slechta, D. A. (2005). Interactions of chronic lead exposure and intermittent stress: Consequences for brain catecholamine systems and associated behaviors and HPA axis fu nction. Toxicological Sciences, 87 (2), 469

- 482. http://dx.doi.org/10.1093/toxsci/kfi269

Virgolini, M. B., Rossi

- George, A., Lisek, R., Weston, D. D., Thiruchelvam, M., & Cory

- Slechta, D. A. (2008). CNS effects of developmental Pb exposure are enhanced by combined maternal and offspring stress. NeuroToxicology, 29 (5), 812

- 827. http://dx.doi.org/10.1016/j.neuro.2008.03.003

Virgolini, M. B., Rossi

- George, A., Weston, D., & Cory

- Slechta, D. A. (2008). Influence of low level maternal Pb exposure and prenatal stress on offspring stress challenge responsivity. NeuroToxicology, 29 (6), 928

- 939.
h ttp://dx.doi.org/10.1016/j.neuro.2008.09.010

Vogetseder, A., Picard, N., Gaspert, A., Walch, M., Kaissling, B., & Le Hir, M. (2008). Proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells. American Journal of Physiology: Cell Physiology,
294 (1), C22

- C28. http://dx.doi.org/10.1152/ajpcell.00227.2007May 2011 5

- 450 DRAFT

– DO NOT CITE OR QUOTE Waalkes, M. P., Diwan, B. A., Ward, J. M., Devor, D. E., & Goyer, R. A. (1995). Renal tubular tumors and atypical hyperplasias in B6C3F1 mice exposed to lead acetate during gestation and lactation occur with minimal chronic nephropathy. Cancer Research, 55 (22), 5265

- 5271. http://www.ncbi.nlm.nih.gov/pubmed/7585586

Waalkes, M. P., Liu, J., Goyer, R. A., & Diwan, B. A. (2004). Metallothionein

- I/II double knockout mice are hypersensitive to
lead

- induced kidney carcinogenesis: Role of inclusion body formation. C ancer Research, 64 (21), 7766

- 7772. http://dx.doi.org/10.1158/0008

- 5472.CAN

- 04

- 2220

Walkowiak, J., Altmann, L., Kramer, U., Sveinsson, K., Turfeld, M., Weishoff

- Houben, M., & Winneke, G. (1998). Cognitive and
sensorimotor functions in 6

- year

- old children in relation to lead and mercury levels: adjustment for intelligence and contrast sensitivity in computerized testing. Neurotoxicology and Teratology, 20 (5), 511

- 521. http://dx.doi.org/10.1016/S0892

- 0362(98)00010

- 5

Wananukul, W., Sura, T., & Salaitanawatwong, P. (2006). Polymorphism of delta

- aminolevulinic acid dehydratase and its effect
on blood lead levels in Thai workers. Archives of Environmental and Occupational Health, 61 (2), 67

- 72. http://dx.doi.org/10.3200/AEOH.61.2.67

- 72

Wang, C.

- L., Chuang, H.

- Y., Ho, C.

- K., Yang, C.

- Y., Tsai, J.

- L., Wu, T.

- S., & Wu, T.

- N. (2002). Relations hip between blood lead concentrations and learning achievement among primary school children in Taiwan. Environmental Research, 89 (1),
12

- 18. http://dx.doi.org/10.1006/enrs.2002.4342

Wang, C. H., Zhang , Y., Liang, J. C., Shan, G., Wang, Y., & Shi, Q. (2006). Impacts of ascorbic acid and thiamine supplementation at different concentrations on lead toxicity in testis. Clinica Chimica Acta, 370 (1

- 2), 82

- 88. http://dx.doi.org/10.1016/j.cca.2006.01.023

Wang, C. Y., Lin, Y. W., & Yang, J. L. (2008). Activation of protein kinase C alpha signaling prevents cytotoxicity and mutagenicity following lead acetate in CL3 human lung cancer cells. Toxicology, 250 (1), 55

- 61. http://dx.doi.org/10.1016/j.tox.2008.06.001

Wang, F. T., Hu, H., Schwartz, J., Weuve, J., Spiro, A. S., III, Sparrow, D., . . . Wright, R. O. (2007). Modifying effects of the HFE polymor phisms on the association between lead burden and cognitive decline. Environmental Health Perspectives, 115 (8), 1210

- 1215. http://dx.doi.org/10.1289/ehp.9855

Wang, G., Chen, X.

- Q., Lipsky, M. M., Whittaker, M. H., & Fowler, B. A. (2005). Effect of combined lead, cadmium and arsenic exposure on the expression of cellular protective factors in rat kidneys at LOEL dose levels. Toxicologist, 85 (1S), 237. http://www.toxicology.org/ai/pub/Tox/2005Tox.pdf

Wang, G. S., & Fowler, B. A. (2008). Roles of biomarkers in evaluating interactions among mixtures of lead, cadmium and
arsenic. Toxicology and Applied Pharmacology, 233 (1), 92

- 99. http://dx.doi.org/10.1016/j.taap.2008.01.017

Wang, L., Chen, D. W., Wang, H., & Liu, Z. P. (2009). Effects of lead and/or cadmium on the expression of metallothionein in the kidney of rats. Biological Trace Element Rese arch, 129 (1

- 3), 190

- 199. http://dx.doi.org/10.1007/s12011

- 008

- 8288

- 3

Wang, L., Wang, H., Hu, M. Z., Cao, J., Chen, D. W., & Liu, Z. P. (2009). Oxidative stress and apoptotic changes in primary
cultu res of rat proximal tubular cells exposed to lead. Archives of Toxicology, 83 (5), 417

- 427. http://dx.doi.org/10.1007/s00204

- 009

- 0425

- z

Wang, L., Wang, Z., & Liu, J. (2010). Protective effect of N

- ac etylcysteine on experimental chronic lead nephrotoxicity in
immature female rats. Human and Experimental Toxicology, 29 (7), 581

- 591. http://dx.doi.org/10.1177/0960327109357270

Wang, Q., Luo, W. J., Z hang, W. B., Dai, Z. M., Chen, Y. M., & Chen, J. Y. (2007). Iron supplementation protects against lead

induced apoptosis through MAPK pathway in weanling rat cortex. NeuroToxicology, 28 (4), 850

- 859. http://dx.doi.org/10.1016/j.neuro.2007.04.004May 2011 5

- 451 DRAFT

– DO NOT CITE OR QUOTE Wang, Q., Zhao, H. H., Chen, J. W., Hao, Q. L., Gu, K. D., Zhu, Y. X., . . . Ye, L. X. (2010). delta

- Aminolevulinic acid dehydratase activity, urinary d elta

- aminolevulinic acid concentration and zinc protoporphyrin level among people with low level of lead exposure. International Journal of Hygiene and Environmental Health, 213 (1), 52

- 58. http:// dx.doi.org/10.1016/j.ijheh.2009.08.003

Wang, Y.

- Y., Sui, K.

- X., Hong, L. I., & Ma, H.

- Y. (2009). The effects of lead exposure on placental NF

- kappaB expression and the consequences for gestation. Reproductive Toxicology, 27 (2), 190

- 195. http://dx.doi.org/10.1016/j.reprotox.2008.12.006

Wasserman, G., Graziano, J. H., Factor

- Litvak, R., Popovac, D., Morina, N., Musabegovic, A., . . . Stein, Z. (1992). Independent effects of lead exposure and iron deficiency anemia on developmental outcome at age 2 years. Journal of Pediatrics,
121 , 695

- 703.

Wasserman, G. A., Factor

- Litvak, P., Liu, X., Todd, A. C., Kline, J. K., Slavkovich, V., . . . Graziano, J. H. (2003). The relationship between blood lead, bon e lead and child intelligence. Neuropsychology, Development, and Cognition. Section C: Child Neuropsychology, 9 (1), 22

- 34. http://dx.doi.org/10.1076/chin.9.1.22.14497

Wasserman, G. A., Graziano, J. H., Factor

- Litvak, P., Popovac, D., Morina, N., Musabegovic, A., . . . Stein, Z. (1994). Consequences of lead exposure and iron supplementation on childhood development at age 4 years. Neurotoxicology and Teratology, 16 (3), 233

- 240. http://dx.doi.org/10.1016/0892

- 0362(94)90044

- 2

Wasserman, G. A., Liu, X., Pine, D. S., & Graziano, J. H. (2001). Contribution of maternal smoking during pregnancy and lead exposure to early child behavior problems. Neu rotoxicology and Teratology, 23 (1), 13

- 21. http://dx.doi.org/10.1016/S0892

- 0362(00)00116

- 1

Wasserman, G. A., Staghezza

- Jaramillo, B., Shrout, P., Popovac, D., & Graziano, J. (1998). The effect o f lead exposure on behavior problems in preschool children. American Journal of Public Health, 88 , 481

- 486. http://dx.doi.org/10.2105/AJPH.88.3.481

Waterman, S. J., El

- Fawal, H. A. N., & Snyder, C. A. ( 1994). Lead alters the immunogenicity of two neural proteins: A potential mechanism for the progression of lead

- induced neurotoxicity. Environmental Health Perspectives, 102 (12), 1052

- 1056.

Watts, S. W., Chai, S., & Webb, R. C. (1995). Lead acetate

- induce d contraction in rabbit mesenteric artery: interaction with calcium and protein kinase C. Toxicology, 99 (1

- 2), 55

- 65. http://dx.doi.org/10.1016/0300

- 483X(94)03003

- K

Weaver, V., & Jaar, B. (2010). UpToDate: Lead nephropathy and lead

- related nephrotoxicity, from http://www.uptodate.com/contents/lead

- nephropathy

- and

- lead

- related

- nephrotoxicity

Weaver, V. M

. (2010). [Further analysis of Korean lead worker data set with a focus on determining the functional form of the

exposure

- response relationship].

Weaver, V. M., Ellis, L. R., Lee, B. K., Todd, A. C., Shi, W., Ahn, K. D., & Schwartz, B. S. (2008). Associat ions between patella lead and blood pressure in lead workers. American Journal of Industrial Medicine, 51 (5), 336

- 343. http://dx.doi.org/10.1002/ajim.20573

Weaver, V. M., Griswold, M., Todd, A. C., Jaar, B. G., Ahn, K. D., Thompson, C. B., & Lee, B. K. (2009). Longitudinal
associations between lead dose and renal function in lead workers. Environmental Research, 109 (1), 101

- 107. http://dx.doi.org/10.1016/j.envres.2008.09.005

Weaver, V. M., Jarr, B. G., Schwartz, B. S., Todd, A. C., Ahn, K.

- D., Lee, S.

- S., . . . Lee, B.

- K. (2005). Associations among lead dose biomarkers, uric acid, and renal function in Korean lead workers. Environmental Health Perspectives, 113 (1), 36

- 42. http://dx.doi.org/10.1289/ehp.7317May 2011 5

- 452 DRAFT

– DO NOT CITE OR QUOTE Weaver, V. M., Kim, N. S., Jaar, B. G., Schwartz, B. S., Parsons, P. J., Steuerwald, A. J., . . . Lee, B.

- K. (2011). Associations of
low

- level urine cadmium with kidney functio n in lead workers. Occupational and Environmental Medicine, 68 (4), 250

- 256. http://dx.doi.org/10.1136/oem.2010.056077

Weaver, V. M., Lee, B.

- K., Ahn, K.

- D., Lee, G.

- S., Todd, A. C., Stewart, W. F., . . . Schwartz, B. S. (2003). Associations of lead
biomarkers with renal function in Korean lead workers. Occupational and Environmental Medicine, 60 , 551

- 562. http://dx.doi.org/10.1136/oem.60.8.551

Weaver , V. M., Schwartz, B. S., Ahn, K.

- D., Stewart, W. F., Kelsey, K. T., Todd, A. C., . . . Lee, B.

- K. (2003). Associations of renal function with polymorphisms in the "delta"

- aminolevulinic acid dehydratase, vitamin D receptor, and nitric oxide synthase genes in Korean lead workers. Environmental Health Perspectives, 111 (13), 1613

- 1619. http://www.ncbi.nlm.nih.gov/pubmed/14527840

Wedrychowski, A., Schmidt, W. N., & Hnilica, L. S. (1986). The in vivo cro ss

- linking of proteins and DNA by heavy metals. Journal of Biological Chemistry, 261 (7), 3370

- 3376. http://www.ncbi.nlm.nih.gov/pubmed/3512554

Wei, H., Wei, D., Yi, S., Zhang, F., & Ding, W. (In Pres s). Oxidative stress induced by urban fine particles in cultured EA.hy926
cells. Human and Experimental Toxicology

. http://dx.doi.org/10.1177/0960327110374207

Weisskopf, M. G., Hu, H., Sparrow, D., L enkinski, R. E., & Wright, R. O. (2007). Proton magnetic resonance spectroscopic evidence of glial effects of cumulative lead exposure in the adult human hippocampus. Environmental Health Perspectives, 115 (4), 519

- 523. http://dx.doi.org/10.1289/ehp.9645

Weisskopf, M. G., Jain, N., Nie, H. L., Sparrow, D., Vokonas, P., Schwartz, J., & Hu, H. (2009). A prospective study of bone lead concentration and death from all causes, cardiovascular diseases, and cancer in the department of veterans affairs normative aging study. Circulation, 120 (12), 1056

- 1064. http://dx.doi.org/10.1161/circulationaha.108.827121

Weisskopf, M. G., Proctor, S. P., Wright, R. O ., Schwartz, J., Spiro, A., III, Sparrow, D., . . . Hu, H. (2007). Cumulative lead exposure and cognitive performance among elderly men. Epidemiology, 18 (1), 59

- 66. http://dx.doi.org/10.109 7/01.ede.0000248237.35363.29

Weisskopf, M. G., Weuve, J., Nie, H., Saint

- Hilaire, M. H., Sudarsky, L., Simon, D. K., . . . Hu, H. (2010). Association of cumulative lead exposure with Parkinson's Disease. Environmental Health Perspectives, 118 (11), 1609

- 1613. http://dx.doi.org/10.1289/ehp.1002339

Weisskopf, M. G., Wright, R. O., Schwartz, J., Spiro, A., III, Sparrow, D., Aro, A., & Hu, H. (2004). Cumulative lead exposure and prospective change in cognition among elderly men: The VA Normative Aging Study. American Journal of Epidemiology, 160 (12), 1184

- 1193. http://dx.doi.org/10.1093/aje/kwh333

Wells, E. M., Navas

- Acien, A., Herbstman, J. B., Ap elberg, B. J., Silbergeld, E. K., Caldwell, K. L., . . . Goldman, L. R. (2011). Low level lead exposure and elevations in blood pressure during pregnancy. Environmental Health Perspectives, 119

, 664

- 669. http://dx.doi.org/10.1289/ehp.1002666

Westerink, R. H., & Vijverberg, H. P. (2002). Ca2+

- independent vesicular catecholamine release in PC12 cells by nanomolar
concentrations of Pb2+. Journal of Neurochemistry, 80 (5), 861

- 867. http://dx.doi.org/10.1046/j.0022

- 3042.2001.00751.x

Wetmur, J. G. (1994). Influence of the common human delta

- aminolevulinate dehydratase polymorphism on lead body burden. Environmental Health Perspectives, 102 (Suppl 3), 215

- 219. http://www.ncbi.nlm.nih.gov/pubmed/7843101

Wetmur, J. G., Lehnert, G., & Desnick, R. J. (1991). The delta

- aminolevulinate dehydratase polymorphism: Higher blood lead
levels in lead workers a nd environmentally exposed children with the 1

- 2 and 2

- 2 isozymes. Environmental Research,
56 (2), 109

- 119. http://dx.doi.org/10.1016/S0013

- 9351(05)80001

- 5May 2011 5

- 453 DRAFT

– DO NOT CITE OR QUOTE Weuve, J., Kelsey, K. T., Schwartz, J., Bellinger, D., Wright, R. O., Rajan, P., . . . Hu, H. (2006). Delta

- aminolevulinic acid dehydratase polymorphism and the relation between low level lead exposure and the Mini

- Mental Status Examination in older men: The Normative Aging Study. Occupational and Environmental Medicine, 63 (11), 746

- 753. http://dx.doi.org/10.1136/oem.2006.027417

Weuve, J., Korrick, S. A., Weisskopf, M. A., Ryan, L. M., Schwartz, J., Nie, H. L., . . . Hu, H. (2009). Cumulati ve exposure to lead in relation to cognitive function in older women. Environmental Health Perspectives, 117 (4), 574

- 580. http://dx.doi.org/10.1289/ehp.11846

White, L. D., Cory

- Slechta, D. A., Gilbert, M. E ., Tiffany

- Castiglioni, E., Zawia, N. H., Virgolini, M., . . . Basha, M. R. (2007). New and evolving concepts in the neurotoxicology of lead. Toxicology and Applied Pharmacology, 225 (1), 1

- 27. http ://dx.doi.org/10.1016/j.taap.2007.08.001

White, R. F., Campbell, R., Echeverria, D., Knox, S. S., & Janulewicz, P. (2009). Assessment of neuropsychological trajectories in longitudinal population

- based studies of children. Journal of Epidemiology and Commu nity Health, 63 , I15

- I26. http://dx.doi.org/10.1136/jech.2007.071530

Wiebe, J. P., & Barr, K. J. (1988). Effect of prenatal and neonatal exposure to lead on the affinity and number of estradiol

recep tors in the uterus. Journal of Toxicology and Environmental Health, 24 (4), 451

- 460. http://dx.doi.org/10.1080/15287398809531176

Wiemann, M., Schirrmacher, K., & Busselberg, D. (1999). Interference of lead with the calcium release activated calcium flux of
osteoblast

- like cells. Calcified Tissue International, 65 (6), 479

- 485. http://dx.doi.org/10.1007/s00223990 0736

Williams, B. L., Pennock

- Román, M., Suen, H. K., Magsumbol, M. S., & Ozdenerol, E. (2007). Assessing the impact of the local
environment on birth outcomes: A case for HLM. Journal of Exposure Science and Environmental Epidemiology, 17

, 445

- 457. http://dx.doi.org/10.1038/sj.jes.7500537

Williams, P. L., Sergeyev, O., Lee, M. M., Korrick, S. A., Burns, J. S., Humblet, O., . . . Hauser, R. (2010). Blood lead levels and delayed onset of puberty in a lon gitudinal study of Russian boys. Pediatrics, 125 (5), 1088

- 1096. http://dx.doi.org/10.1542/peds.2009

- 2575

Wise, J. P., Sr., Stearns, D. M., Wetterhahn, K. E., & Patierno, S. R. (1994). Cell

- enhanced dis solution of carcinogenic lead chromate particles: the role of individual dissolution products in clastogenesis. Carcinogenesis, 15 (10), 2249

- 2254. http://dx.doi.org/10.1093/carcin/15.10.2249

Wise, J . P., Sr., Wise, S. S., LaCerte, C., Wise, J. P., & Aboueissa, A. M. (2011). The genotoxicity of particulate and soluble chromate in sperm whale (physeter macrocephalus) skin fibroblasts. Environmental and Molecular Mutagenesis, 52 (1),
43

- 49. http://dx.doi.org/10.1002/em.20579

Wise, S. S., Holmes, A. L., Ketterer, M. E., Hartsock, W. J., Fomchenko, E., Katsifis, S., . . . Wise, J. P., Sr. (2004). Chromium is the proximate clastogenic species for lead chromate

- induced clastogenicity in human bronchial cells. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 560 (1), 79

- 89. http://dx.doi.org/10.1016/j.mrgentox.2004.02.009

Wise, S. S ., Holmes, A. L., Moreland, J. A., Xie, H., Sandwick, S. J., Stackpole, M. M., . . . Wise, J. P., Sr. (2005). Human lung cell growth is not stimulated by lead ions after lead chromate

- induced genotoxicity. Molecular and Cellular Biochemistry, 279 (1

- 2), 75

- 84. http://dx.doi.org/10.1007/s11010

- 005

- 8217

- 0

Wise, S. S., Holmes, A. L., Qin, Q., Xie, H., Katsifis, S. P., Thompson, W. D., & Wise, J. P., Sr. (2010). Comparative genotoxicity and cytotoxicity of four hexavalent chromium compounds in human bronchial cells. Chemical Research in Toxicology,
23 (2), 365

- 372. http://dx.doi.org/10.1021 /tx900363jMay 2011 5

- 454 DRAFT

– DO NOT CITE OR QUOTE Wise, S. S., Holmes, A. L., & Wise, J. P., Sr. (2006). Particulate and soluble hexavalent chromium ate cytotoxic and genotoxic to human lung epithelial cells. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 610 (1

- 2), 2

- 7. http://dx.doi.org/10.1016/j.mrgentox.2006.06.005

Wise, S. S., Holmes, A. L., Xie, H., Thompson, W. D., & Wise, J. P., Sr. (2006). Chronic exposure to particulate chromate induces spindle assembly checkpoint bypass in human lung cells. Chemical Research in Toxicology, 19 (11), 1492

- 1498. http://dx.doi.org/10.1021/tx0601410

Wise, S. S., Shaffley, F., LaCerte, C., Goertz, C. E. C., Dunn, J. L., Gulland, F. M. D., . . . Wise, J. P. (2009). Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to Steller sea lion lung cells. Aquatic Toxicology, 91 (4), 329

- 335. http://dx.doi.org/10.1016/j.aquatox.2008.12.004

Wiwanitkit, V., Suwansaksri, J., & Soogarun, S. (2008). White blood cell sister chromatid exchange among a sample of Thai subjects exposed to lead : Lead

- induced genotoxicity. Toxicological and Environmental Chemistry, 90 (4), 765

- 768. http://dx.doi.org/10.1080/02772240701712758

Wolf, J., & Daley, A. J. (2007). Microbiological aspects of bacter ial lower respiratory tract illness in children: Atypical pathogens. Paediatric Respiratory Reviews, 8 (3), 212

- 220. http://dx.doi.org/10.1016/j.prrv.2007.07.004

Wolff, M. S., Britton, J. A., Bogusk i, L., Hochman, S., Maloney, N., Serra, N., . . . Forman, J. (2008). Environmental exposures and puberty in inner

- city girls. Environmental Research, 107 (3), 393

- 400. http://dx.doi.org/10.1016/j. envres.2008.03.006

Wozniak, K., & Blasiak, J. (2003). In vitro genotoxicity of lead acetate: Induction of single and double DNA strand breaks and
DNA

- protein cross

- links. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 535 (2), 127

- 139. http://dx.doi.org/10.1016/S1383

- 5718(02)00295

- 4

Wright, J. P., Dietrich, K. N., Ris, M. D., Hornung, R. W., Wessel, S. D., Lanphear, B. P., . . . Rae, M. N. (2008). Association of prenatal and childhood blood lead concentrations with criminal arrests in early adulthood. PLoS Medicine, 5 (5), 732

- 740. http://dx.doi.org/10.1371/journal.pmed.0050101

Wright, R. J. (2009). Moving towards mak ing social toxins mainstream in chidlren's environmental health. Current Opinion in Pediatrics, 21 (2), 222

- 229. http://dx.doi.org/10.1097/MOP.0b013e3283292629

Wright, R. O., Schwartz, J., Wright, R. J., Bollati, V., Tarantini, L., Park, S. K., . . . Baccarelli, A. (2010). Biomarkers of lead exposure and DNA methylation within retrotransposons. Environmental Health Perspectives, 118 (6), 790

- 795.
h ttp://dx.doi.org/10.1289/ehp.0901429

Wright, R. O., Tsaih, S. W., Schwartz, J., Spiro, A., McDonald, K., Weiss, S. T., & Hu, H. (2003). Lead exposure biomarkers and
mini

- mental status exam scores in older men. Epidemiology, 14 (6), 713

- 718. http://dx.doi.org/10.1097/01.EDE.0000081988.85964.db

Wu, J., Basha, M. R., Brock, B., Cox, D. P., Cardozo

- Pelaez, F., McPherson, C. A., . . . Zawia, N. H. (2008). Alzheimer's disease (AD)

- like pathology in aged monkeys after infantile exposure to environmental metal lead (pb): Evidence for a developmental origin and environmental link for AD. Journal of Neuroscience, 28 (1), 3

- 9. http://dx.doi.or g/10.1523/jneurosci.4405

- 07.2008

Wu, J. F., Basha, R., & Zawia, N. H. (2008). The environment, epigenetics and amyloidogenesis. Journal of Molecular Neuroscience, 34 (1), 1

- 7. http://dx.doi.org/10.1007/s12031

- 007

- 0009

- 4

Wu, M. T., Kelsey, K., Schwartz, J., & Sparrow, D. (2003). A "delta"

- aminolevulinic acid dehydratase (ALAD) polymorphism may modify the relationship of low

- level lead exposure to uricemia and renal function: The Normative Aging Study. Environmental Health Perspectives, 111 (3), 335

- 341. http://dx.doi.org/10.1289/ehp.5504May 2011 5

- 455 DRAFT

– DO NOT CITE OR QUOTE Wu, T.

- N., Shen, C. Y., Lai, J. S., Goo, C. F., Ko, K. N., Chi, H. Y., . . . Liou, S. H. (2000). Effects of lead and noise exposures on hearing ability. Archives of Environmental Health, 55 (2), 109

- 114. http://dx.doi.org/10.1080/00039890009603396

Wu, T., Buck, G. M., & Mendola, P. (2003). Blood lead levels and sexual maturation in U.S. girls: The Third National Health and Nutrition Examination Survey, 1988

- 1994. Environmental Health Perspectives, 111 (5), 737

- 741. http://dx.doi.org/10.1289/ehp.6008

Xie, H., Holmes, A. L., Wise, S. S., Huang, S. P., Peng, C., & Wise, J. P. (2007). Neoplastic transformation of human bronchial cells by lead chromate particles. American Journal of Respiratory Cell and Molecular Biology, 37 (5), 544

- 552. http://dx.doi.org/10.1165/rcmb.2007

- 00580C

Xie, H., Wise, S. S., & Wise, J. P. (2008). Deficient repair of particulate hexavalent chromium

- induced DNA double strand breaks leads to neoplastic transformation. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 649 (1

- 2),
230

- 238. http://dx.doi.org/10.1016/j.mrgentox.2007.09.008

Xie, Y., Chiba, M., Shinohara, A., Watanabe, H., & Inaba, Y. (19 98). Studies on lead

- binding protein and interaction between lead and selenium in the human erythrocytes. Industrial Health, 36 (3), 234

- 239. http://dx.doi.org/10.2486/indhealth.36.234

Xu, J., Ji, L. D., & Xu, L. H. (2006). Lead

- induced apoptosis in PC 12 cells: Involvement of p53, Bcl

- 2 family and caspase

- 3. Toxicology Letters, 166 (2), 160

- 167. http://dx.doi.org/10.1016/j.toxlet.2006.06.643

Xu, J., Lian, L., W, C., Wang, X., Fu, W., & Xu, L. (2008). Lead induces oxidative stress, DNA damage and alteration of p53 Bax
and Bcl

- 2 expressions in mice. Food and Chemical Toxicology, 46 (5), 1488

- 1494. http://dx.doi.org/10.1016/j.fct.2007.12.016

Xu, S.

- Z., & Rajanna, B. (2006). Glutamic acid reverses Pb super(2+)

- induced reductions of NMDA receptor subunits in vitro. NeuroToxicology, 27 (2), 169

- 175. http://dx.doi.org/10.1016/j.neuro.2005.08.005

Yamanaka, K., Chun, S. J., Boillee, S., Fujimori

- Tonou, N., Yamashita, H., Gutmann, D. H., . . . Cleveland, D. W. (2008). Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nature Neuroscience,
11 (3), 251

- 253. http://dx.doi.org/10.1038/Nn2047

Yanez, L., Garcia

- Nieto, E., Rojas, E., Carrizales, L., Mejia, J., Calderon, J., . . . Diaz

- Barriga, F. (2003). DNA damage in blood cells from children exposed to arsenic and lead in a mining area. Environmental Research, 93 , 231

- 240. http://dx.doi.org/10.1016/j.envres.2003.07.005

Yang, Y., Raine, A., Lencz, T., Bihrle, S., LaCasse, L., & Colletti, P. (2005). Volume reduction in prefrontal gray matter in unsuccessful criminal psychopaths. Biologic al Psychiatry, 57 (10), 1103

- 1108. http://dx.doi.org/10.1016/j.biopsych.2005.01.021

Yazbeck, C., Thiebaugeorges, O., Moreau, T., Goua, V., Debotte, G., Sahuquillo, J., . . . Huel, G. (2009). Mat ernal blood lead levels and the risk of pregnancy

- induced hypertension: The EDEN cohort study. Environmental Health Perspectives,
117 (10), 1526

- 1530. http://dx.doi.org/10.1289/ehp.0800488

Ye, S.

- H. (1993). Hypoxanthine phosphoribosyl transferase assay of lead mutagenicity on keratinocytes. Acta Pharmacologica Sinica, 14 (2), 145

- 147. http://www.ncbi.nlm.nih.gov/pubmed/8352008

Yetkin

- Ay , Z., Cadir, B., Uskun, E., Bozkurt, F. Y., Delibas, N., Gultepe, F. M., & Ergurhan

- Ilhan, I. (2007). The periodontal status of indirectly lead

- exposed apprentices working in autorepair workshops. Toxicology and Industrial Health,
23 (10), 599

- 606. http://dx.doi.org/10.1177/0748233708090906

Yiin, S. J., & Lin, T. H. (1995). Lead

- catalyzed peroxidation of essential unsaturated fatty acid. Biological Trace Element Research, 50 (2), 167

- 172. http://dx.doi.org/10.1007/BF02789419May 2011 5

- 456 DRAFT

– DO NOT CITE OR QUOTE Yin, Y., Zhang, T., Dai, Y., Bao, Y., Chen, X., & Lu, X. (2008). The effect of plasma lead on anembryonic pregnancy. Annals of the New York Academy of Sciences, 1140 , 184

- 189. http://dx.doi.org/10.1196/annals.1454.042

Yu, C.

- C., Lin, J.

- L., & Lin

- Tan, D.

- T. (2004). Environmental exposure to lead and progression of chronic renal diseases: A four

year prospective longitudinal study

. Journal of the American Society of Nephrology: JASN, 15 , 1016

- 1022. http://dx.doi.org/10.1097/01.ASN.0000118529.01681.4F

Yu, D. Y., Li, W. F., Deng, B., & Mao, X. F. (2008). Effects of le ad on hepatic antioxidant status and transcription of superoxide dismutase gene in pigs. Biological Trace Element Research, 126 (1

- 3), 121

- 128. http://dx.doi.org/10.1007/s12011

- 008

- 8198

- 4

Yu, J., Fuj ishiro, H., Miyataka, H., Oyama, T. M., Hasegawa, T., Seko, Y., . . . Himeno, S. (2009). Dichotomous effects of lead acetate on the expression of metallothionein in the liver and kidney of mice. Biological and Pharmaceutical Bulletin,
32 (6), 1037

- 1042. http://dx.doi.org/10.1248/bpb.32.1037

Yuan, W., Holland, S. K., Cecil, K. M., Dietrich, K. N., Wessel, S. D., Altaye, M., . . . Lanphear, B. P. (2006). The impact of early childhood lead exposure on brain organization: A functional magnetic resonance imaging study of language function.
Pediatr ics, 118 (3), 971

- 977. http://dx.doi.org/10.1542/peds.2006

- 0467

Yucesoy, B., Turhan, A., Ure, M., Imir, T., & Karakaya, A. (1997a). Effects of occupational lead and cadmium exposure on some immunoregula tory cytokine levels in man. Toxicology, 123 (1

- 2), 143

- 147. http://dx.doi.org/10.1016/S0300

- 483X(97)00107

- 8

Yucesoy, B., Turhan, A., Ure, M., Imir, T., & Karakaya, A. (1997b). Simultaneous effec ts of lead and cadmium on NK cell activity and some phenotypic parameters. Immunopharmacology and Immunotoxicology, 19 (3), 339

- 348. http://dx.doi.org/10.3109/08923979709046980

Yun, S., & Hoyer, S. ( 2000). Effects of low

- level lead on glycolytic enzymes and pyruvate dehydrogenase of rat brain in vitro: Relevance to sporadic Alzheimer's disease? Journal of Neural Transmission, 107 (3), 355

- 368. http: //dx.doi.org/10.1007/s007020050030

Zawia, N. H., & Harry, G. J. (1995). Exposure to lead

- acetate modulates the developmental expression of myelin genes in the rat
frontal lobe. International Journal of Developmental Neuroscience, 13 (6), 639

- 644. http://dx.doi.org/10.1016/0736

- 5748(95)00032

- C

Zawia, N. H., Sharan, R., Brydie, M., Oyama, T., & Crumpton, T. (1998). Sp1 as a target site for metal

- induced perturbations of transcriptional regulation of developmental brain gene expression. Developmental Brain Research, 107 (2), 291

- 298. http://dx.doi.org/10.1016/S0165

- 3806(98)00023

- 6

Zeisel, S., & Blusztajn, J. (1994). Choline and human nutritio n. Annual Review of Nutrition, 14 (1), 269

- 296. http://dx.doi.org/10.1146/annurev.nu.14.070194.001413

Zelikoff, J. T., Li, J. H., Hartwig, A., Wang, X. W., Costa, M., & Rossman, T. G. (1988 ). Genetic toxicology of lead compounds. Carcinogenesis, 9 (10), 1727

- 1732. http://dx.doi.org/10.1093/carcin/9.10.1727

Zeller, I., Knoflach, M., Seubert, A., Kreutmayer, S., Stelzmüller, M., Wallnoefe r, E., . . . Bernhard, D. (2010). Lead contributes to arterial intimal hyperplasia through nuclear factor erythroid 2

- related factor

- mediated endothelial interleukin 8 synthesis and subsequent invasion of smooth muscle cells. Arteriosclerosis Thrombosis an d Vascular Biology, 30 (9),
1733

- 1740. http://dx.doi.org/10.1161/ATVBAHA.110.211011

Zeng, H., Chattarji, S., Barbarosie, M., Rondi

- Reig, L., Philpot, B., Miyakawa, T., . . . Tonegawa, S. (2001). For ebrain

- specific calcineurin knockout selectively impairs bidirectional synaptic plasticity and working/episodic

- like memory. Cell,
107 (5), 617

- 629. http://www.ncbi.nlm.nih.gov/pubmed/11733061May 2011 5

- 457 DRAFT

– DO NOT CITE OR QUOTE Zentner, L. E., Rondó, P. H., & Mastroeni, S. S. (2006). Lead contamination and anthropometry of the newborn baby. Journal of Tropical Pediatrics, 52 (5), 369

- 371. http://dx.doi.org/10.1093/tropej/fml009

Zeyrek, D., Soran, M., Cakmak, A., Kocyigit, A., & Iscan, A. (2009). Serum copper and ainc levels in mothers and cord blood of
their newborn infants with neural tube defects: A case

- control study. Indian Pediatrics, 46 (8), 675

- 680. http://www.ncbi.nlm.nih.gov/pubmed/19430086

Zhang, A., Park, S. K., Wright, R. O., Weisskopf, M. G., Mukherjee, B., Nie, H., . . . Hu, H. (2010). HFE H63D polymorphism as a modifier of the effect of cumulative lead exposu re on pulse pressure: The Normative Aging Study. Environmental Health Perspectives, 118 (9), 1261

- 1266. http://dx.doi.org/10.1289/ehp.1002251

Zhang, D., Hu, X., Qian, L., O'Callaghan, J. P., & Hong, J. S. (2010). Astrogliosis in CNS pathologies: Is there a role for microglia? Molecular Neurobiology, 41 (2

- 3), 232

- 241. http://dx.doi.org/10.1007/s12035

- 010

- 8098

- 4

Zhang, L.

- F., Peng, S.

- Q., & Wang, S. (2 009). Decreased aortic contractile reaction to 5

- hydroxytryptamine in rats with long

- term hypertension induced by lead (Pb2+) exposure. Toxicology Letters, 186 (2), 78

- 83. http://dx.doi.org/10.101 6/j.toxlet.2009.01.004

Zhang, L. F., Peng, S. Q., Sheng, W., Li, B. L., Gang, H., & Dong, Y. S. (2007). Direct effects of lead (Pb2+) on the relaxation of in vitro cultured rat aorta to acetylcholine. Toxicology Letters, 170 (2), 104

- 110. http://dx.doi.org/10.1016/j.toxlet.2007.02.004

Zhang, L. F., Peng, S. Q., & Wang, S. (2005). Influence of lead (Pb2+) on the reactions of in vitro cultured rat aorta to 5

- hydroxytryptamine. Toxicology Letters, 159 (1), 71

- 82. http://dx.doi.org/10.1016/j.toxlet.2005.04.009

Zhao, Y., Wang, L., Shen, H. B., Wang, Z. X., Wei, Q. Y., & Chen, F. (2007). Association between delta

- aminolevulinic acid dehydratase ( ALAD) polymorphism and blood lead levels: A meta

- regression analysis. Journal of Toxicology and Environmental Health, 70 (23), 1986

- 1994. http://dx.doi.org/10.1080/15287390701550946

Zhao, Z. Y., Li, R., Sun, L., Li, Z. Y., & Yang, R. L. (2004). Effect of lead exposure on the immune function of lymphocytes and erythrocytes in preschool children. Journal of Zhejiang University

- Science, 5 (8), 1001

- 1004. http://dx.doi.org/10.1007/BF02947614

Zheng, C., Sköld, M., Li, J., Nennesmo, I., Fadeel, B., & Henter, J. (2007). VEGF reduces astrogliosis and preserves neuromuscular junctions in ALS transgenic mice. Biochemical and Biophysical Research Communications, 363 (4),
989

- 993. http://dx.doi.org/10.1016/j.bbrc.2007.09.088

Zimmermann, M. B., Muthayya, S., Moretti, D., Kurpad, A., & Hurrell, R. F. (2006). Iron fortification reduces blood lead levels in children in Bangalore, India. Pediatrics, 117 (6), 2014

- 2021. http://dx.doi.org/10.1542/peds.2005

- 2440

Zoeger, N., Roschger, P., Hofstaetter, J. G., Jokubonis, C., Pepponi, G., Falkenberg, G., . . . Wobrauschek, P. (2006). Lead accumulation in tidemark of articular cartilage. Osteoarthritis and Cartilage, 14 (9), 906

- 913. http://dx.doi.org/10.1016/j.joca.2006.03.001

Zuo, P. J., Qu, W., Cooper, R. N., Goyer, R. A., Diwan, B. A., & Waalkes, M. P. (2009). Potential role of alpha

- synuclein and
metallothionein in lead

- induced inclusion body formation. Toxi cological Sciences, 111 (1), 100

- 108. http://dx.doi.org/10.1093/toxsci/kfp132

Zuscik, M. J., Ma, L., Buckley, T., Puzas, J. E., Drissi, H., Schwarz, E. M., & O'Keefe, R. J. (2007). Lead induces chondroge nesis and alters transforming growth factor

- beta and bone morphogenetic protein signaling in mesenchymal cell populations. Environmental Health Perspectives, 115 (9), 1276

- 1282. http://dx.doi.org/10.1289/ehp .10028