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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 Ca2+ Homeostasis ________________________________________________ 5-3

5.2.2.2. Disruption 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. Cell 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 resulting 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) among 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._________________________________________________5-51
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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. Regression 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 characteristics 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 stressors 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. Summary of effects of maternal and lifetime Pb exposure on FI performance 

watera

. __________________________________________________________5-83

5.3.2.3. Toxicological Studies on the Effects of Chelation _________________________________ 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 Pb concentration). ______________________5-95

5.3.3. Neurobehavioral Effects_____________________________________________________________ 5-98

5.3.3.1. Epidemiologic Studies of Behavioral Effects in Children ___________________________ 5-98

Figure 5-19. 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. ________________________________5-103
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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 contributors 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. Sensory 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 GLE 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 Homeostasis ___________________________________________ 5-129

5.3.6.3. Oxidative Stress __________________________________________________________ 5-129

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. Additional 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. ____________________________________________5-158
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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 stress. ______________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. Summary _______________________________________________________________ 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 Determination __________________________________________________ 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_________________________________________5-204
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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. Associations 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 Renal 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 subtypesa among various blood 

Pb groups.______________________________________________________5-247

5.6.2.2. Lymphocyte Activation ____________________________________________________ 5-248

5.6.2.3. Delayed-type Hypersensitivity _______________________________________________ 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 

(μg/dL). ________________________________________________________5-256

Figure 5-50. Comparison of the relative abundance of B cells among groups with 

increasing blood Pb level (μg/dL). ___________________________________5-256

Table 5-24. Additional characteristics and quantitative dataa for results presented in 

Figures 5-49 and 5-50_____________________________________________5-257
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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 respiratory 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 E2 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. Membrane 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. Reproductive 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 effects 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 ______5-304
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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 effects 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. Summary of epidemiologic studies of cancer incidence and mortality_________5-350

5.10.1.1. Overall Cancer Mortality ___________________________________________________ 5-353
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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 ___________________________ 5-359

5.10.3.3. Mechanisms of Action _____________________________________________________ 5-365

5.10.3.4. Effects of Lead 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 determinations for Pb. _____________________________5-369

Chapter 5. References ___________________________________________________________________________ 5-370
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Chapter 5. Integrated Health Effects 

of LeadExposure

5.1. Introduction

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

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

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

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

5 assessments of the epidemiologic and toxicological evidence for the effects of Pb exposure on major 

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

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

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

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

10 available, including those related to the hepatic system (Section 5.9.1), gastrointestinal system (Section 

11 5.9.2), endocrine system (Section 5.9.3), bone and teeth (Section 5.9.4), ocular health (Section 5.9.5), and 

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

13 on cancer (Section 5.10).

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

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

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

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

18 scientific discipline (i.e., epidemiology, toxicology). Each major section (e.g., neurological, 

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

20 regarding causality. Based upon the framework described in Chapter 1, a determination of causality is 

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

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

23 endpoints.

 

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 (ISA) and the Integrated Risk Information System (IRIS).
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5.2. Modes of Action

5.2.1. Introduction

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

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

3 targeted. A mode of action is the sequence of key events (i.e., empirically observable precursor steps) that 

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

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

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

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

8 modes of action (Figure 5-1). The following section draws information from all of the health effects 

9 sections in the current document and identifies the major modes of action operating at the molecular, 

10 cellular, and tissue/organ level. In turn, individual health effect sections bridge these effects to toxicities 

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

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

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

14 the possible modes of action of Pb.

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.
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5.2.2. Altered Ion Status

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

2 functions in biological systems, including roles as charge carriers, intermediates in enzymatically3 catalyzed reactions, and as structural elements in the proper maintenance of tertiary protein conformations 

4 (Garza et al., 2006). It is through disruption of these biological functions that Pb effects its toxic action, 

5 ultimately adversely affecting such tightly regulated processes as cell signaling, intracellular ion 

6 homeostasis, ion transport, energy metabolism, and enzymatic function.

5.2.2.1. Disruption of Ca2+ Homeostasis

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

8 cell function, including energy metabolism, signal transduction, hormonal regulation, cellular motility, 

and apoptosis (Carafoli, 2005). Ca2+ 9 homeostasis is maintained through a tightly regulated balance of 

cellular transport and intracellular storage (Pentyala et al., 2010). Disruption of Ca2+ 10 homeostasis by Pb 

11 has been observed in a number of different cell types and cell-free environments, indicating that this is a 

12 major mode of action for Pb-induced toxicity on a cellular level.

Ca2+ 13 homeostasis is particularly important in bone cells, as the skeletal system serves as the major 

dynamic reservoir of Ca2+ 14 in the body (Long et al., 1992; Wiemann et al., 1999). Bone cells also are 

unique in that they exist in a microenvironment that is high in both Ca2+ 15 and Pb concentrations, 

16 potentially increasing their relative susceptibility to Pb-induced toxicity (Long et al., 1992). A series of 

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

osteoblastic bone cells to Pb disrupts intracellular Ca2+ levels ([Ca2+ 18 ]i). Exposure of osteoblasts to 1, 5, or 

25 μM Pb for 40-300 minutes resulted in prolonged increases in [Ca2+ 19 ]i of 36, 50 and 120% over baseline, 

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

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

increased [Ca2+]i. Pretreatment of Pb-exposed cells with PTH increased [Ca2+ 22 ]i above concentrations 

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

hormonal control. A similar additive increase in [Ca2+ 24 ]i was also observed when bone cells were co25 treated with epidermal growth factor (EGF) and Pb versus Pb alone (Long & Rosen, 1992). Pb-induced 

increases in [Ca2+ 26 ]i were blocked by a protein kinase C (PKC) inhibitor, indicating that PKC activation 

may serve as the mechanism by which Pb perturbs [Ca2+ 27 ]i (Schanne et al., 1997). Schirrmacher et al. 

(1998) also observed alterations in Ca2+ 28 homeostasis in osteoblasts exposed to 5 μM Pb for 50 minutes 

due to potential disruption of Ca2+ 29 -ATPases. However, Wiemann et al. (1999) demonstrated that exposure 

to 5 or 12.5 μM Pb inhibited the Ca2+ release activated calcium (CRAC) influx of Ca2+ 30 independent of 

any inhibitory effect on Ca2+ 31 - ATPases.
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Ca2+ 1 homeostasis has also been shown be to disturbed in erythrocytes due to Pb exposure 

2 (Quintanar-Escorza et al., 2010; Quintanar-Escorza et al., 2007; Shin et al., 2007). In blood samples taken 

from Pb-exposed workers (blood Pb level = 74.4 ± 21.9 μg/dL), the [Ca2+ 3 ]i was approximately 2.5-fold 

4 higher than that seen in nonexposed workers (blood Pb level = 9.9 ± 2 μg/dL) (Quintanar-Escorza et al., 

2007). The increase in [Ca2+ 5 ]i was associated with higher osmotic fragility and modifications in 

6 erythrocyte shape. When erythrocytes from 10 healthy volunteers were exposed to Pb at concentrations of 

0.2 to 6.0 μM for 24 or 120 hours, dose-related increases in [Ca2+ 7 ]i were observed across all 

8 concentrations for both durations of exposure (Quintanar-Escorza et al., 2010). Subsequent exposures of 

9 erythrocytes to either 0.4 or 4.0 μM Pb (corresponding to 10 or 80 μg/dL in exposed workers (Quintanar10 Escorza et al., 2007)) for 12-120 hours demonstrated duration-related increases at durations >12 hours. 

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

12 for 24 hours. Co-incubation with a vitamin E analog mitigated these effects, indicating that the increase in 

[Ca2+]i 13 is dependent on the oxidative state of the erythrocytes. Shin et al. (2007) observed that incubation 

of human erythrocytes with 5 μM Pb for 1 hour resulted in a 30-fold increase in [Ca2+]i 14 in vitro, inducing 

15 the pro-coagulant activity of exposed erythrocytes. Induction of pro-coagulant activity in erythrocytes 

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

17 increased osmotic fragility could substantially reduce erythrocyte life span and ultimately lead to anemic 

18 conditions.

Similar to effects seen in erythrocytes, Ca2+ 19 homeostasis has been observed in platelets and white 

blood cells. Dowd and Gupta (1991) observed statistically significant increases in [Ca2+]i 20 in human 

platelets exposed to as little as 1 μM Pb for 3.5 hours. The observed increase in Ca2+ 21 levels was attributed 

to increased influx of external Ca2+, possibly through receptor-operated Ca2+ 22 channels. In mouse splenic 

lymphocytes exposed to Pb, [Ca2+ 23 ]i was increased at exposure levels as low as 1 μM when incubated for 

10 minutes or greater (S. Li et al., 2008). These increases in Ca2+ appeared to be reversible as [Ca2+ 24 ]i

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

effects of Pb exposure, indicating a role for calmodulin in disruption of Ca2+ 26 homeostasis in lymphocytes. 

In rat tail arteries exposed to 1.2 μM Pb acetate for 1 hour, intracellular stores of Ca2+ 27 increased over 

controls, possible through increased transmembrane influx of Ca2+ 28 (Piccinini et al., 1977). 

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

resulted in increased release of Ca2+ 30 into the media (Pentyala et al., 2010). Further, Pb exposure also 

decreased the activity of the microsomal Ca2+-ATPase, thus decreasing the sequestration of Ca2+ 31 into 

microsomes. The results of this study suggest that disruption of microsomal release and re-uptake of Ca2+ 32

may alter Ca2+ 33 homeostasis, ultimately leading to altered signal transduction and neuronal dysfunction. 

However, Ferguson et al. (2000) observed that [Ca2+ 34 ]i was decreased in rat hippocampal neurons in 

35 response to exposure to 100 nM Pb for 1-48 hours, although the observed decreases were not time-
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dependent. The decrease in [Ca2+]i was shown to be due to increased efflux of Ca2+ 1 out of the neuron via a 

calmodulin-regulated mechanism, possibly through stimulated Ca2+ efflux via Ca2+ 2 -ATPase.

5.2.2.2. Disruption of Ion Transport Mechanisms

As described above, deregulation of Ca2+ 3 homeostasis results in negative effects in multiple organ 

systems. Under normal conditions, cytosolic concentrations of free Ca2+ 4 are maintained at low levels (0.1 

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

component of the maintenance of Ca2+ homeostasis is transmembrane transport of Ca ions via Ca2+ 6 -

7 ATPase and voltage-sensitive gates (Carafoli, 2005). Pb has been shown to disrupt the normal movement 

of Ca2+ 8 ions, as well as other physiologically important ions through interactions with these transport 

9 mechanisms.

Multiple studies have reported the effects of Pb exposure on Na+-K+-ATPase, Ca2+ 10 -ATPase, and 

Mg2+ 11 -ATPases in animal models. Decreases in the activity of all three ATPases were observed in the 

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

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

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

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

generation of free radicals in Pb-exposed animals. A decrease in the activity of Na+-K+ 16 -ATPase was 

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

18 hydrolytic cleavage of phosphorylated intermediates in the K-related branch of the pump (Gramigni et al., 

2009). Similar decreases in Na+-K+ 19 -ATPase activity were observed in synaptosomes isolated from rats 

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

21 1996) or 15 mg/kg Pb injected intraperitoneally for 7 days (blood Pb = 112.5 μg/dL) (Struzynska, 

Dabrowska-Bouta, et al., 1997). The activity of Ca2+ 22 -ATPase in the sarcoplasmic reticulum of rabbits 

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

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

25 ATPase was reported to be altered in the testes of rats exposed to 300 mg/L Pb acetate gestationally, and 

26 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 Pb27 depleted (20 ± 5 μg/kg) or control (1 mg/kg) diet during gestation and lactation, no difference was 

observed in the activity of Na+-K+-ATPase and Ca2+-Mg2+ 28 -ATPase in the P0 generation (Eder et al., 1990). 

29 However, the F1 generation of Pb-depleted rats displayed decreased activities in both enzymes (meaning 

30 that animals with higher exposure to Pb, i.e., “control” animals, had higher enzymatic activities). A 

similar increase in the activity Na+-K+ 31 -ATPase was observed in rats exposed to 20 mg/kg Pb 

32 intraperitoneally for 14 consecutive days (Jehan & Motlag, 1995). Co-exposure of Pb with zinc and 

33 copper greatly reduced the increase in ATPase activity observed. Although the precise mechanism was not 
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investigated, Navarro-Moreno et al. (2009) reported that Ca2+ 1 uptake was diminished in proximal renal 

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

3 7.6 μg/dL).

4 In vitro studies of ATPase activities in human erythrocyte ghosts have also shown that Pb affects 

5 the transport of metal ions across membranes. Calderon-Salinas et al. (1999) observed that 10-50 mM Pb 

and Ca2+ 6 were capable of inhibiting the passive transport of each other in human erythrocyte ghosts 

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

8 electrogenic transport pathway (Sakuma et al., 1984). Further study by this group (Calderon-Salinas, 

Quintanar-Escorza, et al., 1999) demonstrated that Pb can noncompetitively block the transport of Ca2+ 9 by 

inhibiting the activity of Ca2+-Mg2+ 10 -ATPase at concentrations of 1-5 mM. Mas-Oliva (1989) demonstrated 

that the activity of Ca2+-Mg2+ 11 -ATPase in human erythrocyte ghosts was inhibited by incubation with 0.1-

12 100 μM Pb. The inhibitory action was most likely due to direct reaction with sulfhydryl groups on the 

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

concentrations. Grabowska and Guminska (1996) observed that the activity of Na+-K+ 14 -ATPase was 

decreased in erythrocyte ghosts exposed to concentrations as low as 10 μg/dL Pb; activity of Ca2+- Mg2+ 15 -

ATPase was less sensitive to Pb exposure and Mg2+ 16 -ATPase activity was not affected.

17 In a study investigating ATPase activities in occupationally-exposed workers in Nigeria, Abam et 

al. (2008) observed that the activity of erythrocyte membrane-bound Ca2+- Mg2+ 18 -ATPase was decreased 

19 by roughly 50% in all occupational groups (range of mean ± SD blood Pb level across nine occupational 

20 groups = 28.75 ± 11.31 - 42.07 ± 12.01 μg/dL) compared to nonexposed controls (blood Pb = 12.34 ± 

2.44 (males) and 16.85 ± 6.01 μg/dL (females)). Increased membrane concentrations of Ca2+ 21 and 

22 magnesium were also observed, indicating that Pb prevented the efflux of those cations from the cell, 

23 most likely by substituting for those metals in the active site of the ATPase. Huel et al. (2008) found that 

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

Ca2+ 25 -ATPase activity in plasma membranes of erythrocytes isolated from cord blood; newborn hair Pb 

levels were more strongly associated with cord Ca2+ 26 pump activity than cord blood Pb

27 Pb has also been shown to disrupt cation transport mechanisms through direct action on voltage28 sensitive cation channels. Audesirk and Audesirk (1991, 1993) demonstrated that extracellular free Pb 

inhibits the action of multiple voltage-sensitive Ca2+ 29 channels, with free Pb IC50 (half maximal inhibitory 

30 concentration) values of 0.7 μM for L-type channels and 1.3 μM for T-type channels in neuroblastoma 

31 cells, and IC50 values as low as 0.03 μM for L-type channels in cultured hippocampal neurons. Sun and 

Suszkiw (1995) confirmed the inhibitory action of extracellular Pb on Ca2+ 32 channels, demonstrating an 

IC50 value of 0.3 μM in adrenal chromaffin cells. The observed disruption of the Ca2+ 33 channels most 

likely reflects competition between Pb and Ca2+ for the extracellular Ca2+ 34 binding domain of the channel. 

Research by other laboratories supported these findings: Pb inhibited the action of multiple Ca2+ 35 channels 

36 in human embryonic kidney cells transfected with L-, N-, and R-type channels (IC50 values of 0.38 μM, 
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1 1.31 μM, and 0.10 μM, respectively) (Peng et al., 2002) and P-type channels in cultured hippocampal 

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

enhance Ca2+ currents through attenuation of the Ca2+ dependent deactivation of Ca2+ 3 channels at an EC50

value of 0.2 nM, possibly through blocking the intracellular Ca2+ binding domain, or through Ca2+ 4

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

Pb also disrupts the action of Ca2+ 6 -dependent potassium channels. Alvarez et al. (1986) observed 

7 that Pb promoted the efflux of potassium from inside-out erythrocyte vesicles in a dose-dependent manner 

8 at concentrations of 1-300 μM, either through action on a Mg modulatory site or through direct 

interaction with the Ca2+ 9 binding site. Fehlau et al. (1989) also demonstrated Pb-induced activation of the 

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

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

12 modulation of potassium permeability is due to alterations in channel gating. Silken et al. (2001) observed 

13 that Pb activated potassium channels in erythrocytes from the marine teleost Scorpaena porcus in a dose14 dependent manner after a 20-minute incubation; minor loss of potassium was seen at Pb concentrations of 

15 1-2 μM, whereas exposure to 20-50 μM Pb resulted in approximately 70% potassium loss. Competitive 

16 and inhibitory binding assays suggest that Pb directly activates potassium channels in S. porcus.

Disruption of Neurotransmitter Release

Pb has been shown to inhibit the evoked release of neurotransmitters by inhibiting Ca2+ 17 transport 

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

19 1984). However, concentrations of Pb as low as 5 μM were also observed to actually increase the 

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

confirmed that Pb demonstrates Ca2+ 21 -mimetic properties in enhancing neurotransmitter release from cells 

in the absence of Ca2+ and Ca2+ 22 -induced depolarization. Tomsig and Suszkiw (1993, 1995) reported that 

23 Pb exposure induced the release of norepinephrine (NE) from bovine adrenal chromaffin cells, and was 

considerably more potent at doing so than Ca2+ (K0.5 of 4.6 nM for Pb versus 2.4 μM for Ca2+ 24 ). Activation 

25 of protein kinase C (PKC) was observed to enhance the Pb-induced release of NE. Westrink and Vijverber 

(2002) observed that Pb acted as a high affinity substitute for Ca2+ 26 , and triggered enhanced catecholamine 

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

Ca2+ 28 -induced evoked release of neurotransmitters combined with the ability of Pb to enhance spontaneous 

29 releases could result in higher noise in the synaptic transmission of nerve impulses in Pb-exposed 

30 animals. In rats exposed to Pb at concentrations of 0.1-1.0% in drinking water beginning at GD15-16 and 

31 continuing to 120 days postnatal, decreases in total potassium-stimulated hippocampal GABA release 

32 were seen at exposure levels of 0.1-0.5% (blood Pb = 26.8 ± 1.3 - 61.8 ± 2.9 μg/dL) (Lasley & Gilbert, 

33 2002). Maximal effects were observed at 0.2% Pb in drinking water, but effects were less evident at 0.5%, 
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and were absent at 1.0%. In the absence of Ca2+ 1 , potassium-induced release was increased in the two 

2 highest exposure concentrations, suggesting a Pb-induced enhancement of evoked release of GABA. The 

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

low concentrations, but a Ca2+ 4 mimic in regard to independently evoking exocytosis and release at higher 

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

6 release of neurotransmitters may involve Pb-induced activation of CaMKII-dependent phosphorylation of 

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

8 intracellularly mediated effects of Pb on spontaneous release of neurotransmitters, Pb-induced inhibition 

of evoked transmitter releases is largely due to extracellular blockage of the voltage-sensitive Ca2+ 9

10 channels. 

5.2.2.3. Displacement of Metal Ions and Perturbed Protein Function

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

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

13 Metal binding sites on proteins are generally ion-specific and are influenced by multiple factors, including 

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

15 Kirberger & Yang, 2008). The coordination chemistry that normally regulates metal-protein binding 

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

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

18 2008). However, due to differences in its physical properties, Pb induces abnormal conformational 

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

et al., 2005), and these structural changes elicit altered protein function. It is known that [Ca2+]i 20 is an 

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

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

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

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

cellular processes, including growth and differentiation (Goldstein, 1993). PKC contains a C2 Ca2+ 25 -

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

activity (Garza et al., 2006). Markovac and Goldstein (1988b) observed that, in the absence of Ca2+ 27 , 

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

The activation of PKC by Pb was more potent than Ca2+ 29 -dependent activation by five orders of 

30 magnitude. Long et al. (1994) confirmed these findings, reporting that Pb had a Kact 4800 times smaller 

than Ca2+ (55 pM versus 25 μM, following a 3 minute exposure). However, Ca2+ 31 had a higher maximal 

activation of PKC than Pb. This possibly indicates the presence of multiple Ca2+ 32 -binding sites on the 

protein, and that Pb may bind the first site more efficiently than Ca2+ 33 , but not subsequent sites. Tomsig 
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1 and Suszkiw (1995) further demonstrated the ability of Pb to activate PKC at picomolar concentrations in 

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

was only partial (approximately 40% of the maximum activity induced by Ca2+ 3 ) and tended to decrease at 

4 concentrations greater than one nanomolar. 

Contrary to the above findings, Markovac and Goldstein (1988a) observed that Pb and Ca2+ 5

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

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

8 investigated in whole vessel preparations, no activation was observed, but PKC did become redistributed 

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

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

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

12 was increased at concentrations greater than 100 nM (Belloni-Olivi et al., 1996). The observation that 

neither Ca2+ 13 nor diacylgycerol was increased due to exposure indicates that Pb-induced activation of PKC 

14 is due to direct interaction with the protein. Pb-induced alterations in PKC have also been observed in 

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

16 (Chai & Webb, 1988; Watts et al., 1995) and human erythrocytes from Pb-exposed workers (blood Pb = 

17 5.4 to 69.3 μg/dL) (K.-Y. Hwang et al., 2002), and decreased activity in mouse macrophages and the rat 

18 brain cortex at micromolar concentrations (Lison et al., 1990; Murakami et al., 1993). 

Calmodulin is another important protein essential for proper Ca2+ 19 -dependent cell signaling. 

Calmodulin contains an “EF-hand” Ca2+ 20 binding domain, and is dependent on the cation for proper 

21 activity (Garza et al., 2006). Calmodulin regulates events as diverse as cellular structural integrity, gene 

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

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

24 including activation of calmodulin-dependent phosphodiesterase activity after 10 minutes incubation 

25 (minimal activation at 100 nM, EC50 = 0.5-1.0 μM), stimulation of brain membrane phosphorylation at Pb 

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

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

(1983) reported the affinity of Pb for calmodulin’s Ca2+-binding sites was approximate to that of Ca2+ 28

itself (Kd ~ 20 μM), whereas Richardt et al. (1986) observed that Pb was slightly more potent than Ca2+ 29 at 

30 binding calmodulin (IC50 = 11 and 26 μM, respectively). Both studies indicated that Pb was much more 

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

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

in, calmodulin compared to Ca2+ 33 (EC50 values of 400-550 pM (threshold = 100 pM) and 450-500 nM 

(threshold = 100 nM), respectively). Pb, in the absence of Ca2+ 34 , was also observed to activate calmodulindependent cyclic nucleotide phosphodiesterase activity at much lower concentrations compared to Ca2+ 35

36 (EC50 value 430 pM [threshold = 300 pM versus EC50 1200 nM (threshold = 200 nM; 50-minute 
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incubation]). When incubated with physiological concentrations of Ca2+ 1 , Pb induced phosphodiesterase 

2 activity at concentrations as low as 50 pM. Pb activated calcineurin, a phosphatase with widespread 

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

of Ca2+ 4 (incubation time = 30 minutes), but inhibited its activity at concentrations greater than 200 pM 

5 (Kern & Audesirk, 2000). Thus, picomolar concentrations of intracellular Pb appear to amplify the 

activity of calmodulin and thus can be expected to alter intracellular Ca2+ 6 signaling in exposed cells (Kern 

7 et al., 2000). Mas-Oliva (1989) observed that low-dose (<1 μM, 20-minute incubation) stimulatory effects 

of Pb exposure on the activity of Ca2+-Mg2+ 8 -ATPase was due to Pb binding to calmodulin and subsequent 

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

for 1 to 48 hours resulted in increased activation of a calmodulin-dependent Ca2+ 10 extrusion mechanism.

Pb has also been observed to alter the activity of other proteins that rely on Ca2+ 11 binding for normal 

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

and bone growth and has three Ca2+ 13 -binding sites (Dowd et al., 2001). Incubation of osteocalcin in 

solution with Ca2+ and Pb resulted in the competitive displacement of Ca2+ 14 by Pb (Dowd et al., 1994). Pb 

was found to bind to osteocalcin more than 1000-times more tightly than Ca2+ 15 (Kd = 1.6 ± 0.42 nM versus 

16 0.007 mM, respectively), and analysis with NMR indicated Pb induced similar, though slightly different, 

secondary structures in osteocalcin, compared to Ca2+ 17 . The authors hypothesized that the observed 

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

19 exposure reduced osteocalcin adsorption to hydroxyapatite (Dowd et al., 1994). Further research by this 

group confirmed that Pb bound osteocalcin approximately 10,000-times more tightly than Ca

2+ 20 (Kd = 

21 0.085 μM versus 1.25 mM, respectively) (Dowd et al., 2001). However, the authors reported that Pb 

22 exposure actually caused increased hydroxyapatite adsorption at concentrations 2-3 orders of magnitude 

lower than seen with Ca2+. Additionally, Pb can displace Ca2+ in numerous other Ca2+ 23 -binding proteins 

important in muscle contractions, renal Ca2+ 24 transport and neurotransmission, including troponin C, 

25 parvalbumin, CaBP I and II, phospholipase A2, and syntapotagmin I, at concentrations as low as the 

26 nanomolar range (Bouton et al., 2001; Osterode & Ulberth, 2000; Richardt et al., 1986). 

Pb can displace metal cations other than Ca2+ 27 that are requisite for protein function. One of the most 

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

29 aminolevulinic acid dehydratase (ALAD). ALAD contains four zinc-binding sites and all four need to be 

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

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

32 inhibition of the enzyme in the erythrocytes of Pb-exposed workers and adolescents (blood Pb level >10 

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

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

35 weeks (blood Pb level = 6.56 ± 0.98 μg/dL) (M. K. Lee et al., 2005). Further experiments indicated that 
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1 lower concentrations of zinc result in greater inhibition of enzyme activity by Pb, suggesting a 

2 competitive inhibition between zinc and Pb at a single site (Simons, 1995). 

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

4 expression, including GATA proteins and transcription factors TFIIIA, Sp1, and Erg-1 (Ghering et al., 

5 2005; Hanas et al., 1999; M. Huang et al., 2004; Zawia et al., 1998) (G. R. Reddy & Zawia, 2000). Pb 

was found to form tight complexes with the cysteine residues in GATA proteins (βPb = 6.4 × 109 M-1 6 for 

single zinc fingers and β2

Pb2 = 6.4 × 1019 M-2 7 ), and was able to displace bound zinc from the protein under 

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

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

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

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

12 reported that exposure to Pb caused the dissociation of TFIIIA-DNA adducts and that NMR spectroscopy 

13 indicated that altered TFIIIA activity is the result of a Pb-induced abnormal protein conformation. 

14 Pb exposure modulated the DNA-binding profiles of the transcription factors Sp1 and Erg-1 in rat 

15 pups exposed to 0.2% Pb acetate via lactation, resulting in a shift in DNA-binding towards early 

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

17 shifts in Sp1 DNA-binding profiles were shown to be associated with abnormal expression of genes 

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

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

20 zinc finger motif, thus displacing zinc and resulting in an increase in the DNA-binding efficiency of the 

21 synthetic peptide (Razmiafshari et al., 2001; Razmiafshari & Zawia, 2000). However, in DNA-binding 

22 assays utilizing recombinant Sp1 (which has three zinc finger motifs, opposed to only one in the synthetic 

23 peptide), incubation with as little as 37 μM Pb resulted in the abolishment of Sp1’s DNA-binding 

24 capabilities (Razmiafshari & Zawia, 2000).

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

26 adenine and hypoxanthine/guanine phosphoribosyltransferase in erythrocyte lysates of rats exposed to 

27 0.1% Pb in drinking water for 9 months (blood Pb = 7.01 ± 1.64 μg/dL) and in human erythrocyte lysates 

28 exposed to 100 nM Pb for as little as 5 minutes (Baranowska-Bosiacka et al., 2009), and cGMP 

29 phosphodiesterase at picomolar concentrations in homogenized bovine retinas (D. Srivastava et al., 1995). 

30 Pb was also reported to inhibit pyrimidine 5’-nucleotidase through competitive inhibition of magnesium 

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

32 et al., 2006).

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

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

35 implications for numerous cellular and physiological processes, including cell signaling, growth and 
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1 differentiation, gene expression, energy metabolism, and biosynthetic pathways. Table 5-1 provides a list 

2 of enzymes and proteins whose function may be perturbed by Pb exposure.

Table 5-1. Enzymes and proteins potentially affected by exposure to Pb and the metal 

cation cofactors necessary for their proper physiological activity

Metalloprotein/Enzyme Direction of Action Metal Cation; Reference

Enzymes

Aminolevulinic acid dehydratase ↓ Zn; Simons (1995)

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

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

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

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

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

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

8 swollen appearance with frequent rupture of the outer membrane, distorted cristae, loss of cristae, 

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

10 mitochondria (Goyer, 1968; Goyer et al., 1968; Marchlewicz et al., 2009; Navarro-Moreno et al., 2009; L. 

11 Wang et al., 2010).
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1 Transmembrane mitochondrial ion transport mechanisms are perturbed by exposure to Pb. Pb 

inhibits the uptake of Ca2+ 2 into mitochondria (Parr & Harris, 1976), while simultaneously stimulating the 

efflux of Ca2+ out of the organelle (Simons, 1993a), thus disrupting intracellular/mitochondrial Ca2+ 3

4 homeostasis. Pb exposure has also been shown to decrease the mitochondrial transmembrane potential in 

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

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

7 incubated with 10 nM to 10 μM for 15 minutes (L. H. He et al., 2000). Further research indicated that 

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

opening of a mitochondrial transmembrane pore (MTP), possibly by directly binding to the metal (Ca2+ 9 )-

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

11 MTP is the first step of the mitochondrial-regulated apoptotic cascade pathway in many cells (Lidsky & 

12 Schneider, 2003; Rana, 2008). He et al. (2000) additionally observed cytochrome c release from 

13 mitochondria, and caspase-9 and -3 activation following exposure of rod cells to Pb. Induction of 

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

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

Altered Energy Metabolism

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

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

18 (state 3) respiration was lower compared to controls (Goyer et al., 1968). The rate of ATP formation in 

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

20 state 3 respiration and respiratory control ratios (state 3/state 4 [succinate or pyruvate/malate-activated]) 

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

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

23 (Fowler et al., 1980). Statistically significant Pb-induced decreases in ATP and adenylate energy charge 

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

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

26 differentiated PC-12 cells incubated with as little as 1 μM Pb for 48 hours (Prins et al., 2010). The 

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

28 in the expression of the voltage-dependent anion channel (VDAC), which maintains cellular ATP levels in 

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

30 exposure of osteoblasts to 10 μM Pb. Parr and Harris (1976) reported that Pb inhibited coupled and 

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

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

33 of Pb on the energy supply to the actions of classic respiratory inhibitors, low temperature and chemical 
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1 uncouplers. Bragadin et al. (1998) supported this view by demonstrating that alkylated Pb compounds 

2 acted as a chemical uncoupler of respiration by abolishing the proton gradient necessary for oxidative 

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

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

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

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

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

8 Pb. 

9 Pb has also been shown to decrease glycolysis in osteoblasts exposed to 10 μM Pb and in human 

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

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

12 obtained from Pb workers directly exposed to Pb, compared to controls exposed to lower concentrations 

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

14 vitro exposure of human umbilical cord erythrocytes to 100-200 μg/dL Pb for 20 hours was observed to 

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

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

17 increase in purine degradation products (adenosine, etc.). Similar decreases in cellular nucleotide pools 

18 were observed when rats were exposed to 1% Pb in drinking water for four weeks (Baranowska-Bosiacka 

19 & Hlynczak, 2004). In erythrocytes, nucleotides are synthesized via salvage pathways such as the adenine 

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

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

22 (Baranowska-Bosiacka et al., 2009).

23 Disruptions in erythrocyte energy metabolism have been observed in workers occupationally 

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

25 workers with an average duration of exposure of 8.4 years (blood Pb not reported). Morita et al. (1997)

26 evaluated the effect of Pb on NAD synthetase in the erythrocytes of Pb-exposed workers (blood Pb = 34.6 

27 ± 20.7 μg/dL) and observed an apparent dose-dependent decrease in NAD synthetase activity with 

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

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

Altered Heme Synthesis

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

31 synthase (i.e., δ-aminolevulinic acid dehydratase), a cytoplasmic enzyme requiring zinc for enzymatic 

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

mitochondrial iron-sulfur containing enzyme that incorporates Fe2+ 33 into protoporphyrin IX to create 
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1 heme. Farant and Wigfield (1987, 1990) observed that Pb inhibits the activity of porphobilinogen 

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

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

4 ferrochelatase in rat liver mitochondria and observed that 10 μM Pb (30 minute incubation) reduced 

5 NAD(P)H-dependent heme synthesis by half when ferric, but not ferrous, iron was used. Pb inhibits the 

insertion of Fe2+ 6 into the protoporphyrin ring and instead, Zn is inserted into the ring creating zinc 

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

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

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

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

11 2006) and animals (blood Pb = 24.7 μg/dL) (Rendón-Ramirez et al., 2007). 

5.2.3. Protein Binding

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

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

14 proteins are also able to bind Pb and protect against its toxic effects through sequestration. The ability of 

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

16 formation of intranuclear inclusion bodies in the liver and kidney. Further research into the composition 

17 of intranuclear inclusion bodies and the identification of specific Pb-binding proteins has been conducted 

18 since that time.

5.2.3.1. Intranuclear and Cytoplasmic Inclusion Bodies

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

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

21 accompanied by altered mitochondrial structure and reduced rates of oxidative phosphorylation. Pb has 

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

23 bodies, and to be concentrated within the subsequently induced intranuclear inclusion bodies following 

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

25 Leonard, et al., 1970; Goyer, May, et al., 1970; McLachlin et al., 1980; Navarro-Moreno et al., 2009; 

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

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

28 or 7 months (Deveci, 2006; Navarro-Moreno et al., 2009). Intranuclear and cytoplasmic inclusions have 

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

30 1973; J. Singh et al., 1999). Pb found within nuclei has also been shown to bind to the nuclear membrane 

31 and histone fractions (Sabbioni & Marafante, 1976).
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1 Upon denaturing intranuclear inclusion bodies with strong denaturing agents, Moore et al. (1973)

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

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

4 migrates as a single band on acrylamide gel electrophoresis. In contrast with Moore and Goyer’s findings, 

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

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

7 found in control rats, indicating that the protein was induced by Pb exposure. This finding was in 

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

9 (Choie et al., 1975; McLachlin et al., 1980). In addition to its presence in kidneys of Pb-exposed animals, 

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

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

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

13 that p32/6.3 was enriched in the basal ganglia, diencephalon, hippocampus, cerebellum, brainstem, spinal 

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

15 residues. Selvin-Testa et al. (1991) and Harry et al (1996) reported that pre- and postnatal exposure of rats 

16 to 0.2-1.0% Pb in drinking water increased the levels of another brain protein, glial fibrillary acidic 

17 protein (GFAP), in developing astrocytes and that this increase may be indicative of a demand for 

18 astrocytes to sequester Pb.

5.2.3.2. Cytosolic Lead Binding Proteins

19 Numerous studies have also identified cytosolic Pb-binding proteins. Two binding proteins, with 

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

21 postmitochondrial cytosolic fraction after injection with 50 mg Pb. The two proteins were also found in 

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

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

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

donation of a zinc cation to ALAD (Goering & Fowler, 1984, 1985). Cadmium and zinc, but not Ca2+ 25 or 

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

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

28 (Mahaffey et al., 1981; Mahaffey & Fowler, 1977; Mistry et al., 1986). Additional cytosolic Pb-binding 

29 proteins have been identified in the kidneys of Pb-exposed rats and humans, including the cleavage 

30 product of α2-microglobulin, acyl-CoA binding protein (MW = 9 kDa), and thymosin β4 (MW = 5 kDa) 

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

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

33 of exposed rats and human brain homogenates exposed in vitro (DuVal & Fowler, 1989; Goering et al., 
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1 1986; Quintanilla-Vega et al., 1995). One protein (MW = 12 kDa) was shown to alleviate hepatic ALAD 

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

3 Cytosolic Pb-binding proteins have been shown to be high in glutamic acid, aspartic acid, and cysteine 

4 residues (DuVal & Fowler, 1989; Fowler et al., 1993). Some evidence exists that cytosolic Pb-binding 

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

6 (Y. Qian et al., 2000; Y. Qian et al., 2005).

5.2.3.3. Erythrocytic Lead Binding Proteins

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

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

9 Lolin & O'Gorman, 1988; Ong & Lee, 1980a, 1980b; Raghavan & Gonick, 1977). However, Bergdahl et 

10 al. (1997) observed the principal Pb-binding protein to be 240 kDa and identified it as ALAD. Two 

11 smaller Pb-binding proteins were observed, but not identified (MW = 45 and <10 kDa). ALAD levels are 

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

13 exposure in both exposed humans (blood Pb = 30-75 μg/dL) and rats (Pb exposure = 25 mM in drinking 

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

15 ALAD is a polymorphic gene with three isoforms: ALAD 1-1, ALAD 1-2, or ALAD 2-2. Carriers 

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

17 ALAD-1 allele (Astrin et al., 1987; H.-S. Kim et al., 2004; Pérez-Bravo et al., 2004; Scinicariello et al., 

18 2007; C. M. Smith, Hu, et al., 1995; Wetmur, 1994; Wetmur et al., 1991; Y. Zhao et al., 2007). Some 

19 newer studies, however, either observed lower blood Pb levels in carriers of the ALAD-2 allele or no 

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

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

22 The ALAD-2 protein binds Pb more tightly than the ALAD-1form: in workers carrying the ALAD23 2 gene, 84% of blood Pb was bound to ALAD versus 81% in carriers of the ALAD-1 gene (p = 0.03) 

24 (Bergdahl, Grubb, et al., 1997). This higher affinity for Pb in ALAD-2 carriers may sequester Pb and 

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

26 the observation that carriers of the ALAD-2 gene have higher levels of hemoglobin (Scinicariello et al., 

27 2007), decreased plasma levulinic acid (B. S. Schwartz, Lee, Stewart, Sithisarankul, et al., 1997), 

28 decreased levels of zinc protoporphyrin (H.-S. Kim et al., 2004; Scinicariello et al., 2007), lower cortical 

29 bone Pb (C. M. Smith, Wang, et al., 1995), and lower amounts of DMSA-chelatable Pb (B. S. Schwartz et 

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

31 findings that ALAD-2 polymorphisms reduced the bioavailability of Pb are somewhat equivocal. Wu et 

32 al. (2003) observed that ALAD-2 carriers had lower blood Pb level (5.8 ± 4.2 μg/dL) than carriers of the 

33 ALAD-1 gene (blood Pb level = 6.2 ± 4.1 μg/dL), and that ALAD-2 carriers demonstrated decreased 
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1 renal function at lower patellar Pb concentrations than those observed to decrease renal function in 

2 ALAD-1 carriers. This potentially indicates that ALAD-2 carriers have enhanced Pb bioavailability. 

3 Weaver et al. (2003) observed that ALAD-2 polymorphisms were associated with higher DMSA4 chelatable Pb concentrations, when normalized to creatinine levels. Further, Montenegro et al. (2006)

5 observed among individuals with ALAD 1-1 or ALAD 1-2/2-2 genotypes a significant increase in the 

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

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

8 with ALAD 1-2/2-2 genotypes increases the probability of adverse health effects in these individuals.

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

10 whole blood (Bergdahl et al., 1998). The 45 and <10 kDa Pb-binding proteins bound approximately 12-

11 26% and <1% of the blood Pb, respectively. At blood Pb concentrations greater than 40 μg/dL, greater 

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

13 kDa protein as pyrimidine-5’-nucleotidase and the <10 kDa protein as acyl-CoA binding protein. Smith et 

14 al. (1998) previously identified acyl-CoA binding protein as a Pb-binding protein found in the kidney.

15 Studies also observed the presence of an inducible, low-molecular weight (approximately 10 kDa) 

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

17 1981; Raghavan & Gonick, 1977). The presence of this low molecular weight protein seemed to have a 

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

19 lowered expression of this protein or low levels of Pb bound to it (Raghavan et al., 1980, 1981). The 

20 presence of low molecular weight Pb-binding proteins in exposed workers was confirmed by Lolin and 

21 O’Gorman (1988) and Church et al. (1993a, 1993b). Further Lolin and O’Gorman (1988) reported that the 

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

23 ALAD’s Pb-binding capacity identified by Bergdahl et al. (Bergdahl et al., 1998). Xie et al. (1998)

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

25 ALAD only at higher blood Pb levels. Church et al. (1993a, 1993b) observed the presence of a 6-7 kDa 

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

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

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

29 metallothionein.

5.2.3.4. Metallothionein

30 Metallothionein is a low-molecular metal-binding protein, most often zinc or copper, that is rich in 

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

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

33 production of Pb- and Zn-metallothionein in mice exposed via i.p. or i.v. injection at 30 mg/kg (Maitani et 
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1 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. 

2 injection at 24 μmol/100g (Ikebuchi et al., 1986). The induced Pb-metallothionein consisted of 28% half3 cysteine and reacted with an antibody for Zn-metallothionein II (Ikebuchi et al., 1986). In contrast, 

4 exposure of rats to Pb via drinking water (200 or 300 mg/L) failed to induce metallothionein in the 

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

6 (1987a, 1987b) observed that pretreatment of rats with zinc before injection with Pb resulted in Pb and 

7 zinc co-eluting with zinc-thionein, and that zinc-thionein I and II were able to bind Pb in vitro (Goering & 

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

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

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

11 DuVal and Fowler (1989) who demonstrated rat brain Pb-binding proteins attenuated Pb-induced 

12 inhibition of ALAD. 

13 Metallothionein has been reported to be important in the amelioration of Pb-induced toxicity 

14 effects. Liu et al. (1991) reported that zinc-metallothionein reduced Pb-induced membrane leakage and 

15 loss of potassium in cultured hepatocytes incubated with 600-3,600 μM Pb. Metallothionein-null mice 

16 exposed to 1,000, 2,000, or 4,000 ppm Pb for 20 weeks suffered renal toxicity described as nephromegaly 

17 and decreased renal function compared to Pb-treated wild-type mice (Qu et al., 2002). Interestingly, 

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

19 Pb than the wild-type mice (Qu et al., 2002). Metallothionein levels were induced by Pb exposure in non20 null mice. Exposure to Pb (1,000, 2,000, or 4,000 ppm), both for 104 weeks as adults and from GD8 to 

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

22 kidneys of metallothionein-null rats compared to wild type mice (Tokar et al., 2010; Waalkes et al., 2004). 

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

24 in the formation of inclusion bodies and mitigation of Pb-induced toxic effects, and that those with 

25 polymorphisms in metallothionein coding genes may be at greater susceptibility to Pb. In support of this 

26 theory, Chen et al. (2010) observed that Pb-exposed workers with a mutant metallothionein allele had 

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

28 more susceptible to Pb toxicity.

5.2.4. Oxidative Stress

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

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

superoxide (O2

- 31 ), hydroxyl radical (OH ̇), and hydrogen peroxide (H2O2), that can attack and denature 

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

33 been shown to cause oxidative damage to the heart, liver, kidney, reproductive organs, brain, and 
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1 erythrocytes, which may be responsible for a number of Pb-induced pathologies (Gonick et al., 1997; 

2 Khalil-Manesh, Gonick, Cohen, Bergamaschi, et al., 1992; Khalil-Manesh et al., 1994; Salawu et al., 

3 2009; Sandhir & Gill, 1995; Shan et al., 2009; Vaziri, 2008b). The origin of ROS produced after Pb 

4 exposure is likely a multipathway process, resulting from oxidation of δ-aminolevulinic acid (ALA), 

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

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

7 disruption of functional metal ions within oxidative stress proteins, such as superoxide dismutase (SOD), 

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

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

10 antioxidant enzymes and increased lipid peroxidation (Chapter 7).

5.2.4.1. δ-ALA Oxidation

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

carrier-mediated mechanisms including a vanadate-sensitive Ca2+ 12 pump. Once Pb enters erythrocytes, it is 

13 predominantly found in the protein-bound form, with hemoglobin and δ-ALA dehydratase (δ-ALAD) 

14 both identified as targets (Bergdahl, Grubb, et al., 1997). Through its sulfhydryl and metal ion disrupting 

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

16 including δ-ALA synthetase, δ-ALAD, and ferrochelatase. Pb is able to disrupt the Zn ions requisite for 

17 the activity of δ-ALAD, the rate limiting step in heme synthesis, leading to enzyme inhibition at 

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

19 inhibit the activity of δ-ALAD in humans with a threshold value around 5 μg/dL (Ahamed et al., 2005; 

20 Sakai & Morita, 1996). A significant negative correlation (r = -0.6) was found between blood Pb levels in 

21 adolescents (4-20 μg/dL) and blood δ-ALA activity (Ahamed et al., 2006). This inhibition of δ-ALAD 

22 results in the accumulation of δ-ALA in blood and urine, where δ-ALA undergoes tautomerization and 

23 autoxidation. Oxidized δ-ALA generates ROS through reduction of ferricytochrome c and electron 

24 transfer from oxyHb, metHb, and other ferric and ferrous complexes (Hermes-Lima et al., 1991; Monteiro 

et al., 1991). The autoxidation of δ-ALA produces O2

- 25 , OH ̇, H2O2, and an ALA radical (Monteiro et al., 

26 1989; Monteiro et al., 1986). 

5.2.4.2. Membrane and Lipid Peroxidation

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

28 lead to membrane and lipid peroxidation. It is possible that ROS produced from δ-ALA oxidation, as 

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

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

31 a catalyst for these events (Adonaylo & Oteiza, 1999; Quinlan et al., 1988). Increased lipid peroxidation 
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1 measured as TBARS from liposomes, microsomes, and erythrocytes was shown after Pb (Pb(NO3)2) 

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

3 1988). The extent of peroxidation of lipids varies based on the number of double bonds present in 

4 unsaturated fatty acids, since double bonds weaken the C-H bonds on the adjacent carbon, making H 

5 removal easier (Yiin & Lin, 1995). After Pb exposure (4-12 μg/dL, 24 hours, in vitro), the production of 

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

to the number of double bonds of the fatty acid. In the absence of Fe2+ 7 , Pb does not promote lipid 

8 peroxidation, however it may accelerate peroxidation by H2O2 (Quinlan et al., 1988). This could be due to 

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

10 peroxidation. 

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

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

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

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

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

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

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

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

19 in altered membrane fluidity (Donaldson & Knowles, 1993). Changes in membrane fluidity will disturb 

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

21 alter membrane permeability and function.

22 A number of recent studies report increased measures of lipid peroxidation in various organs, 

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

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

25 (Dogru et al., 2008; Ergurhan-Ilhan et al., 2008; D. A. Khan et al., 2008; Mohammad et al., 2008; Patil, 

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

27 2006; Quintanar-Escorza et al., 2007). One study found a correlation between the MDA levels and blood 

28 Pb levels even in the unexposed workers who had lower (i.e., <12 μg/dl) blood Pb levels (Quintanar29 Escorza et al., 2007). Other studies found increased evidence of lipid peroxidation among the general 

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

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

32 between blood Pb levels between 4-20 μg/dL in adolescents and blood MDA level. Similar results have 

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

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

35 treated (50 μg, 1-4 hours) rat brain homogenates (Rehman et al., 1995) and in specific brain regions, 

36 hippocampus and cerebellum, after Pb exposure (500 ppm, 8 weeks) to rats (Bennet et al., 2007). Overall, 
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1 there was a correlation between the blood Pb level and measures of lipid peroxidation often measured by 

2 MDA levels.

3 Interestingly, studies in many species of plants, invertebrates, and vertebrates exhibit increased 

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

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

6 exposure.

5.2.4.3. NAD(P)H Oxidase Activation

NAD(P)H oxidase is a membrane bound enzyme that requires Ca2+ 7 in order to catalyze the 

production of O2

- 8 from NAD(P)H and molecular oxygen (Leseney et al., 1999). Two studies provide 

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

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

the NAD(P)H subunit gp91 11 phox in the brain, heart, and renal cortex of Pb treated rats (100 ppm in 

12 drinking water, 12 weeks). This upregulation was present in Pb-treated (1-10 ppm) human coronary artery 

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

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

15 this protein.

5.2.4.4. Antioxidant Enzyme Disruption

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

17 decreased activity of antioxidant defense enzymes. Pb has been shown to alter the function of several 

18 antioxidant enzymes, including SOD, CAT, glucose-6-phosphate dehydrogenase (G6PD), and the 

19 enzymes involved in glutathione metabolism, GPx, glutathione-S-transferase (GST), and glutathione 

20 reductase (GR). These changes in the antioxidant defense system could be due to the high affinity of Pb 

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

22 consequence of increased oxidative damage by Pb.

23 Studies of the effects of Pb on the activities of SOD and CAT give divergent results. These 

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

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

26 2000). SOD exists in multiple isoforms that require copper and zinc (SOD1 and SOD3) (Antonyuk et al., 

27 2009) or manganese (SOD2) (Borgstahl et al., 1992). A number of studies have found decreased activity 

28 of these enzymes (Ergurhan-Ilhan et al., 2008; Mohammad et al., 2008; Pandya et al., 2010; Patil, 

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

30 2006; D. Y. Yu et al., 2008), whereas others observe increased activity (Ahamed et al., 2008; M. K. Lee et 

31 al., 2005). Pb exposure (500 ppm, 1, 4, and 8 weeks) in rats showed that organ SOD and CAT responded 
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1 differently depending on the dose and tissue investigated. Activity of SOD and CAT varied based on the 

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

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

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

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

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

7 result of metal ion substitution by Pb causing enzyme inactivation.

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

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

10 Glutathione will donate an electron while in its reduced state (GSH), which leads to conversion to the 

11 oxidized form, glutathione disulfide (GSSG). Pb binds to the thiol and can both interfere with the 

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

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

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

15 Chetty et al., 2005; Flora et al., 2007; Nakagawa, 1989, 1991; Pandya et al., 2010). Chronic Pb exposure 

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

17 thus often manifesting as an increase in Pb-induced GSH (Corongiu & Milia, 1982; Daggett et al., 1998; 

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

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

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

21 levels.

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

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

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

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

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

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

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

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

30 (Bokara et al., 2009).

31 GSH is used as a cofactor for peroxide reduction and detoxification of xenobiotics by the enzymes 

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

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

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

35 Nakagawa, 1991; Schrauzer, 1987; D. Y. Yu et al., 2008). Similar to other antioxidant enzymes, 

36 compensatory upregulation of these enzymes is described after Pb treatment (Bokara et al., 2009; 
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1 Conterato et al., 2007; Daggett et al., 1998; Ergurhan-Ilhan et al., 2008). However, other studies have 

2 shown that these enzymes may not be able to compensate for the increased Pb-induced ROS, further 

3 contributing to the oxidative environment (Farmand et al., 2005).

4 Recently, γ-glutamyltranferase (GGT) within its normal range has been regarded as an early and 

5 sensitive marker of oxidative stress. This may be because cellular GGT metabolizes extracellular GSH to 

6 be used in intracellular GSH synthesis. Thus, cellular GGT acts as an antioxidant enzyme by increasing

7 the intracellular GSH pool. However, the reasons for the association between GGT and oxidative stress 

8 have not been fully realized (D. H. Lee et al., 2004). Occupational Pb exposure (blood Pb level, 29.1 

9 μg/dL) results in increased serum GGT levels (D. A. Khan et al., 2008). Interestingly, low blood Pb levels 

10 found in a sample of the U.S. population (NHANES III) were positively associated with serum GGT 

11 levels showing a dose-response relationship at levels <7 μg/dL (D. H. Lee et al., 2006).

5.2.4.5. Nitric Oxide Signaling

12  ̇NO, also known as endothelium-derived relaxing factor, is a potent endogenous signaling 

13 molecule involved in vasodilation. Acute and chronic Pb exposure decreases the biologically active  ̇NO, 

14 not through reduction in  ̇NO-production capacity (Vaziri & Ding, 2001; Vaziri, Ding, et al., 1999), but as 

15 a result of inactivation and sequestration of  ̇NO by ROS (Malvezzi et al., 2001; Vaziri, Liang, et al., 

1999). Endogenous  ̇NO can interact with ROS, specifically O2

- 16 , produced following exposure to Pb to 

form the highly cytotoxic reactive nitrogen species, peroxynitrite (ONOO- 17 ). This reactive compound can 

18 damage cellular DNA and proteins, resulting in the formation of nitrotyrosine among other products. 

19 Overabundance of nitrotyrosine in plasma and tissues is present after exposure to Pb (Vaziri, Liang, et al., 

20 1999).  ̇NO is also produced by macrophages in the defense against certain infectious agents, including 

21 bacteria. Studies have found that Pb exposure can significantly reduce production of  ̇NO in immune cells 

22 (J.-E. Lee et al., 2001; Pineda-Zavaleta et al., 2004; L. Tian & Lawrence, 1995), possibly leading to 

23 reduced host resistance (L. Tian & Lawrence, 1996).

24 Production of  ̇NO is catalyzed by a family of enzymes called nitric oxide synthases (NOS), 

25 including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS), that require a 

26 heme prosthetic group and a zinc cation for enzymatic activity (Messerschmidt et al., 2001). 

27 Paradoxically, the reduction in  ̇NO availability in vascular tissue following Pb exposure is accompanied 

28 by significant upregulation in NOS isotypes (Gonick et al., 1997; Vaziri & Ding, 2001; Vaziri, Ding, et 

29 al., 1999). A direct inhibitory action of Pb on NOS enzymatic activity has been rejected (Vaziri, Ding, et 

30 al., 1999). Instead, the upregulation of NOS occurs as compensation for the decreased  ̇NO resulting from 

31 ROS inactivation (Vaziri & Ding, 2001; Vaziri et al., 2005; Vaziri & Wang, 1999).
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Soluble Guanylate Synthase

1 Many biological actions of  ̇NO, such as vasorelaxation, are mediated by cyclic guanosine 

2 monophosphate (cGMP), which is produced by soluble guanylate cyclase (sGC) from the substrate 

3 guanosine triphosphate (GTP). Soluble guanylate cyclase is a heterodimer requiring one molecule of 

4 heme for enzymatic activity (Boerrigter & Burnett, 2009). In vascular smooth muscle cells (VSMC), sGC 

5 serves as the  ̇NO receptor. Marked reduction in plasma concentrations and urinary excretion of cGMP is 

6 observed after Pb exposure (5 ppm for 30 days and 0.01% for 12 months) (Khalil-Manesh, Gonick, 

7 Weiler, et al., 1993; M. Marques et al., 2001). In addition, Pb exposure downregulated the protein 

8 abundance of sGC in vascular tissue (Courtois et al., 2003; Farmand et al., 2005; M. Marques et al., 

9 2001). This downregulation in sGC was prevented by antioxidant therapy suggesting that oxidative stress 

10 also plays a role in Pb-induced downregulation of sGC (M. Marques et al., 2001).

5.2.5. Inflammation

11 Misregulated inflammation represents one of the major effects of Pb-induced immunotoxicity. It is 

12 important to note that this can manifest in any tissue where immune cell mobilization and tissue insult 

13 occurs. Enhanced inflammation and tissue damage occurs through the modulation of inflammatory cell 

14 function and production of pro-inflammatory cytokines and metabolites. Overproduction of ROS and an 

15 apparent depletion of antioxidant protective enzymes and factors (e.g., selenium) accompany this 

16 immunomodulation (Chetty et al., 2005). 

17 Traditional immune mediated inflammation can be seen with bronchial hyper-responsiveness, 

18 asthma, and respiratory infections after exposure to Pb. But it is important to recognize that any tissue or 

19 organ can be affected by immune-mediated inflammatory dysfunction given the distribution of immune 

20 cells as both permanent residents and infiltrating cell populations (Carmignani et al., 2000; Mudipalli, 

21 2007). Pb spheres implanted in the brains of rats produced neutrophil-driven inflammation with apoptosis 

22 and indications of neurodegeneration (Kibayashi et al., 2010). Pb also induces renal tubulointerstitial

23 inflammation (18 μg/dL or 100 ppm for 14 weeks) (Ramesh et al., 2001; Rodriguez-Iturbe et al., 2005), 

24 which has been coupled with activation of the redox sensitive nuclear transcription factor kappa B 

25 (NFκB) and lymphocyte and macrophage infiltration (23-27 μg/dL, 100 ppm for 14 weeks) (Bravo et al., 

26 2007). These events could be in response to the oxidative environment developed from Pb, since Pb27 induced inflammation and NFκB activation can be ameliorated by antioxidant therapy (Rodriguez-Iturbe 

28 et al., 2004). 

29 Inflammation can be mediated by the production of chemical messengers such as prostaglandins 

30 (PG). Pb exposure has been reported to increase arachidonic acid (AA) metabolism, thus elevating the 

31 production of PGE2, PGF2, and thromboxane in humans (48 μg/dL) and cell models (0.01 μM, 48 h) 

32 (Cardenas et al., 1993; Chetty et al., 2005; Flohe et al., 2002; Knowles & Donaldson, 1997; J. J. Lee & 
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1 Battles, 1994). Dietary Pb supplementation (500 ppm, 19 days) can increase the percentage of cell 

2 membrane AA, the precursor of cyclooxygenase and lipoxygenase metabolism to PGs and leukotrienes 

3 (Knowles & Donaldson, 1990). Additionally, Pb (1 μM, 20 μg/dL) may promote the release of AA via 

4 activation of phospholipase A2, as shown in isolated VSMC (Dorman & Freeman, 2002).

5 Inflammation may be the result of increased pro-inflammatory signaling or may exacerbate these 

6 signaling pathways. Pb can elevate the expression of NFκB, as well as expression of activator protein-1 

7 (AP-1) and cJun (Bravo et al., 2007; Korashy & Ei-Kadi, 2008; Korashy & El-Kadi, 2008; Pyatt et al., 

8 1996; Ramesh et al., 1999). Pb exposure (25 μM) to dendritic cells stimulates phosphorylation of the 

9 Erk/MAPK pathway, but not p38, STAT3 or 5, or CREB (D. Gao et al., 2007)

5.2.5.1. Cytokine Production

10 There are three modes of major effects of Pb on immune cytokine production. First, Pb can act on 

11 macrophages to elevate the production of pro-inflammatory cytokines such as TNF-α and IL-6 (S. Chen 

12 et al., 1999; Y.-J. Cheng et al., 2006; Dentener et al., 1989). This can result in local tissue damage during 

13 the course of immune responses affecting such targets as the liver. Second, when Pb acts on dendritic 

14 cells, it skews the ratio of IL-12/IL-10 such that Th1 responses are suppressed and Th2 responses are 

15 promoted (S. Chen et al., 2004; Miller et al., 1998). Third, when acquired immune responses occur 

16 following exposure to Pb, Th1 lymphocyte production of cytokines is suppressed (e.g. IFN-γ) (Heo et al., 

17 1996; Lynes et al., 2006). In contrast, Th2 cytokines such as IL-4, IL-5, and IL-6 are elevated (D. Gao et 

18 al., 2007; D. Kim & Lawrence, 2000). The combination of these three modes of cytokine changes induced 

19 by Pb creates a hyperinflammatory state among innate immune cells and acquired immunity is skewed 

20 toward Th2 responses.

21 Iavicoli et al. (2006) reported below-background blood Pb concentrations produced significant 

22 changes in cytokine levels. At the lowest dietary Pb concentration (0.8 μg/dL), IL-2 and IFN-γ were 

23 elevated over the controls, indicating an enhanced Th1 response. However, as the dietary and blood Pb 

24 concentrations increased (resulting in blood Pb level 12-61 μg/dL), a Th2 phenotype was observed with 

25 suppressed IFN-γ and IL-2 and elevated IL-4 production. These findings support the notion that the 

26 immune system is remarkably sensitive to Pb-induced functional alterations and that nonlinear effects 

27 may occur at extremely low Pb exposures. TGF-β production is also altered by Pb exposure to cells (1 

28 μM, 3 days) (Zuscik et al., 2007). IL-2 is one of the more variable cytokines relative to Pb-induced 

29 changes. Depending upon the protocol it can be slightly elevated in production or unchanged. Recently,

30 Gao et al (2007) found that Pb-treated dendritic cells (25 μM) promoted a slight but significant increase in 

31 IL-2 production among lymphocytes. Proinflammatory cytokines have been measured in other organs and 

32 cell types after Pb exposure. Elevation of IL-1β and TNF-α were observed in the hippocampus after Pb 

33 exposure and increased IL-6 was found in the forebrain (Struzynska et al., 2007).
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1 Consistent with animal studies, epidemiologic studies have also demonstrated Pb-associated 

2 decreases in Th1-type cytokines and increases in Th2-type cytokines. Among adults without occupational 

3 Pb exposures in Incheon, Korea with blood Pb levels ranging from 0.337 to 10.47 μg/dL, Kim et al. 

4 (2007) found associations of blood Pb with serum levels of TNF-α and IL-6 that were larger among men 

5 with blood Pb levels ≥ 2.51 μg/dL (median). In models that adjusted for age, sex, BMI, and smoking 

6 status, a 1 μg/dL increase in blood Pb was associated with a 23% increase (95% CI: 4,55) in log of TNF-α 

7 and a 26% increase in log of IL-6 (95% CI: 0,55).

8 Results from studies of occupationally-exposed adults also suggested that Pb exposure may be 

9 associated with decreases in Th1 cytokines and increases in Th2 cytokines; however, analysis were 

10 mostly limited to comparisons of levels among different occupational groups with different mean blood 

11 Pb levels (Di Lorenzo et al., 2007; Valentino et al., 2007; Yucesoy et al., 1997a). The exception was the 

12 study of male foundry workers, pottery workers, and unexposed workers by Valentino et al. (2007). 

13 Multiple regression analyses were performed with age, BMI, smoking, and alcohol consumption included

14 as covariates. Although effect estimates were not provided, statistically significant associations were 

observed between blood Pb and IL-10 and TNF-α, with R2 15 values of 0.249 and 0.235, respectively. 

16 Exposed workers also had higher levels of most Th2 cytokines (IL-2, IL-6, IL-10, and TNF-α) and lower 

17 levels of the Th1 cytokine IFN-γ. Levels of IL-2, IL-6, and IL-10 showed an increasing trend from the 

18 lowest to highest blood Pb group. In contrast with most other studies, both exposed worker groups had 

19 lower IL-4 levels compared with controls. In a similar analysis, DiLorenzo et al. (2007) separated 

20 exposed workers into intermediate (9.1-29.4 μg/dL) and high (29.4-81.1 μg/dL) blood Pb level groups, 

21 with unexposed workers comprising the low exposure group (blood Pb levels 1-11 μg/dL). Excluded from 

22 this study were exposed workers from the highest end of the distribution of blood Pb levels in DiLorenzo 

23 et al. (2006). Mean TNF-α levels showed a monotonic increase from the low to high blood Pb group, 

24 which was suggestive of a concentration-dependent relationship. Levels of granulocyte colony25 stimulating factor (G-CSF) did not differ between the intermediate and high blood Pb groups among the 

26 Pb recyclers; however, G-CSF levels were higher in the Pb recyclers than in the unexposed controls. 

27 Furthermore, among all subjects, blood Pb showed a strong, positive correlation with G-CSF. Yucesoy et 

28 al. (1997a) found statistically significant lower serum levels of the Th1 cytokines, IL-1β and IFN-γ, in 

29 workers compared with controls; however levels of the Th2 cytokines, IL-2 and TNF-α levels, were 

30 similar between groups. Overall, exposure to Pb increases the production of pro-inflammatory cytokines, 

31 skews the ratio of Th1 and Th2 cytokines to favor Th2 responses, and suppresses lymphocyte cytokine 

32 production.
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5.2.6. Endocrine Disruption

5.2.6.1. Hypothalamic-Pituitary-Gonadal Axis

1 Pb is a potent endocrine disrupting chemical that causes reproductive and developmental effects at 

2 moderate levels of exposure in both male and female animal models. Pb may act both at multiple points 

3 along the hypothalamic-pituitary-gonadal (HPG) axis and directly at gonadal sites. The HPG axis 

4 functions in a closely regulated manner to produce circulating sex steroids and growth factors required for 

5 normal growth and development. Chronic Pb exposure has been shown to reduce serum levels of follicle6 stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and estradiol (Biswas & Ghosh, 

7 2006; Dearth et al., 2002; E. F. Krieg, Jr., 2007; Ronis et al., 1998; Rubio et al., 2006; Sokol & Berman, 

8 1991). This is likely through the inhibition of LH secretion and the reduction in the expression of the 

9 steroidogenic acute regulatory protein (StAR) (B. M. Huang et al., 2002; B. M. Huang & Liu, 2004; 

10 Ronis et al., 1996; V. Srivastava et al., 2004). StAR is the rate-limiting step essential in maintaining 

11 gonadotropin-stimulated steroidogenesis, which results in the formation of testosterone and estradiol. Pb 

12 (prenatal exposure resulting in blood Pb level 3 μg/dL) decreases basal StAR synthesis, but not 

13 gonadotropin-stimulated StAR synthesis, suggesting that Pb may not directly affect ovarian 

14 responsiveness to gonadotropin stimulation (V. Srivastava et al., 2004). Instead, Pb may act on the 

15 hypothalamic-pituitary level to alter LH secretion, which is necessary to drive StAR production and 

16 subsequent sex hormone synthesis. Release of LH and FSH from the pituitary is controlled by 

17 gonadotropin-releasing hormone (GnRH). Pb exposure (10 μM, 90 min) in rat brain median eminence 

18 cells can block GnRH release (Bratton et al., 1994). Pb may also interfere with release of pituitary 

19 hormones through interference with cation-dependent secondary messenger systems, which mediate 

20 hormone release and storage.

21 Endocrine disruption may also be a result of altered hormone binding to endocrine receptors. 

22 Prenatal and postnatal Pb exposure (20 ppm) is able to decrease the number of estrogen receptors found in 

23 the uterus and receptor binding affinity (Wiebe & Barr, 1988). Altered hormone binding ability may be 

24 due to the ion binding properties of Pb, resulting in changes in receptor tertiary structure that will disrupt 

25 ligand binding. In addition, Pb-induced changes in hormone levels that act as inducing agents for receptor

26 synthesis may affect the number of hormone receptors produced. 

27 Some of these endocrine disrupting effects of Pb have been related to the generation of ROS. 

28 Treatment with antioxidants is able to counteract a number of the endocrine disrupting effects of Pb, 

29 including apoptosis and decreased sperm motility and production (P. C. Hsu, Liu, et al., 1998; Madhavi et 

30 al., 2007; Rubio et al., 2006; Salawu et al., 2009; Shan et al., 2009; C. H. Wang et al., 2006). Direct 

31 generation of ROS in epididymal spermatozoa was observed after Pb exposure (i.p. 20 or 50 mg/kg, 6 
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1 wk) (P. C. Hsu, Hsu, et al., 1998). In addition, lipid peroxidation in the seminal plasma was significantly 

2 increased in a group of Pb-exposed workers with blood Pb >40 μg/dL (A. Kasperczyk et al., 2008).

3 The liver is often associated with the HPG axis due in part to its production of insulin-like growth 

4 factor 1 (IGF-1). Pb exposed humans (>4 μg/dL), animals (14 μg/dL), and gonadal cells (0.05 mg/mL) 

5 show a decrease in plasma IGF-1 (Dearth et al., 2002; Huseman et al., 1992; Kolesarova et al., 2010; Pine 

6 et al., 2006), which may be the result of decreased translation or secretion of IGF-1 (Dearth et al., 2002). 

7 IGF-1 also induces LH-releasing hormone release, such that IGF-1 decrements may explain decreased LH 

8 and estradiol levels. IGF-1 production is stimulated by growth hormone (GH) secreted from the pituitary 

9 gland and could be the result of GH depletion.

10 A number of studies have revealed that Pb exposure affects the dynamics of growth. Decreased 

11 growth after Pb exposure could be the result of Pb induced decreased GH levels (Berry et al., 2002; 

12 Camoratto et al., 1993; Huseman et al., 1987; Huseman et al., 1992). This decrease in GH could be a 

13 result of decreased release of GH releasing hormone (GHRH) from the hypothalamus or disrupted GHRH 

14 binding to its receptor, which has been reported in vitro after Pb treatment (IC50 free Pb in solution 52 

15 pM, 30 minutes) (Lau et al., 1991). GH secretion may also be altered from decreased testosterone, a result 

16 of Pb exposure. 

5.2.6.2. Hypothalamic-Pituitary-Thyroid Axis

17 The effects of Pb on the hypothalamic-pituitary-thyroid (HPT) axis are mixed. Pb exposure impacts 

18 a variety of players in the thyroid hormone system. A number of human studies have shown a negative 

19 association between elevated blood Pb and thyroxine (T4) and free T4 levels without alteration in 

20 triiodothyronine (T3), suggesting that long-term Pb exposure may depress thyroid function (Dundar et al., 

21 2006; Robins et al., 1983; Tuppurainen et al., 1988). However, animal studies on thyroid hormones have 

22 shown mixed results. Pb exposed cows were reported to have an increase in plasma T3 and T4 levels 

23 (Swarup et al., 2007), whereas mice and chickens manifested decreased serum T3 concentrations after Pb 

24 exposure accompanied by increased lipid peroxidation (Chaurasia et al., 1998; Chaurasia & Kar, 1997). 

25 Decreased serum T3 and increased lipid peroxidation were both restored by vitamin E treatment, 

26 suggesting the disruption of thyroid hormone homeostasis could be a result of altered membrane 

27 architecture and oxidative stress (Chaurasia & Kar, 1997).

28 Decreased T4 and T3 may be the result of altered pituitary release of thyroid stimulating hormone 

29 (TSH). However, several studies have reported higher TSH levels in high level Pb-exposed subjects 

30 (Abdelouahab et al., 2008; Gustafson et al., 1989; Lopez et al., 2000; B. Singh et al., 2000), which would 

31 result in increased T4 levels. Overall, results on the effects of Pb on the HPT axis are inconclusive.
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5.2.7. Cell Death and Genotoxicity

1 A number of studies have attempted to characterize the genotoxicity of inorganic Pb in human 

2 populations, laboratory animals, and cell cultures. Endpoints investigated include DNA damage (single3 and double-strand breaks, DNA-adduct formation), mutagenicity, clastogenicity (sister chromatid 

4 exchange, micronucleus formation, chromosomal aberrations), and epigenetic changes (changes in gene 

5 expression, mitogenesis). It is important to note that numerous studies have utilized exposure to Pb 

6 chromate to investigate genotoxicity endpoints; some studies have specifically attributed the observed 

7 increases in DNA damage and clastogenicity to the chromate ion while others have not. Due to the 

8 uncertainty whether observed genotoxic effects are due to chromate or Pb in studies using this form of 

9 inorganic Pb, only studies utilizing other forms of inorganic Pb (e.g., Pb nitrate, acetate) are discussed 

10 below. 

5.2.7.1. DNA Damage

11 A number of studies in human populations have observed positive associations between Pb 

12 exposure and DNA damage, as measured as DNA strand breaks. Most of these associations have been 

13 observed in occupationally-exposed populations (Danadevi et al., 2003; de Restrepo et al., 2000; Fracasso 

14 et al., 2002; Grover et al., 2010; Hengstler et al., 2003; Minozzo et al., 2010; Palus et al., 2003; Shaik & 

15 Jamil, 2009). It is important to note that occupationally-exposed workers have very high blood Pb levels, 

16 and in one study (de Restrepo et al., 2000) the association between blood Pb and DNA damage was only 

17 observed in workers with blood Pb greater than 120 μg/dL. Also, the studies were equivocal in regard to 

18 how blood Pb levels correlated with DNA damage: Fracasso et al. (2002) observed that DNA damage 

19 increased with increasing blood Pb levels (blood Pb levels, <25, 25-35, and >35 μg/dL), whereas Paulus 

20 et al. (2003) and Minozzo et al. (2010) observed no correlation (mean blood Pb = 50.4 (range = 28.2-

21 65.5) and 59.43 ± 28.34 μg/dL, respectively). Lastly, workers occupationally-exposed to Pb are also 

22 potentially exposed to other genotoxic materials, making it difficult to rule out confounding co-exposures. 

23 For example, Hengstler et al. (2003) examined workers exposed to Pb, cadmium, and cobalt and observed 

that neither blood or air Pb (4.4 (IQR: 2.84-13.6) μg/dL; 3.0 (IQR: 1.6-50.0) μg/m3 24 ) was associated with 

25 DNA damage when examined alone, but that blood Pb influenced the occurrence of single strand DNA 

26 breaks when included in a multiple regression model along with cadmium in air and blood and cobalt in 

27 air. Two studies were found that investigated Pb-induced DNA damage resulting from nonoccupational 

28 exposures. Mendez-Gomez (2008) observed that children living at close and intermediate distances to a 

29 Pb smelter 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 

30 of 4.6 (0.1-8.7) μg/dL for children living distant to the smelter. DNA damage was significantly increased 

31 in children living nearest the smelter, compared to the intermediate distant children, but was not 

32 significantly different from children living farthest away from the smelter. Multivariate analysis (which 
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1 considered the children’s urinary As levels, highest in children farthest from the smelter), revealed no

2 significant associations between DNA damage and blood Pb. Further, DNA repair ability was also 

3 observed to be nonrelated to blood Pb. Alternatively, Yanez et al. (2003) observed that children living 

4 close to a mining complex (blood Pb level= 11.6, range = 3.0 to 19.5 μg/dL) did have increased levels of 

5 DNA damage compared to control children that lived further away from the mining facility (blood Pb 

6 level = 8.3 (3.0-25.0) μg/dL).

7 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, 

8 single strand DNA breaks in white blood cells were observed but did not increase with increasing dose 

9 (K. D. Devi et al., 2000). The three highest doses had responses which were similar in magnitude and 

10 were actually lower than the responses to lower doses tested. In mice exposed to Pb (0.68 μg/dL) via 

11 inhalation for up to 4 weeks, differential levels of DNA damage were observed in different organ systems, 

12 with only the lung and the liver demonstrating statistically greater DNA damage compared to the 

13 respective organ controls after acute exposure (Valverde et al., 2002). Statistically elevated levels of DNA 

14 damage were observed in the kidneys, lungs, liver, brain, nasal cavity, bone marrow, and leukocytes of 

15 mice exposed over a period of 4 weeks, although variability was high in all groups, and the magnitude of 

16 the DNA damage was characterized as weak and did not increase with increasing durations of exposure. 

17 Xu et al. (2008) exposed mice to 10-100 mg/kg Pb acetate via gavage for four weeks and observed a 

18 dose-dependent increase in DNA single strand breaks in white blood cells that was statistically significant 

19 at 50 and 100 mg/kg. The authors characterized the observed DNA damage as severe. Pb nitrate induced 

20 DNA damage in primary spermatozoa in Pb-exposed rats (blood Pb level = 19.5 and 21.9 μg/dL, 

21 respectively) compared to control rats (Nava-Hernandez et al., 2009). The level of DNA damage was not 

22 dose-dependent, and was comparable in both exposure groups. Narayana and Al-Bader (2011) observed 

23 no increase in DNA damage in the livers of rats exposed to 0.5 or 1% Pb nitrate in drinking water for 60 

24 days. Interestingly, although the results were not statistically significant and highly variable, DNA 

25 fragmentation appeared to be lower in the exposed animals.

26 Studies investigating Pb-induced DNA damage in human cell cultures were contradictory. Pb 

27 acetate did not induce DNA strand breaks in HeLa cells when exposed to 500 μM for 20-25 hours or 100 

28 μM for 0.5-4 hours (Hartwig et al., 1990; R. D. Snyder & Lachmann, 1989). Pb nitrate, administered to 

29 lymphoma cells at 1-10 mM for 6 hours, did not result in any DNA-protein crosslinks (M. Costa et al., 

30 1996). Pb acetate was observed by Wozniack and Blasiak (2003) to result in DNA single and double 

31 strand breaks in primary human lymphocytes exposed to 1-100 μM for 1 hour, although the pattern of 

32 damage was peculiar. DNA damage was greater in cells exposed to 1 or 10 μM, compared to those 

33 exposed to 100 μM. DNA-protein crosslinks were only observed in the 100 μM exposure group, 

34 suggesting that the decreased strand breaks observed in the high dose group may be a result of increased 

35 crosslinking in this group. Shaik et al. (2006) also observed DNA damage in human lymphocytes exposed 

36 to 2.1-3.3 mM Pb nitrate for 2 hours. Although there was a dose-dependent increase in DNA damage from 
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1 2.1 to 3.3 mM, no statistics were reported and no unexposed control group was included making it 

2 difficult to interpret these results. Gastaldo et al. (2009) observed that exposure of human endothelial cells 

3 to 1-1000 μM Pb nitrate for 24 hours resulted in a dose-dependent increase in DNA double strand breaks.

4 Studies in animal cell lines were equally as ambiguous as those using human cell lines. Zelikoff et 

5 al. (1988) and Roy and Rossman (1992) reported that Pb acetate (concentration not stated and 1 mM, 

6 respectively) did not induce single or double DNA strand breaks or DNA-protein or DNA-DNA 

7 crosslinks in CHV79 cells exposed to Pb acetate. However, both Xu et al. (2006) and Kermani et al. 

8 (2008) reported Pb acetate induced DNA damage in PC12 cells exposed to 0.1, 1, or 10 μM for 24 hours 

9 and in bone marrow mesenchymal stem cells exposed to 60 μM for 48 hours, respectively. Wedrychowski 

10 et al. (1986) reported that DNA-protein crosslinks were induced in a dose-dependent manner in hepatoma 

11 cells exposed to 50-5000 μM Pb nitrate for 4 hours. Pb acetate and Pb nitrate increased the incidence of 

12 nick translation in CHV79 cells when a bacterial DNA polymerase was added.

13 Pb acetate did not induce single strand DNA breaks in HeLa cells exposed to 500 μM for 20-25 

14 hours (Hartwig et al., 1990). However, exposure to both Pb acetate and UV light resulted in increased 

15 persistence of UV-induced strand breaks, compared to exposure to UV light alone. Similar effects were 

16 seen in hamster V79 cells: UV-induced mutation rates and SCE frequency was exacerbated by co17 incubation with Pb acetate. Taken together, these data suggest that Pb acetate interferes with normal DNA 

18 repair mechanisms triggered by UV exposure alone. Pb nitrate was observed to affect different DNA 

19 double strand break repair pathways in human endothelial cells exposed to 100 μM for 24 hours. 

20 Exposure to Pb inhibited nonhomologous end joining (NHEJ) repair, but increased two other repair 

21 pathways, MRE11-dependent and Rad51-related repair (Gastaldo et al., 2007). Interestingly, exposure of 

22 lung carcinoma cells to 100, 300, or 500 μM Pb acetate for 24 hours resulted in an increase in nucleotide 

23 excision repair efficiency (J. P. Li et al., 2008), though this result is difficult to interpret. Roy and 

24 Rossman (1992) observed an increase in UV-induced mutagenicity when CHV79 cells were co-exposed 

25 to 400 μM Pb acetate (a nonmutagenic dose of Pb acetate), indicating an inhibition of DNA repair. 

26 Treatment of Chinese hamster ovary cells to 0.5-500 μM Pb acetate resulted in a dose-dependent 

27 accumulation of apurinic/apyrimidinic site incision activity, indicating that DNA repair was adversely 

28 affected (McNeill et al., 2007).

5.2.7.2. Mutagenicity

29 Only one human study was found that investigated Pb-induced mutagenicity. Van Larebeke et al. 

30 (2004) investigated the frequency of mutations in the hypoxanthine phosphoribosyltransferase (HPRT) 

31 gene in Flemish women without oxxupational Pb exposures or a number of other heavy metals and 

32 organic contaminants. Blood Pb (range 78.2-251.0 nM) was statistically significantly positively 

33 associated with HPRT mutation frequency in the total population. Also, women with high blood Pb (i.e., 
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1 greater than the population median, not reported) demonstrated a greater mutation frequency compared to 

2 women with lower blood Pb.

3 Pb-induced mutagenicity was investigated in four studies using human cell cultures. Ye (1993)

4 exposed human keratinocytes to 0.1-100 μM/mL Pb acetate for 2-24 hours. This study did not measure 

5 HPRT mutations directly, but rather measured the amount of tritium incorporated into DNA as an 

6 indicator of mutation. In the presence of 6-thioguanine, tritium incorporation was increased in exposed 

7 cells, indicating weak mutagenicity. Hwua and Yang (1998) reported that Pb acetate was not mutagenic in 

8 human foreskin fibroblasts exposed to 500-2000 μM for 24 hours. Pb acetate remained nonmutagenic in 

9 the presence of 3-aminotriazole, a catalase inhibitor, indicating that oxidative metabolism did not play a 

10 part in potential mutagenicity of Pb. Exposure to Pb acetate alone did not induce mutagenicity in lung 

11 carcinoma cells (100-500 μM for 24 hours) or fibroblasts (300-500 μM for 24 hours) (J. P. Li et al., 2008; 

12 C. Y. Wang et al., 2008). However, pretreatment with PKC inhibitors before Pb treatment did result in

13 statistically significant increases in mutagenicity in both cell lines.

14 Results from investigations into Pb-induced mutagenicity using animal cell lines were as equivocal 

15 as the findings from human cell line studies, although differences in mutagenicity may be reflective of 

16 specific Pb compounds used. Pb acetate was observed to be nonmutagenic (HPRT assay) in Chinese 

17 hamster V79 cells exposed to 1-25 μM of the compound for 24 hours (Hartwig et al., 1990), but elicited a 

18 mutagenic response in CHV79 cells (gpt assay) exposed to 1700 μM for 5 days (N. K. Roy & Rossman, 

19 1992). Pb acetate was observed to be nonmutagenic (HPRT assay) in Chinese hamster ovary cells 

20 exposed to 5 μM for 6 hours (McNeill et al., 2007). The observation of mutagenicity in the second study 

21 is complicated by the concurrent observation of severe cytotoxicity at the same dose. Pb nitrate was 

22 alternatively found to be nonmutagenic in CHV79 cells (gpt assay) exposed to 0.5-2000 μM for 5 days 

23 (N. K. Roy & Rossman, 1992), or mutagenic in the same cell line (HPRT assay) exposed to 50-5000 μM 

24 for 5 days (Zelikoff et al., 1988). However, mutagenicity was only observed at 500 μM, and was higher 

25 than that observed at higher doses. Pb sulfate was also observed to be mutagenic in CHV79 cells (HPRT 

26 assay) exposed to 100-1000 μM for 24 hours, but as with Pb nitrate, it was not dose-dependent (Zelikoff 

27 et al., 1988). Pb chloride was the only Pb compound tested in animal cell lines that was consistently 

28 mutagenic: three studies from the same laboratory observed dose-dependent mutagenicity in the gpt assay 

29 in Chinese hamster ovary cells exposed to 0.1-1 μM Pb chloride for one hour (Ariza et al., 1998; Ariza & 

30 Williams, 1996, 1999). 

5.2.7.3. Clastogenicity

31 Clastogenicity is the ability of a compound to induce chromosomal damage, and is commonly 

32 observed as sister chromatid exchange, micronuclei formation, or incidence of chromosomal aberrations 
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1 (i.e., breaks or gaps in chromosomes). The potential for Pb to be clastogenic has been investigated in 

2 numerous studies as described below.

Sister Chromatid Exchange

3 An association between blood Pb (mean blood Pb = 10.48 - 86.9 μg/dL ) and sister chromatid 

4 exchange (SCE) was observed in a number of occupational studies (Anwar & Kamal, 1988; Bilban, 1998; 

5 Duydu et al., 2005; Duydu et al., 2001; X. P. Huang et al., 1988; Palus et al., 2003; Pinto et al., 2000; 

6 Wiwanitkit et al., 2008). However, there are numerous methodological issues that limit firm conclusions 

7 from being drawn, most notably that occupational co-exposures to other genotoxic materials were 

8 possible, although some studies excluded workers with exposures to known mutagens (X. P. Huang et al., 

9 1988; Pinto et al., 2000). In most studies that attempted to investigate the dose-response relationship in 

10 workers, no association was observed between increasing blood Pb levels and the incidence of SCE 

11 (Duydu et al., 2001; Palus et al., 2003; Pinto et al., 2000). However, Huang et al. (1988) did observe 

12 increased SCE in exposed workers in the two highest blood Pb groups (52.1 and 86.9 μg/dL), although 

13 the association was only statistically significant in the 86.9 μg/dL group. Pinto et al. (2000) did report an 

14 association with duration of exposure (range of years exposed = 1.6-40). Two studies reported no 

15 correlation between occupational exposure to Pb and incidence of SCE (T. Rajah & Ahuja, 1995; T. T. 

16 Rajah & Ahuja, 1996). However, these two studies may have suffered from limited statistical power to 

17 observe an effect as they only included very small Pb exposed populations. Mielzynska et al. (2006)

18 investigated the incidence of SCE in children exposed to Pb and PAHs in Poland. Children had an average 

19 blood Pb concentration of 7.69 μg/dL and 7.87 SCEs/cell. Male children had higher blood Pb 

20 concentrations compared to females, but lower numbers of SCEs. No control population was included in 

21 this study, and thus interpretation of these findings is difficult.

22 Pb exposure has been observed to induce SCE in multiple laboratory animal studies. In mice 

23 exposed to up to 100 mg/kg Pb acetate intraperitoneally, Pb induced SCE at 50 and 100 mg/kg (Fahmy, 

24 1999). Pb nitrate, also administered i.p., induced the formation of SCE in a dose-dependent manner (10-

25 40 mg/kg) in the bone marrow of exposed mice (Dhir et al., 1993). Nayak et al. (1989) exposed pregnant 

26 mice to 100-200 mg/kg Pb nitrate via i.v. injection and observed an increase in SCE in dams at 150 and 

27 200 mg/kg; no SCEs were observed in the fetuses. Tapisso et al. (2009) exposed rats to 21.5 mg/kg Pb 

28 acetate (1/10th the LD50) via intraperitoneal injection on alternating days for 11 or 21 days, for a total of 5 

29 or 10 exposures. Induction of SCE in the bone marrow of exposed rats was increased over controls in a 

30 significant duration-dependent manner. It is important to note that all three of these studies utilized an 

31 injection route of exposure that may not be relevant to exposures encountered by the general population 

32 (e.g., drinking water exposure). 
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1 Only two studies were found that investigated SCE formation in human cell lines due to Pb 

2 exposure. Statistically significant, dose-dependent increases in SCE were observed in human lymphocytes 

3 obtained from a single donor when incubated with 1, 5, 10, or 50 μM Pb nitrate (Ustundag & Duydu, 

4 2007). Melatonin and N-acetylcysteine were reported to ameliorate these effects, indicating Pb may 

5 induce SCE through increased oxidative stress. Pb chloride was also observed to increase SCE levels in 

6 human lymphocytes exposed to 3 or 5 ppm (Turkez et al.). 

7 Studies investigating SCE in rodent cells were more equivocal than those in human cells. Pb 

8 sulfate, acetate, and nitrate were found to not induce SCE in Chinese hamster V79 cells (Hartwig et al., 

9 1990; Zelikoff et al., 1988). Both of these studies only examined 25-30 cells per concentration, reducing 

10 their ability to detect Pb-induced SCE. Cai and Arenaz (1998), on the other hand, used 100 cells per 

11 treatment and observed that exposure to 0.05-1 μM Pb nitrate for 3-12 hours resulted in a weak, dose12 dependent increase in SCE in Chinese hamster ovary cells. Lin et al. (1994) also observed a dose13 dependent increase in SCE in Chinese hamster cells exposed to 3-30 μM Pb nitrate for 2 hours.

Micronucleus Formation

14 Micronucleus formation in Pb-exposed workers was investigated in numerous occupational studies 

15 (Bilban, 1998; Grover et al., 2010; M. I. Khan et al., 2010; Minozzo et al., 2004; Minozzo et al., 2010; 

16 Palus et al., 2003; Pinto et al., 2000; Shaik & Jamil, 2009; Vaglenov et al., 1998; Vaglenov et al., 2001). 

17 The workers in the occupational studies generally had high blood Pb levels (>20 μg/dL) making 

18 comparisons to the general population difficult, although Pinto et al. (2000) observed increased 

19 micronuclei in exposed workers with an average blood Pb concentration of only 10.48 μg/dL. Studies that 

20 analyzed workers according to the magnitude of their blood Pb levels reported no correlation between Pb 

21 exposure and the observation of micronuclei (Minozzo et al., 2004; Minozzo et al., 2010; Palus et al., 

22 2003; Pinto et al., 2000), although Pinto et al. (2000), Grover et al. (2010), and Minozzo et al. (2010) did 

23 report an association between micronuclei formation and duration of exposure. Only one study was found 

24 that investigated micronucleus formation in a nonworker population; Mielzynska et al. (2006) examined 

25 associations of blood Pb and urinary PAH metabolite levels with micronuclei formation in children in 

26 Poland. Children, with an average blood Pb concentration of 7.69 μg/dL, were observed to have 4.44 

27 micronucleated cells per 1000 cells analyzed. Although no control group was included in this study, a 

28 statistically significant positive correlation was observed between blood Pb concentrations and 

29 micronuclei frequency, and children with blood Pb greater than 10 μg/dL had significantly more 

30 micronucleated cells than children with blood Pb less than 10 μg/dL.

31 Micronucleus formation in response to Pb exposure has been observed in rodent animal studies. 

32 Celik et al. (2005) observed that exposure of female rats to 140, 250, or 500 g/kg Pb acetate once per 

33 week for 10 weeks resulted in statistically significantly increased numbers of micronucleated 
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1 polychromatic erythrocytes (PCEs) compared to controls. Similarly, Alghazal et al. (2008) exposed rats to 

2 100 mg/L Pb acetate daily for 125 days and observed statistically significant increases in micronucleated 

3 PCEs in both sexes. Tapisso et al. (2009) exposed rats to 21.5 mg/kg Pb acetate (1/10th the LD50) via i.p. 

4 injection on alternating days for 11 or 21 days, for a total of 5 or 10 exposures. Formation of micronuclei 

5 in the bone marrow of exposed rats was increased over controls in a significant duration-dependent 

6 manner. Two further studies investigated formation of micronuclei in the bone marrow of exposed mice: 

7 Roy et al. (1992) exposed mice to 10 or 20 mg/kg Pb nitrate i.p. and observed a dose-dependent increase 

8 in micronuclei, whereas Jagetia and Aruna (1998) observed an increase in micronuclei in mice exposed to 

9 0.625-80 mg/kg Pb nitrate i.p., though the increase was not dose-dependent. Mice exposed to 1 g/L Pb 

10 acetate via a more environmentally relevant route of exposure, drinking water, for 90 days had 

11 statistically significant increases in micronucleated PCEs (C. C. Marques et al., 2006). 

12 Three studies were found that reported increased micronucleus formation in human cell lines 

13 treated with Pb. Dose-dependent micronucleus formation was observed in human lymphocytes when 

14 exposed to either 1, 5, 10, or 50 μM Pb nitrate or 3 or 5 ppm Pb chloride (Turkez et al.; Ustundag & 

15 Duydu, 2007). Gastaldo et al. (2007) also observed a dose-dependent increase in micronuclei in human 

16 endothelial cells exposed to 1-1000 μM Pb nitrate for 24 hours. Only two animal cell culture studies 

17 investigating micronuclei formation were found. One study observed that micronuclei were not induced in 

18 Chinese hamster cells exposed to 3-30 μM Pb nitrate for 2 hours (R. H. Lin et al., 1994), whereas the 

19 other observed that Pb acetate induced a dose-dependent increase in Chinese hamster cells when 

20 administered at 0.05-10 μM for 18 hours (Bonacker et al., 2005).

Chromosomal Aberrations

21 Chromosomal aberrations (e.g., chromosome breaks, nucleoplasmic bridges, di- and acentric 

22 chromosomes, and rings) were examined in a number of occupational studies (Bilban, 1998; De et al., 

23 1995; Grover et al., 2010; X. P. Huang et al., 1988; Pinto et al., 2000; Shaik & Jamil, 2009). 

24 Methodological limitations outlined in previous sections, including potential for occupational co-exposure 

25 to genotoxic substances and generally high blood Pb levels (>20 μg/dL) that are difficult to interpret in 

26 the context of the general population, also pertain to the present findings. No correlation was observed 

27 between increasing blood Pb levels and incidence of chromosomal aberrations, although an association 

28 was observed between duration of exposure and chromosomal damage (Grover et al., 2010; Pinto et al., 

29 2000). Two studies reported no association between occupational exposure to Pb and chromosomal 

30 aberrations (Andreae, 1983; Anwar & Kamal, 1988). Smejkalova (1990) observed chromosomal damage 

31 and aberrations in children living in a heavily Pb-contaminated area of Czechoslovakia and an area with 

32 less contamination, although the difference between the two was not statistically significant. Although 

33 blood Pb levels were statistically significantly higher in the Pb-contaminated children, they were 
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1 generally comparable (low 30s versus high 20s μg/dL, respectively), indicating there may not be enough 

2 of an exposure difference to detect a significant difference in aberration rates. 

3 The majority of animal studies investigating Pb-induced genotoxicity focused on the ability of Pb 

4 to produce chromosomal damage. Fahmy (1999) exposed mice to 25-400 mg/kg Pb acetate i.p., either as a 

5 single dose or repeatedly for 3, 5, or 7 days. Chromosomal damage was observed to increase in bone 

6 marrow cells (100-400 mg/kg) and spermatocytes (50-400 mg/kg) in a dose-dependent manner after both 

7 exposure regimens. Pb nitrate was also observed to produce dose-dependent chromosomal damage in 

8 mice exposed i.p. to a single dose of 5, 10, or 20 mg/kg (Dhir, Sharma, et al., 1992). In a similar 

9 experiment, Dhir et al. (1990) exposed mice to 10, 20, or 40 mg/kg Pb nitrate and saw an increase in 

10 chromosomal aberrations, although there was no dose-response as the response was similar in all doses 

11 tested. Nayak et al. (1989) exposed pregnant mice to 100-200 mg/kg Pb nitrate via i.v. injection and 

12 observed no chromosomal gaps or breaks in dams or fetuses, but did report some karyotypic 

13 chromosomal damage and weak aneuploidy at the low dose. In a similar experiment, low levels of 

14 chromosomal aberrations were observed in dams and fetuses injected with 12.5-75 mg/kg Pb nitrate, but 

15 there was no dose-response reported and few cells were analyzed (Nayak, Ray, & Persaud, 1989). In rats 

16 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, 

17 Pb-induced chromosomal aberrations were observed (Chakraborty et al., 1987). The above studies all 

18 suffer from the use of a route of exposure that may not be relevant to human environmental exposures. 

19 However, studies utilizing drinking water or dietary exposures also observed increases in chromosomal 

20 damage. Aboul-Ela (2002) exposed mice to 200 or 400 mg/kg Pb acetate by gavage for 5 days and 

21 reported that chromosomal damage was present in the bone marrow cells and spermatocytes of animals 

22 exposed to both doses. Dhir et al. (1992) also observed a dose-dependent increase in chromosomal 

23 damage in mice exposed via gavage, albeit at much lower doses: either 5 or 10 mg/kg. Nehez et al. (2000)

24 observed a Pb-induced increase in aneuploidy and percent of cells with damage after exposure to 

25 10 mg/kg administered by gavage 5 days a week for 4 weeks. In the only study that investigated dietary 

26 exposure, El-Ashmawy et al. (2006) exposed mice to 0.5% Pb acetate in feed, and observed an increase in 

27 abnormal cells and frequency of chromosomal damage.

28 Only three studies were found that investigated the ability of Pb to induce chromosomal damage in 

29 human cell lines and all three reported negative findings. Wise et al. (2004; 2005) observed that Pb 

30 glutamate was not mutagenic in human lung cells exposed to 250-2,000 μM for 24 hours. Shaik et al. 

31 (2006) observed that Pb nitrate did not increase chromosomal aberrations in primary lymphocytes 

32 (obtained from healthy volunteers) when incubated with 1.2 or 2 mM for 2 hours. Four studies utilizing 

33 animal cell lines generally supported the finding of no Pb-induced chromosomal damage in human cell 

34 lines. Pb nitrate was found to induce no chromosomal damage in Chinese hamster ovary cells exposed to 

35 500-2000 μM for 24 hours (J. P. Wise, Sr. et al., 1994), 3-30 μM for 2 hours (R. H. Lin et al., 1994), or 

36 0.05-1 μM for 3-12 hours (Cai & Arenaz, 1998). Wise et al. (1994) did observe increased chromosomal 
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1 damage in Chinese hamster ovary cells exposed to 1,000 μM Pb glutamate for 24 hours, but did not see 

2 any damage in cells exposed to higher concentrations (up to 2,000 μM).

5.2.7.4. Epigenetic Effects 

3 Epigenetic effects are heritable changes in gene expression resulting without changes in the 

4 underlying DNA sequence. A prime example of an epigenetic effect is the abnormal methylation of DNA, 

5 which could lead to altered gene expression and cell proliferation and differentiation. 

DNA Methylation

6 A single i.v. injection of 75 μmol/kg Pb nitrate resulted in global hypomethylation of hepatic DNA 

7 in rats (Kanduc et al., 1991). The observed hypomethylation in the liver was associated with an increase 

8 in cell proliferation. Two additional studies in humans observed that DNA methylation patterns in adults 

9 and cord blood were inversely correlated with bone Pb levels (Pilsner et al., 2009; R. O. Wright et al., 

10 2010). Changes in DNA methylation patterns could potentially lead to dysregulation of gene expression 

11 and altered tissue differentiation.

Mitogenesis

12 Only a few studies have investigated the potential epigenetic effects of Pb exposure in human 

13 populations. One such epigenetic effect investigated was mitogenesis that induces cells to proliferate 

14 when they should not. Three studies (Minozzo et al., 2004; Minozzo et al., 2010; T. Rajah & Ahuja, 1995)

15 were found that reported that Pb reduced mitogenesis in Pb-exposed workers (blood Pb = 35.4 μg/dL, 

16 59.4 μg/dL, and not reported, respectively). The observation of decreased cell division in exposed 

17 workers may indicate that cells suffered DNA damage and died during division, or that division was 

18 delayed to allow for DNA repair to occur. It is also possible that Pb exerts an aneugenic effect and arrests 

19 the cell cycle. 

20 Many studies have investigated the ability of Pb to induce mitogenesis in animal models, and have 

21 consistently shown that Pb nitrate can stimulate DNA synthesis and cell proliferation in the liver of 

22 animals exposed to 100 μM/kg via i.v. injection (Columbano et al., 1987; Columbano et al., 1990; Coni et 

23 al., 1992; Ledda-Columbano et al., 1992; Nakajima et al., 1995). Shinozuka et al. (1996) observed that 

24 Pb-induced hepatocellular proliferation was similar in magnitude to that induced by TNF-α at 100 μM/kg, 

25 and Pb was observed to induce TNF-α in glial and nerve cells and NF-κB, TNF-α, and iNOS in liver cells 

26 in exposed animals at 12.5 mg/kg and 100 μM/kg, respectively (Y.-J. Cheng et al., 2002; Menegazzi et al., 

27 1997). Only one study was found that observed a mitogenic effect after inhalation: exposure to 0.01M Pb 

28 acetate for 4 weeks resulted in increased cellular proliferation in the lungs (Fortoul et al., 2005).
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1 A great amount of research has been conducted investigating the potential effects of Pb on 

2 mitogenesis in human and animal cell cultures. In human cell cultures, Pb acetate inhibited cell growth in 

3 hepatoma cells (0.1-100 μM for 2-6 days) (Heiman & Tonner, 1995) and primary oligodendrocyte 

4 progenitor cells (1 μM for 24 hours) (W. Deng & Poretz, 2002), and had no observable effects on growth 

5 in glioma cells (0.01-10 μM for 12-72 hours) (M. Y. Liu et al., 2000). Pb glutamate had no effect on cell 

6 growth in human lung cells, but did increase the mitotic index (250-1,000 μM for 24 hours) (S. S. Wise et

7 al., 2005). The increase in the mitotic index was attributed to an arrest of the cell cycle at M-phase, and 

8 was not attributed to an actual increase of cell growth and proliferation. Gastaldo et al. (2007) also 

9 reported S and G2 cell cycle arrests in human endothelial cells following exposure to 100 μM Pb nitrate 

10 for 24 hours. Conflicting results with regard to DNA synthesis were reported, with a dose-dependent 

11 inhibition of DNA synthesis reported in hepatoma cells (1-100 μM for 72 hours) (Heiman & Tonner, 

12 1995), but an induction of synthesis observed in astrocytoma cells (1-50 μM for 24 hours) (Lu et al., 

13 2002). 

14 In rat fibroblasts and epithelial cells, Pb acetate, chloride, oxide, and sulfate were all observed to 

15 inhibit cell growth (10 μM -1 mM for 1-7 days and 0.078-320 μM for 48 hours, respectively) (Apostoli et 

16 al., 2000; Iavicoli et al., 2001). Iavicoli et al. (2001) observed that in addition to inhibiting cell growth in 

17 rat fibroblasts, Pb acetate caused GS/M and S-phase arrest. Pb acetate decreased cell proliferation in 

18 mouse mesenchymal stem cells when administered at 0-100 μM for 48 hours (Kermani et al., 2008). Pb 

19 nitrate was alternatively reported to increase (R. H. Lin et al., 1994) and decrease (Cai & Arenaz, 1998)

20 the mitotic index in Chinese hamster ovary cells exposed to 1μM Pb nitrate. Lin et al. (1994) did not 

21 consider cell cycle arrest when measuring the mitotic index, and did not observe a decrease at higher 

22 concentrations; in fact, the highest concentration tested, 30 μM, had a mitotic index equal to the untreated 

23 control cells. 

5.2.7.5. Gene Expression

24 Two animal studies have investigated the ability of Pb to alter gene expression in regard to phase I 

25 and II metabolizing enzymes. Suzuki et al. (1996) exposed rats to 100 μg/kg Pb acetate or nitrate via i.p. 

26 injection and observed an induction of glutathione transferase P (GST-P) with both Pb compounds. The 

27 induction of GST-P by Pb was observed to operate on the transcriptional level and to be dependent on the 

28 direct activation of the cis-element GPEI enhancer. Degawa et al. (1993) reported that i.v. exposure to 20, 

29 50, or 100 μmol/kg Pb nitrate selectively inhibited CYP1A2 levels. Pb was shown to not inhibit CYP1A2 

30 by direct enzyme inhibition, but rather to decrease the amount of Cyp1A2 mRNA. In contrast, Korashy 

31 and El Kadi (2004) observed that exposure of murine hepatoma cells to 10-100 μM Pb nitrate for 24 

32 hours increased the amount of Cyp1A1 mRNA while not influencing the activity of the enzyme. 

33 NAD(P)H:quinone oxidoreductase and glutathione S-transferase Ya activities and mRNA levels were 
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1 increased after exposure to Pb. Incubation of primary human bronchial epithelial cells with 500 μg/L Pb 

2 acetate for 72 hours resulted in the up-regulation of multiple genes associated with cytochrome P450 

3 activity, glutathione metabolism, the pentose phosphate pathway, and amino acid metabolism (Glahn et

4 al., 2008).

5 One additional animal study provides further evidence that exposure to Pb compounds can perturb 

6 gene expression. Zawia and Harry (1995) investigated whether the observed Pb-induced disruption of 

7 myelin formation in rat pups exposed postnatally was due to altered gene expression. In pups exposed to 

8 0.2% Pb acetate via lactation from PND1-20, the expression of proteolipid protein (PLP), a major 

9 structural constituent of myelin, was statistically significantly elevated at PND20, compared to controls. 

10 The expression of another structural element of myelin, myelin basic protein (MBP), was similarly 

11 elevated in exposed animals, although not significantly so. The expression of both genes returned to 

12 control levels 5 days following the termination of exposure. These data suggest that altered gene 

13 expression in structural myelin proteins due to Pb exposure may be responsible for observed alterations in 

14 abnormal conduction of nerve impulses.

5.2.7.6. Apoptosis

15 Occupational exposure to Pb and induction of apoptosis was investigated in three studies. One 

16 study directly reported that exposure to Pb increased apoptosis compared to nonexposed controls 

17 (Minozzo et al., 2010), whereas the other two reported that two early indicators of apoptosis, karyorrhexis 

18 and karyolysis, were elevated in exposed workers (Grover et al., 2010; M. I. Khan et al., 2010). Pb nitrate 

19 was also observed to induce apoptosis in the liver of exposed animals (Columbano et al., 1996; Nakajima 

20 et al., 1995). Apoptosis was observed in rat fibroblasts exposed to Pb acetate and rat alveolar 

21 macrophages exposed to Pb nitrate (Iavicoli et al., 2001; Shabani & Rabbani, 2000). Observation of Pb22 induced apoptosis may represent the dysregulation of genetically-controlled cell processes and tissue 

23 homeostasis.

5.2.8. Summary

24 The diverse health effects of Pb are mediated through multiple, interconnected modes of action. 

25 Each of the modes of action discussed has the potential to contribute to the development of a number of 

26 Pb-induced health effects (Table 5-2). Evidence for the majority of these modes of action is observed at 

27 low blood Pb level in humans and animals, between 2 and 5 μg/dL, and at doses as low as the picomolar 

28 range in animals and cells. Dose captures Pb exposure concentration in invitro systems or in animal 

29 studies when no blood Pb level was reported. These observable effect levels are limited by the data and 

30 methods available and do not imply that these modes of action are not acting at lower exposure levels or 

31 that these doses represent the threshold of the effect.
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Table 5-2. Related health effects resulting from the MOAs of Pb and the lowest level 

eliciting the MOA reported as blood Pb level and dose delivered

MOA

Related Health Effects 

(Section) ISA

Lowest Level at which

MOA Observed 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) Ahamed 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 0.01μM Kim et al. (2007) Chetty et al. (2005)

Endocrine 

Disruption

Reproductive Effects and Birth Outcomes (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 Ca2+ 1 homeostasis, altered ion transport 

2 mechanisms, and perturbed protein function through displacement of metal cofactors) appears to be the 

3 major unifying mode of action underlying all subsequent modes of action (Figure 5-1). Pb will interfere 

with endogenous Ca2+ 4 homeostasis, necessary as a cell signal carrier mediating normal cellular functions. 

[Ca2+ 5 ]i have been shown to increase in a number of cell types including bone, erythrocytes, brain cells, 

and white blood cells, due to the increased flux of extracellular Ca2+ 6 into the cell. This disruption of ion 

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 Ca2+ 8 channels. Pb can interfere with these proteins through direct 

9 competition between Pb and the native metals present in the protein metal binding domain or through

disruption of proteins important in Ca2+ 10 -dependent cell signaling, such as PKC or calmodulin. Disruption 

of ion transport not only leads to altered Ca2+ 11 homeostasis, it can also result in perturbed neurotransmitter 

12 function. Pb-exposure decreases evoked release of neurotransmitters, while simultaneously increasing 

spontaneous release of neurotransmitters through Ca2+ 13 mimicry. Pb is able to displace metal ions, such as 

Zn, Mg, and Ca2+ 14 , from proteins due to the flexible coordination number of Pb and multiple ligand 

15 binding ability, leading to abnormal conformational changes to proteins and altered protein function. 

16 Evidence for this metal ion displacement and protein pertubation has been shown at picomolar 

17 concentrations of Pb. Additional effects of altered cellular ion status are the inhibition of heme synthesis 

18 and decreased cellular energy production due to perturbation of mitochondrial function.

19 Although Pb will bind to proteins within cells through interactions with side group moieties, thus 

20 potentially disrupting cellular function, protein binding of Pb may represent a mechanism by which cells 

21 protect themselves against the toxic effects of Pb. Intranuclear and intracytosolic inclusion body 

22 formation has been observed in the kidney, liver, lung, and brain following Pb exposure. A number of 

23 unique Pb binding proteins have been detected, constituting the observed inclusion bodies. The major Pb 
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1 binding protein in blood is ALAD with carriers of the ALAD-2 allele potentially exhibiting higher Pb 

2 binding affinity. Additionally, metallothionein is a important protein in the formation of inclusion bodies 

3 and mitigation of the toxic effects of Pb.

4 A second major mode of action of Pb is the development of oxidative stress, due in many instances 

5 to the antagonism of normal metal ion functions. The origin of oxidative stress produced after Pb 

6 exposure is likely a multipathway process, resulting from oxidation of δ-ALA, NAD(P)H oxidase 

7 activation, membrane and lipid peroxidation, and antioxidant enzyme depletion. Through the inhibition of 

8 δ-ALAD due to displacement of Zn, accumulated δ-ALA goes through an auto-oxidation process to 

9 produce ROS. Additionally, Pb can induce the production of ROS through the activation of NAD(P)H 

10 oxidase. Pb-induced ROS can interact with membrane lipids to cause a membrane and lipid peroxidation 

cascade. Enhanced lipid peroxidation can also result from Pb potentiation of Fe2+ 11 initiated lipid 

12 peroxidation and alteration of membrane composition after Pb exposure. Increased Pb-induced ROS will 

13 also sequester and inactivate biologically active ·NO, leading to the increased production of the toxic 

14 product nitrotyrosine, increased compensatory NOS, and decreased sGC protein. Pb-induced oxidative 

15 stress not only results from increased ROS production but also through the alteration and reduction in 

16 activity of the antioxidant defense enzymes. The biological actions of a number of these enzymes are 

17 antagonized due to the displacement of the protein functional metal ions by Pb. 

18 In a number of organ systems Pb-induced oxidative stress is accompanied by misregulated 

19 inflammation. Pb exposure will modulate inflammatory cell function, production of pro-inflammatory 

20 cytokines and metabolites, inflammatory chemical messengers, and pro-inflammatory signaling cascades. 

21 Cytokine production is skewed toward the production of pro-inflammatory cytokines like TNF-α and IL-6 

22 as well as toward the promotion of a Th2 response and suppression of a Th1 response accompanied by 

23 production of related cytokines.

24 Pb is a potent endocrine disrupting chemical. Pb will disrupt the HPG axis evidenced by a decrease 

25 in serum hormone levels, such as FSH, LH, testosterone, and estradiol. Pb interacts with the 

26 hypothalamic-pituitary level hormone control causing a decrease in pituitary hormones, altered growth 

27 dynamics, inhibition of LH secretion, and reduction in StAR protein. Pb has also been shown to alter 

28 hormone receptor binding likely due to interference of metal cations in secondary messenger systems and 

29 receptor ligand binding and through generation of ROS. Pb also may disrupt the HPT axis by alteration of 

30 a number of thyroid hormones, possibly due to oxidative stress. However the results of these studies are 

31 mixed and require further investigation.

32 Genotoxicity and cell death has been investigated after Pb exposure in humans, animals, and cell 

33 models. High level Pb exposure to humans leads to increased DNA damage, however lower blood Pb 

34 levels have caused these effects in experimental animals and cells. Reports vary on the effect of Pb on 

35 DNA repair activity, however a number of studies report decreased repair processes following Pb 

36 exposure. There is evidence of mutagenesis and clastogenicity in highly exposed humans, however weak 
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1 evidence has been shown in animals and cells based systems. Human occupational studies provide limited 

2 evidence for micronucleus formation (>10 μg/dL), supported by Pb-induced effects in both animal and 

3 cell studies. Animal studies have also provided evidence for Pb-induced chromosomal aberrations. The 

4 observed increases in clastogenicity may be the result of increased oxidative damage to DNA due to Pb 

5 exposure, as co-exposures with antioxidants ameliorate the observed toxicities. Limited evidence of 

6 epigenetic effects is available, including DNA methylation, mitogenesis, and gene expression. Altered 

7 gene expression may come about through Pb displacing Zn from multiple transcriptional factors, and thus 

8 perturbing their normal cellular activities. Consistently positive results have provided evidence of 

9 increased apoptosis following Pb exposure. 

10 Overall, Pb-induced health effects can occur through a number of interconnected modes of action 

11 that generally originate with the alteration of ion status.

5.3. Neurological Effects

5.3.1. Introduction

12 The central nervous system undergoes rapid differentiation in utero and in early life, which makes 

13 the developing fetus and infant particularly susceptible to neurological effects associated with 

14 environmental exposures (Landrigan et al., 1999; Rice & Barone, 2000). Based on evidence from diverse

15 prospective and cross-sectional studies focusing primarily on effects associated with Pb exposure levels 

16 less than 10 μg/dL, the 2006 Pb AQCD concluded that the “overall weight of the available evidence 

17 provides clear substantiation of neurocognitive decrements being associated in young children with 

18 blood-Pb concentrations” (U.S. EPA, 2006). Several individual studies observed inverse associations 

19 between blood Pb levels and IQ that persisted at blood Pb levels in the range of 2-8 μg/dL (Lanphear et 

20 al., 2000; J. Schwartz, 1994). This association was substantiated in a pooled analysis of children, 5 to 10 

21 years of age, participating in seven prospective studies (Boston, MA; Cincinnati, OH; Rochester, NY; 

22 Cleveland, OH; Mexico City, Mexico; Port Pirie, Australia; and Kosovo, Yugoslavia) (Lanphear et al., 

23 2005). The 2006 AQCD described associations of blood Pb with a broad range of additional, related 

24 neurodevelopmental endpoints, including academic achievement and performance, motor skills, mood, 

25 and antisocial and delinquent behavior (U.S. EPA, 2006).

26 Toxicological studies not only provided coherence with similarly consistent findings for Pb27 induced impairments in learning, behavior and attention, and sensory acuities, but also provided 

28 biological plausibility by characterizing mechanisms for Pb-induced neurological effects. In particular, 

29 toxicological evidence for Pb exposure interfering with neuronal metabolism at the cellular and 

30 histological level (e.g., synaptic architecture during development, neurotransmitter release, glia, neurite 

31 outgrowth, the blood brain barrier, and oxidative stress), provided biological plausibility for blood Pb 
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1 levels of children being associated with deficits in multiple functional domains such as cognitive function, 

2 motor function, memory, mood, and behavior. Additional biological plausibility was provided by 

3 observations of associations of childhood blood Pb levels with decreased neuronal density and neuronal 

4 loss measured in adulthood, as assessed by magnetic resonance imaging techniques (Cecil et al., 2005; 

5 Meng et al., 2005; Trope et al., 2001; Trope et al., 1998; Yuan et al., 2006). 

6 A key finding across several epidemiologic studies of children was a larger estimated effect for a 

7 given incremental increase in blood Pb levels on neurocognitive deficits in children with lower blood Pb 

8 levels compared with children with higher blood-Pb levels (Bellinger & Needleman, 2003; Canfield, 

9 Henderson, et al., 2003; Kordas et al., 2006; Lanphear et al., 2005; Rothenberg & Rothenberg, 2005; 

10 Tellez-Rojo et al., 2006). Among these studies were two analyses of the pooled cohort data, both of which 

11 demonstrated a supralinear relationship between blood Pb level and IQ with a steeper negative slope 

12 observed at blood Pb levels <10 μg/dL (Lanphear et al., 2005; Rothenberg & Rothenberg, 2005).

13 Consistent with epidemiologic findings, toxicological studies also observed nonlinear Pb exposure14 response relationships for outcomes such as behavioral responses, neuronal activation, and dopamine 

15 release.

16 Another area of focus included the comparison of various lifestages of Pb exposure in terms of risk 

17 of neurodevelopmental deficits. Toxicological studies demonstrated that in utero with or without early 

18 postnatal exposure to Pb was the most sensitive window for Pb-dependent neurological effects. 

19 Epidemiologic studies observed neurocognitive deficits in association with prenatal, peak childhood, 

20 cumulative childhood, and concurrent blood Pb levels. Among studies of children that examined multiple 

21 lifestages of exposure, several found that concurrent blood Pb was associated with the largest decrement 

22 in IQ (Baghurst et al., 1992; Canfield, Henderson, et al., 2003; A. Chen et al., 2005; Lanphear et al., 

23 2005), with some studies finding that the magnitude of association increased with age (Dietrich, Berger, 

24 Succop, et al., 1993; Factor-Litvak et al., 1999; Ris et al., 2004; G. A. Wasserman et al., 1994). Several 

25 studies of children compared effect estimates for Pb levels measured in blood, deciduous tooth, or tibial 

26 bone and found that compared with blood Pb levels, tooth or bone Pb levels were associated with an equal 

27 or larger magnitude of neurodevelopmental deficits (Bellinger et al., 1991; Greene & Ernhart, 1993; 

28 Needleman et al., 1979; G. A. Wasserman et al., 2003). These findings pointed to an effect of cumulative 

29 childhood exposure. A common limitation of epidemiologic studies of children was the high correlation 

30 among Pb exposure metrics at different ages, making it difficult to distinguish among effects of Pb 

31 exposure at different ages (Lanphear et al., 2005) and to ascertain which developmental periods of Pb 

32 exposure were associated with the greatest risk of neurodevelopmental decrements. The issue of 

33 persistence of the neurological effects of Pb exposure also was considered, with some evidence 

34 suggesting that the associations of blood Pb levels with neurodevelopmental outcomes persisted into 

35 adolescence and young adulthood in the absence of marked reductions in blood Pb level (Needleman et 

36 al., 1990; Ris et al., 2004; Tong et al., 1996).
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1 In epidemiologic studies of adults, neurological effects (e.g., impaired memory, attention, reaction 

2 time, visuomotor tasks and reasoning, alterations in visual or brainstem evoked potentials, postural sway, 

3 and nerve conduction) were mostly clearly indicated in occupationally-exposed workers in association 

4 with blood Pb levels in the range of 14 to 40 μg/dL (Baker et al., 1979; Cantarow & Trumper, 1944). 

5 Studies of environmentally-exposed adults produced mixed findings; however, bone Pb levels (Weisskopf 

6 et al., 2004; R. O. Wright et al., 2003) were associated with decrements in neurocognitive function more 

7 so than were blood Pb levels (E. F. Krieg, Jr et al., 2005; Nordberg et al., 2000). These findings suggested 

8 that cumulative Pb exposures, including higher past exposures, may be important contributors to 

9 neurological effects in adults.In addition to cognitive function, blood and bone Pb levels also were 

10 associated with greater self-reported anxiety and depression scores among environmentally-exposed 

11 adults (Rhodes et al., 2003). These findings in adults were strongly supported by parallel findings in 

12 female and male rodents for Pb-induced depression and emotional changes, respectively. Studies of adults 

13 also reported associations of blood Pb with risk of amyotrophic lateral sclerosis (ALS) and essential 

14 tremor, although the body of literature was smaller and evidence was less consistent than that for 

15 cognitive function. Whereas toxicological studies demonstrated Pb-induced neurodegeneration and 

16 formation of neurofibrilary tangles commonly associated with Alzheimer’s disease pathophysiology, 

17 blood Pb level generally was not associated with Alzheimer’s disease in adults. 

18 As discussed throughout this section, recent epidemiologic and toxicological studies continue to 

19 demonstrate associations between exposure to or biomarkers of Pb and neurological effects and expand 

20 upon the previous body of evidence by demonstrating similar effects (e.g., cognitive function, 

21 impairments in behavior) at lower blood Pb levels (1-5 μg/dL). Whereas previous evidence was 

22 inconsistent, new studies in children report positive associations between blood Pb levels and attention 

23 deficit hyperactivity disorder (ADHD). New toxicological studies expand evidence for prenatal and 

24 postnatal Pb exposure effects on learning, memory, and attention and provide insight into the contribution 

25 of stress to this paradigm. New or expanded areas of toxicological research related to Pb exposure include 

26 mood disorders, neurofibrilary tangle formation, and adult dementia after early life Pb exposures. 

27 Historically important areas of toxicological research are further expanded with recent publications of Pb28 dependent effects on neurotransmitters, synapses, glia, neurite outgrowth, the blood brain barrier, and 

29 oxidative stress. The data detailed in the subsequent sections continue to enhance the understanding of 

30 neurological and neurobehavioral outcomes associated with Pb exposure.
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5.3.2. Neurocognitive Function and Learning

5.3.2.1. Epidemiologic Studies of Cognitive Function in Children

Full-scale IQ in Children

1 Several longitudinal cohort studies were initiated in the 1980s in order to address limitations of 

2 cross-sectional studies, including establishing a temporal association between exposure and outcome, 

3 examining persistence of neurocognitive deficits to older ages, and comparing risk estimates among blood 

4 Pb levels at different lifestages. Moreover, cooperation among investigators to adopt similar assessment 

5 protocols facilitated pooled and meta-analyses and comparison of results across populations that differed 

6 in blood Pb levels, race/ethnicity, and SES. Individual cohort studies in diverse populations were 

7 consistent in demonstrating that blood Pb levels in the range of 5 to 10 μg/dL (Bellinger et al., 1987; 

8 Bellinger & Needleman, 2003; Bellinger et al., 1991; Canfield, Henderson, et al., 2003; J. Schwartz, 

9 1994; Tellez-Rojo et al., 2006), were associated with losses in full-scale IQ (FSIQ) pointsin children. 

10 These findings were substantiated in a pooled analysis of seven prospective studies (Boston, MA; 

11 Cincinnati, OH; Rochester, NY; Cleveland, OH; Mexico City, Mexico; Port Pirie, Australia; and Kosovo, 

12 Yugoslavia) by Lanphear et al. (2005) as well as multiple meta-analyses that included both cross-sectional 

13 and prospective studies (Needleman & Gatsonis, 1990; Pocock et al., 1994; J. Schwartz, 1994) (Figure 5-

14 2 and Table 5-3).

15 The analysis pooling data from seven prospective studies included 1333 children with mean 

16 (5th-95th percentile) blood Pb levels of 12.4 μg/dL (4.1-34.8 μg/dL) (Lanphear et al., 2005). In 

17 multivariate models that adjusted for study site, maternal IQ, Home Observation for the Measurement of 

18 Environment (HOME) inventory, birth weight, and maternal education, IQ measured at schoolage (mean 

19 6.9 years) was inversely associated with concurrent, peak, average lifetime, and early childhood blood Pb 

20 levels, with the largest decrement in IQ estimated for concurrent blood Pb levels (-0.23 points [95% CI: -

21 0.32, -0.14] at a blood Pb level of 1 μg/dL).

22 In Lanphear et al. (2005), various models were investigated to characterize the shape of the blood 

23 Pb dose-response relationship. Consistent with findings from individual studies, Lanphear et al. (2005)

24 found that a log-linear model best fit the data, with a greater decrease in IQ estimated for an increase in 

25 concurrent blood Pb from <1-10 μg/dL (6.2 points [95% CI: 3.8, 8.6]) than an increase from 10 to 20 

26 μg/dL (1.9 points [95% CI: 1.2, 2.6]). Sensitivity analyses, in which one study was successively excluded, 

27 revealed that no single study was responsible for driving the results. Although HOME score was not 

28 available in the Rochester study, exclusion of that cohort’s data resulted in a less negative effect estimate. 

29 However, the change was less than 3%.
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1 Studies published since the 2006 Pb AQCD continue to demonstrate associations between 

2 increasing blood Pb level and decrements in FSIQ (Figure 5-2 and Table 5-3). Whereas most studies 

3 demonstrated decrements in FSIQ in association with blood Pb levels ranging from 5 to10 μg/dL, Kim et 

4 al. (2009) was particularly noteworthy for demonstrating an association in a population with lower blood 

5 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 

6 tested using the Korean Educational Development Institute-WISC, which assesses vocabulary, arithmetic, 

7 picture arrangement, and block design. In a linear regression analysis adjusted for age, sex, maternal and 

8 paternal education, yearly income, prenatal smoking, postnatal environmental tobacco smoke exposure, 

9 birth weight, and maternal age at birth, a 1 μg/dL increase in blood Pb level was associated with a -1.03 

10 point decrease (95% CI: -1.71, -0.36) in FSIQ. Increasing blood Pb levels also was associated in 

11 performance and verbal IQ (PIQ and VIQ, respectively). Although several important confounders were 

12 considered, there was no direct assessment of the home environment and the primary caregiver’s IQ in 

13 this study which are notable limitations.

14 Kim et al. (2009) also examined effect modification of the blood Pb-FSIQ relationship by blood 

15 manganese (Mn) levels. The mean (range) blood Mn level was 14.3 μg/dL (5.3-29.02), respectively. 

16 Blood Pb and Mn levels were not correlated (r = -0.03, p = 0.64). To examine effect modification, 

17 children were divided into two groups: blood Mn level above and below the median (14 μg/dL). 

18 Multivariate linear regression models predicting FSIQ, VIQ, and PIQ used concurrently measured blood 

19 Pb level as the predictor variable in the low and high Mn groups. The associations for blood Pb level with 

20 FSIQ and VIQ were larger in magnitude for children in the high Mn group (e.g., -5.3 FSIQ points [95% 

21 CI: -10.1, -5.3] per 1 μg/dL increase in blood Pb level), compared with children in the low Mn group

22 (e.g., -4.0 FSIQ points [95% CI: -9.9, 1.80] per 1 μg/dL increase in blood Pb level) (Figure 5-3). 
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Reference

Exposure 

Period

Mean (SD)

Blood Pb 

(μg/dL)

FSIQ Age 

(Years)

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 al. (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 (25th-50th) 7-8

Tong et al. (1996) Cumulative 17.8 (5.8) 11-3

Factor-Litvak et al. (1999) Cumulative 30 and 8a 7

Wasserman et al. (2003) Cumulative 31.6 and 6.3a 10-12

Surkan et al. (2007) Concurrent 5-10 vs. 1-2b 6-10

Pooled/Meta-Analyses

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 children with blood Pb levels 5-10 μg/dL, with children with blood Pb levels 1-2 

μg/dL serving as the reference 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. 
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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 AfricanAmerican 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)

Linear 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, -

0.17)

Canfield et 

al. (2003)

172 children born 1994- 1995 in Rochester, NY 

followed from 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 followed 

from birth (1987-1992) 

followed until age 10-15 

yr.

Prenatal (3rd 

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- 50th: 17.4, 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

WISC-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 

occupational 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) 

meanc: 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 et 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).

Figure 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. 

1 Min and colleagues (2009) examined the relationship between Pb exposure assessed at age 4 years 

2 and children’s IQ and academic achievement at 4, 9, and 11 years of age in a sample of 278 urban 

3 children originally enrolled in a prospective study on the effects of prenatal poly-drug exposure 

4 (determined by assay of infant meconium or urine, maternal urine, or maternal self-report). The study 

5 population was primarily African-American (86%) and low SES (98%); 39% of mothers had not finished 

6 high school, and 14% were married at the time of enrollment. The mean blood Pb level at age 4 years was 

7 7.0 μg/dL (SD 4.1, range 1.3-23.8); 36% had blood Pb levels <5 μg/dL, and 19% had levels >10 μg/dL. 

8 The researchers utilized restricted cubic spline functions for blood Pb level to test for a nonlinear 

9 relationship between blood Pb levels and FSIQ. Although the cubic spline term did not attain statistical 

10 significance, analyses suggested a steeper slope at lower Pb levels (up to 7 μg/dL) These findings were 

11 consistent with those from the pooled analysis (Lanphear et al., 2005) and other studies (Tellez-Rojo et 

12 al., 2006).

13 Also similar to previous studies with repeated assessments of cognitive function over time, Min et 

14 al. (2009) found that the association between concurrent blood Pb level and FSIQ persisted with 
May 2011 5-52 DRAFT – DO NOT CITE OR QUOTE

1 increasing age. The magnitude of the inverse association between blood Pb level and FSIQ was consistent 

2 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 

3 0.50, 0.41, and 0.54 at ages 4, 9, and 11 years, respectively (Figure 5-2 and Table 5-3). The findings of 

4 this study also indicated that specific cognitive domains may be more sensitive to Pb exposure at different 

5 stages of development. Non-verbal reasoning decrements assessed using the Wechsler Preschool and 

6 Primary Scales of Intelligence-Revised (WPPSI-R) Performance IQ (PIQ) and Wechsler Intelligence 

7 Scales (WISC)-IV Perceptual Reasoning Index were consistently associated with increasing blood Pb 

8 level even at younger ages while verbal decrements did not become apparent until assessments at 11 years 

9 of age. Lower reading scores were associated with increased Pb exposure at 9 and 11 years while math 

10 scores were not affected until age 11 years. An important consideration that may limit the generalizability 

11 of these findings is the high prevalence of prenatal exposure to cocaine (51% of subjects) and alcohol

12 (77% of subjects). However, accounting for prenatal drug exposure did not attenuate or modify (i.e., no 

13 interaction effects) the negative associations between blood Pb level and cognitive outcomes. 

14 Surkan et al. (2007) used data originally collected for the New England Children’s Amalgam Trial 

15 (NECAT), a study of 6 to 10 year old English-speaking children from urban Boston, Massachusetts and 

16 rural Farmington, Maine designed to assess the effect of amalgam dental fillings on children’s 

17 neurodevelopment. At baseline (prior to placement of amalgam fillings), blood Pb levels were measured, 

18 and children were administered an extensive battery of neuropsychological tests including tests of 

19 memory, learning, visual-motor ability, reading, reaction time. In analyses that excluded 3 children with 

20 blood Pb level >10 μg/dL, children with blood Pb levels of 5 to 10 μg/dL had significantly lower WISC21 III Full FSIQ scores (-6.07 points (95% CI: -10.7, -1.36]) compared with children who had levels of 1 to 2 

22 μg/dL (referent group), adjusting for age, race/ethnicity (Black or Hispanic vs. non-Hispanic white), birth 

23 weight, SES, and primary caregiver IQ. 

Specific Indices of Cognitive Function in Children

24 In addition to FSIQ, an index of global cognitive function, blood Pb levels also were associated 

25 with specific cognitive abilities, including attention, executive function, language, memory and learning, 

26 and visuospatial processing in previous studies of children and adolescents (Bellinger et al., 1991; 

27 Bellinger & Stiles, 1993; Canfield, Kreher, et al., 2003; Chiodo et al., 2004; Dietrich et al., 1991; Dietrich 

28 et al., 1992; Kordas et al., 2006; Lanphear et al., 2000; Needleman et al., 1979; Ris et al., 2004; Tellez29 Rojo et al., 2006). Studies often find associations with several endpoints, and because several tests of 

30 neurocognitive function are interrelated, it is difficult to ascribe the effects of Pb exposure to a specific 

31 domain of neurocognitive function. For example, in U.S. representative analysis of NHANES III (1988-

32 1994) data, which included 4853 children ages 6-16 years with a geometric mean blood Pb level of 1.9 

33 μg/dL, Lanphear et al. (2000) found that a 1 μg/dL increase in blood Pb levels was associated with 
May 2011 5-53 DRAFT – DO NOT CITE OR QUOTE

1 decreases in arithmetic (-0.70 points [95% CI: -1.0, -0.37]), reading (-0.99 points [95% CI: -1.4, -0.62]), 

2 block design (-0.10 points [95% CI: -0.18, -0.02]), and digit span (-0.05 [95% CI: -0.09, -0.01]) subtests. 

3 Finding that blood Pb levels are associated with a spectrum of neurocognitive indices provides biological 

4 plausibility for associations observed between blood Pb levels and IQ. Furthermore, these tests of 

5 attention, learning, and memory in humans have parallel tests in animals, and compared with evidence for 

6 IQ, evidence for these specific tests may improve understanding of the coherence between findings in 

7 humans and animals (Rice, 1996).

8 Studies published since the 2006 Pb AQCD continue to observe associations between increasing 

9 blood Pb level and decrements in these specific indices of cognitive function. Compared with studies of

10 IQ, studies of specific cognitive indices consistently find associations at lower blood Pb levels 

11 (population means: 1.2 to 7 μg/dL and quantities of blood Pb levels as low as 2 μg/dL) (Figure 5-4 and 

12 Table 5-4). Recent studies of cognitive function also expanded on previous evidence by providing 

13 information on effect modification by genetic, nutritional, and caregiving quality (E. F. Krieg, Jr. et al., 

14 2010; Pilsner et al., 2010; Solon et al., 2008; Surkan et al., 2008).
May 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 errorsb

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. 1d End of grade scorec

3 vs. 1d

Miranda et al. (2009) Early childhood 2 vs. 1d End of grade score

3 vs. 1d

Froehlich et al. (2007) Concurrent 6.1 (4.9) Spatial memoryb

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 (standardized) 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. bStandard 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. dEffect estimate compares 

test scores of children in different categories of blood Pb concentration.

Figure 5-4. Associations 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 (μg/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 

education, 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, parental 

education, maternal IQ, child 

IQ, birth weight, urinary 

cotinine

No omission errorsb

No comission errorsb

Word reading scorec

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 (μg/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, raceethnicity, 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 4th grade 

children, 2000-2004

7 counties in central 

North 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

4th grade end-ofgrade scorec -0.08 (-0.16, 0), blood Pb 2 

μg/dL vs. 1 μg/dLd

-0.13 (-0.21, -0.05), blood 

Pb 3 μg/dL vs. 1 μg/dLd

Miranda et al. 

(2009)

57,678 4th 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

4th grade end-ofgrade score-0.04 (-0.07, -0.001), blood 

Pb 2 μg/dL vs. 1 μg/dLd

-0.05 (-0.09, -0.02) , blood 

Pb 3 μg/dL vs. 1 μg/dLd

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 memoryb

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, 0.09)

-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 1st 

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)

Studies 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 μg/dL 

vs. 1-2 μg/dLd

Chandramouli et 

al. (2009)

488 children born 

1991-1992 followed 

from birth to age 7-8 

yr

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 μg/dL vs. 0-2 μg/dLd

-0.49 (-0.79, -0.31), blood 

Pb 5-10 μg/dL vs. 0-2 μg/dLd

-0.44 (-0.93, -0.21), blood 

Pb >10 μg/dL vs. 0-2 μg/dLd
May 2011 5-56 DRAFT – DO NOT CITE OR QUOTE

Study Population/

Location

Blood Pb 

Levels (μg/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, Philippines

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 

premature, 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 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. 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.

1 Krieg et al. (2010) examined an older subset of children (ages 12-16 years) from NHANES III 

2 (1988-1994) that were previously examined in Lanphear et al. (2000). Similar to Lanphear et al. (2000), 

3 Krieg et al. (2010) found associations of increasing concurrent blood Pb level with decrements in block 

4 design, digit span, reading score, and arithmetic score in these older children (Figure 5-4 and Table 5-4). 

5 In another study of Korean children (ages 8-11 years) with similar blood Pb levels (mean: 1.9 μg/dL [SD: 

6 0.67]), Cho and colleagues (2010) found concurrent blood Pb levels to be associated with decreases in in 

7 tests of attention (errors in responding to targets); however associations were not statistically significant 

8 in models that adjusted for urinary cotinine levels (Figure 5-4 and Table 5-4). Although associations of 

9 blood Pb levels with word and color naming were negative, effect estimates were associated with wide 

10 95% CIs.

11 Krieg et al. (2010) additionally provided additional information on effect modification by vitamin 

12 D receptor (VDR) variants. Although there were not differences in blood Pb levels among the various 

13 haplotypes of VDR, various polymorphisms and haplotypes modified the association between blood Pb 

14 level and a range of neurocognitive tests. The VDR regulates calcium absorption and metabolism, and 

15 effect modification by VDR variants is consistent with the well-established mode of action of Pb in 

16 mimicking calcium in shared transport and metabolic pathways. However, several inconsistencies were

17 observed by Krieg et al. (2010) in that a particular variant was associated with a lower Pb-associated 

18 decrement in performance for some tests and a greater Pb-associated decrement in performance for other 

19 tests. For example, among children ages 12-16 years, the VDR rs2239185 CC genotype was associated 

20 with the largest blood Pb-associated decrease in digit span score and reading score. The slopes for digit 

21 span score (95% CI) per 1 μg/dL increase in blood Pb level were -1.5 (-2.2, -0.71) for the CC genotype 

22 and -0.26 (-0.99, 0.46) for the TT genotype. Conversely, the TT genotype was associated with the greatest 

23 Pb-associated decrease in arithmetic score. The slopes (95% CIs) for math score per 1 μg/dL increase in 

24 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. 
May 2011 5-57 DRAFT – DO NOT CITE OR QUOTE

1 Effect modification by VDR rs731236 was more consistent across cognitive tests, with larger decrements 

2 in blood Pb-associated cognitive performance among children with the CC genotype.

3 Multiple studies in different Mexico City mother-child dyads recently reported on associations 

4 between blood Pb levels (e.g., maternal, cord blood, or child postnatal Pb levels) and mental development 

5 in children at age 24 or 36 months (H. Hu et al., 2006; Pilsner et al., 2010; Surkan et al., 2008). Hu et al. 

6 (2006) assessed the impact of timing of exposure prenatally among 146 mother-child dyads meeting the 

7 following criteria: born at 37 weeks or greater gestational age, at least one valid Pb measurement during 

8 pregnancy, complete information on maternal age and IQ, and child’s blood Pb level at 24 months when 

9 the 24-month Bayley Mental Development Index (MDI) was ascertained. Comparing whole blood and 

10 plasma Pb levels collected at each of 3 trimesters, this group found that 1st trimester blood Pb (or plasma 

11 Pb) was the best predictor of subsequent 24-month Bayley Scale MDI scores. Another study excluding 

12 the first trimester demonstrated an inverse association between IQ assessed at age 6-10 years and third 

13 trimester maternal blood Pb level, but not with maternal blood Pb levels measured at other times during 

14 pregnancy or within child Pb blood levels averaged over 6 to 10 years (Schnaas et al., 2006).

15 Surkan et al. (2008) found negative associations (statistically nonsignificant) of concurrent blood 

16 Pb levels with Bayley MDI and Psychomotor Development Index overall in a population of 379 Mexico 

17 City children between ages 12 and 36 months. However, when data were stratified by maternal self18 esteem, negative associations were observed among children with mothers with in the lowest three 

19 quartiles of self-esteem but not among children with mothers in the highest quartile of self-esteem (Figure 

20 5-4 and Table 5-4). These findings indicate that higher maternal psychosocial functioning (e.g., stress, 

21 anxiety, depression, self-esteem) may contribute to better caregiving, which in turn may improve 

22 neuropsychological functioning of the child. These limited data in humans are well-supported by findings 

23 from animal studies that have shown that environmental enrichment can reverse the effects of early stress 

24 experiences on reactions such as depressed behavior, HPA activation, and immunosuppression (Francis et 

25 al., 2002; Laviola et al., 2008; Laviola et al., 2004; Moreley-Fletcher et al., 2003). With specific regards 

26 to Pb exposure, Schneider et al. (2001) and Guilarte et al. (2003) demonstrated that the social isolation or 

27 enrichment can exacerbate or protect against, respectively, the neurological effects from Pb exposure. It is 

28 worth mentioning in this context that the potential programming effects of stress on childhood health 

29 outcomes may occur at an even more fundamental level, i.e., through epigenetic programming (Dolinoy 

30 & Jirtle, 2008). Pb exposure of animals and blood Pb levels in humans have been associated with altered 

31 DNA methylation patterns which in turn, may be associated with altered gene expression patterns 

32 (Section 5.3.6.11 and Section 5.10.4).

33 In a recent study in Mexico City, Mexico, investigators found increasing cord blood Pb levels to be 

34 associated with lower MDI scores among children at age 24 months (-0.051 points [95% CI: -0.087, -

35 0.016] in standardized score per 1 μg/dL increase in blood Pb level) (Pilsner et al., 2010). Investigators 

36 additionally examined effect modification by variants in the methylenetetrahydrofolate reductase 
May 2011 5-58 DRAFT – DO NOT CITE OR QUOTE

1 (MTHFR) gene. The MTHFR enzyme is involved in folate metabolism, specifically, catalyzing the 

2 conversion of 5,10-methylenetetrahydrofolate to 5-methylenehydrofolate, which, in turn, is involved in 

3 homocysteine methylation to the amino acid methionine. The transfer of methyl groups that results from 

4 folate metabolism is important for biological processes including Phase II detoxification reactions and 

5 epigenetic regulation of gene expression. The MTHFR gene has common functional variants, including 

6 the C677T SNP, which produces an enzyme with lower metabolic activity and is associated with lower 

7 serum folate levels (Kordas et al., 2009). Although Pilsner et al. (2010) found that both cord blood Pb 

8 levels and the MTHFR 677T allele were associated with lower child MDI score at age 24 months, they 

9 did not find a statistically significant interaction between blood Pb level and the MTHFR 677T allele. 

10 Results from stratified analyses were not reported, thus differences in the magnitude of association 

11 between genotypes could not be compared. 

12 Instead of analyzing MTHFR genetic variants to represent folate metabolism, Solon et al. (2008)

13 measured red blood cell folate levels among children in the Phillippines. Not only did investigators find 

14 an association between increasing blood Pb level and lower cognitive test performance (Table 5-4), but 

15 they found effect modification by red blood cell folate levels. Among children with folate levels less than 

16 or equal to 230 μg/mL, blood Pb level had a negative marginal effect on MDI (-0.80 to -2.44 points), 

17 whereas among children with higher folate levels, blood Pb level did not have a negative marginal impact.

18 Thus, in contrast with those from Pilsner et al. (2010), findings from Solon et al. (2008) indicate that 

19 children with folate deficiencies may be at increased susceptibility to Pb effects on cognitive function.

Academic Performance in Children

20 Although the preponderance of evidence for Pb-associated neurodevelopmental deficits is for IQ 

21 and specific indices of cognitive function, academic achievement and school performance are corollaries 

22 to aptitude that may be more objective measure of one’s abilities and skills and have important 

23 implications for success later in life. Aptitude tests are used to predict future performance of an individual 

24 on a task or test. Achievement tests and school performance, in comparison, assess an individual’s actual 

25 knowledge in subject areas the individual has studied and measure the acquired knowledge of that subject. 

26 Studies reviewed in the 2006 Pb AQCD consistently demonstrated associations of Pb biomarkers with 

27 measures of academic achievement and performance including scores on math or vocabulary tests, class 

28 rank, teacher’s assessment of academic functioning, and high school completion. Several studies found 

29 that blood or dentin Pb levels measured at an early age (ages 2-8 years) were associated with outcomes at 

30 older ages (ages 8-18 years), suggesting early exposure to Pb may have persistent effects (Bellinger et al., 

31 1992; Leviton et al., 1993; Needleman et al., 1990). Results from the longitudinal study by Bellinger et al. 

32 (1992) was particularly noteworthy for examining associations of blood Pb at several ages with scores on 

33 the Kaufman Test of Educational Achievement at age 10 years. Only blood Pb level at age 2 years showed 
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1 a statistically significant association with lower predicted academic achievement. Additionally, the 

2 association was robust to adjustment for IQ, indicating that blood Pb levels may be associated with 

3 reduced performance on academic tasks not reflected in indices of IQ. Several studies also found 

4 associations between concurrent blood Pb levels and academic achievement (Al-Saleh et al., 2001; 

5 Kordas et al., 2006; Lanphear et al., 2000; C.-L. Wang et al., 2002). Among recent studies, academic 

6 performance was examined less frequently; however, findings are consistent with the extant body of 

7 evidence.

8 Miranda et al. (2007) linked blood Pb surveillance data collected between 0 and 5 years with end9 of-grade (EOG) testing data at 4th grade for 7 of the largest counties in North Carolina. Approximately 

10 22-30% of children in these counties were screened for Pb poisoning, and the total sample size in the 

11 analysis was approximately 8,600 children for both math and reading achievement tests. For both reading 

12 and math, achievement test scores were inversely associated with early childhood blood Pb screening data 

13 (Figures 5-4 and 5-5 and Table 5-4).

Source: Used with permission from Elsevier Science, Miranda et al. (2007).

Figure 5-5. Comparing model results for 4th-grade EOG mathematics 

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.

14 Similar results were obtained in a subsequent study expanded to the entire state of North Carolina 

15 (Miranda et al., 2009) (Figure 5-4 and Table 5-4). Investigators additionally used quantile regression to 
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1 estimate effects for conditional percentiles of EOG (e.g., what is the 10th percentile of EOG test scores 

2 conditioned on early childhood blood Pb levels) rather than conditional means. Compared with linear 

3 regression, quantile regression is more robust in response to outliers and predicts outcomes at the top and 

4 bottom tails of the distribution of the outcome rather than at the mean. The distributions in EOG scores 

5 for children with blood Pb levels greater than or equal to 10 μg/dL were more spread out than those for 

6 children with lower blood Pb levels. With increasing blood Pb levels, the lower tail of the EOG 

7 distribution was stretched out more so than the middle or upper tail of the distribution. For example, in 

8 comparisons of children with blood Pb levels of 5 μg/dL versus children with blood Pb levels of 1 μg/dL, 

9 children in the 5th percentile of EOG have a greater decrease in EOG score compared with children in the 

10 95th percentile of EOG (Figure 5-6). These findings indicate that children residing at the lowest 

11 performance regions of the EOG distribution may be differentially affected by blood Pb levels as 

12 compared with children in the middle or higher regions. Similarly, using quantile regression, Miranda et 

13 al. (2009) showed that, in addition to elevated blood Pb levels, cumulative social risk (lower parental 

14 education, being enrolled in a school lunch program) further enhanced the negative effects on academic 

15 achievement in these children.

Source: Used with permission from Elsevier Science, Miranda et al. (2009)

Figure 5-6. Reduction in EOG achievement test scores at each percentile 

of the test distribution.Note greater effect of Pb at low end of 

the distribution.
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1 Similar to Miranda et al. (2009), Chandramouli et al. (2009) observed associations between early 

2 childhood blood Pb levels (age 30 months) and later academic performance (Standard Assessment Tests at 

3 age 7 years) among participants of the Avon Longitudinal Study of Parents and Children conducted in the 

4 U.K (Figure 5-4 and Table 5-4). While these aforementioned recent studies found negative associations 

5 for early childhood blood Pb levels, unlike the longitudinal assessment by Bellinger et al. (1992), they did 

6 not have available blood Pb measurements at other lifestages to compare associations with blood Pb 

7 levels at other lifestages. Therefore, results from these recent studies do not preclude associations with 

8 blood Pb levels at other lifestages. Among children (ages 6-10 years) participating in NECAT, increasing 

9 concurrent blood Pb level was associated with poorer performance on the Wechsler Individual 

10 Achievement Test, even when adjusted for IQ (Surkan et al., 2007). In analyses adjusted for child IQ, 

11 caregiver IQ, age, SES, race, and birth weight, children with concurrent blood Pb levels 5-10 μg/dL 

12 scored 5.2 (95% CI: 0.95, 9.45) points and 4.0 (95% CI: 0.43, 7.6) points lower on reading and math 

13 composite scores on the respectively, compared to children with levels of 1-2 μg/dL. Blood Pb levels 

14 were similarly associated with other tests of cognitive function (e.g., FSIQ, working memory, cognitive 

15 flexibility and a number of executive functioning domains [i.e., ability to formulate, test, and adapt 

16 hypotheses]). Children with blood Pb levels 3-4 μg/dL had lower scores compared with children with 

17 blood Pb levels 1-2 μg/dL; however, differences were not statistically significant.

Age-based Susceptibility to Lead-associated Neurodevelopmental Deficits

18 Plasticity is a consequence of environmental exposures during critical life periods affecting key 

19 physiological systems that orchestrate underlying developmental processes (Feinberg, 2007). Exposure to 

20 environmental toxins during prenatal and/or early postnatal development may alter the normal course of 

21 morphogenesis and maturation that occurs in utero and early in life, resulting in changes that affect 

22 structure or function of the central nervous system via altered neuronal growth and/or 

23 synaptogenesis/pruning structure (Landrigan et al., 1999; Rice & Barone, 2000). This hypothesis is well24 supported by findings in animals that prenatal Pb exposure alters brain development via changes in 

25 synaptic architecture (Section 5.3.6.5) and neuronal outgrowth (Section 5.3.6.10) and leads to 

26 impairments in memory and learning (Section 5.3.2.2) and emotional and depressive changes postnatally 

27 (Section 5.3.3.4). Unlike other organ systems, the unidirectional nature of CNS development limits the 

28 capacity of the developing brain to compensate for cell loss, and environmentally-induced cell death can 

29 result in a permanent reduction in cell numbers (Bayer, 1989). Hence, when normal development is 

30 altered, the early effects may persist into adult life even in the absence of current exposure, magnifying 

31 the public health impact. Supporting evidence is provided by toxicological studies that find that Pb 

32 exposure during neonatal development but not in adulthood leads to neurodegenerative amyloid plaque 

33 formation in the brains of aged rodents and monkeys (Section 5.3.5.2).
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1 With repeated assessment of children prenatally to later childhood and early adulthood, the 

2 prospective cohort studies have aimed to distinguish among the effects of blood Pb levels at different 

3 periods of development. In the collective body of evidence, neurocognitive decrements have been 

4 associated with prenatal, early childhood, childhood average, and concurrent blood Pb levels. In these 

5 studies, the identification of developmental periods when children are most sensitive to Pb-associated 

6 neurocognitive decrements has been complicated by the high degree of correlation in children’s blood Pb 

7 levels over time and the confounding of age and peak blood Pb levels (Dietrich, Berger, & Succop, 1993; 

8 Lanphear et al., 2005; Needleman et al., 1990).

9 As described in detail in the 2006 Pb AQCD, several studies with varying lengths of follow-up 

10 demonstrated associations of prenatal blood Pb levels with neurodevelopmental deficits thoughout

11 childhood and into early adulthood (U.S. EPA, 2006). The prenatal period may be susceptible lifestage of 

12 Pb exposure not only because of the nervous system developmental process occurring as described above 

13 but also because of factors that result in elevated Pb exposures. Substantial fetal Pb exposure may occur 

14 from mobilization of maternal skeletal Pb stores even related to remote exposures (Gulson et al., 2003; H. 

15 Hu & Hernandez-Avila, 2002). Pb can cross the placenta to affect the developing fetal nervous system 

16 (Rabinowitz, 1988). Maternal and umbilical cord blood Pb levels generally are highly correlated, 

17 indicating that a newborn infant’s blood Pb levels reflects that of the mother (Schell et al., 2003).

18 Associations with prenatal blood Pb levels were demonstrated most consistently for cognitive 

19 function and behavior assessed between infancy and age 3 years (Figures 5-7 and 5-8 and Table 5-5). 

20 Among studies examining associations of early-life blood Pb measures (maternal, cord, or neonatal 

21 blood), results were mixed as to whether prenatal (Bellinger et al., 1984; H. Hu et al., 2006) or concurrent 

22 blood Pb levels (G. Wasserman et al., 1992; G. A. Wasserman et al., 1998) were associated with a greater 

23 decrement in cognitive function. Several studies found that prenatal or neonatal blood Pb levels were 

24 associated with neurodevelopmental decrements assessed in neonates (within 30 days) or early in infancy 

25 (within 3 months), which indicated that relatively short-durations of Pb exposure may be associated with 

26 negative neurological effects (Dietrich et al., 1987; Ernhart et al., 1986; Rothenberg et al., 1989; Shen et 

27 al., 1998). 

28 A recent analysis of 444 children participating in the Krakow Prospective Cohort Study 

29 corroborated previous findings for prenatal exposure and found associations at lower umbilical cord blood 

30 Pb levels than those in previous studies. Children in Jedrychowski et al. (2009b) had a median (range) of 

31 cord blood Pb levels of 1.23 (0.44-6.9 μg/dL), and increasing umbilical cord blood Pb levels was 

32 associated with lower 36-month Bayley MDI (Figure 5-4 and Table 5-4). Investigators also observed a 

33 larger magnitude of effect in males compared with females (Figure 5-9). These findings are consistent 

34 with the hypothesis that the developing male central nervous system may be more vulnerable than 

35 females’ to environmental insults resulting in later behavioral problems (Moffitt et al., 2001).
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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 (1st 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, 8.5b Concurrent

Bellinger et al. (1987) ≥15 vs. <3c MDI, 24 mo Prenatal

Assessment at School Age

Wasserman et al. (1994) 14.4 (10.5) GCI, 4 yr Prenatal

39.9, 9.6b 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 (2nd quartile) FSIQ, 7 yr Prenatal

16.6 (2nd quartile) Early childhood, 2 yr

15.7 (2nd quartile) Lifetime avg

Bellinger et al. (1992) >10 vs. <3c 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 = Early 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 = Full-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.
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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 yrs

Leviton et al. (1993) 4.8-6.3 (2nd quartile) Hyperactivity, Prenatal

2.0-2.9 (2nd quartile)a Girls, 8 yrs Early childhood, 6 yr

Bellinger et al. (1994) 1.98 (0.38) Behavioral Prenatal

3.4 (2.4)b Problems, 8 yrs 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 Prenatal

NR Behavior, Early childhood, 6.5 yr

NR 15-17 yrs 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 blood 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 lifestages with 

behavioral indices in children. 
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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 1st 

trimester mean 

(range):

7.1 (1.5-43.6)

Early childhood (12 

mo) mean (SD): 5.2 

(3.4)

Concurrent mean 

(SD): 4.8 (3.7)

Log linear regression 

model adjusted for 

concurrent blood Pb, sex, 

maternal age, current 

weight, height-for-age Z 

score, maternal IQ

Bayley MDI 

assessed at age 

24 mo

Prenatal 1st trimester: -4.1

(-8.1, -0.17)

Prenatal (avg): -3.5 (-7.7, 

0.63)

12 month: -2.4 (-6.2, 1.49)

Concurrent: -1.0 (-3.9, 1.9)

Gomaa et al. 

(2002)

197 children followed 

prenatally to age 24 mo

Mexico City, Mexico

Prenatal (cord blood) 

mean (SD): 6.7 (3.4)

Log linear regression 

model adjusted for 

maternal IQ, maternal 

age, sex, parental 

education, marital status, 

breastfeeding duration, 

child hospitalization status

Bayley MDI 

assessed at age 

24 mo

Prenatal: -2.1 (-3.9, -0.39)

Jedrychowski et 

al. (2009b)

444 children born 2001-

2004 followed prenatally to 

age 36 mo

Krakow, Poland

Prenatal (cord blood) 

geometric mean 

(range): 1.29 (0.44-5)

Linear regression model 

adjusted for maternal 

education, birth order, 

prenatal ETS, sex

Bayley MDI 

assessed at age 

36 mo

Prenatal: -2.9 (-5.0, -0.75)

Wasserman et al. 

(1992)

392 children followed 

prenatally to age 24 mo

Kosovo, Yugoslavia (K. 

Mitrovica, Pristina)

Prenatal (cord blood) 

mean (SD): 14.4 

(10.4)

Concurrent means:

K. 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 

assessed at age 

6, 12, 18, 24 mo

Prenatal: -4.8 (-7.3, -2.3), 

blood Pb levels ≥ 15 μg/dL 

vs. blood Pb levels <3b

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)

Concurrent: -4.1 (-6.2, -2.0)
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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) 

μg/g

Concurrent mean 

(SD): 6.4 (4.1)

Log linear regression 

model adjusted for family 

social class, maternal IQ, 

marital status, preschool 

attendance, HOME score, 

out of home care, number 

of residence changes, 

recent medication use, 

number of adults in 

household, sex, race, 

birth weight, birth order

McCarthy GCI 

assessed at age 

57 mo

Early childhood blood: -3.0 

(-5.7, -0.2)

Early childhood tooth: -2.5 

(-10.2, 5.2)

Concurrent blood: -2.3 (-

6.0, 1.4)

Dietrich et al. 

(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 ≤ 0.01c

Neonatal: -0.38, p ≤ 0.01c

Concurrent: -0.19, p ≤ 0.01c

Lifetime avg: -0.16, p ≤ 

0.01c

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 

adjusted 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 

age 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 μg/dL vs. <3 

μg/dLb

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, Rochester, and 

Yugoslavia cohorts

Median (5th-95th)

Early childhood: 12.7 

(4.0-34.5)

Peak: 18.0 (6.2-47.0)

Lifetime avg: 12.4 

(4.1-34.8)

Concurrent: 9.7 

(3.5-33.2)

Log linear regression 

model adjusted for HOME 

score, birth weight, 

maternal IQ, maternal 

education

FSIQ measured 

at ages 4.8-10 yr

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)
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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 crosssectional studies (3499 

children)

Early childhood (2 yr) 

range in means: 

6.8-21.2

Meta-analysis of 

combining effect 

estimates from individual 

studies

FSIQ assessed 

using various 

tests at ages 5-10 

yr

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 model 

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)

Prenatal mean (SD): 

16.1 (2.6)

Concurrent mean 

(SD): 25.8 (19.1)

Hierarchical log linear 

regression analyses 

adjusted for town, sex, 

ethnicity, maternal 

education, HOME score

Anxiety/depression 

assessed using 

Child Behavior 

Checklist at 3 yr

Prenatal: 1.16 (0.02, 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 2nd

quartile: 4.8-6.3

Early childhood 

(tooth) second 

quartile: 2.0-2.9 μg/g

Log linear regression 

model adjusted for singleparent family, gestational 

age <37 wk, mother not a 

college graduate, selfidentification as black, 

only child, daycare during 

first 3 yr

Hyperactivity 

assessed using 

Boston Teacher 

Questionnaire at 

age 8 yr

Prenatal, girls: 0.26 (-0.69, 

1.13)

Early childhood, girls: 0.10 

(-0.92, 1.1)

Bellinger et al. 

(1994)

1,782 children followed 

from birth (1979-1980) 

to age 8 yr

Boston area, MA

Prenatal (cord blood) 

mean (SD): 6.8 (3.1)

Early childhood 

(tooth) mean (SD): 3.4 

(2.4) ppm

Log linear regression 

analyses adjusted for 

prepregnant weight, race, 

delivery by cesarean 

section, marital status, 

paternal and maternal 

education, sex, birth 

weight, maternal smoking, 

prenatal care beginning 

after the first trimester, 

recipient of public 

assistance, number of 

children in family, child 

currently on medication

Problem behaviors 

(t-scores) 

assessed using 

Teacher Report 

Form of the Child 

Behavior Profile at 

age 8 yr

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 followed 

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 Environment, GCI = General Cognitive Index, 

FWS = Filtered Word Test, KABC = Kaufman Assessment 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 category serving as the reference group. c

Sufficent data were not provided in order to calculate 95% CI.
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Source: Used with permission from Elsevier Science, Jedrychowski et al. (2009a).

Figure 5-9. Regression of fitted MDI score at 36 months on logtransformed concentration of cord blood Pb level by sex.

1 In studies that examined cognitive and behavioral indices measured in schoolaged children (ages 4-

2 17 years), statistically significant, negative associations with prenatal or neonatal (10 days after birth) 

3 blood Pb measures were observed less frequently (Figures 5-7 and 5-8 and Table 5-5). Multiple studies 

4 conducted in the Cincinnati cohort found that blood Pb levels measured 10 days after birth but not 

5 maternal blood Pb during pregnancy were associated with impairments in cognitive function, auditory 

6 processing, and motor function as well as increased behavioral problems in children between ages 4 and 6 

7 years (Dietrich, Berger, & Succop, 1993; Dietrich, Berger, Succop, et al., 1993; Dietrich et al., 1991; 

8 Dietrich et al., 1992). In these Cincinnati studies, concurrent blood Pb levels generally were estimated to 

9 have similar magnitudes of effect. In most of the studies examining associations of prenatal or neonatal 

10 blood Pb levels with neurodevelopmental outcomes in later childhood, early or cumulative childhood 

11 blood Pb levels were associated with the greater decrements in function. A larger effect estimate for peak 

12 blood Pb levels was corroborated in meta-analysis of results from five cohort studies (Pocock et al., 1994)

13 (Figure 5-7 and Table 5-5). These findings may indicate the lack of persistence of early Pb exposures. 

14 Further, associations of cord blood Pb levels with neurodevelopmental measures in infancy may reflect 

15 associations with blood Pb levels in infancy, which are expected to be similar to those during the prenatal 

16 period. Cord blood Pb level may be a good surrogate of early postnatal blood Pb levels.

17 Early childhood blood Pb levels also were associated with diverse neurodevelopmental effects

18 assessed later in childhood and into early adulthood in both recent (reviewed earlier in Section 5.3.2.1, 

19 Figure 5-4, and Table 5-4) and previous studies that did not compare various lifestages of Pb exposure 
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1 (Cecil et al., 2005; Tong et al., 2000; Yuan et al., 2006). This lag effect may be the result of a 

2 toxicological process in which some period of time is required for past Pb exposure to affect CNS 

3 function. Alternatively, Pb exposure may affect higher-order neurodevelopmental processes that are more 

4 reliably assessed at later ages when children’s processes modalities are more highly differentiated. Early 

5 testing may lead to false-negative results and fail to identify a child who is at risk for later 

6 neurodevelopmental dysfunction. In some studies that have reported associations between early7 childhood blood Pb levels and neurodevelopmental decrements later in life, children’s blood Pb levels had

8 not markedly changed over time Thus, the early-childhood blood Pb levels may have been serving as 

9 surrogates of concurrent or cumulative blood Pb levels.

10 As depicted in Figures 5-7 and 5-8 and Table 5-5, several studies estimated larger decreases in 

11 neurodevelopmental endpoints for concurrent or lifetime average blood Pb levels than blood Pb levels at 

12 other lifestages. These findings were substantiated in the analysis pooling data from seven prospective 

13 studies, in which concurrent, peak, average lifetime, and early childhood blood Pb levels were all 

14 negatively associated with IQ, with the largest magnitude of decrease associated with concurrent blood Pb 

15 levels (Lanphear et al., 2005). Childhood average blood Pb levels (Dietrich, Berger, & Succop, 1993; 

16 Dietrich, Berger, Succop, et al., 1993; Lanphear et al., 2005) and tooth Pb levels (Bellinger et al., 1994)

17 have been associated with neurodevelopmental effects, indicating that biomarkers of cumulative 

18 childhood Pb exposure also may contribute to neurodevelopmental effects in children. Associations with 

19 concurrent blood Pb level were also demonstrated consistently in studies without comparisons to 

20 exposures at other lifestages (Figures 5-2 and 5-4 and Tables 5-3 and 5-4).

21 Some studies have aimed to improve assessment of age-based susceptibility by examining children 

22 with different degrees of changes in blood Pb levels over time (i.e., children whose blood Pb level ranking 

23 changed over time) (Bellinger et al., 1990; A. Chen et al., 2005; Hornung et al., 2009; Tong et al., 1998). 

24 Except for Tong et al. (1998), these studies have demonstrated stronger effects of concurrent blood Pb 

25 levels (Figure 5-10 and Table 5-6). Tong et al. (1998) found that early-life blood Pb level was associated 

26 with a larger deficit in IQ. As part of the Port Pirie, Australia cohort study, investigators separately 

27 examined intellectual attainment in groups of children with different degrees of decline in blood Pb levels

28 between ages 2 and 11-13 years. Although the mean blood Pb level in the study population declined 

29 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 

30 among children. In comparisons of tertiles of change in blood Pb level between age 2 and 11-13 years, 

31 investigators found that intellectual attainment scores at ages 2, 4, 7, and 11-13 years did not significantly 

32 differ between children with the largest declines (>16 μg/dL) in blood Pb level and children with a lower 

33 decline (<10 μg/dL). These findings indicated a stronger effect of higher blood Pb levels early in life even 

34 among groups with lower concurrent levels.
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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 < 3a

Concurrent, Prenatal 3-10a

Concurrent, Prenatal ≥ 10a

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 = Fullscale 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.
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Table 5-6. Additional characteristics and quantitative 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 μg/dL decline: 0.03 

(-0.15, 0.21)b

10.2-16.2 μg/dL decline: 0.04 

(-0.15, 0.23)b

>16.2 μg/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 levelc

Prenatal <3 μg/dL: -0.16 

(-0.43, 0.11)

Prenatal 3-10 μg/dL: -0.14 

(-0.57, 0.29)

Prenatal ≥ 10 μg/dL: -0.46 

(-0.81, -0.11)

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 μg/dL, Low 

age 7 (<7.2 μg/dL): 0d

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 

(5th-95th):

Peak: 13.6 (4.6-

34.4)

Early childhood: 

8.9 (3.0-23.8)

Lifetime mean: 

8.5 (3.0-22.1)

Concurrent: 6.0 

(1.9-17.9)

Linear regression model adjusted 

for city, HOME score, birth weight, 

maternal IQ, maternal education

FSIQ assessed using 

WISC-R at age 6 yr

0.5 ratio of blood Pb level at 

age 6 to age 2: 0 (reference)

2.0 ratio of blood Pb level at 

age 6 to age 2 yr: -0.70 (-1.0, -0.40)

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.

cEffects are estimated for concurrent blood Pb level (continuous variable) in children in different categories of prenatal blood Pb level: <3 μg/dL, 3-10 μg/dL, and ≥ 10 μg/dL. 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), and high levels at age 2 (>11.4 

μg/dL) and age 7 (>7.2 μg/dL). Cutoffs were based on the median blood Pb levels.

1 In several different U.S. cohorts of children, larger decrements on neurocognitive function were 

2 estimated for concurrent blood Pb levels (Bellinger et al., 1990; A. Chen et al., 2005; Hornung et al., 

3 2009) (Figure 5-10 and Table 5-6). In the Boston cohort, Bellinger et al. (1990) found that at age 57 

4 months, cognitive performance, as assessed by McCarthy GCI, was similar between children with higher 

5 (≥ 10 μg/dL) and lower (<3 μg/dL) prenatal blood Pb levels. Additionally, increasing concurrent blood Pb 

6 levels (age 57 months) were associated with the largest decline in GCI scores over time (score at age 57 

7 months – score at age 24 months) among children with high prenatal blood Pb levels (≥ 10 μg/dL), which 

8 indicated an effect among children with both high early and concurrent blood Pb levels (Figure 5-10 and 

9 Table 5-6). These findings indicated that by age 5 years, children with higher prenatal blood Pb levels 

10 appear to recover the Pb-associated decrements in cognitive function unless concurrent blood Pb levels 
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1 remain high. The investigators also demonstrated that positive home and caregiving environment (e.g., 

2 HOME score >52, higher SES, higher maternal IQ) may also protect against decrements in cognitive 

3 function associated with higher postnatal Pb exposures.

4 As part of the multicenter Treatment of Lead-Exposed Children (TLC) trial, Chen et al. (2005)

5 evaluated how change in blood Pb over time was related to IQ at later ages. The TLC was a clinical trial 

6 designed to examine the effect of chelation using succimer to prevent cognitive impairment in 780 urban 

7 children enrolled at 12 to 33 months of age with elevated blood Pb concentrations (20-44 μg/dL). Blood 

8 Pb and IQ were assessed at ages 24 and 36 months and 3, 5, and 7 years. Concurrent blood Pb level above

9 the median level (>7.2 μg/dL) was associated with a larger decrease in IQ, regardless of whether prenatal 

10 blood Pb levels were low or high (less than or greater than the median of 11.4 μg/dL, respectively). 

11 Pooling the Cincinnati and Rochester cohorts (n = 397), Hornung et al. (2009) also created a new 

12 indicator of Pb exposure: the ratio of blood Pb level at 6 years of age to that at 2 years of age. The greatest 

13 decrease in cognitive and behavioral development was observed for children with blood Pb ratios greater 

14 than 1 (indicating an increase in blood Pb level from 2 to 6 years of age) (Figures 5-10 and 5-11 and Table 

15 5-6). Presumably areas under the curve would be similar among children with blood Pb level ratios of 1, 

16 greater than 1, and less than 1, indicating that cumulative blood Pb levels would not be predictive. It is 

17 important to note that in these aforementioned studies, blood Pb levels were higher than those currently 

18 measured in among children in the U.S. Additionally, children in these study populations experienced 

19 larger decreases in blood Pb levels over time. It is unclear whether these findings would apply to children 

20 Blood Pb levels in the U.S. who currently are within the same age range and who would be expected to 

21 have smaller decreases in blood Pb levels over time.
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Source: Hornung et al. (Hornung 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 years 

of age: peak at 2 years (blue diamonds), peak at 5 years (black 

triangles), and constant blood Pb level level (white squares). 

1 In the collective body of epidemiologic evidence of children, it is difficult to ascertain which 

2 lifestage of Pb exposure is associated with the greatest susceptibility to Pb-associated neurodevelopmental 

3 effects. Associations have been observed with prenatal, early-childhood, lifetime average, and concurrent 

4 blood Pb levels as well as childhood tooth Pb levels. The assessment of age-based susceptibility is 

5 complicated further by the fact that blood Pb levels in children, although highly affected by recent dose, 

6 are also influenced by Pb stored in bone due to rapid growth-related bone turnover in children relative to 

7 adults. Thus, concurrent blood Pb level in children also may reflect cumulative dose (Section 4.3.5). 

8 Nonetheless, while the evidence indicates that prenatal and early-childhood blood Pb levels are associated 

9 with neurodevelopmental deficits, subsequent exposures that are reflected in concurrent, cumulative 

10 blood Pb levels or tooth Pb levels also are demonstrated to contribute to neurodevelopmental deficits 

11 throughout schoolage and into adolescence. Additional results from Hornung et al. (2009) and recent 

12 studies described earlier in the section support the conclusion from the 2006 AQCD that concurrent blood 

13 Pb level appears to be the best predictor of neurodevelopmental effects in children. These findings are 

14 consistent with the understanding that the nervous system continues to develop throughout childhood. 

15 Thus, the course of cognitive development may be modified in children, depending on concurrent blood 

16 Pb levels or positive caregiving environment. 
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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.)
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Table 5-7. Summary of findings from neurotoxicological exposure-response 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, males.

3 Stangle et al. (2007) 13 & 31 Cognition; 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: Corticosterone-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-Maternal 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 Alzheimer 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 hippocampal 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 Neurotransmitter; 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. (2008)

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

1 The 2006 AQCD reported deficits in the Morris water maze with Pb exposure. The Morris water 

2 maze tests memory and learning by having a mouse swim and locate or remember the location of a 
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1 platform submerged in opaque water. New research since the 2006 AQCD continues to show Pb-induced 

2 impaired Morris water maze performance. Data on neurocognition and learning as well as other 

3 neurotoxicological endpoints with dose responsive data are shown in Figure 5-12 and accompanying 

4 Table 5-7. Dams received Pb acetate dissolved in drinking water (0.1%, 0.5%, and 1% with corresponding 

5 blood Pb level of 4, 8 and 10 μg/dL at postnatal day [PND] 21) throughout gestation and lactation. 

6 Beginning at weaning, Pb exposed pups were subjected to Morris water maze performance testing. Pb 

7 exposed pups had significant increases in escape latency and number of crossings of the platform area at 

8 0.5% and 1% Pb acetate exposure (blood Pb levels of 8 and 10 μg/dL, respectively, indicating impaired 

9 memory and learning (Li et al., 2009). The pups in Li et al. (2009) were not separated by sex. Another 

10 study found that dietary supplementation with various supplements listed below or with 

11 methioninecholine concomitant with Pb exposure in weanling males shortened the escape latency of Pb12 exposed pups to more closely resemble the escape latency of control pups (G. Fan et al., 2009; Fan et al., 

13 2010). Zinc or methionine were effective dietary supplements in the G. Fan et al. 2009 study (2009); 

14 glycine, taurine, vitamin C, vitamin B1, tyrosine had no effect on the Morris water maze results. These 

15 data on the effect of Pb on learning and memory in the Morris water maze confirm findings by two other 

16 labs. Jett et al. (1997) showed increased escape latency in adult rats exposed to Pb via direct injections 

17 into the dorsal hippocampus. Kuhlmann et al. (1997) used maternal Pb diet exposure (gestation and 

18 lactation), continuous Pb exposure (gestation through adulthood) or post-weaning Pb exposure and only 

19 found only significant impairments in the maternal and continuous exposure groups. This new study 

20 confirms the findings of earlier studies that learning and memory are significantly impaired in rodents 

21 who are exposed to Pb early in life.

Working Memory

22 Working memory is the ability to temporarily keep information in mind while using the 

23 information to perform a related or unrelated task. The Morris water maze is able to measure working 

24 memory in addition to learning. Using this test, the 2006 AQCD found working memory was significantly 

25 affected in chronic developmentally exposed (Pb Acetate in feed 10 days prior to mating through PND 

26 21) female offspring at PND 21 (Jett, Kuhlmann, Farmer, et al., 1997). Delayed spatial alternation (DSA) 

27 is another test used to measure working memory. With DSA, an animal receives rewards based on 

28 responses at two separate levers. Work from the 2006 AQCD showed that Pb-exposed animals had 

29 deficits under DSA testing. These deficits included increased response errors, decreased percent of correct 

30 responses, and perseverance at one lever (repeatedly pressing the same lever without moving between the 

31 two locations). These results have been consistently shown with non-human primate studies (continuous

32 Pb exposure or juvenile to adult exposure) and less consistently shown with rats (juvenile only or juvenile 

33 to adult exposure). Working memory is a subcategory of executive function or goal-oriented problem 
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1 solving. Deficits in working memory are thought to underlie associations between blood Pb levels and 

2 ADHD in humans. 

Response Inhibition

3 Response inhibition is another measure of executive function and is measured with multiple tests 

4 that measure premature responses, decreased pause time between two scheduled events and increased 

5 perseverance. These tests include Differential Reinforcement of Low Rates of Responding (DRL), DSA, 

6 Fixed Interval testing (FI), FI with Extinction (FI-Ext), Fixed Ratio-FI (FR-FI), and Signal Detection with 

7 Distraction. Multiple studies from the 2006 AQCD and earlier literature have shown that early life Pb 

8 exposure contribute to response inhibition across the spectrum of these aforementioned tests. Monkeys 

9 with moderate blood Pb levels (11-13 μg/dL) learned the DRL task more slowly but eventually acquired 

10 reinforcement rates equal to controls. Newer data from female rats exposed to Pb (Stangle et al., 2007)

11 continued to show animals with premature responses after Pb exposure or response inhibition decrements. 

Learning Ability, Schedule-Controlled Behavior

12 The 2006 AQCD discussed learning or cognition as measured with schedule controlled-behaviors 

13 including fixed interval (FI) and fixed ratio (FR) operant conditioning and found that FI response rate was 

14 affected differentially with low level and high level Pb exposures increasing and decreasing FI response 

15 rate in females, respectively. This curvilinear response has since been further explored in more recent 

16 work, much of which also includes the effect of psychological stress on Pb exposure. 

Learning Ability with Stress

17 The combined paradigm of Pb exposure and stress experienced by a person or a laboratory animal 

18 is now being studied by multiple investigators who are focusing on the common pathway of HPA axis 

19 alteration and altered brain neurotransmitter levels. Data on stress and Pb with dose responsive data 

20 endpoints are shown in Figure 5-12 and accompanying Table 5-7. Cory-Slechta and colleagues have 

21 conducted multiple investigations in this area. Most recently, they have shown enhanced learning deficits 

22 in female rats (offspring) following lifetime Pb exposure combined with maternal restraint or prenatal 

23 stress (Cory-Slechta et al., 2010). This exposure paradigm used dams who were exposed to Pb for 2 

24 weeks prior to mating through lactation and pups from a mixed sex litter received drinking water Pb (50 

25 ppm) exposure through the remainder of their lifetime resulting in blood Pb levels of dams and pups 

26 ranging from 5-13 μg/dL.

27 Pb plus stress-related outcomes were followed in female offspring of dams who were exposed to 

28 Pb from 2 months prior to mating through lactation, i.e., developmental Pb exposure (2 exposure groups: 
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1 50 or 150 ppb Pb acetate drinking water solutions) (Virgolini, Rossi-George, Lisek, et al., 2008). Dams 

2 underwent restraint stress at gestational 16-17. Marked increases in response rates on FI performance in 

3 the Pb-stress female offspring versus control was found in animals whose mean blood Pb level was 11 

4 μg/dL (50 ppb Pb acetate). Because these animals did not show effects with maternal stress or Pb 

5 exposure alone, this effect was potentiated in the animals exposed to Pb plus additional stress. 

6 Similarly, lifetime Pb exposure (50 or 150 ppm, blood Pb level 11-16 and 25-33 μg/dL, 

7 respectively) plus stress (maternal or offspring) also induced FI aberrations at the post-reinforcement 

8 pause (PRP) period in female offspring, another potentiated effect (Rossi-George et al., 2011) (Table 5-8). 

9 Within the FI schedule, the PRP represents timing capacity or proper temporal discrimination. Namely, 

10 the PRP is the period during which the animal must wait or pause before depressing the lever for a 

11 reward. In this case, Pb plus stress exposed animals start responding too early due to a decreased pause or 

12 PRP interval. Abberant FI performance in infants and children has been used as a marker for impulsivity. 

13 Separately, overall FI response rate was significantly increased in Pb exposure alone and with maternal or 

14 offspring stress at the 50 ppm exposure dose. At 150 ppm, stress (maternal or offspring) increased FI 

15 response rate but Pb alone had no effect on FI. Biochemical analysis of possible mechanistic contributions 

16 to these aberrations revealed alterations in frontal cortex norepinephrine, reductions in dopamine 

17 homeostasis in the nucleus accumbens and enhancement of the striatial monoamine system. This study on 

18 the effect of lifetime Pb exposure with or without stress on FI testing itself or during the PRP component 

19 of FI testing further confirm learning deficits and provide possible mechanistic explanations.

20 Pb exposure over various exposure windows has been shown to affect corticosterone levels in 

21 rodents. Maternal Pb exposure (150 ppm drinking water from 2 months prior to mating through lactation 

22 with restraint stress as detailed above) induced increased basal corticosterone in female and male 

23 offspring at 9 months of age; no interactions of Pb and stress were seen in this model (Cory-Slechta et al., 

24 2004). By 14 months of age, these offspring had reduced corticosterone concentrations versus control 

25 animals, indicating a possible acceleration of age-related decreases in basal corticosterone levels (Cory26 Slechta et al., 2008) that were enhanced with maternal stress. Postnatal exposure of male rodents to Pb 

27 (PND 21-5 months of age) showed significant decrements in baseline corticosterone; this effect produced 

28 a U-shaped concentration-response curve with significant decrements in basal corticosterone levels in the 

29 50 ppm exposure group versus control (Virgolini et al., 2005). In summary, developmental (gestational 

30 and lactational) and post-weaning exposure to Pb induced permanent changes in the HPA axis 

31 (corticosterone levels) in both sexes which are dynamic as the animal ages. 
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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 

different from 0NS control; # denotes significantly different

from corresponding Pb-NS value; + differs from 50-NS.

1 Mechanistic understanding of the cognitive deficits seen with Pb and/or stress exposure was 

2 explored in a recent study. HPA hypofunction following dam Pb exposure (pup gestational and lactational 

3 Pb exposure) with or without maternal stress was reported (Rossi-George et al., 2009). This study used 

4 the same model of developmental Pb exposure as is detailed in the preceding paragraph. Outcomes were 

5 followed in both male and female offspring. At 5-6 months of age, basal corticosterone in females was 

6 significantly increased at 150 ppm and unaffected at 50 ppm (Figure 5-13). In males, basal corticosterone 

7 of the 50 ppm group was significantly decreased and 150ppm was unaffected. These authors also 

8 explored the function of the glucocorticoid negative feedback loop and found that Pb and/or maternal 

9 stress significantly impacted this negative feedback. This negative feedback loop was more greatly 

10 impacted at the lower dose (50 ppm v 150 ppm Pb acetate, blood Pb level at PND 21 of 19 and 32 μg/dL, 

11 respectively). To test the effect of an outside stress on the HPA axis, mice in this study were subjected to 

12 vehicle injections. The corticosterone response to this vehicle injection stress was prolonged in a non13 linear dose response manner in both sexes with the most profound effects seen at the lower 50 ppm dose. 

14 Maternal stress also prolonged the corticosterone stress response to vehicle injection and enhanced the Pb 

15 effect in males. To test the negative feedback of the HPA axis, exogenous dexamethasone (DEX) was 

16 administered to suppress endogenous corticosterone. The DEX test revealed HPA axis hypofunction. 
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1 Specifically Pb and Pb plus maternal stress initially reduced the ability of DEX to suppress 

2 corticosterone. With time, the effect of this DEX test in males induced prolonged corticosterone 

3 suppression or failure to return to baseline as seen in control animals. In summary, dam Pb exposure 

4 induced negative feedback hypofunction in both sexes with an inverse U dose response function. A 

5 similar recent study explored the effect of lifetime Pb exposure on the HPA axis, looking at basal 

6 corticosterone levels in male and female offspring at two time periods (2 months old and 10 months old; 

7 before and after behavioral testing, respectively) in adulthood (Rossi-George et al., 2011). Pb and stress 

8 have no effect on basal corticosterone levels in males at either time period (Figure 5-13). At the first time 

9 period, Pb exposure elevated basal corticosterone levels in a dose-dependent fashion in females, and Pb 

10 plus stress attenuated the PB-dependent elevations in corticosterone to baseline levels (Figure 5-13). At 

11 the second time period, Pb and stress accelerated the age-dependent decrease in basal corticosterone 

12 levels in females (Figure 5-13).

13 These two studies of lifetime exposure (Rossi-George et al., 2009) reported different basal stress 

14 hormone levels with Pb exposure. Males with lifetime Pb exposure had no significant corticosterone 

15 response to Pb exposure; whereas males with dam Pb exposure had significant decreases in corticosterone 

16 at 5 months of age in the 50 ppm exposure group only (not seen in 150 ppm Pb exposure group). On the 

17 other hand, females had dose-dependent corticosterone responses to Pb exposure in both exposure models 

18 (lifetime Pb exposure and dam Pb exposure.
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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 stressors 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). 
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1 Maternal stress alone also led to HPA axis negative feedback hypofunction. Pb plus maternal stress 

2 enhanced negative feedback in males and attenuated this effect in females. Pb exposure with or without 

3 maternal stress prolonged the effect of DEX-dependent corticosterone supression in males. These data 

4 together show that HPA axis alterations could be an underlying mechanism linking commonalities 

5 between the contribution of Pb and stress to adverse health outcomes. 

6 Schedule-control behavior is often measured using FI or FR testing. Because the FI animals are 

7 regularly handled by laboratory personnel and participate in tests of cognition, their baseline level of 

8 stress may be skewed from that of a laboratory animal that constantly remains in a cage without daily 

9 handling. Because effects on the HPA axis are of interest to Pb researchers, the baseline corticosterone 

10 levels of animals who have participated in behavior testing (FI) and those who have not (NFI) have been 

11 compared. Specifically, the corticosterone differences between FI and NFI animals after developmental 

12 Pb exposure (dam-only Pb exposure) have been measured. Virgolini et al. (2008) found that basal 

13 corticosterone levels were significantly different between FI and NFI animals. Also, the combination of 

14 dam Pb exposure with maternal stress was explored in FI and NFI animals. At the baseline age of 4-5 

15 months, NFI animals who were not behaviorally trained displayed significant differences from FI 

16 animals. Pb exposure with or without stress did not induce differences in corticosterone levels in FI 

17 females. The corticosterone level of male FIs was affected by Pb and stress exposure (Figure 5-13). In the 

18 FI males, the 50 ppb Pb exposure group (50Pb) had decreased corticosterone versus control (no Pb 

19 exposure) and the 150 ppb Pb exposure group (150Pb) had elevated corticosterone versus control. Male 

20 NFI animals demonstrated a U shaped dose response corticosterone curve with 50Pb significantly less 

21 than control or 150Pb. In the NFI males, stress did not affect corticosterone levels or interact with the 

22 effect of Pb. NFI females exposed to 150Pb had significantly elevated corticosterone versus control (no 

23 Pb exposure). When drawing conclusions about the effects of Pb exposure, these data demonstrate that 

24 behaviorally trained animals have an altered HPA axis and response to Pb exposure versus animals who 

25 are housed under conditions without daily handling by caregivers. 

26 Another study looked at female rats with lifetime Pb exposure combined with prenatal stress and 

27 found enhanced learning deficits(drinking water 50 ppm Pb acetate, offspring blood Pb 7-13 μg/dl) 

28 (Cory-Slechta et al., 2010). Learning was evaluated with multiple schedule of repeated learning (RL) and 

29 performance testing. Repeated learing was impaired but performance was not affected with Pb exposure. 

30 The impaired RL was further enhanced with prenatal stress. There were significant associations between 

31 Pb/stress and coticosterone concentration, dopamine from the frontal cortex, dopamine turnover in the 

32 nucleus accumbens, and total number of responses required to learn a sequence. Also Pb exposed 

33 offspring with and without maternal stress exposure had significant decreases in hippocampal nerve 

34 growth factor (NGF) versus control. Thus, this study demonstrates that lifetime Pb exposure with or 

35 without prenatal stress induced learning deficits in female mice. In a similar study, the authors proposed 

36 that associations of Pb and stress with learning deficits (F1 testing in females) may be related to 
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1 aberrations in corticosterone and dopamine (Rossi-George et al., 2011). Earlier work has shown that dam 

2 or prenatal stress (PS) affects the HPA axis of the offspring. A newer study was conducted to determine 

3 the influence of low level dam Pb exposure and prenatal stress on offspring stress challenge responsivity 

4 (intermittent stress as an adult) (Rossi-George et al., 2011). In a similar study, the authors proposed that 

5 associations of Pb and stress with learning deficits (F1 testing in females) may be related to aberrations in 

6 corticosterone and dopamine (Rossi-George et al., 2011). Dam Pb exposure (50 or 150 ppm Pb acetate) 

7 followed by intermittent stressors (cold, novelty or restraint) to offspring as adults induced significant 

8 changes in FI response rate. Females were more sensitive to the adult intermittent stressors at the higher 

9 dose of Pb (150 ppm) with significant increases in FI response rate and decreased PRP, i.e. increased 

10 impulsivity (Figure 5-14). Males were more sensitive (decreased FI response rate due to decreased run 

11 rate) to the restraint stress at the lower Pb dose (50 ppm). At the higher dose of Pb, males were more 

12 sensitive to the cold stress (increased FI response rate and increased run rate) (Figure 5-15). 

13 Corticosterone levels were followed in this study and showed dose dependent correlations with FI 

14 outcomes in females but were independent of dose in males.

Table 5-8. Summary of effects of maternal and lifetime Pb exposure on FI performance 

watera.

Pb (ppm) Maternal Pbb Lifetime Pbc

Overall rate PRP Overall rate PRP

0 ppm

0-PS NO SIGNIFICANT EFFECTd NO SIGNIFICANT EFFECT NO SIGNIFICANT EFFECT NO SIGNIFICANT EFFECT

0-OS NO SIGNIFICANT EFFECT *↓-23% NO SIGNIFICANT EFFECT NO SIGNIFICANT EFFECT

50 ppm

50-NS NO SIGNIFICANT EFFECT NO SIGNIFICANT EFFECT *↑95% NO SIGNIFICANT EFFECT

50-PS NO SIGNIFICANT EFFECT NO SIGNIFICANT EFFECT *↑79.2% *↓-42%

50-OS *↑64.9% NO SIGNIFICANT EFFECT *↑74.7% *↓-39.3%

150 ppm

150-NS *↑42.4% *↓-30.3% NO SIGNIFICANT EFFECT NO SIGNIFICANT EFFECT

150-PS NO SIGNIFICANT EFFECT *↓-25.7% *↑90.7% *↓-44.7%

150-OS *↑59.2% NO SIGNIFICANT EFFECT *↑78.5% NO SIGNIFICANT EFFECT
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Note: *Dam blood Pb levels ranged from 5-13 μg/dL over gestation and lactation; offspring blood Pb ranged from 7-13 μg/dL from early life time 

points out to ten months of age. Thus, this study demonstrates that lifetime Pb exposure with or without prenatal stress induced learning deficits in 

female mice. Mechanistically, these authors propose that associations of Pb and stress with learning deficits may be related to 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 Elsevier Science, Rossi-George et al. (2011) (Table 1).

Cognitive Flexibility

1 Cognitive flexibility is the ability to reallocate mental resources when situations 

2 change (Monsell, 2003). Discrimination reversal learning is used to measure cognitive 

3 flexibility, which is a subclass of executive function. The 2006 AQCD reported reversal 

4 learning deficits in monkeys with blood Pb of 11-20 μg/dL. Rats also showed similar 

5 deficits but the authors attributed the changes to learning related problems instead of 

6 cognitive flexibility (Garavan et al., 2000; Hilson & Strupp, 1997). Interestingly, recent 

7 work has shown that NMDA receptors and D2-like receptors, two known targets of Pb, are 

8 involved in discrimination reversal learning (Herold, 2010). Another test of cognitive 

9 flexibility is called concurrent random interval (RI-RI) scheduling in which depression on 

10 two response levers is reinforced at different frequencies. The 2006 AQCD reported 

11 monkeys with cognitive flexibility impairment under RI-RI (Newland et al., 1994). 

Selective Attention

12 Few animal toxicology studies measure selective attention. Those that do employ signal detection 

13 with distraction, a test looking for increased omissions after exposure to an external distraction. The 

14 newest publication in this area showed no effect with this test after juvenile through adolescence exposure 

15 in female rats (Stangle et al., 2007). The two dose groups yielded blood Pb levels of 13 μg/dl and 31ug/dl 

16 (Stangle et al., 2007). 

5.3.2.3. Toxicological Studies on the Effects of Chelation

17 Earlier work in the animal toxicology literature has shown that succimer or chelation treatment of 

18 Pb exposed lab animals was able to normalize various aberrant Pb-induced behaviors including activity 

19 level, habituation (Gong & Evans, 1997) and forced-swim immobility (P. W. Stewart et al., 1996). A more 

20 recent study looked at the effect of succimer treatment on various neurobehaviroal and neurocognitive 

21 outcomes in control and neonatally Pb-exposed female animals (PND 1-30 Pb acetate exposure, 300 ppm 

22 dam through lactation and either 30 or 300 ppm pup water) by drinking water, generating a moderate Pb 

23 (m-Pb) exposure and a high Pb (h-Pb) exposure group. Pb blood levels at PND52 in the control, m-Pb, h-
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1 Pb, m-Pb+succimer, and h-Pb+succimer are 1.5, 12.6, 31, 2.8, and 8.5 μg/dL, respectively; brain Pb levels 

2 at the same time for the same groups are 41, 1040, 3690, 196, and 1370 ng/g dry weight, respectively. 

3 Succimer treatment significantly attenuated the m-Pb induced impaired learning ability. Effects on arousal 

4 that were significantly affected in h-Pb rats were significantly attenuated with succimer treatment. 

5 Succimer treatment in the h-Pb animals only slightly improved learning ability and did not improve the 

6 impaired inhibitory control (Stangle et al., 2007). These are important findings because they provide 

7 evidence that certain adverse neurobehavioral or cognitive outcomes associated with Pb exposure appear 

8 to be reversible with chelation therapy.

9 A 3-week course of Pb acetate (PND 1-17, dam drinking water) plus or minus succimer/chelator 

10 (PND 31–52) treatment was performed to determine if succimer could alleviate behavioral deficits in rats 

11 exposed to Pb for the first 4 weeks of life. Pb-exposed animals had altered reactivity and increased reward 

12 omission and errors. Pb-exposed animals receiving chelation therapy had normalized reactivity to reward 

13 omission and errors (Beaudin et al., 2007). Adverse Pb-induced behavioral outcomes were attenuated with 

14 chelation therapy in Pb-exposed animals.

15 Another study (G. Fan et al., 2009) looked at methionine choline supplementation in Pb exposed 

16 animals to understand its effect on Pb disposition in various tissues (blood, bone, brain) and how this 

17 might contribute to neurocognitive or neurobehavioral changes. As a sulfur source, methionine is a 

18 chelator and a free-radical scavenger. Choline is important for cell membranes and neurotransmitter 

19 synthesis (Zeisel & Blusztajn, 1994). In this model, methionine choline attenuated Pb-dependent memory 

20 and learning defecits (Section 5.3.2.2). Exposure of weanling male rats to Pb acetate in drinking water 

21 (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 

22 0.63 μg/g. Methionine choline supplementation significantly attenuated blood Pb and bone Pb but 

23 produced a non-significant attenuation of brain Pb (0.51 μg/g) in mice that had significant improvements 

24 in learning and memory (Section 5.3.2.2). 

25 Also, in another study the metal chelators DP-109 and DP-460 are neuroprotective in the ALS 

26 mouse neurodegenerative model or Tg(SOD1-G93A) (Petri et al., 2007).

27 In summary, succimer or chelation therapy appears to be able to restore Pb-dependent impairments 

28 of learning and arousal as well as being neuroprotective in a dose dependent fashion. In these studies 

29 succimer use was most efficacious at lower doses of Pb exposure. Chelation does not restore Pb30 dependent impaired inhibitory control. Chelation with the supplement methionine choline affected the 

31 disposition of Pb in various tissues, significantly attenuating blood and bone Pb levels and non32 significantly attenuating brain Pb. 
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5.3.2.4. Epidemiologic Studies of Cognitive Function in Adults

Adults without Occupational Lead Exposures

1 The 2006 AQCD cited more consistent associations of bone Pb levels but not concurrent blood Pb 

2 levels with cognitive performance in environmentally-exposed adults (U.S. EPA, 2006). Studies published 

3 since the 2006 Pb AQCD continue to support the previous findings. Several studies analyzed data from 

4 NHANES III (1991-1994) and investigated effect modification by age and genetic variants. Krieg et al. 

5 (2009) overall did not find blood Pb level to be associated consistently with poorer performance on 

6 cognitive testing among 2,090 adults 20-59 years of age nor among 1,796 adults 60 years of age and 

7 older. There were also few statistically significant differences in the association with blood Pb between 

8 ALAD genotypes (E. F. Krieg, Jr. et al., 2009). Among the 20-59 year-old adults, a borderline significant 

9 difference was observed for mean reaction time, however, among subjects in the CC and CG ALAD 

10 genotype groups combined (ALAD2 carriers in the terminology above) (n=161), reaction time improved 

11 (i.e., faster reaction time) with increasing blood Pb level. In contrast, ALAD2 subjects had a greater 

12 increase in the number of errors on a symbol-digit substitution task (p = 0.07) in association with 

13 increasing blood Pb level. In the same study, contrasting observations were made in adolescent NHANES 

14 participants. Effect modification by ALAD2 is not entirely clear as it may increase susceptibility to Pb15 associated health effects by increasing blood Pb levels or diminish Pb bioavailability by maintaining it in 

16 a sequestered state in the bloodstream. Krieg et al. (2010) also found differences in the association 

17 between blood Pb level and scores on a symbol-digit substitution test by the VDR variants, rs731236 and 

18 VDR rs2239185, as well as VDR haplotype. Similar to observations in children (Section 5.3.2.1), results 

19 were not consistent across the various tests. However, blood Pb level generally was associated with 

20 greater decrements in cognitive performance among adults with the CC genotypes of VDR variants.

21 Shih et al. (2006) studied participants in the Baltimore Memory Study (BMS), a longitudinal study 

22 of men and women 50-70 years of age residing in Baltimore, MD with a mean (SD) blood Pb level of 

23 3.46 (2.23) μg/dL. A total 1,140 out of 2,351 (48.5%) subjects participated from neighborhoods that 

24 represented a diversity of race and SES. Of these, 991 had complete data for blood and tibia bone Pb, 

25 cognitive testing, and covariates. After excluding the three participants with the highest bone Pb values, 

26 the two participants with the two most negative bone Pb values, and one person with a blood Pb level 10 

27 standard deviations greater than the mean, the analytic sample was 985. Scores on individual cognitive 

28 tests from a battery of 20 in-person administered tests were grouped into different cognitive domains 

29 (language, processing speed, eye-hand coordination, executive function, verbal memory and learning, 

30 visual memory, and visuoconstruction) by transforming individual test scores into z-scores and averaging. 

31 Negative associations were observed more consistently for tibia Pb levels than for blood Pb levels (Figure 

32 5-16 and Table 5-9). Tibia bone Pb levels were associated with worse performance on tests in all domains 
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1 in models adjusted for age, sex, testing technician, and presence of the apolipoprotein (APO)E-ε4 allele. 

2 The magnitudes and statistical significance of associations were attenuated with additional adjustment for 

3 education, race, and wealth. In thesefully-adjusted models, all domains except language and processing 

4 speed were negatively associated with tibia bone Pb, but only the association with visuoconstruction was 

5 borderline statistically significant. Analysis of a quadratic term for tibia Pb indicated no evidence of 

6 nonlinearity, thus results for linear models were presented. In linear models, visuoconstruction scores 

7 decreased by 0.0044 SDs (95% CI: -0.0091, 0.0003) per 1 μg/g bone increase in tibia Pb level. The mean 

8 (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 

9 first such study with a large proportion of African-Americans (n=395). In a subsequent analysis, 

10 increasing tibia Pb levels were associated with a greater decrease in cognitive performance among 

11 subjects living in neighborhoods with a greater number psychosocial hazards (Glass et al., 2009) (Figure 

12 5-16 and Table 5-9).
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Reference Data (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. (2010) 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 NPHd

High NPHd

Eye-hand coordination Middle NPHd

High NPHd

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: Black diamonds = blood Pb, blue circles = tibia Pb, white circles = patella Pb. ALAD = aminolevulinate dehydratase, VDR = vitamin D receptor, NPH = neighborhood 

psychosocial hazard. aEffect estimates for Model B are presented. bBlood Pb levels represent levels in ALAD wildtype and ALAD2 carriers, respectively. cEffect estimates for the 

following strata: African-Americans with tibia Pb levels ≤ 15 μg/dL, African-Americans with tibia Pb levels >15 μg/dL, whites with tibia Pb levels ≤ 15 μg/dL, and whites with tibia 

Pb levels >15 μg/dL. dEffect estimates for the interaction between tibia Pb levels and NPH tertile, with the lowest NPH tertile serving as the reference group. eTibia Pb levels 

represent levels in ALAD wildtypes and ALAD2 carriers, respectively. fPatella Pb levels represent 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.
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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 within 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 adults, 

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 total latency, sec)

Serial digit learning score

Word recall

Story 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, education, 

family income, race-ethnicity, 

test language (≥60 yr)

Symbol Digit Substitution

(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 yr

VDR haplotype CC

VDR haplotype CT

VDR haplotype TC

VDR haplotype TT

Ages 20-59 yr

VDR haplotype CC

VDR haplotype CT

VDR haplotype TC

VDR haplotype TT

Ages ≥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, 

Baltimore, 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 mean (SD):

5.3 (2.9) 

μg/dL (ALAD 

wildtype)

4.8 (2.7) 

μg/dL 

(ALAD2 

carriers)

Linear regression adjusted 

for blood Pb main effect, 

ALAD genotype, age at 

cognitive test, education, 

alcohol consumption, 

cumulative 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, WIASR

-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)c
May 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 

supplements, 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 status, 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)

0.006 (-0.03, 0.04) -0.08 (-0.12, -0.04)

-0.03 (-0.06, 0.01)

-0.08 (-0.11, -0.04) -0.014 (-0.05, 0.02)

-0.022 (-0.17, -0.07)

-0.044 (-0.09, 0.003)

BandeenRoche et 

al. (2009)

1140 adults, 

ages 50-70 yr

Baltimore 

Memory 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 coordination

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)

0.02 (-0.05, 0.10) -0.02 (-0.06, 0.01)

0.04 (-0.002, 0.08)

-0.008 (-0.08, 0.06)

0.002 (-0.09, 0.09)

0.04 (-0.003, 0.09) -0.07 (-0.13, -0.005)

0.001 (-0.007, 0.09) -0.0009 (-0.011, 0.09)

0.003 (-0.003, 0.08)

-0.004 (-0.011, 0.03)

Glass et 

al. (2009)

1001 adults, 

mean age 59 

yr

Baltimore 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

0.01 (-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)d
May 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 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 

System, WIAS-R

Tibia

Patella

Tibia

Patella

Tibia

Patella

Tibia

Patella

-0.17 (-0.33, -0.007)

0.01 (-0.10, 0.12)

-0.07 (-0.23, 0.08)

-0.01 (-0.12, 0.10)

0.05 (-0.10, 0.21)

0.07 (-0.40, 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 letter 

fluency

Assessed using Telephone 

Interview for Cognitive 

Status and East Boston 

Memory Test

Tibia

Patella

Tibia

Patella

-0.04 (-0.09, 0.005)

-0.01 (-0.05, 0.03) -0.05 (-0.10, -0.003)

-0.03 (-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 

measurement and first 

cognitive test, yr between 

cognitive tests

Visuospatial, pattern 

comparison

(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 (1st- 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 expenditure 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)

-0.03 (-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 HFE variants

-0.20 (-1.0, 0.70)e

-1.40 (-3.3, 0.40)e -6.3 (-10.4, -2.1)e
May 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 trials

Lowest tertile

Medium tertile

Highest tertile

Lowest tertile

Medium tertile

Highest tertile

Assessed using 

CANTAB and Montreal 

Cognitive Assessment

Calcaneus

Tibia

Calcaneus

Tibia

87.56f

86.67

80.67, p = 0.027

85.42f

87.08

82.44, p = 0.25

2.54f

2.61

2.72, p = 0.21

2.62f

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 tertile of NPH serving 

as the reference group.

e

Effect estimate refers to the change in cognitive function score over time. f

Results refer to mean cognitive function scores among tertiles of bone Pb.

1 Weuve et al. (2009) studied the association of blood and bone Pb levels with cognitive function 

2 among a subset of 587 women from the Nurses Health Study from whom blood and bone Pb

3 measurements were taken between the ages of 47 and 74 years. The mean (SD) blood Pb level in this 

4 group was 2.9 (1.9) μg/dL. The women had had Pb measured as part of their participation in two separate 

5 sub-studies, one of osteoporosis and the other of hypertension. The inclusion criteria included residence in

6 the Boston area and absent of major chronic diseases at the time of Pb measurement. Cognitive function 

7 was assessed via a battery of telephone administered tests (Telephone Interview of Cognitive Status, digit 

8 span backwards, alphabetizing span, animal naming [category fluency], “f” naming [letter fluency], and a 

9 composite verbal memory score) an average of 5 years after Pb measurement. As in the aforementioned 

10 studies of adults, negative associations were observed more consistently for tibia and patella Pb levels 

11 than for blood Pb levels (Figure 5-16 and Table 5-9). Contrary to expectation, scores on the “f” naming 

12 test were positively associated with patella and tibia bone Pb levels. In separate models, the “f” naming 

13 test was omitted from a composite index of all cognitive tests, and a one SD (10 μg/g bone) increase in

14 tibia Pb level was associated with 0.051-point decrease (95% CI: -0.010, -0.003) in the standardized 

15 composite score. This effect size of tibia bone Pb level was approximately equivalent to the effect size for

16 3 years of age among these women. The magnitude of effect was smaller for patella Pb levels (-0.033 

17 [95% CI: -0.080, 0.014) per 1 SD unit increase in patella Pb level)

18 Several analyses of Normative Aging Study (NAS) have contributed to greater understanding of the 

19 relationship between biomarkers of Pb dose and neurocognitive effects in adults. The NAS is conducted 

20 at the VA Outpatient Clinic in Boston, MA and is a multidisciplinary longitudinal investigation of the 
May 2011 5-93 DRAFT – DO NOT CITE OR QUOTE

1 aging process that was established in 1961 and originally involved 2,280 men residing in the Greater 

2 Boston area aged 21 to 80 years with no current or past chronic medical conditions. Participants are 

3 evaluated every 3-5 years with in-person clinical assessments, self-administered questionnaires. In-person 

4 administered cognitive tests included those in the general domains of attention/executive function, short5 term memory, visuospatial, and verbal/language. About 60-70% of participants, depending on the specific 

6 cognitive test, took individual tests twice approximately 3.5 years apart. The average (SD) age of the men 

7 at baseline was 69 (± 7) years, and the median blood Pb level was 5 μg/dL. Beginning in 1991, 

8 measurement of bone Pb levels began, with 68% of active NAS subjects participating. All analyses were 

9 adjusted for age, age squared, education, smoking, and alcohol intake. In one study of Mini-Mental State 

10 Examination (MMSE) tests scores among 720 men 45 years of age and older, blood Pb levels were 

11 negatively associated with MMSE score among ALAD2 carriers (Weuve et al., 2006). A 3 μg/dL 

12 increment in blood Pb level (the interquartile range) was associated with a 0.26 point lower mean MMSE 

13 score (95% CI: -0.54, -0.01) among ALAD2 carriers and a 0.04 point lower score (95% CI: -0.16, -0.07) 

14 among noncarriers. A subsequent study in the same cohort considered performance on a battery of oher 

15 cognitive tests and did not find a consistent pattern of modification of the association between blood or 

16 bone Pb levels and cognitive function by ALAD genotype (Rajan et al., 2008). Nonetheless, Rajan et al. 

17 (2008) found tibia Pb levels to be associated more consistently with lower cognitive performance 

18 compared with blood or patella Pb levels (Figure 5-16 and Table 5-9).

19 The NAS also examined effect modification by hemochromatosis (HFE) gene variants. In models 

20 adjusted for age, years of education, nonsmoker, former smoker, pack-years, nondrinker, alcohol 

21 consumption, English as first language, computer experience, and diabetes, an interquartile range increase 

22 in tibia bone Pb level (15μg/g) was associated with a 0.22 point steeper annual decline (95% CI: -0.39, -

23 0.05) in MMSE score among men with any variant HFE allele (either H63D or C282Y). The magnitude 

24 of association was less negative among those with only HFE wildtype alleles (F. T. Wang et al., 2007)

25 (Figure 5-17). As indicated in Figure 5-17, a nonlinear association was observed between tibia Pb levels 

26 and change in MMSE score, with a steeper decline per unit increase in tibia Pb level at higher tibia Pb 

27 levels. Effect modification by nongenetic factors also were examined in the NAS cohort. Increasing bone 

28 Pb levels were associated with greater decreases in cognitive function among individuals with higher 

29 individual-level perceived stress (Peters et al., 2007).
May 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 among 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, English 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.

1 Two large NAS studies examined the relationship of blood and bone Pb levels with the change in 

2 cognitive function over time. Weisskopf et al. (2007) expanded on an earlier study of Pb biomarkers and 

3 cognitive function (Payton et al., 1998) The average (SD) age of the men at baseline was 69 (±7) years, 

4 and the median blood Pb concentration was 5 μg/dL. Two measurements of cognitive function, collected 

5 approximately 3.5 years apart, were available 60-70% of participants. All analyses were adjusted for age, 

6 age squared, education, smoking, and alcohol intake. There was little association between blood Pb levels 

7 and cognitive test scores, except possibly for vocabulary scores, although this association was greatly 

8 weakened when the five men with the highest blood Pb levels (>15 μg/dL) were excluded. For bone Pb

9 analyses, the authors used repeated measures analysis with an interaction term between bone Pb level and

10 time in order to estimate the association between bone Pb level and decline in cognitive test score over 

11 time. There were no main effect associations with bone Pb levels; however, increases in patella and tibia 

12 bone Pb levels were associated with decreases in cognitive performance over time. The only test for 

13 which this reached statistical significance (p<0.05), however, was increased response latency on a pattern 

14 comparison test. Contrary to expectation, bone Pb levels were associated with fewer errors on the same 

15 pattern comparison test. The authors proposed that this may be related to slowing reaction time to 

16 improve accuracy. When the 9 men with the highest bone Pb levels were removed, the association was no 

17 longer statistically significant.
May 2011 5-95 DRAFT – DO NOT CITE OR QUOTE

1 In an examination of the patella Pb concentration-reaction time relationship, Weisskopf et al. 

2 (2007) found a statistically significant nonlinear association, with latency times on the pattern comparison 

3 test becoming worse over time (i.e., larger values or slower response latencies) up to approximately 60 

4 μg/g bone mineral, but the change over time leveling off at higher concentrations (Figure 5-18). Below 60 

5 μg/g, a 20 μg/g difference in patella Pb level was associated with a decrease in pattern comparison test 

6 score of approximately 0.15 ms. Intriguingly, however, the cognitive tests where the association with 

7 bone Pb was significantly worse among ALAD-2 carriers (constructional praxis and pattern comparison) 

8 were the same tests that were significantly associated with bone Pb in an earlier study (Weisskopf, 

9 Proctor, et al., 2007). 

Source: Used with permission from Williams & Wilkins, Weisskopf et al. (2007).

Note: Models are adjusted 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 subjects 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 Pb

concentration).

10 In a somewhat similar approach to that taken in the NAS, BMS investigators took advantage of 

11 repeat cognitive testing of study subjects (91% of the original cohort returned for a second round of 

12 testing and 83% for a third round each at approximately 14-month intervals) to analyze associations of 

13 blood and bone Pb levels with changes in cognitive performance over time cohort (Bandeen-Roche et al., 

14 2009). An interquartile range increase in tibia Pb level (12.7 μg/g) was associated with a 0.019 units per 
May 2011 5-96 DRAFT – DO NOT CITE OR QUOTE

1 year decrease in eye-hand coordination z-score. The association was somewhat stronger among African2 Americans than it was among whites (Figure 5-16 and Table 5-9). Of the other tests, only change in 

3 verbal memory and learning suggested some association with tibia Pb levels. In analyses of what they 

4 term persistent effects (analogous to the main effects analyses of longitudinal data in the NAS), a similar 

5 pattern was found as that reported by Shih et al. (2006) in the original BMS cross-sectional analyses. 

6 Increasing tibia Pb levels were associated with decreases in cognitive performance; however, the effect 

7 sizes decreased as more covariates were added to models. In models adjusted for age, sex, race, and 

8 education, performance on executive function, verbal memory and learning, and visual memory test 

9 decreased with increasing tibia Pb levels, with p-values ranging from 0.16 to 0.26. In contrast to the 

10 results of the longitudinal analyses described above, race-stratified analyses of persistent effects indicated 

11 that tibia Pb levels were associated with greater decreases in performance on tests of eye-hand 

12 coordination, executive functioning, and verbal memory and learning among whites compared with 

13 African-Americans.

14 Other studies generally confirm the same pattern as described in the larger studies above. One 

15 study of 188 rural Chinese men and women found a weakly negative association between plasma Pb

16 levels and a composite cognitive score based on a battery of in-person administered tests (S. J. Gao et al., 

17 2008). It should be noted, though, that Pb in plasma makes up a very small fraction of all Pb in blood and 

18 is a different, and much less used, biomarker than Pb in whole blood. The relevance of this Pb fraction is 

19 not entirely clear. Pb in plasma is not bound to erythrocytes, as is about 99% of blood Pb. Thus, it has 

20 been postulated that plasma Pb may be more toxicologically active (Chuang et al., 2001; Hernandez-Avila 

21 et al., 1998). In another study of 47 men and women in Rochester, NY (55-67 years of age), subjects in 

22 the higher two tertiles of calcaneal bone Pb level had lower scores on a delayed matching-to-sample task 

23 (van Wijngaarden et al., 2009) (Table 5-9). The pattern was similar for a paired associates learning task, 

24 although the results did not reach statistical significance. In analyses of tibia Pb levels, subjects in the 

25 highest tertile of tibia Pb level performed worse on cognitive tests (Table 5-9).

26 In summary, among adults without occupational exposures to Pb, there is weak evidence for an

27 association between blood Pb levels and cognitive function. The strongest evidence of association 

28 between blood Pb levels and cognitive function in adults was provided by the various NHANES analyses 

29 of various age and genetic variant subgroups (E. F. Krieg, Jr. & Butler, 2009; E. F. Krieg, Jr. et al., 2009; 

30 E. F. Krieg, Jr. et al., 2010). These NHANES analyses did not have bone Pb measurements for 

31 comparison. Consistent with the conclusion of the 2006 Pb AQCD, recent studies continued to 

32 demonstrate associations between bone Pb levels and cognitive deficits in adults (Figure 5-16 and Table 

33 5-9). Recent studies also demonstrated that bone Pb levels may be associated specifically with cognitive 

34 decline over time. Among recent studies that analyzed both blood and bone Pb levels, bone Pb levels, in 

35 particular tibia Pb levels, were associated with greater decreases in cognitive performance than were 

36 blood Pb levels across of the various cognitive tests that were performed. The discrepant findings for 
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1 blood and bone Pb levels indicate that biomarkers of cumulative Pb exposure, including higher levels in 

2 the past, may the best predictors of neurocognitive function in adults. The effects associated with 

3 cumulative Pb exposure are also demonstrated by observations that tibia Pb levels were associated with 

4 larger decreases in cognitive performance than were patella Pb levels. Tibia bone has a slower rate of 

5 turnover compared with patella bone and is an indicator of longer-term Pb exposure.

Adults with Occupational Lead Exposures

6 The 2006 Pb AQCD concluded that the evidence for an association between blood Pb levels and 

7 cognitive function in adults was most consistent among adults occupationally exposed to Pb. Results from 

8 a few recent studies of occupationally-exposed adults support the previous conclusions. Dorsey et al. 

9 (2006) followed-up on a cohort of Pb-exposed workers in Korea with a mean age of 43.4 years, on whom 

10 patella bone Pb measurements were made. This group represented a typically highly-exposed 

11 occupational group with an average blood Pb level of 30.9 μg/dL. In this cohort, both blood and tibia Pb

12 levels previously were associated with poorer performance on a battery of neurocognitive tests (B. S. 

13 Schwartz et al., 2005; B. S. Schwartz et al., 2001). Dorsey et al. (2006) found patella Pb levels to be 

14 associated with poorer performance in the domains of manual dexterity, sensory PNS function, and 

15 depression symptoms. In this occupational cohort, however, the associations between patella Pb levels 

16 and cognitive function were not as strong as the associations with either blood or tibia Pb levels.

17 A follow-up study of the original 1982 Lead Occupational Study was conducted in 2001-2004 

18 among 83 of the original 288 Pb-exposed workers and 51 of the original 181 controls (Khalil, Morrow, et 

19 al., 2009). Those originally in the exposed workers group had last worked in a job with Pb exposure from 

20 0.02 to 16 years (median = 6) prior to follow-up testing. While the follow-up participation was somewhat 

21 low, participants did not appear to differ from nonparticipants on most baseline cognitive tests except for 

22 performing slightly better on aspects of the grooved pegboard test and recall on a paired associates 

23 learning task. This suggests that the follow-up participation was not biased to poor performers. At follow24 up, the former Pb-exposed workers performed significantly (p <0.05) worse than the controls in total 

25 cognitive score and in the spatial and general intelligence domains. They also performed worse in all 

26 other domains (e.g., motor, executive, and memory) although not significantly so. A similar pattern was 

27 observed in analyses using tibia Pb levels measured at the follow-up visit as the exposure variable. 

28 Associations also were seen with blood Pb levels (median among the exposed: 12 μg/dL), although these 

29 generally did not reach statistical significance. Among the former Pb workers, tibia Pb levels were

30 associated with a greater decrease in total score and scores for spatial and executive domains between 

31 baseline and follow-up. Tibia Pb level were associated inversely with other domains as well, although 

32 they were not statistically significant.
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1 Two additional studies aimed to characterize factors that either mediate or modify the association 

2 between Pb biomarkers and cognitive function. A study among 61 current Pb smelter workers with an 

3 average age of 40 years and blood Pb of 29.1 μg/dL found that both a time-weighted integrated blood Pb

4 measure (p = 0.09) and tibia Pb level (p = 0.08) were associated with longer times to complete the 

5 grooved pegboard test (Bleecker, Ford, Vaughan, et al., 2007). In another study to examine the modifying 

6 effects of cognitive reserve (assessed by performance on the Wide Range Achievement Test-R for 

7 reading) on the Pb-cognitive function association among 112 Pb smelter workers, a time-weighted 

8 integrated blood Pb measure (an index of cumulative exposure) was associated with decrements in motor 

9 performance (p<0.05), and among those with low cognitive reserve, tests of attention tasks and a digit 

10 symbol task as well (Bleecker, Ford, Celio, et al., 2007). 

11 Iwata et al. (2005) examined the association between blood Pb level and aspects of postural sway 

12 among 121 Pb-exposed workers in Japan with blood Pb levels between 6 and 89 μg/dL (mean: 40 μg/dL). 

13 In multiple regression analyses adjusted for age, height, and smoking and drinking status, increasing 

14 blood Pb level was associated with increases in sagittal sway with eyes open (p <0.05) and eyes closed (p 

15 <0.01) and transversal sway with eyes closed (p <0.05). The authors calculated a benchmark dose level

16 (Budtz-Jorgensen et al., 2001; NRC, 2000) of 14.3 μg/dL from a linear dose-response model of their data. 

17 Although the data were slightly better fit with a supralinear dose-response function, the linear function 

18 was also statistically significant.

19 Apolipoprotein E is a transport protein for cholesterol and lipoproteins. The gene appears to 

20 regulate synapse formation (connections between neurons) and may be particularly critical in early 

21 childhood. A genetic variant, called the ApoE-ε4 allele is a haplotype between 2 exonic SNPs and is 

22 perhaps the most widely studied genetic variant with respect to increasing risk of neurologic disease. 

23 Carriers of the E4 variant are at twofold increased risk of developing Alzheimer’s disease, although the 

24 majority of such individuals still do not develop the disease. A study of occupationally-exposed adults 

25 found that among individuals with at least one ApoE-ε4 allele, Pb was associated with greater decrements 

26 in tests such as digit symbol, pegboard assembly, and complex reaction time (W. F. Stewart et al., 2002).

5.3.3. Neurobehavioral Effects

5.3.3.1. Epidemiologic Studies of Behavioral Effects in Children

27 Several epidemiologic studies reviewed in the 2006 Pb AQCD reported associations between blood 

28 Pb levels and problems with behavior and social conduct that ranged from inattentiveness to self-reported 

29 delinquent behaviors to criminal activities (Bellinger et al., 1994; Bellinger & Rappaport, 2002; Burns et 

30 al., 1999; Dietrich et al., 2001; Needleman et al., 2002; Needleman et al., 1996; G. A. Wasserman et al., 

31 1998). Recent studies continue to demonstrate similar associations and provide new evidence for 
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1 associations of blood Pb levels with ADHD diagnosis and diagnostic indices (Figure 5-19 and Table 5-

2 10). Noncognitive effects of Pb are more complex to study relative to IQ tests. However, domain-specific 

3 neuropsychological assessments are advantageous as they may provide greater insight into the underlying 

4 CNS damage that may be associated with exposures (e.g., structural, neural system, neurotransmitter) (R. 

5 F. White et al., 2009). Most of these studies found that blood or dentin Pb levels measured at an early age 

6 (e.g., 2-6 years of age) were associated with behavioral problems later in childhood and early adulthood 

7 (7-22 years of age). Most of these studies examined associations with blood Pb levels assessed at one 

8 time point; however, even the prospective studies with serial measurements of blood Pb levels, found 

9 associations with both prenatal and early childhood blood Pb levels (Dietrich et al., 2001; J. P. Wright et 

10 al., 2008) and lifetime average blood Pb levels (Burns et al., 1999). Therefore, uncertainty remained over 

11 what was the critical time period of Pb exposure for increasing risk of behavioral problems and 

12 misconduct. In many of the studies noted above, blood Pb level also was associated with IQ and other 

13 endpoints of cognitive function, thus it was unclear whether blood Pb-associated neurocognitive deficits, 

14 poor school performance, and attention problems, in turn, progressed to antisocial and delinquent 

15 behavior later in life.

16
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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 ADHD index

Braun et al. (2006)

a 1.1-1.3 (3rd quintile) Concurrent ADHD Dx

0.8-1.0 (2nd quintile) Concurrent ADHD Dx

1.4-2.0 (4th quintile)a Concurrent ADHD Dx

>2.0 (5th quintile)a Concurrent ADHD Dx

Froehlich et al. (2009) >2.0a Concurrent ADHD Dx, prenatal smoke exposure

Braun et al. (2008) 0.8-1.0 (2nd quartile)a Concurrent Conduct disorder

1.0-1.4 (3rd quartile)a Concurrent Conduct disorder

1.5-10 (4th 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 deficit 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 behavioral 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 

using Conners’ ADHD/DSMIV 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)
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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 regression 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, 

clustering in school and 

classroom

ADHD index

At age 3-7 yr assessed 

using Conners’ ADHD/DSMIV 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 

3rd quintile: 1.1-

1.3

Logistic regression model 

adjusted for postnatal ETS, 

prenatal ETS, age, sex, race, 

childcare attendance, health 

insurance coverage, ferritin 

levels

ADHD Dx or medication use 

at age 4-15 yr

1.4 (0.4, 3.4), blood Pb 

0.8-1.0 μg/dL vs. blood 

Pb <0.8 μg/dLb

2.1 (0.7, 6.8), blood Pb 

1.1-1.3 μg/dL vs. blood 

Pb <0.8 μg/dLb

2.7 (0.9, 8.4), blood Pb 

1.4-2.0 μg/dL vs. blood 

Pb <0.8 μg/dLb

4.1 (1.2, 14.0), blood Pb 

> 2.0 μg/dL vs. blood Pb 

<0.8 μg/dLb

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 μg/dL plus 

prenatal ETS exposure 

vs. blood Pb level <0.8 

μg/dL and no prenatal 

ETS exposureb

Braun et 

al. (2008)

Children ages 8-15 yr

U.S. NHANES 2001-2004

2nd quartile: 0.8- 1.0Logistic 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 yr7.24 (1.06, 49.47), blood 

Pb level 0.8-1.0 μg/dL 

vs. blood Pb <0.8 μg/dLb

12.37 (2.37, 64.56), 

blood Pb level 1.1-1.4 

μg/dL vs. blood Pb <0.8 

μg/dLb

8.64 (1.87, 40.04), blood 

Pb level 1.5-10 μg/dL vs. 

blood Pb <0.8 μg/dLb

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 Behavior

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.

1 Burns et al. (1999) and Silva et al. (1988) aimed to characterize the direct association of blood Pb 

2 level with behavior by adjusting for child IQ in their models. They found positive associations, suggesting 

3 that blood Pb level may have an independent effect on behavior. However, because a decrement in IQ 

4 may on the causal pathway to behavioral problems, including both IQ and behavioral problems may result 

5 in an underestimate of the effect on behavior. Hence, to account for the relationship between IQ and 

6 behavior, Chen and colleagues (2007) used structural equation modeling (specifically path analysis) to 

7 estimate the direct effects of blood Pb level on behavioral problems as well as indirect effects mediated 
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1 through child’s IQ (assessed using the WPPSI-R at age 5 years and the WISC-III) at age 7 years). Among 

2 5- and 7-year-old participants from the TLC trial (described in Section 5.3.2.1), conduct problems were 

3 assessed using the Conners’ Parent Rating Scale-Revised, and other behaviors were assessed with the 

4 Behavior Assessment System for Children-Teacher Rating Scale (BASC-TRS) and Behavior Assessment 

5 System for Children-Parent Ratings Scale (BASC-PRS). In models that adjusted for sex, race, clinical 

6 site, language (English or Spanish), parent’s education, parent’s employment, age at blood Pb testing, 

7 caregiver’s IQ, and treatment group, peak blood Pb levels at age 2 years were not associated with conduct 

8 problems at age 5 years or any of the BASC scores at age 7 years. Concurrent blood Pb levels had 

9 statistically significant direct effects on behavioral symptoms index, externalizing problems and school 

10 problems as assessed by BASC-TRS and externalizing problems (outbursts of behavior) as assessed by 

11 BASC-PRS (Figures 5-20 and 5-21).

12 Chen et al. (2007) also found significant indirect effects (mediated through child’s IQ) of blood Pb 

13 levels at age 7 years with all measurements except the BASC-TRS externalizing problems and BASC14 PRS internalizing problems (repressing problems). However, because blood Pb level was estimated to 

15 have direct effects on several behavioral endpoints and, in for some endpoints, a larger magnitude of 

16 direct effect, the authors inferred IQ to only partially mediate associations between blood Pb levels and 

17 behavioral outcomes (Figures 5-20 and 5-21). The study also had limitations, including the lack of 

18 adjustment for HOME score and lack of information on other covarying family and neighborhood 

19 characteristics that may be relevant (i.e., social stressors). Also, these findings may not be generalizable to 

20 the general population given that the children in the study population had been referred for chelation 

21 therapy at enrollment because of high blood levels. In this study, only concurrent blood Pb levels were 

22 examined, and it is uncertain whether the observed associations were due to the residual effect of high 

23 blood Pb levels (20-44 μg/dL) four years earlier.
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Source: Used with permission from the American Academy of Pediatrics, Chen et al. (2007)

Note: Values 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: Values 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 concentrations at age 7 on BASC-PRS.

1 The 2006 Pb AQCD summarized indirect evidence for associations of blood Pb level with 

2 behavioral features of ADHD including distractibility, poor organization, lacking persistence in

3 completing tasks, and daydreaming (Bellinger et al., 1994; Fergusson et al., 1993; Needleman et al., 1979; 
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1 G. A. Wasserman et al., 2001; G. A. Wasserman et al., 1998). Prior studies examining the association of 

2 blood Pb level with a diagnosis of ADHD were limited by small sample size, and results were 

3 inconclusive (David et al., 1972; Gittleman & Eskenazi, 1983). In addition, many earlier studies of 

4 inattention and impulsivity included children with higher blood Pb levels than those observed in 

5 contemporary children.

6 A recent analysis of data from NHANES (1999-2002) found a positive relationship between blood 

7 Pb level and ADHD (parent-report of a diagnosis of ADHD or use of stimulant medication) (Braun et al., 

8 2006). This analysis included 4,704 children aged 4-15 years. These authors reported a monotonic 

9 increase in odds of ADHD from the lowest to highest quintile of blood Pb level (Figure 5-22). Children in 

10 the fifth quintile of blood Pb level (>2.0 μg/dL) had the highest odds of ADHD compared with those in 

11 the lowest quintile (<0.8 μg/dL) (OR: 4.1 [95% CI: 1.2, 14]). Children in the other three higher quintiles 

12 of blood Pb level also had increased odds of ADHD relative to the reference group; however, the 

13 associations were not statistically significant (Tables 5-19 and 5-10).

14 In the same NHANES dataset, however, restricted to children ages 8-15 years, Froehlich and 

15 colleagues (2009) demonstrated the joint effects of prenatal tobacco smoke (maternal report) and blood 

16 Pb levels. They found independent effects on ADHD for prenatal tobacco smoke exposure (OR: 2.4 [95% 

17 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 

18 >1.3 μg/dL compared with children with blood Pb levels < 0.8 μg/dL. A statistically significant 

19 interaction also was found. Compared to children in the lowest tertile of blood Pb levels with no exposure 

20 to prenatal tobacco smoke, children in the highest tertile of blood Pb level with exposure to prenatal 

21 tobacco smoke had the greatest odds of ADHD (OR: 8.1 [95% CI: 3.5, 18.7]).

Source: Braun et al. (2006)

Note: Adjusted 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.
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1 Pb has long been known to impact dopaminergic neurons. Pb will inhibit depolarization-evoked 

2 neurotransmitter release and stimulate spontaneous neurotransmitter release, including effects specific to 

3 dopaminergic neurons (Section 5.3.6.9). This background would make dopamine related genes strong 

4 candidates as effect modifiers of Pb-associated neurodevelopmental effects. There are several known 

5 functional variants in dopamine related genes, including the dopamine 2 receptor, dopamine 4 receptor 

6 (DRD4), and the dopamine transporter. The DRD4 gene has a 48 base pair long repeat in exon 3. The 

7 longer repeat alleles (DRD4.7) appear to have less binding affinity for dopamine and have been 

8 associated with ADHD. However, DRD4.7 also has been associated with sustained attention, response 

9 inhibition, and quicker response time. Thus, it is not clear whether DRD4.7 would increase or decrease 

10 susceptibility to Pb-associated ADHD. Froehlich et al. (2007) studied 174 children from the Rochester 

11 prospective birth cohort and measured DRD4 genotype, blood Pb level, and several functional domains 

12 using the Cambridge Neuropsychological Testing Automated Battery. Increasing blood Pb levels were 

13 associated with poorer rule learning and reversal, spatial span, and planning. These negative associations 

14 were larger in magnitude among boys and those lacking DRD4.7.

15 In a population of children in a similar age range (8-11 years) and similar blood Pb levels (mean 

16 1.9 μg/dL) as in the NHANES studies, Cho et al. (2010) found a statistically significant relationship of 

17 blood Pb levels with parent- and teacher-rated ADHD symptoms (i.e., inattentiveness, hyperactivity, and 

18 total score).

19 Nicolescu et al. (2010) examined the relationship between blood Pb level and ADHD-related 

20 behaviors among 83 children, ages 8-12 years, living near a metal-processing plant in Romania. These 

21 authors examined associations of Pb, mercury, and aluminum biomarkers with performance on four 

22 different attention tasks on the computerized German Kinder-KITAP as well as behavior ratings using the 

23 parent and teacher reports on the Connors scale. Only concurrent blood Pb levels, and not other metals, 

24 were consistently associated with increased odds for ADHD-related behaviors (OR: 1.16 [95% CI: 0.98, 

25 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 

26 when analyses were repeated taking out the 5 children with blood Pb levels at or above 10 μg/dL, 

27 associations with both questionnaire-based ADHD ratings and KITAP-performance remained statistically 

28 significant.

29 Roy and colleagues (2009) examined associations between blood Pb level and a range of 

30 behavioral problems in 756 children, ages 3-7 years, in Chennai, India. In this population, mean (SD) 

31 blood Pb level was 11.4 (5.3) μg/dL. Anxiety, social problems, inattention, hyperactivity, and ADHD as 

32 well as executive function were assessed based on the teacher’s report of the Conners’Teacher Rating 

33 Scales-39, Conners’ADHD/Diagnostic and Statistical Manual for Mental Disorders, 4th Edition Scales, 

34 and the Behavior Rating Inventory of Executive Function questionnaire. In generalized estimating 

35 equations, increasing blood Pb level was associated with higher anxiety, social problems, and ADHD 

36 index scores (Figure 5-19 and Table 5-10).
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1 Recent studies add to the collective body of evidence demonstrating associations between blood Pb 

2 level and a variety of conduct problems including conduct disorder (Braun et al., 2008), oppositional 

3 defiant disorder (Nigg et al., 2008) and more serious behaviors such as criminal behavior (J. P. Wright et 

4 al., 2008). These findings were corroborated in a recent meta-analysis on Pb and conduct problems 

5 (Marcus et al., 2010) that included 19 studies with a total of 8,561 children and adolescents (mean ages 

6 ranging from 3.5 years to 18.4 years). An overall medium effect size was estimated (r across studies was 

7 0.19, p<0.001). Moreover, the meta-analysis demonstrated a consistent relationship between increasing 

8 blood Pb levels and conduct problems despite considerable heterogeneity across studies (e.g., ways in 

9 which conduct problems were defined and assessed, participant ages, participant blood Pb levels). In the 

10 meta-analysis, covariates such as SES, birth weight, parental IQ, and family environment did not 

11 attenuate the relationship between blood Pb level and conduct problems. Interestingly, a larger magnitude 

12 of effect was estimated for hair Pb levels compared with bone or blood Pb levels. Although the authors 

13 suggested that hair Pb may be a better indicator of cumulative Pb exposure compared to bone Pb levels, 

14 due to the high turnover of bone in throughout childhood and into adolescence, an empirical basis for 

15 interpreting hair Pb measurements in terms of body burden or exposure has not been firmly established 

16 (Section 4.3.4.2).

17 In addition to examining ADHD in the NHANES population, Braun and colleagues (2008) also 

18 examined conduct disorder defined using DSM-IV criteria. The prevalence of conduct disorder was 

19 higher among males, older children (13-15 years, relative to those less than 13 years), and children with 

20 higher blood Pb levels. In analyses adjusted for child’s age, gender race/ethnicity, SES, mother’s age at 

21 child’s birth, prenatal tobacco smoke exposure (based on maternal report), and child’s cotinine levels, 

22 children in the highest quartile of blood Pb level (>1.5 μg/dL) had increased odds of conduct disorder 

23 relative to children in the lowest quartile (0.2-0.7 μg/dL) (OR: 8.7 [95% CI: 1.9, 40]). Poisson regression 

24 models showed that compared with children in the lowest quartile of blood Pb level, children in the 

25 highest quartile had 1.73 (95% CI: 1.2, 2.4) times as many conduct disorder symptoms.

26 In their longitudinal study of children in the U.K., Chandramouli et al. (2009) found associations of 

27 blood Pb level at age 30 months not only with hyperreactivity but also with antisocial behavior at ages 7 

28 and 8 years. Child behavior was assessed using three methods: parent- and teacher-report on the Strengths 

29 and Difficulties Questionnaire at age 7 years, the Development and Well-being Assessment at age 8 years, 

30 and the Anti-social Behaviour Interview at age 8 years. Attention was measured using the Test of 

31 Everyday Attention for Children at age 8 years. Blood Pb levels showed statistically significant positive 

32 association with antisocial behavior. Similar to Chen et al. (2007) and Burns et al. (1999), increasing 

33 blood Pb level was associated with antisocial behavior, independently of IQ.

34 Wright et al. (2008) recently examined the relationship between prenatal and postnatal blood Pb 

35 levels and arrests for criminal offenses at ages 19-24 years in the CLS. In this birth cohort, prenatal and 

36 postnatal blood Pb levels previously have been reported to be associated with self- and parent-reported 
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1 delinquent and social acts at ages 16-17 (Dietrich et al., 2001). Data on criminal arrests for participants

2 and their mothers were obtained from a computer search of Hamilton County, Ohio criminal justice 

3 records. They also examined the absolute change in arrest rates between participants with higher levels of 

4 blood Pb compared to those with lower blood Pb levels defining attributable risk as the average difference 

5 in annual arrest rates between participants at the 95th percentile of blood Pb and those at the 5th 

6 percentile. Mean blood Pb concentrations were 8.3 μg/dL (range 1 to 26) for the prenatal period (maternal 

7 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

8 to 33) at age 6 yr. In models that adjusted for maternal IQ, sex, SES score, and maternal education, the 

9 relative risks (RRs) for total arrests per 1 μg/dL increment in blood Pb level were 1.07 (95% CI: 1.01, 

10 1.13) for prenatal blood Pb level, 1.01 (95% CI: 0.97, 1.05) for average childhood blood Pb level, and 

11 1.05 (95% CI: 1.01, 1.09) for blood Pb level at age 6 years. Relative risks for violent criminal arrests were 

12 also larger for prenatal blood Pb levels and age 6-year blood Pb levels. The RRs for arrests involving 

13 violent crimes per 1 μg/dl increment in blood Pb level were 1.06 (95% CI: 0.97, 1.15) for prenatal blood 

14 Pb level, 1.05 (95% CI: 1.01, 1.10) for average childhood blood Pb level, and 1.08 (95% CI: 1.03, 1.14) 

15 for blood Pb level at age 6 years. Although interactions terms of blood Pb by sex were not statistically 

16 significant, the attributable risk for males was considerably higher for males (0.85 arrests/year [95% CI:

17 0.48, 1.47]) than for females (0.18 [95% CI: 0.09, 0.33]). Similar to findings from Dietrich et al. (2001), 

18 prenatal and early childhood blood Pb levels were associated positively with risk of criminial behavior in 

19 early adulthood. Results from the two studies combined suggest that in addition to the prenatal period, 

20 early childhood blood Pb levels may also predict criminal behavior in adulthood. However, it is important 

21 to note that in these CLS studies, concurrent blood Pb levels were not analyzed.

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

Adults

22 Effects of blood Pb levels on emotional regulation in adults have received far less attention than 

23 that in children and cognitive function in adults. Nonetheless, evaluation of mood states is an integral part 

24 of the World Health Organization’s (WHO) neurocognitive test battery and, indeed, it has been suggested 

25 that the assessment of mood with the Profile Of Mood States may be particularly sensitive to toxicant 

26 exposures (1987). With respect to Pb exposure, several early studies of Pb-exposed workers (mean blood 

27 Pb levels ranging from 23.5 to 64.5 μg/dL) found higher prevalence of symptoms of mood disorders and 

28 anxiety among Pb-exposed workers than unexposed controls (mean blood Pb levels ranging from 15-38 

29 μg/dL) (Baker et al., 1984; Baker et al., 1985; Lilis et al., 1977; Maizlish et al., 1995; Parkinson et al., 

30 1986; B. S. Schwartz et al., 2005). In one of the few previous studies of adults without occupational 

31 exposure to Pb and more relevant to blood and bone Pb levels measured currently in the U.S. among 

32 adults without occupational exposures, an association was observed between both blood (mean: 6.3 μg/dL 
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1 [SD: 4.16] and bone (tibia mean: 21.9 μg/g [SD: 13.5]) Pb levels and depression and anxiety symptoms 

2 (Brief Symptom Inventory, BSI) among men in the NAS (Rhodes et al., 2003).

3 More recently, subsequent assessments of the NAS men have provided understanding of effect 

4 modification of the associations of blood, patella, and tibia Pb levels with psychiatric symptom 

5 dimensions by ALAD genotype (Rajan et al., 2007). In addition to corroborating associations of Pb with 

6 BSI symptoms reorted by Rhodes et al. (2003), Rajan et al. (2007) found that of eight symptom 

7 dimensions considered, associations of tibia bone Pb levels with phobic anxiety, positive symptom total, 

8 and anxiety scale were modified by ALAD genotype, with interaction terms attaining statistical 

9 significance for phobic anxiety and positive symptom total. For all psychiatric symptoms, the association 

10 with tibia Pb was worse among ALAD 1-1 carriers, which was the opposite genotype observed to have 

11 larger Pb-associated decrements in cognitive performance (Section 5.3.2.4).

12 A study of 1,987 adults age 20-39 years participating in NHANES 1999-2004 was the largest study 

13 of psychiatric disorders and the study with the lowest blood Pb levels (mean 1.61 μg/dL [SD: 1.72]) 

14 (Bouchard et al., 2009). Investigators examined cases of major depressive disorder (MDD), panic disorder 

15 and generalized anxiety disorder (GAD) as determined using the WHO Composite International 

16 Diagnostic Interview, which follows criteria defined in DSM. Compared with those in the lowest quintile 

17 of blood Pb level (<0.7 μg/dL), adults in the highest quintile (≥ 2.11 μg/dL) had increased odds of MDD 

18 (OR: 2.32 [95% CI: 1.13, 4.75]) and panic disorder (OR: 4.94 [95% CI: 1.32, 18.48]). Odds ratios were 

19 even larger in analyses excluding current smokers. While those in higher quintiles of blood Pb level (>0.7 

20 μg/dL) had increased odds to GAD, the associations were not statistically significant. Although studies in 

21 adults without occupation Pb exposure are sparse, consistent with studies of occupationally-exposed 

22 adults and experimental evidence, they demonstrate associations of blood (as low as 2.11 μg/dL) and 

23 bone Pb level (population mean tibia Pb level 22 μg/g) with psychiatric outcomes.

5.3.3.3. Toxicological Studies of Neurobehavioral Outcomes

24 Pb is a known risk factor for neurobehavioral changes with preferentially targeted sites including 

25 the prefrontal cerebral cortex, cerebellum, and hippocampus; affected functions include cognition, 

26 execution of motor skills, and memory/behavior, respectively. As discussed in earlier AQCDs, young 

27 animals are especially susceptible to the effects of Pb due to differences in structure of the nervous system 

28 and to the ongoing development with greater Pb absorption and retention. Pb exposure has been 

29 documented to induce neurobehavioral changes in exposed animals including effects on learning, social 

30 behavior, memory, attention, motor function, locomotor ability and vocalization. At the cellular level, Pb 

31 impairs axon and dendritic development and contributes to neurochemical changes in proteins, 

32 membranes, redox/antioxidant balance, and neurotransmission through a multitude of mechanisms, many 

33 of which involve the ability of Pb to mimic calcium. Very early research on neurobehavioral endpoints 
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1 failed to capture the disposition of Pb and its resulting body burden or blood Pb and is thus difficult to use 

2 in risk assessment. 

3 Since then, the 1986 AQCD reported the literature on rodent and nonhuman primate Pb-induced 

4 aberrant operant conditioning tasks in rodents and non-human primates (blood Pb 11 μg/dL to 15 μg/dL) 

5 with other studies yielding hyperactive or inappropriate Pb-mediated responses (U.S. EPA, 1986) possibly 

6 of hippocampal origin with a curvilinear response, decreasing at higher doses possibly due to impairment 

7 of motor function at the high doses (Crofton et al., 1980; Ma et al., 1999). Pb exposure in lab animals 

8 contributed to distractibility, reduced adaption capacity to changes in behavior, impaired ability to inhibit 

9 inappropriate responses and perservation (U.S. EPA, 2006). Pb is known to impair learning (blood Pb 11 

10 μg/dL) measured using Fixed Interval tasks (FI) defined as scheduled reinforcement delivered after a 

11 fixed period since the last reward, with premature responses not rewarded. The 2006 AQCD found FI 

12 response rates (blood Pb 58 to 94 μg/dL) were sensitive to Pb exposure, which was primarily accounted 

13 for by decreased interresponse times. Inter-response rates and overall run rate are the two subcomponents 

14 of FI response rate. Spatial and non-spatial discrimination reversal or reversal of a previously learned 

15 habit is significantly affected after developmental Pb exposure and is exacerbated with distracting stimuli; 

16 discrimination reversal has been shown to be especially sensitive to Pb exposure. Repeat-acquisition 

17 testing revealed that these deficits are likely not due to sensory or motor impairment at this dose. The 

18 results from different studies testing the effect of Pb on memory are mixed with impaired memory shown 

19 at blood Pb level of 10 μg/dL and improved memory in other studies. Low dose Pb does not appear to 

20 affect short-term memory. Memory tests may give incorrect results when opportunities exist for impaired 

21 attention to contribute to test results (U.S. EPA, 2006). Together, the data from the 2006 AQCD showed 

22 that social behavior and learning in rodents and nonhuman primates is significantly affected by Pb 

23 exposure (blood Pb 15-40 μg/dL). 

24 In the new literature, gestationally-Pb exposed (GLE) male mice (low and high dose Pb, 10 μg/dL 

25 and 42 μg/dL blood Pb at PND10) were significantly less active than control mice and low dose GLE 

26 mice were significantly less active than high dose GLE mice, demonstrating a non-linearity of GLE dose 

27 responsiveness (Leasure et al., 2008). A similar Pb dose response non-linearity (baseline corticosterone) 

28 was seen in male mice exposed post-weaning to Pb (Virgolini et al., 2005). Activity level of GLE female 

29 mice versus control was unaffected (Leasure et al., 2008). Amphetamine induced motor activity was 

30 monitored in male and female GLE mice at 1 year of age. Amphetamine-induced activity of male low and 

31 high dose GLE offspring was significantly elevated versus control; GLE females had no change in 

32 sensitivity to amphetamine-induced motor activity (Leasure et al., 2008). 

33 Rotarod performance measures endurance, balance and coordination in mice. GLE male mice had 

34 significantly shorter mean latencies to fall from the rotarod compared with controls; females were 

35 unaffected. Further, low dose GLE male mice had significantly poorer rotarod performance than high 

36 dose GLE male mice (fell off quicker, 10 μg/dL and 42 μg/dL blood Pb at PND10), showing non-linearity 
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1 of dose-responsiveness to GLE (Leasure et al., 2008). Other rotarod experiments at higher doses of Pb 

2 exposure and at various speeds of rotarod rotation yielded mixed results (Grant et al., 1980; Kishi et al., 

3 1983; Overmann, 1977). 

4 Herring gull chicks exposed to a single IP bolus dose (100 mg/kg Pb acetate) of Pb on post-natal 

5 day two at a dose created to be similar to that which wild herring gulls are exposed to in the wild were 

6 found to have neurobehavioral deficits, including learning deficits. Pb-exposed chicks displayed multiple 

7 deficits related to impaired survival skills including decreased time spent begging the parent for food, 

8 decreased accuracy at pecking for food in the parent bird’s mouth, decreased time spent in the shade 

9 (behavioral thermoregulation), decreased learning in food location testing, decreased recognition of 

10 familiar individuals (caretaker or sibling), and slower development of motor skills (treadmill test) versus 

11 control birds (Burger & Gochfeld, 2005). The impaired thermoregulation with Pb exposure agrees with 

12 earlier work in rat pups who also showed impaired thermoregulatory behavior, i.e., impaired ultrasonic 

13 vocalization (Davis, 1982). These studies in herring gull chicks demonstrate that a single early life 

14 exposure to Pb can induce neurobehavioral deficits that affect survival skills. 

5.3.3.4. Toxicological Studies of Mood Alterations

15 Neurotoxicological studies often focus on motor, sensory, behavioral or cognitive outcomes and 

16 often fail to evaluate psychological pathologies. Recent epidemiologic studies have reported that prenatal 

17 or early life blood Pb levels or ALAD changes, a biomarker of Pb exposure, may be a risk factor for 

18 development of mood disorders in adulthood (i.e. schizophrenia, major depression or panic disorders) 

19 (Bouchard et al., 2009; Opler et al., 2004). With this in mind, animal studies of Pb exposure during 

20 pregnancy and lactation and outcomes in offspring look to address the role of developmental Pb exposure 

21 on emotional state and mood disorder-like behavior in adult offspring. Wistar rats exposed to 10mg of Pb 

22 acetate daily by gavage during pregnancy (G) or pregnancy plus lactation (G+L) produced pups that were 

23 then tested in the open field test or the forced swimming test also known as Porsolt’s test. Blood Pb levels 

24 in the pups at PND70 was 5-7 μg/dL. The open field test can measure emotion and exploratory behavior; 

25 Pb (G+L) treated male rats had increased emotionality with the open field test. Pb exposed (G+L) female 

26 offspring had a significant increased depressive phenotype in the forced swim test (de Souza Lisboa et al., 

27 2005). It is interesting to note that this is one of many Pb-induced changes that seem to be sex-specific. 

28 Depression may seem initially like an unexpected comorbidity for immune inflammatory 

29 dysfunction, but many forms of depression are linked with the same cytokine imbalances that occur with 

30 Pb-induced innate immune dysfunction (Maes; T. W. W. Pace & Miller, 2009). Some researcher use 

31 sickness behavior and its associated malaise as a model for depression. Examples from animal models 

32 include the study by Dyatlov and Lawrence (2002) with Pb exposure in mice. In this study, sickness 

33 behavior, which is due to an interaction of the immune system and the CNS in Pb-exposed mice, was 
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1 potentiated by Pb-exposure (blood Pb level 17 μg/dL) and this correlated with depletions in specific 

2 thymic T-cell populations. Pb-exposure also potentiated the infection-dependent elevation in IL-1β, a 

3 cytokine which has been shown to inhibit hippocampal glutamate release in young and not aged animals. 

4 Sickness behavior was induced with Listeria monocytogenes infection; Pb exposure was from birth 

5 through lactation and continued for a brief period after weaning until the experiment was terminated. 

6 Sickness behavior is characterized by overall malaise, decreased food intake, immobility and changes in 

7 core body temperature. Pb potentiated the sickness behavior in exposed animals.

Figure 5-23. Animal toxicology evidence of possible Pb-dependent 

contributors to the development of mood disorders.

8 Schizophrenia is associated with a shortened lifespan in humans as reflected by 

9 increased standardized mortality ratio (McGrath et al., 2008). An environmental origin of 

10 schizophrenia was proposed years ago (Tsuang, 2000) but the specific link between prenatal 

11 Pb exposure, using ALAD as a biomarker, and schizophrenia is just beginning to appear in 

12 the literature and has been described in two publications (Opler et al., 2004; Opler et al., 

13 2008). Because of this, the animal toxicology literature is beginning to explore the 

14 mechanisms that may contribute to schizophrenia development and has proposed two 

15 explanations. These are Pb-induced NMDA receptor (NMDAR) hypofunction and Pb16 induced decreases in hippocampal neurogenesis (Figures 5-23, 5-24, and 5-25). Pb may bind 

17 a divalent cation site in the NMDAR and allosterically inhibit glycine binding 

18 (Hashemzadeh-Gargari & Guilarte, 1999); human studies of patients with schizophrenia 

19 have shown aberrations at this site (Coyle & Tsai, 2004). These findings are consistent with 

20 the glutamatergic hypothesis of schizophrenia which shows that NMDAR noncompetitive 

21 antagonist use in patients with schizophrenia exacerbates their psychotic symptoms and that 

Epidemilogic

associations

Immune

Elevated Cytokines

NMDA Receptor Sickness Behavior

Hypofunction

Decreased 

hippocampal

neurogenesis

Structural

Neurotransmitters

Behavioral

Humans Rodents

More 

emotional Depression 
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1 administration of antagonist to non-psychotic subjects can induce a schizophrenic 

2 phenotype. The second mechanistic hypothesis for Pb-associated schizophrenia induction is 

3 decreased hippocampal degenerate gyrus (DG) neurogenesis, which is seen in patients with 

4 schizophrenia (Kempermann et al., 2008; Reif et al., 2006), in animal models of 

5 schizophrenia {M, 2007, 670321} and in animal models with developmental Pb exposure 

6 (Jaako-Movits et al., 2005; Verina et al., 2007) (Figures 5-23, 5-24, and 5-25). Animal 

7 models of schizophrenia (i.e., phencyclidine administration) show decreased hippocampal 

8 DG neurogenesis that can be reversed by treatment with clozapine, which is often used in 

9 schizophrenia (Maeda et al., 2007). These DG pathways are also NMDAR-dependent. 

10 Studies cited in this section are further detailed in other sections of the ISA. Thus, a Pb11 dependent contribution to mood disorders exists in the toxicology literature with support 

12 from behavioral, neurochemical and ultrastructural data.

Figure 5-24. Schematic representation of the contribution of Pb exposure 

to the development of a phenotype consistent with 

schizophrenia.

13 As recently reviewed by Wright (2009), social stress and physical environmental toxins impact 

14 overlapping biological processes which determine adaptive plasticity in early neurodevelopment. 

15 Development of CNS organization into functional neuronal and synaptic networks can be determined by 

16 environmental signals which modify neuronalgenesis, synaptic formation and synaptic pruning (LeDoux, 

17 2003). Environmental factors can promote or disrupt this process depending on whether they are positive 

18 (social supports, good nutrition, etc.) or negative (psychosocial stress, chemical toxicants, malnutrition, 

19 trauma, etc.). While plasticity allows recovery from short-term toxic exposures, the neural mechanisms 

Rodent 

Studies

Pb exposure and Schizophrenia

Associated in Human Cohorts

(Epidemiologic)

NMDA Receptor

Hypofunction

(Neurochemical)

Glutamatergichypothesis of 

schizophrenia

Human Populations or Cohorts

Decreased Hippocampal

Neurogenesis

(Structural)

Pharmacologicallyinduced Schizophrenia in 

Animal models

Pb exposure NMDAR 

Antagonism
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1 underlying the plasticity of the developing brain exposed to chronic toxic exposures could induce 

2 permanent structural or organizational changes via altered neuronal growth and/or 

3 synaptogenesis/pruning. While historically research has focused on how social and physical 

4 environmental factors independently affect children’s health, evolving theory and methodologies 

5 underscore the importance of studying integrated effects (L. D. White et al., 2007; R. J. Wright, 2009). 

6 Recent studies highlight how social conditions influence susceptibility to future environmental exposures 

7 and, when contemporaneously exposed, how social-physical environmental interactions may account for 

8 more variance in explaining risk than main effects of either factor alone (R. J. Wright, 2009).

Source: Used with permission from Elsevier Science, 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.
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5.3.4. Sensory Acuity

5.3.4.1. Epidemiologic Studies of Children

1 Although not as widely examined as cognitive and behavioral outcomes, several studies 

2 demonstrated associations of blood Pb level with increased hearing thresholds and decrements in auditory 

3 processing in children (U.S. EPA, 2006). Such evidence has been limited largely to studies described in 

4 the 2006 Pb AQCD, including large U.S. studies, including NHANES II (J. Schwartz & Otto, 1987) and 

5 the Hispanic Health and Nutrition Examination Survey (HHANES) (J. Schwartz & Otto, 1991). In these 

6 studies, concurrent blood Pb level (median 8 μg/dL) from 6 to 18 μg/dL was associated with a 2-db loss 

7 in hearing and an increase in the percentage (15%) of children with a substandard hearing threshold 

8 (2,000 Hz). Blood Pb level also was associated with increases in hearing threshold across several 

9 frequencies in a population of children in Poland with similar blood Pb levels (median 7.2 μg/dL [range: 

10 1.9 to 28]) (Osman et al., 1999). In the HHANES and Polish studies, associations persisted in analyses 

11 restricted to subjects with blood Pb levels below 10 μg/dL. In the CLS, blood Pb level was associated 

12 with impaired auditory processing, albeit at higher concentrations. In this cohort, the mean (SD) lifetime 

13 average blood Pb level was 17.4 μg/dL (8.8), and a 1 μg/dL increase in lifetime average blood Pb was 

14 associated with a 0.07-point (p <0.05) lower score on the Filtered Word test, indicative of incorrect 

15 identification of filtered or muffled words (Dietrich et al., 1992). Despite the higher blood Pb levels in the 

16 CLS, the observed associations with auditory function were consistent with those with related endpoints, 

17 including cognitive deficits (Section 5.3.2.1) and behavioral problems (Section 5.3.3.1). 

5.3.4.2. Epidemiologic Studies of Adults

18 Rather than evidence for effect on hearing thresholds, among adults, evidence of association 

19 between blood Pb levels and auditory function comprised changes in auditory evoked brainstem 

20 potentials (U.S. EPA, 2006). Two studies of hearing thresholds came to somewhat different conclusions. 

21 One study examined 183 Pb-workers with blood Pb levels from 1 to 18 μg/dL and among multiple 

22 frequencies examined, found correlations between increasing blood Pb level and increased hearing 

23 threshold at 4 kHz (Forst et al., 1997). A second study included 220 Pb-battery workers with higher blood 

24 Pb levels (mean: 56.9 μg/dL [SD: 25.3]) (Wu et al., 2000). Although hearing impairment was associated 

25 with a measure of cumulative Pb exposure based on years of work and ambient Pb measurements, no 

26 association was found with blood Pb levels at the time of hearing testing in analyses adjusted for age, 

27 gender, and duration of employment. These findings may indicate that in an occupational setting with 

28 high Pb exposure, any one blood Pb measure may not be the best biomarker for cumulative exposure over 

29 the duration of work.
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1 Studies published since the 2006 Pb AQCD have produced findings consistent with previous 

2 studies. While studies of Pb-exposed workers continued to dominate, an NAS study provided new 

3 evidence for increasing bone Pb levels being associated with hearing loss in adult males without 

4 occupational Pb exposures (Park et al., 2010). A hospital-based case-control study recruited workers 

5 referred for hearing testing (average hearing thresholds above 25 dB) as cases and workers with normal 

6 hearing thresholds who were having occupational health examinations for other reasons as controls 

7 (Chuang et al., 2007). The 121 cases had a geometric mean blood Pb level of 10.7 μg/dL, and the 173 

8 controls had a geometric mean blood Pb level of 3.9 μg/dL. In models that adjusted for age, smoking, 

9 alcohol consumption, years of noise exposure, as well as Mn, As, and Se levels in blood, blood Pb levels 

10 was associated with a statistically significant higher average hearing threshold (0.5-6 kHz). 

11 Similar findings were reported in a study of 259 steel plant workers with no parental history of ear12 related problems, no congenital abnormalities, no occupational organic solvent exposure, and hearing loss 

13 difference no more than 15 dB between both ears (Y.-H. Hwang et al., 2009). The participants had an 

14 average blood Pb level of 54.3 μg/dL (SD: 34.6). Average noise levels also were measured in work areas 

15 and dichotomized at 80dB. In analyses adjusted for age and work area noise (dichotomized at 80dB), 

16 workers with blood Pb ≥ 7 μg/dL had a statistically significant increased odds (range of ORs: 3.06 to 

17 6.26) of hearing loss at frequencies of 3, 4, 6, and 8 kHz compared to workers with blood Pb levels ≤ 4 

18 μg/dL.

19 Park et al. (2010) analyzed data from 448 men in the NAS with an audiometric hearing test within 

20 5 years of bone Pb measurements (all but 5 of these men had audiometric testing before the bone Pb

21 measurement) and who did not have unilateral hearing loss. In cross-sectional analyses adjusted for age, 

22 race, education, body mass index, pack-years of cigarettes, diabetes, hypertension and occupational noise 

23 (based on a job-exposure estimate from occupations), and presence of a noise notch (indicative of noise24 induced hearing loss), higher patella bone Pb level was associated with a statistically significant higher 

25 hearing thresholds for frequencies greater than 1 kHz. In analyses of pure tone average hearing loss, a 21 

26 μg/g increase in patella bone Pb level (interquartile range) was associated with an OR of 1.48 (95% CI: 

27 1.14, 1.91) after controlling for all covariates. Similar, but slightly weaker associations were seen with 

28 tibia bone Pb levels. Audiometric data collected from the same men approximatly 20 years earlier 

29 (median observations/participant: 5; median follow-up: 23 years) were used to assess the association 

30 between tibia bone Pb levels and the change in hearing thresholds over that time. Increasing tibia Pb level 

31 was associated with faster rate of increase in hearing threshold for frequencies of 1, 2, and 8 kHz and a 

32 pure tone average. For the pure tone average, a 15 μg/g increase in tibia bone Pb level (interquartile 

33 range) was associated with an increased in hearing threshold of 0.05 dB per year. Blood Pb was not 

34 examined in this study. Together with those from studies of Pb-exposed workers, findings from the NAS 

35 study primarily indicate that biomarkers of cumulative Pb exposure are associated with increased hearing 

36 thresholds in adults. Because only bone Pb levels were examined in the NAS study, further investigation 
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1 is required to characterize potential differences between measures of cumulative and recent Pb exposures 

2 in the effects on hearing in adults without occupational exposures.

5.3.4.3. Toxicological Studies of Sensory Organ Function

3 Pb affects multiple aspects of the nervous system including the sensory organs. The 1986 and 2006 

4 Pb AQCDs detailed the effects of Pb on the retina, CNS visual processing areas, and the auditory system 

5 and described possible or known mechanisms of action where available. The new research examines 

6 effects on these systems at even lower Pb exposures and blood Pb levels.

The Effect of Lead on Vision

7 The selective action of Pb on retinal rod cells and bipolar cells is well documented in earlier 

8 AQCDs and research in this area continues to date (Fox et al., 1997; Fox & Sillman, 1979). Pb exposure 

9 during perinatal development and adulthood has also been shown to affect the visual cortex (L. G. Costa 

10 & Fox, 1983) and subcortical neurons (Cline et al., 1996). The 1986 AQCD mentioned that neonatal rats 

11 exposed to Pb out to PND21, through gestational and lactational exposure, had significant alterations in 

12 visual evoked responses and impaired visual acuity; it was hypothesized that a decreased number of 

13 cholinergic receptors and alterations in the ratio of inhibitory to excitatory systems in the cerebrospinal 

14 axis may be the underlying mechanisms leading to these retinal changes (U.S. EPA, 1986). A 1996 

15 publication detailing environmentally relevant doses of Pb administered to tadpoles showed that Pb 

16 inhibited the growth of developing neurons in the subcortical retinotectal pathway, the main efferent from 

17 the retina (Cline et al., 1996). The 2006 AQCD evolved to detail Pb-induced impairment of retinal 

18 function in non-human primates as well as focusing on mechanisms of action for specific physiological 

19 retinal changes using both in vitro and in vivo evidence, where available. With Pb-induced retinal effects, 

20 decreased maximal ERG amplitude or sensitivity and increased mean ERG latency was linked to 

21 increased retinal cGMP both in vitro and in vivo. Delayed dark adaptation and increased response 

22 thresholds at scotopic backgrounds were linked to in vivo apoptotic endpoints including rod bipolar cell 

23 death, increased Bax (apoptosis protein) translocation, increased cytochrome c release (apoptosis trigger), 

24 and decreased rhodopsin; in vitro evidence also included retinal apoptosis from the calcium/Pb signal 

25 localized to the mitochondrial permeability transition pore. Other endpoints seen in Pb-induced impaired 

26 retinal function included competitive inhibition of cGMP phosphodiesterase (PDE) in vitro and decreased 

27 stimulated cGMP PDE in vivo; also decreased retinal Na+/K+ ATPase activity has been reported both in 

28 vivo and in vitro. The effects of early life Pb exposure on the retina in monkeys was detailed in work by 

29 Reuhl et al. (1989) in the 2006 AQCD. Chronic Pb exposure from birth to age 6 produced 

30 cytoarchitectural changes in visual projection areas of the brain of rodents; maximum blood Pb level in 

31 the low and high dose group reached 20 μg/dL and 220μg/dL, respectively. Liliental et al. (1988) found 
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1 decreased amplitudes and increased latencies in visual evoked potentials from electroretinograms. Pb 

2 affected amplitude under dark conditions (dark adaptation, B waves affected) and latencies under bright 

3 conditions; blood Pb levels were 40 and 50 μg/dL in the 350 or 600 ppm Pb groups, respectively. Earlier 

4 work in rodents found that a moderate to high postnatal Pb exposure induced ERG subnormality (Fox & 

5 Chu, 1988; Fox & Farber, 1988; Otto & Fox, 1993). Thus, the historical animal toxicology literature 

6 shows multiple effects on vision from Pb exposure (Table 5-11, high dose). 

Table 5-11. Summary of toxicological studies of Pb on the retina.

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 affected, females not affected

No increase in retinal apoptosis

Increased progenitor cell proliferation

Increased neurogenesis of rods

Larger size of retina

7 The current literature for this ISA has more work by the Fox lab showing retinal effects in rodents 

8 after human equivalent gestational Pb exposure (GLE, gestationally out to PND10); this is 

9 developmentally equivalent to the in utero retinal development period in humans. Pb exposure during 

10 various developmental windows and at specific doses has been shown to significantly affect 

11 electroretinographs (ERG) both in Pb-exposed humans and rodents (Fox et al., 2008; Rothenberg et al., 

12 2002) (Table 5-11 and Figure 5-26). Consistent with low dose Pb exposure associated ERG 

13 supernormality in children (Rothenberg et al., 2002), Fox et al. (2008) found low and moderate ose 

14 gestational Pb-exposure (GLE) induced persistent supernormal scotopic ERGs in rodents. Low and 

15 moderate GLE also induced increased rod and rod bipolar cell neurogenesis (proliferation) and increased 

16 thickness and cell number of the outer and inner neuroblastic layers of the retina (ONL and INL) (Fox et 

17 al., 2008; Giddabasappa et al., 2011). Rodents with moderate dose GLE (blood Pb level 25 μg/dL) had 

18 27-fold increased retinal progenitor cell proliferation (Giddabasappa et al., 2011). Extension of the in vivo 

19 studies to isolated cultured cells showed GLE increased and prolonged proliferation of retinal progenitor 

20 cells (Giddabasappa et al., 2011). Nitric oxide has been shown to regulate retinal progenitor cell 

21 proliferation in chick embryos (Magalhaes et al., 2006). 

22 Because Pb exposure has been shown to impair NOS activity in other organs (Section 5.2.4.5), 

23 these authors postulate that impaired NO production may be linked to aberrant retinal cell proliferation 

24 (Giddabasappa et al., 2011). GLE did not significantly affect apoptosis during retinal development 

25 (Giddabasappa et al., 2011) but it did contribute to increased proliferation of retinal cells. GLE induced 

26 decreased DA synthesis and use in a dose-dependent fashion (Fox et al., 2008) (Figure 5-27). 
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Note: *p <0.05

Source: Fox et al. (2008)

Figure 5-26. Retinal a-wave and b-wave ERG amplitude in GLE adult males. 

Note: *p <0.05

Source: Fox et al. (2008)

Figure 5-27. Retinal dopamine metabolism in adult control and GLE rats. 

1 Many outcomes in this study showed an inverse U Pb dose-response curve as is shown with the 

2 high dose exposures having vastly different effects from low dose GLE. GLE exposure at high doses 

3 produced ERG subnormality, rod cells loss, and decreased rod neurogenesis (Fox et al., 2008) (Table 5-

4 11). The high dose GLE rodents showed dose-dependent decreased DA synthesis and use (Figure 5-27). 

5 These new animal toxicology data confirm the epidemiologic data showing ERG supernormality at low 

6 dose GLE. They provide further insight into retinal changes showing increased proliferation of Pb-
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1 exposed retinal progenitor cells without changes in apoptosis. GLE induced dose-dependent decrements 

2 in retinal DA use and synthesis.

Lead-Induced Auditory Effects

3 The 2006 Pb AQCD mentioned auditory effects on non-human primates who were exposed to Pb 

4 throughout gestation and out to age 8-9 (blood Pb levels 33-56 μg/dL during Pb exposure period). 

5 Auditory evoked potentials, which are used as a general test to assess neurological auditory function, 

6 revealed Pb related effects that persisted even after Pb exposure had ceased and blood Pb level had 

7 returned to baseline levels. In Pb-exposed animals, half of the pure tone detection thresholds were outside 

8 of the control range at certain frequencies (Rice, 1997), which is consistent with data from humans 

9 developmentally exposed to Pb. Thus, these authors found that early life Pb exposure impaired auditory 

10 function. The cochlear nerve in both developing and mature humans appears to be especially sensitive to 

11 the Pb insult. At low to moderate Pb exposures, elevated thresholds and increased latencies are seen in 

12 brainstem auditory evoked potentials. There is coherence between the animal and the human literature on 

13 the effects of chronic Pb exposure on auditory function. 

5.3.5. Neurodegenerative Diseases

5.3.5.1. Epidemiologic Studies of Adults

14 The 2006 Pb AQCD described several studies examining associations of blood and bone Pb levels 

15 with neurodegenerative diseases such as Alzheimer’s disease and dementia. Two NAS studies found 

16 associations between increasing bone Pb levels and decreasing MMSE scores (Weisskopf et al., 2004; R. 

17 O. Wright et al., 2003), which pointed to an association with dementia, given that the MMSE is widely 

18 used as a screening tool for dementia. Overall, studies had sufficient limitations, and findings were 

19 inconclusive (U.S. EPA, 2006). New studies on dementia are not available to assess further the 

20 association of Pb biomarkers with dementia. Similarly, new studies examining Alzheimer’s disease are 

21 not available, and as in 2006, the evidence is inconclusive regarding the association between Pb 

22 biomarkers and Alzheimer’s disease. In contrast, there has been additional investigation of ALS, 

23 Parkinson’s disease (PD), and essential tremor, which is described below.

Amyotrophic Lateral Sclerosis

24 Most studies of the association between Pb and ALS have relied on indirect methods of assessing 

25 Pb exposure and overall, have produced inconsistent results. The 2006 Pb AQCD reviewed two case26 control studies that measured blood Pb levels. One study found no difference in mean blood Pb levels 
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1 between the 16 ALS cases (mean: 12.7 μg/dL) and 39 controls (mean: 10.8 μg/dL) (Vinceti et al., 1997). 

2 Another study that examined blood and bone Pb levels in a New England-area population found increased 

3 odds of ALS among subjects with blood Pb levels ≥ 3 μg/dL (Kamel et al., 2002). In analyses of tibia or 

4 patella Pb tertiles, subjects in the highest two tertiles (≥ 10 μg/g patella Pb and ≥ 8 μg/g tibia Pb) had 

5 elevated odds of ALS; however, associations were not statistically significant. In analyses of Pb 

6 biomarkers as continuous variables, odds ratios for all three biomarkers were similar; however, only 

7 associations for blood Pb level were statistically significant. Kamel et al. (2002) also found that an 

8 estimate of cumulative Pb exposure based on occupational history to be significantly associated with ALS 

9 (Kamel et al., 2002). Thus, the stronger findings for blood Pb level were surprising given that bone Pb

10 level is a better biomarker of cumulative Pb exposure than is blood Pb level. One explanation for these 

11 findings is that the association could be the result of reverse causality since the half-life of blood Pb is 

12 only about 30 days, and blood was collected from people who already had ALS. If, for example, reduced 

13 physical activity among those with ALS led to more bone turnover, then more Pb would be released from 

14 bones into circulation leading to elevations in blood Pb levels among cases as a result of effects of the 

15 disease. 

16 Since the 2006 Pb AQCD, additional studies have been conducted in the New England-area case17 control study. One study indicated that the association between blood Pb level and ALS was not modified 

18 by the ALAD genotype (Kamel et al., 2005). Another study examined survival with ALS among 100 of 

19 the original 110 ALS cases (Kamel et al., 2008). Higher tibia Pb levels were associated with longer 

20 survival time. Findings were similar for patella and blood Pb levels, however, they were associated with 

21 smaller increases in survival time. These paradoxical findings raise the concern that in a case-control 

22 study of ALS, the association between bone Pb levels and ALS may be biased because the case group 

23 may comprise more individuals with longer survival time. Consequently, their bone Pb levels may be 

24 higher because they reflect a longer period of cumulative exposure. However, because the strongest 

25 findings for survival were found for tibia Pb, it might be expected a bias would be most apparent in a 

26 study examining associations of tibia Pb levels and ALS incidence. However, this was not observed in the 

27 one study that had bone and blood Pb biomarkers (Kamel et al., 2002). 

28 Another case-control study examined blood Pb levels and odds of ALS among 184 cases (33 were 

29 either progressive muscular atrophy or primary lateral sclerosis, mean blood Pb level: 2.41 μg/dL ) and 

30 194 controls (mean blood Pb level: 1.76 μg/dL) (Fang et al., 2010). The cases were recruited from the 

31 National Registry of U.S. Veterans with ALS, and controls were recruited from among U.S. Veterans 

32 without ALS frequency matched by age, gender, race, and past use of the Veterans Administration system 

33 for health care. A doubling of blood Pb levels was associated with an OR (95% CI) of 2.6 (1.9, 3.7). 

34 Associations did not differ substantially by indicators of bone turnover but were slightly higher among 

35 ALAD 1-1 carriers. The association with blood Pb level was similar in analyses that excluded the 

36 progressive muscular atrophy and primary lateral sclerosis cases. The similar results by degree of bone 
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1 turnover suggests that reverse causation is not likely driving the association between blood Pb level and 

2 ALS. Whether other types of reverse causality are occurring, however, cannot be ruled out. This study did 

3 not have measures of bone Pb and therefore could not assess the association with biomarkers of 

4 cumulative Pb exposure.

5 In summary, several studies have found associations of blood and bone Pb levels with ALS; 

6 however, the issues of reverse causality and bias due to survival time make it difficult to draw firm 

7 conclusions.

Parkinson’s Disease

8 A few studies, some ecological (Aquilonius & Hartvig, 1986; Rybicki et al., 1993) and some case9 control relying on questionnaire data or occupational history (Gorell et al., 1997; Gulson et al., 1999; 

10 Tanner et al., 1989) have indicated associations between exposure to heavy metals, particularly Pb, and 

11 risk of PD, although the evidence is limited and far from conclusive. Coon et al. (2006) expanded on

12 earlier studies that had found more than a twofold increased risk of PD among adults occupationally13 exposed to Pb for more than 20 years (Gorell et al., 1997; Gulson et al., 1999) by examining associations 

14 with bone and whole-body Pb levels. Coon et al. (2006) included 121 PD patients and 414 age-, sex-, and 

15 race-, frequency-matched controls all receiving health care services from the Henry Ford Health System.

16 Subjects in the highest quartile of both tibia (OR: 1.62 [95% CI: 0.83, 3.17] for levels ≥ 15 μg/g) and 

17 calcaneus (OR: 1.50 [95% CI: 0.75, 3.00] for levels ≥ 25.29 μg/g) bone Pb levels were at elevated odds of 

18 PD compared to those in the lowest quartile. The highest OR for PD was estimated for subjects in the 

19 highest quartile of whole-body lifetime exposure to Pb, compared to the lowest quartile of exposure (OR: 

20 2.27 [95% CI:1.13, 4.55] for levels ≥ 80.81 μg/g).

21 The second study to explore the association between biomarkers of Pb and PD was published 

22 recently (Weisskopf et al., 2010). This study was based in the Boston, MA area and had more than twice 

23 as many cases as Coon et al. (2006): 330 cases and 308 controls. Subjects in the highest quartile of tibia 

24 Pb level (>16.0 μg/g) had elevated odds of PD compared to those in the lowest quartile (OR: 1.91 [95% 

25 CI: 1.01, 3.60]). In this study, cases and controls were recruited from several different sources including 

26 movement disorder clinics and community-based cohorts, which could have introduced some biases. 

27 However, when analyses were restricted to cases recruited from movement disorder clinics and to their 

28 spouse, in-law, or friend as controls, the results were even stronger (OR: 3.21 [95% CI: 1.17, 8.83]). 

29 Although the use of spouse, in-law, and friend controls can introduce bias, this is expected to be towards 

30 the null as these groups are likely to share many exposures.
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Essential Tremor

1 The 2006 Pb AQCD described two studies that found associations between blood Pb levels and 

2 odds of PD in New York City-based populations (Louis et al., 2005; Louis et al., 2003). Since then, Dogu 

3 et al. (2007) reported on a case-control study of 105 essential tremor cases from a movement disorder 

4 clinic in Turkey and 105 controls (69 spouses and 36 other relatives living in the same district). Blood Pb 

5 levels in this study were comparable to those found in the earlier New York based studies: the median 

6 blood Pb was 2.7 μg/dL among cases and 1.5 μg/dL among controls. After adjusting for age, sex, 

7 education, cigarette smoking (yes versus no), cigarette pack-years, and ethanol use (yes vs. no), a 1 μg/dL 

8 increase in blood Pb level was associated with more than a fourfold increase in odds of essential tremor

9 (OR: 4.19 [95% CI: 2.59, 6.78]). This OR was much larger than that obtained in the New York study (OR: 

10 1.19 [95% CI: 1.03, 1.37]) (Louis et al., 2003). The magnitude of association in Dogu et al. (2007) is even 

11 more striking because so many of the controls were spouses who are expected to share many 

12 environmental exposures as cases. Most of the essential tremor cases were retired at the time of the study, 

13 but past occupation that could have contributed to Pb exposure (possibly stored in bone from where it 

14 could have contributed to blood Pb levels at the time of the study) was not assessed. One of the earlier 

15 New York studies also found a very high OR for essential tremor among ALAD2 carriers (per 1 μg/dL 

16 increase in blood Pb level, OR: 71.8 [95% CI: 1.08, 4789.68); however, the very wide 95% CI indicated 

17 lack of precision in the effect estimate.

18 Several studies of essential tremor have reported a very strong association between blood Pb levels

19 and essential tremor, although studies employing biomarkers of Pb have had relatively small sample sizes 

20 and have produced imprecise effect estimates.

5.3.5.2. Toxicological Studies

21 In epidemiological studies, Pb level (bone and blood) is associated with increased odds of ALS 

22 (Kamel et al., 2005) and paradoxically with longer survival time in patients diagnosed with ALS (Kamel 

23 et al., 2008). Chronic Pb exposure (Pb acetate in drinking water at 200 ppm from weaning onward, blood 

24 Pb level 27 μg/dL) reduced astrocyte reactivity and induced increased survival time in the superoxide 

25 dismutase transgenic (SOD1 Tg) mouse model of severe ALS (Barbeito et al., 2010). In this model, Pb 

26 exposure does not significantly increase the onset of the ALS disease in SOD1 Tg mice, but Pb exposure 

27 was associated with longer survival time in SOD1 Tg mice (Barbeito et al., 2010). Baseline levels of 

28 VEGF are elevated in astrocytes from the ventral spinal cord of untreated SOD1 Tg control mice versus 

29 control, non-transgenic animals. VEGF was not induced in Pb treated non-transgenic astrocytes. Further, 

30 Pb-exposed SOD1 Tg mice had significant elevations of astrocyte VEGF versus vehicle treated SOD1 

31 animals (Barbeito et al., 2010). Control animals exposed to Pb showed no elevation in VEGF expression 

32 above control vehicle-treated animals (Barbeito et al., 2010). Other research has suggested that ALS 
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1 initiation is dependent on motor neuron function and ALS progression is dependent on astrocyte and 

2 microglia function (Boillee et al., 2006; Yamanaka et al., 2008). 

3 Consistent these findings, other have reported that VEGF administration to the SOD1 Tg mice 

4 significantly reduced glial reactivity, a marker or neuroinflammation (Zheng et al., 2007). Using a cell 

5 based co-culture system of neurons and astrocytes, Barbeito et al (2010) found that an up-regulation of 

6 VEGF production by astrocytes in the Pb-exposed SOD1 Tg mice is protective against motor neuron 

7 death in the SOD1 Tg cells (Barbeito et al., 2010). Chronic Pb exposure in a mouse model of ALS was 

8 associated with increased survival time and correlated with higher spinal cord VEGF levels, making 

9 astrocytes less cytotoxic to surrounding motor neurons (Barbeito et al., 2010). Also, in another study the 

10 metal chelators DP-109 and DP-460 are neuroprotective in the ALS mouse model or Tg(SOD1-G93A) 

11 (Petri et al., 2007).

12 Improper activation of microglia and release of inflammatory cytokines and metabolites can 

13 contribute to neurodegeneration (L. Qian & Flood, 2008; D. Zhang et al., 2010). These two cell types are 

14 known to accumulate or sequester Pb in the nervous system. Researchers have implicated dysfunctional 

15 astrocytes as playing an important role in the chain of misregulated inflammation leading to 

16 neurodegenerative conditions (Barbeito et al., 2010; De Keyser et al., 2008).

Cell Death Pathways

17 Earlier work has documented that Pb exposure could induce cell death or apoptosis in various 

18 models including rat brain (Tavakoli-Nezhad et al., 2001), retinal rod cells (L. H. He et al., 2000; 2003), 

19 cerebellar neurons (Oberto et al., 1996), and PC12 cells (Sharifi & Mousavi, 2008). This study addresses 

20 chronic (40 days) Pb exposure-induced hippocampal apoptosis in young (exposure starting at 2-4 weeks 

21 of age) and old (exposure starting at 12-14 weeks of age) male rats exposed to 500 ppm Pb by drinking 

22 water (blood Pb, 98 μg/dL); apoptosis was verified by light and electron microscopy, and increased pro23 apoptotic Bax protein levels (Sharifi et al., 2010). Another study followed the developmental profile of 

24 changes in various apoptotic factors in specific brain regions of animals exposed to Pb acetate (0.2% dam 

25 drinking water) during lactation. Male offspring blood Pb at the end of lactation or PND20 was 80 μg/dL. 

26 These data showed hippocampal mRNA for various apoptotic factors including caspase-3, Bcl-x and 

27 Brain-derived neurotrophic factor (BDNF) was significantly upregulated on PND12, PND15 and PND20. 

28 The cortex of these male pups also showed upregulation of Bcl-x and BDNF on PND 15 and PND20 

29 (Chao et al., 2007). The cerebellum did not have elevated apoptotic mRNA levels in this model. This 

30 study shows temporal and regional changes in activation of death protein message levels in male 

31 offspring. Thus, the new data continue to confirm that Pb exposure induced apoptosis in brains of exposed 

32 animals. 
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Lead-Induced Neuronal Plaque Formation

1 Studies from the 2006 Pb AQCD highlighted the importance of Pb exposure in early life in 

2 promoting Alzheimer’s like pathologies in the adult brain. Pb has been recognized for decades as having a 

3 more profound effect on children than adults who receive the same exposure. In the last decade, the 

4 developmental origins of adult disease (DoAD) paradigm and the similar Barker hypothesis have reported 

5 that early life exposures can result in aberrant adult outcomes. Epidemiology data show that blood Pb 

6 level effects on neurological outcomes fit this paradigm. Recent evidence in the toxicological literature 

7 points to latent effects in rodent and non-human primate models of gestational and/or early life exposure 

8 to Pb including neurodegeneration similar to Alzheimer’s like pathologies, obesity (in males only), retinal 

9 aberrations (male only), and neurobehavioral aberrations. Immune outcomes fitting the DoAD paradigm 

10 involve tissue inflammation and loss of organ function. Bolin et al. (2006) demonstrated the connection 

11 between developmental exposure to Pb in the rat with early life programming and the resulting 

12 inflammation-associated DNA damage with neurodegenerative loss in the adult brain. Wu and colleagues 

13 (2008) had similar findings in a study using infantile exposure to Pb in monkeys. The investigations 

14 reinforce the need to directly examine the long-term effects of developmental exposure to xenobiotics 

15 rather than relying on adult exposure information to predict probable health risks from prenatal, neonatal 

16 or juvenile exposure (Dietert & Piepenbrink, 2006). Mechanistically, some of these pathologies have been 

17 associated with changes in the epigenome. Multiple Pb studies point to sensitive windows of exposure; 

18 early life or developmental exposures are far more sensitive than adult exposures. Alzheimer’s disease is 

19 characterized by amyloid-beta peptide (Ab) accumulation, hyper-phosphorlyation of the tau protein, 

20 neuronal death and synaptic loss. The toxicological evidence for Pb in contributing to the AD pathology 

21 follows.

22 The fetal basis of amyloidogenesis has been explored extensively by Zawia’s laboratory in both 

23 rodents and non-human primates. Amyloid deposits in the brain are seen in patients with Alzheimer’s 

24 disease and in the aged brain of individuals with Down Syndrome who display an Alzheimer’s-like 

25 pathology. Mechanistically, amyloid plaques originate from the cleavage of the amyloid precursor protein 

26 (APP) to Ab, which comprises the plaque. By exposing rodents to Pb as neonates or as aged animals, it 

27 was determined that neonatal Pb exposure is a sensitive window for induction of the amyloidogenesis in 

28 the aged animal brains and that exposure to Pb in old animals did not contribute to plaque formation. 

29 Following cortical APP gene expression over the lifetime of male rodents exposed neonataly via lactation 

30 to Pb (PND1-PND20 exposure, dam drinking water Pb acetate 200 ppm, pup PND20 blood Pb level 46 

31 μg/dL and cortex 0.41 μg/g wet weight of tissue), one sees a biphasic significant increase above control 

32 animals in APP expression with the first increased phase manifesting neonatally and the second phase 

33 manifesting in old age (82 weeks of age) (M.R. Basha et al., 2005). A concomitant biphasic response is 

34 seen in specificity protein 1 (Sp1), a transcription factor known to be related to APP expression. Ab, the 
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1 amyolid plaque constituent, was also significantly elevated in these aged animals developmentally 

2 exposed to Pb. A separate subset of rodents exposed to Pb only as aged adults (18-20 weeks of age) were 

3 unresponsive in APP or Sp1 expression or Ab production after Pb exposure, indicating the developmental 

4 window and not adult exposure as the susceptible period for Pb-dependent amyloidogenesis. The Zawia 

5 lab (J. Wu et al., 2008) has confirmed similar amyloid findings in brains of monkeys that were exposed to 

6 Pb as infants (PND1-PND400), i.e., significantly higher message levels of APP, and Sp1 and significantly 

7 higher protein expression of APP and Ab in aged female monkey cortex tissue (23 year old Macaca 

8 fascicularis) from a cohort of animals established in the 1980s (Rice, 1990, 1992). After weaning and 

9 when still being dosed with Pb, the monkeys had blood Pb level of 19-26 μg/dL. As a caveat, by the time 

10 neonatally Pb-exposed animals become aged and manifest with amyloid plaques, blood Pb level and brain 

11 cortex Pb levels have returned to control or baseline levels. Thus, the rodent and non-human primate 

12 toxicology studies concur and show that developmental Pb exposure induced elevations in neuronal 

13 plaque proteins in the aged animals. 

14 Transcription factors are essential in the regulation of the developing brain. Pb exposure is known 

15 to perturb DNA binding of transcription factors including SP1 at essential sites like zinc finger proteins.

16 In Long-Evans hooded rat pups exposed during lactation, these Pb-induced developmental perturbations 

17 of SP1 DNA binding can be ameliorated by exogenous zinc supplementation (M. R. Basha et al., 2003). 

18 The mechanism by which Ab peptide formation is affected by Pb exposure has been explored by 

19 Behl et al. It has been shown that the choroid plexus is able to remove beta-amyloid peptides from the 

20 brain extracellular matrix and that Pb impairs this function and may be mediated by the 

21 metalloendopeptidase, insulin-degrading enzyme (IDE), which metabolizes Ab (Behl et al., 2009).

22 Further studies with developmental Pb exposure (gestational plus lactational, dam drinking water 

23 solutions of 0.1%, 0.5% or 1%, blood Pb level 400, 800 and 1,000 μg/L) showed that the hippocampus 

24 contained neurofibriallary changes as early as PND21. These changes manifested with Tau hyper25 phosphorylation, and increased tau and beta amyloid hippocampal protein levels in Pb-exposed offspring 

26 (Li et al., 2010). The multiple new studies on Pb-dependent changes in the neurofibrillary proteins show 

27 that developmental Pb exposure induced significant increases in neuronal plaque associated proteins, 

28 indicating that early life Pb exposures may contribute to dementia in adulthood. 

29 Data from the animal toxicology literature point to an early life window in which Pb exposure can 

30 contribute to pathological brain changes consistent with those seen in Alzheimer’s disease including Ab 

31 peptide accumulation and activation of Ab supporting transcription factors as well as tau 

32 hyperphosphorylation. 
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5.3.6. Studies of Mechanisms of the Neurological Effects of 

Lead

5.3.6.1. Effects on Brain Physiology and Activity

1 A growing body of epidemiologic evidence demonstrates associations of Pb biomarkers with 

2 electrophysiologic changes in the brain. By providing insight into the underlying mechanisms by which 

3 Pb exposure may disrupt brain function, findings from these studies have provided biologically plausible 

4 evidence for the effects of Pb exposure on cognitive, psychological and behavioral consequences 

5 observed in children and adults. Much of the early work was conducted by Otto and colleagues (Otto et 

6 al., 1985; Otto & Fox, 1993), which found associations of blood Pb level with auditory and visual evoked 

7 potentials. Rothenberg et al. (1994) and Rothenberg et al. (2000) reported similar findings; however, the 

8 direction of association differed between prenatal (maternal) and postnatal (ages 1-4 years) blood Pb

9 level. Postnatal blood Pb level was associated with a decrease in interpeak intervals in auditory evoked 

10 potentials at age 5-7 years. Prenatal blood Pb level showed a biphasic relationship, with a negative 

11 association at blood Pb levels of 1-8 μg/dL and a positive association at blood Pb levels of 8-30 μg/dL. 

12 These findings provide mechanistic support for observations of Pb-associated changes in sensory acuities

13 (Section 5.3.4.1).

14 Studies using magnetic resonance imaging (MRI) or spectroscopy (MRS) as clinical outcome 

15 measures have been limited in number and sample size, but have shown associations of blood Pb level

16 with alterations in brain physiology such as reduced levels of N-acetylaspartate, creatine, or choline in 

17 young adults (Cecil et al., 2005; Meng et al., 2005; Trope et al., 2001; Trope et al., 1998; Yuan et al., 

18 2006). These changes have been linked to decreased neuronal density or loss and alteration in myeline. 

19 Notably, Trope et al. (2001; 1998) and Meng et al. (2005) reported that all subjects had normal MRIs with 

20 no evidence of structural abnormalities. Thus, the clinical significance of the observed physiological 

21 changes is unclear. Additionally, these studies compared subjects with relatively high childhood blood 

22 levels (23-65 μg/dL) to those with childhood blood Pb levels <10 μg/dL. Therefore, it is unclear whether 

23 physiological changes would be observed in association with lower blood Pb levels. Cecil et al. (2005)

24 and Yuan et al. (2006) conducted functional MRI in 42 adult (ages 20-23 years) participants from the CLS 

25 cohort during a verb generation language task and found that mean childhood blood Pb level was 

26 associated with decreased activation in the left frontal gyrus and left middle temporal gyrus, regions 

27 traditionally associated with semantic language function. Although these findings were in adults, they 

28 were consistent with findings in the same cohort of associations of blood Pb level with other indices of 

29 language in childhood.

30 Since the 2006 Pb AQCD, studies examining MRI data have been limited to CLS cohort 

31 participants as adults (ages 19-24), and recent results continue to support associations of childhood blood 
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1 Pb levels with physiological changes in the brain in adulthood. These recent studies expanded on previous 

2 studies by including larger sample sizes and aiming to characterize Pb effects more precisely by 

3 examining blood Pb levels at different periods in childhood and aiming to link changes in brain activity to 

4 neurodevelopmental deficits. Brubaker et al. (2009) used diffusion tensor imaging to examine 

5 associations between mean childhood blood Pb and white matter integrity in 91 young adults,

6 hypothesizing that childhood Pb exposure may alter adult white matter architecture via deficits in axonal 

7 integrity and myelin organization. Fractional anisotropy (FrA), mean diffusivity (MD), axial diffusivity 

8 (AD), and radial diffusivity (RD) were measured on an exploratory voxel-wise basis. In adjusted 

9 analyses, mean childhood blood Pb levels were associated with decreased FrA throughout white matter. 

10 Regions of the corona radiata demonstrated highly significant Pb-associated decreases in FrA and AD and 

11 increases in MD and RD. The genu, body, and splenium of the corpus callosum demonstrated highly 

12 significant Pb-associated decreases in RD, smaller and less significant decreases in MD, and small areas 

13 with increases in AD. The results of this analysis suggest multiple insults appear as distinct patterns of 

14 white matter diffusion abnormalities in the adult brain which may be indicative of altered myelination and 

15 axonal integrity. Additionally, childhood blood Pb levels appear to differentially affect neural elements, 

16 which may be related to the stage of myelination development at various periods of exposure.

17 Another study of 157 CLS participants provided evidence of region-specific reductions in adult 

18 gray matter volume in association with childhood blood Pb levels and by examining associations between 

19 MRI-assessed brain volume and historic neuropsychological assessments, provided insight into the 

20 potential clinical significance of changes in brain physiology associated with blood Pb levels (Cecil et al., 

21 2008). Using conservative, minimum contiguous cluster size and statistical criteria (700 voxels, 

22 unadjusted p <0.001), approximately 1.2% of the total gray matter was significantly and inversely 

23 associated with mean childhood blood Pb level. The most affected regions included frontal gray matter, 

24 specifically the anterior cingulate cortex and the ventrolateral prefrontal cortex (i.e., areas traditionally 

25 related to executive functions, mood regulation, and decision-making). Comparing neuropsychological 

26 factor scores with gray matter volume, investigators found that fine motor factor scores positively 

27 correlated with gray matter volume in the cerebellar hemispheres; adding blood Pb level as a variable to 

28 the model attenuated this correlation. These findings are notable in light of other studies linking brain 

29 volume changes with altered function and suggest that MRI changes association with blood Pb levels may 

30 be indicative of decrements in neurocognitive and neurobehavioral function. Schwartz and colleagues 

31 (2007) showed that larger RO1 volumes were associated with better cognitive function in 5 or 6 cognitive 

32 domains (visuoconstruction, processing, speed, visual memory, executive functioning, and eye-hand 

33 coordination). More recent studies by Raine and colleagues suggest that deficits in cortical volume or 

34 activity found in select brain regions, including the prefrontal gray matter, may predispose individuals to 

35 impulsive, aggressive, or violent behavior. 
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1 In a subsequent analysis, Brubaker et al. (2010) investigated the developmental trajectory of 

2 childhood blood Pb levels on adult gray matter. Adjusted voxel-wise regression analyses were performed 

3 for associations between adult gray matter volume loss and yearly mean blood Pb levels from 1 to 6 years 

4 of age in the entire cohort and by sex. Investigators observed significant inverse associations between

5 gray matter volume loss and yearly mean blood Pb levels from 3 to 6 years of age. The extent of 

6 prefrontal gray matter loss associated with yearly mean blood Pb levels increased with advancing age of 

7 the subjects. These results indicate that blood Pb levels later in childhood are associated with greater 

8 losses in gray matter volume than are childhood mean or maximum blood Pb levels. This study 

9 demonstrates that maximum blood Pb levels do not fully account for gray matter changes, particularly in 

10 the frontal lobes of young men. Notably, although they did not consider Pb, Yang et al. (2005) reported 

11 volume reduction in gray matter in psychopaths, adding additional evidence that these physiological 

12 changes may be related to overt deleterious outcomes. Consistent with Wright et al. (2008), Cecil et al. 

13 (2008) found that gray matter volume loss associated with childhood blood Pb levels was much larger in 

14 CLS male adults than female adults. In an expanded analysis of the developmental trajectory of childhood 

15 blood Pb levels on adult gray matter, Brubaker et al. (2010) found that the inverse associations between

16 gray matter volume loss and yearly mean blood Pb levels were more pronounced in the frontal lobes of 

17 men than women for blood Pb levels measured at all ages.

18 Whereas the aforementioned CLS studies examined associations of childhood blood Pb levels, a 

19 recent analysis of the NAS participants indicated that biomarkers of cumulative, long-term Pb exposure 

20 also may be associated with changes in brain structure and function in older adults. Weisskopf et al. 

21 (2007) found increasing bone Pb level to be associated with increased myoinositol (mI)/Cr ratio with 

22 increasing bone Pb concentration among 31 elderly men from the NAS. A higher mI/Cr ratio may be 

23 indicative of glial activation and is a signal reportedly seen in the early stages of HIV-related dementia 

24 and Alzheimer’s disease.

25 Studies of Pb-workers also found associations of blood and bone Pb levels with changes in brain 

26 structure and physiology. Stewart et al. (2006) studied 532 former organolead workers with a mean age of 

27 56 years and found that an estimate of past peak tibia bone Pb (mean: 23.9 μg/g) was significantly 

28 associated with more white matter lesions (WML). Higher estimated peak tibia Pb also was associated 

29 with smaller total brain volume and volumes of frontal and total gray matter, parietal white matter, the 

30 cingulate gyrus, and insula. In this same group, Caffo et al. (2008) found evidence that the association 

31 between tibia Pb level and cognitive function, in particular, visuo-construction domain tasks, and to a 

32 lesser degree, executive function and eye-hand coordination, were mediated by the association between 

33 tibia Pb levels and brain region volume changes. In a similar study of 61 current Pb smelter workers with 

34 an average age of 40 years, higher estimates of cumulative Pb exposure were also associated with WML, 

35 and there was evidence that this association accounted for, in part, the association between higher 

36 cumulative Pb exposure and worse performance on the grooved pegboard test (Bleecker, Ford, Vaughan, 
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1 et al., 2007). Another small study of 15 current occupationally Pb-exposed workers (mean blood Pb level: 

2 63.5 μg/dL) and 19 non-Pb exposed controls (mean blood Pb level: 8.74 μg/dl) found smaller 

3 hippocampal volumes in MRI scans among the Pb workers (Jiang et al., 2008). A lower ratio of the brain 

4 metabolites N-acetyl-aspartate (NAA) and creatine (Cr), indicative of neuronal density, was also found 

5 among the Pb workers, as well as an increased lipid to creatine ratio, indicative of cell membrane or white 

6 matter damage. Similarly, a study of 22 Pb paint factory workers and 18 controls found lower NAA/Cr 

7 ratios among the Pb exposed workers as well as lower choline/Cr, possibly indicative of reduced cell 

8 membrane turnover (Hsieh et al., 2009). 

5.3.6.2. Cholesterol and Lipid Homeostasis

9 Various pathological conditions are associated with elevated plasma free fatty acids or elevated 

10 cholesterol. Adult male rats exposed to Pb acetate (200, 300 or 400 ppm) in their drinking water for 12 

11 weeks manifested with Pb-induced cholesterogenesis and phospholipidosis in brain tissue (Ademuyiwa et 

12 al., 2009). Pb-dependent changes in brain cholesterol produced an inverse U dose response curve with the 

13 highest brain cholesterol at 200 ppm followed by 300 ppm Pb. Animals exposed to 400 ppm Pb did not 

14 have significant changes in brain cholesterol. Mechanistically, Pb exposure has been shown to depress the 

15 activity of cholesterol-7-a-hydroxylase, an enzyme involved in bile acid biosynthesis (Kojima et al., 

16 2005); bile acids are the route by which cholesterol is eliminated from the body. Pb exposure produced 

17 significant increases in brain triglycerides with an 83% increase at 300 ppm and a 108% increase at 400 

18 ppm. At 200 ppm, Pb exposure induced a non-significant decrease in brain triglycerides. Pb exposure 

19 across all three dose groups induced significantly increased brain phospholipids. Interestingly, plasma free 

20 fatty acids were significantly elevated in a dose-dependent fashion; plasma triglycerides and cholesterol 

21 were unaffected by Pb exposure. The molar ratio of brain cholesterol to phospholipids, an indicator of 

22 membrane fluidity (Abe et al., 2007), was significantly increased at 200 and 300 ppm Pb exposure 

23 indicating increased membrane fluidity. Brain Pb in all dose groups was below the limit of detection (0.1 

24 ppm). Blood Pb at 0, 200, 300, and 400 ppm were 7, 41, 61, and 39 μg/dL, respectively. In summary, Pb 

25 exposure significantly increased brain cholesterol, triglycerides, and phospholipids as well as significantly 

26 increased plasma free fatty acids. These effects were sometimes more prominent at lower doses of Pb. 

27 Future characterization of molecular and cellular pathways affected by Pb exposure may bring insight to 

28 this Pb-dependent phospholipidosis and cholesterogenesis. 

5.3.6.3. Oxidative Stress

29 Pb has been shown to induce oxidative stress in multiple animal models and this oxidative stress 

30 can contribute to DNA damage, which can be measured with the biomarker 8-hydroxy-2’-

31 deoxyguanosine (8-oxo-dG). The contribution of reactive oxygen or nitrogen species to these Pb induced 
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1 changes was assayed by looking at the ratio of 8-oxo-dG to 2-deoxyguanosine (2-dG). 2-dG is a DNA 

2 nucleoside enzyme that can generate 8-oxo-dG from a parent compound forming a DNA adduct or 

3 biomarker during conditions of nitrosative or oxidative stress. The 8-oxo-dG to 2-dG ratio data from 

4 rodent male offspring recapitulated the amyloid data with significant biphasic elevations in 

5 developmentally Pb-exposed animals (0.2% Pb acetate in dam drinking water from PND1-20) versus 

6 control, non-Pb exposed animals at early (PND5) and late life time points (80 weeks of age) (Bolin et al., 

7 2006). Activity of the base-excision DNA repair enzyme oxoguanine glycosylase or Ogg1 was unaffected 

8 by Pb exposure (Bolin et al., 2006). Interestingly, the monkey data were the same as the rodent data. The 

9 ratio of 8-oxo-dG to 2-dG in the brains of aged monkeys (23 years) after being exposed as infants, was 

10 significantly elevated above controls (J. Wu et al., 2008). Similar to the amyloid data, the oxidative stress 

11 markers showed no significant changes above baseline when animals were exposed to Pb as aged adults 

12 (Bolin et al., 2006; J. Wu et al., 2008). Thus, the data for biomarkers of oxidative stress concur with the 

13 amyloidogenesis data with both demonstrating kinetically similar biphasic significant elevations in 

14 markers of oxidative stress and amyloidogenesis with early life Pb exposure.

15 Because the brain has the highest energy demand and metabolism of any organ, energy homeostasis 

16 is of utmost importance. Pb has been shown to inhibit various enzymes involved in energy production or 

17 glucose metabolism including glyceraldehydes-3 phosphate dehydrogenase, hexokinase, pyruvate kinase, 

18 and succinate dehydrogenase (Regunathan & Sundaresan, 1984; Sterling et al., 1982; Verma et al., 2005; 

19 Yun & Hoyer, 2000). Mitochondria produce ATP or energy through oxidative phosphorylation. Aberrant 

20 mitochondrial function can decrease the energy pool and contribute to ROS formation via electron 

21 transport chain disruption. ATP depletion can also affect synaptic and extracellular neuotransmission. The 

22 mitochondrial Na/K ATPase is important in maintaining the inner mitochondrial membrane potential 

23 (delta omega sub m) and the health of the mitochondria. To address the effect of Pb exposure on these 

24 mitochondrial parameters, mice were mated, produced offspring and nursed the offspring until PND8 at 

25 which time the brains were collected from the pups (Baranowska-Bosiacka et al., 2011). Cerebellar 

26 granular cells were harvested from these PND 8 control and Pb-exposed animals (0.1% Pb acetate in dam 

27 drinking water, blood Pb level 4 μg/dL and cerebella Pb 7.2 μg/g dry weight). These neuronal cells were 

28 cultured for 5 days in vitro, at which point various mitochondrial parameters were measured. With Pb 

29 exposure, reactive oxygen species were significantly increased in both the cortical granule cells and in the 

30 mitochondria. Intracellular ATP concentration and adenylate energy charge values were significantly 

31 decreased in cells of Pb-exposed mice versus control. Neuronal Na/K ATPase activity was significantly 

32 lower in cortical granule cells from Pb-exposed mice versus controls. Mitochondrial mass was unaffected 

33 with Pb treatment, but mitochondrial membrane potential was significantly decreased with Pb exposure. 

34 Pb-exposed crayfish who are placed under hypoxic conditions adapt to the situation by decreasing their 

35 metabolism (Morris et al., 2005), manifesting with whole organism findings consistent with these cell

36 data. These data show impaired mitochondrial function and energy production in neuronal cells from mice 
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1 with gestational and lactational Pb exposure with concomitant increases in mitochondrial and cellular 

2 ROS production. 

5.3.6.4. Nitrosative Signaling and Nitrosative Stress

3 The nitric oxide system is increasingly being recognized as a signaling system in addition to its 

4 more classical role as a marker of cellular stress. In studies of learning and memory using the Morris 

5 water maze, hippocampal changes in NO were noted after completion of the test. Pb exposure has been 

6 repeatedly shown to increase the escape latency in Pb-exposed animals (Section 5.3.2.2). Chetty (2001)

7 initially reported decreased hippocampal nNOS with perinatal Pb exposure. Namely, with repeated swim 

8 tests, control animals more quickly find a submerged platform, i.e. escape, than do Pb-exposed animals. 

9 After either 4 or 8 weeks of Pb exposure to weanling male rats (blood Pb level 0.3 umol/L), hippocampal 

10 NOS and NO are significantly decreased. Dietary supplementation concomitant with 8 weeks of Pb 

11 exposure induced significant increases in hippocampal NOS (taurine or glycine) and decreases in 

12 hippocampal NOS (vitamin C, methionine, tyrosine, or vitamin B1). These animals also had significant 

13 changes in hippocampal NO with supplementation. NO increased with taurine and decreased with vitamin 

14 C, tyrosine or glycine co-exposure with Pb. Dietary supplementation after 4 weeks of Pb exposure in 

15 weanling males (4 week blood Pb level 2.3 umol/L & 8 week Blood Pb level 0.39 umol/L), induced 

16 significant increases in NO with the supplements tyrosine, methionine, or ascorbic acid. Zinc 

17 supplementation in this model had no effect on the NO system. The conclusions of this study are that 

18 various combinations of nutrients significantly attenuate Pb-dependent decreases in NO/NOS. 

19 Specifically, nutrients prevented (8 weeks Pb plus concomitant exposure to methionine, zinc, ascorbic 

20 acid, and glycine) or restored (4 weeks Pb exposure followed by 4 weeks nutrient exposure, taurine and 

21 thiamine) Pb-dependent decrements in NO/NOS concentrations (G. Fan et al., 2009). 

5.3.6.5. Synaptic Changes

22 Work in earlier criteria documents as well as earlier publications in the scientific literature point to 

23 an effect of developmental Pb exposure on synapse development, which mechanistically may contribute 

24 to multiple Pb related aberrant outcomes, including changes in long-term potentiation (LTP) and 

25 facilitation. Earlier work has shown that developmental Pb exposure is responsible for altered density of 

26 dendritic hippocampal spines (Király & Jones, 1982; Petit & LeBoutillier, 1979), aberrant synapse 

27 elimination (Lohmann & Bonhoeffer, 2008) and abnormal long-term and short-term plasticity 

28 (MacDonald et al., 2006). Newer research using the Drosophila larval neuromuscular junction model has 

29 shown that stimulation with multiple action potentials (also called AP trains) induced significant increases 

30 in intracellular calcium and significant delays in calcium decays back to baseline levels at the pre31 synaptic neuronal bouton in developmentally Pb exposed larvae versus control. Pb-exposed larvae had 
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1 reduced activity of the plasma membrane calcium ATPase, which is responsible for extravasations of 

2 calcium from the synaptic terminal (T. He et al., 2009). Intracellular calcium in Pb exposed larvae was no 

3 different from controls under resting conditions or in neurons with stimulation by a single action 

4 potential. Pb media concentrations in these experiments were 100 or 250 μM with the low dose body 

5 burden (100 μM) of Pb calculated to be 13-48 μM per larvae. Facilitation of a neuronal terminal is 

6 defined as the increased ability to transmit an impulse down a nerve due to prior excitation of the nerve. 

7 After stimulation of the axon, facilitation of the excitatory post-synaptic potential, which is dependent on 

8 residual terminal calcium, was significantly elevated in Pb exposed larvae versus control (T. He et al., 

9 2009). The data from this synapse study demonstrate that developmental Pb exposure affected the plasma 

10 membrane calcium ATPase, induced changes in the intracellular calcium levels during impulse activation, 

11 and produced changes in facilitation of the neuronal networks of Drosophila. Thus, the neuromuscular 

12 junction is a potential site of Pb interaction. 

13 A study by Li et al. (2009) focused on inflammatory endpoints and synaptic changes after 

14 gestational plus lactational dam drinking water Pb exposure (solutions of 0.1%, 0.5% or 1%, offspring 

15 blood Pb level 40, 80 and 100 μg/dL, respectively at PND 21). Hippocampal TNF-alpha was significantly 

16 elevated with Pb exposure and proteins that comprise the SNARE complex were all changed with Pb 

17 exposure. The SNARE complex of synaptic proteins includes SNAP-25, VAMP-2 and Syntaxin 1a and is 

18 essential in exocytotic neurotransmitter release at the synapse (Li et al., 2009). Thus, Li et al. (2009)

19 found significant difference in hippocampal synaptic protein composition and increased pro-inflammatory 

20 cytokine levels in the brains of Pb-exposed offspring.

21 Neurotransmission is an energy-dependent process with calcium-dependent ATP releases found at 

22 the synaptic cleft. At the synapse, ATP is metabolized by ecto-nucleotidases. In heme synthesis, Pb is 

23 known to substitute for the cation zinc in another nucleotidase, pyrimidine 5′-nucleotidase, and is thus 

24 used as a biomarker of Pb exposure. Acute exposure (96h) of male and female zebrafish to Pb acetate (20 

25 μg/L) in their water induced significant decreases in ATP hydrolysis in brain tissue. This dose is deemed 

26 to be an environmentaly relevant dose. With a chronic exposure (30 days), Pb acetate promoted the 

27 inhibition of ATP, ADP and AMP hydrolysis; these data are consistent with findings in rodents 

28 (Baranowska-Bosiacka et al., 2011). The authors hypothesize that at 30 days, this change in nucelotide 

29 hydrolysis was likely due to post-translational modification because message level of enzymes 

30 responsible for the hydrolysis, NTPDase1 and 5′-nucleotidase, were unchanged (Senger et al., 2006). 

31 Thus, Pb is shown to affect nucleotidase activity in the central nervous system of zebrafish, possibly 

32 contributing to aberrant neurotransmission.

33 Another enzyme important in synaptic transmission at cholinergic junctions in the CNS and at 

34 neuromuscular junctions peripherally is acetylcholinesterase (AChE). After 24 hours of exposure to 

35 environmentally relevant concentrations of PbAcetate (20 ug/L water), AChE activity was significantly 

36 inhibited in zebrafish brain tissue. In Pb-exposed fish, AChE activity returned to baseline by 96 hours and 
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1 maintained baseline activity after chronic exposure of 30 days. Thus, Pb is also able to affect synaptic 

2 homeostasis of AChE in the brain of exposed zebrafish (Richetti et al., 2010). 

3 Pb is known to act as an antagonist of the NMDA receptor. The NMDAR is essential for proper 

4 pre-synaptic neuronal activity and function. Primary cultures of mouse hippocampal cells were exposed to 

5 Pb (10 or 100 μM solutions in media) during the period of synaptogenesis (Neal et al., 2010). This 

6 exposure induced the loss of two proteins necessary for presynaptic vesicular release, synaptophysin 

7 (Syn) and synaptobrevin (Syb), without affecting a similar protein synaptotagmin (Syt). This deficit is 

8 seen in both GABAergic and glutamatergic neurons. Pb also induced an increase in number of 

9 presynaptic contact sites. But, these sites may be non-functional as they lack the protein receptor 

10 complexes necessary for proper vesicular exocytosis. Another factor involved in growth and signaling of 

11 pre-synaptic neurons is BDNF, which is synthesized and released by post-synaptic neurons. BDNF is 

12 regulated by the NMDAR and acts in a retrograde fashion, participating in pre-synaptic maturation. In 

13 this model, both pro-BDNF and BDNF release were significantly attenuated with Pb exposure. Further, 

14 exogenous BDNF administration rescued the aforementioned Pb-dependent pre-synaptic effects. Thus, 

15 this cell culture model shows that Pb-dependent pre-synaptic aberrations are controlled by NMDAR16 dependent BDNF effects on synaptic transmission.

5.3.6.6. Blood Brain Barrier

17 Two barrier systems exist in the body to separate the brain or the central nervous system from the 

18 blood. These two barriers are the blood brain barrier (BBB) and the blood cerebrospinal fluid barrier 

19 (BCB). The blood brain barrier, formed by tight junctions at endothelial capillaries forming the zonulae 

20 occludens (occludins, claudins, and cytoplasmic proteins), separates the brain from the blood and its 

21 oncotic and osmotic forces, allowing for selective transport of materials across this barrier. Pb exposure 

22 during various developmental windows is known to affect the blood brain barrier even at low Pb 

23 concentrations resulting in increased permeability (Dyatlov et al., 1998; Moorhouse et al., 1988; 

24 Struzynska, Walski, et al., 1997; Sundstrom et al., 1985). Because the BBB is under-developed early in 

25 life, prenatal and perinatal Pb exposure results in higher brain Pb accumulation than does similar 

26 exposures later in life (Moorhouse et al., 1988). Earlier AQCDs have shown that the chemical form of Pb, 

27 and its ability to interact with proteins and other blood components affects its ability to penetrate the BBB 

28 (U.S. EPA, 2006). Pb compromises the function of the BCB, and decreased the CSF level of transthyretin, 

29 a thryoid binding protein made in the choroid plexus. The choroid plexus and cerebral endothelial cells 

30 that form these BBB and BCS tight junctions are known to accumulate Pb more than other cell types and 

31 regions of the CNS. Hypothyroid status can contribute to impaired learning and IQ deficits. More recent 

32 research with weanling rats fed Pb through Pb acetate drinking water exposure manifested histologically 

33 with leaky cerebral vasculature as detected with lanthum nitrate staining of the brain parenchyma, an 
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1 indication of BBB impairment, that was ameliorated or that resembled controls after iron 

2 supplementation. These weanlings also had significant Pb-induced decreases in the BBB tight junction 

3 protein occludin in the hippocampus, brain cortex, and cerebellum that were rescued to control levels with 

4 iron supplementation (Q. Wang et al., 2007). These data demonstrate that Pb induced a leaky BBB in 

5 weanling rats with associated decreases in the junctional protein occludin; dietary supplementation with 

6 iron was able to ameliorate these Pb-induced impairments of the BBB in male rats. 

5.3.6.7. Cell Adhesion Molecules

7 Classic cell adhesion molecules including NCAM and the cadherins are junctional or cell surface 

8 proteins that are critical for cell recognition and adhesion. Cell adhesion molecules, particularly the 

9 cadherins, are calcium-dependent and thus interaction from competing cations like Pb can contribute to 

10 nervous system barrier function disruption, tissue development dysregulation, immune dysfunction, and 

11 affect learning and memory (Prozialeck et al., 2002). 

5.3.6.8. Glial Effects

12 Astroglia and oligodendroglia are supporting cells in the nervous system that maintain the 

13 extracellular space in the brain and provide support and nutrition to neurons via nutrient transport, 

14 strucutral support to neurons, and myelination, among other effects. Glial cells are known to serve as Pb 

15 sinks in the developing and mature brain (Tiffany-Castiglioni et al., 1989) by sequestering Pb. This glial 

16 sequestration of Pb has been shown to decrease brain glutamine concentrations at a dose of 0.25 ± 1.0 μM

17 Pb acetate via Pb-dependent reduction in glutamine synthestase activity in the astroglia; astroglia take up 

18 glutamate after its release and convert it to glutamine. Pb causes hypomyelination and demyelination (F. 

19 Coria et al., 1984) mediated through the oligodendrocytes with younger animals being more susceptible to 

20 the effects of (Tiffany-Castiglioni et al., 1989). Unfortunately, Pb accumulation in young glial cells may 

21 contribute to a lifelong exposure to this Pb sink in the brain as it is released over time where it can 

22 damage surrounding neurons (Holtzman et al., 1987).

Glial transmitters

23 To determine the contribution of the gliotransmitter serine to Pb mediated changes in long-term 

24 potentiation (LTP), Sun et al. (2007) performed in vitro patch clamp monitoring of rat hippocampal CA1 

25 section LTPs collected from pups exposed to Pb Acetate in utero, lactationally and through drinking water 

26 out to PND28. D-serine supplementation relieved the chronic Pb exposure dependent impaired magnitude 

27 of hippocampal LTP (H. Sun et al., 2007), which is known to be regulated by the NMDAR (Bear & 

28 Malenka, 1994). The use of 7-chlorokynurenic acid, an antagonist of the glycine binding site of the 

29 NMDAR-the binding site of D-serine, effectively abolished D-serine’s rescue of the LTP. NMDAR-
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1 independent LTP hippocampal neurotransmission with slices from Pb-exposed mossy-CA3 synapses was 

2 not rescued by exogenous D-serine supplementation. These data indicate that glial transmission may 

3 provide promising therapeutic targets for intervention after Pb exposure or with other affective or 

4 cognitive disorders known to manifest with aberrant NMDAR-dependent neurotransmission.

5.3.6.9. Neurotransmitters

5 Pb has been shown to compete with calcium for common binding sites and second messenger 

6 activation. When Pb activates a calcium-dependent system in the nervous system, it can contribute to 

7 aberrant neurotransmitter regulation and release because this system intimately relies on calcium signals 

8 for its homeostasis. Pb-dependent alterations in neurotransmission are discussed in further detail below.

Monoamine Neurotransmitters and Stress

9 Combined exposures of maternal stress and Pb exposure can synergistically enhance behavioral 

10 and neurotoxic outcomes in exposed offspring, sometimes even potentiating an effect that would 

11 otherwise be sub-threshold. Virgolini et al. (2008) found that effects on the central nervous system by 

12 developmental Pb exposure (2 months prior to mating through lactation, 50 or 150 ppm Pb acetate 

13 drinking water exposure, blood Pb level 11 μg/dL and 35 μg/dL, respectively) are enhanced by combined 

14 maternal and offspring stress. Offspring neurotransmitter concentrations were significantly affected with 

15 Pb exposure, but the most interesting findings were those of potentiated effects or effects that were not 

16 seen with Pb exposure alone or stress alone. These potentiated effects were only seen when Pb was 

17 combined with stress (maternal [MS] and/or offspring stress [OS]). Potentiation of serotonin (5HT) levels 

18 in females was significant in the frontal cortex in females and in the nucleus accumbens (NAC) in the 

19 male offspring (50 and 150 ppm Pb drinking water exposure) (Cory-Slechta et al., 2009). Regional 5HT 

20 levels were unaffected in offspring with no stressors and Pb exposure. Thus, Pb alone did not significantly 

21 affect 5HT levels. 5HIAA concentration was significantly increased with Pb exposure alone in the 

22 striatum of male offspring at 150 ppb Pb exposure; with the remaining exposures, Pb plus stress 

23 potentiated striatal and frontal cortex 5HIAA in males. Potentiated 5HIAA levels in females were 

24 significant in the NAC at both Pb doses; stress alone also significantly increased 5HIAA levels in females 

25 with no Pb exposure. Pb-induced changes in brain neurochemistry with or without concomitant stress 

26 exposure are complex with differences varying by brain region, neurotransmitter type and sex of the 

27 animal. 
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Monoamine Neurotransmitters and Auditory Function

1 The monoamine neurotransmitters include DA, 5HT, and NE. Earlier work has shown that perinatal 

2 rat Pb exposure induced increased tyrosine hydroxylase, increased DA and increased cerebral cortex 

3 catecholamine neurotransmission (Bielarczyk et al., 1996; C. B. Devi et al., 2005; Leret et al., 2002). 

4 Earlier publications detailing the window of exposure, duration of exposure and dose of Pb used have 

5 varying effects on monoamine transmitters. In more recent work, these neurotransmitters, among others, 

6 have been implicated in auditory function in the brainstem in various integration centers there including 

7 the lateral superior olive (LSO), and the superior olivary complex (SOC). The SOC is vital for sound 

8 detection in noisy settings among other functions. Low level Pb exposure has been associated with altered 

9 processing of auditory temporal signals in animal studies (Finkelstein et al., 1998; Lurie et al., 2006). 

10 Because Pb alters auditory processing, the monoamine system is a potential target for Pb-mediated 

11 interactions. Blood Pb levels for control, very low Pb (VLPb) and low Pb (LPb) exposure groups are 1.4, 

12 8.0, and 42.2 μg/dL, respectively. Developmental Pb exposure from the formation of breeding pairs to 

13 PND21, which is at the end of auditory development in the mouse, led to significant decreases in 

14 immunostaining of LSO and SOC brainstem sections for monoamine vesicular transporter VMAT2, and 

15 for 5HT and dopamine beta-hyrdoxylase (DbH), a marker for NE. This immunochemistry was significant 

16 for both VLPb and LPb exposure for VMAT2 and DbH but 5HT only had significant decrements with 

17 VLPb. Immunostaining for TH and transporters including VGLUT1, VGAT, VAChAT indicated that they 

18 were unaffected by developmental Pb exposure. These data provide evidence that specific regions of the 

19 brainstem relating to auditory integration with interaction from the monoamine neurotransmitter system 

20 are affected by developmental Pb exposure (Fortune & Lurie, 2009).

Dopamine

21 The 2006 AQCD detailed low dose Pb-dependent decreased dopaminergic cell activity in the 

22 substantia nigra and ventral segmental areas. Earlier studies with moderate to high dose postnatal or adult 

23 Pb exposure have reported changes in dopamine (DA) metabolism, DA and DOPAC, a DA metabolite. 

24 Thus, these were measured in various brain regions of year old males to determine if GLE affected DA 

25 metabolism. Low and high dose GLE in male rodents induced significant elevations in the DOPAC to DA 

26 ratio, and DOPAC concentration in the forebrain. In the forebrain, DA was significantly decreased in low 

27 dose GLE males and significantly elevated in GLE high dose males compared to controls. In the striatum, 

28 DOPAC was significantly elevated in both low and high dose GLE exposed males, but DA concentration 

29 was only significantly elevated in high dose GLE males. The striatum ratio of DOPAC to DA was not 

30 significantly different from control. These new data expand upon the monoamine literature base which 

31 reports perinatal rat low concentration Pb exposure induced increased sensitivity of the dopamine 

32 receptors (D2 and D3) (Cory-Slechta et al., 1992; Gedeon et al., 2001), produced higher DA levels (C. B. 
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1 Devi et al., 2005; Leret et al., 2002), and enhanced catecholamine neurotransmission in the cerebral 

2 cortex, cerebellum, and hippocampus (C. B. Devi et al., 2005). 

3 The interaction of dopamine and the nitric oxide system in the striatum was studied after prenatal 

4 Pb exposure. Blood Pb was not reported in this study, but similarly treated Wistar rat pups in other studies 

5 report blood Pb levels at parturition in range of 50-100 μg/dL (Grant et al., 1980). 7-nitroinidazole (7-NI), 

6 a selective inhibitor of nNOS, enhanced amphetamine-evoked dopamine release in the rat striatum 

7 (Nowak et al., 2008). Prenatal Pb exposure attenuated 7-NI’s facilitatory effect on dopamine release in the 

8 striatum. This interaction is ROS-independent; using spin trap measurements, there were no significant 

9 concentration changes in hydroxyl radical with Pb exposure (Nowak et al., 2008). Thus, the neuronal NO 

10 system appears to be involved in specific aspects of Pb-dependent dopaminergic changes. 

Dopamine and Vision

11 In various experimental animal models, the loss of retinal dopamine or zinc is associated with 

12 supernormal rod-mediated scotopic electroretinograms (ERGs), pointing to the retina as a sensitive target 

13 of low dose Pb exposure (19 μg/dL). In the human and non-human primate literature, GLE is associated 

14 with increased amplitude (supernormality) of ERGs (Lilienthal et al., 1994; Lilienthal et al., 1988; 

15 Rothenberg et al., 2002) and in the animal toxicology literature postnatal Pb exposure induces 

16 subnormality of the ERGs. New research in the animal toxicology literature recapitulated the low dose 

17 human literature, showing that low-and moderate (LPb or MPb) level gestational Pb exposure in the rat 

18 produced supernormal ERGs with associated significant increases in retinal neurogenesis and significant 

19 decreases in retinal dopamine use and dopamine turnover, DA and DOPAC:DA ratio, respectively. High 

20 gestational Pb exposure (HPb) produced significant subnormal ERGs, similar to the findings with 

21 postnatal human Pb exposures. Rats (dams) were exposed to Pb acetate in drinking water starting 2 weeks 

22 prior to mating and throughout gestation and lactation until PND 10, a period of developmental exposure 

23 that is equivalent to gestational exposure in humans with peak blood Pb PND1-10 of 12, 24, and 46 μg/dL 

24 in LPb, MPb, and HPb, respectively. LPb and MPb gestational exposure induced increased cellularity or 

25 retinal thickness in the outer nuclear layer, inner nuclear layer and total retina (Leasure et al., 2008). In 

26 conclusion, the retina is a sensitive site to low-dose Pb exposure and gestational Pb exposure produced 

27 dose-dependent decreases in DA use and turnover; inverted U shaped Pb dose response curves were 

28 reported for retinal endpoints including ERG and retinal thickness. 

NMDA

29 NMDA receptors (NMDAR) have been shown to contribute to synaptic plasticity and Pb-exposure 

30 at different developmental stages is known to contribute to aberrations in LTP or LTD in the hippocampus 

31 via reduced NMDA current, among other mechanisms (L. Liu et al., 2004). The 2006 AQCD detailed that 
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1 Pb induced decreases in stimulated glutamate release that affected LTP. Further, it detailed that the Pb2 dependent decreased magnitude and increased threshold of the LTP in the hippocampus is biphasic or 

3 non-linear. NMDAR subtypes have been shown to be significantly decreased with developmental Pb 

4 exposure (Guilarte & McGlothan, 1998). Recent work looking at supplement use, found Pb-dependent 

5 decreases in message and protein level of NMDAR subunit NR1 was rescued with methioninecholine 

6 coexposure in these weanling male rats (Fan et al., 2010). Fan et al. (Fan et al., 2010) found that Pb7 dependent suppression of the NMDAR subunits NR2A and NR2B were not rescued with 

8 methioninecholine treatment. Other recent mechanistic studies have found that pretreatment of primary 

9 fetal brain neuronal rat cultures with glutamic acid, a NMDAR agonist, reversed Pb-dependent reductions 

10 in NMDAR subunits (S.-Z. Xu & Rajanna, 2006) whereas pretreatment with the NMDA antagonist MK11 801 exacerbated Pb-induced NMDAR defecits (S.-Z. Xu & Rajanna, 2006). Thus, glutamic acid or 

12 methioninecholine may offer therapeutic possibilities for Pb-induced neuronal NMDAR decrements. 

13 The Guilarte lab has made extensive contributions to the Pb animal toxicology literature and a 

14 recent publication details the effect of low dose developmental/lifetime Pb exposure on changes in 

15 hippocampal neurogenesis in adulthood (Verina et al., 2007), an emerging area of research affecting long16 term potentiation, spatial learning, neuronal outgrowth, and possibly mood disorders like schizophrenia. 

17 NMDAR mediates the integration of new neurons into existing neuronal pathways in the adult 

18 hippocampal DG, which is important to learning and memory. Lifetime Pb chow exposure (dam Pb

19 acetate exposure 10 days prior to mating through pregnancy out to PND50 or PND78) induced significant 

20 decrements in hippocampal granule cell neurogenesis or proliferation of new cells in adult rats. Also, Pb 

21 exposed animals had significant decreases in brain volume in the stratum oriens (SO) region of the 

22 hippocampus, specifically significant decreases in the mossy fiber terminals of the SO. A marker for 

23 immature or newly formed neurons showed a significant decrease in the length-density of these cells in 

24 the outer portion of the DG in Pb-exposed animals. These findings show that exposure to environmentally 

25 relevant doses of Pb induced significant aberrations in adult hippocampus granule cell neurogenesis and 

26 morphology, providing mechanistic explanations for Pb-induced neuronal aberrations. Guilarte et al. 

27 (2003) demonstrated that Pb exposure of rats from an enriched environment was associated with reversal 

28 of learning impairment, increased expression of hippocampal NMDA receptor subunit 1, and increased 

29 induction of brain derived neurotrophic factor mRNA (Guilarte et al., 2003).

5.3.6.10. Neurite Outgrowth

30 The 2006 AQCD reported Pb decreased neurite outgrowth at 20μg/dL noting that Pb interfered 

31 with neurite outgrowth via protein kinase mediated pathways (MAPK/ERK); earlier work has 

32 documented decreased primary DA neuron outgrowth with 0.001 μM Pb exposure (Lidsky & Schneider, 

33 2004). More recent studies have shown that dam exposure to low dose Pb (blood Pb level 4 μg/dL) of 
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1 dams significantly decreased pup hippocampal neurite outgrowth (pup blood Pb level 12 μg/dL) and 

2 reduced the expression of hippocampal polysialylated neural cell adhesion molecule (PSA-NCAM), 

3 NCAM, and sialytransferase; PSA-NCAM is transiently expressed in newly formed neurons (Q. S. Hu et 

4 al., 2008) during the period of neurite outgrowth from embyrogenesis until the early postnatal period and 

5 is down-regulated in the adults except in areas known to exhibit synaptic plasticity (Seki & Arai, 1993). 

6 NCAM is important for memory formation, plasticity and synapse formation and early life Pb exposure in 

7 laboratory rodents affects its expression. 

5.3.6.11. Epigenetics

8 DNA methyltransferase activity was significantly decreased in cortical neurons from both monkey 

9 (aged animals) and mouse brains (fetal cells exposed to Pb in culture, 0.1 μM Pb) after Pb exposure (J. F. 

10 Wu et al., 2008). DNA methyltransferases catalyze the transfer of a methyl group to DNA and are 

11 important in epigenetics (i.e., silencing of genes like tumor suppressors) and imprinting. 

5.3.7. Examination of the Lead Concentration-Response 

Relationship

12 With each successive Pb AQCD and supplement, epidemiologic and toxicological studies find that 

13 progressively lower blood Pb levels are associated with cognitive deficits and behavioral impairments. 

14 For example, among children, such decrements were observed in association with blood Pb levels in the 

15 range of 10-15 μg/dL in the 1986 Addendum and 1990 Supplement and 10 μg/dL and lower in the 2006 

16 AQCD (U.S. EPA, 2006). Furthermore, in the 2006 AQCD, several individual studies, pooled analyses, 

17 and meta-analyses estimated a supralinear blood Pb concentration-response relationship in children, i.e., 

18 greater decrements in cognitive function per incremental increase in blood Pb level among children in 

19 lower strata of blood Pb levels compared with children in higher strata of blood Pb levels (Figure 5-28 

20 and Table 5-12). While the majority of epidemiologic evidence indicated differences in effect estimates 

21 above and below 10 μg/dL, several studies of children with mean blood Pb levels less than 5 μg/dL 

22 estimated larger effects for children with <5 μg/dL (compared with children with blood Pb levels 5-10 

23 μg/dL, and >0 μg/dL) (Tellez-Rojo et al., 2006), <2.5 μg/dL (compared with children with blood Pb 

24 levels <5 μg/dL, <.5 μg/dL, <10 μg/dL, and all subjects) (Lanphear et al., 2000), and <1.2 μg/dL 

25 (maternal plasma Pb, compared with plasma Pb levels >1.2 μg/dL) (H. Hu et al., 2006). Using data from 

26 NHANES 1999-1994, Lanphear et al. (2000) examined differences in effect estimates among multiple 

27 strata of blood Pb levels and found the largest deficit in reading score per 1 μg/dL increment in blood Pb 

28 among children with blood Pb levels less than 1 μg/dL. As lower concentrations of Pb exposure are being 

29 used experimentally, the toxicological literature reports nonlinear concentration-response relationships for 
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1 some endpoints and similar to the epidemiologic literature, shows larger effects in lower Pb exposure 

2 groups.

3
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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 >1.226a

<1.226a

Kordas et al. (2006) Concurrent Math score All

<10

Lanphear et al. (2005) Concurrent FSIQ ≥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 1st 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 quantitative 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

Lanphear 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 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 

>10

<10 -0.58b-1.56b
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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 1st

trimester plasma 

Pb median: 1.226

Linear regression model 

adjusted for plasma Pb in 

2nd trimester, plasma Pb in 

3rd 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

-4.0b

-15.0b

Kordas et al. 

(2006)

602 children in 1st grade

Torreon, Mexico

Concurrent mean 

(SD): 11.4 (6.1)

Linear regression model 

adjusted for sex, age, 

hemoglobin, family 

possessions, forgetting 

homework, house 

ownership, crowding, 

maternal education, birth 

order, family structure, 

arsenic exposure, tester, 

school

Math achievement 

test in 1st grade

all

<10

-0.17 (-0.28, -0.06)

-0.42 (-0.92, 0.08)

Lanphear et 

al. (2005)

1333 children pooled from 

Boston, Cincinnati, 

Cleveland, Mexico City, 

Port Pirie, Rochester, and 

Yugoslavia cohorts

Median (5th95th)

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 

effect 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.

1 Using data pooled from seven prospective studies, Lanphear et al. (2005) fit various types of 

2 models to the data and observed that a cubic spline, log-linear model, and piece-wise linear model all 

3 supported a more negative concentration-response relationship at lower blood Pb levels. A linear model 

4 was found to be inadequate as the polynomial terms for concurrent blood Pb were statistically significant. 

5 These findings were corroborated by a separate analysis by Rothenberg and Rothenberg (2005) which 

6 found that the log-linear model fit the relationship between blood Pb level and IQ better than did a linear 

7 model.

8 Studies of adults have not widely examined the shape of the relationship between blood or bone Pb 

9 level and cognitive performance. In the various NHANES analyses, only log-linear models were used to 

10 fit the data (E. F. Krieg, Jr. & Butler, 2009; E. F. Krieg, Jr. et al., 2009; E. F. Krieg, Jr. et al., 2010). Other 

11 studies examined nonlinearity with the use of quadratic terms, penalized splines, or visual inspection of 

12 bivariate plots (Bandeen-Roche et al., 2009; Shih et al., 2006; Weisskopf, Proctor, et al., 2007). While 

13 there was some evidence for nonlinearity for some cognitive tests (Figures 5-17 and 5-18), the majority of 

14 results suggested linear associations. Shih et al. (2006) found that a quadratic term for tibia Pb was not 
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1 statistically significant and found a linear model fit adequately the relationship between tibia Pb level and 

2 various tests of cognitive performance. In contrast to most studies, Wang et al. (2007) found that among 

3 HFE variant carriers, there was a steeper decline in MMSE score at higher tibia Pb levels (20-25 μg/g, 

4 Figure 5-17).

5 Attenuation of the concentration-response relationships at higher exposure or dose levels has been 

6 reported in the occupational literature, and explanations have included greater exposure measurement 

7 error, competing risks, saturation of biological mechanisms at higher levels, larger proportion of 

8 susceptible populations at lower exposure levels, and variations in other risk factors among exposure 

9 levels (Stayner et al., 2003). Other explanations for nonlinearity include different mechanisms operating

10 at different exposure levels, confounding by omitted or misspecified variables, and the lower incremental 

11 effect of Pb due to covarying risk factors such as low SES, poor caregiving environment, and higher 

12 exposure to other environmental factors.

13 The contribution of these factors to the supralinear relationship between blood Pb levels and 

14 neurocognitive function has not been examined widely in epidemiologic studies to-date. However, in 

15 several populations, higher blood Pb levels have been measured in susceptible groups such as those with 

16 higher poverty, greater exposure to tobacco smoke, lower parental education, and lower birth weight, 

17 which argues against a larger proportion of susceptible populations at lower blood Pb levels (Lanphear et 

18 al., 2000; Lanphear et al., 2005). It has been suggested that in populations of low SES, poorer caregiving 

19 environment, and greater social stress, the incremental effect of Pb exposure may be attenuated due to the 

20 overwhelming effects of these other risk factors (J. Schwartz, 1994). Several studies have found 

21 significant associations of these sociodemographic risk factors with neurocognitive deficits, and Miranda 

22 et al. (Miranda et al., 2009) found that indicators of SES (i.e., parental education and enrollment in a 

23 free/reduced fee lunch program) accounted for larger decrements in EOG scores than did blood Pb level 

24 (Figure 5-6). Few studies have compared Pb effect estimates among groups in different sociodemographic 

25 strata, and the limited data are mixed. Greater Pb-associated neurocognitive deficits in low-SES groups 

26 were reported by Bellinger et al. (1990). In a meta-analysis of eight studies, Schwartz (1994) found a 

27 smaller decrement in IQ per 1 μg/dL increase in blood Pb level for studies in disadvantaged populations 

28 (-2.7 points [95% CI: -5.3, -0.07]) than for studies in advantaged populations (-4.5 points [95% CI: -5.6, -

29 2.8]). It is important to note that blood Pb is associated with deficits in neurocognitive function in both 

30 higher and lower SES groups; however, it is unclear what differences there are between groups in the 

31 decrement per unit increase in blood Pb and whether these differences can explain the nonlinear dose32 response relationship.

33 Rothenberg and Rothenberg (2005) formally assesed the influence of residual confounding on the 

34 nonlinear blood Pb concentration-response relationship by comparing model fit between linear and spline 

35 transformations (df = 2) of covariates such as maternal IQ, HOME score, and maternal education. 

36 Inclusion of covariates as spline functions did not significantly improve model fit either with a linear 
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1 blood Pb term or log blood Pb term, which indicated that their inclusion as linear functions was adequate. 

2 These findings demonstrate that the improved model fit with log-specification of blood Pb level was not 

3 due to residual confounding by covariates.

4 Consistent with the epidemiologic literaure, toxicological studies also find nonlinear relationships 

5 between Pb exposure and neurological effects in animals. In particular, multiple studies have shown U- or 

6 inverse U-shaped curves with lower exposures of Pb having different or often the opposite effect from 

7 higher doses. U-shaped Pb exposure-responses include rotoarod performance, adult forebrain dopamine 

8 levels, amphetamine-induced motor activity, and latency to fall from rotarod (Leasure et al., 2008). 

9 Inverted U-shaped Pb dose-responses include histological findings such as the numbers of rod 

10 photoreceptors and bipolar cells, forebrain dopamine use, activity level, and adult body weight (Leasure 

11 et al., 2008) as well as ERG wave amplitudes (Fox et al., 2008) and hippocampal neurogenesis (Fox et al., 

12 2008; Gilbert et al., 2005).

13 Because toxicological studies typically do not have confounding, exposure measurement error or 

14 other epidemiologic influences, i.e., susceptibility, they have permitted assessment of a mechanistic basis 

15 for nonlinear Pb exposure- or dose-response relationships. Several lines of evidence support the 

16 possibility of low-dose and high dose-Pb acting through differential activation of mechanisms. For 

17 example, in mice, lower Pb exposure (50 ppm) is associated with differential responses of the 

18 neurotransmitters dopamine and norepinephrine compared with control treatment and higher doses (150 

19 ppm) (Leasure et al., 2008; Virgolini et al., 2005). These differential responses of neurotransmitter 

20 systems to lower versus higher Pb exposures may provide mechanistic understanding of the nonlinearity 

21 of Pb-induced behavioral changes in animals and may also explain the nonlinear blood Pb-neurocognitive 

22 and neurobehavioral associations reported widely among children. Additional evidence points to 

23 differences in hormonal homeostasis by Pb exposure level. In male mice with chronic Pb exposure 

24 (PND21-9 months of age), basal corticosterone levels are significantly lower in the 50 ppm exposure 

25 group than in the control or 150 ppm Pb exposure group.

26 Additional mechanistic understanding comes from differences in histological changes found in Pb27 exposed animals. Compared with higher Pb exposure, lower Pb exposure stimulates greater induction of 

28 c-fos, a marker of neuronal activation and action potential firing (Lewis & Pitts, 2004). These findings 

29 may underlie the nonlinear association between Pb exposure and learning and the U-shaped behavioral 

30 dose-responsiveness seen with amphetamine-induced motor activity in males after GLE (Leasure et al., 

31 2008).

32 Sensory organ findings in animals also show vastly different outcomes with low versus higher Pb 

33 exposure. Higher Pb exposure produces subnormal retinal ERGs and lower Pb exposure produces 

34 supernormal ERGs in both children (Rothenberg et al., 2002) and rodents (Fox & Chu, 1988; Fox & 

35 Farber, 1988; Fox et al., 1991). Inverted U-shaped dose-response curves have been seen for rod 

36 photoreceptor numbers or neurogenesis (Giddabasappa et al., 2011) and retinal thickness (Fox et al., 
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1 2010). Thus, these dichotomous histological findings are coherent with the functional retinal test or the 

2 ERG where higher Pb exposure produces subnormal ERGs and lower exposure Pb produces supernormal 

3 ERGs.

4 Hierarchical enzyme activity also may explain nonlinear Pb concentrations-response relationships. 

5 The phosphatase enzyme calcineurin has been shown to be inhibited by higher Pb exposure and 

6 stimulated by lower Pb exposure (Kern & Audesirk, 2000). At lower Pb exposure, Pb displaces calcium at 

7 its binding sites on calmodulin and by acting as a calmodulin agonist at calcineurin’s catalytic A subunit, 

8 stimulates calcineurin activity. At higher Pb exposure, Pb can bind directly to a separate calcium-binding 

9 B subunit, overriding the calmodulin-dependent effect and turning off the activity of calcineurin. 

10 Interestingly, mice with modulated calcineurin expression exhibit aberrant behavior related to 

11 schizophrenia or impaired synaptic plasticity and memory (Zeng et al., 2001). This example of the 

12 stimulatory effects of Pb at lower exposure and inhibitory effects at higher exposure gives another 

13 example of biological plausibility for the nonlinear concentration-response relationship reported for Pb in 

14 multiple studies.

15 The supralinear concentration-response relationship widely documented for Pb is consistent with 

16 the lack of a threshold for Pb-associated neurological effects as a smaller effect estimate would be 

17 expected at lower blood Pb levels if a threshold existed. Schwartz (1994) explicitly assessed evidence for 

18 a threshold in the Boston prospective cohort data by regressing IQ and blood Pb level on potential 

19 confounders including age, race, maternal IQ, SES, and HOME score and fitting a nonparametric 

20 smoothed curve to the residuals of both regression models (variation in IQ or blood Pb level not explained 

21 by covariates). A 7-point decrease in IQ was observed over the range of blood Pb residuals below 0, 

22 which corresponds to the mean blood Pb level in the study (6.5 μg/dL). Thus, in the Boston study, the 

23 association between blood Pb level and IQ was clearly demonstrated at blood Pb levels below 5 μg/dL.

24 An important limitation of previous studies in terms of characterizing the concentration-response 

25 relationship, in particular, identifying whether a threshold exists, has been the limited examination of 

26 effects in populations or blood Pb strata with blood Pb levels more comparable to the current U.S. 

27 population mean. While Schwartz (1994) did not find evidence for a threshold in the Boston study data, 

28 the mean blood Pb in that population was 6.5 μg/dL, and 56% of subjects had a blood Pb level >5 μg/dL. 

29 Recent studies indicate a downward shift in the distribution of blood Pb levels (i.e., 50% of subjects in the 

30 2001-2004 NHANES population had a blood Pb <1 μg/dL (Braun et al., 2008). Additionally, more 

31 sensitive quantification methods have improved the detection limits, for example, from 0.6 μg/dL to 0.025 

32 μg/dL in NHANES. This has allowed categorization of children in mulitple blood Pb quantiles below 1 

33 μg/dL (Braun et al., 2008). Consequently, the examination of populations with large proportions of 

34 subjects at very low blood Pb levels has improved the ability to discern a threshold for Pb-associated 

35 neurological effects. Several recent studies reported associations between blood Pb levels and deficits in 

36 neurocognitive and neurobehavioral endpoints in populations with mean blood Pb levels <2 μg/dL (Braun 
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1 et al., 2008; Braun et al., 2006; Cho et al., 2010; E. F. Krieg, Jr. et al., 2010). In comparisons of various 

2 quantiles of blood Pb, Chandramouli et al. (2009) observed a lower SAT score among children in the U.K. 

3 with blood Pb levels 2-5 μg/dL compared with children with blood Pb levels 0-2 μg/dL. Likewise, 

4 Miranda et al. (2009) reported lower EOG scores in children in North Carolina with blood Pb levels of 2 

5 μg/dL compared with children with blood Pb levels of 1 μg/dL. In the 2001-2004 NHANES population, 

6 Braun et al. (2008) found higher odds ratios for conduct disorder and ADHD among children with blood 

7 Pb levels 0.8-1.0 μg/dL (2nd quartile) compared with children with blood Pb levels 0.2-0.7 μg/dL (1st 

8 quartile). Collectively, these new findings in children, as summarized in this document, do not provide 

9 evidence for a threshold for the neurological effects of Pb in the ranges of blood Pb levels examined to10 date.

11 It is important to note, however, that the lack of a reference population with blood Pb levels 

12 reflecting pre-industrial Pb exposures limits the ability to identify a threshold. Estimates of “background” 

13 blood Pb levels have been garnered from the analysis of ancient bones in pre-industrialized societies. 

14 These studies suggest that the level of Pb in blood in preindustrial humans was approximately 0.016 

15 μg/dL (Flegal & Smith, 1992), approximately 65-fold lower than that currently measured in U.S. 

16 populations and lower than the levels at which neurological effects have been observed (1 μg/dL). Thus, 

17 the current evidence does not preclude the possibility of a threshold existing in the large range of blood 

18 levels between 1 μg/dL and preindustrial “background” levels.

5.3.8. Summary and Causal Determination

19 The 2006 Pb AQCD concluded that the collective body of epidemiologic studies provides clear and 

20 consistent evidence for the effects of Pb exposure on neurocognitive function in children. This conclusion 

21 was substantiated by the coherence of findings across studies of diverse design and populations (varying 

22 distributions of blood Pb levels, SES, parental intelligence, and quality of caregiving) that blood Pb 

23 levelswere associated with a broad spectrum of neurocognitive and neurobehavioral indices, including 

24 cognitive function (IQ), higher-order processes such as language and memory, academic achievement, 

25 behavior and conduct, delinquent and criminal activity, sensory acuities, and changes in brain structure 

26 and activity as assessed by MRI or MRS (Figure 5-29). Toxicological studies not only provided coherence 

27 with similarly consistent findings for Pb-induced impairments in parallel tests of learning, behavior and 

28 attention, and sensory acuities, but also provided biological plausibility by characterizing mechanisms for 

29 Pb-induced neurological effects (Figure 5-29). These mechanisms included Pb-induced inhibition of 

30 neurotransmitter release, decline in synaptic plasticity, decreases in neuronal differentiation, and 

31 decreases in the integrity of the blood-brain-barrier. Both epidemiologic studies in children and 

32 toxicological studies reviewed in the 2006 Pb AQCD demonstrated neurocognitive deficits in association 

33 with blood Pb levels at or below 10 μg/dL, and evidence from both disciplines also supported a nonlinear 
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1 concentration-response relationship, with greater cognitive or behavioral decrements per unit increase in 

2 blood Pb level estimated for lower blood Pb levels or estimated for lower Pb exposures. Among adults, 

3 although associations of blood Pb level with the spectrum of neurological effects (e.g., impairments in 

4 memory, attention, mood, balance, and motor function) were most consistently observed in 

5 occupationally-exposed adults with blood Pb levels ≥ 14 μg/dL, studies of adults without occupational Pb 

6 exposures indicated associations between biomarkers of cumulative Pb exposure, serial blood Pb or bone 

7 Pb measurements, and decrements in cognitive function.

8 Building on the strong body of evidence presented in the 2006 AQCD, recent studies continue to 

9 support associations of Pb biomarkers or exposures with neurological effects (Figure 5-29). Although 

10 fewer in number, recent studies in children corroborate findings from the several previous longitudinal 

11 and cross-sectional studies in demonstrating associations between blood Pb levels and FSIQ. In the 

12 cumulative body of evidence, negative associations between blood Pb level and IQ are best substantiated 

13 at mean blood Pb levels in the range of 5-10 μg/dL; however, an association was observed in a recent 

14 study with a mean blood Pb level of 1.73 μg/dL. A majority of recent epidemiologic studies in children 

15 has focused on examining specific indices of neurocognitive function such as reading and verbal skills, 

16 memory, learning, and visuospatial processing and has demonstrated associations with blood Pb levels as 

17 low as 2 μg/dL (population mean or quantiles). The consistently positive associations observed between 

18 blood Pb levels and this diverse set of neurocognitive indices provides coherence with findings for IQ, a 

19 global measure of cognitive function that reflects the integration of these individual domains. Additional 

20 coherence for findings in children is derived from evidence in animals that blood Pb levels of 11.6 μg/dL

21 and higher are associated with changes in learning and memory (Figure 5-29). Recent toxicological 

22 studies continue to demonstrate that in utero and early postnatal exposure to Pb is the most sensitive 

23 window for Pb-dependent neurological effects. In the 2006 Pb AQCD, uncertainty was noted among 

24 studies in children regarding the relative importance of prenatal, early life, concurrent, and lifetime 

25 measures of Pb exposures. It also was noted that distinguishing among the effects of Pb exposures at 

26 different lifestages is difficult in epidemiologic studies due to the high correlations among children’s 

27 blood Pb levels over time. Although blood Pb levels at all of these lifestages are associated with 

28 neurocognitive deficits in children, stronger effects generally are estimated for concurrent blood Pb 

29 levels, and recent evidence indicates that among both children with relatively lower or higher early 

30 childhood blood Pb levels, concurrent blood Pb levels are more strongly associated with neurocognitive 

31 deficits. In addition to performance on various neurocognitive tests, recent epidemiologic studies in 

32 children link blood Pb levels (in quantiles as low as 2 μg/dL) with factors that may be indicators of 

33 children’s life success, including the level of educational attainment and end-of-grade score. In particular, 

observations of lower 4th 34 grade end-of-grade score among children with blood Pb levels of 2 μg/dL

35 compared with children with blood Pb levels of 1 μg/dL indicate that a threshold may not exist for the 

36 neurodevelopment effects of Pb in children.
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1 Recent studies in children continue to support associations of blood Pb levels (population means or 

2 quantiles of 3-11 μg/dL) with a range of behavioral problems from anxiety and distractability to conduct 

3 disorder and delinquent behavior (Figure 5-29). Whereas previous evidence was not compelling, new 

4 evidence indicates associations between blood Pb levels and ADHD diagnosis and contributing diagnostic 

5 indices. In particular, a recent NHANES analysis demonstrated associations at blood Pb levels between 1 

6 and 2 μg/dL). These findings for ADHD are well-supported by observations in animals of Pb-induced 

7 increased response rates and impulsivity. Additional coherence is provided by evidence in aquatic and 

8 terrestrial species for Pb affecting behaviors that decrease the ability of organisms to escape predators or 

9 capture prey (Chapter 7.1, 7.2). Both epidemiologic studies in children and adults as well as toxicological 

10 studies demonstrate associations of Pb biomarkers or exposure with deficits in visual acuity and hearing 

11 and auditory processing. New evidence from toxicological studies demonstrates the effects of lower blood 

12 Pb levels (<15 μg/dL) with retinal changes in male offspring. Combined evidence for Pb-associated 

13 neurocognitive deficits, inattention, conduct disorder, and effects on sensory function provides plausible 

14 mechanisms by which Pb exposure may contribute to academic underachievement and to more serious 

15 problems of delinquent behavior.

16 Studies in adults without occupational exposure to Pb have not provided consistent evidence for 

17 associations or blood or bone Pb levels with the range of neurological effects. Levels of Pb in bone, 

18 particularly in tibia, which is an indicator of cumulative Pb exposure, including higher exposures in the 

19 past, are better predictors of cognitive performance rather than a single blood Pb measurement. One 

20 explanation for the overall weaker body of evidence may be that cognitive reserve may compensate for 

21 the effects of Pb exposure on learning new information. Compensatory mechanisms may be overwhelmed 

22 with age, which may provide an explanation for more consistent associations between biomarkers of 

23 cumulative Pb exposure (serial blood measurements, tibia Pb levels) and neurocognitive deficits. Among 

24 recent studies of adults, blood Pb levels and bone Pb levels have been associated with essential tremor 

25 and Parkinson’s Disease, respectively. These findings are well-supported by toxicological evidence for 

26 Pb-induced decreased dopaminergic cell activity in the substantia nigra, which contributes to the primary 

27 symptoms of Parkinson’s disease. Biological plausibility also is provided by observations of 

28 developmental Pb exposures of monkeys and rats resulting in neurodegeneration in aged brains. Recent 

29 evidence also indicates associations between early-life ALAD activity, a biomarker of Pb exposure, and 

30 schizophrenia later in adulthood. Consistent with these findings, toxicological studies have observed Pb31 induced emotional changes in males and depression changes in females. It is not surprising that Pb 

32 exposure may increase the risk of different neurological endpoints in children and adults given the 

33 predominance of different neurological processes operating at different ages, in particular, neurogenesis 

34 and brain development in children and neurodegeneration in adults. 

35 Several host and environmental factors may modify the association between Pb exposure and 

36 neurological effects in children. Interactions of blood Pb levels with race/ethnicity and SES continue to be 
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1 poorly characterized. Although the 2006 Pb AQCD cited mixed epidemiologic evidence for effect 

2 modification by sex, recent epidemiologic evidence points to males having increased susceptibility for 

3 Pb-associated neurological effects. Toxicological studies continue to demonstrate increased susceptibility 

4 of males for endpoints such as sensory function, balance, stress hormone homeostasis, and brain 

5 membrane composition. Although limited, evidence suggests that risk of Pb-associated neurocognitive 

6 deficits in children also may be modified by variants in genes for apolipoprotein E and dopamine 

7 receptors. In addition to host factors, recent studies suggested that associations between blood Pb levels 

8 and neurological effects in children are greater with coexposures to environmental tobacco smoke and 

9 manganese. Historical animal toxicology findings demonstrate interactions between Pb exposure and 

10 stress. Namely, Pb-exposed animals reared in cages with enriched environments (toys) perform better in 

11 the Morris water maze than their Pb-exposed littermates who were reared in isolation. New findings 

12 indicate a potentiating effect of stress on behavior and memory with lower Pb exposures. In comparison,

13 epidemiologic evidence for such interactions has been sparse. However, consistent with historical animal 

14 studies, a recent study indicated that positive social environment of children as characterized by maternal 

15 self-esteem, attenuates the negative association between blood Pb level and cognitive function. While 

16 effect modification by these host and environmental factors has not been examined widely in 

17 epidemiologic studies, new studies provide information on potentially susceptible populations that may 

18 benefit from early intervention to reduce the risk of neurological effects. Furthermore, the robust evidence 

19 for varying susceptibilities to Pb-induced neurological effects provides a basis to integrate 

20 mechanistically the findings of toxicology and epidemiology.

21 Extensive evidence from toxicological studies clearly substantiates the biological plausibility for 

22 epidemiologic findings by characterizing mechanisms underlying neurological effects. Pb exposure of 

23 animals induces dopamine changes in animals (Figure 5-29). Dopamine plays a key role in cognitive 

24 functions mediated by the prefrontal cortex and also motor functions mediated by the substantia nigra, 

25 and animal toxicological findings provide mechanistic support for associations in humans between blood 

26 Pb levels and neurocognitive deficits and in adults for associations with Parkinson’s Disease. Current 

27 toxicological research has been expanded to document that early-life Pb exposure can contribute to 

28 neurodegeneration and neurofibrillary tangle formation in the aged brain. Pb induces complex 

29 neurochemical changes in the brain that differ by region of the brain, neurotransmitter type, age and sex 

30 of the organism. These changes remain aberrant over time but are dynamic in nature. The effect of Pb on 

31 NMDA receptors and the contribution of this paradigm to mood disorders is detailed. Synapse formation, 

32 adhesion molecules, and nitrosive stress continue to be areas of research with known Pb-associated 

33 adverse outcomes. Finally, the new area of epigenetics details that Pb exposure affects methylation 

34 patterns in rodent brains. These toxicological data complement the expanding epidemiologic data and 

35 often provide coherence between the two fields.
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1 In summary, recent evidence substantiates and expands upon the established epidemiologic and 

2 toxicological literature of neurologic effects associated with Pb exposure. In epidemiologic studies of 

3 children, consistently positive associations of blood Pb levels with deficits in neurocognitive function, 

4 attention, and sensory acuities support observed associations with school performance as assessed by end5 of-grade scores and level of educational attainment, which in turn, may explain associations with 

6 delinquent and criminal behavior. In particular, observations of lower academic achievement and ADHD 

7 among children in quantiles of blood Pb levels in the range of 1 to 2 μg/dL have not indicated that a 

8 threshold exists for the neurodevelopment effects of Pb in children. Epidemiologic findings are 

9 strengthened by the coherence and biological plausibility provided by toxicological findings for similar or 

10 parallel endpoints and for the mechanisms underlying the neurological effects (Figure 5-29). The 

11 collective body of evidence integrated across epidemiologic and toxicological studies and across the 

12 spectrum of neurological endpoints is sufficient to conclude that there is a causal relationship between 

13 Pb exposures and neurological effects.
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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).
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5.4. Cardiovascular Effects

5.4.1. Introduction

1 Both human and animal studies provide consistent evidence for an association of increased BP and 

2 arterial hypertension with chronic exposure to Pb resulting in adult blood Pb levels below 5 μg/dL. In 

3 addition, studies have suggested a connection between measures of Pb exposure and other cardiovascular 

4 diseases in adults such as ischemic heart disease, cerebrovascular disease, peripheral vascular disease, and 

5 cardiovascular disease related mortality. Toxicological studies explore the underlying mechanisms by 

6 which Pb exposure can lead to human cardiovascular health outcomes. Such studies have demonstrated 

7 that the Pb content in heart tissue reflects the increases in blood Pb levels (Lal et al., 1991). In general, 

8 associations between blood Pb and bone Pb (particularly in the tibia) with health outcomes in adults 

9 indicate acute effects of recent dose and chronic effects of cumulative dose, respectively. In some 

10 physiological circumstances of increased bone remodeling or loss (e.g., osteoporosis and pregnancy), Pb 

11 from bone of adults may also contribute substantially to blood Pb concentrations. Additional details on 

12 the interpretation of Pb in blood and bone are provided in Section 4.3.5. Additionally, as the 

13 cardiovascular and renal systems are intimately linked, cardiovascular effects can arise secondarily to Pb14 induced renal injury (Section 5.5).

15 The previous Pb AQCD (U.S. EPA, 2006) concluded that both epidemiologic and animal 

16 toxicological studies support the relationship between increased Pb exposure and increased cardiovascular 

17 outcome, including increased BP, increased incidence of hypertension, and cardiovascular morbidity and 

18 mortality. Meta-analysis of these human studies found that each doubling of blood Pb level (between 1 

19 and >40 μg/dL) was associated with a 1 mmHg increase in systolic BP and a 0.6 mmHg increase in 

20 diastolic BP (Nawrot et al., 2002). On a population-wide basis, the measured effect size translates into a 

21 large number of events for a moderate population size and thus has important health consequences for the 

22 occurrence of stroke, myocardial infarction, and sudden death. It was also noted that most of the reviewed 

23 studies using cumulative Pb exposure measured by bone Pb also showed increased BP (Y. Cheng et al., 

24 2001; H. Hu et al., 1996) or increased hypertension with increasing bone Pb (B.-K. Lee et al., 2001). Over

25 a range of bone Pb concentrations (<1.0 to 96 μg/g), every 10 μg/g increase in bone Pb was associated

26 with increased odds ratio of hypertension between 1.28 and 1.86, depending upon the study. Two studies 

27 observed averaged increased systolic BP of ~0.75 mmHg for every 10 μg/g increase in bone Pb 

28 concentration over a range of <1 to 52 μg/g. Since bone Pb measures Pb accumulation over time, duration 

29 of past exposure to Pb plays a role in increased BP.

30 The previous Pb AQCD also provided compelling evidence for a number of mechanisms leading to 

31 increased BP, and the development of hypertension and other cardiovascular diseases observed after Pb 

32 exposure. The strongest evidence supported the role of oxidative stress in the pathogenesis of Pb-induced 
May 2011 5-153 DRAFT – DO NOT CITE OR QUOTE

1 hypertension. Additionally, several studies focused on other pathways or cellular, molecular, and tissue 

2 events promoting the Pb-induced increase in BP. These mechanisms include inflammation, adrenergic and 

3 sympathetic activation, renin-angiotensin-aldosterone system (RAAS) activation, vasomodulator 

4 imbalance, and vascular cell dysfunction. Studies continue to support the observed increase in BP and 

5 hypertension development following Pb exposure, as well as build on the evidence for the biological 

6 pathways of these effects. This section reviews the published studies pertaining to the cardiovascular 

7 effects of Pb exposure in experimental animals, isolated vascular tissues, cultured vascular cells, and 

8 humans. Emphasis has been placed on studies published since the 2006 AQCD (U.S. EPA, 2006), 

9 however the large body of evidence that existed prior to that review has been summarized and 

10 incorporated into the current review. 

5.4.2. Blood Pressure and Hypertension

5.4.2.1. Epidemiology

11 The most commonly used indicator of cardiovascular morbidity is increased BP and its derived 

12 index, hypertension. Hypertension diagnoses in these studies require that the patient or subject have 

13 diastolic and/or systolic BP above certain cut-points or be taking anti-hypertensive medicines. These BP 

14 cut-points have historically been established by reference to informed medical opinion and as medical 

15 knowledge improves BP cut-points defining hypertension have been lowered over time. Consequently, 

16 different studies using “hypertension” as a cardiovascular outcome may assign different cut-points, 

17 depending on the year and location of the study and the individual investigator. All of the new studies in 

18 the current review used the same criteria for hypertension (e.g., systolic BP at or above 140, diastolic at or 

19 above 90 or taking anti-hypertensive medications). Studies in the medical literature show that increasing 

20 BP is associated with increased rates of cardiovascular disease including coronary disease, stroke, 

21 peripheral artery disease, and cardiac failure. Coronary disease (i.e. myocardial infarction, angina 

22 pectoris, sudden death) is the most lethal sequela of hypertension (Chobanian et al., 2003; Ingelsson et al., 

23 2008; Kannel, 2000a, 2000b; Neaton et al., 1995; Pastor-Barriuso et al., 2003; Prospective Studies 

24 Collaboration, 2002). 

25 Several recent general population and occupational cohort studies examined the associations of 

26 blood Pb and/or bone Pb with BP (Figure 5-30 and Table 5-13) as well as the associations of these Pb 

27 exposure metrics with hypertension (Figure 5-31 and Table 5-14). In a cross-sectional analysis, Martin et 

28 al. (2006) examined the association of blood and tibia Pb with BP and hypertension in a community-based 

29 cohort study of older adults (n = 964). Four models evaluated associations for BP and hypertension 

30 considering SES and race/ethnicity. Blood Pb but not tibia Pb was a strong and significant predictor of BP 

31 in all models with an approximately 1 mmHg increase in systolic BP with each 1 μg/dL increase in blood 
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1 Pb level and an approximately 0.5 mmHg increase in diastolic BP per 1 μg/dL increase in blood Pb level.

2 Tibia Pb but not blood Pb was associated with hypertension in logistic regression models. The authors 

3 applied propensity analysis to their models to better account for the effect of risk factors such as 

4 race/ethnicity, age and SES that were strongly associated with tibia Pb level. The propensity score 

5 analysis and model adjustment did not substantially change the numerical findings and conclusions (e.g. 

6 tibia Pb and hypertension were positively associated independent of race/ethnicity and socioeconomic 

7 status). No evidence for effect modification by race/ethnicity was found. Overall, the results suggest that 

8 Pb has an acute effect on BP as a function of recent dose and a chronic effect on hypertension risk as a 

9 function of cumulative exposure.

Reference Strata Pb Distributiona Exposure Metricb

Martin et al. (2006) 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: aPb distribution is the median (IQR) estimated to make comparable. bEffect estimates were 

standardized to 1 μg/dL blood Pb or 10 μg/g bone Pb.

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).
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Table 5-13. Additional characteristics and quantitative data for associations of blood and 

bone Pb with BP measures 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 technician, serum total 

cholesterol. SES, race/ ethnicity 

also included in select models 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):

Visit 1: 20.3 (9.6), Women

Visit 2: 20.8 (10.8), 

Women

Visit 3: 19.8 (10.7), 

Women

Visit 1: 35.0 (13.5), Men

Visit 2: 36.5 (14.2), Men

Visit 3: 35.4 (15.9), Men

Tibia Pb, mean (SD):

Visit 1: 28.2 (19.7), 

Women

Visit 2: 22.8 (20.9), 

Women

Visit 1: 41.7 (47.6), Men

Visit 2: 37.1 (48.1), Men

Patella Pb, mean (SD):

Visit 3 49.5 (38.5) Women

Visit 3 87.7 (117.0)

Multivariable models using GEE 

were used in longitudinal 

analyses. Models were adjusted 

for visit number, baseline age, 

baseline age squared, baseline 

lifetime alcohol consumption, 

baseline body mass index, sex, 

baseline BP lowering medication 

use, alcohol consumption, body 

mass index, sex, BP lowering 

medication use.

Model 1 (short-term)

Blood Pb concurrent

β=0.08 (-0.01, 0.16)

Blood Pb (longitudinal)

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): 30.9 (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: 

NR
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Study Population

/Location Parameter Pb Data Statistical Analysis Effect Estimate 

(95% CI)

Scinicariello et 

al. (2010)

6,016 NHANES III 

participants ≥ 17 yr BP Blood Pb:Overall Mean (SE): 2.99 

(0.09) μg/dL

Non-Hispanic Whites: 

2.87 (0.09)

Non-Hispanic Blacks 3.59 

(0.20)

Mexican American 3.33 

(0.11)

Multivariable linear regression of 

log-transformed blood Pb level 

adjusted for age, sex, education, 

smoking status, alcohol intake, 

BMI, serum creatinine levels, 

serum calcium, glycosylated 

hemoglobin, and hematocrit

Ln blood Pb

SBP

Non-Hispanic whites: 

β±SE=1.05±0.37 (p=0.01)

Non-Hispanic 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 interactions 

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 yr) enrolled 

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; height; 

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 μg/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 VANAS 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: Referent
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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 Inverse 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 mediator/modifier 

and moderator in the 

analysis, no effects 

presented

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 logistic 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 during 

pregnancy

Log-transformed blood Pb

at mid-pregnancy 

SBP r = 0.08; p = 0.03)

DBP (r = 0.07; p = 0.03) 

Significant correlations 

also observed after 24 

weeks of gestation and 

after 36 weeks of 

gestation.

Elmarsafawy 

et al. (2006)

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.
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Reference Study Strata Blood Pb (μg/dL) Comparison

Scinicariello et al. (2010) NHANES III Non-Hispanic 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 1b

Non-Hispanic 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 1b

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 1b

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-Hispanic 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-Hispanic 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. (2006)

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 homozygotes (e.g., ALAD1-1). c

Effect estimates were standardized to 1 μg/dL 

blood Pb. d

Effect estimates were standardized to 1 μg/g bone Pb.

Figure 5-31. Odds ratio (95% CI) for associations of blood and bone Pb 

with hypertension measures.
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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 Statistical Analysis Effect Estimate 

(95% CI)

Scinicariello et 

al. (2010)

6,016 NHANES 

III participants ≥ 

17 y

Hypertension

(current 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 logistic 

regression model adjusted for 

race/ethnicity, age, sex, 

education, smoking status, 

alcohol intake, BMI, serum 

creatinine levels, serum 

calcium, glycosylated 

hemoglobin, and hematocrit

Non-Hispanic whites:

Q1 Reference

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 NHANES 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)

Logistic regression models 

adjusted for age, education, 

smoking status, cigarette 

smoking, BMI, hematocrit, 

alcohol consumption, physical 

activity, antihypertensive 

medication use, and diagnosis 

of type-2 diabetes

OR’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) 

Men <50 yr 

0.98 (0.80, 1.22)

Men >50 yr 

1.20 (1.02, 1.41), 

Women <50 yr 

1.23 (1.04, 1.46), 

Women >50 yr 

1.09 (1.94, 1.26).
May 2011 5-160 DRAFT – DO NOT CITE OR QUOTE

Study Population/

Location Parameter Pb Data Statistical Analysis Effect Estimate 

(95% CI)

Muntner et al. 

(2005)

9,961 NHANES 

(1999-2002) 

participants

Hypertension

(current use of 

antihypertensive 

medication, SBP 

≥ 140 mmHg, or 

DBP ≥ 90 mmHg)

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 adjusted 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 

antihypertensive 

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: 1.20-1.70 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, parity, 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 

Korea (12/1999- 6/2001)

Hypertension

(mean SBP ≥ 140 

mmHg, DBP ≥ 90 

mmHg; and/or 

use of 

antihypertensive 

medications; or 

physician 

diagnosis) 

Blood Pb: 

Mean (SD): 31.9 (14.8) μg/dL

Patella Pb:

Mean (SD): 37.5 (41.8) μg/g

Logistic regression models 

adjusted for age, gender, BMI, 

diabetes, antihypertensive and 

analgesic medication use, Pb 

job duration, work status, 

tobacco and alcohol use

None of the Pb exposure 

metrics examined were 

(blood, patella, and ln 

patella) were significantly 

associated with 

hypertension (results not 

reported) 

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/m3

Seasonal mean at birth: 

0.0950 μg/m3

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/m3 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/m3 increase:1.04 

(1.04, 1.05)
May 2011 5-161 DRAFT – DO NOT CITE OR QUOTE

Study Population/

Location Parameter Pb Data Statistical 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 models adjusted for age, BMI, family 

history of hypertension, history 

of smoking, dietary sodium 

intake, and cumulative alcohol 

ingestion

Low calcium group (<800 

mg/d):

Blood Pb: 1.07 (1.00, 

1.15)

Tibia Pb: 1.02 (1.00, 1.04)

Patella Pb: 1.01 (1.00, 

1.03)

High calcium group (>800 

mg/d):

Blood Pb: 1.03 (0.97, 

1.11)

Tibia Pb: 1.01 (0.97, 1.04)

Patella Pb: 1.01 (0.99, 

1.03)

References not included in Figure 5-31 are included in this table.

1 In another cross-sectional analysis, Perlstein et al. (2007) examined the association of BP and pulse 

2 pressure (PP) among predominantly white older adults in the greater Boston area. The subjects in this 

3 study had at least one bone Pb measurement during the years 1991-1997 and were not on antihypertensive 

4 medication at the time of the measurement. A statistically significant association between blood Pb and 

5 DBP was observed in adjusted models but the correlations of BP with tibia Pb were not significant. Men 

6 with tibia Pb above the median had a significantly higher mean PP compared to men with tibia Pb below 

7 the median (4.2 mmHg [95%CI: 1.9, 6.5]). The trend toward increasing PP with increasing tibia Pb was

8 significant although none of the confidence intervals for PP referenced to the lowest quintile of tibia Pb

9 excluded the null value.

10 Peters et al. (2007) examined the modification by self-reported stress of the associations of Pb 

11 exposure (tibia and patella Pb) with BP and hypertension in a cohort of subjects enrolled in the VA-NAS. 

12 Cross-sectional analyses of the effect of bone Pb and stress on BP and hypertension were conducted. 

13 Increased but nonsignificant associations between hypertension status and SBP with bone Pb were 

14 observed. Interaction of stress with tibia Pb (tibia Pb β=3.77 [CI: 0.46, 7.09]) and stress with patella Pb 

15 was significant (patella Pb β=2.60 [CI: -0.95, 6.15]) in systolic BP models (neither bone, self-reported 

16 stress, nor the interaction predicted DBP). Figure 5-32 shows the association between SBP and tibia Pb, 

17 comparing those with high and low self-reported stress. Peters et al. (2007) also used Cox proportional 

18 hazards models to assess the interaction of stress and bone Pb level on the association of bone Pb with the 

19 development of hypertension among those free of hypertension at baseline. The results of this analysis 

20 showed interactions between both tibia and patella Pb and the incidence of hypertension (RR of 

21 developing hypertension among those with high stress: 2.66 [CI: 1.43, 4.95] per SD increase in tibia Pb 

22 and 2.64 [CI: 1.42, 4.92] per SD increase in patella Pb). These provide information regarding factors that 

23 moderate or modify Pb effects on cardiovascular health. Gump et al. (2005) described significantly 

24 greater total peripheral vascular resistance (TPR) associated with increased blood Pb among 122 children 

25 (mean age 9.5 years) under acute stress, assessed by minor tracing or reaction time tasks which are 

26 consistent with α and β adrenergic activation. In a new analysis (Gump et al., 2007) significant effects of 
May 2011 5-162 DRAFT – DO NOT CITE OR QUOTE

1 SES on stress-induced reactivity of BP and TPR are reported. A significant SES by blood Pb level 

2 interaction is also reported suggesting possibly heightened effects of blood Pb level on stress changes in 

3 TPR and BP in low SES groups.

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 selfreported stress.

4 Elmarsafawy et al. (2006) examined the modification of Pb effect by dietary calcium, with 467 

5 subjects from the VA-NAS. Responses on a semi-quantitative dietary frequency questionnaire with one6 year recall were converted to estimated calcium intake. Hypertension was modeled using logistic 

7 regression and included interaction terms between Pb (tibia, patella and blood Pb) and a dichotomized 

8 calcium intake variable (split at 800 mg/day). They also constructed alternative models stratified on the 

9 calcium variable. Nonsignificant increases in hypertension associated with elevated blood, tibia, and 

10 patella Pb were observed in both low and high calcium groups. The only significant interaction reported 

11 was between BMI and calcium in a tibia Pb model. The authors report that in linear regression models of 

12 BP stratified by calcium status, SBP increased 0.40 (95% CI: 0.11, 0.70) mmHg for every 1 μg/g increase 

13 in tibia Pb concentration in the high calcium group, and 0.19 (95% CI: 0.01, 0.37) mmHg in the low 

14 calcium group.

15 Glenn et al. (2006) simultaneously modeled the multiple Pb dose measures of individuals over 

16 repeated time periods, assessing cross-sectional as well as longitudinal relationships. The initial blood Pb 

17 level was used as a baseline covariate and the difference in blood Pb level between visits were computed 

18 for subsequent visits. The bone Pb measures were used to indicate historical exposure and cumulative 

19 exposure. Four models were specified: Model 1 was conceptualized to reflect short-term exposure; Model 

20 2 to reflect longer-term exposure controlling for recent dose; Model 3 to reflect longer-term exposure 

21 controlling for cross-sectional influence of cumulative dose; and Model 4 to reflect both short-term 
May 2011 5-163 DRAFT – DO NOT CITE OR QUOTE

1 change with recent dose and longer-term change with cumulative dose. Concurrent and longitudinal 

2 measures of blood Pb were associated with SBP in Model 1 (short-term exposure) and Model 4 (short3 and longer-term exposures). No associations with tibia Pb at baseline were observed while historical tibia 

4 Pb metric was negatively associated with SBP in each of the models. This study suggests that Pb exposure 

5 may act continuously on systolic BP and reduction in exposure may contribute to reductions in BP, while 

6 cumulative Pb burden may contribute to hypertension incidence by other mechanisms over longer time 

7 periods. Elevated BP may reflect an immediate response to Pb at a biochemical site of action as a 

8 consequence of recent dose or a persistent effect of cumulative doses over a lifetime.

9 In a separate analysis of the third year cross-sectional results of the same occupationally-exposed 

10 group, Weaver et al. (2008) examined associations between patella Pb and blood Pb level and SBP, DBP, 

11 and hypertension to determine interactions of the patella Pb effects with ALAD and vitamin D receptor 

12 (VDR) polymorphisms. None of the Pb exposure metrics were associated with DBP. Patella Pb alone was 

13 not significantly associated with SBP, while blood Pb, either alone or with patella Pb was positively and 

14 significantly associated with SBP. The patella Pb-age and blood Pb-age interactions were not significant. 

15 There were no significant effects of blood Pb or patella Pb on hypertension status, or effect modification 

16 by age or sex. Further, interactions between polymorphisms of the VDR and of ALAD with blood Pb and 

17 patella Pb on SBP were not significant. Mean blood Pb level was high (30.9 μg/dL) compared to non18 occupational groups. 

19 Weaver et al. (2010) provided the results of further analysis of the Korean worker cohort (V. M. 

20 Weaver et al., 2008), with a focus on determining functional form of the concentration-response 

21 relationships. The coefficient indicates that every doubling of blood Pb level is associated with a systolic 

22 BP increase of 1.76 mmHg. The J test, a statistical test for determining which, if either, of two functional 

23 forms of the same variable provides superior fit to data in non-nested models (Davidson & MacKinnon, 

24 1981) returned a p-value of 0.013 in favor of the natural log blood Pb level over the linear blood Pb level 

25 specification. This analysis indicates that systolic BP increase in this cohort is better described as a 

26 logarithmic function of blood Pb level within the blood Pb level range of the study than by a linear 

27 function.

28 Yazbeck et al. (2009) conducted a cross-sectional study examining a community-based group of 

29 pregnant women to determine the association of Pregnancy Induced Hypertension (PIH) with blood Pb 

30 level and unlike most other studies adjusted their model for metal blood concentrations of cadmium, 

31 manganese, and selenium. PIH was defined as systolic BP >140 mmHg and/or diastolic BP >90 mmHg

32 during at least two clinic visits after week 22 of gestation. Patients with pre-existing chronic hypertension

33 were excluded. An association between blood Pb and PIH was observed (OR 3.29 [95% CI: 1.11, 9.74]) 

34 between PIH cases (2.2 ± 1.4 μg/dL blood Pb) and normotensive patients (1.9 ± 1.2 μg/dL blood Pb). 

35 Cadmium and selenium concentrations were comparable between PIH and no PIH groups. Adjustment for 

36 the metals slightly attenuated but did not eliminate the association between blood Pb levels and the risk of 
May 2011 5-164 DRAFT – DO NOT CITE OR QUOTE

1 PIH. They observed no significant interactions among blood Pb level, any of the other elements, and 

2 maternal characteristics in predicting the risk of PIH. Interaction between selenium and Pb concentrations 

3 was not significant, and the putative protection effects of selenium through antioxidative properties were 

4 not confirmed in this study. 

5 Chen et al. (2006) reported an ecological study of air Pb concentration and PIH, aggregated by all 

6 50 U.S. states and the District of Columbia. The PIH data were taken from the live births and infant 

7 deaths up to 1 year of age compiled by the National Center for Health Statistics (CDC, 2000) for the year 

8 1998. Associations between state level air Pb and state level PIH were reported (OR 1.04 [95% CI: 1.03, 

1.04] per 0.05 μg/m3 9 Pb). No individual level data were used in the analysis. Wells et al. (2011) measured 

10 the influence of cord blood Pb on BP in 285 women at admission to the Johns Hopkins Hospital in 

11 Baltimore, MD, during labor and delivery. Women in the fourth quartile of blood Pb elevations (>0.96 

12 μg/dL) had significantly higher systolic and diastolic BP (upon admission and for maximum BP) 

13 compared to women in the first quartile (<0.46 μg/dL). The authors used Benchmark Dose Software 

14 V2.1, developed by the EPA, to estimate benchmark dose (BMD) and the associated lower confidence 

15 limit for benchmark dose (BMDL) for one standard deviation (SD) increase in BP, which is 

16 approximately equivalent to a 10% increase above the mean for the first quartile blood Pb “controls”. The 

17 BMD approach is used here only as a means of characterizing the exposure level where effects might be 

18 found. These BMDL results indicate that the 95% lower bound confidence limit on the venous blood Pb 

19 level that is associated with a 1 SD increase is about 1.85 μg/dL for all BP outcomes. While these reported 

20 results are similar to those in the 2006 Pb AQCD as well as those found 25 years ago but with blood Pb 

21 levels an order of magnitude lower, the authors did not provide enough information to allow for 

22 verification of the BMD analysis.

23 Zhang et al. (2010) examined the effect of polymorphisms of the hemochromatosis gene (HFE) on 

24 the bone Pb effect on PP among older adult men participating in the VA-NAS. Subjects had up to three PP 

25 measurements during the 10 year study period. The overall results demonstrated a strong relationship 

26 between bone Pb and PP in this study, similar to the cross-sectional PP study of many of the same subjects 

27 of the VA-NAS group, without genotyping, reviewed above (Perlstein et al., 2007). The effect of bone Pb 

28 (tibia and patella) among those with the H63D variant was greater compared to those with the wild-type 

29 or the C282Y variant. In another gene-environment interaction analysis, Scinicariello et al. (2010) used 

30 NHANES III (1988-1994) data to examine the interaction between ALAD genotype and blood Pb in 

31 relation to BP in a cross-sectional analysis. A significant interaction between log blood Pb level and 

32 ALAD1-2/2-2b among non-Hispanic whites and non-Hispanic blacks was observed. In addition, 

33 associations of blood Pb with SBP and DBP across race/ethnicity strata were presented. The strongest 

34 associations were observed among non-Hispanic blacks. Scinicariello et al. (2010) also examined the 

35 association of blood Pb level with hypertension. Significant associations between blood Pb level and 

36 hypertension were observed among non-Hispanic blacks and nonsignificant increases were observed 
May 2011 5-165 DRAFT – DO NOT CITE OR QUOTE

1 among non-Hispanic whites and Mexican Americans (with the exception of Q2 association for Mexican 

2 Americans.) In addition, non-Hispanic white ALAD2 carriers in the highest blood Pb level quartile had a 

3 significantly higher association with hypertension compared with ALAD1 homozygous individuals. 

4 Muntner et al. (2005) also used the NHANES data (1999-2002) to examine the cross-sectional 

5 effect of blood Pb on hypertension, peripheral artery disease (PAD), and chronic kidney disease. The PAD 

6 results are discussed later in Section 5.4.3.4 and chronic kidney disease results are discussed in Section 

7 5.5.2.2). Blood Pb increased regularly with age among those with blood Pb measurements (1.28 μg/dL 

8 [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 

9 group.) Associations were observed between blood Pb level and hypertension across race/ethnicity groups 

10 with significant trends observed for non-Hispanic blacks and Mexican Americans. Park et al. (2009)

11 examined the association of blood Pb as well as bone Pb, which was predicted from blood Pb, with 

12 hypertension. The predicted bone Pb metrics were derived from models using VA-NAS data and applied 

13 to the NHANES (1988-1994) population. Blood Pb was associated with hypertension overall in the 

14 NHANES part of this study, with larger associations among black men and women as well as older adults. 

15 Associations with estimated bone Pb were also observed. 

5.4.2.2. Toxicology

16 An array of studies have provided evidence that extended exposure to low levels of Pb (<5 μg/dL) 

17 can result in delayed onset of hypertension in experimental animals that persists long after the cessation of 

18 Pb exposure (U.S. EPA, 2006). Tsao et al. (2000) found significantly increased systolic and diastolic BP 

19 in rats with blood Pb levels relevant to human exposure (2.15 μg/dL). As this was the lowest Pb level 

20 tested, no evidence of a threshold was evident. After Pb exposure is removed, blood, heart, aorta, and 

21 kidney Pb levels decreased quickly within the first three months (H.-R. Chang et al., 2005). Pb-induced 

22 elevated systolic BP persisted for one month following Pb exposure cessation, followed by obvious 

23 decreases in BP until 4 months after Pb exposure. Between 4 and 7 months after Pb exposure, the still24 elevated BP did not decrease further, thus never returning to control BP levels. Decreases in BP were 

25 closely correlated with decreases in blood Pb level after exposure cessation. Prenatal Pb exposure in rats 

26 given a low calcium diet also resulted in increased arterial pressure (Bogden et al., 1995). 

27 Experimental animal studies continue to provide evidence to conclude that Pb exposure results in 

28 delayed, yet sustained arterial hypertension. Increased systolic BP developed in rats after exposure to 90 -

29 10,000 ppm Pb (as Pb acetate in drinking water) for various time periods resulting in blood Pb level 

30 between 19.3-240 μg/dL (Badavi et al., 2008; Bagchi & Preuss, 2005; Bravo et al., 2007; Grizzo & 

31 Cordellini, 2008; Heydari et al., 2006; Reza et al., 2008; Vargas-Robles et al., 2007; L.-F. Zhang et al., 

32 2009). However, past studies have shown statistically significant elevations in BP in rats with lower blood 

33 Pb level (Figure 5-33). Consistent with measurements of systolic BP, Pb exposure (100 ppm for 14 
May 2011 5-166 DRAFT – DO NOT CITE OR QUOTE

1 weeks; blood Pb level 24 μg/dL) also caused an increase in intra-aortic mean arterial pressure (Bravo et 

2 al., 2007). One study tested low levels of Pb exposure (30 ppm; blood Pb level 7.6 μg/dL) and did not 

3 find a statistically significant increase in systolic BP despite elevated blood Pb level after 8 weeks of 

4 treatment, however the data do represent a trend of increasing BP (Rizzi et al., 2009). Additionally, pups 

5 of Pb exposed dams (1,000 ppm through pregnancy and lactation) exhibited increased blood Pb level 

6 (58.7 μg/dL) and increased arterial systolic BP after weaning (Grizzo & Cordellini, 2008) suggesting a 

7 role for childhood Pb exposure leading to adult disease.
May 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. (2006) 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 weeks 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 associated with changes in 

SBP from the current ISA and 2006 Pb AQCD. 

1 Pb induced hypertension persists long after cessation of Pb exposure. Bagchi and Preuss (2005)

2 found that elevated systolic BP was maintained for 210 days after Pb exposure cessation. Chang et al. 

3 (2005) reported a partial reversibility of effect after cessation of Pb exposure, where Pb-induced elevated 

4 BP decreased but did not return to control levels 7 months post Pb exposure. However, chelation therapy 

5 using Na2CaEDTA was able to return systolic BP to levels comparable to untreated rats (Bagchi & Preuss, 

6 2005). Studies reporting the effect of Pb (as blood Pb level) on systolic BP in unanesthetized adult rats 

7 since 1992 report a positive increase in BP with increasing blood Pb level (Figure 5-34).

 

 

 

 

 

 

 

 

 

0 5 10 15 20 25 30 35

 

Dose with Statistically Significant Effect

Dose with No Statistically Significant Effect
May 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

1 The previous Pb AQCD examined a number of mechanisms leading to Pb-induced hypertension, 

2 including oxidative stress, hormonal and blood pressure regulatory system dysfunction, vasomodulation, 

3 and cellular alterations. Further examination of these possible mechanisms of hypertension from Pb 

4 exposure in experimental animals and cells has been conducted and are provided below.

Oxidative Stress Response

Reactive Oxygen Species and Nitric Oxide

5 Studies discussed in the previous Pb AQCD suggest a role for oxidative stress in the pathogenesis 

6 of Pb-induced hypertension, mediated by the inactivation of nitric oxide ( ̇NO) and downregulation of 

7 soluble guanylate cyclase (sGC) (Attri et al., 2003; Dursun et al., 2005; Gonick et al., 1997; Khalil8 Manesh et al., 1994; Khalil-Manesh, Gonick, Weiler, et al., 1993; Vaziri et al., 1997). Pb-induced 

9 reduction of biologically active  ̇NO is not due to a reduction in  ̇NO-production capacity (Vaziri & Ding, 

10 2001; Vaziri, Ding, et al., 1999); instead it is a result of inactivation and sequestration of  ̇NO by ROS 

100

120

140

160

180

200

0 5 10 15 20 25 30 35 40

Blood 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. 2009
May 2011 5-169 DRAFT – DO NOT CITE OR QUOTE

1 (Malvezzi et al., 2001; Vaziri, Liang, et al., 1999). Oxidative stress from Pb exposure in animals may be 

2 due to upregulation of NAD(P)H oxidase (Ni et al., 2004; Vaziri et al., 2003), induction of Fenton and 

3 Haber-Weiss reactions (Ding et al., 2000; Ding et al., 2001), and failure of the antioxidant enzymes, CAT 

4 and GPx, to compensate for the increased ROS (Farmand et al., 2005; Vaziri et al., 2003). Many 

5 biological actions of  ̇NO, such as vasorelaxation, are mediated by cGMP, which is produced by sGC 

6 from the substrate GTP. Oxidative stress also plays a role in Pb-induced downregulation of sGC (Courtois 

7 et al., 2003; Farmand et al., 2005; M. Marques et al., 2001). The reduction of the vasodilator  ̇NO leads to 

8 increased vasoconstriction and BP.

9 Pb-induced oxidative stress also induces renal tubulointerstitial inflammation which plays a crucial 

10 role in models of hypertension (Rodriguez-Iturbe et al., 2005; Rodriguez-Iturbe et al., 2004). 

11 Tubulointerstitial inflammation from treatment with Pb has been coupled with activation of the redox 

12 sensitive NFκB (Ramesh et al., 2001). Pb-induced hypertension, inflammation, and NFκB activation can 

13 be ameliorated by antioxidant therapy (Rodriguez-Iturbe et al., 2004). There is mixed evidence to suggest 

14 that Pb-induced hypertension may also be promoted by activation of PKC leading to enhanced vascular 

15 contractility (Valencia et al., 2001; Watts et al., 1995). 

16 Recent studies continue to provide evidence for the role of ROS and  ̇NO metabolism in Pb17 induced hypertension and vascular disease. Increased SBP after Pb exposure has been accompanied by 

increased superoxide (O2

-

) and O2

- 18 positive cells (Bravo et al., 2007; Vargas-Robles et al., 2007) , elevated 

19 urinary malondialdehyde (MDA) (Bravo et al., 2007), and increased 3-nitrotyrosine (Vargas-Robles et al., 

2007). Inhibition of NAD(P)H oxidase, an enzyme that generates O2

- 20 and hydrogen peroxide, was able to 

21 block Pb-induced (1 ppm) aortic contraction to 5-HT (L. F. Zhang et al., 2005). Increased SBP, intra22 aortic mean arterial pressure, and MDA after Pb exposure (100 ppm; blood Pb level 23.7-27 μg/dL) were 

23 also prevented by treatment with the immunosuppressive, mycophenolate mofetil (MMF) (Bravo et al., 

24 2007). MMF has been shown to inhibit endothelial NAD(P)H oxidase, which could explain the decrease 

25 in oxidative stress and BP. Red grape seed extract was also able to protect rats from Pb-induced (100 

26 ppm) increased BP and heart rate, perhaps through the antioxidant properties of the extract (Badavi et al., 

27 2008).

28 Exposure to Pb can also affect the activity and levels of antioxidant enzymes. Male and female rats 

29 exposed to Pb for 18 weeks (100-1,000 ppm) had altered responses in antioxidant enzyme found in heart 

30 tissue (Alghazal, Lenártová, et al., 2008; Sobekova et al., 2009). Pb exposure (>100ppm) in female rats 

31 increased the activity of cardiac SOD, GST, GR, and GPx and increased cardiac TBARS (1,000 ppm). Pb 

32 exposure in male rats did not affect the activity of SOD or production of TBARS, however decreased the 

33 activity of GST and GR (>100 ppm). Male and female rats also accumulated different amounts of Pb in 

34 the cardiac tissue after similar exposure (♂ 100 ppm: 205% of control, 1,000 ppm: 379%; ♀ 100 ppm: 

35 246%, 1,000 ppm: 775%), which could explain the sex differences observed.
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1 Oxidative stress can trigger a cascade of events that promote cellular stress, renal inflammation, 

2 and hypertension. As was shown previously (Rodriguez-Iturbe et al., 2004), Pb exposure can increase 

3 renal NFκB, which was associated with tubulointerstitial damage and infiltration of lymphocytes and 

4 macrophages (Bravo et al., 2007). These events could also be ablated by MMF treatment, likely due to its 

5 anti-inflammatory and antioxidant properties. Pb is also able to induce inflammation in human endothelial 

6 cells as a model for vessel intima hyperplasia (Zeller et al., 2010). The proinflammatory cytokine, 

7 interleukin-8 (IL-8) protein and mRNA were increased, dose and time dependently, after in vitro Pb 

8 exposure (5-50 μM). Enhanced IL-8 production was mediated through activation of the transcription 

9 factor Nrf2 (but not NFκB, hypoxia inducible factor-1, or aryl hydrocarbon receptor), as shown through 

10 increased nuclear translocation and Nrf2 cellular knockdown experiments. Additionally, measures of 

11 endothelial stress, NQO1 and HO-1 protein, were induced by Pb exposure (Zeller et al., 2010).

12 Oxidative stress affects vascular reactivity and tone through inactivation and sequestration of  ̇NO, 

13 causing a reduction in biologically active  ̇NO. Recent studies confirm these past conclusions on the 

14 interplay of ROS and  ̇NO metabolism in the cardiovascular effects of Pb. Elevated SBP and altered 

15 vasorelaxation after Pb exposure is accompanied by a decrease in total nitrates and nitrites (NOx) 

16 (Heydari et al., 2006; L. F. Zhang et al., 2007). Serum NOx levels in Pb-treated rats remained depressed 

17 for 8 weeks and then reversed after 12 weeks, despite continued elevation in SBP (Heydari et al., 2006). 

18 This return of serum NOx levels could be a result of compensatory increases in endothelial NOS (eNOS) 

19 attempting to replenish an over-sequestered  ̇NO supply. With this in mind, studies have shown increased 

20 eNOS protein expression after chronic Pb exposure in kidney (L. F. Zhang et al., 2007) and isolated 

21 cultured aorta (Vargas-Robles et al., 2007). No change in inducible NOS was observed in isolated 

22 cultured aorta after 1 ppm Pb exposure (L. F. Zhang et al., 2007).

23  ̇NO, also known as endothelium-derived relaxing factor, is a potent endogenous vasodilator. 

24 Studies continue to investigate the effects of Pb on  ̇NO dependent vascular reactivity. Perinatal Pb 

25 exposure (1000 ppm through pregnancy and lactation, blood Pb level 58.7 μg/dL) resulted in a greater 

26 increase in maximal contraction to L-NAME, which decreases  ̇NO production, with a greater effect in 

27 the endothelium than the smooth muscle cells (Grizzo & Cordellini, 2008). Additionally, blocking NOS 

28 with L-NAME abolished the relaxant response evoked by ACh, which triggers the release of  ̇NO from 

29 the endothelial cell, in aortic rings of perinatally exposed rats. Cyclooxygenase (COX) inhibition 

30 decreased the EC50 of the ACh response in Pb treated animals. This study suggests that pups exposed to 

31 Pb through pregnancy and lactation have an altered vascular reactivity that is endothelium dependent and 

32 occurs due to the altered release of  ̇NO and a COX-derived vasoconstrictor (Grizzo & Cordellini, 2008). 

33 Similarly, a recent study provides evidence that acute Pb exposure increases rat tail artery reactivity 

34 in an endothelium dependent manner due to a COX-derived vasoconstrictor and in part free radicals and 

35  ̇NO (Silveira et al., 2010). Acute exposure of rat tail artery to Pb (100 μM, 1 h) increased reactivity to 

36 phenylephrine. Pb exposure decreased ACh induced relaxation, suggesting damage to the endothelium. Pb 
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1 did not affect smooth muscle integrity since sodium nitroprusside (SNP)-induced vasorelaxation was 

2 unchanged. Inhibition of NOS increased the Pb pressor response whereas COX inhibition eliminated the 

3 response to PHE. Treatment with the SOD mimetic Tempol decreased, but did not eliminate, the Pb 

4 pressor response (Silveira et al., 2010). A second study showed that Pb (90 ppm) exposure did not change 

5 the rat thoracic aortic ring relaxation response curves to the  ̇NO donor, SNP (Rizzi et al., 2009). 

6 Conversely, Skoczynska and Stojek (2005) found that Pb exposure (50 ppm; blood Pb level 11.2 

7 μg/dL) enhanced  ̇NO-mediated vasodilation by ACh in rat mesenteric arteries and NOS inhibition 

8 enhanced the ACh relaxant response. In rat renal interlobar arteries, Pb exposure blunted the increase in 

9 AngII mediated contraction from NOS inhibition by L-NAME (Vargas-Robles et al., 2007).

Vascular Reactivity 

10 Alteration of the adrenergic system from Pb exposure, which can increase peripheral vascular 

11 resistance, and thereby arterial pressure, may be one cause of Pb-induced hypertension. Pb exposure in 

12 animals can increase stimulation of the sympathetic nervous system (SNS), as shown by increased plasma 

13 norepinephrine and plasma catecholamines (Carmignani et al., 2000; H.-R. Chang et al., 1997), and 

14 decreased β adrenergic receptor density and β agonist-stimulated cAMP production in the aorta and heart 

15 (H.-R. Chang et al., 1997; Tsao et al., 2000). These stimulatory effects on the SNS paralleled effects on 

16 BP, cardiac contractility, and carotid blood flow. Increased Pb induced arterial pressure and heart rate 

17 were abrogated by ganglionic blockade (C.-C. Lai et al., 2002) and gradually decreased 7 months after Pb 

18 exposure cessation along with Pb-induced SNS alterations (H.-R. Chang et al., 2005).

19 Increased BP can be caused by vascular narrowing leading to increased total peripheral resistance, 

20 resulting from activation of the SNS. In this neural mechanism, activation of the SNS leads to 

21 vasoconstriction, whereas inhibition leads to vasodilation. It has been suggested that Pb leads to increased 

22 vascular reactivity to catecholamines (i.e. epinephrine, norepinephrine (NE), and dopamine), hormones of 

23 the SNS. Indeed, the isolated mesenteric vessel bed from Pb treated rats (50 ppm blood Pb level: 11.2 

24 μg/dL, but not 100 ppm blood Pb level: 17.3 μg/dL) exhibited increased reactivity to NE (Skoczynska & 

25 Stojek, 2005). Similarly, 100 ppm Pb did not affect the NE induced contractile response after 10 months 

26 of exposure (L.-F. Zhang et al., 2009), suggesting a small range of doses affecting pressor response to NE. 

27 Catecholamines act primarily through the adrenergic and dopaminergic receptors. Antagonists of α1-

28 adrenergic, α2-adrenergic, β-adrenergic, and dopamine D1 receptors abolish Pb-induced aortic contraction 

29 (Fazli-Tabaei et al., 2006; Heydari et al., 2006). Phenylephrine-induced aortic contractions were enhanced 

30 by treatment with Pb (100 ppm; blood Pb level: 26.8 μg/dL), indicating a specific role for the α1-

31 adrenergic receptor. Additionally, Pb blunted the isoproterenol-induced relaxation, supporting a role for 

32 the β-adrenoceptors (Heydari et al., 2006; Vassallo et al., 2008).
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1 Recently, there has been mixed evidence for Pb disrupting vascular reactivity to other pressor 

2 agents. One study found that Pb (50 ppm; 12 weeks; blood Pb level: 11.2 μg/dL) increased acetylcholine 

3 (ACh) induced relaxation in rats (Skoczynska & Stojek, 2005). Additionally, studies have shown no 

4 change in ACh induced vasorelaxation after Pb exposure (Grizzo & Cordellini, 2008; Rizzi et al., 2009). 

5 However, Zhang et al. (2007) and Silveira et al. (2010) found that Pb (1 ppm and 100 μM, 1 h) blunted 

6 ACh induced relaxation in isolated rat thoracic aorta and tail artery, respectively. Another study 

7 investigated the influence of Pb on vasoconstriction from 5-hydroxytryptamine (5-HT). Pb (1 ppm) 

8 treatment of isolated rat thoracic aorta increased 5-HT induced contraction, which was endothelium 

9 dependent, but not due to 5-HT2B receptor expression (L. F. Zhang et al., 2005). Follow-up of this study 

10 in whole animals found, on the contrary, that Pb (100 ppm; blood Pb level: 28.4 μg/dL) decreased the 

11 maximum contractile response to 5-HT, but did not affect 5-HT plasma levels or 5-HT2B receptor 

12 expression (L.-F. Zhang et al., 2009). 

13 Studies continue to investigate the role of  ̇NO, also known as endothelium-derived relaxing factor, 

14 in Pb induced changes in vascular reactivity. A recent study provides evidence that acute Pb exposure 

15 increases rat tail artery reactivity in an endothelium dependent manner due in part to free radicals and 

16  ̇NO (Silveira et al., 2010). Acute exposure of rat tail artery to Pb (100 μM, 1 hour) increased reactivity to 

17 phenylephrine. Pb exposure decreased ACh induced relaxation, suggesting damage to the endothelium. 

18 However, Pb did not affect smooth muscle integrity since SNP-induced vasorelaxation was unchanged. 

19 Similarly, Pb (90 ppm) exposure did not change the rat thoracic aortic ring relaxation response curves to 

20 the  ̇NO donor, SNP (Rizzi et al., 2009). Inhibition of NOS increased the Pb pressor response to PHE 

21 (Silveira et al., 2010). Another study showed that blocking NOS with L-NAME, abolished the relaxant 

22 response evoked by ACh, which triggers the release of  ̇NO from the endothelial cell, in aortic rings of 

23 perinatally exposed rats (1,000 ppm through pregnancy and lactation, blood Pb level 58.7 μg/dL) (Grizzo 

24 & Cordellini, 2008). Additionally, perinatal Pb exposure resulted in a greater increase in maximal 

25 contraction to L-NAME, which decreases  ̇NO production, with a greater effect in the endothelium than 

26 the smooth muscle cells. This study suggests that pups exposed to Pb through pregnancy and lactation 

27 have an altered vascular reactivity that is endothelium dependent and occurs due in part to the altered 

28 release of  ̇NO (Grizzo & Cordellini, 2008). In addition, Pb exposure (100 ppm, 12 weeks) increased the 

29 renal vascular response to AngII in isolated perfused kidneys from Pb exposed rats (Vargas-Robles et al., 

30 2007). NOS inhibition by L-NAME increased AngII-induced vasoconstriction in control but not Pb31 exposed arteries, suggesting impaired  ̇NO availability (Vargas-Robles et al., 2007). Conversely, 

32 Skoczynska and Stojek (2005) found that Pb exposure (50 ppm; blood Pb level 11.2 μg/dL) enhanced 

33  ̇NO-mediated vasodilation by ACh in rat mesenteric arteries and NOS inhibition enhanced the ACh 

34 relaxant response.
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Renin-Angiotensin-Aldosterone and Kininergic Systems

1 The adrenergic system also affects the renin-angiotensin-aldosterone system (RAAS), which is 

2 responsible for fluid homeostasis and blood pressure regulation, and has been shown to be affected by Pb 

3 exposure. Meta-analysis found that Pb exposure (30-40 μg/dL) increases plasma renin activity and renal 

4 tissue renin in young rats, but not old (Vander, 1988). Exposure of experimental animals to Pb also 

5 induced increases in plasma, aorta, heart, and kidney angiotensin converting enzyme (ACE) activity; 

6 plasma kininase II, kininase I, and killikrein activities; and renal angiotensin II (AngII) positive cells 

7 (Carmignani et al., 1999; Rodriguez-Iturbe et al., 2005; Sharifi et al., 2004). ACE activity declined over 

8 time while arterial pressure stayed elevated, suggesting that the RAAS may be involved in the induction, 

9 but not the maintenance of Pb-induced hypertension in rats.

10 Recent studies continue to implicate the RAAS in the development of Pb-induced hypertension, 

11 especially during early exposure in young animals. Low level Pb (100 ppm, 14 weeks; blood Pb level 

12 23.7-27 μg/dL) exposure increased renal cortical AngII content and the number of tubulointerstitial 

13 AngII-positive cells (Bravo et al., 2007). This heightened intrarenal angiotensin corresponded with 

14 sodium retention and increased SBP and was ablated by the anti-inflammatory antioxidant, MMF. 

15 Similarly, early high level Pb (1% Pb, 40 days; blood Pb level >240 μg/dL after exposure, 12-13 μg/dL 

16 after chelation after 1 year) accumulation resulted in sustained hypertension (Bagchi & Preuss, 2005). 

17 Treatment with the AngII receptor blocker, Losartan, resulted in a greater decrease in SBP in Pb exposed 

18 rats than control rats that continued into later periods of follow-up (day 283). Increased SBP after early 

19 exposure to Pb corresponded with increased water intake, urine output, potassium excretion, and 

20 decreased urinary sodium and urine osmolality. These functional changes in renal behavior are consistent 

21 with the actions of a stimulated RAAS. AngII, a main player in the RAAS, induces arteriolar 

22 vasoconstriction leading to increased BP. Pb exposure increased the vascular reactivity to AngII (Vargas23 Robles et al., 2007). These studies point to the activation of the RAAS in the course of Pb-induced 

24 hypertension.

Vasomodulators 

25 The balance between production of vasodilators and vasoconstrictors is important in the regulation 

26 of blood pressure and cardiovascular function. The previous AQCD reported that the effects of Pb on 

27 vasomodulators are contradictory. Urinary excretion of the vasoconstrictor, thromboxane (TXB2), and the 

28 vasodilatory prostaglandin, 6-keto-PGF1α, were unchanged in rats with Pb-induced hypertension (Gonick 

29 et al., 1998). However, in vitro Pb exposure promoted the release of the prostaglandin substrate, 

30 arachidonic acid, in vascular smooth muscle cells (VSMC) via activation of phospholipase A2 (Dorman & 

31 Freeman, 2002). Plasma concentration and urinary excretion of the vasoconstrictive peptide, endothelin 

32 (ET) 3 was increased after low (100 ppm), but not high level (5000 ppm) Pb exposure in rats (Gonick et 
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1 al., 1997; Khalil-Manesh et al., 1994; Khalil-Manesh, Gonick, Weiler, et al., 1993). Antagonism of the ET 

2 receptor A blunted the downregulation of sGC and cGMP production by Pb in isolated rat artery 

3 segments, suggesting that some of the hypertensive effects of Pb exposure may be mediated through ET 

4 (Courtois et al., 2003). Additionally, Pb-exposed animals exhibited fluid retention and a dose dependent 

5 decline in the vasodilator, atrial natriuretic factor (ANF) (Giridhar & Isom, 1990). These studies suggest 

6 that Pb may interfere with the balance between vasodilators and vasoconstrictors forming the hormonal 

7 regulation of vascular contraction and blood pressure.

8 The imbalance in vasomodulators is one explanation for the concentration-dependent 

9 vasoconstriction observed after Pb exposure (Piccinini et al., 1977; Valencia et al., 2001; Watts et al., 

10 1995). Vasoconstriction after Pb exposure was not reported in all studies (Shelkovnikov & Gonick, 2001)

11 and is likely varied depending on the type of vessel used, the Pb concentration employed, and the animal 

12 species being studied. Studies have reported attenuation of acetylcholine- and NO-mediated vasodilation 

13 (M. Marques et al., 2001; Oishi et al., 1996) in some, but not all vascular tissues and in some, but not all 

14 studies (Purdy et al., 1997). These effects have been variably attributed to Pb mediated activation of PKC 

and direct action on the VSMCs through the Ca2+ 15 mimetic properties of Pb among other possibilities 

16 (Piccinini et al., 1977; Valencia et al., 2001; Watts et al., 1995).

17 One recent study investigated the role of the endothelial derived vasoconstrictor, ET-1, in Pb18 induced hypertension. ET-1 from the endothelium acts on the ETA-type receptors located on the vascular 

19 smooth muscle layer and may be involved in vascular reactivity by  ̇NO and COX derivatives. Pb 

20 exposure (1 ppm, 24 hours) to rat aortic segments decreased expression of sGC-β1 subunit, an enzyme 

21 involved in  ̇NO-induced vasodilation, and increased expression of COX-2 in an endothelium dependent 

22 manner (Molero et al., 2006). Even though Pb treatment did not alter ET-1 or ETA-type receptor protein 

23 expression in this system, blocking the ETA-type receptors partially reversed Pb-induced changes in sGC 

24 and COX-2 in vascular tissue. This study suggests that the endothelium and ET-1 may contribute to Pb25 induced hypertension through activation of ETA-type receptors that alter expression of COX-2 and sGC26 β1 subunit, which affects  ̇NO signaling.

27 COX-2 blockade has been shown to prevent Pb-induced downregulation of sGC expression 

28 (Courtois et al., 2003). Inhibition of COX-2 also decreases the Pb induced pressor response to ACh 

29 (Grizzo & Cordellini, 2008) and PHE (Silveira et al., 2010) in experimental animals. These studies 

30 suggest that Pb induced vascular reactivity may depend on the participation of a COX-derived 

31 vasoconstrictor, such as prostaglandins, prostacyclins, or thromboxanes.

5.4.2.4. Summary

32 The 2006 Pb AQCD reported a clear positive association between blood Pb level and BP. The effect 

33 was modest, but highly significant, as determined by a meta-analysis (Nawrot et al., 2002) of over 30 
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1 studies comprising over 40,000 subjects (Figure 5-35), reporting that each doubling of blood Pb was 

2 associated with a 1 mmHg increase in systolic BP and a 0.6 mmHg increase in diastolic BP. Recent 

3 studies support this conclusion at lower blood Pb levels (<2 μg/dL) and add to the evidence base on 

4 susceptibility factors and bone Pb associations with BP and hypertension at levels <20 μg/g. Associations 

5 of bone and/or blood Pb with systolic BP and hypertension were higher among non-whites, those 

6 reporting high stress, and those with the HFE H63D and ALAD genotypes.

7 A recent study in an ethnically diverse community-based cohort of women and men aged 50-70 

8 years of age suggests that Pb has an acute effect on BP as a function of recent dose measured by blood Pb

9 and a chronic effect on hypertension risk as a function of cumulative exposure measured by tibia Pb 

10 (Martin et al., 2006). This study verified other studies by demonstrating that with each increase of 1 

11 μg/dL blood Pb level, systolic BP would increase 1 mmHg and diastolic BP would increase 0.5 mmHg. 

12 Additionally, recent epidemiologic studies provided evidence for associations between blood Pb and BP 

13 and hypertension at relatively low blood Pb level; a positive relationship was found in the NHANES data 

14 (1999-2002) at a geometric mean blood Pb level of 1.64 μg/dL (Muntner et al., 2005). Animal 

15 toxicological studies also provide support for effects of low blood Pb level on increased BP with 

16 statistically significant increases shown as low as 2 μg/dL (Tsao et al., 2000). Collectively, all animal 

17 toxicological studies providing blood Pb level and BP measurements report positive increases in BP with 

18 increasing blood Pb level (Figure 5-34). New studies also demonstrate reversibility of Pb-induced 

19 increased BP following Pb exposure cessation or chelation.
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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.

Figure 5-35. Change in SBP, association size in mmHg with 95% CI, 

associated with a doubling in the blood Pb concentration. 

1 Epidemiologic studies continue to investigate the relationship between bone Pb and increased BP. 

2 A recently published meta-analysis of epidemiological studies used bone Pb as an exposure index and BP 

3 or hypertension as the outcome (Figure 5-36) (Navas-Acien et al., 2008). The eight studies (three 

4 prospective, five cross-sectional) showed positive relationships between tibia Pb and systolic BP but not 
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1 diastolic BP. A pooled estimate for an increase in systolic BP of 0.26 mmHg (95% CI: 0.02, 0.50) for the 

2 cross-sectional studies was reported. The estimate for the longitudinal studies was 0.33 mmHg (95% CI: -

3 0.44, 1.11). With the exception of one study, positive associations of bone Pb with hypertension were also 

4 reported. Pooled estimates of 1.04 (95% CI: 1.01, 1.07) per 10 μg/g increase in tibia Pb and 1.04 (95% 

5 CI: 0.96, 1.12) per 10 μg/g increase in patella Pb were reported. 

6 Recent epidemiological studies have also emphasized the interaction between long-term Pb 

7 exposure and factors that moderate or modify the Pb effect, like chronic stress and metabolic syndrome, 

8 on BP and hypertension. Bone Pb coupled with high stress was associated with a strong and reliable 

9 increased risk of developing hypertension in an originally nonhypertensive group (Peters et al., 2007). 

10 Also, long duration Pb exposure interacted with components of the metabolic syndrome to drive HRV in 

11 directions associated with increased cardiovascular events (Park et al., 2006). 
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Source: 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 Pb levels and the prevalence of hypertension and Cheng et al (2001) reported the crosssectional association between bone Pb levels and SBP in study participants free of hypertension at 

baseline.

Note: The studies are ordered by increasing mean bone Pb levels. The area of each square is 

proportional to the inverse of the variance of the estimated change or log relative risk. Horizontal 

lines represent 95% confidence intervals. Diamonds represent summary estimates from inversevariance 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 μg/g increase in bone Pb 

levels. 

1 Recent epidemiologic studies investigated the interaction of genotypes with effects of Pb on the 

2 cardiovascular system. Significant evidence was presented for modification of the effect of blood Pb level 

3 on BP by ALAD genotype (Scinicariello et al., 2010). Additionally, polymorphisms in the 

4 hemochromatosis gene modified the pulse pressure response to bone Pb exposure, where pulse pressure 

5 represents a good predictor of cardiovascular morbidity and mortality and an indicator of arterial stiffness 

6 (A. Zhang et al., 2010). Park et al. (2009) provided further evidence of gene variants, specifically those 

7 related to iron metabolism, impacting the effect of long-term Pb exposure on the cardiovascular system, 

8 evaluated by QT interval changes.
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1 Not only has Pb exposure been shown to increase BP and hypertension, but Pb exposure can 

2 contribute to the development of other cardiovascular diseases. Recent epidemiologic and toxicological 

3 studies provide evidence for increased atherosclerosis, thrombosis, ischemic heart disease, peripheral 

4 artery disease, arrhythmia, and cardiac contractility.

5 Animal toxicological evidence continues to build on the evidence supporting the mechanisms 

6 leading to these cardiovascular alterations. Enhanced understanding of Pb-induced oxidative stress 

7 including  ̇NO inactivation, endothelial dysfunction leading to altered vascular reactivity, activation of the 

8 RAAS, and vasomodulator imbalance provides biological plausibility for the consistently positive 

9 associations observed between blood and bone Pb and cardiovascular effects.

5.4.3. Vascular Effects and Cardiotoxicity

10 Not only has Pb exposure been shown to increase BP and alter vascular reactivity, but Pb can alter 

11 cardiac function, initiate atherosclerosis, and increase cardiovascular mortality. Past toxicological studies 

12 have reported that Pb can increase atheromatous plaque formation in pigeons, increase arterial pressure, 

13 decrease heart rate and blood flow, and alter cardiac energy metabolism and conduction (Prentice & 

14 Kopp, 1985; Revis et al., 1981). Epidemiologic studies discussed in the previous AQCD provided limited 

15 evidence to support the association of ischemic heart disease (IHD) and peripheral artery disease (PAD) 

16 with increased blood Pb.

5.4.3.1. Effects on Vascular Cell Types

17 The endothelium layer is an important constituent of the blood vessel wall, which regulates 

18 macromolecular permeability, vascular SMC tone, tissue perfusion, and blood fluidity. Damage to the 

19 endothelium is an initiating step in development of atherosclerosis, thrombosis, and tissue injury. Given 

20 that chronic Pb exposure promotes a number of these diseases, numerous studies have investigated the 

21 role of Pb on endothelial dysfunction. The endothelial layer makes up only a small part of the vascular 

22 anatomy; the majority of the vessel wall is composed of vascular SMC, which work in concert with the 

23 EC in contraction and relaxation of the vessel, local BP regulation, and atherosclerotic plaque 

24 development. Since Pb has been shown repeatedly to result in hypertension and vascular disease, studies 

25 continue to investigate the effects of Pb on SMC.

26 Pb exposure (50 μM, 2 weeks) stimulated SMC invasiveness in isolated human arteries leading to 

27 the invasion of medial SMC into the vessel intima and development of intimal hyperplasia, a key step in 

28 atherosclerotic progression (Zeller et al., 2010). In addition, Pb exposure (50 μM, 12 hours) promoted 

29 SMC elastin expression and increased arterial extracellular matrix in isolated human arteries. SMC 

30 invasiveness was also increased in culture by treatment with supernatant of Pb-treated human EC (50 

31 μM), suggesting that Pb-exposed EC secrete an activating compound. This compound was confirmed to 
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1 be interleukin-8 (IL-8). Pb exposure (5-50 μM) was able to dose-dependently increase IL-8 synthesis and 

2 secretion in human umbilical vein EC cultures through activation of the transcription factor Nrf2. 

3 Neutralization of IL-8 could block SMC invasion and arterial intima thickening (Zeller et al., 2010). This 

4 study provides evidence that Pb exposure stimulates EC to secrete IL-8 in an Nrf2-dependent manner that 

5 stimulates SMC invasion from the vessel media to intima leading to a vascular thickening and possibly 

6 atherogenesis.

7 A number of cardiovascular diseases, including atherosclerosis, are characterized by increased 

8 inflammatory processes. Numerous studies have shown that Pb exposure is able to induce an 

9 inflammatory environment in humans and animals by increasing inflammatory mediators like 

10 prostaglandin E2 (PGE2). Human aortic vascular SMC treated with Pb (1 μM, 1-12 hours) exhibited 

11 increased secretion of PGE2 time-dependently through enhanced gene transcription (W. C. Chang et al.). 

12 This was preceded by a Pb-induced increase in gene expression of the rate limiting enzymes in the 

13 regulation of prostaglandins, cytosolic phospholipase A2 (cPLA2) and COX-2. The induction of these 

14 enzymes was mediated by activation of ERK1/2, MEK1, and MEK2. Further investigation into the 

15 entrance of Pb into the cell revealed that inhibition of the store-operated calcium channels (SOC) could 

16 only partially suppress cPLA2 and COX activation by Pb; however inhibition of epidermal growth factor 

17 receptor (EGFR) attenuated Pb-induced cPLA2 and COX activation and PGE2 secretion. Overall this 

18 study suggests that Pb can induce proinflammatory events in vascular SMC in the form of increased PGE2

19 secretion and cPLA2 and COX-2 expression through activation of EGFR via ERK1/2 pathways.

20 Damage to the endothelium is a hallmark event in the development of atherosclerosis. Past studies 

21 have shown that Pb exposure results in de-endothelialization, impaired proliferation, and inhibition of 

22 endothelium repair processes after injury (Fujiwara et al., 1997; Kaji et al., 1995; Kishimoto et al., 1995; 

23 Ueda et al., 1997). However, Pb exposure does not lead to nonspecific cytotoxicity at low exposure levels 

24 (2-25 μM) as shown by the lack of release of lactate dehydrogenase (LDH) from Pb-treated bovine aortic 

25 EC (Shinkai et al., 2010). Instead, Pb results in specific cytotoxicity (caspase3/7 activation) through 

26 endoplasmic reticulum (ER) stress that can be protected against by the ER chaperones glucose-regulated 

27 protein 78 (GRP78) and glucose-regulated protein 94 (GRP94). GRP78 and GRP94 play key roles in the 

28 adaptive unfolded protein response that serves as a marker of and acts to alleviate ER stress. Exposure of 

29 Pb to EC induces GRP78 and GRP94 gene (2-25 μM) and protein (GRP78 (5-25 μM) and GRP94 (10-25 

30 μM)) expression through activation of the IRE1-JNK-AP-1 pathways (Shinkai et al., 2010). This study 

31 suggests that the functional damage caused by Pb exposure to EC may be partly attributed to induction of 

32 ER stress.
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5.4.3.2. Cholesterol

1 As blood cholesterol rises so does the risk of coronary heart disease. Early occupational studies 

2 (Ademuyiwa, Ugbaja, Idumebor, et al., 2005; Bener et al., 2001; Kristal-Boneh et al., 1999) at higher than 

3 current blood Pb levels reported higher total cholesterol levels related to Pb exposure, and mixed results 

4 for HDL, LDL, and triglycerides. More recently, Poreba et al. (2010), in an occupational study, reports no 

5 significant differences in parameters of lipid metabolism between Pb exposed and unexposed 

6 hypertensive patients. Other Pb studies (Menke et al., 2006) adjust models for total cholesterol to control 

7 for this coronary heart disease risk factor and note that mean total cholesterol was higher at higher blood 

8 Pb levels. In developing models to predict bone Pb levels, Park et al. (2009) noted that total and high9 density lipoprotein cholesterol were selected as 2 of 18 predictors for the bone Pb level model. Their 

10 findings suggested that total and HDL cholesterols, as part of a larger groups of predictors, may be critical 

11 contributors to such prediction models. The major risk factor that lipids represent for heart disease make 

12 relating lipid levels to lead exposures an interesting but challenging hypothesis to test.

5.4.3.3. Heart Rate Variability

13 Pb has been shown to not only affect vascular contractility but also cardiac contractility. Park et al. 

14 (2006) investigated the interaction of key markers of the metabolic syndrome and bone Pb effect on heart 

15 rate variability (HRV) in a group of 413 older adults with patella Pb measurements from the VA-NAS.

16 Metabolic syndrome was defined to include three or more of the following: waist circumference >102 cm, 

17 hypertriglyceridemia (>150 mg/dL), low HDL cholesterol (<40 mg/dL in men), high blood pressure 

18 >130/85 mmHg, and high fasting glucose (>110 mg/dL). Those using antihypertensive medication or 

19 diabetes medications were counted as high BP or high fasting glucose, respectively. The strongest 

20 relationships with bone Pb were among those with three or more metabolic abnormalities. Tests for trend 

21 by number of metabolic abnormalities was significant at p<0.05 for patella Pb. These results suggest 

22 multiplicative effects of long duration Pb exposure on key predictors of CVD. Park et al. (2006) also 

23 reported the penalized spline fits to bone Pb in models assessing only main effects of bone Pb. The 

24 optimal degree of smoothing determined by the generalized cross-validation criterion for all HRV 

25 measures was 1, which indicated that the associations were nearly linear. The spline fits and associated 

26 statistics showed that the (nonsignificant) bone Pb main effects on HRV measures were linear. However, 

27 the relationship with LF/HF was linear in log(LF/HF).

28 Park et al. (2009) followed up a previous report (Y. Cheng et al., 1998), which found prolongation 

29 of corrected QT interval (QTc) with increasing bone Pb in men <65 years, but not in men ≥ 65. In the 

30 recent work, authors stratified multiple regression models on polymorphisms in three genes known to 

31 alter iron metabolism, hemochromatosis (HFE), transferrin (TF), and heme oxygenase-1 (HMOX-1), and 

32 related QTc intervals to blood, tibia and patella Pb level. They also used interaction models with cross-
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1 product terms between genotype and the Pb biomarker. The distributions of all genotypes but the HFE 

2 mutant, H63D, were in Hardy-Weinberg equilibrium. Subjects homozygous for the other HFE mutant, 

3 C282Y, had higher bone Pb concentrations and those homozygous for H63D and heterozygous for both 

4 C282Y and H63D had lower bone Pb. The HMOX-1 variant alone, compared to the wild type, showed a 

5 significant interaction with tibia Pb (increased QTc [11.35 msec] for each 13 μg/g increase in bone Pb in 

6 L-allele variants). No other gene variant alone showed significant QTc differences from wild types, either 

7 for tibia and patella Pb or for (linear) blood Pb. Lengthening of QTc with increased tibia and blood Pb 

8 was more pronounced with an increase in the total number of gene variants, driven by a joint effect 

9 between HFE variant and HMOX-1 L allele. Tests for linear trend in QTc by increasing number of gene 

10 variants from 0 to 3 were significant for both tibia and blood Pb. This study provided further suggestive 

11 evidence of gene variants impact on long-term Pb exposure on the cardiovascular system. 

12 Increased incidence of arrhythmia and atrioventricular conduction block increased in rats after 12 

13 weeks of Pb exposure (100 ppm; blood Pb level 26.8 μg/dL) (Reza et al., 2008). Also, Pb exposure after 8 

14 weeks increased heart rate and systolic BP. These corresponded with increased cardiac contractile force 

15 and prolonged ST interval, without alteration in QRS duration or coronary flow. In contrast, another study 

16 found that Pb (100 μM) exposure dose-dependently reduced myocardial contraction using rat right 

ventricular strips by reducing sarcolemmal Ca2+ 17 influx and myosin ATPase activity (Vassallo et al., 2008). 

18 This study also found that Pb exposure could change the response to inotropic agents and blunted the 

19 force produced during contraction. Conversely, past studies have found that Pb exposure increases 

intracellular Ca2+ 20 content (Favalli et al., 1977; Lal et al., 1991; Piccinini et al., 1977), which could result 

21 in increased cardiac output and hypertension.

5.4.3.4. Peripheral Artery Disease 

22 Peripheral artery disease (PAD) is an indicator of atherosclerosis and measured by the ankle 

23 brachial index, which is the ratio of BP between the posterior tibia artery and the brachial artery. PAD is 

24 typically defined as an ankle brachial index of less than 0.9. Muntner et al. (2005), whose results 

25 describing the association of blood Pb and hypertension in the NHANES 1999-2002 data set were 

26 discussed previously, also examined the association of blood Pb with PAD. They observed an increasing 

27 trend in the prevalence of PAD with increasing Pb level. The OR for PAD comparing the fourth quartile 

28 of blood Pb (>2.47 μg/dL) to the first quartile of blood Pb (<1.06 μg/dL) was 1.92 (95% CI: 1.02, 3.61). 

29 These results are consistent with a previous NHANES analysis of the association of blood Pb with PAD 

30 conducted by Navas-Acien et al. (2004).

31 Navas-Acien et al. (2004) reported a significant trend of increasing OR with increasing quartile of 

32 blood Pb or Cd. However, the authors reported a significant association of blood Cd with PAD and a non33 significant association with blood Pb. These authors tested both Pb and Cd in separate models, tested the
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1 metals simultaneously, and tested the interaction between the metals. The correlation coefficient between

2 natural log Pb and natural log Cd was 0.32 (p <0.001). When blood Pb and blood Cd were in the same 

3 model together, the ORs were diminished slightly but both had significant trends of increasing OR with 

4 increasing quartile of the metal. Their interaction was not significant. In a later analysis, Navas-Acien et 

5 al. (2005) used the same 1999-2000 NHANES data set that they used in their 2004 paper, but constructed 

6 PAD models using a suite of urine metal concentrations. Power was reduced in this study because only 

7 659-736 subjects (compared to 2,125) had spot urine metal tests in the data set. Urine Cd, but not urine 

8 Pb, was reliably elevated in all models in PAD subjects, while there were indications of elevations in 

9 antimony and tungsten. Spot urine Pb measurements are less reliable compared to blood Pb 

10 measurements. In another NHANES analysis Navas-Acien et al. (2005) reported associations between 

11 PAD and urinary Pb level that were sensitive to adjustment for urinary creatinine.

5.4.3.5. Ischemic Heart Disease

12 A few studies discussed in the previous Pb AQCD reported association between Pb exposure and 

13 increased risk of cardiovascular outcomes associated with IHD, including left ventricular hypertrophy (J. 

14 Schwartz, 1991) and myocardial infarction (Gustavsson et al., 2001). Recently, Jain et al. (2007) reported 

15 the incidence of IHD (physician confirmed MI, angina pectoris) among older adult males enrolled in the 

16 VA-NAS during the period of September, 1991 to December, 2001. All subjects had blood Pb and bone

17 Pb measurements with no IHD at enrollment. Fatal and nonfatal cases were combined for analysis. Blood, 

18 tibia, and patella Pb levels were log-transformed. Significant hazard ratios were reported for the 

19 association of IHD with blood Pb level and patella Pb level. When blood Pb and patella Pb were included 

20 simultaneously in the model, HR were only moderately attenuated (HR= 1.24 [95% CI: 0.80, 1.93] per 

21 SD increase in blood Pb and HR = 2.62 [95% CI: 0.99, 6.93] per SD increase in patella Pb). When blood 

22 Pb and tibia Pb were included simultaneously in the model the estimates were only moderately attenuated 

23 (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 

24 increase in tibia Pb). This suggests that both contribute independently to IHD. In an ecological study, 

25 Marchwinska-Wyrwal et al. (2010) report simple associations between 15-year air Pb averages in 15 

26 Silesian cities in Poland with similar health and socioeconomic conditions but widely varying air 

27 contamination, including air Pb and air Cd. They report associations between air Pb and CVD by city 

28 when each city was weighted equally. 

29 IHD, characterized by reduced blood supply to the heart, may result from increased thrombosis. A 

30 recent study suggests that Pb exposure promotes a procoagulant state that would contribute to thrombus 

31 formation (Shin et al., 2007). Pb exposure (i.v. 25 mg/kg, 1 hour) in a rat model of venous thrombosis 

32 resulted in increased thrombus formation. Additionally, increased blood coagulation (5 μM) and thrombin 

33 generation (2-5μM) was observed in a dose-dependent manner after Pb exposure to human erythrocytes. 
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1 This enhanced procoagulant activity in Pb-treated erthyrocytes was the result of increased outer cell 

2 membrane phosphatidylserine (PS) exposure (human RBC: 2-5 μM; rat RBC: 5 μM). Similar to these in 

3 vitro results, PS externalization on erythrocytes was increased in Pb exposed rats (50-100 mg/kg). 

4 Increased PS exposure was likely the result of increased intracellular calcium (5 μM), enhanced 

5 scramblase activity (5-10 μM), inhibited flippase activity (5-10 μM), and ATP depletion (1-5 μM) after Pb 

6 exposure (Shin et al., 2007).

5.4.3.6. Atherosclerosis

7 Studies provide evidence for increased atherosclerosis and intimal medial thickening (IMT) after 

8 Pb exposure. The association between stroke subtypes and severity of cerebral atherosclerosis was 

9 examined in relation to a single blood Pb level and total 72-hour urine Pb amount (body Pb store – EDTA 

10 mobilization test) in a cross-sectional study of 153 patients (mean age 63.7) receiving digital subtraction 

11 angiography in Chang Gung Memorial Hospital in Taiwan from 2002 to 2005 (T.-H. Lee et al., 2009). 

12 Body Pb stores were positively associated with the severity of artery stenosis in the intracranial carotid 

13 system but not the extracranial and vertebrobasilar systems. As development of atherosclerosis is a 

14 lifelong process, cumulative body Pb stores may be more sensitive than single blood Pb in the prediction 

15 of atherosclerosis. 

16 Zeller et al. (2010) examined human radial and internal mammary arteries exposed to Pb in culture 

17 and reported a dose-dependent increase in arterial intimal thickness (nonsignificant at 5 μM, significant at 

18 50 μM, 2 weeks) and intimal extracellular matrix accumulation (50 μM). Also, Pb exposure promoted 

19 endothelial cell (EC) proliferation (5 and 50 μM, 72 hours) and SMC elastin expression (50 μM, 12 

20 hours), as discussed above (Section 5.4.3.1) (Zeller et al., 2010). A second study showed that Pb exposure 

21 (100 ppm, 10 months; blood Pb level 28.4 μg/dL) to rats also increased the aortic media thickness, media22 lumen ratio, and medial collagen content (L.-F. Zhang et al., 2009). These morphological changes to the 

23 vessel do not only imply initiation of arteriosclerosis. These vascular changes could be to blame for 

24 decreased contractile response of the vessel due to altered visco-dynamic vessel properties or could be an 

25 effect of Pb-induced hypertension. 
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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 VANAS 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-cases 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 from 13 

Silesian cities,

Poland

CVD Air Pb (avg yearly rate):

127.8 - 359.9 ng/m3 Linear regression models β= 1.52 (0.76)

Peripheral artery disease

Muntner et al. 

(2005)

9,961 NHANES 

(1999-2002) 

participants

PAD Blood Pb:

Q1: <1.06 μg/dL, 

Q2: 1.06-1.63 μg/dL 

Q3: 1.63-2.47 μg/dL 

Q4: >2.47 μg/dL

Logistic regression models 

adjusted for age, 

race/ethnicity, sex, diabetes 

mellitus, BMI, cigarette 

smoking, alcohol 

consumption, high school 

education, health insurance 

status

OR (95% CI): 

1.00 (Reference), 

1.00 (0.45, 2.22), 

1.21 (0.66, 2.23), 

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, 

HFnorm, LF, 

LFnorm, LF/HF)

Patella Pb:

Median (IQR): 23.0 (15-34) 

μg/g

Estimateda

: 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) 

Log LF/HF: 3.3 (–10.7, 19.5)

Per 17 μg/g tibia Pb

Patella Pb: Model 2 Change 

(95%CI)

HF: –0.6 (–3.1, 1.9) 

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.
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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)

QTcb interval Blood Pb:

Median (IQR): 5 (4-7) μg/dL

Patella 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

1 There are a limited number of studies that investigate the association between Pb exposure and 

2 cardiovascular effects other than hypertension (Table 5-15). These studies have presented associations 

3 between various measures of Pb, representing distinct exposure time periods, and atherosclerosis, IHD, 

4 PAD, and HRV. Also, limited, mixed evidence of occupational exposure to Pb and altered cholesterol 

5 have been reported. Additionally, studies in isolated vascular tissues and cells provided mechanistic 

6 evidence to support the biological plausibility of these other vascular effects and cardiotoxicity. A recent 

7 study provided evidence for the interaction between Pb exposure and gene variants for iron metabolism 

8 on the prolonged QT interval (Park, Hu, et al., 2009). Blood Pb (>2.5 μg/dL) was associated with greater 

9 risk for PAD in two NHANES analyses (Muntner et al., 2005; Navas-Acien et al., 2004). In addition, in 

10 the VA-NAS cohort of older adult men, blood Pb (≥ 5 μg/dL) and patella Pb levels were associated with 

11 increased morbidity from IHD (Jain et al., 2007). A recent study involving both human and toxicological 

12 studies elucidated the mechanisms for observed Pb-mediated arterial IMT, an early event in Pb-induced 

13 atherogenesis (Zeller et al., 2010). Studies in isolated tissues and cells found that Pb stimulated the 

14 synthesis and secretion of IL-8 in EC, which was responsible for stimulating SMC invasion into the 

15 vessel intimal layer. Pb exposure also increased extracellular matrix and elastin, primary sites for lipid 

16 deposition in the vessel wall. Overall, the relatively few studies that investigate associations between 

17 biomarkers of Pb exposure and other cardiovascular events provide supportive evidence for the role of Pb 

18 in the development of these diseases, yet further research is needed to understand these relationships.

5.4.4. Mortality Associated with Long-Term Lead Exposure

19 The previous Pb AQCD (U.S. EPA, 2006) stated that collectively the then available analyses of 

20 NHANES II and III data suggest a significant effect of Pb on cardiovascular mortality in the general U.S. 

21 population but cautioned that these findings should be replicated before these estimates for Pb-induced 

22 cardiovascular mortality could be used for quantitative risk assessment purposes. This involved two 

23 NHANES analyses that examined the association of blood Pb with all cause and cause-specific mortality 
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1 (Lustberg & Silbergeld, 2002; Schober et al., 2006). As blood Pb levels in adults are comprised of both 

2 recent Pb exposure and Pb mobilization from bone, it is unclear whether the mortality associated with 

3 blood Pb levels are due to current or cumulative Pb exposures. Given the decline in ambient air Pb 

4 concentrations and population blood Pb levels, it is likely that study subjects had a much higher Pb 

5 exposure in their past than what they are experiencing currently. Using NHANES II data, Lustberg and 

6 Silbergeld (2002) found significant increases in all-cause, circulatory and cancer mortality, comparing 

7 those with blood Pb levels from 20-29 μg/dL to those with blood Pb levels less than 10 μg/dL. Using 

8 NHANES III data, Schober et al. (2006) found significant associations for all cause mortality, 

9 cardiovascular, and cancer mortality comparing those with blood Pb levels from 5-9 μg/dL and above 10

10 μg/dL to those with blood Pb levels less than 5 μg/dL. 

11 Three new studies make substantial additions to the evidence base. A further analysis of the 

12 NHANES III database by a different research group using different methods provides further information 

13 addressing uncertainties from other earlier analyses. Two longitudinal prospective studies in different 

14 cohorts conducted by different researchers with different methods in different parts of the U.S. provide 

15 coherence to the evidence base. Menke et al. (2006) examined the associations of all-cause and cause16 specific mortality using NHANES III data. Subjects at least 18 years of age were followed up to 12 years 

17 after they were surveyed and 1,661 deaths were identified. Those with blood Pb levels from 3.63 to 10 

18 μg/dL had significantly higher risks of all-cause (1.25 [95% CI: 1.04, 1.51]), cardiovascular (1.55 [95% 

19 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

20 compared to those with blood Pb levels less than 1.93 μg/dL and non-significantly increased risk of 

21 cancer mortality. Hazard ratios were not higher comparing those with blood Pb levels from 1.94 to 3.62 

22 μg/dL to those with blood Pb levels <1.93 μg/dL. However, trends of increasing hazard with increasing 

23 blood Pb tertile were significant (p <0.017) for all models of all CVD presented. Menke et al. (2006)

24 evaluated several of the model covariates (e.g. diabetes, hypertension, and GFR) in a subgroup analysis 

25 (Figure 5-37). The authors reported that there were no interactions between blood Pb and other adjusted 

26 variables and found a high consistency of HRs across models with a varying number of control variables 

27 (indicating little residual confounding). In the previous NHANES III analysis of the association of blood 

28 Pb with mortality, Schober et al. (2006) included participants greater than 40 years of age (N = 9,686) and 

29 adjusted for covariates including age, sex, ethnicity and smoking rather than the full suite of covariates 

30 evaluated by Menke et al. (2006). Schober et al. (2006) reported significant associations comparing those 

31 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, 

32 1.98]), CVD (1.55 [95% CI: 1.16, 2.07]), and cancer (1.69 [95% CI: 1.14, 2.52]) mortality and generally 

33 non-significant increases comparing those with blood Pb levels from 5-9 μg/dL to those with blood Pb 

34 levels <5 μg/dL. 
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Source: Used with permission from Lippincott Wiliams & Wilkins, Menke et al. (2006)

Note: Hazard ratios were calculated for a 3.4-fold increase in blood Pb level with log-blood Pb as a 

continuous variable. This increase corresponds to the difference between the 80th and 20th 

percentiles of the blood Pb distribution (4.92 μg/dL versus 1.46 μg/dL, respectively).

Figure 5-37. Multivariate adjusted relative hazards of all-cause and 

cardiovascular mortality. 

1 Both Menke et al. (2006) and Schober et al. (2006) present mortality curves that plot the hazard 

2 ratios against blood Pb level. Figure 5-38 shows the mortality curves for both stroke and MI reported by 

3 Menke et al. (2006), which reach a peak around 6-7 μg/dL. The curves were fitted by restricted quadratic 

4 splines with knots at the 10th percentile (1.00 μg/dL; 0.048 μM/L), the 50th percentile (2.67 μg/dL; 0.287 

5 μM/L), and the 80th percentile (5.98 μg/dL) blood Pb levels. 
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Source: Used with permission from Lippincott Wiliams & Wilkins, Menke et al. (2006)

Note: A histogram 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 level between 1 μg/dL and 10 μg/dL. 

1 Schober et al. (2006) examined proportional hazard assumptions, tested for linear trend across 

2 blood Pb tertiles, and evaluated log-transformed continuous blood Pb level as a 5-knot cubic spline 

3 (position of knots not stated). The test for linear trend by blood Pb tertile was significant at the p<0.01

4 level. The results of the spline fit of the continuous blood Pb level term to relative hazard of all 

5 cardiovascular diseases reported by Schober et al. (2006) is shown in Figure 5-39. In contrast to the curve 

6 presented by Menke et al. (2006) the relative hazard axis and the blood Pb axis are both linear. Dashed 

7 lines are 95% CIs and the referent blood Pb level was 1.5 μg/dL. Despite differences in the age groups 

8 included, follow-up time, and categorization of blood Pb levels, results reported by Menke et al. (2006)

9 and Schober et al. (2006) consistently conclude that higher blood Pb is associated with increased CVD 

10 mortality. 
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Source: Schober et al. (2006)

Note: The solid line shows the fitted five-knot spline relationship; the dashed lines are the pointwise 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 μg/dL (12.5th percentile). 

1 In addition to the NHANES analyses described above, two studies of older adult males (Weisskopf 

2 et al., 2009) and older adult females (Khalil, 2010; Khalil, Wilson, et al., 2009) were conducted. 

3 Weisskopf et al. (2009) used data from the VA-NAS to determine the association of blood, tibia, and 

4 patella Pb with mortality. The authors identified 241 deaths over an average observation period of 8.9 

5 years (7,673 person-years) in study subjects. The strongest associations were observed between mortality 

6 and patella Pb concentration. Non-significant increases in CVD mortality with tibia Pb and no association 

7 between blood Pb and mortality were observed. Tibia bone Pb concentration is thought to reflect a longer 

8 cumulative exposure period than patella bone Pb because the residence time of Pb in trabecular bone is 

9 shorter than in cortical bone. Ischemic heart disease contributed most to the relationship between patella 

10 Pb and all CVD death with HR of 2.69 (95% CI: 1.42, 5.08). Although there was high correlation between 

11 tibia and patella Pb (Pearson r = 0.77), trabecular bone Pb may have more influence on circulating blood 

12 Pb level, and thus organ concentration of Pb, than cortical bone Pb because of its shorter residence time in 

13 bone. In contrast to the NHANES analyses, blood Pb was not significantly related to cardiovascular 

14 mortality in this study. This discrepancy may be related to differences in sample size and resulting power, 

15 modeling strategies (e.g. linear versus log-linear blood Pb level terms), or age range of the study 

16 populations. The youngest subjects at baseline in the Weisskopf et al. (2009) study were approximately

17 50-55 years old, compared to the youngest in the Menke et al. (2006) and Schober et al. (2006) studies, 

18 which were 17 and 40 years, respectively. Further the blood Pb tertile analysis of the Weisskopf study 
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1 could have been affected if the majority of a hypothesized non-linear effect was contained largely in the 

2 lowest (reference) blood Pb tertile. 

3 Weisskopf et al. (2009) also conducted an exposure-response analysis. The test for linear trend of 

4 HR by bone Pb tertile was significant in both tibia and patella Pb models. The linear relationship using 

5 tertile patella Pb was confirmed in other models using continuous patella Pb and non-linear penalized 

6 spline terms, where higher order components were non-significant. The number of knots and their 

7 placement within the Pb variable, which can influence these results though the number and placement of 

8 knots of the penalized spline, were determined by an iterative best fit procedure to the data.

9 Concentration-response relationships shown in Figure 5-40 are approximately linear for patella Pb on the 

10 log hazard ratio scale for all CVD, but appear non-linear for ischemic heart disease (p <0.10). Peak HR is 

11 shown around 60 μg/g, beyond which HR tends to decrease, though the confidence limits are wide. 

Source: Used with permission 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 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. 
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1 The association of Pb with mortality has also been examined among women enrolled in the Study 

2 of Osteoporotic Fractures (SOF) (Khalil, Wilson, et al., 2009). This prospective cohort (N = 533) enrolled 

3 female volunteers from two locations across the U.S. (Baltimore, MD and Monongahela Valley, PA). All4 cause mortality comparing those with blood Pb levels >8 μg/dL to those with blood Pb levels <8 μg/dL

5 was significantly increased (1.59 [95% CI: 1.02, 2.49]). Combined cardiovascular disease mortality (1.78 

6 [95% CI: 0.92, 3.45]), coronary heart disease mortality (3.08 [95% CI: 1.23, 7.70]), but not stroke 

7 mortality (1.13 [95% CI: 0.34, 3.81]) HR was increased among the women enrolled in this study. In 

8 addition, Khalil et al. (2010) provided both tertile and quintiles analyses, as well as exposure-response 

9 results shown in Figure 5-41. 

Source: Khalil et al. (2010)

Figure 5-41. Multivariate adjusted relative hazard (left axis) of mortality 

associated with blood Pb levels between 1 μg/dL and 15 μg/dL. 

5.4.4.1. Summary

10 The mortality results in this review support and expand upon findings from the previous Pb AQCD 

11 (U.S. EPA, 2006), which included two NHANES mortality studies (Lustberg & Silbergeld, 2002; Schober 

12 et al., 2006). The recent NHANES mortality study discussed above (Menke et al., 2006) addressed many 
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1 of the limitations of the these earlier studies, including control for a wider range of potential confounders, 

2 testing for interactions with Pb, consideration of exposure-response relationships, extensive model 

3 evaluations, and including sub-categories of CVD. Further, an association with increased mortality was 

4 observed at lower population blood Pb concentrations. The mean blood Pb level of the NHANES III 

5 population was 2.58 μg/dL. The Pb attributable risk of increased cardiovascular mortality in the 

6 NHANES III analysis of Menke et al. (2006) reached its maximum at blood Pb levels between of 6 and 7 

7 μg/dL. In addition, the first evidence that bone Pb, a metric of cumulative Pb exposure, is associated with 

8 increased mortality was reported. Several studies report associations between the accumulated Pb in bone

9 and higher CVD morbidity, which are consistent with the mortality findings.

10 Quantitative differences in Pb-associated hazard for death between studies may be influenced by 

11 age range of the study groups, follow up time to death, variation in model adjustment, central tendency 

12 and range of the Pb exposure measure, assumptions of linearity of the Pb exposure term, and choice of 

13 exposure metric. Quantitative differences in Pb-associated mortality across NHANES II and NHANES III 

14 studies or between different NHANES III may be explained by the use of continuous or ordered blood Pb 

15 exposure variables and different data selection strategies. Further, studies using ordered categories of 

16 blood Pb level may obtain different results, as the range of blood Pb level represented in the reference 

17 category will affect the calculated coefficients of the remaining percentiles or groups. 

18 Specifically, Menke et al. (2006) is the strongest study presently published for estimating the 

19 national effects of Pb on cardiovascular disease-related mortality. The study uses the nationally 

20 representative NHANES III (1988 – 1994) sample allowing results to be generalized to the segment of the 

21 US population included in the sample. The results provide confirmation of earlier published studies and 

22 address some of the key weaknesses noted in those studies. Weisskopf et al. (2009) is the first published 

23 mortality study using bone Pb as an exposure index. The study is a prospective study with nearly 100% 

24 successful follow-up of deaths. This rigorous study found increased cardiovascular disease mortality with 

25 trabecular bone Pb. The Khalil et al. (2010; 2009) Study of Osteoporotic Fractures provides supporting 

26 results in a different study cohort consisting of white females aged 65-87 years. Further, a number of prior 

27 studies have already found association between accumulated Pb reflected in bone Pb measurements and 

28 higher CVD morbidity (see CVD morbidity section), to which is added new findings of increased 

29 mortality due to CVD from long-term Pb exposure (bone Pb). Despite the differences in design and 

30 methods across studies, effects on mortality were consistently observed (Figure 5-42 and Table 5-16). In 

31 studies that broke out CVD-related mortality into sub-categories, MI, stroke, and IHD mortality, death32 causes related to higher BP and hypertension were all significantly elevated as Pb exposure increased.
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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 yr < 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 yr < 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 Dichotomous

women CVD 1986 – 1988 ≥8 Dichotomous

Mean age = 70 yr 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

Men <4 Q1 Reference

Mean age = 67.3 yr 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).
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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 Analysis Effect Estimate 

(95% CI)

Menke et al. 

(2006)

13,946 adult 

participants of 

NHANES III , 

≥ 17 yr (1988- 1994)

All cause and cause-specific 

mortality 

(through 2000)

CVD:ICD-9 390-434; ICD-10 

I00-I99), MI (ICD-9 410-414 

and 429.2; ICD-10 I20-I25), 

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, Model 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 yr

All cause and cause-specific 

mortality 

Ordered 

categorical blood 

Pb level:

<5 μg/dL

5-9 μg/dL

≥10 μg/dL

Also ln(blood 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 yr, 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 hazard

regression analysis adjusted for 

age, smoking, education. 

Additional models adjusted for 

alcohol intake, physical activity, 

BMI, total cholesterol, serum HDL, 

diabetes mellitus, race, and 

hypertension

All-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, 

smoking, 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)

Stroke: 1.13 (0.34, 3.81)

Cancer: 1.64 (0.73, 

3.71)
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Study Population/

Location Parameter Pb Data Statistical Analysis Effect Estimate 

(95% CI)

Neuberger et al. 

(2009)

Residents at or 

near Tar Creek 

Superfund site, 

Ottawa County, OK (exposed pop. 

5,852, unexposed 

pop. 16,210) 

Cause-specific mortality Not reported Standardized mortality ratio 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, 

112.5)

Stroke:

Both sexes: 121.6 

(119.2, 123.9)

Men: 146.7 (107.4, 

195.7) Women: 106.5 

(80.2, 138.6)

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 5-42 are included in this table.

5.4.5. Air Lead-PM Studies

5.4.5.1. Hospital Admissions

1 In addition to blood Pb, some recent epidemiologic studies have used Pb measured in PM10 and 

2 PM2.5 air samples to represent Pb exposures. Some studies have analyzed Pb individually, whereas others 

3 have applied source apportionment techniques to analyze Pb as part of a group of correlated components. 

4 A common limitation of air-Pb studies is the variable size distribution of Pb-bearing PM (Section 3.5.3) 

5 and its relationship with blood Pb levels. Relative to studies of Pb biomarkers, time-series studies provide 

6 weak evidence for association between PM2.5-Pb concentrations and cardiovascular hospital admissions 

7 and mortality in older adults. In a time-series study of 106 U.S. counties, Bell et al. (2009) found that an 

8 increase in lag 0 PM2.5-Pb was associated with an increased risk of cardiovascular hospital admissions. 

9 Quantitative results were not presented; however, the 95% CI was wide and included the null value. Ostro 

et al. (2007) found that a 5 ng/m3 10 (interquartile range) increase in lag 3 PM2.5-Pb was associated with a 

11 1.89% increase (95% CI: -0.57, 4.40%) in cardiovascular mortality in six California counties in the cool 

12 season. In both of these studies, statistically significant positive associations were observed for other PM 

13 components such as nickel, vanadium, and zinc also. In the absence of detailed data on correlations 

14 among components or results adjusted for copollutants, it is difficult to exclude confounding by these 

15 other components.

16 To address correlations among PM chemical components, some studies have applied source 

17 apportionment techniques to group components into common source categories. In these source-factor 

18 studies, it is difficult to attribute findings to a particular component in a group. In a study of 20 counties 

19 near Atlanta, GA, Sarnat et al. (2008) found that PM2.5-Pb mass was explained by a “woodsmoke” factor, 

20 which was associated with a 2.4% (95% CI: 1.5, 3.3%) increased risk of cardiovascular hospital 
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1 admission (lag 0, per interquartile range increase). Less than 10% of variation in PM2.5-Pb mass was 

2 explained by “woodsmoke,” thus the association may not be attributable specifically to Pb. Andersen et 

3 al. (2007) found that in Copenhagen, Denmark, variation in PM10-Pb was explained by a “vehicle” factor 

that also included copper and iron. A 0.6 μg/m3 4 increase in the 3-day lagged sum of “vehicle” factor 

5 pollutants was not associated with increased risk of cardiovascular hospital admissions among adults age 

6 65 years and older (RR: 0.999, [95% CI: 0.993, 1.004]). 

5.4.5.2. Mortality

7 Time-series studies of PM2.5-Pb have reported positive associations with all-cause mortality. In the 

8 Harvard Six Cities Study, Laden et al. (2000) found a 1.16% (95% CI: 0.20, 2.9%) increased risk in allcause mortality per 461.4 ng/m3 9 (5th-9th percentile) increase in PM2.5-Pb. In six California counties, 

Ostro et al. (2007) found that a 5 ng/m3 10 (interquartile range) increase in PM2.5-Pb was associated with a 

11 1.74% (95% CI: 0.24, 3.26%) increased risk in all-cause mortality during the cool season. The limitations 

12 of air-Pb studies have been described previously (Section 5.5.5.1), and are relevant to the interpretation of 

13 these findings for all-cause mortality.

5.4.6. Summary and Causal Determination

14 The 2006 Pb AQCD concluded that there was a relationship between increased blood Pb and bone 

15 Pb and increased adverse cardiovascular outcomes in adults, including increased BP and increased 

16 incidence of hypertension (U.S. EPA, 2006). This was substantiated by the coherence of effects observed 

17 across epidemiologic and toxicological studies. The large evidence base of epidemiologic studies 

18 conducted by many researchers in many locations using different designs found a clear positive 

19 association between blood Pb level and BP. Meta-analysis of these studies found that each doubling of 

20 blood Pb level (between 1 and >40 μg/dL) was associated with a 1 mmHg increase in systolic BP and a 

21 0.6 mmHg increase in diastolic BP (Nawrot et al., 2002). In addition, most of the reviewed studies using 

22 cumulative Pb exposure measured by bone Pb levels also showed increased BP. Similarly, toxicological 

23 studies provided evidence for exposure to low levels of Pb resulting in increased BP in experimental 

24 animals that persists long after the cessation of Pb exposure. Also, animal toxicological studies provided 

25 mechanistic evidence to support the biological plausibility of Pb-induced hypertension, including 

26 oxidative stress, altered sympathetic activity, and vasomodulator imbalance. Studies in the 2006 Pb 

27 AQCD also suggested a connection between Pb exposure and other cardiovascular diseases such as 

28 ischemic heart disease, cerebrovascular disease, peripheral vascular disease, and cardiovascular disease 

29 related mortality, however this evidence was limited. 

30 Building on the strong body of evidence presented in the 2006 Pb AQCD, recent studies continue 

31 to support associations between long-term Pb exposure and cardiovascular effects with recent 
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1 epidemiologic studies informing past uncertainties (e.g. confounding, low Pb exposures). A recent study 

2 in an ethnically diverse community-based cohort of women and men aged 50-70 years of age suggests 

3 that Pb has an acute effect on BP as a function of recent dose measured by blood Pb and a chronic effect 

4 on hypertension risk as a function of cumulative exposure measured by tibia Pb (Martin et al., 2006). This 

5 study verified other studies by demonstrating that with each increase of 1 μg/dL blood Pb level, systolic 

6 BP would increase 1 mmHg and diastolic BP would increase 0.5 mmHg. Additionally, recent 

7 epidemiologic studies provided evidence for associations between blood Pb and hypertension at relatively 

8 low blood Pb levels; a positive relationship was found in the NHANES data set at a geometric mean 

9 blood Pb level of 1.64 μg/dL (Muntner et al., 2005). Animal toxicological studies also provide support for 

10 effects of low blood Pb level on increased BP with statistically significant increases shown as low as 2 

11 μg/dL (Tsao et al., 2000). Collectively, all animal toxicological studies providing blood Pb level and BP 

12 measurements report positive increases in BP with increasing blood Pb level (Figure 5-34). New studies 

13 also demonstrate reversibility of Pb-induced increased BP following Pb exposure cessation or chelation.

14 Epidemiologic studies continue to investigate the relationship between bone Pb, representing 

15 cumulative Pb exposure, and increased BP. Navas-Acien et al. (2008) published a meta-analysis of 

16 epidemiological studies examining this association. Studies passing the detailed inclusion criteria all 

17 showed positive relationships between bone Pb measures and BP and all but one that characterized 

18 hypertension showed positive risk or odds ratios associated with bone Pb. Recent epidemiological studies 

19 have also emphasized the interaction between long-term Pb exposure and factors that moderate or modify 

20 the Pb effect, like chronic stress and metabolic syndrome, on BP and hypertension. Bone Pb coupled with 

21 high stress was associated with a strong and reliable increased risk of developing hypertension in an 

22 originally nonhypertensive group (Peters et al., 2007). Also, long duration Pb exposure interacted with 

23 components of the metabolic syndrome to drive HRV in directions associated with increased 

24 cardiovascular events (Park et al., 2006).

25 Recent epidemiologic studies investigated the interaction of genotypes with effects of Pb on the 

26 cardiovascular system. Significant evidence was presented for modification of the effect of blood Pb level 

27 on BP by ALAD genotype (Scinicariello et al., 2010). Additionally, polymorphisms in the 

28 hemochromatosis gene modified the pulse pressure response to bone Pb exposure, where pulse pressure 

29 represents as a good predictor of cardiovascular morbidity and mortality and an indicator of arterial 

30 stiffness (A. Zhang et al., 2010). Park et al. (2009) provided further evidence of gene variants, specifically 

31 those related to iron metabolism, impacting the effect of long-term Pb exposure on the cardiovascular 

32 system, evaluated by QT interval changes.

33 Not only has Pb exposure been shown to increase BP and hypertension, but Pb exposure can 

34 contribute to the development of other cardiovascular diseases. Recent epidemiologic and toxicological 

35 studies provide evidence for increased atherosclerosis, thrombosis, ischemic heart disease, peripheral 

36 artery disease, arrhythmia, and cardiac contractility (blood Pb levels >2.5 μg/dL).
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1 Animal toxicological evidence continues to build on the evidence supporting the mechanisms 

2 leading to these cardiovascular alterations. Enhanced understanding of Pb-induced oxidative stress 

3 including  ̇NO inactivation, endothelial dysfunction leading to altered vascular reactivity, activation of the 

4 RAAS, and vasomodulator imbalance provides biological plausibility for the consistently positive 

5 associations observed between blood and bone Pb and cardiovascular effects.

6 New evidence extends the potential continuum of Pb-related cardiovascular effects by 

7 demonstrating associations between Pb exposure and both cardiovascular and all-cause mortality. All8 cause mortality was positively associated with increased blood Pb level. The recent analysis of the 

9 nationally representative NHANES III (1988-1994) sample reported positive associations with 

10 cardiovascular mortality, with stronger associations with myocardial infarction and stroke mortality 

11 (Menke et al., 2006). These findings were supported by a community-based cohort of women age 65-87 

12 years, in which effect estimates were increased for mortality from cardiovascular disease and coronary 

13 heart disease (Khalil, Wilson, et al., 2009). Weisskopf et al. (2009) published the first mortality study 

14 using bone Pb as an exposure index. This prospective study found that trabecular bone Pb levels were 

15 associated with increased mortality from cardiovascular disease and ischemic heart disease with hazard 

16 ratios of 5.6 and 8.4, respectively.

17 In summary, new studies evaluated in the current review support or expand upon the strong body of 

18 evidence presented in the 2006 AQCD that Pb exposure is causally associated with cardiovascular health 

19 effects. Both epidemiologic and toxicological studies continue to demonstrate a consistently positive 

20 relationship between Pb exposure and increased BP or hypertension development in adults and this 

21 relationship is observed at adult blood Pb levels (mean 2 μg/dL) lower than that reported in the 2006 

22 AQCD. While some studies evaluate exposure-response relationships, the information is inconclusive. 

23 Recent studies investigate cumulative Pb exposure measures and suggest that bone Pb related strongly to 

24 hypertension risk. Evidence of Pb increasing the risk of developing other cardiovascular diseases has also 

25 been shown. By demonstrating Pb-induced oxidative stress including  ̇NO inactivation, endothelial 

26 dysfunction leading to altered vascular reactivity, activation of the RAAS, and vasomodulator imbalance, 

27 toxicological studies have characterized the mode of action of Pb and provided biological plausibility for 

28 the consistently positive associations observed in epidemiologic studies between blood and bone Pb and 

29 cardiovascular effects. These observed associations between Pb exposure and cardiovascular morbidity 

30 are supported by recent reports of increased cardiovascular mortality. Collectively, the evidence integrated 

31 across epidemiologic and toxicological studies as well as across the spectrum of cardiovascular health 

32 endpoints is sufficient to conclude that there is a causal relationship between Pb exposures and 

33 cardiovascular health effects.
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5.5. Renal Effects

5.5.1. Introduction

1 This section summarizes key findings with regard to effects of Pb on the kidney in animal

2 toxicology and epidemiologic studies. After chronic Pb exposure, pathological changes in the renal 

3 system include proximal tubule (PT) cytomegaly, renal cell apoptosis, mitochondrial dysfunction, 

4 aminoaciduria, increased electrolyte excretion, ATPase dysfunction, oxidant redox imbalance, altered 

5 glomerular filtration rate (GFR), chronic kidney disease (CKD) development, and altered  ̇NO 

6 homeostasis with ensuing elevated BP. 

7 The cardiovascular and renal systems are intimately linked. Homeostatic control at the kidney level 

8 functions to regulate water and electrolyte balance via filtration, re-absorption and excretion and is under 

9 tight hormonal control. Pb exposure damages the kidneys and its vasculature and systemic hypertension 

10 ensues with effects on the cardiovascular and renal systems (Section 5.4). Chronic increases in vascular 

11 pressure can contribute to glomerular and renal vasculature injury, which can lead to progressive renal 

12 dysfunction and kidney failure. In this manner, Pb-induced hypertension has been noted as one cause of 

13 Pb-induced renal disease. However, the relationship between BP and renal function is more complicated. 

14 Not only does hypertension contribute to renal dysfunction but damage to the kidneys can also cause 

15 increased BP. Long-term control of arterial pressure is affected by body fluid homeostasis which is 

16 regulated by the kidneys. In examining the physiological definition of BP (i.e., mean BP equates to 

17 cardiac output multiplied by total peripheral resistance [TPR]) the role of the kidneys in BP regulation is 

18 highlighted. Cardiac output is driven by left ventricular and circulating blood volume. TPR is driven by 

19 vasomodulation and electrolyte balance. Thus, it is possible to dissect the causes of hypertension from 

20 features of primary kidney disease. Increased extracellular fluid volume results in increased blood volume 

21 which enhances venous return of blood to the heart and increases cardiac output. Increased cardiac output 

22 not only directly increases BP, but also increases TPR due to a compensatory autoregulation or vessel 

23 constriction. In addition, damage to the renal vasculature will alter the intra-renal vascular resistance 

24 thereby altering kidney function and affecting the balance between renal function and BP. The interrelated 

25 nature of these systems can lead to further exacerbation of vascular and kidney dysfunction following Pb 

26 exposure. As kidney dysfunction can increase BP and increased BP can lead to further damage to the 

27 kidneys, Pb-induced damage to both systems may result in a cycle of further increased severity of disease.

28 In general, associations between blood Pb and bone Pb (particularly in the tibia) with health 

29 outcomes in adults indicate acute effects of recent dose and chronic effects of cumulative dose, 

30 respectively. In some physiological circumstances of increased bone remodeling or loss (e.g., osteoporosis 

31 and pregnancy), Pb from bone of adults may also contribute substantially to blood Pb concentrations. 

32 Blood Pb in children, although highly affected by recent dose, is also influenced by Pb stored in bone due 
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1 to rapid growth-related bone turnover in children relative to adults. Thus, blood Pb in children is also 

2 reflective of cumulative dose. Additional details on the interpretation of Pb in blood and bone are 

3 provided in Section 4.3.5.

5.5.1.1. Kidney Outcome Measures

4 The primary function of the kidneys is to filter waste from the body while maintaining appropriate 

5 levels of water and essential chemicals, such as electrolytes, in the body. Therefore, the gold standard for 

6 kidney function assessment involves measurement of the GFR through administration of an exogenous 

7 radionuclide or radiocontrast marker (e.g., 125I-iothalamate, iohexol) followed by timed sequential blood 

8 samples or, more recently, kidney imaging, to assess clearance through the kidneys. This procedure is 

9 invasive and time-consuming. Therefore, serum levels of endogenous compounds are routinely used to 

10 estimate GFR in large epidemiology studies and clinical settings. Creatinine is the most commonly used 

11 endogenous compound; blood urea nitrogen (BUN) has also been used. Increased serum concentration or 

12 decreased kidney clearance of these markers both indicate decreased kidney function. The main limitation 

13 of endogenous compounds identified to date is that non-kidney factors impact their serum levels. 

14 Specifically, since creatinine is metabolized from creatine in muscle, muscle mass and diet affect serum 

15 levels resulting in variation in different population subgroups, e.g., women and children compared to 

16 men, which are unrelated to kidney function. Measured creatinine clearance, involving measurement and 

17 comparison of creatinine in both serum and urine, can address this problem. However, measured 

18 creatinine clearance utilizes timed urine collections, traditionally over a 24-hour period, and the challenge 

19 of complete urine collection over an extended time period makes compliance difficult. 

20 Therefore equations to estimate kidney filtration that utilize serum creatinine but also incorporate 

21 age, sex, race, and, in some, weight, in an attempt to adjust for differences in muscle mass have been 

22 developed. Although these are imperfect surrogates for muscle mass, such equations are currently the 

23 preferred outcome assessment method. Traditionally, the Cockcroft-Gault equation (Cockcroft & Gault, 

24 1976), which estimates creatinine clearance, a GFR surrogate, has been used. In the last decade, the 

25 abbreviated Modification of Diet in Kidney Disease (MDRD) Study equation (Levey et al., 1999; Levey 

26 et al., 2000), which estimates GFR, has become the standard in the kidney epidemiology and clinical 

27 communities. With widespread use of the MDRD equation, it became clear that it underestimates GFR at 

28 levels in the normal range. Therefore, the CKD-Epidemiology Collaboration (CKD-EPI) equation was

29 recently developed to be more accurate in this range (Levey et al., 2009). This is a decided advantage in 

30 nephrotoxicant research since most participants in occupational and many even in general population 

31 studies have GFRs in a range that is underestimated by the MDRD equation.

32 Both the MDRD and CKD-EPI equations use serum creatinine and the inability to adjust for 

33 muscle mass has led to evaluation of alternative serum biomarkers such as cystatin C, a cysteine protease 
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1 inhibitor that is filtered, reabsorbed, and catabolized in the kidney (Fried, 2009). It is produced and 

2 secreted by all nucleated cells thus avoiding the muscle mass confounding with serum creatinine (Fried, 

3 2009). Despite this, recent research indicates that serum cystatin C varies by age, sex, and race (Kottgen 

4 et al., 2008) and a cystatin C-based eGFR equation that includes age, sex, and race was recently 

5 developed (Stevens et al., 2008).

6 Most of the kidney outcome measures discussed above were developed for use in the clinical 

7 setting. Unfortunately, they are insensitive for detection of early kidney damage, as evidenced by the fact 

8 that serum creatinine remains normal after kidney donation. Therefore, in the last two decades, the utility 

9 of early biological effect (EBE) markers as indicators of preclinical kidney damage has been of interest. 

10 These can be categorized as markers of function (i.e., low molecular weight proteins that should be 

11 reabsorbed in the PT such as β2-microglobulin and retinol-binding protein [RBP]); biochemical alteration 

12 (i.e., urinary eicosanoids such as prostaglandin E2, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, 

13 and thromboxane B2); and cytotoxicity (e.g., N-acetyl-β-D-glucosaminidase [NAG]) (Cardenas et al., 

14 1993). Elevated levels may indicate an increased risk for subsequent kidney dysfunction. However, most 

15 of these markers are research tools only, and their prognostic value remains uncertain since prospective 

16 studies of most of these markers in nephrotoxicant-exposed populations are quite limited to date. 

17 Recently, microalbuminuria has been identified as a PT marker, not just glomerular as previously thought

18 (Comper & Russo, 2009). Kidney EBE markers are a major recent focus for research in patients with 

19 acute kidney injury (AKI) and markers such as neutrophil gelatinase-associated lipocalin (NGAL) and 

20 kidney injury molecule-1 (Kim-1), developed in AKI research, may prove useful for chronic 

21 nephrotoxicant work as well (Devarajan, 2007; M. A. Ferguson et al., 2008). 

5.5.2. Nephrotoxicity and Renal Pathology Related to Lead 

Effects

5.5.2.1. Toxicology

Renal Function and Interstitial Fibrosis

22 Past studies have shown that chronic continuous or repeated Pb-exposure can result in interstitial 

23 nephritis and focal or tubular atrophy. After an initial 3 months of Pb exposure (in a longitudinal 12-

24 month exposure study to either 0.01% [low dose] or 0.5% [high dose] Pb acetate in drinking water, male

25 rats), elevated GFR, consistent with hyperfiltration, and renal hypertrophy were observed; high dose 

26 animals also had increased NAG and GST (Khalil-Manesh, Gonick, Cohen, Bergamaschi, et al., 1992; 

27 Khalil-Manesh, Gonick, & Cohen, 1993; Khalil-Manesh, Gonick, Cohen, Alinovi, et al., 1992). At 6 

28 months of exposure, GFR decreased, albuminuria was present, and pathology ensued with focal tubular 
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1 atrophy and interstitial fibrosis formation. This pathology remained consistent out to 12 months, and at 12 

2 months glomeruli developed focal and segmental sclerosis. Similarly, GFR remained decreased after 12 

3 months of exposure. The evidence provided by toxicological studies that showed a difference in GFR 

4 with acute Pb exposure (hyperfiltration) versus chronic exposure (decreased GFR) provided biological 

5 plausibility to epidemiological studies that observed a similar phenomenon by age in humans with Pb 

6 exposure. These dichotomous changes in GFR (acute versus chronic Pb exposure) are consistent between 

7 the toxicological and epidemiology literature.

8 Biomarkers of Pb-induced renal toxicity have been developed including the enzymes lysosomal 

9 NAG, GST, brush border antigens (BB50, BBA, HF5), and Tamm-Horsfall protein. GST functions as a 

10 biomarker since renal ALAD is protected by the kidney antioxidant GSH. Urinary NAG and GST levels 

11 increase after 3 months of high dose Pb exposure (blood Pb level 125 μg/dL) (Dehpour et al., 1999; 

12 Khalil-Manesh, Gonick, Cohen, Alinovi, et al., 1992), whereas only urinary NAG was increased 

13 following low dose Pb exposure (blood Pb level 29 μg/dL) (Khalil-Manesh, Gonick, & Cohen, 1993). 

14 Occupational studies found that urinary NAG correlated best with recent blood Pb changes. 

15 The adverse effects of chronic Pb exposure as detailed above are partially rescued with chelation 

16 therapy such as DMSA (Khalil-Manesh, Gonick, Cohen, Bergamaschi, et al., 1992). Improvements 

17 include increased GFR, decreased albuminuria, and decreased inclusion body numbers but little change in 

18 tubulointerstitial scarring. Administration of an Indian herb to Pb exposed mice, as is discussed in further 

19 detail in the antioxidant section (Section 5.5.5), produced similar findings. There was a function rescue 

20 however Pb-induced pathology remained (Jayakumar et al., 2009). Thus, administration of various 

21 compounds (chelators, antioxidants) to Pb-exposed animals produced hemodynamic rescue. 

22 Recent studies have confirmed the previously observed increase in serum creatinine following 

23 chronic Pb exposure in rats. Annabi Berrahal et al. (2011) reported on the effects of age-dependent 

24 exposure to Pb on nephrotoxicity in male rats. Pups were exposed to Pb lactationally (as a result of dams 

25 consuming water containing 50 ppm Pb acetate) until weaning. Thereafter the offspring were exposed to 

26 the same solution from weaning (day 21) until sacrifice. Male pups were sacrificed at age 40 days 

27 (puberty; blood Pb level 12.7 μg/dL) and at age 65 days (post-puberty; blood Pb level 7.5 μg/dL). Serum 

28 creatinine was elevated at both 40 days and 65 days (0.54 and 0.60 mg/dL compared to control values of 

29 0.45 mg/dL [p<0.001]).Various parameters of Pb-dependent renal dysfunction are listed in Table 5-17

30 below. Other investigators have also shown that chronic Pb exposure has adverse effects on renal 

31 function. Pb exposed male rats (500 ppm Pb acetate in drinking water for 7 months) had elevated urinary 

32 pH, proteinuria, as well as glucose and blood in the urine (Navarro-Moreno et al., 2009).

33 Qiao et al. (2006) measured the effect of Pb on the expression of the renal fibrosis-related nuclear 

34 factor-kappa B (NFκB), transforming growth factor (TGF-β) and fibronectin in Sprague-Dawley rat 

35 kidney. Pb was administered at a dose of 0.5% Pb acetate, continuously for either one, two or three 

36 months. All growth factors increased by the end of three months of treatment but only NFκB increased 
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1 progressively at each time period. These changes were hypothetically related to the development of Pb2 induced renal fibrosis in rats, but, no histology was performed.

3 Roncal et al. (2007) found that Pb accelerated arteriolopathy and tubulointerstitial injury in non-Pb4 related CKD. Sprague-Dawley rats were administered Pb acetate at 150 ppm for 4 weeks, followed by 

5 remnant kidney surgery (left kidney mass reduced by 2/3 and right kidney removed) and then

6 continuation of Pb exposure for an additional 12 weeks. Pb-treated rats had higher systolic BP, lower 

7 creatinine clearance, and higher proteinuria than controls. Most striking was development of worse 

8 arteriolar disease, peritubular capillary loss, tubulointerstitial damage, and macrophage infiltration. Pb

9 treatment was associated with significant worsening of pre-glomerular vascular disease, as characterized 

10 by an increase in the media-to-lumen ratio. There was also a higher percentage of segmental sclerosis 

11 within glomeruli and a tendency for a higher number of sclerotic glomeruli. Additionally, a loss of 

12 peritubular capillaries, as reflected by a reduction in thrombomodulin staining, was observed. This was 

13 associated with worse tubular injury (osteopontin staining) due to more interstitial fibrosis (type III 

14 collagen staining) and a greater macrophage infiltration in the interstitium. The increase in macrophages 

15 was associated with higher renal MCP-1 mRNA. Low level Pb exposure concomitant with existing renal 

16 insufficiency due to surgical kidney resection accelerated vascular disease and glomerular pathology. 

17 These findings are consistent with the previous work of Bagchi et al. (2005) also showing that Pb exposed 

18 animals with non-Pb-related CKD (remnant surgery) had kidney dysfunction including impairment of the 

19 renin-angiotensin system (Losartan challenge), elevated systolic BP, and alterations in renal excretion of 

Pb, K+, and Na+ 20 . Thus, this model shows that low blood Pb level may exacerbate pre-existing underlying 

21 kidney disease. 

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

PND40

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+0.25a 4.55+0.27 6.04+0.29a

Plasma Urea

(mg/L) 0.37+0.019 0.47+0.021a 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+0.53a

a

p <0.001

Source: Used with permission from John Wiley & Sons, Berrahal et al. (2011)
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Histological Changes 

1 Historical studies discussed in previous Pb AQCDs have identified Pb-dependent renal damage by 

2 the presence of dense intranuclear inclusion bodies, which are capable of sequestering Pb (Goyer, May, et 

3 al., 1970). Chelators like CaNa2EDTA removed these inclusion bodies from affected nuclei (Goyer et al., 

4 1978). Multiple endpoints indicate dysfunction in the PT after Pb exposure. Pb-induced formation of 

5 intranuclear inclusion bodies in the PT is protective; Pb is sequestered such that it is not in its 

6 bioavailable, free, toxicologically active form. Intranuclear inclusion bodies are seen in the kidney with 

7 acute Pb exposures but present to a lesser degree with chronic exposures (See Section 5.2.3 for further 

8 discussion). Other PT ultrastructural changes in Pb-induced nephropathy include changes to the PT 

9 epithelium, endoplasmic reticulum dilation, nuclear membrane blebbing, and autophagosome enlargement

10 (Fowler et al., 1980; Goyer, Leonard, et al., 1970). Symptoms similar to the PT transport-associated 

11 Fanconi syndrome appear with Pb exposure, albeit often at high doses of Pb. These symptoms, which 

12 include increased urinary electrolyte excretion (zinc), decreased Na-K-ATPase activity, mitochondrial 

13 aberrations, and aminoaciduria, have also been reported in Pb exposed children. 

14 New studies since the 2006 Pb AQCD are consistent with the historical findings and build upon the 

15 literature base by including the role of antioxidants in histological preservation. Massanyi et al. (2007)

16 reported on Pb induced alterations in male Wistar rat kidneys after single i.p. doses of Pb acetate (50, 25, 

17 and 12.5 mg/kg); kidneys were removed and analyzed 48 hours after Pb administration. Qualitative 

18 microscopic analysis detected dilated Bowman’s capsules and dilated blood vessels in the interstitium 

19 with evident hemorrhagic alterations. Quantitative histomorphometric analysis revealed increased relative 

20 volume of interstitium and increased relative volume of tubules in the experimental groups. The diameter 

21 of renal corpuscles and the diameter of glomeruli and Bowman’s capsule were significantly increased.

22 Measurement of tubular diameter showed dilatation of the tubule with a significant decrease of the height 

23 of tubular epithelium compatible with degenerative renal alterations. These findings extend the 

24 observations of Fowler et al. (1980) and Khalil-Manesh et al. (1992; 1992); in particular, the enlarged 

25 glomeruli are consistent with the early hyperfiltration caused by Pb.

26 A recent study has also reported inclusion body formation in the nuclei, cytoplasm, and

27 mitochondria of PT cells of Pb exposed rats (50 mg Pb/kg bw i.p., every 48 h for 14 days) (Navarro28 Moreno et al., 2009). These inclusion bodies were not observed in chronically Pb-exposed rats (500 ppm 

29 Pb in drinking water, 7 months). However, chronic Pb exposure resulted in morphological alterations 

30 including loss of PT apical membrane brush border, collapse and closure of the PT lumen, and formation 

31 of abnormal intercellular junctions.

32 Vogetseder et al. (2008) examined the proliferative capacity of the renal PT (particularly the S3 

33 segment) following i.v. administration of Pb to juvenile and adult male Wistar rats. Proliferation induction 

34 was examined by detection of Bromo-2’-deoxyuridine (BrdU), Ki-67 (labels S, G2, and M phase cells), 
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1 and cyclin D1 (an essential cell cycle progression protein). The cycling marker Ki-67 revealed a much 

2 higher proliferation rate in the S3 segment in control juvenile rats (4.8 ± 0.3%) compared with control 

3 adult rats (0.4 ± 0.1%). Pb administration (3.8 mg /100 g bw) increased the proportion of Ki-67-positive 

4 cells to 26.1 ± 0.3% in juvenile rats and 31.9 ± 0.3% in adult rats. Thus, the increased proliferation caused 

5 by Pb was age independent. The proliferation induction caused by Pb administration may be a result of 

reduced cell cycle inhibition by p27kip-1 6 . Acute Pb treatment increased the incidence of cyclin D1 in the 

7 BrdU-positive cells suggesting Pb was able to accelerate reentry into the cell cycle and cause proliferation 

8 in the PT. Pb-dependent proliferation has also been reported in the retina with gestational Pb exposure 

9 (Giddabasappa et al., 2011).

10 Ademuyiwa et al. (2009) examined Pb-induced phospholipidosis and cholesterogenesis in rat 

11 tissues. Sprague-Dawley rats were exposed to 200, 300 and 400 ppm Pb acetate for 12 weeks. The Pb

12 exposure resulted in induction of phospholipidosis in kidney tissue, accompanied by depletion of renal 

13 cholesterol. The authors suggested that induction of cholesterogenesis and phospholipidosis in kidney 

14 may be responsible for some of the subtle and insidious cellular effects of Pb-mediating nephrotoxic 

15 manifestations. Drug-induced PT phospholipidosis is seen clinically with use of the potentially 

16 nephrotoxic aminoglycoside drugs, including gentamicin (Baronas et al., 2007). 

17 Various antioxidants have been shown to attenuate histopathological changes to the 

18 kidney. Ozsoy et al. (In Press) found L-carnitine to be protective in a model of experimental 

19 Pb toxicity in female rats. Markers of histopathological change in the kidney, including 

20 tubule dilatation, degeneration, necrosis, and interstitial inflammation were rescued by L21 carnitine treatment in females. Male rats exposed to Pb (0.2% for 6 weeks) also displayed 

22 tubular damage, whereas concomitant treatment with Pb and an extract of Achyranthes 

23 aspera ameliorated the observed damage (Jayakumar et al., 2009). El-Nekeety et al. (2009)

24 found an extract of the folk medicine plant Aquilegia vulgaris to be protective against Pb 

25 acetate-induced kidney injury in Sprague-Dawley rats. Rats were treated with Pb (20 ppm; 2 

26 weeks) and extract (administered before, during, or after Pb). Pb treatment resulted in 

27 tubular dilatation, vacuolar and cloudy epithelial cell lining, interstitial inflammatory cell 

28 infiltration, hemorrhage, cellular debris, and glomerulus hypercellularity. Concomitant 

29 exposure of Pb and extract produced histology indiscernible from control. Post treatment 

30 with extract partially rescued the Pb induced histopathology. El-Neweshy and El-Sayed

31 studied the influence of vitamin C supplementation (20 mg/kg pretreatment every other day) 

32 on Pb-induced histopathological alterations in exposed male rats (20 mg/kg by intragastric 

33 feeding once daily for 60 days). Control rats showed normal histology, while Pb-treated rats 

34 exhibited karyomegaly with eosinophilic intranuclear inclusion bodies in the epithelial cells 

35 of the proximal tubules. Glomerular damage and tubular necrosis with invading 

36 inflammatory cells were also seen. Rats treated with Pb acetate plus vitamin C exhibited 
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1 relatively mild or no karyomegaly with eosinophilic intranuclear inclusion bodies in the 

2 proximal tubules. Normal glomeruli were noted in animals exposed to Pb and vitamin C. 

3 These findings are presented in more detail in Section 5.5.5 but they consistently show that 

4 some antioxidants are capable of preventing or rescuing Pb-dependent histopathological 

5 changes.

Alteration of Renal Vasculature and Reactivity

6 As discussed in Section 5.5.1, changes in renal vasculature function or induction of hypertension 

7 can contribute to further renal dysfunction. Pb will increase BP through the promotion of oxidative stress 

8 and altered vascular reactivity. Also, Pb has been shown to act on known vasomodulating systems in the 

9 kidney. In the kidney, two vascular tone mediators,  ̇NO and ET-1, are affected by Pb exposure.

10 Antioxidants attenuated Pb-dependent oxidative/nitrosative stress in the kidney and abrogated the Pb11 induced increased BP (Vaziri, Ding, et al., 1999). Administration of the vasoconstrictor endothelin-1 (ET12 1) affected mean arterial pressure (MAP) and decreased GFR (Novak & Banks, 1995). Acute high dose 

13 Pb exposure completely blocked the ET-1-dependent GFR decrease but had no effect on MAP. Depletion 

14 of the endogenous antioxidant glutathione using the drug buthionine sulfoximine, a GSH synthase 

15 inhibitor, increased BP and increased kidney nitrotyrosine formation without Pb exposure, demonstrating 

16 the importance of GSH in maintenance of BP (Vaziri et al., 2000). Multiple studies have shown that Pb 

17 exposure depletes GSH stores. Catecholamines are vascular moderators that are also affected by Pb 

18 exposure (Carmignani et al., 2000). The effect on BP with Pb exposure is especially relevant to the kidney 

19 because it is both a target of Pb deposition and a mitigator of BP. These historic data detail the interaction 

20 of known modulators of vascular tone with Pb.

21 Recently, Vargas-Robles et al. (2007) examined the effect of Pb exposure (100 ppm Pb acetate for 

22 12 weeks) on BP and angiotensin II vasoconstriction in isolated perfused kidney and interlobar arteries. 

23 Vascular reactivity was evaluated in the presence and absence of L-NAME in both Pb-treated and control 

24 animals. Pb exposure significantly increased BP (134 ± 3 versus 100 ± 6 mm Hg), eNOS protein 

25 expression, oxidative stress, and vascular reactivity to angiotensin II. L-NAME potentiated the vascular 

26 response to angiotensin II in the control group, but had no effect on the Pb-treated group. Conversely, 

27 passive microvessel distensibility, measured after deactivation of myogenic tone by papaverine, was 

28 significantly lower in the arteries of Pb-exposed rats. Nitrites released from the kidney under the influence 

29 of angiotensin II in the Pb group were lower as compared to the control group whereas 3-nitrotyrosine 

30 was higher in the Pb group. The authors conclude that Pb exposure increases vascular tone through nitric 

31 oxide-dependent and independent mechanisms, increasing renal vascular sensitivity to vasoconstrictors.
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Apoptosis and/or Ischemic Necrosis of Tubules and Glomeruli

1 Apoptosis or programmed cell death in excess can cause cell atrophy while an insufficiency can 

2 lead to uncontrolled cell proliferation, such as cancer. Pb exposure has been shown to cause 

3 morphological changes to the kidney structure. Some of these Pb-induced changes are a result of cellular 

4 apoptosis or necrosis. Past studies have shown Pb-induced necrosis in proximal tubule cells (Fowler et al., 

5 1980). Pb-induced apoptosis is known to act through the mitochondria (Rana, 2008). Pb-induced calcium 

6 overload may depolarize the mitochondria, resulting in cytochrome c release, caspase activation, and 

7 apoptosis. The apoptosis is mediated by Bax translocation to the mitochondria and can be blocked by 

8 overexpression of Bcl-xl. Also, Pb-induced ALA accumulation can generate ROS, which may damage 

9 DNA leading to apoptosis. 

10 Mitochondria are targets of Pb toxicity and often involved in apoptosis. Pb can induce uncoupling 

11 of oxidative phosphorylation, decreased substrate utilization, and modification of mitochondrial ion 

12 transport. ATP energetics are affected when ATP-Pb chelates are formed and ATPase activity is decreased. 

13 ROS formation can contribute to these mitochondrial changes and to other changes within the kidney. 

14 Antioxidant supplementation after Pb exposure can remedy some adverse outcomes. All of these 

15 outcomes, in conjunction with Pb-dependent depletion of antioxidants (e.g. GSH) and elevation of lipid 

16 peroxidation point to possible susceptibility of the kidney to apoptosis or necrosis. Literature in this area 

17 is emerging.

18 Rodriguez-Iturbe et al. (2005) reported that chronic exposure to low doses of Pb (100 ppm in 

19 drinking water for 14 weeks) results in renal infiltration of immune cells, apoptosis, NFκB activation and 

20 overexpression of tubulointerstitial angiotensin II.

21 Navarro-Moreno et al. (2009) examined the effect of 500 ppm Pb in drinking water over 7 months 

22 on the structure (including intercellular junctions), function, and biochemical properties of PT cells of

23 Wistar rats. Pb effects in epithelial cells consisted of an early loss of the apical microvillae, followed by a 

24 decrement of the luminal space and the respective apposition and proximity of apical membranes, 

25 resulting in the formation of atypical intercellular contacts and adhesion structures. Inclusion bodies were 

26 found in nuclei, cytoplasm, and mitochondria. Lipid peroxidation (TBARS measurement) was increased 

27 in the Pb-treated animals as compared to controls. Calcium uptake was diminished and neither proline nor 

28 serine incorporation that was present in controls was noted in the PT of Pb-exposed animals. The authors 

29 speculate that Pb may compete with calcium in the establishment and maintenance of intercellular 

30 junctions.

31 Tubular necrosis was also observed in rats exposed to Pb acetate (100 ppm s.c.) for 30 days (El32 Sokkary et al., 2005). Histological sections of kidneys from Pb treated rats showed tubular degeneration 

33 with some necrotic cells. Similarly, El-Neweshy and El-Sayed reported glomerular damage and tubular 

34 necrosis with invading inflammatory cells after Pb treatment (20 mg/kg by intragastric feeding once daily 
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1 for 60 days) to male rats. The incidence of necrosis was decreased in both of these studies by pretreatment 

2 with either melatonin or vitamin C. Pretreatment with melatonin (10 mg/kg), an efficacious free radical 

3 scavenger and indirect antioxidant, resulted in a near normal tubular structure. The authors conclude that 

4 melatonin protected the liver and kidneys from the damaging effects of exposure to Pb through inhibition 

5 of lipid peroxidation and stimulation of endogenous antioxidative defense systems (El-Sokkary et al., 

6 2005). Vitamin C supplementation (20 mg/kg pretreatment every other day) protected the renal 

7 architecture and histology (El-Neweshy & El-Sayed).

8 Wang et al. (2009) examined the effect of Pb acetate (0.25, 0.5 and 1 μM) on cell death in cultured 

9 rat primary PT cells. A progressive loss in cell viability, due to both apoptosis and necrosis, were seen in 

10 cells exposed to Pb. Apoptosis predominated and could be ameliorated with concomitant N-acetylcysteine

11 exposure, whereas necrosis was unaffected. Elevation of ROS levels and intercellular calcium, depletion 

12 of mitochondrial membrane potential, and intracellular glutathione levels were seen during Pb exposure. 

13 Pb-dependent apoptosis was demonstrated morphologically (Hoechst 33258 staining) with 

14 condensed/fragmented chromatin and apoptotic body formation. CAT and SOD activities were 

15 significantly elevated, reflecting the response to accumulation of ROS.

16 Table 5-18 presents the acute and chronic renal effects of Pb exposure observed in recent and past 

17 animal toxicology studies.

Table 5-18. Acute and chronic effects of Pb on the kidney/renal system – evidence from 

animal toxicology studies.

Acute Chronic

Mitochondrial dysfunction

Renal cell apoptosis 

Nuclear Inclusion Body Formation

Proximal Tubule Cytomegaly

Glomerular Hypertrophy

Increased GFR

Mitochondrial dysfunction

Renal cell apoptosis

Oxidant redox imbalance

Altered NO homeostasis

ATPase dysfunction

Aminoaciduria

Increased electrolyte excretion 

Elevated blood pressure

Decreased GFR

5.5.2.2. Epidemiology in Adults

18 A number of significant advances in research on the impact of Pb on the kidney in the 20 years 

19 following the 1986 Pb AQCD (U.S. EPA, 1986) were noted in the 2006 Pb AQCD (U.S. EPA, 2006). 

20 These included research in general and CKD patient populations at much lower levels (5-10 μg/dL) of Pb 

21 exposure than previously studied. Furthermore, chelation therapy in CKD patients was evaluated, also at 

22 levels of exposure not previously thought to be amenable to chelation. Through these lines of research, it 

23 became clear that chronic Pb nephropathy, characterized by tubulointerstitial nephritis due to chronic, 

24 high-level Pb exposure (Bonucchi et al., 2007), is a small portion of the contribution that Pb makes to 
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1 kidney dysfunction overall in the population. Pb, at much lower doses than those causing chronic Pb 

2 nephropathy, acts as a cofactor with other more established kidney risks to increase the risk for CKD and 

3 disease progression in susceptible patients. The populations at risk for kidney dysfunction (diabetics and 

4 hypertensives) are increasing worldwide, particularly in countries where obesity is epidemic. Pb exposure 

5 continues to decline in many industrialized countries, although less so among minority populations, 

6 which, notably, are also higher risk groups for CKD. Thus, the extent of the public health impact of Pb on 

7 the kidney depends on the balance of these two factors.

8 In the 2006 Pb AQCD (U.S. EPA, 2006), several key issues could not be completely resolved based 

9 on the Pb-kidney literature published to date. These included the lowest Pb dose at which adverse kidney 

10 effects occur, whether associations at current Pb levels are due to higher past exposures, the impacts of Pb 

11 on the kidney in children, the use of paradoxical Pb-kidney associations on risk assessment in the 

12 occupational setting, and the impact of co-exposure to other environmental nephrotoxicants, such as 

13 cadmium. In the intervening five years, relevant data for several of these challenges have been published. 

General Population Studies

14 The 2006 Pb AQCD reported studies that examined the effect of Pb exposure on kidney function in 

15 general populations. This was a new approach to Pb-kidney research in the two decade time period 

16 covered by the 2006 Pb AQCD. The studies in this category provided critical evidence that the effects of 

17 Pb on the kidney occur at much lower doses than previously appreciated based on occupational exposure 

18 data. The landmark Cadmibel Study was the first large environmental study of this type that adjusted for 

19 multiple kidney risk factors (Staessen et al., 1992). It included 965 men and 1,016 women recruited from 

20 cadmium exposed and control areas in Belgium. Mean blood Pb was 11.4 μg/dL (range 2.3-72.5) and 7.5 

21 μg/dL (range 1.7-60.3) in men and women, respectively. After adjustment, log transformed blood Pb was 

22 negatively associated with measured creatinine clearance. A 10-fold increase in blood Pb was associated 

23 with a decrease in creatinine clearance of 10 and 13 mL/min in men and women, respectively. Blood Pb 

24 was also negatively associated with estimated creatinine clearance. 

25 Four studies assessing the kidney impact of Pb exposure have been published in the Normative 

26 Aging Study (NAS) population (R. Kim et al., 1996; Payton et al., 1994; Tsaih et al., 2004; M. T. Wu et 

27 al., 2003). Participants in this study were originally recruited in the 1960s in the Greater Boston area. 

28 Inclusion criteria included male gender, age 21 to 80 years, and absence of chronic medical conditions. 

29 Longitudinal data contained in two NAS publications remain essential, particularly in light of the dearth 

30 of prospective data on the kidney effects of Pb. The first of these included 459 men whose blood Pb levels 

31 from periodic examinations, conducted every 3 to 5 years during 1979-1994, were estimated based on 

32 measurements in stored packed red blood cell samples adjusted for hematocrit level (R. Kim et al., 1996). 

33 Participants were randomly selected to be representative of the entire NAS population in terms of age and 
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1 follow-up. Kidney status was assessed with serum creatinine. Data from four evaluations were available 

2 for the majority of participants. Mean (SD) age, blood Pb level, and serum creatinine, at baseline, were 

3 56.9 (8.3) years, 9.9 (6.1) μg/dL, and 1.2 (0.2) mg/dL, respectively. In the longitudinal analysis, using 

4 random-effects modeling, ln-transformed blood Pb was associated with change in serum creatinine over 

5 the subsequent follow-up period in the 428 participants whose highest blood Pb level was ≤ 25 μg/dL (β= 

6 0.027 [95% CI: 0.0, 0.054]); associations in the entire group and subsets with different peak blood Pb 

7 levels (≤ 10 or 40 μg/dL) had p-values between 0.07 and 0.13. 

8 This study made two other key contributions. In order to address the question of whether 

9 nephrotoxicity observed at current blood Pb levels is due to higher blood Pb levels from past exposure, 

10 these authors performed a sensitivity analysis in participants whose peak blood Pb levels, dating back to 

11 1979, were ≤ 10 μg/dL. A significant positive association between blood Pb and concurrent serum 

12 creatinine remained. These authors also addressed reverse causality, which attributes increased blood Pb

13 levels to lack of kidney excretion rather than as a causative factor for CKD, by showing in adjusted plots 

14 that the association between blood Pb and serum creatinine occurred over the entire serum creatinine 

15 range, including the normal range where reverse causality would not be expected.

16 Cortical and trabecular bone Pb measurements were obtained in addition to whole blood Pb in 

17 evaluations performed in the Normative Aging Study between 1991 and 1995. Associations between 

18 baseline blood, tibia, and patella Pb and change in serum creatinine over an average of 6 years in 448 men 

19 were reported in a subsequent NAS publication (Tsaih et al., 2004). At baseline 6 and 26% of subjects had 

20 diabetes and hypertension, respectively. Mean blood Pb levels and serum creatinine decreased 

21 significantly over the follow-up period in the group. Pb dose was not associated with change in creatinine 

22 in all participants. However, diabetes was observed to be an effect modifier of the relations of blood and 

23 tibia Pb with change in serum creatinine. For ln blood Pb, the positive association with serum creatinine 

24 was substantially stronger in diabetics (β = 0.076 [95% CI: 0.031, 0.121]) compared to non-diabetics (β = 

25 0.006 [95% CI: -0.004, 0.016]). A similar relationship was observed for tibia Pb. An interaction was also 

26 observed between tibia Pb and hypertension, although it is possible that many of the 26 diabetics were 

27 also included in the hypertensive group and were influential there as well. Reverse causality was 

28 addressed in a sensitivity analysis of participants whose serum creatinine was <1.5 mg/dL; the authors 

29 reported that longitudinal associations did not materially change. These studies are depicted in either 

30 Figure 5-43 and Table 5-19.

31
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1

Study/Age (yr) Pb Levela 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 (95th percentile) 58 (54,63)

Staessen et al. (1992) 7.5 (5.2, 10.9) Belgian women

Avg age (SD) 48 (16)

loge 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 m2

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 μg/dL or bone 

Pb level (IQR) in μg/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. 

2 NHANES data analyses benefit from a number of strengths including large sample size, ability to 

3 adjust for numerous Pb risk factors, and the fact that the study population is representative of the U.S. 

4 non-institutionalized, civilian population. As a result, the impact of Pb on the kidney has been examined 

5 in multiple NHANES datasets obtained over the last few decades (Figure 5-44 and Table 5-19). The 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 
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1 results of these publications, covering different time frames, have been consistent in providing support for 

2 Pb as a CKD risk factor, including NHANES III, conducted from 1988-1994, where hypertensives and 

3 diabetics were observed to be susceptible populations (Muntner et al., 2003) and NHANES 1999-2002 

4 (Muntner et al., 2005). 

5 A recent publication examined this relationship in NHANES data collected from 1999 through 

6 2006 (Navas-Acien et al., 2009). The geometric mean blood Pb level was 1.58 μg/dL in 14,778 adults 

7 aged ≥ 20 years. After adjustment for survey year, sociodemographic factors, CKD risk factors, and blood 

cadmium, the odds ratios for albuminuria (≥ 30 mg/g creatinine), reduced eGFR (<60 mL/min/1.73 m2 8 ), 

9 and both albuminuria and reduced eGFR were 1.19 (95% CI: 0.96, 1.47), 1.56 (95% CI: 1.17, 2.08), and 

10 2.39 (95% CI: 1.31, 4.37), respectively, comparing the highest to the lowest blood Pb quartiles. Thus, in 

11 the subset of the population with the most severe kidney disease (both reduced eGFR and albuminuria), 

12 the risk from Pb was greater. Cadmium was included as a covariate and Pb remained significantly 

13 associated. In fact, the most important contribution of this recent NHANES analysis was the evaluation of 

14 joint Pb and cadmium exposure (discussed below).

15 An important contribution of all three NHANES publications is that they provide evidence that 

blood Pb remains associated with reduced kidney function (<60 mL/min/1.73 m2 16 as estimated with the 

17 MDRD equation cross-sectionally) despite steadily declining Pb levels during the time periods covered. 

18 Additional studies in this category have also reported worse kidney function related to Pb dose (Goswami 

19 et al., 2005; Hernandez-Serrato et al., 2006; L. H. Lai et al., 2008).
May 2011 5-214 DRAFT – DO NOT CITE OR QUOTE

 

 

 

 

 

5

10

2 

-40 -10 10 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/1.73m2

μg/dL Blood Pb % Change per μg/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 difference 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 kidney 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.
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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 % 

Change 

in Kidney Metric 

(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) μg/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 μg/dL

Q1: ≤ 1.1

Q2: 1.2 to 1.6

Q3: 1.7 to 2.4

Q4: >2.4

eGFR <60 

mL/minute/1.73 m2

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 m2

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 μg/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 m2

; 

calculated using the 

Filler and Lepage 

equation)

Linear 

regression

Q4: -0.42 (-0.73, -0.11)

Kim et al. 

(1996)

Adult males Boston, MA; 

1979-1994

459 Median = 8.6 μg/dL Serum creatinine 

concentrations

Randomeffects model≤ 40 μg/dL blood Pb: 

0.016 (0.004, 0.028)

≤ 25 μg/dL blood Pb: 

0.019 (0.006, 0.032)

≤ 10 μg/dL blood Pb:

0.030 (0.011, 0.049)

Payton et al. 

(1994)

Adult males Boston, MA; 

1988-1991

744 Mean (SD) = 8.1 

(3.9) μg/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)

μg/dL

Tibia Pb = 21.5 

(13.5) μg/g

Patella Pb = 32.4 

(20.5) μg/g

Serum 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 % 

Change 

in Kidney Metric 

(95% CI)

Staesson et 

al. (1992)

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 μg/dL Change in serum 

creatinine 

concentrations

Randomeffects model≤ 40 μg/dL blood Pb: 0.01 

(-0.0, 0.02)

≤ 25 μg/dL blood Pb: 0.01

(-0.0, 0.03)

≤ 10 μg/dL blood Pb: 0.02

(-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)

μg/dL

Tibia Pb = 21.5 

(13.5) μg/g

Patella Pb = 32.4 

(20.5) μg/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) μg/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 followup period for each 1 μg/dL 

increment of blood Pb
May 2011 5-217 DRAFT – DO NOT CITE OR QUOTE

Occupational Studies

1 The vast majority of studies in the literature on the impact of Pb on the kidney have been 

2 conducted in the occupational setting. In general, study size and extent of statistical analysis are much 

3 more limited than for general population studies. Publications in only three populations have reported 

4 adjusted results in occupationally exposed workers in the five years since the 2006 Pb AQCD. In a two 

5 year prospective cohort study, generalized estimating equations were used to model change in kidney 

6 function between each evaluation in relation to tibia Pb and concurrent change in blood Pb in 537 current 

7 and former Pb workers (V. M. Weaver et al., 2009). Tibia Pb was evaluated at the beginning of each 

8 follow-up period and Pb measures were adjusted for baseline Pb dose and other covariates. In males, 

9 serum creatinine decreased and calculated creatinine clearance increased over the course of the study; 

10 these changes were largest in participants whose blood Pb declined concurrently or whose tibia Pb was 

11 lower. In females, decreasing serum creatinine was associated with declining blood Pb (as in males),

12 however, increasing blood Pb was associated with a concurrent increase in serum creatinine. Women 

13 (25.9% of the study population) were older and more likely to be former Pb workers than men which may 

14 have been important factors in the effect modification observed by sex. 

15 Chia and colleagues observed a significant, positive association between blood Pb and urine NAG 

16 in linear regression models after adjustment for age, gender, race, exposure duration, ALAD G177C

17 polymorphism and the interaction between ALAD and blood Pb (Chia et al., 2006). Similar positive 

18 associations were observed between blood Pb and a wider range of EBE markers in models that adjusted 

19 for age, gender, race, exposure duration, and the HpyCH4 ALAD SNP (discussed below) (Chia et al., 

20 2005). A study of 155 male workers reported significant, positive correlations between blood and urine Pb 

21 and urine NAG and albumin after controlling for age and job duration (Y. Sun, Sun, Zhou, Zhu, Lei, et al., 

22 2008). An important additional study that analyzed occupational Pb exposure is discussed below under 

23 patient population studies (Evans et al., 2010).

24 Two studies have performed benchmark dose calculations for the effect of Pb on the kidney. Both

25 used only EBE markers and found NAG to be the most sensitive outcome; reported lower confidence 

26 limits on the benchmark doses were 10.1 μg/dL (Y. Sun, Sun, Zhou, Zhu, Lei, et al., 2008), and 25.3 

27 μg/dL (T. A. Lin & Tai-yi, 2007).

28 A number of other publications in the five years since the 2006 Pb AQCD have reported 

29 significantly worse kidney outcomes in unadjusted analyses in occupationally exposed workers compared 

30 to unexposed controls (Patil et al., 2007) and/or significant correlations between higher Pb dose and 

31 worse kidney function (Alinovi et al., 2005; Garcon et al., 2007; D. A. Khan et al., 2008; T. A. Lin & Tai32 yi, 2007; Y. Sun, Sun, Zhou, Zhu, Lei, et al., 2008). One small study found no significant differences

33 (Orisakwe et al., 2007).
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1 Overall, the occupational literature published in the last five years on the kidney impact of Pb 

2 exposure has been more consistent in reporting significant associations than data reviewed for the 2006 

3 Pb AQCD. This may reflect increased reliance on EBE markers as more sensitive outcome measures, 

4 publication bias, or multiple comparisons due to a greater number of outcomes assessed.

5 Publications that include dose-response information provide evidence of Pb-related nephrotoxicity 

6 in the occupational setting across the Pb dose ranges analyzed (Ehrlich et al., 1998; V. M. Weaver, Lee, et 

7 al., 2003). Data in 267 Korean Pb workers in the oldest age tertile (mean age = 52 years) reveal no 

8 threshold for a Pb effect (beta = 0.0011, p = <0.05; regression and lowess lines shown) (V. M. Weaver, 

9 Lee, et al., 2003) (added variable plot shown in Figure 5-45).

Source: Used with permission from the BMJ Publishing Group, Weaver et al. (2003)

Note: Both the adjusted regression line and the line estimated by the smoothing method of the SPLUS 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. Added variable plot of association between serum creatinine 

and blood Pb in 267 Korean Pb workers in the oldest age 

tertile. 

10 A major challenge in interpretation of the occupational literature is the potential for Pb-related 

11 hyperfiltration. Hyperfiltration involves an initial increase in glomerular hypertension which results in 

12 increased GFR. If persistent, increased risk for subsequent CKD occurs. This pattern has been observed in 

13 diabetes, hypertension, and obesity (Nenov et al., 2000). As discussed in the 2006 Pb AQCD, findings 

14 consistent with hyperfiltration have been observed in three occupational populations (Hsiao et al., 2001; 

Adjusted blood Pb level (μg/dL)

Adjusted serum creatinine (mg/dL)
May 2011 5-219 DRAFT – DO NOT CITE OR QUOTE

1 Roels et al., 1994; V. M. Weaver, Lee, et al., 2003), a study of adults who were Pb poisoned as children 

2 (H. Hu, 1991), and a study in European children (De Burbure et al., 2006). Longitudinal data in Pb 

3 exposed rodents provide evidence of a hyperfiltration pattern of increased, followed by decreased GFR, 

4 associated with Pb exposure and are critical in interpretation of the human Pb-kidney literature (Khalil5 Manesh, Gonick, Cohen, Alinovi, et al., 1992). Pb could induce glomerular hypertension resulting in 

6 hyperfiltration by several mechanisms including increased ROS, changes in eicosanoid levels, and/or an 

7 impact on the renin-angiotensin system (Roels et al., 1994; Vaziri, 2008b). Whether hyperfiltration 

8 contributes to pathology in humans is unclear; longitudinal studies are needed. 

9 Regardless, significant findings could be obscured if opposite direction associations are present in 

10 different segments of the study population and interaction models are not performed to address this. In the 

11 Korean Pb workers (V. M. Weaver, Lee, et al., 2003; V. M. Weaver, Schwartz, et al., 2003), significant 

12 associations in opposite directions were observed only when relevant effect modifiers were included in 

13 the model. This is a valid concern for risk assessment, since the factors involved in these inverse 

14 associations in Pb-exposed populations are not well defined at present.

Patient Population Studies

15 CKD as defined by the National Kidney Foundation - Kidney Disease Outcomes Quality Initiative 

16 (NKF-K/DOQI) workgroup (National Kidney Foundation, 2002) is the presence of markers of kidney 

damage or GFR <60 mL/min/1.73 m2 17 for ≥ 3 months. The MDRD equation is the most common one used 

18 in the eGFR determination for this definition. Notably, decreased GFR is not required for the first criteria 

19 and markers of kidney damage are not required for the second criteria.

20 Several key studies in CKD patient populations have been published in the last five years (Table 5-

21 20). One CKD patient study, discussed in the 2006 Pb AQCD, remains the hallmark for prospective 

22 evaluation of susceptible patient populations to determine if CKD progression (kidney function decline) is 

23 greater in participants with higher baseline Pb dose. Yu et al. (2004) followed 121 patients over a four 

24 year period. Eligibility required well-controlled CKD with serum creatinine between 1.5 and 3.9 mg/dL. 

25 Importantly, EDTA-chelatable Pb <600 μg/72 h, a level below that traditionally thought to indicate risk 

26 for Pb-related nephrotoxicity, was required at baseline. Patients with potentially unstable kidney disease 

27 were excluded (i.e., due to systemic diseases such as diabetes). Mean blood Pb and EDTA-chelatable Pb 

28 levels were 4.2 μg/dL and 99.1 μg/72 hours, respectively. In a Cox multivariate regression analysis,

29 chelatable Pb was significantly associated with overall risk for the primary endpoint (doubling of serum 

30 creatinine over the 4-year study period or need for hemodialysis). When the group was dichotomized by 

31 EDTA chelatable Pb level, Kaplan-Meier analysis demonstrated that significantly more patients (15/63) in 

32 the high-normal group (EDTA chelatable Pb level ≥ 80 but <600 μg/72 hours) reached the primary end 

33 point than in the lower EDTA chelatable Pb levels (<80 μg Pb/72 hours) group (2/58). Associations 
May 2011 5-220 DRAFT – DO NOT CITE OR QUOTE

1 between baseline chelatable or blood Pb level and change in eGFR (estimated by the MDRD equation

2 (Levey et al., 1999) were modeled separately using GEE. Based on these models, a 10 μg higher 

chelatable Pb level or 1 μg/dL higher blood Pb level reduced the GFR by 1.3 and 4.0 ml/min/1.73 m2 3 , 

4 respectively, during the 4-year study period. Two recent studies expanded the CKD patient populations in 

5 which this effect was observed to those with diabetic nephropathy (J.-L. Lin, Lin-Tan, Yu, et al., 2006)

6 and with the lowest Pb body burdens studied to date (J.-L. Lin, Lin-Tan, Li, et al., 2006). Results of these 

7 observational studies have been summarized (V. Weaver & Jaar, 2010). 

Table 5-20. Patient population studies: kidney function decline

Study n

Baseline 

mean 

(SD) 

blood Pb 

(μg/dL)

Baseline mean 

(SD) chelatable 

Pb (μg/72 hours)

Baseline mean 

(SD) eGFR 

(ml/min/1.73 

m2

)

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 μg/dL 

higher blood Pb, at baseline, 

associated with 4.0 

mL/min/1.73 m2 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)

8 A recent population-based case-control study examined occupational Pb exposure as a risk factor 

9 for severe CKD (Evans et al., 2010). The study included 926 cases with first time elevations of serum 

10 creatinine >3.4 mg/dL for men and >2.8 mg/dL for women and 998 population-based controls. 

11 Occupational Pb exposure was assessed using an expert rating method based on job histories. Eighty-one 

12 cases and 95 controls were judged to have had past occupational Pb exposure. Of those, 23 cases and 32 

controls were thought to have been exposed to Pb levels ≥ 30 μg/m3 13 (the current US OSHA limit is 50 

14 μg/m3). Using multivariable logistic regression modeling, the adjusted OR for CKD was 0.97 (95% CI:

15 0.68-1.38) in Pb-exposed compared to non-exposed participants. No significant increased odds were 

16 observed for low, medium or high exposed groups using either average or cumulative exposure metrics. 

17 In addition, the CKD patients were followed prospectively for a mean of 2.5 years for the 70 Pb exposed 

18 patients and 2.4 years for the 731 patients without past occupational Pb exposure. Mean eGFRs (using the 

MDRD equation) were 16.0 and 16.6 ml/min/1.73 m2 19 in exposed and non-exposed patients, respectively, 

20 indicating severe disease in both groups. Using mixed-effects multivariable models, eGFRs declined by 

4.27 and 3.39 mL/min/1.73 m2 21 /y in ever and most Pb-exposed CKD patients, respectively, compared with 
May 2011 5-221 DRAFT – DO NOT CITE OR QUOTE

4.55 mL/min/1.73 m2 1 /y in patients without occupational Pb exposure. Thus, in this study, no adverse 

2 kidney effect of occupational Pb exposure was evident.

3 Strengths noted by the authors included virtually complete case ascertainment and minimal loss to 

4 follow-up. Exposure assessment was listed as both a strength and a limitation. Expert rating methods are 

5 commonly used when biological monitoring is not an option and in case-control studies where many 

6 occupational exposures are considered. In Pb-kidney research, this approach is uncommon except in the 

7 case-control setting. However, given the challenges of interpreting blood Pb in dialysis patients (discussed 

8 below), this approach may have advantages in this study of such severe CKD. Two case-control studies 

9 examining occupational risk factors for CKD have been published; one found Pb exposure to be a risk 

10 factor (Nuyts et al., 1995), the other did not although moonshine alcohol consumption was a risk 

11 presumably due to Pb level (Steenland et al., 1990). The prospective observational aspect of this study is 

12 similar in design to the work of Lin and colleagues but differs in several important respects. In this study, 

13 only occupational Pb exposure was considered whereas the work in Taiwan excludes occupational 

14 exposure and uses Pb dose measures. In the past in developed countries, environmental exposures were 

15 substantial. For example, mean tibia Pb levels were 21.5 and 16.7 μg/g bone mineral, in environmentally 

16 exposed 50- to 70-year-old African-Americans and whites, respectively, in Baltimore (Martin et al., 

17 2006). In Korean Pb workers, mean baseline tibia Pb level was only twofold higher (35.0 μg/g) (V. M. 

18 Weaver, Lee, et al., 2003) which illustrates the substantial body burden in middle- and older-aged 

19 Americans from lifetime Pb exposure. Declines in blood Pb levels in Sweden have been reported and 

20 attributed to the leaded gasoline phase-out (Elinder et al., 1986; Strömberg et al., 1995), although blood 

21 Pb levels were lower than those noted during the U.S. phase-out. Finally, the severe degree of CKD in this 

22 population creates a survivor bias at enrollment and limits the eGFR decline possible during follow-up, 

23 thus limiting the ability to identify factors that influence that decline. 

ESRD Patient Studies

24 End stage renal disease (ESRD) is a well established public health concern, which is characterized 

25 by the use of dialysis to perform the normal functions of the kidney. Incidence and prevalence in the US 

26 continue to increase resulting in rates that are the third highest among nations reporting such data (U.S. 

27 Renal Data System, 2009). Studies in patients with CKD requiring chronic hemodialysis (ESRD) have 

28 also been published in the past five years. One study reported much higher blood Pb levels than had been 

29 appreciated by the treating clinicians (Davenport et al., 2009). Of 271 adult patients on regular thrice 

30 weekly dialysis, blood Pb levels ranged from 3 to 36.9 μg/dL; 25.5% had levels >20 μg/dL, 59% had 

31 values of 10-20 μg/dL, and 15.5% were <10 μg/dL. Few details on the statistical analysis were provided 

32 which complicates interpretation of the findings. However, blood Pb was positively correlated with 

33 hemodialysis vintage (months on dialysis; Spearman’s r = 0.38, p-value <0.001); negatively correlated 
May 2011 5-222 DRAFT – DO NOT CITE OR QUOTE

1 with urine output (r = -0.44, p-value <0.001) and higher in patients using single carbon filter and reverse 

2 osmosis water purification devices. Another recent publication reported higher Pb in dialysate than in the 

3 tap water used in its preparation (B. Chen et al., 2009). A systematic review of a wide range of trace 

4 elements in hemodialysis patients reported higher Pb levels in patients compared to controls although the 

5 difference was not large (Tonelli et al., 2009). These data suggest that blood Pb monitoring in dialysis 

6 patients may be useful.

7 Interpretation of Pb dose in patients on dialysis is challenging for several reasons. First, renal 

8 osteodystrophy, the bone disease related to kidney disease, may result in increased release of Pb from

9 bone stores. Thus, interpretation of blood and even bone Pb levels may require adjustment with one or 

10 more of a range of osteoporosis variables. Secondly, as observed above (Davenport et al., 2009), residual 

11 kidney function may have a substantial impact on blood Pb levels in populations with such minimal 

12 excretion. Third, as illustrated in the studies cited above (B. Chen et al., 2009; Davenport et al., 2009), 

13 water and concentrates used in dialysis may be variable sources of Pb. A recent study reported decreased 

14 blood Pb in post-dialysis compared to pre-dialysis samples (Kazi et al., 2008). Thus, substantial 

15 fluctuations in blood Pb are possible while on dialysis. Finally, anemia is common in CKD and Pb is 

16 stored in red blood cells. Thus, measurement of blood Pb in anemia may require adjustment for 

17 hemoglobin; no standardized approach to this currently exists.

18 Given these caveats, a pilot study observed significantly higher median blood Pb levels in 55 

19 African-American dialysis patients compared to 53 age- and sex-matched controls (6 and 3 μg/dL 

20 respectively; P <0.001) (Muntner et al., 2007). This study was unique in that tibia Pb levels were 

21 assessed. Median tibia Pb was higher in ESRD patients although the difference did not reach statistical 

22 significance (17 and 13 μg/g bone mineral, respectively (p = 0.13). In order to determine the potential 

23 impact of renal osteodystrophy, median blood and tibia Pb levels in the dialysis patients were examined 

24 by levels of serum parathyroid hormone, calcium, phosphorus, and albumin and were not found to be 

25 significantly different (Ghosh-Narang et al., 2007). A study of 211 diabetic patients on hemodialysis (J.-L. 

26 Lin et al., 2008) found parathyroid hormone and serum creatinine to be associated with blood Pb level in 

27 crude but not adjusted associations. In contrast, a study of 315 patients on chronic peritoneal dialysis 

28 observed parathyroid hormone to be positively correlated and residual renal function negatively correlated 

29 with logarithmic-transformed blood Pb levels after adjustment (J.-L. Lin et al., 2010). In the prospective 

30 portion of this study, blood Pb levels at baseline were categorized by tertile (range of 0.1 to 29.9 μg/dL 

31 with cut points of 5.62 and 8.66 μg/dL). Cox multivariate analysis, after adjustment for parathyroid 

32 hormone level, residual renal function, and 20 other variables, showed increased all-cause mortality in the 

33 middle and highest compared to the lowest tertiles (hazard ratio= 2.1 [95% CI: 2.0-2.2] and 3.3 [95% CI:

34 1.3-13.5], respectively). Given other recent publications in hemodialysis patients by this group, it would 

35 be valuable to examine this risk after adjustment for serum ferritin (Jenq et al., 2009), hemoglobin A1C

36 (Lin-Tan, Lin, Wang, et al., 2007), and blood cadmium (C. W. Hsu et al., 2009).
May 2011 5-223 DRAFT – DO NOT CITE OR QUOTE

Clinical Trials in Chronic Kidney Disease Patients

1 Randomized chelation trials in CKD patients, uncommon in nephrotoxicant research, provide 

2 unique information on the kidney impact of Pb. These studies have been performed by Lin and colleagues 

3 in Taiwan and involve similar study designs. Initially, patients are observed in order to compare CKD 

4 progression prior to chelation. Then, CKD patients whose diagnostic EDTA chelatable Pb levels are 

5 within certain ranges (generally 60-600 μg/72 hours and thus below the level commonly considered for 

6 chelation) are randomized. The treated group receives weekly chelation with 1 g EDTA intravenously for 

7 up to 3 months. The control group receives placebo infusions. In the follow-up period, chelation is 

8 repeated for defined indications such as increased serum creatinine or chelatable Pb levels above specified 

9 cut-offs. Placebo infusions are repeated in the controls as well. The results of the most recent of these 

10 trials are summarized in Table 5-21 below.

Table 5-21. Clinical randomized chelation trials in chronic kidney disease patients

Reference Group n

Baseline 

mean 

(SD) 

blood Pb 

(μg/dL)

Baseline 

mean (SD) 

chelatable Pb 

(μg/72 hr)

Baseline mean 

(SD) eGFR 

(ml/min/1.73 

m2)

Months of 

treatment/ 

follow-up

Change in 

eGFR per yr

(ml/min/1.73 

m2)

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 μg)

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).

11 This study design requires replication in larger populations at multiple clinical centers. If 

12 confirmed, the effect may be due to removal of Pb. However, chelation may also have a direct beneficial 

13 effect on kidney function, regardless of Pb exposure. Antioxidant effects of CaNa2EDTA which may 

14 improve kidney function directly via improved blood flow to the kidneys have been reported (Jacobsen et 

15 al., 2001; Saxena & Flora, 2004). EDTA benefits in a Pb rodent model appeared to occur via reduced 

16 oxidation (Saxena & Flora, 2004). EDTA administration reduced kidney damage in a rat model of acute 

17 renal failure induced by ischemia (Foglieni et al., 2006). Similarly DMSA has been reported to prevent 

18 renal damage when co-administered during induction of nephrosclerosis in a non-Pb exposed rat model

19 (Gonick et al., 1996). Benefits from chelation reported in rodent models of Pb-related nephrotoxicity 
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1 (Khalil-Manesh, Gonick, Cohen, Bergamaschi, et al., 1992; Sanchez-Fructuoso, Blanco, et al., 2002; 

2 Sanchez-Fructuoso, Cano, et al., 2002) did not appear to occur via reversal of structural damage (Khalil3 Manesh, Gonick, Cohen, Bergamaschi, et al., 1992); again suggesting that improved hemodynamics from 

4 reduction of reactive oxidant species, which could be due to reduced Pb and/or directly from the chelating 

5 agent, may be a mechanism (Gonick et al., 1996). However, the most parsimonious explanation for the 

6 combination of Lin’s observational and experimental chelation work is that Pb is the underlying reason. 

7 Moreover, if the benefit can be replicated, this could be a valid treatment regardless of the mechanism or 

8 whether Pb is involved.

9 The unique body of work in patient populations by Lin and co-workers, both observational and 

10 experimental, has numerous strengths including prospective study design, randomization, Pb dose 

11 assessment that includes bioavailable body burden, longitudinal statistical analysis, and control for 

12 multiple kidney risk factors. However, the generalizability of the results to broader populations is 

13 unknown. In addition, the observed effect of Pb on decline in GFR has been variable; the annual decline 

14 in eGFR per standard deviation (SD) higher Pb dose at baseline was much lower in the 2003 study than in 

15 subsequent publications (see Table 5-21 above). Small sample sizes and differences in renal diagnoses 

16 between groups may be factors in this variability. However, if confirmed in large populations at multiple 

17 centers and shown not to worsen cognition or other effects through Pb mobilization, chelation could yield 

18 important public health benefits.

5.5.2.3. Epidemiology in Children

Lead Nephrotoxicity in Children

19 Both the 2006 and 1986 Pb AQCDs noted that the degree of kidney pathology observed in adult 

20 survivors of untreated childhood Pb poisoning in the Queensland, Australia epidemic (Inglis et al., 1978)

21 has not been observed in other studies of childhood Pb poisoning. Recent publications remain consistent 

22 with that conclusion; a recent study observed an impact of childhood Pb poisoning on IQ but not kidney 

23 outcomes (C. Coria et al., 2009). Chelation was raised as a potential explanation for this discrepancy in 

24 the 2006 Pb AQCD. 

25 With declining Pb exposure levels, recent work has focused on studies in children at much lower 

26 environmental exposure levels. However, insensitivity of the clinical kidney outcome (i.e., GFR) 

27 measures for early kidney damage is a particular problem in children who do not have many of the other 

28 kidney risk factors that adults do, such as hypertension and diabetes. As a result, such studies have 

29 utilized EBE markers. However, data to determine the predictive value of such biomarkers for subsequent 

30 kidney function decline in Pb exposed populations are extremely limited (Coratelli et al., 1988) and may 

31 pose particular challenges in children due to puberty related biomarker changes (Sarasua et al., 2003). 
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1 Three studies that included analysis of clinical kidney outcomes were discussed in the 2006 Pb AQCD. 

2 One found no difference in mean serum creatinine between 62 exposed and 50 control children (Fels et 

3 al., 1998). Two larger studies observed significant associations that were in opposite directions; blood Pb 

4 was positively associated with serum cystatin-C in 200 17-year-old Belgian adolescents but negatively 

5 associated with serum creatinine and cystatin C in 300-600 European children (n varied by outcome)

6 (De Burbure et al., 2006; Staessen et al., 2001).

7 Therefore, one of the key gaps identified in the 2006 Pb AQCD was limited data in children and 

8 adolescents particularly with respect to GFR measures. A recently published NHANES analysis in 

9 adolescents begins to fill this gap (Fadrowski et al., 2010). Associations between blood Pb and kidney 

10 function were investigated in 769 adolescents aged 12-20 years in the US NHANES III, conducted from 

11 1988-1994. Kidney function was assessed with two eGFR equations. One utilized serum cystatin C and 

12 the other used the more traditional marker, serum creatinine. Median blood Pb and cystatin C-based eGFR 

levels were 1.5 μg/dL and 112.9 mL/min/1.73 m2 13 , respectively. Cystatin C-based eGFR was lower (-6.6 

mL/min/1.73 m2 14 [95% CI: -0.7, -12.6]) in participants with Pb levels in the highest quartile (≥ 3.0 μg/dL) 

15 compared with those in the lowest (<1 μg/dL). A doubling of blood Pb level was associated with a -2.9 

mL/min/1.73 m2 16 (95% CI: -0.7 to -5.0) lower eGFR. In contrast, the association between blood Pb and 

17 creatinine-based eGFR, although in the same direction, was not statistically significant. Additional 

18 research in children, including with longitudinal follow-up, a range of outcome assessment methods, and 

19 with exposure only after Pb was banned from gasoline, is warranted. 

5.5.2.4. Associations between Lead Dose and New Kidney Outcome Measures

20 As noted above, in an effort to more accurately estimate kidney outcomes, new equations to 

21 estimate GFR based on serum creatinine have been developed, and the utility of other biomarkers, such as 

22 cystatin C, as well as equations based on them, are being studied. However, few publications have utilized 

23 these state-of-the-art techniques when evaluating associations between Pb or cadmium dose and renal 

24 function. In addition to the study in NHANES adolescents discussed above (Fadrowski et al., 2010), a 

25 cross-sectional study of Swedish women reported that higher blood Pb (median = 2.2 μg/dL) and 

26 cadmium (median = 0.38 μg/L) levels were associated with lower eGFR based on serum cystatin C alone 

27 (without age, sex, and race) after adjustment for socio-demographic and CKD risk factors (Akesson et al., 

28 2005). Associations were comparable to those using creatinine clearance as the kidney outcome for Pb; 

29 however associations between cadmium dose measures were stronger for the cystatin C based outcome. 

30 Staessen et al. (2001) found a significant association between blood Pb level and serum cystatin C in a 

31 cross-sectional study of adolescents; creatinine based measures were not reported. However, in a cross32 sectional study of European children, higher blood Pb levels were associated with lower serum cystatin C 

33 and creatinine; these inverse associations were attributed to hyperfiltration (De Burbure et al., 2006). A 
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1 very recent publication compared associations of blood Pb and eGFR using the traditional MDRD 

2 equation to those with four new equations: CKD-EPI, and cystatin C single variable, multivariable, and 

3 combined creatinine/cystatin C, in 3941 adults who participated in the 1999-2002 NHANES cystatin C 

4 subsample (Spector et al., 2011). Similar to the NHANES adolescent analysis, associations with the 

5 cystatin C outcomes were stronger. After multivariable adjustment, differences in mean eGFR for a 

6 doubling blood Pb were -1.9 (95% CI: -3.2, -0.7), -1.7 (95% CI: -3.0, -0.5), and -1.4 (95% CI: -2.3, -0.5) 

mL/min/1.73 m2 7 , using the cystatin C single variable, multivariable and combined creatinine/cystatin C 

8 equations, respectively, reflecting lower eGFR with increased blood Pb. The corresponding differences 

9 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 

10 MDRD equations, respectively.

5.5.3. Mechanisms of Lead Nephrotoxicity

5.5.3.1. Altered Uric Acid

11 Individuals who have been heavily exposed to Pb are at increased risk for both gout and kidney 

12 disease (Batuman, 1993; Shadick et al., 2000). Pb is thought to increase serum uric acid by decreasing its 

13 kidney excretion (Ball & Sorensen, 1969; Emmerson, 1965; Emmerson & Ravenscroft, 1975). Research 

14 during the past decade indicates that uric acid is nephrotoxic at lower levels than previously recognized 

15 (R. J. Johnson et al., 2003). Therefore, the 2006 Pb AQCD reviewed literature implicating increased uric 

16 acid as one mechanism for Pb-related nephrotoxicity (Shadick et al., 2000; V. M. Weaver et al., 2005).

17 However, this is not the only mechanism, since associations between blood Pb and serum creatinine 

18 remained significant even after adjustment for uric acid (V. M. Weaver et al., 2005). These mechanistic 

19 relations have more than just theoretical importance. Clinically relevant therapies may be possible since 

20 EDTA chelation has been reported to improve both kidney function and urate clearance in patients with

21 kidney insufficiency and gout, even when EDTA-chelatable Pb body burdens were low (J.-L. Lin et al., 

22 2001). 

23 Conterato et al. (2007) followed various parameters of kidney function after acute or chronic Pb 

24 exposure in rats. Acute exposure to Pb acetate consisted of a single i.p. injection of 25 or 50 mg/kg Pb

25 acetate, while chronic exposure was one daily i.p. injection of either vehicle or Pb acetate (5 or 25 mg/kg 

26 ) for 30 days. Acute and chronic exposure at both dose levels increased plasma uric acid levels. 

27 Conversely, Annabi Berrahal et al. (2011) found that plasma uric acid levels decreased after 65 days of Pb 

28 exposure (post-puberty; blood Pb 7.5 μg/dL) (Table 5-17). Plasma urea levels increased after 40 days of 

29 exposure (puberty; blood Pb 12.7 μg/dL). Changes in plasma urea are used as an acute renal marker of 

30 injury.
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5.5.3.2. Oxidative Damage

1 A role for ROS in the pathogenesis of experimental Pb-induced hypertension and renal disease has 

2 been well established (Vaziri, 2008a, 2008b; Vaziri & Khan, 2007). The production of oxidative stress 

3 following Pb exposure is detailed in respect to modes of action of Pb (Section 5.2.4). Past studies have 

4 shown that acute Pb exposure can elevate kidney GST levels, affecting glutathione metabolism (Daggett 

5 et al., 1998; Moser et al., 1995; Oberley et al., 1995).

6 Annabi Berrahal et al. (2011) reported on the effects of age-dependent exposure to Pb on 

7 nephrotoxicity in male rats (Table 5-17). Pups were exposed to Pb lactationally (as a result of dams 

8 consuming water containing 50 ppm Pb acetate) until weaning. Thereafter the male pups were exposed to 

9 the same solution from weaning (day 21) until sacrificed at age 40 days (puberty; blood Pb 12.7 μg/dL) 

10 and at age 65 days (post-puberty; blood Pb 7.5 μg/dL). MDA concentration in kidney was significantly 

11 increased relative to controls to the same degree at both 40 and 65 days, while total sulfhydryl groups 

12 were significantly decreased only at 65 days. These changes reflect an increase in oxidative stress after 

13 exposure to Pb.

14 Conterato et al. (2007) examined the effect of Pb acetate on the cytosolic thioredoxin reductase 

15 activity and oxidative stress parameters in rat kidneys. Acute exposure to Pb acetate consisted of a single 

16 i.p. injection of 25 or 50 mg/kg Pb acetate, while chronic exposure consisted of one daily i.p. injection of 

17 Pb acetate (5 or 25 mg/kg ) for 30 days. Measured were thioredoxin reductase-1, a selenoprotein involved 

18 in many cellular redox processes, SOD, δ-ALAD, GST, GPx, non protein thiol groups (NPSH), CAT, as 

19 well as plasma creatinine, uric acid, and inorganic phosphate levels. Acute exposure at the 25 mg Pb dose 

20 level resulted in increased SOD and thioredoxin reductase-1 activity, while exposure to the 50 mg dose 

21 level increased CAT activity and inhibited δ-ALAD activity in the kidney. Chronic exposure at the 5 mg 

22 dose level of Pb inhibited δ-ALAD and increased GST, NPSH, CAT, and thioredoxin reductase-1. 

23 Chronic exposure to the 25-mg dose level reduced δ-ALAD, but increased GST, NPSH, and plasma uric 

24 acid levels. No changes were observed in TBARS, GPx, creatinine or inorganic phosphate levels after 

25 either acute or chronic exposure. As both acute and chronic exposure to Pb increased thioredoxin 

26 reductase-1 activity, the authors suggest that this enzyme may be a sensitive indicator to exposure at low 

27 Pb dosage.

28 Jurczuk et al. (2006) published a study of the involvement of some low molecular weight thiols in 

29 the peroxidative mechanisms of action of Pb in the rat kidney. Wistar rats were fed a diet containing 500 

30 ppm Pb acetate for a period of 12 weeks and were compared to a control group receiving distilled water 

31 for the same time period. GSH, metallothionein (MT), total and nonprotein SH groups (TSH and NPSH)

32 were measured, as well as the blood activity and urinary concentration of δ-ALA. The concentrations of 

33 GSH and NPSH were decreased by Pb administration, while MT concentration was unchanged. δ-ALAD 

34 in blood was decreased, whereas urinary δ-ALA was increased by Pb administration. Negative 
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1 correlations were found between the kidney GSH concentrations and previously reported concentrations 

2 of Pb and MDA in kidneys of these rats. It is apparent from graphical presentation of the data that GSH 

3 was reduced by more than 50% following Pb administration, while TSH was reduced by approximately 

4 15%. No values for either blood or kidney Pb levels or kidney MDA were reported in this article. In 2007,

5 the same authors (Jurczuk et al., 2007) reported on the renal concentrations of the antioxidants, vitamins 

6 C and E, in the kidneys of the same Pb treated and control rats. Exposure to Pb significantly decreased

7 vitamin E concentration by 13% and vitamin C concentration by 26%. The kidney concentration of 

8 vitamin C negatively correlated with MDA concentration. The authors concluded that vitamins E and C 

9 were involved in the mechanism of peroxidative action of Pb in the kidney, and their protective effect 

10 may be related to scavenging of free radicals

11 El-Neweshy and El-Sayed studied the influence of vitamin C supplementation on Pb-induced 

12 histopathological alterations in male rats. Rats were given Pb acetate, 20 mg/kg by intragastric feeding 

13 once daily for 60 days. Control rats were given 15 mg of sodium acetate per kg once daily, and an 

14 additional group was given Pb acetate plus vitamin C (20 mg/kg every other day) 30 minutes before Pb 

15 feeding. Control rats showed normal histology, while Pb-treated rats exhibited karyomegaly with 

16 eosinophilic intranuclear inclusion bodies in the epithelial cells of the proximal tubules. Glomerular 

17 damage and tubular necrosis with invading inflammatory cells were also seen. Rats treated with Pb

18 acetate plus vitamin C exhibited relatively mild karyomegaly and eosinophilic intranuclear inclusion 

19 bodies of proximal tubules in 5 rats, while an additional 5 rats were normal. Normal glomeruli were noted 

20 in all. Thus vitamin C could be shown to ameliorate the renal histopathological effects of Pb intoxication.

21 Masso-Gonzalez and Antonio-Garcia (2009) studied the protective effect of natural antioxidants 

22 (zinc, vitamin A, vitamin C, vitamin E, and vitamin B6) against Pb-induced damage during pregnancy 

23 and lactation in rat pups. At weaning, pups were sacrificed and kidneys analyzed. Pb-exposed pups had 

24 decreased body weights. Blood Pb level in the control group was 1.43 μg/dL, in the Pb group it was 22.8 

25 μg/dL, in the Pb plus zinc plus vitamins it was 21.2 μg/dL, and in the zinc plus vitamin group blood Pb

26 was 0.98 μg/dL. The kidney TBARS were significantly elevated in Pb exposed pups, while treatment with 

27 vitamins and zinc returned TBARS to control levels. Kidney catalase activity was significantly increased 

28 above control with Pb treatment; however supplement with zinc and vitamins reduced catalase activity 

29 towards normal. Pb exposure inhibited kidney Mn-dependent SOD but not Cu-Zn-dependent SOD 

30 activity. Thus, supplementation with zinc and vitamins during gestation and lactation is effective in 

31 attenuating the redox imbalance induced by developmental, chronic low-level Pb exposure.

32 Bravo et al. (2007) reported further that mycophenolate mofetil (an immunosuppressive agent used 

33 in renal transplantation which inhibits T and B cell proliferation) administration reduces renal 

34 inflammation, oxidative stress and hypertension in Pb-exposed rats. Thus, an inflammatory immune and 

35 oxidative stress component can be seen as contributing to Pb-induced renal effects and hypertension.
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1 Although the majority of studies of the effects of Pb exposure have been on male rats, two studies 

2 have appeared which compare the response of male rats with female rats (Alghazal, Lenártová, et al., 

3 2008; Sobekova et al., 2009). Sobekova et al. (2009) contrasted the activity response to Pb on the 

4 antioxidant enzymes, GPx and GR, and on TBARS in both male and female Wistar rats of equal age. 

5 Males weighing 412 ± 47 g and females weighing 290 ± 19 g were fed diets containing either 100 ppm or 

6 1,000 ppm Pb acetate for 18 weeks. In the male rats, kidney Pb content increased by 492% on the 100 

7 ppm Pb diet and by 7,000% on the 1000 ppm Pb diet. In the female rats, kidney Pb content increased by 

8 410% on the 100 ppm Pb diet and by 23,000% on the 1,000 ppm Pb diet. There was virtually no change 

9 in GPx in the kidney of male rats given the 100 ppm Pb diet but there was a significant reduction in GPx 

10 in the female rats on both the 100 ppm diet and 1000 ppm diet. In male rats, GR was increased from 182 

11 units/gram of protein in control kidneys to 220 units on the 100 ppm Pb diet and 350 units on the 1,000 

12 ppm diet. In female rats, kidney GR decreased from 242 units in control animals to 164 units in animals 

13 on the 100 ppm Pb diet and 190 units in animals on the 1,000 ppm diet. In male rats, kidney TBARS 

14 content increased from 7.5 units/gram protein to 10.0 units (1,000 ppm Pb diet group). In female rats, 

15 there was a reduction in TBARS from 14.4 units per gram protein to 10.0 units in rats on the 100 ppm Pb

16 diet and to 11 units in rats on the 1,000 ppm Pb diet. 

17 Alghazal et al. (2008) compared the activity responses of the antioxidant enzyme, SOD and the 

18 detoxifying enzyme, GST, of the same rats exposed to 100 ppm or 1,000 ppm Pb acetate for 18 weeks. 

19 Similar to the previous study, kidney TBARS were increased only in male rats given the higher dose of 

20 Pb. Kidney SOD activity, on the other hand, was increased in both males and females at the higher dose 

21 of Pb, while GST activity was increased in kidney of males at the higher dose of Pb and decreased at the 

22 lower dose, but was decreased at both doses of Pb in females. Thus there were significant differences in 

23 the response of male and female rats to Pb exposure. Differences could be accounted for in part due to the 

24 greater deposition of Pb in female rat kidneys. Another explanation, offered by the authors, is that male 

25 rats are known to metabolize some foreign compounds faster than females, so that the biological half-life 

26 of xenobiotics in the females is longer.

5.5.3.3. Lead Effect on Renal Gangliosides

27 Gangliosides are constituents of the plasma membrane that are important for control of renal GFR 

28 because they can act as receptors for various molecules and have been shown to take part in cell-cell 

29 interactions, cell adhesion, recognition and signal transduction. Perez Aguilar et al. (2008) studied 

30 changes in renal gangliosides following Pb exposure (600 ppm Pb acetate in their drinking water for 4 

31 months) in adult male Wistar rats. Pb exposure caused an increase in blood Pb from 2.1 to 35.9 μg/dL.

32 There was no change in serum creatinine or in hemoglobin, but there was an increase in urinary δ-ALA. 

33 The following renal gangliosides were measured by immunohistochemistry and by thin layer 
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1 chromatography: GM1, GM2, GM4, and 9-O-acetylated modified form of the GD3 ganglioside (9-O-Ac2 GD3). The ganglioside pattern was mainly characterized by a decrease in the GM1 ganglioside as well as 

3 by a mild increase in GM4 and GM2 gangliosides, while the strongest alteration was observed in the 9-O4 Ac-GD3, which was overexpressed. The latter was observed only in the glomerular zone. This was 

5 associated with a decrease in apoptotic glomerular cells, as assessed by the TUNEL assay. The authors 

6 hypothesized that the increase in GD3-O-acetylation could represent a strategy to attenuate the normal 

7 renal apoptotic process and therefore contribute to cell survival during Pb exposure.

5.5.3.4. Role of Metallothionein

8 Yu et al. (2009) described dichotomous effects of Pb acetate on the expression of MT in the liver 

9 and kidney of mice. Male mice were i.p. injected with Pb acetate in doses of 100, 200, and 300 μmol/kg 

10 and sacrificed 4, 8, and 24 hours after Pb treatment. Administration of Pb increased the levels of MT-1 

11 mRNA in the liver and kidneys, but increased MT protein only in the liver. Treatment of mouse PT cells 

12 in vitro with Pb also resulted in an increase in MT mRNA, but little increase in MT protein. Thus Pb

13 exerts a dual effect on MT expression in the kidney: enhancement of MT gene transcription but 

14 suppression of MT mRNA translation.

15 Zuo et al. (2009) explored the potential role of α-Synuclein (Scna) and MT in Pb induced inclusion 

16 body formation. They used MT-I/II double knockout (MT-null) and parental wild type (WT) cell lines to 

17 explore the formation process of Pb-induced inclusion bodies. Unlike WT cells, MT-null cells did not 

18 form inclusion bodies after Pb exposure. Western blot of the cytosol showed that soluble MT protein in 

19 WT cells was lost during Pb exposure as inclusion bodies formed. However, transfection of MT-1 into 

20 MT-null cells allowed inclusion body formation after Pb exposure. As Scna is a protein with a natural 

21 tendency to aggregate into oligomers, Scna was measured in WT cells and MT-null cells after Pb

22 exposure. Scna protein showed poor basal expression in MT-null cells, and Pb exposure increased Scna 

23 expression only in WT cells. MT transfection increased Scna transcript to WT levels. In both of these cell 

24 lines Pb-induced Scna expression rapidly increased and then decreased over 48 hours as Pb-induced 

25 inclusion bodies were formed. A direct interaction between Scna and MT was confirmed ex vivo by an 

26 antibody pull down assay, where the proteins co-precipitated with an antibody to MT. Pb exposure caused 

27 increased colocalization of MT and Scna proteins. In archival kidney samples of renal cortex from WT 

28 mice chronically treated with Pb, MT was localized to the surface of inclusion bodies. Thus, Scna may be 

29 a component of Pb-induced inclusion bodies and, with MT, may play a role in inclusion body formation.

30 Figure 5-46 (and Table 5-22) presents the recent animal toxicological data for studies investigating 

31 the effects of Pb (as blood Pb level) on various measures of kidney health and function. Dysfunction in 

32 kidney function measures, including urinary flow, ALP, microalbumin, and NAG, was observed at blood 

33 Pb concentrations above 19.7 μg/dL (L. Wang et al., 2010).
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1 10 100

 

 

 

 

 

 

 

Blood Pb Level (μg/dL)

 

 

Biomarker (L. Wang et al., 2010)

Inflammation (Roncal et al., 2007)

Oxidative Stress – Redox (NavarroMoreno et al., 2009)

Oxidative Stress – Redox (L. Wang 

et al., 2010)

Oxidative Stress – Redox (MassóGonzález & 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. Dose-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 Concentration 
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Table 5-22. Additional characteristics for results of toxicological studies presented in 

Figure 5-46

Reference

Blood Pb Level 

with Response 

(μg/dL)

Outcome

Wang 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 lactation

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, microvilli 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, urinary 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 in male 

pups after gestational Pb exposure.

5.5.4. Effects of Exposure to Lead Mixtures

1 The effect of Pb on other cations, specifically calcium, is well established in the kidney literature. 

2 Calcium-mediated processes involving receptors, transport proteins, and second messenger signaling 

3 among other endpoints are significantly affected by Pb exposure.The disposition of Pb in the soft tissues 

4 (kidney and spleen) can change with exposure to Pb and other compounds. Pb plus Cd exposure changed 

5 Pb disposition with increased blood Pb (versus Pb alone group) and decreased metal concentration in the 

6 kidney and liver (versus Pb alone). An iron deficient diet significantly increased Pb deposition in adult 

7 animals (Hashmi et al., 1989), pregnant dams, and maternally exposed fetuses (U. S. Singh et al., 1991).

8 Dietary thiamine plus zinc slightly reduced blood and kidney Pb in exposed animals (Flora et al., 1989). 

9 Selenium, a cofactor for GPx, attenuated Pb-dependent lipid peroxidation and abrogates the Pb-dependent 

10 attenuation of GR and SOD. Concomitant exposure to the cations aluminum and Pb protects Pb-exposed 

11 animals from ensuing nephropathy (Shakoor et al., 2000). In summary, Pb is known to affect processes 

12 mediated by endogenous divalent cations. In addition, exposure to other metals or divalent cations can 

13 modulate Pb disposition and its effects in the body.
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5.5.4.1. Lead and Cadmium

1 Cd shares many similarities with Pb; it is a ubiquitous PT nephrotoxicant at high exposure levels 

2 and accumulates in the body. Despite this, few studies have evaluated associations between low-level Cd

3 exposure and CKD or the impact of joint exposure of these or other metals on CKD. As discussed in the 

4 2006 Pb AQCD, Cd, at the lower exposure levels common in the U.S. and other developed countries, has 

5 a substantial impact on associations between Pb exposure and the kidney EBE marker, NAG, even in the 

6 presence of occupational level Pb exposure. In one report, mean NAG, although higher in the Pb-exposed 

7 group compared to controls, was correlated with urine Cd and not blood or tibia Pb (Roels et al., 1994). In 

8 another occupational population where both metals were significantly associated with NAG, a 0.5 μg/g 

9 creatinine increase in Cd had the same effect on NAG as a 66.9 μg/g bone mineral increase in tibia Pb (V. 

10 M. Weaver, Lee, et al., 2003).

11 The 2006 Pb AQCD noted that data examining the dose-response relation between environmental 

12 Cd and the kidney were too scarce to determine the impact of Cd exposure on relations between Pb 

13 exposure and other kidney outcomes. A recent publication in NHANES data collected from 1999 through 

14 2006 addresses this need; (results pertaining solely to Pb were discussed in Section 5.5.2.2) (Navas-Acien 

15 et al., 2009). Geometric mean blood Cd level was 0.41 μg/L in 14,778 adults aged ≥ 20 years. After 

16 adjustment for survey year, sociodemographic factors, CKD risk factors, and blood Pb, the odds ratios for 

albuminuria (≥ 30 mg/g creatinine), reduced eGFR (<60 mL/min/1.73 m2 17 ), and both albuminuria and 

18 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), 

19 respectively, comparing the highest with the lowest blood Cd quartiles. Both Pb and Cd remained

20 significantly associated after adjustment for the other although effect modification was not observed. 

21 However, the odds ratios comparing participants in the highest with the lowest quartiles of both metals 

22 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 

23 (95% CI: 1.58, 10.65) for albuminuria and reduced eGFR together. These findings are consistent with 

24 other recent publications (Akesson et al., 2005; Hellstrom et al., 2001), support consideration of both 

25 metals as CKD risk factors in the general population, and provide novel evidence of increased risk in 

26 those with higher environmental exposure to both metals.

27 However, a very recent study suggests that interpretation of low-level Cd associations with GFR 

28 measures may be much more complex. Conducted in Pb workers to address the fact that few studies have 

29 examined the impact of low-level Cd exposure in workers who are occupationally exposed to other 

30 nephrotoxicants such as Pb, Cd dose was assessed with urine Cd, which is widely considered the optimal 

31 dose metric of cumulative Cd exposure. In 712 Pb workers, mean (SD) blood and tibia Pb, urine Cd, and 

32 eGFR using the MDRD equation were 23.1 (14.1) μg/dl, 26.6 (28.9) μg/g, 1.15 (0.66) μg/g creatinine, 

and 97.4 (19.2) ml/min/1.73m2 33 , respectively (V. M. Weaver et al.). After adjustment for age, sex, BMI, 

34 urine creatinine, smoking, alcohol, education, annual income, diastolic BP, current or former Pb worker 
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1 job status, new or returning study participant, and blood and tibia Pb, higher ln-urine Cd was associated 

with higher calculated creatinine clearance, eGFR (β = 8.7 ml/min/1.73 m2 2 [95% CI: 5.4, 12.1]) and ln3 NAG, but lower serum creatinine. These unexpected paradoxical associations have been reported in two 

4 other publications (De Burbure et al., 2006; Hotz et al., 1999) and have been observed in two other 

5 populations. Potential explanations for these paradoxical results included a normal physiologic response 

6 in which urine Cd levels reflect renal filtration; the impact of adjustment for urine dilution with creatinine 

7 in models of kidney outcomes; and Cd-related hyperfiltration.

8 Wang et al. (2009) studied the effects of Pb and/or Cd on oxidative damage to rat kidney cortex 

9 mitochondria. In this study young female Sprague Dawley rats were fed for 8 weeks with either Pb

10 acetate (300 ppm), Cd chloride (50 ppm), or Pb and Cd together in the same dosage. Lipid peroxidation 

11 was assessed as MDA content. Renal cortex pieces were also processed for ultrastructural analysis and for 

12 quantitative rtPCR to identify the mitochondrial damage and to quantify the relative expression levels of 

13 cytochrome oxidase subunits (COX-I/II/III). Cytochrome oxidase is the marker enzyme of mitochondrial 

14 function, and COX-I, II, and III are the three largest mitochondrially encoded subunits which constitute 

15 the catalytic functional core of the COX holoenzyme. Mitochondria were altered by either Pb or Cd

16 administration, but more strikingly by Pb plus Cd administration, consisting of disruption and loss of 

17 mitochondrion cristae. Kidney cortex MDA levels were increased significantly by either Pb or Cd, given 

18 individually, but more so by Pb plus Cd. COX-I/II/III were all reduced by either Pb or Cd administration, 

19 but more prominently by Pb plus Cd administration. This study adds to our knowledge of the synergistic 

20 effects of Pb and Cd on kidney mitochondria.

5.5.4.2. Lead, Cadmium, and Arsenic

21 Wang and Fowler (2008) present a general review of the roles of biomarkers in evaluating 

22 interactions among mixtures of Pb, Cd, and arsenic. Past studies have found that that addition of Cd to 

23 treatment of rats with Pb or Pb and As significantly reduced the histological signs of renal toxicity from 

24 each element alone; on the other hand, animals exposed to Cd in addtion to Pb or Pb and As showed an 

25 additive increase in the urinary excretion of porphyrins, indicating that, although measured tissue burdens 

26 of Pb were reduced, the biologically available fraction of Pb is actually increased (Mahaffey et al., 1981; 

27 Mahaffey & Fowler, 1977). 

28 Stress proteins were examined after exposure to mixtures of Pb and other metals. Induction of MT 

29 was strongest in groups with Cd treatment. However, co-exposure to Pb and As induced higher levels of 

30 MT protein than either Pb or As exposure alone in kidney tubule cells. Heat shock proteins (Hsps) are 

31 commonly altered under the situation of exposure to metal mixtures. Both in vitro low dose studies and in 

32 vivo studies showed that Hsps were induced in a metal/metalloid, dose and time-specific manner (G. 
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1 Wang et al., 2005). Additive or more than additive interactions occur among Pb, Cd and As under 

2 combined exposure conditions.

5.5.4.3. Lead and Zinc

3 Zinc has been investigated as a protective compound against the effects of Pb. Pb treatment (35 

4 mg/kg i.p. for 3 days) caused a significant fall in hemoglobin content, significant increases in lipid 

5 peroxidation and decreased level of reduced glutathione in liver, together with diminished total protein 

6 content in liver and kidney. Zinc (10 mg/kg i.p.) and ascorbic acid (10, 20 and 30 mg/kg i.p.) treatment 

7 showed a moderate therapeutic effect when administered individually, but more pronounced protective 

8 effects after combined therapy (Upadhyay et al., 2009).

9 Jamieson et al. (2008) studied the effect of dietary zinc content on renal Pb deposition. Weanling 

10 Sprague Dawley rats were assigned to marginal zinc (MZ, 8 mg Zn/kg diet), zinc adequate control (CT, 

11 30 mg Zn/kg ), zinc-adequate diet-restricted (DR, 30 mg Zn/kg), or supplemental zinc (SZn, 300 mg 

12 Zn/kg) groups, with or without Pb acetate, 200 ppm for 3 weeks. Pb exposure did not result in 

13 nephromegaly or histological alterations. The marginal zinc rats had higher renal Pb (35%) and lower 

14 renal zinc (16%) concentrations than control rats. On the other hand, supplemental zinc was more 

15 protective than the control diet against renal Pb accumulation (33% lower). Standard procedures for 

16 indirect immunoperoxidase staining were used to determine MT localization in the kidney. Pb had no 

17 effect on MT staining intensity, distribution, or relative protein amounts. Western blot analysis confirmed 

18 that MT levels were responsive to dietary zinc but not to Pb exposure.

5.5.4.4. Lead and Mercury

19 Stacchiotti et al. (2009) studied stress proteins and oxidative damage in a renal derived cell line 

20 exposed to inorganic mercury and Pb. The time course of the expression of several heat shock proteins, 

21 glucose-regulating proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to 

22 subcytotoxic doses of inorganic mercury (HgCl2, 1-40 μM) and Pb (PbCl2, 2-500 μM) were analyzed. 

23 Reactive oxygen and nitrogen species were detected by flow cytometric analysis. Endogenous total GSH 

24 content and the enzymatic activity of GST were determined in cell homogenates. Western blot analysis 

25 and immunohistochemistry were used for quantification of heat shock proteins and metallothionein.

26 Reverse transcription PCR was used for quantification of metallothionein. The higher doses of mercury 

27 (20 μM and 40 μM) were shown to markedly inhibit growth of the cell line while the higher doses of Pb

28 (60 μM to 500 μM) inhibited cell growth to a lesser degree. After 24 hours of exposure at 20 μM mercury, 

29 the cells presented abnormal size and pyknotic nuclei, swollen mitochondria and both apoptosis and overt 

30 necrosis. In the presence of 60 or 300 μM Pb, the cells lost cell-cell and cell-matrix contacts, showed a 

31 round size, irregular nuclear contour and often mitotic arrest, but no apoptosis or overt necrosis at 24 
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1 hours. Mercury induced a significant increase in both and reactive nitrogen species, the reactive nitrogen 

2 species maximal at 24 hours, and the ROS at 48 hours. Pb (60 or 300 μM) did not cause an increase in 

3 reactive oxygen or reactive nitrogen species beyond the levels seen in control cells. Total GSH 

4 significantly increased in cells grown in the presence of Pb; the effect was dose-dependent and GSH 

5 reached its maximal value at a dose of 300 μM Pb. The effect of mercury was biphasic, 10 μM 

6 significantly enhancing GSH by 600%, while the amount of GSH detected after 20 μM mercury only 

7 increased by 50% compared to control. GST activity was enhanced by both Pb and mercury. Heat shock 

8 proteins Hsp25 and Hsp72 were up-regulated by mercury but there was no effect on Grp78 as compared 

9 to control. On the contrary, Pb treatment only upregulated Grp78. Mercury induced a time-dependent 

10 effect on metallothionein mRNA expression, which reached its maximal value 3 hours after beginning 

11 treatment and reverted to control values at 24 hours. With Pb, on the other hand, mRNA transcription was 

12 dose- and time-dependent. The transcripts remained overexpressed compared to controls up to 72 hours. 

13 The results of this study with regard to the Pb effect on metallothionein synthesis clearly differ from the 

14 study of Jamieson et al. (2008) that found no increase in metallothionein following Pb exposure. This 

15 discrepancy remains to be clarified.

5.5.5. Impact of Treatment with Antioxidants on Renal Lead 

Accumulation and Pathology

5.5.5.1. Treatment with Antioxidants

16 Wang et al. (2010) assessed the protective effect of N-acetylcysteine (NAC) on experimental 

17 chronic Pb nephrotoxicity in immature female rats. NAC is a potent oxygen free radical scavenger, a

18 metal chelator, and the precursor to the antioxidant glutathione. Sprague-Dawley rats received Pb acetate 

19 (300 ppm in drinking water) and/or NAC (100 mg/kg/day, by i.p. injection) for 8 weeks to investigate the 

20 protective effect of NAC on Pb-induced renal damage and oxidative stress. Serum and renal cortical Pb

21 levels were markedly increased in the Pb treated animals, but reduced in the Pb plus NAC treated 

22 animals. There were time-related increases in urinary alkaline phosphatase, urinary GGT, urinary NAG, 

23 urinary total protein, urinary β-2 microglobulin, and urinary microalbumin, which were all decreased by 

24 NAC. Serum urea nitrogen was significantly increased by Pb administration and reduced towards normal 

25 by Pb plus NAC. Alterations in proximal tubular structures were observed in most kidney samples from 

26 Pb-treated rats, but animals treated with combination Pb plus NAC showed well-preserved cell structures 

27 and organelles. Indices of oxidative stress (MDA, SOD, GSH, GPx, and CAT) were altered by Pb

28 treatment and restored to or towards normal by Pb plus NAC treatment (MDA increased and the 

29 remainder decreased). Thus NAC can be shown to have both an anti-oxidative and a chelator effect on Pb

30 intoxication.
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1 Saxena et al. (2005) investigated the beneficial role of monoesters of meso-2, 3-

2 dimercaptosuccinic acid in the mobilization of Pb and recovery of tissue oxidative injury in rats. 

3 Dimercaptosuccinic acid (DMSA) is known as a Pb chelator and as an antioxidant by virtue of its 

4 possession of thiol groups. In this study, DMSA, and two of its analogues, monomethyl 

5 dimercaptosuccinic acid (MmDMSA) and mono-cyclohexyl dimercaptosuccinic acid (MchDMSA) were 

6 assessed as to their capacity to reduce Pb concentration in blood and soft tissues and to recover Pb7 induced oxidative stress in male Wistar rats who were exposed to Pb acetate (0.1% in drinking water) for 

8 20 weeks. Rats were then treated orally with five days of DMSA or its two analogues at a dose of up to 

9 100 mg/kg once daily. Exposure to Pb caused a rise in blood Pb levels to approximately 25 μg/dL. 

10 Exposure to Pb also caused a significant decrease in blood ALAD activity and GSH levels, accompanied 

11 by inhibition of kidney ALAD and an increase in δ-aminolevulinic acid synthatase (ALAS) activity in 

12 liver and kidneys. Pb exposure also resulted in increased blood and soft tissue (brain, liver, and kidney) 

13 Pb and TBARS levels and decreased GSH levels. These were restored by treatment with DMSA and its 

14 analogues, particularly MchDMSA. 

15 Abdallah et al. (2010) explored the effect of Pb toxicity on coenzyme Q levels in rat tissues. 

16 Coenzyme Q acts as an electron and proton carrier in mitochondria and functions as an antioxidant in its 

17 reduced form (ubiquinol). Both coenzyme Q9 and coenzyme Q10 were measured in rat tissues as 

18 coenzyme Q9 is the predominant form found in the rat. Male albino rats were injected i.p. with Pb acetate 

19 in a dose of 5 mg/kg daily for 6 weeks. No blood Pb levels were reported. TBARS were elevated above 

20 controls in serum, liver, kidney and brain while non-protein sulfhydryl groups (indicative of GSH) were 

21 decreased in serum and kidney. Both oxidized and reduced coenzyme Q9 levels were significantly 

22 reduced in kidneys from Pb-treated rats as contrasted to controls (48.6 ± 5.6 versus 95.5 ± 10.1 nmol/g 

23 tissue, oxidized, and 35.4 ± 3.0 versus 61.4 ± 5.1 nmol/g tissue, reduced). On the other hand, levels of 

24 oxidized and reduced coenzyme Q10 were unchanged. Thus the reduced levels of coenzyme Q 

25 attributable to Pb intoxication may participate in the diminished antioxidant defense mechanism.

26 El-Sokkary et al. (2005) evaluated the effect of melatonin against Pb-induced hepatic and renal 

27 toxicity in male rats. Melatonin is known to be efficacious as a free radical scavenger and indirect 

28 antioxidant. Three groups of animals were used: control, Pb acetate-treated (100 ppm) and Pb acetate and

29 melatonin (10 mg/kg) given subcutaneously for 30 days. Lipid peroxidation was measured as the sum of 

30 MDA plus 4-hydroxyalkenals (4-HAD). Pb increased kidney lipid peroxidation products, but these were 

31 reduced towards normal by melatonin. Both SOD and GSH levels were reduced by Pb, and were 

32 increased by melatonin. Histological section of kidneys of Pb treated rats showed tubular degeneration 

33 with some apparently necrotic cells, while melatonin treated rats demonstrated a near normal structure.

34 The authors conclude that melatonin protected the liver and kidneys from the damaging effects of 

35 exposure to Pb through inhibition of lipid peroxidation and stimulation of endogenous antioxidative 

36 defense systems.
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1 Ozsoy et al. studied the protective effects of L-carnitine on experimental Pb toxicity in rats. Female 

2 two-month old rats were fed 0.5 mg/kg Pb acetate alone or with daily injections of 0.5 mg/kg L-carnitine 

3 for 60 days. Control animals were injected with physiological saline. Pb caused an increase in serum 

4 creatinine and histopathological changes in the kidney, consisting of tubule dilatation, degeneration and 

5 necrosis and interstitial inflammation. In the Pb and L-carnitine group serum creatinine was reduced to 

6 control values and the histopathological changes were reversed. Immunological staining for Cu/Zn-SOD 

7 was elicited by Pb feeding and reduced by L-carnitine. The authors attribute the beneficial effects of L8 carnitine to its antioxidant effect.

9 Reddy et al. (2010) used Sprague-Dawley rats that were treated with 10 mg/kg/day of Pb acetate 

10 and/or thiamine (25 mg/kg/day) for 7 weeks. Thiamine treatment normalized the Pb-induced alterations in 

11 blood ALAD activity and urinary NAG activity.

12 Kharoubi et al. (2008) described the prophylactic effects of Wormwood (Artemisia absinthium L.) 

13 plant extracts on kidney function on Pb-exposed animals. Male Wistar rats were exposed to Pb acetate 

14 (750 ppm in drinking water) for 11 weeks, and then received Wormwood extract (200 mg/kg) for 4 

15 weeks. Significant differences in blood and urinary Pb concentration were observed between the Pb group 

16 and the Wormwood group (blood Pb 55.6 μg/dL compared to 22.3 μg/dL). Pb induced lipid peroxidation 

17 (TBARS and protein carbonyls in the kidney), but these levels were reduced by Wormwood extract. 

18 Wormwood extract also attenuated the effects of Pb on renal function. These results indicated that 

19 Wormwood extract had significant antioxidant activity and protected the kidney from Pb-induced toxicity.

20 Jayakumar et al. (2009) evaluated the effect of a methanolic extract of the Indian herb, Achyranthes

21 aspera, in preventing Pb-induced nephrotoxicity in rats. Male albino Wistar rats, received Pb acetate 

22 (0.2% for 6 weeks) or Pb acetate plus A. aspera (200 mg/kg for 6 weeks) simultaneously. A. aspera

23 partially prevented the increases in kidney weight, BUN, serum uric acid, and serum creatinine caused by 

24 Pb administration. The levels of urinary marker enzymes, GGT, β-glucuronidase, NAG, Cathepsin D, and 

25 LDH, which were reduced by Pb administration, were increased to or towards normal by A. aspera. 

26 Kidney histology revealed that Pb-treated animals showed tubular damage, whereas the Pb plus A. aspera

27 -treated animals showed a reduction in tubular damage. 

28 The effectiveness of various plant or bacterial extracts as antioxidants in the kidney was explored 

29 in two separate publications. El-Nekeety et al. (2009) evaluated the protective effect of an extract of the 

30 folk medicine plant Aquilegia vulgaris against Pb acetate-induced oxidative stress in Sprague-Dawley 

31 rats. The experimental group was treated with Pb acetate, 20 ppm, and/or an extract of A. vulgaris, 100 

32 ppm, for 2 weeks prior to Pb acetate. Pb acetate increased serum urea, and decreased serum total protein 

33 and albumin. These changes were reversed by treatment with the extract. Histological examination of 

34 kidneys of rats treated with Pb showed tubular dilatation, interstitial inflammatory cells, hemorrhage, 

35 cellular debris, and hypercellularity in the glomerulus, with apoptotic nuclei in renal tubular epithelial 

36 cells. The rats treated simultaneously with Pb and the extract showed essentially normal renal tubules and 
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1 glomeruli while rats treated with Pb and then the extract showed improvement in tubular structure, but 

2 interstitial fibrosis was still present. This experiment confirmed that exposure to Pb generates free 

3 radicals, and that an extract of A. vulgaris resulted in restoration of the different parameters tested. The 

4 second experiment in this group was by Ponce-Canchihuaman et al. (2010) who evaluated the antioxidant 

5 activity of the cyanobacterium Spirulina maxima against Pb acetate-induced hyperlipidemia and oxidative 

6 damage in the liver and kidney of male rats. Male Wistar rats were exposed to Pb acetate by i.p. injection 

7 (25 mg/rat on a weekly basis for 3 weeks and a 5% supplement of Spirulina was given in food). The 

8 findings in the kidney were similar to those in the liver (see Section 5.9.1). Thus Pb-induced oxidative 

9 stress and renal damage can be attenuated by treatment with Spirulina extract.

10 Finally, there is a need to examine whether the chelator, CaNa2EDTA, acts also as an antioxidant 

11 and promotes increased vasodilatation and thus increased renal blood flow by enhancing the delivery of 

12 NO. This question arises because of the observations of Lin et al. (2006) that repeated injections of 

13 CaNa2EDTA leads to improvement in kidney function in patients with chronic renal failure, even in 

14 individuals with very low body Pb stores. Jacobsen et al. (2001),examined the anti-oxidative effects of 

15 Gallic acid, EDTA, and an emulsifier in mayonnaise enriched with 16% fish oil. EDTA was shown to be 

16 an efficient antioxidant in the fish oil enriched mayonnaise as it strongly inhibited the formation of free 

17 radicals and volatile oxidation compounds. The authors suggest that the antioxidative effect appears to be 

18 due to its ability to chelate free iron in egg yolk at the oil-water interface. 

5.5.5.2. Treatment with Antioxidants plus Chelators 

19 Santos et al. (2006) assessed the potentiating effects of chelators (2,3-dimercaptopropanol [BAL], 

20 2,3-dimercaptopropane-l-sulfonic acid [DMPS], and meso-2,3-dimercaptosuccinic acid [DMSA]) given 

21 simultaneously with Pb acetate on δ-ALAD activity, both in vivo and ex vivo. Ex vivo, human blood was 

22 pre-incubated with BAL or DMSA (10 μM) or DMPS (1 μM) then Pb acetate added to the reaction 

23 mixture. In vivo, mice were given daily injections of 50 mg/kg Pb acetate for 15 days and then injected 

24 with 1/3 of LD50 of the chelating agents. In human blood the inhibitory effect of Pb acetate (1 and 100 

25 μM) was markedly increased in the presence of BAL and DMPS, whereas DMSA ameliorated the enzyme 

26 inhibition caused by 1 μM Pb acetate. In vivo Pb acetate inhibited δ-ALAD activity by 42%. Parallel to 

27 the ex vivo results, BAL and DMPS, but not DMSA, increased the inhibitory potency of Pb in blood. In

28 the kidney, BAL and DMSA but not DMPS increased inhibitory activity. The authors conjecture that the 

29 chelators may deplete the cells of zinc, an essential element for δ-ALAD activity. Supporting the 

30 chelation effect seen is the Santos study is work by Bradberry and Vale (2009), Hamidinia et al. (2006), 

31 and Aslani et al. (Aslani et al., 2010) who found decreased kidney Pb content post-chelation.
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5.5.6. Summary and Causal Determination

1 The 2006 Pb AQCD concluded that “in the general population, both circulating and cumulative Pb 

2 was found to be associated with a longitudinal decline in renal function,” evidenced by increased serum 

3 creatinine and decreased creatinine clearance or eGFR associated with blood and bone Pb levels (U.S. 

4 EPA, 2006). Data in general and patient populations provided consistent evidence of low-level Pb 

5 nephrotoxicity (Akesson et al., 2005; R. Kim et al., 1996; Tsaih et al., 2004; C.-C. Yu et al., 2004); effects 

6 on eGFR were observed in human hypertensives at mean blood Pb level of 4.2 μg/dL (Muntner et al., 

7 2003). These findings were substantiated by the coherence of effects observed across epidemiologic and 

8 toxicological studies. Both human and animal studies have observed hyperfiltration; in animals during the 

9 first 3 months after Pb exposure, effects were characterized by increased GFR and increased kidney 

10 weight due to glomerular hypertrophy. However, chronic exposure resulted in decreased GFR, interstitial 

11 fibrosis, and kidney dysfunction. Additionally, toxicological studies found that early effects of Pb on 

12 tubular cells were generally reversible, but continued exposure resulted in chronic irreversible damage. 

13 Toxicological studies provided mechanistic evidence to support the biological plausibility of Pb-induced 

14 renal effects, including oxidative stress leading to  ̇NO inactivation. Despite the strong body of evidence 

15 presented in the 2006 Pb AQCD, uncertainty remained on the public health significance of such effects in 

16 the general population, the implications of hyperfiltration, and reverse causality.

17 Recent epidemiologic studies in adult general and patient populations, with few exceptions, 

18 continue to be consistent in observing associations between blood and bone Pb levels and worse kidney 

19 function and provide important evidence that nephrotoxicity occurs at current population levels of 

20 biomarkers of Pb exposure. These studies benefit from a number of strengths that vary by study but 

21 include comprehensive assessment of Pb dose with bone Pb as a measure of cumulative body burden and 

22 chelatable Pb as a measure of bioavailable Pb; prospective study design; and statistical approaches that 

23 utilize a range of exposure and outcome measures, while adjusting for numerous kidney and Pb risk 

24 factors. Large sample sizes provide strength to the general population studies. Reexamination of a study 

25 from the 2006 Pb AQCD provided data to conclude that a 10-fold increase in blood Pb (e.g., from 1 to 10 

26 μg/dL) would result in an 18 mL/min decrease in estimated creatinine clearance or a 25% decrease from 

27 the mean, and that an increase in blood Pb from the 5th to the 95th percentile (3.5 μg/dL) had the same 

adverse impact on eGFR as an increase of 4.7 years in age or 7 kg/m2 28 in body mass index (Akesson et al., 

29 2005). In populations with lower blood Pb levels, a downward shift in kidney function of the entire 

30 population due to Pb may not result in CKD in identifiable individuals; however, that segment of the 

31 population with the lowest kidney reserve may be at increased risk for CKD when Pb is combined with 

32 other kidney risk factors. At blood Pb levels that are common in the general U.S. population, Pb increases 

33 the risk for clinically relevant effects particularly in susceptible populations such as those with underlying 
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1 chronic medical diseases that increase CKD risk such as diabetes mellitus and hypertension and co2 exposure to other environmental nephrotoxicants. 

3 Absence of impact in the occupational setting cannot be used as a rationale for discounting Pb4 related nephrotoxicity at lower environmental levels. Research in the occupational setting has 

5 traditionally been far less consistent than in environmentally exposed populations. A number of 

6 explanatory factors for this inconsistency, all due to limitations of the occupational literature, were 

7 discussed in the 2006 Pb AQCD. The observation of paradoxical or inverse associations (higher Pb dose 

8 with lower serum creatinine, and/or higher eGFR or calculated or measured creatinine clearance) in 

9 several of these studies cannot be resolved solely by utilizing stronger research techniques. Irrespective of 

10 the mechanism, these associations have risk assessment implications. If associations are in opposite 

11 directions in different subgroups of the population and the relevant effect modifier is not considered, null 

12 associations will be observed.

13 Important data on the kidney effects of Pb on children were reported in a recent NHANES analysis 

14 in adolescents that observed an association between higher blood Pb and lower cystatin C-based eGFR

15 (Fadrowski et al., 2010). These findings are consistent with a rodent model in which a low dose of Pb (50 

16 ppm) administered from birth resulted in renal impairment (elevated serum creatinine as compared to 

17 control rats), but these observations require confirmation by measurement of GFR and renal pathology

18 (Berrahal et al., 2011). These limited studies add to the strength of the association between blood Pb and 

19 altered renal function in children despite the need for additional research.

20 CKD results in substantial morbidity and mortality, and, even at earlier stages than those requiring 

21 kidney dialysis or transplantation, is an important risk factor for cardiac disease. As kidney dysfunction 

22 can increase BP and increased BP can lead to further damage to the kidneys, Pb-induced damage to either 

23 or both renal or cardiovascular systems may result in a cycle of further increased severity of disease. Pb 

24 exposure has been causally linked to both increased BP and other cardiovascular effects (Section 5.4). 

25 Interestingly, animal studies have shown Pb-induced vascular injury in the kidney associated with 

26 increased glomerular sclerosis, tubulointerstitial injury, increased collagen staining, and an increase in 

27 macrophages associated with higher levels of MCP-1 mRNA (Roncal et al., 2007). It is possible that the 

28 cardiovascular and renal effects of Pb observed are mechanistically linked and are contributing to the 

29 progression of the diseases. 

30 Recently available animal toxicological studies strengthen the evidence regarding the mechanisms 

31 leading to these renal alterations, especially, similar to the cardiovascular system, the influence of Pb32 induced oxidative stress. The mode of action of Pb in the kidneys has been extended to the field of 

33 immunology, where it was shown that low Pb exposure results in infiltration of lymphocytes and 

34 macrophages associated with increased expression of NFκB in proximal tubules and infiltrating cells.

35 Additionally, recent evidence expands on the evidence of acute effects of Pb, including mitochondrial 
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1 dysfunction, renal cell apoptosis, and glomerular hypertrophy. These mechanisms are useful in 

2 understanding the occurrence of acute hyperfiltration followed by chronic kidney dysfunction.

3 Current evidence does not allow for the identification of a threshold for Pb-related nephrotoxicity; 

increased odds of CKD (characterized by eGFR <60 mL/min/1.73 m2 4 ) were apparent in NHANES 

5 analyses that included data collected as recently as 2006 (Navas-Acien et al., 2009). The odds of reduced 

6 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% 

7 (95% CI: 1.17, 2.08) at blood Pb >.4 μg/dL.

8 In summary, new studies evaluated in the current review support or expand upon the strong body of 

9 evidence presented in the 2006 Pb AQCD that biomarkers of Pb exposure are associated with renal health 

10 effects. Epidemiologic studies continue to demonstrate a consistently positive relationship between blood 

11 Pb level and kidney dysfunction at blood Pb levels (mean <2 μg/dL) comparable to those occurring in the 

12 current U.S. population with no evidence for a threshold across the range of levels studied. By 

13 demonstrating Pb-induced oxidative stress and describing mechanisms of acute changes following Pb 

14 exposure, toxicological studies provide biological plausibility for the associations observed in 

15 epidemiologic studies between Pb and kidney dysfunction. Collectively, the evidence integrated across 

16 epidemiologic and toxicological studies as well as across the spectrum of kidney health endpoints is 

17 sufficient to conclude that there is a causal relationship between Pb exposures and renal health 

18 effects.

5.6. Immune System Effects

5.6.1. Introduction

19 With respect to studies conducted in laboratory animal and in vitro models, Pb is one of the most 

20 extensively researched and studied immunotoxicants. Experimental studies of the effects of Pb exposure 

21 on host resistance date back to the 1960s while those focusing on Pb-induced immune functional 

22 alterations, including developmental immunotoxicity (DIT), were first conducted during the 1970s. 

23 Despite the long history of Pb-associated immunotoxicity research, the immune-based effects in animals 

24 with blood Pb levels in the range of current U.S. population levels (i.e., <10 μg/dL), particularly early in 

25 life, are a relatively recent finding from within the last 10-15 years (Dietert & McCabe, 2007). Over the 

26 same time period, similar advances in the understanding of Pb-associated changes in immunological 

27 parameters in humans without occupation Pb exposures have substantiated the immunomodulatory effects 

28 of Pb. 

29 In the 2006 Pb AQCD (U.S. EPA, 2006), both toxicological studies in animals and epidemiologic 

30 studies of humans provided strong evidence that the immune system was one of the more sensitive 

31 systems affected by Pb exposure. However, rather than producing overt cytotoxicity or pathology, Pb 
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1 exposure of experimental models and blood Pb levels in humans were found to be associated with 

2 alterations in the abundance and function of a variety of immune cells (Figure 5-47). In both toxicological 

3 and epidemiologic studies, macrophages and T lymphocytes were observed to be particularly sensitive to 

4 Pb, but Pb-associated changes were also reported in B lymphocytes and neutrophils. Several of these 

5 changes were observed at blood Pb levels <10 μg/dL or the equivalent in humans and experimental 

6 models, levels at which neurological effects also were observed. 

7 Alterations in these aforementioned immune cells can lead to changes changes in cell-to-cell 

8 interactions, multiple signaling pathways, and inflammation that affect both innate and acquired 

9 immunity, that in turn, influence risk of developing infectious, allergic and autoimmune diseases as well 

10 as exacerbating inflammatory responses in other organ systems (Figure 5-47).

Figure 5-47. Immunological pathways by which Pb exposure may increase 

risk of immune-related diseases.

11 Studies conducted in animal and in vitro models have provided consistent evidence for Pb inducing 

12 effects on the range of immune effects presented in this continuum. Among the hallmarks reported for Pb13 induced changes in functional pathways are: (1) a suppression of T-derived lymphocyte helper (Th)1-

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

Overproduction 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 diseases
May 2011 5-244 DRAFT – DO NOT CITE OR QUOTE

1 driven cell-mediated immunity (as measured by a delayed-type hypersensitivity [DTH] response); (2) an 

2 increase in Th2-driven immunoglobulin E (IgE) antibody production; and (3) a proinflammatory shift in 

3 macrophage function. The latter was characterized by increased production of reactive oxygen 

4 intermediates reactive oxygen species (ROS), prostaglandin E2 (PGE2), and inflammatory cytokines such 

5 as tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 and decreased production of IL-12 and nitric 

6 oxide (NO). In animal studies, these effects of Pb were more prominent with in utero Pb exposures and in 

7 males. In the 2006 Pb AQCD, epidemiologic evidence was available for relatively fewer immune 

8 endpoints (e.g., proinflammatory shifts in immune cell function); however, coherence was observed 

9 between toxicological and epidemiologic findings for Pb-associated changes in circulating IgE levels and 

10 T and B cell abundance and activation (U.S. EPA, 2006). Although many epidemiologic studies indicated 

11 associations between blood Pb levels and immune system effects, several limitations of study design and 

12 analytic methods were noted, including cross-sectional analyses; small sample size; inconsistent 

13 adjustment for potential confounders such as age, sex, smoking, and comorbid conditions; and limited 

14 assessment of the magnitude of association between blood Pb levels and changes in immune function.

15 Consistent with inhibition of Th1 activity, toxicological evidence presented in the 2006 Pb AQCD 

16 linked Pb exposure of animals to impaired host resistance and increased risk of certain infections (U.S. 

17 EPA, 2006). Consistent with inducing a hyperinflammatory state and local tissue damage, Pb exposure 

18 was found to induce generation of autoantibodies, indicating an elevated risk of autoimmune reactions. 

19 Additionally, the demonstrated shift toward a Th2 response suggested that Pb could elevate the risk of 

20 atopy and allergic responses. While toxicological studies provided the evidence for biological plausibility, 

21 the epidemiologic evidence was too sparse to draw conclusions regarding associations between blood Pb 

22 levels and these broader indicators of immune dysfunction in humans. 

23 Studies published since the 2006 AQCD support the previous findings of Pb-induced immune 

24 effects and demonstrate similar effects at lower blood Pb levels (<2-5 μg/dL). Recent studies also expand 

25 on the array of immunological parameters affected by Pb exposure as presented in Figure 5-47. For 

26 example, new toxicological evidence indicates that Pb modulates function of dendritic cells. Results from 

27 new toxicological and epidemiologic studies strengthen the link between Pb-associated effects on immune 

28 cells and immune- and inflammatory-based diseases by providing evidence for changes in intermediary 

29 signaling and inflammatory pathways (Figure 5-47). New epidemiologic studies examine signaling 

30 molecules such as proinflammatory cytokines and NO to produce findings parallel with toxicological 

31 studies. Another important advance is the increasing knowledge of the broader role of Pb-associated 

32 immune modulation in mediating Pb effects in nonlymphoid tissues (e.g., in the neurological, 

33 reproductive, and respiratory systems). Although primarily cross-sectional in design, recent epidemiologic 

34 studies address many limitations of earlier studies through greater examination of children and adults 

35 without occupation Pb exposures with blood Pb levels more comparable to those currently measured in 
May 2011 5-245 DRAFT – DO NOT CITE OR QUOTE

1 the U.S. population and greater consideration of confounding by age, sex, smoking, and comorbid 

2 conditions. 

5.6.2. Cell-Mediated Immunity

5.6.2.1. T Cells

3 Toxicological and epidemiologic evidence reviewed in the 2006 Pb AQCD consistently 

4 demonstrated Pb effects on decreasing T cell populations (U.S. EPA, 2006), which mediate responses to 

5 antigens and infectious agents. Consistent with previous findings, in a recent study of Wistar rats 

6 administered 200 ppm Pb acetate, the percentages of CD4+ (T helper) and CD8+ (cytotoxic T) T cells 

7 were decreased (with CD4-CD8- cells elevated) in the submaxillary lymph nodes (p <0.05), with 

8 intraperitoneal (i.p.) exposure but not oral exposure (Teijon et al., 2010). The 2006 Pb AQCD also 

9 described numerous toxicological studies in which Pb exposure shifted the development and/or activation 

10 of CD4+ T cell populations such that production of Th2 cytokines was favored and production of Th1 

11 cytokines was suppressed (U.S. EPA, 2006). Recent studies expand information on the potential 

12 mechanisms underlying T cell activation. In cultures of human CD4+ T cells, Pb (1 μM, 30 minutes) has 

13 been shown to activate transcription factor NFκB (regulates T cell activation) (Pyatt et al., 1996) and to 

14 increase, in a dose-dependent manner (10 and 50 μM PbCl2, 24 hours), the expression of MHC class II 

15 surface antigens (HLA-DR), which mediates the CD4+ response to exogenous antigens (Guo et al., 

16 1996b). Heo et al. (2007) provide evidence for the direct effects of Pb on T cells by showing that Pb (25 

17 μM) blocked production of the Th1 cytokine interferon-γ (IFN-γ) in cultures of stimulated mouse T cells 

18 by suppressing translation of the protein. This blockage was rescued with the addition of IL-12. Kasten19 Jolly et al. (2010) found that Pb may not necessarily skew towards a Th2 phenotype via a direct effect on 

20 T cells. In this study, developmental Pb exposure of mice (0.1mM Pb acetate in drinking water of dams 

21 from GD8 to PND21, resulting in pup blood Pb levels 10-30 μg/dL) induced gene expression of IL-4 and 

22 suppressed production of IFN-γ in splenic cells. These changes occurred in the absence of STAT4 or 

23 STAT6, the preferential signaling pathways for T cells and occurred with concomitant increases in 

24 adenylate cyclase 8 and phosphatidylinositol 3-kinase, indicating that Pb may promote Th2 activity via T 

25 cell-independent pathways.

26 Epidemiologic studies provide evidence for blood Pb levels being associated with a shift in 

27 production from Th1 to Th2 cytokines in humans (Section 5.6.5.4); however, the extant evidence for 

28 effects on T cells in humans is derived largely from older studies describing changes in the abundance of 

29 several T cell subtypes, including CD3+, CD4+, and CD8+, that may affect cell-to-cell interactions 

30 required in acquired immunity responses to antigens. Among studies of subjects without occupational Pb 

31 exposures, decreases in the abundance of CD3+ (Figure 5-48 and Table 5-23), CD4+, and CD8+ T cells
May 2011 5-246 DRAFT – DO NOT CITE OR QUOTE

1 often were observed among children with blood Pb levels in the range of 5 to 44 μg/dL (Lutz et al., 1999; 

2 Sarasua et al., 2000; Z. Y. Zhao et al., 2004). However, Karmaus et al. (2005) observed decreases in 

3 several T cell subtypes in children with blood Pb levels 2.2-2.8 μg/dL compared with children with blood 

4 Pb levels <2.2 μg/dL, indicating that decreases in T cell abundance also may occur at blood Pb levels that 

5 are more comparable to those of the current U.S. general population. In association with higher blood Pb 

6 levels (≥ 10 μg/dL), Zhao et al. (2004), Fischbein et al. (1993), and Mishra et al. (2010) observed a 

7 decrease in the ratio of CD4+/CD8+ cells, indicative of a compromised response to viral infections. These 

8 findings are consistent with the documented effects of Pb exposure on diminished host resistance and 

9 associations with bacterial and viral infections (Section 5.6.4.1). Changes in the CD4+/CD8+ ratio have 

10 not been examined in populations with lower blood Pb levels. In a large multicity U.S. study conducted in 

11 subjects living near a Pb smelting operation plus demographically-matched controls, Sarasua et al. (2000)

12 indicated that the associations between blood Pb levels and T cells may vary with age. Investigators

13 analyzed blood Pb level as a continuous variable and found that among children 6-35 months in age, a 1 

14 μg/dL increase in blood Pb level was associated with decreases in the percentage of CD3+ (-0.18 [95% 

15 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; 

16 however, in older children (36-71 months, 6-15 years) and adults (16-75 years), many effect estimates 

17 were positive.

18 Despite the consistency of findings across studies, it is not clear what may be the impact of the 

19 small magnitudes of change in T cell abundance on the cell-to-cell interactions that mediate downstream 

20 acquired immune responses. For example, in populations without occupational Pb exposures, Pb21 associated decreases in the relative abundance of CD3+ cells range between 1 and 9% (Table 5-23). 

22 Larger decreases (20-35%) are observed in studies of occupationally-exposed males with higher blood Pb 

23 levels than those expected in the general population (>25 μg/dL) (Fischbein et al., 1993; Undeger et al., 

24 1996).
May 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 results in subjects

without occupational Pb exposures, and bars in dark or light blue represent results in subjects with 

occupational Pb exposures.

Figure 5-48. Comparisons of the relative abundance of CD3+ T cells among 

groups with increasing blood Pb level (μg/dL).

Table 5-23. Comparison of serum abundance of T-cell subtypesa 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 mo6 yr

Springfield, MO

<10

10-14

15-19

20-44

68.4

69.7

64.6

65.3
May 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

23.7e 20.623.2e 1.411.09e

Adults without Occupational Pb Exposures

Sarasua et 

al. (2000)

c 433 children 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

46.9e

45.6

29.6

21.4e

25.7

23

25.4

1.95

1.38

0.95e

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

858.8e 977.6829.1

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

31e

26

27

26

2.6

1.4

1.3e

44

43

40

61

73

70e

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 Activation

1 Pb exposure produces an expansion of alloreactive T and B lymphocytes. This occurs as a result of 

2 reversing the normal suppression that is mediated by a macrophage-like subpopulation. As discussed in 

3 Section 5.6.5.2, changes in NO production appear to be involved in this process (Farrer et al., 2008). 

4 Additionally, Pb alters antigen processing that occurs in antigen presenting cells (APCs, e.g., primarily 

5 dendritic cells and macrophages), which appear to shift signals to T cells skewing both the nature of the 

6 subsequent response and the spectrum of activities among those expanded populations of lymphocytes 

7 (Farrer et al., 2005). Gao et al. (2007) reported that dendritic cells that matured in the presence of Pb 

8 promoted enhanced alloreactive T cell proliferation compared to control dendritic cells. An additional 

9 effect of Pb has been described relative to activation of T cells. Using the local lymph node assay 

10 (LLNA), Carey et al. (2006) found that PbCl2 was able to provide a costimulatory signal to antigens that 

11 could activate T cells. The exact mechanistic basis for this is not known.
May 2011 5-249 DRAFT – DO NOT CITE OR QUOTE

1 Epidemiologic findings are mostly limited to studies of occupationally-exposed adults and to 

2 associations with blood Pb level >10 μg/dL. In the only study of children in Missouri, the percentage of 

3 activated T cells (as indicated by the cell surface marker HLA-DR) was higher in children with blood Pb 

4 level 15-19 μg/dL than in children with blood Pb level <15 μg/dL; however, activated cells were not 

5 elevated in children with blood Pb levels 20-44 μg/dL (Lutz et al., 1999). In contrast with toxicological 

6 findings, the direction of lymphocyte proliferation (in response to mitogens and/or to specific antigens) in 

7 association with occupational Pb exposure is unclear. Pb promotes the activation of Th2 cells and 

8 suppresses Th1 cells, therefore, the differential activation of specific subtypes may be not discernable in 

9 studies that measure overall lymphocyte proliferation. Whereas some studies have reported similar levels 

10 of lymphocyte proliferation (≤ 1% difference) between Pb-exposed workers and unexposed controls (N. 

11 Cohen et al., 1989; Queiroz, Perlingeiro, et al., 1994), others have reported lower lymphocyte 

12 proliferation among Pb-exposed workers (8-25%) (Alomran & Shleamoon, 1988; Fischbein et al., 1993; 

13 Kimber et al., 1986; Mishra et al., 2003). With the exception of Fischbein et al. (1993), these latter studies 

14 included subjects with high blood Pb levels (>60 μg/dL). Additionally, with much of the evidence limited 

15 to comparisons of mean proliferative responses between Pb-exposed and -unexposed workers, it is 

16 difficult to apply findings to populations with lower blood Pb levels. In one of the few analyses of 

17 correlation between blood Pb levels and lymphocyte proliferation, Mishra et al. (2003) found a lack of 

18 correlation, suggesting the influence of another occupationally-related factor on lymphocyte proliferation. 

5.6.2.3. Delayed-type Hypersensitivity

19 The DTH assay is commonly used as an indicator of the T cell-mediated adaptive immune 

20 response, i.e., induration and erythema resulting from the activation of T cells and recruitment of 

21 monocytes to the site of antigen deposition. The DTH response is largely Th1-dependent in that Th1 

22 cytokines drive the production of antigen-specific T cells directed against the antigen (sensitizing phase) 

23 and the recruitment of antigen-specific T cells and monocytes to the site of antigen deposition (elicitation 

24 phase). In the 2006 Pb AQCD and several recent reviews, a suppressed DTH response was identified as 

25 one of the most consistently observed and well-established immunomodulatory effects of Pb exposure in 

26 animal models (Dietert & McCabe, 2007; Mishra, 2009; U.S. EPA, 2006). A majority of the evidence for 

27 Pb suppressing the DTH response is provided by the historical literature in which effects were observed in 

28 association with both gestational (Bunn, Ladics, et al., 2001; Bunn, Parsons, et al., 2001a, 2001b; S. Chen 

29 et al., 2004; S. Chen et al., 1999; Faith et al., 1979; J.-E. Lee et al., 2001; Miller et al., 1998) and postnatal 

30 (Laschi-Loquerie et al., 1984; M. J. McCabe, Jr. et al., 1999; Muller et al., 1977) Pb exposures of animals. 

31 In some studies, the suppressed DTH response was accompanied by a decreased production of IFN-γ (S. 

32 Chen et al., 1999; J.-E. Lee et al., 2001), which is the primary cytokine that stimulates recruitment of 

33 macrophages, a key component of the DTH response. The concomitant decrease in IFN-γ demonstrated 
May 2011 5-250 DRAFT – DO NOT CITE OR QUOTE

1 further that Pb-induced suppression of the DTH response reflects the inhibition of Th1 functional 

2 activities.

3 One of the most salient findings collectively was that DTH was suppressed in animals with blood 

4 Pb levels ranging from <2 to 5 μg/dL (Bunn, Ladics, et al., 2001; Bunn, Parsons, et al., 2001a, 2001b; 

5 Miller et al., 1998; Muller et al., 1977). In some studies that examined Pb exposures at different stages of 

6 gestation, exposures later in gestation suppressed DTH (Bunn, Parsons, et al., 2001b; J.-E. Lee et al., 

7 2001). These latter findings may reflect the status of thymus and T cell development. A recent study 

8 contributed to the robust evidence by indicating a role for dendritic cells in the Pb-induced suppression of 

9 the DTH response. Gao et al. (2007) exposed bone marrow-derived dendritic cells in vitro to PbCl2 (25 

10 μM, 10 days) then the antigen ovalbumin (OVA) and injected the cells into naïve mice. The Pb-exposed 

11 dendritic cells inhibited the OVA-specific DTH footpad response in mice compared with mice exposed to 

12 control dendritic cells.

13 The capacity of Pb to suppress the DTH response is strongly supported by mechanistic studies in 

14 which Pb suppresses Th1 cytokine production (Section 5.6.5.4). Further, coherence is provided by 

15 associations observed between Pb exposure or blood Pb levels and other responses related to the 

16 inhibition of Th1-driven adaptive immune responses, including increased susceptibility to developing 

17 certain infections and tumors (Section 5.6.4.1). 

5.6.2.4. Macrophages and Monocytes

18 Macrophages and monocytes, the blood form of tissue macrophages, are among the most sensitive 

19 targets of Pb-induced immune effects. The 2006 Pb AQCD emphasized the large number of toxicological 

20 studies showing the effects of Pb on a wide range of alterations in macrophage function to promote a 

21 hyperinflammatory phenotype (U.S. EPA, 2006). These changes include enhanced production of ROS, 

22 suppressed production of NO, enhanced production of TNF-α, excessive metabolism of arachidonic acid 

23 into immunosuppressive metabolites (e.g., prostaglandin E2), impaired phagocytic activity and lysosomal 

24 function, and potentially altered receptor expression [e.g., toll-like receptors]). These studies are 

25 described in detail in Section 5.6.5.2. Because macrophages are major resident populations in most tissues 

26 and organs and are also highly mobile in response to microbial signals and tissue alterations, their 

27 functional impairment in response to Pb exposure may serve as a link between Pb-induced immune 

28 effects and impaired host defense, tissue integrity, and organ homeostasis in numerous physiological 

29 systems and organs (Section 5.6.4.5). Recent studies by Bussolaro et al. (2008), Kasten-Jolly et al. (2010), 

30 and Mishra et al. (2006) using mouse macrophages exposed in vitro to Pb reinforced the capacity of Pb to 

31 induce a broad spectrum of functional alterations in macrophages such as lipopolysaccharide (LPS)-

32 induced production of NO. Although not examined widely, epidemiologic evidence suggests that Pb 

33 exposure may be associated with changes in macrophage function in humans. Pineda-Zavaleta et al. 
May 2011 5-251 DRAFT – DO NOT CITE OR QUOTE

1 (2004) examined children in Lagunera, Mexico, attending schools at varying distances from an active Pb

2 smelter (range of blood Pb levels: 3.5-47.5 μg/dL) and found that blood Pb levels were associated with a 

3 hyperinflammatory state in macrophages as indicated by a decrease in NO production and increase in 

4 superoxide anion production (Section 5.6.5.2). Studies of occupationally-exposed adults mostly observe 

5 that Pb exposure is associated with impaired function of macrophages. Adjusting for age, race, smoking, 

6 and workshift, Pinkerton et al. (1998) found lower abundance of monocytes among Pb smelter workers 

7 (7.8%) than among unexposed controls (8.5%). Fischbein et al. (1993) found lower abundance of HLA8 DR+ cells in firearms instructors with blood Pb level <25 μg/dL (8.8%) and ≥ 25 μg/dL (8.7%) than 

9 among unexposed controls (15.2%). HLA-DR+ is an indicator of activated function state of APCs and is 

10 upregulated in response to cell signaling.

5.6.2.5. Neutrophils

11 Although neutrophils were found not to be a significant direct target of Pb in the 2006 Pb AQCD 

12 (U.S. EPA, 2006), the modulation of their activity by Pb may have important consequences on the 

13 dysregulation of inflammation and ability to response to infectious agents. Studies of cultured human 

14 polymorphonuclear cells (PMNs) (Governa et al., 1987) and occupationally-exposed adults (Bergeret et 

15 al., 1990; Queiroz et al., 1993; Queiroz, Costa, et al., 1994; Valentino et al., 1991) have found reduced 

16 PMN functionality, as indicated by reduced chemotactic response, phagocytic activity, respiratory 

17 oxidative burst activity, or reduced ability to kill ingested antigen, among Pb workers compared with 

18 controls. Important limitations to applying these epidemiologic findings broadly include male-only study 

19 populations, relatively high blood Pb levels of workers (range of mean levels: 33.1-71 μg/dL) and the 

20 lack of direct examination of associations between blood Pb level and neutrophil function.

21 In both studies of animals and occupationally-exposed adults, Pb exposure has been associated 

22 with an increase in neutrophil counts, which has been interpreted as a compensatory response to Pb23 induced impairment in neutrophil chemotactic activity and a hyperinflammatory response. In a study by

24 Kibayashi et al. (2010) to investigate host responses to gunshot wounds in the brain, neutrophils were a 

25 major responding cell. Implantation of Pb spheres (compared with glass spheres in the controls) in male 

26 Wistar rats led to major neutrophil infiltration with inflammatory-related damage that included apoptosis 

27 and indications of neurodegeneration.

28 In a group of 68 ceramic, Pb recycling, or bullet manufacturing workers and 50 controls selected 

29 among food plant workers, DiLorenzo et al. (2006) observed that a 1 μg/dL increase in blood Pb level 

30 was associated with an increase in ANC of 21.8 cells/μL (95% CI: 11.2, 32.4 cells/μL) adjusted for age, 

31 BMI, and smoking status. The geometric mean (range) of blood Pb levels was 20.5 μg/dL (3.2-120) 

32 among workers and 3.5 μg/dL (1-11) among controls. Eight workers with medium to high Pb exposures 

(exact blood Pb levels not reported) had neutrophilia (n >7,500 cells/mm3 33 ) versus no controls, suggesting 
May 2011 5-252 DRAFT – DO NOT CITE OR QUOTE

1 that chronic, higher-level Pb exposures can lead to a biologically meaningful excess of circulating 

2 neutrophils. Additionally, in analyses comparing three blood Pb level groups, controls, workers with 

3 blood Pb levels ≤ 30 μg/dL, and workers with blood Pb levels >30 μg/dL, ANC was observed to increase 

4 monotonically, supporting a blood Pb dose-dependent relationship. When the three blood Pb groups were 

5 further stratified by current smoking, two-way ANOVA indicated positive interaction between blood Pb 

6 level and current smoking. Higher blood Pb level was associated with higher ANC only when current 

7 smokers were compared. Among nonsmokers, ANCs were similar across blood Pb groups.

8 Coherence for the effects of Pb exposure on neutrophils is provided by findings that blood Pb level 

9 is associated with mediators of neutrophil proliferation, survival, maturation, and functional activation. 

10 These mediators include cytokines such as TNF-α (Section 5.6.5.4) and complement. The complement 

11 system is a component of the innate immune system that controls various cell-mediated immune 

12 responses such as chemotaxis of macrophages and neutrophils and phagocytosis of antigens. The effects 

13 of Pb exposure on complement have not been widely examined; however the limited data suggest Pb may 

14 suppress complement activity. Both Ewers et al. (1982) and Undeger et al. (1996) measured lower 

15 complement C3 protein among Pb-exposed workers compared with unexposed controls, with Ewers et al. 

16 (1982) additionally observing an inverse association between blood Pb level and C3 among workers. 

17 However, the implications of these findings are limited due to the high blood Pb levels in these 

18 occupationally-exposed groups (range of blood Pb levels: 18.6-85.2 μg/dL and 38-100 μg/dL, 

19 respectively) and by the lack of adjustment for potential confounding variables.

5.6.2.6. Dendritic Cells

20 Since the 2006 Pb AQCD (U.S. EPA, 2006), new evidence from both an ex vivo and in vitro model

21 suggests that the effects of Pb exposure on suppressing Th1 activity and promoting Th2 activity may be a 

22 consequence of the direct action of Pb on the function of dendritic cells (a major APC). Prior research on 

23 the effects of Pb in favoring Th2 over Th1 activity emphasized the direct measurement of Th1 vs. Th2 T24 cell populations and cytokine profiles. But new research techniques (D. Gao & Lawrence, 2010) have 

25 provided an opportunity to look upstream at how dendritic cells may be involved in mediating the effects 

26 of Pb on acquired immunity. Gao et al. (2007) used bone marrow cultures exposed to Pb to examine the 

27 impact of Pb on dendritic cell maturation and function. They found that Pb (25 μM, 10 days) altered the 

28 course of dendritic cell maturation by changing the ratio of cell surface markers, such as the CD86/CD80 

29 ratio, that promote Th2 cell development. Additionally, upon activation with LPS, Pb-matured dendritic 

30 cells produced less IL-6, TNF-α, and IL-12 (stimulates growth and differentiation of T cells) than control 

31 cells but the same amount of IL-10 (inhibits production of Th1 cytokines). The effect of Pb in altering the 

32 cytokine expression profile of dendritic cells, in particular, the lower IL-12/IL-10 ratio, may serve as an 

33 important signal to shift naïve T cell populations towards a Th2 phenotype. Strengthening the role of 
May 2011 5-253 DRAFT – DO NOT CITE OR QUOTE

1 dendritic cells in mediating Pb immune effects were ex vivo results from the same study that Pb-naïve 

2 Balb/c mice implanted with Pb-treated dendritic cells were skewed towards Th2 activity as indicated by 

3 inhibited DTH (Section 5.6.2.3) and IgG2a antibody (Section 5.6.3) responses (D. Gao et al., 2007). 

5.6.2.7. Natural Killer (NK) Cells

4 The collective body of toxicological and epidemiologic evidence indicates that the innate immune 

5 NK cells are not affected to a large extent by Pb exposure. This conclusion is underscored by 

6 epidemiologic observations that blood Pb levels are associated with T or B cell abudance but are not 

7 associated with NK cell abundance or the level of functional activity (Karmaus et al., 2005; Pinkerton et 

8 al., 1998; Sarasua et al., 2000). Likewise, similar means of NK cell abundance or functional activity have 

9 been observed in Pb-exposed workers and unexposed controls (Fischbein et al., 1993; Kimber et al., 

10 1986; Mishra et al., 2003; Pinkerton et al., 1998; Undeger et al., 1996; Yucesoy et al., 1997b). Consistent 

11 with epidemiologic findings, in a recent in vitro study comparing the toxicity of metals for different 

12 populations of immune cells, Fortier et al. (2008) found that PbCl2 (7.5-20.7 μg/dL), did not significantly 

13 affect NK cytotoxicity compared with the DMSO vehicle; however, PbCl2 did not affect other immune 

14 parameters (e.g., monocyte phagocytic activity or lymphocyte proliferation as well.

5.6.3. Humoral Immunity

15 The 2006 Pb AQCD indicated that Pb exposure was associated with enhanced humoral immune 

16 responses as characterized by the proliferation of B cells and increased production of Ig antibodies (U.S. 

17 EPA, 2006). Both toxicological and epidemiologic studies (Figure 5-49 and Table 5-24) have 

18 demonstrated Pb-associated increases in IgE production, which is strongly implicated in mediating 

19 allergic responses and inflammation in allergic asthma. In animal studies, Pb exposures induced 

20 concomitant increases in IgE and IL-4 production by T cells (S. Chen et al., 1999; J. E. Snyder et al., 

21 2000), consistent with the hypothesis that Th2-mediated mechanisms can induce class switching of B 

22 cells for the production of IgE. Additional coherence and biological plausibility for Pb effects on humoral 

23 immunity have been provided by epidemiologic observations for increases in B cell abundance in 

24 association with increasing blood Pb level or occupational Pb exposure (Figure 5-50 and Table 5-24). 

25 Earlier toxicological and epidemiologic studies also found similar associations of Pb with increases in 

26 other classes of Igs including IgG, IgM, and IgA. 

27 Recent toxicological evidence continues to support the role of T cell-mediated mechanisms in Pb28 induced activation of B cells and production of Ig antibodies. Carey et al. (2006) treated Balb/c mice with 

29 subsensitizing doses of a T cell-independent [Trinitrophenyl-Ficoll (TNP-Ficoll)] or T cell-dependent 

30 [TNP-ovalabumin (TNP-OVA)] hapten-protein conjugate with or without co-exposures to PbCl2. Seven 

31 days later, they examined the effects of PbCl2 on the LLNA response to TNP-Ficoll or TNP-OVA. PbCl2
May 2011 5-254 DRAFT – DO NOT CITE OR QUOTE

1 exposure (25-50 μg, injected) increased the numbers of T and B cells in the lymph node against both 

2 TNP-Ficoll and TNP-OVA. Further, in a dose-dependent manner, PbCl2 induced statistically significant 

3 elevations of IgM, IgG2a, and IgG1 antibody producing cells in the lymph node. While the increase in 

4 IgM-producing cells against TNP-Ficoll indicated a T-cell independent mechanism, the increases in 

5 IgG2a- and IgG1- producing cells against both antigens indicated a Th1- and Th2-mediated mechanism, 

6 respectively. Despite seeing increases in both IgG1- and IgG2a-producing cells, the authors concluded 

7 that Pb skewed the response toward Th2 and had considerable potential for promoting allergic 

8 sensitization against T-dependent antigens. 

9 Other animal studies provided strong support for Pb exposure stimulating humoral responses 

10 preferentially via Th2-mediated mechanisms. Gao et al. (2006) found that Pb exposure (50 μg, i.p., 

11 3 times/week, 3 weeks) of Balb/c mice elevated IgG1 over that of Th1-driven IgG2a. Similar results were 

12 reported when Pb-exposed dendritic cells were used to initiate antibody production (D. Gao & Lawrence, 

13 2010). In a highly-specialized strain of IFN-γ knockout mice (lacking the capacity to produce IFN-γ), Pb 

14 exposure had the reverse effect of increasing the IgG2a/IgG1 ratio. These results were surprising given 

15 evidence that IFN-γ usually directs secretion of IgG2a; however, the authors suggested that in these 

16 knockout-mice, Pb may initiate a Th1 response via an IFN-γ independent pathway to enhance IgG2a 

17 production (D. Gao et al., 2006). In a microarray study examining the DIT of Pb in Balb/c mice, Kasten18 Jolly et al. (2010) found that early-life Pb exposure (0.1mM Pb acetate in drinking water of dams from 

19 GD8 to PND21, resulting in pup blood Pb levels 10-30 μg/dL) produced statistically significant increases 

20 in the expression of genes encoding Ig antibodies or those involved in B lymphocyte function and 

21 activation. These genes included those for the heavy chain of IgM, IL-4, IL-7 and IL-7 receptor, IL-21, 

22 RAG-2, CD antigen 27, B-cell leukemia/lymphoma 6, RNA binding motif protein 24, Histocompatibility 

23 class II antigen A (beta 1), Notch gene homolog 2, and histone deacetylase 7A.

24 In epidemiologic studies, associations between increasing blood Pb level and increasing serum IgE 

25 level are demonstrated primarily in children (Annesi-Maesano et al., 2003; Karmaus et al., 2005; Lutz et 

26 al., 1999; L. Sun et al., 2003). Most studies indicate that the association between blood Pb level and IgE is 

27 nonmonotonic (Figure 5-49 and Table 5-24). Whereas many studies were focused on examining 

28 differences between children with blood Pb levels below and above 10 μg/dL (Figure 5-49 and Table 5-

29 24), Karmaus et al. (2005) demonstrated 28% increases in IgE among children with blood Pb levels 2.84-

30 3.41 and >3.4 μg/dL compared with children with blood Pb level <2.2 μg/dL. Another strength of this 

31 study was the adjustment for potential confounding by various organochlorine compounds, age, number 

32 of infections in the previous 12 months, and serum lipids. A clear blood Pb dose-dependent relationship 

33 was not observed as the mean IgE was lower among children in the second quartile of blood Pb levels 

34 (2.2-2.8 μg/dL) than among children in the lowest quartile (<2.2 μg/dL). Annesi-Maesano et al. (2003)

35 provided additional information on potential critical developmental windows of Pb exposure. Cord blood 

36 IgE was associated with infant hair Pb level but not with cord or placental Pb level. From these findings, 
May 2011 5-255 DRAFT – DO NOT CITE OR QUOTE

1 the authors inferred a stronger effect of Pb exposure integrated over the entire gestational period rather 

2 than exposures closer to birth. Additionally, the magnitude of association was larger in the subgroup with 

3 nonallergic mothers, pointing to the possible stronger effect of family history of allergy on IgE levels at 

4 birth compared with blood Pb level. Sarasua et al. (2000) was unique among studies of children in 

5 examining associations of blood Pb level with IgA, IgG, and IgM. Associations were consistently positive 

6 (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, 

7 and IgM, respectively, per 1 μg/dL increase in blood Pb level, adjusted for age, sex, and location) and 

8 tended to be larger in magnitude in the youngest age group (6-35 months), suggesting an increased 

9 susceptibility of exposure at younger ages. In this study, associations with IgE were not examined.

10 In studies of adults, one without (Pizent et al., 2008) and one with occupational Pb exposure (Heo 

11 et al., 2004), investigators reported higher IgE levels in association with higher blood Pb levels. In a study 

12 of urban adults in Zagreb, Croatia with blood Pb levels between 0.56 and 7.4 μg/dL, a statistically 

13 significant, positive association between blood Pb level and IgE was found in women but not men (Pizent 

14 et al., 2008). Several covariates were considered in a stepwise multiple regression, including age, 

15 smoking intensity, and alcohol consumption. Among women not on hormone replacement therapy or oral 

16 contraceptives, a 1 μg/dL increase in blood Pb level was associated with a 0.60 increase in log IgE (95% 

17 CI: 0.58, 1.18). Investigators did not report an effect estimate in men because it did not attain statistical 

18 significance. The study included 166 women and 50 men, thus, it was difficult to ascertain whether there 

19 was suggestion of association in men but lack of power to indicate statistical significance. Although blood 

20 Pb levels were lower in women (mean: 2.16 μg/dL, range 0.56-7.35 μg/dL) than in men (mean: 3.17 

21 μg/dL, range 0.99-7.23), both groups had levels similar to those reported in studies of children. Among 

22 battery manufacturing workers (mostly males), Heo et al. (2004) found a monotonic increase in IgE levels 

23 among workers with blood Pb levels <0, 10-29, and ≥ 30 μg/dL.

24 A majority of the epidemiologic evidence for the effects of Pb on humoral immunity comprises 

25 comparisons of serum IgA, IgG, and IgM levels between Pb-exposed and -unexposed workers (Alomran 

26 & Shleamoon, 1988; Anetor & Adeniyi, 1998; Ewers et al., 1982; Kimber et al., 1986; Pinkerton et al., 

27 1998; Queiroz, Perlingeiro, et al., 1994; Sarasua et al., 2000; Undeger et al., 1996). In contrast with 

28 toxicological findings, epidemiologic evidence reflects more mixed associations, with studies reporting 

29 higher, lower, and similar Ig levels in Pb-exposed workers compared with -unexposed controls. Among 

30 studies reporting lower Ig levels in Pb-exposed workers were a few with high mean blood Pb levels 

31 among exposed workers (56.3 and 74.8 μg/dL) (Anetor & Adeniyi, 1998; Undeger et al., 1996).
May 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 (μg/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. (1996) 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 increasing blood Pb level (μg/dL).
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Table 5-24. Additional characteristics and quantitative dataa 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

59c

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

63.7d

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 

666f

680f

630

103

108

105

124f

50.1 

55.0

58.2

61.4f

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 

1094f

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

262f

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

535.8g

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

11.2g

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

854.6g

140.4

93.3g

210.3

168.1

635.9i

545.5

a

IgE data are presented as IU/mL. 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 organochlorine exposures. B cell data represent the 

number of cells/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 lowest 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.
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5.6.4. Immune-based Diseases

5.6.4.1. Host Resistance

1 The capacity of Pb to reduce host resistance to bacteria has been known for almost 40 years and 

2 was supported by several toxicological studies described in the 2006 Pb AQCD (U.S. EPA, 2006). 

3 Biological plausibility has been provided by observations of Pb affecting mechanisms underlying 

4 diminished host resistance, e.g., decreased capacity for Th1-driven acquired immune antiviral responses 

5 (indicated by the overproduction of PGE2) and increased inflammatory responses in target tissue resulting 

6 in further damage to host protective barriers. Gupta et al. (2002) demonstrated that elevated Pb exposure 

7 of mice (>125 mg/kg Pb, 13.0 μg/dL blood Pb) reduced host resistance to viral infections as indicated by 

8 an increased viral titre and increased mortality. Host resistance to bacteria such as Listeria requires 

9 effective Th1-driven responses including the production of IL-12 and IFN-γ (Lara-Tejero & Pamer, 2004),

10 but these are suppressed by Pb (D. Gao et al., 2007). The lack of IFN-γ can inhibit appropriate and timely 

11 macrophage activation. However, beyond this, suppression of NO production and along with it, the 

12 microbicidal metabolite peroxynitrite, can compromise host resistance to some bacteria (U.S. EPA, 2006). 

13 Recently, mechanisms through which Pb impacts both innate immune cells and natural host defense 

14 barriers to increase the likelihood of serious bacterial infections have been delineated further. Kasten-Jolly 

15 et al. (2010) showed that developmental exposure of mice to Pb resulted in an upregulation of splenic 

16 RNA of caspase-12, a cysteine protease that inhibits the clearance of bacteria both systemically and in the 

17 gut mucosa (Saleh et al., 2006). 

18 With limited investigation, the effect of Pb on host resistance to parasitic agents is uncertain. The 

19 2006 Pb ACQD described one study in which Pb-exposed (30-100 mM) mouse macrophages had 

20 diminished ability to kill Leishmania enrietti parasites (Mauel et al., 1989); however, given the well21 established effect of Pb in promoting Th2 activity, it is plausible that Pb could enhance host resistance to 

22 parasites that require robust Th2 responses (e.g., helminths) (U.S. EPA, 2006). In a recent study, Pb 

23 enhanced host resistance to malaria (Koka et al., 2007). However, this was related to the capacity of Pb to 

24 induce eryptosis and the rapid removal of malaria-infected erythrocytes and not to Pb-induced alterations 

25 in immune function.

26 The collective body of epidemiologic data is sparse; however, several studies have found an 

27 association between blood Pb levels and respiratory infections. In a Boston-area study of 1978 children 

28 (ages 4-8 years), Rabinowitz et al. (1990) reported that compared with children with blood Pb levels <10 

29 μg/dL, children with blood Pb levels ≥ 10 μg/dL were more likely to have parental report “other 

30 respiratory infections” (not defined by authors) (OR: 1.5 [95% CI: 1.0, 2.3]), severe ear infections (OR: 

31 1.2 [95% CI: 1.0, 1.4]), and illnesses other than cold or influenza (OR: 1.3 [95% CI: 1.0, 1.5]) (Figure 5-

32 51 and Table 5-25). Analyses did not adjust for any potential confounders. Likewise, without considering 
May 2011 5-259 DRAFT – DO NOT CITE OR QUOTE

1 confounders, Karmaus et al. (2005) found that children with more than 10 infections in the previous 12 

2 months had higher blood Pb levels (mean: 3.3 μg/dL) compared with children with 1 to <5 infections 

3 (mean: 2.8 μg/dL) or 5-10 infections (mean: 2.6 μg/dL) in the same time period. A unique study was 

4 conducted in children in Cordoba, Argentina, in which Pb exposure was assessed by measuring Pb in total 

5 deposition samples and in lichen biomonitors from sites near 4 city health clinics (Carreras et al., 2009). 

6 Lichen has been recognized for the uptake, accumulation, and sequestration of environmental chemicals 

7 from the air. Pb content in lichen and colocated total deposition samples were highly correlated, 

8 indicating that the lichen was a suitable indicator of environmental concentrations. In an ecologic analysis 

9 that did not consider potential confounders, clinics near sites with higher levels of Pb in total deposition 

10 and lichen samples had higher frequency of visits by children for pharyngitis, tonsilitis, and laryngitis. 

11 Because other metals, including manganese, iron, copper, and nickel were also associated with respiratory 

12 illnesses, it was difficult to characterize the independent effects of Pb. Similar to studies in children, 

13 among adults, frequency of self-reported colds or influenza was greater among Pb battery or smelter plant 

14 workers (28.8%) than among unexposed controls (16.1%); however, a statistical analysis was not 

15 performed on the data (Ewers et al., 1982). Thus, while several studies in humans indicate associations 

16 between indicators of Pb exposure and infectious illnesses, they are limited by weak analytic methods and 

17 lack of consideration for potential confounders. Evaluation of the relationship between Pb exposure and 

18 host resistance in humans would be improved by more longitudinal investigation and rather than group 

19 comparisons, analyses of associations between blood Pb levels and viral or bacterial infections.

5.6.4.2. Asthma and Allergy

20 Toxicological studies and to varying degrees, epidemiologic studies, have demonstrated Pb effects 

21 on multiple immunological pathways, including elevated production of Th2 cytokines such as IL-4, 

22 increased IgE antibody production (Figure 5-49), and Pb-induced inflammatory cell dysfunction. These 

23 are well-recognized pathways in the development of allergy and allergic disease, including asthma. It has 

24 been suggested that low exposure to Pb exerts immunostimulating effects in contrast to higher exposure, 

25 which has been implicated in suppressing immune function (Mishra et al., 2006). Coherent with the 

26 mechanistic evidence, several epidemiologic studies indicate associations between blood Pb levels and 

27 asthma and allergy (Figure 5-51 and Table 5-25). In univariate analyses of children near Boston, MA, 

28 Rabinowitz et al. (1990) reported a positive association between high blood Pb level (>10 μg/dL) and 

29 asthma (RR: 1.3 [95% CI: 0.8, 2.0]) but not eczema (RR: 1.0 [95% CI: 0.6, 1.6]). 

30
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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.
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Table 5-25. Additional characteristics and quantitative data for results presented in 

Figure 5-51.

Reference Population Blood Pb level mean or group 

(μg/dL) Outcome

Odds 

ratio 

(95% CI)a

Rabinowitz et al. 

(1990)

Boston area, MA

1768 children

≥ 10 vs. <10b 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. <10b 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. <10b 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 <5c

African American ≥5 vs. African American 

<5d

African American ≥10 vs. African 

American <5d

African American <5 vs. Caucasian <5c

African American ≥5 vs. Caucasian <5c

African American ≥10 vs. Caucasian <5c

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 blood 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 with blood Pb level <5 μg/dL as the reference group. Model covariates include sex, birth weight, and 

annual income.

1 Positive associations between blood Pb level and asthma- and allergy-related conditions also were 

2 observed in large studies with multivariate analyses. An additional common strength of these studies was 

3 the prospective follow-up of subjects that allowed investigators to establish temporality between the 

4 measurement of blood Pb level and onset of disease. In a study of 4,634 children (ages 1-3 years) in 

5 southeastern Michigan that controlled for annual income, birth weight, and sex, an elevated risk of 

6 incident asthma requiring a doctor visit or medication (indicator of severe asthma) was reported in 

7 association with blood Pb levels ≥ 5 μg/dL most strongly among Caucasian children (relative risk [RR]: 

8 2.7 [95% CI: 0.9, 8.1]) (Joseph et al., 2005) (Figure 5-51 and Table 5-25). In analyses restricted to African 

9 American children, blood Pb level was weakly associated with asthma requiring medical care (Figure 5-

10 51 and Table 5-25). In analyses that used Caucasian children with blood Pb level <5 μg/dL as the 

11 reference group, blood Pb level was associated with statistically significant increases in the risk of asthma 

12 among African children in all blood Pb level categories, which indicated a racial/ethnic effect rather than 

13 a Pb effect. However, among African American children, the RR was much higher in the ≥ 10 μg/dL 

14 blood Pb category (RR: 3.0 [95% CI: 1.2, 7.1]) than in the ≥ 5 μg/dL (RR: 1.5 [95% CI: 1.2, 1.8]) or <5 
May 2011 5-262 DRAFT – DO NOT CITE OR QUOTE

1 μg/dL blood Pb categories (RR: 1.8 [95% CI: 1.3, 2.4]), which pointed to a possible race/ethnicity by 

2 blood Pb level interaction. These findings should be interpreted with caution due to the small number of 

3 asthmatics requiring medical care in the high blood Pb level categories (5 Caucasian children with blood 

4 Pb ≥ 5 μg/dL and 9 African American children with blood Pb level ≥ 10 μg/dL).

5 While the aforementioned studies examined concurrent blood Pb levels, findings from a recent 

6 prospective birth cohort study demonstrated that prenatal blood Pb levels (cord blood Pb level mean: 1.16 

7 μg/dL [95% CI: 0.12, 1.22]) was associated with an increased risk of allergic sensitization at age 5 years 

8 (Jedrychowski et al., 2011). In models that adjusted for sex, parity, maternal age, maternal education, 

9 maternal atopy, and environmental tobacco smoke exposure, measures of prenatal Pb exposure (cord or 

10 maternal blood Pb) were associated with greater risk of positive skin prick test (SPT) to dust mite, dog, or 

11 cat allergen compared with concurrent blood Pb levels (Figure 5-51 and Table 5-25). The greater risk 

12 associated with prenatal blood Pb measures was substantiated by the weak correlation observed between 

13 umbilical cord and age 5-year blood Pb levels (r = 0.29). Larger risks were estimated for Pb than for other 

14 risk factors, including blood levels of mercury, polycyclic aromatic hydrocarbon DNA adducts, and 

15 residential levels of dust mite or pet allergen.

16 Interestingly, in a study of 216 adults without occupational Pb exposures, Pizent et al. (2008) found 

17 that among women, the association between blood Pb level and total IgE was statistically significant, 

18 whereas the association with positive SPT to common inhaled allergens was not. An increase in IgE 

19 mediates the acute inflammatory response to allergens. Among men, not only was the opposite observed, 

20 but the association between blood Pb level and positive SPT was negative (OR: 0.92 [95% CI: 0.86, 0.98] 

21 in stepwise regression models that considered age, smoking intensity, and alcohol consumption). 

22 Interpretation of the findings is difficult because only statistically significant effect estimates were 

23 reported; thus it is not possible to ascertain whether there were suggestions of association in women or 

24 whether there were discrepant findings for the related outcomes of IgE and positive SPT.

5.6.4.3. Other Respiratory Effects

25 Increased bronchial responsiveness (BR) is a characteristic feature of asthma and other respiratory 

26 diseases and can result from activation of innate immune responses and increased airway inflammation. 

27 Compared with findings for IgE or asthma, evidence of association between blood Pb levels and BR is 

28 weak (J. Y. Min et al., 2008; Pizent et al., 2008). In a study of 525 middle-aged adults in Seoul, Korea, 

29 Min et al. (2008) found an association between blood Pb levels and BR. Study subjects had a mean (SD) 

30 blood Pb level of 2.90 (1.59) μg/dL. A 1 μg/dL increase in blood Pb level was associated with an increase 

31 in BR index (log [% decline in forced expiratory volume in 1 second (FEV1)/log of final methacholine 

32 concentration in mg/dL]) of 0.018 (95% CI: 0.004, 0.03), adjusting for age, sex, height, smoking, lung 

33 function, and asthma diagnosis (J. Y. Min et al., 2008). In contrast to Min et al. (2008), Pizent et al. (2008)
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1 observed a negative association between blood Pb level and nonspecific BR in men (2.4% decrease [95% 

2 CI: -4.2, -0.52%] in percent change FEV1 post-histamine challenge per 1 μg/dL increase in blood Pb 

3 level). Although this counterintuitive finding was not discussed by investigators, it was consistent with 

4 the negative association found between blood Pb level and SPT among men in this study.

5 In less rigorous analyses that compared occupational groups with relatively low Pb exposures, 

6 associations of blood Pb levels with lung function were less clearly indicated (A. Y. M. Jones et al., 2008; 

7 A. Y. M. Jones et al., 2006). In a comparison of male drivers of buses with (mean blood Pb level: 5.0 

8 μg/dL) and without (mean blood Pb level: 3.7 μg/dL) air conditioning in Hong Kong, China, drivers of 

9 non-air conditioned buses had lower exposures to PM10, lower blood Pb levels but lower forced vital 

10 capacity and similar FEV1 (A. Y. M. Jones et al., 2006). In this study, the authors attributed the slightly 

11 higher blood Pb levels among air conditioned bus drivers to the poor efficiency in the filters and higher 

12 PM10 levels measured on those buses versus the non-air conditioned buses. In a comparison of roadside 

13 vendors and adjacent shopkeepers, blood Pb levels and various lung function parameters were similar 

14 between groups (A. Y. M. Jones et al., 2008). Neither study directly examined associations between blood 

15 Pb levels and lung function.

16 In addition to blood Pb, several recent epidemiologic studies have used Pb measured in PM10 and 

17 PM2.5 air samples to represent Pb exposures. Some studies have analyzed the Pb component individually, 

18 whereas others have applied source apportionment techniques to analyze Pb as part of a group of 

19 correlated components. A majority of air-Pb studies has found associations with asthma-related morbidity 

20 in children and respiratory-related hospitalizations and mortality in older adults (Table 5-26). Despite the 

21 concordance between the findings of air-Pb and blood Pb studies, a common limitation of air-Pb studies is 

22 the variable size distribution of Pb-bearing PM (Section 3.5.3) and its relationship with blood Pb levels. 

23 Additionally, in these air-Pb studies, other PM components such as elemental carbon, copper, and zinc 

24 also were associated with respiratory effects. In the absence of detailed data on correlations among 

25 components or results adjusted for copollutants, it is difficult to exclude confounding by these other 

26 components.
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Table 5-26. Associations of air-Pb with respiratory effects

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-PM2.5

Mean: 5.1 ng/m3 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/m3

increaseb

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 MarchMay 2004

Lag 1 Pb-PM10

Mean (SD): 51 ng/m3

(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 

increaseb

-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-PM2.5

Mean (IQR): 4 (4) 

ng/m3

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/m3 increase

1.011 (0.99, 1.033)

Statistically significant, 

quantitative results not 

reported

Bell et al. (2009) 106 U.S. counties

Adults, ages ≥ 65 yr

1999-2005

Lag 0 Pb-PM2.5

Mean (SD): 4.89 ng/m3

(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” PM10 

factorc 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 models)

Respiratory hospital 

admissions (adults 

ages ≥ 65 yr)

Asthma (children 

ages 5-18 yr) 

RR per 0.6 μg/m3

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” PM2.5 

factorc comprising Pb 

(minor contribution), 

potassium, organic 

carbon, ammonium

Mean: 1.6 mg/m3 (cool 

season), 0.8 mg/m3

(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 25th

and 75th 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 studies. b

Investigators did not provide sufficient information to calculate 95% CIs. c

Souce apportionment techniques were applied to group correlated components into common source categories.

5.6.4.4. Autoimmunity

1 The 2006 AQCD described animal studies in which Pb exposure induced the generation of 

2 autoantibodies (Bunn et al., 2000; El-Fawal et al., 1999; Hudson et al., 2003; Waterman et al., 1994). 

3 Whereas some evidence linked this risk of autoimmunity to a shift towards Th2 responses, other evidence 

4 pointed to a shift towards Th1 responses. While recent studies did not examine Pb-induced production of 

5 autoantibodies, some provided indirect evidence by indicating that the changes induced by Pb had broader 

6 implications for increasing risk of autoimmunity. For example, Kasten-Jolly et al. (2010) examined the 
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1 impact of developmental Pb exposure of mice on changes in gene expression in the spleen. They found 

2 that Pb upregulated digestive and catabolizing enzymes that could lead to the generation of self-peptides, 

3 which in conjunction with other Pb-induced immunomodulatory effects, had the potential to induce the 

4 generation of autoantibodies. In Carey et al. (2006), the activation of neoantigen-specific T cells in PbCl2-

5 exposed mice also indicated the potential for autoantibody generation. Evidence of Pb-associated 

6 autoimmune responses in humans is limited to a study of male Pb battery workers with blood Pb levels 

7 ranging from 10 to 40 μg/dL (El-Fawal et al., 1999). In this study, the Pb-exposed workers had higher 

8 levels of IgM and IgG autoantibodies to neural proteins compared with unexposed controls (blood Pb 

9 levels not reported) (El-Fawal et al., 1999).

5.6.4.5. Specialized Cells in Other Tissues

10 Resident macrophages in tissues represent a significant target for Pb-induced immune effects, and 

11 alteration in the function of these cells can contribute to organ/tissue dysfunction, cell death, tissue 

12 pathology and tissue-specific autoimmune reactions. Among the specialized populations are microglia and 

13 astrocytes in the brain, Kupffer cells in the liver, alveolar macrophages in the lung, keratinocytes and 

14 Langerhans cells in the skin, osteoclasts in the bone, and preadipocytes in adipose tissue. The effects of 

15 Pb on these cells are important as they may contribute to disease in nonlymphoid tissues (Figure 5-52). 

16 Because these specialized cells are not always recognized as macrophages, the resulting diseases and 

17 conditions are not always recognized as being linked with Pb-induced immune dysfunction.
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Figure 5-52. Specialized macrophages in nonlymphoid tissue may serve as 

a significant link between Pb and disease in multiple organ 

systems.

1 Fan et al. (2009) reported that Kupffer cells undergo significant changes in phenotypic expression 

2 (e.g., CD68 and ferritin light chain), organization, and functional activity connected to Pb-induced 

3 apoptosis in the liver. In the central nervous system, subacute exposure of Wistar rats to Pb (15 mg/kg of 

4 Pb acetate, i.p.) during early postnatal maturation was observed to produce chronic glial activation with 

5 coexisting features of inflammation and neurodegeneration (Struzynska et al., 2007). Among the 

6 cytokines detected in the brains of these Pb-treated rats were IL-1β, TNF-α and IL-6. In bone, resident 

7 tissue macrophages regulate osteoblast function (M. K. Chang et al., 2008), and osteoblasts are known 

8 targets of Pb. This can contribute to later life diseases such as arthritis [reviewed in Zoeger et al. (2006)]. 

9 Pb-induced elevation of TGF-β production is also involved in chondrogenesis in bone. 

10 Resident immune cells in reproductive organs are also potential targets of Pb. Pace et al. (2005)

11 reported that Pb exposure in mice contributed to poor reproductive performance that was concomitant 

12 with altered homeostasis of the testicular macrophage population in that organ. The authors proposed that 

13 increased oxidative damage and apoptosis among these macrophages and reduced potential to maintain 

14 organ homeostasis contributed to the observed pattern of male sterility.

Pb

Osteoblastsand 

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

macrophages
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5.6.4.6. Tumors

1 While toxicological evidence supports high doses of Pb directly promoting tumor formation or 

2 inducing mutagenesis and genotoxicity (Section 5.10), evidence for involvement of the immune system is 

3 limited. Kerkvliet and Baecher-Steppan (1982) observed that male C57Bl/6 mice exposed to 130 and 

4 1300 ppm of Pb acetate in drinking water had enhanced moloney sarcoma virus-induced tumor growth 

5 compared with control animals. These findings indicated that Pb-induced immunomodulation affecting 

6 tumors likely results from a combination of suppressed Th1 responses and increased inflammation 

7 leading to excessive release of ROS into tissues. The promotion of cancer is a relatively common 

8 outcome in chemical-induced immuntoxicology, particularly when early life exposures are involved 

9 (Dietert).

5.6.5. Mechanisms of Lead-Induced Immunomodulation

5.6.5.1. Inflammation

10 Misregulated inflammation represents one of the major immune-related effects of Pb and a major 

11 mode of action for Pb effects in multiple organ systems (Section 5.2.5). It is important to note that 

12 inflammation can manifest in any tissue where immune cell mobilization and tissue insult occurs (as with 

13 an infection). Enhanced inflammation and tissue damage occurs through the modulation of inflammatory

14 cell function and production of proinflammatory cytokines and metabolites. Among the problems 

15 presented by this immunomodulation are the overproduction of ROS and an apparent depletion of 

16 antioxidant protective enzymes and factors (e.g., selenium). Chetty et al. (2005) reported that Pb-induced 

17 inflammatory damage involves the depletion of glutathione. While several processes have been proposed 

18 to explain the mechanisms of Pb-induced oxidative damage, the exact combination of processes involved 

19 remains to be determined (Section 5.2.4). 

20 Traditional immune-mediated inflammation can be seen with asthma, respiratory infections, and 

21 BR in association with Pb exposure. Nonetheless, inflammation also is a general response to tissue injury 

22 whether mediated by infection, toxic insult, or other stresses. Thus, Pb-induced misregulation of 

23 inflammation could exacerbate disease and damage in almost any organ given the distribution of immune 

24 cells as both permanent residents and infiltrating cell populations. As described in Section 5.2.5, 

25 misregulation of inflammation represents a potential mode of action for Pb induced effects on the liver, 

26 kidney, and vasculature. 

27 In epidemiologic studies, whereas Pb-associated changes in proinflammatory cell function (Section 

28 5.6.2) and cytokine production (Section 5.6.5.4) have been demonstrated, less certain are the effects of Pb 

29 exposure on nonspecific indicators of inflammation that may be related to multisystemic effects as have 

30 been demonstrated in toxicological and in vitro studies. Using 1999-2004 NHANES data for adults 40 
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1 years of age or older, Songdej et al. (2010) examined the relationship between blood Pb levels (mean: 

2 1.89 μg/dL) and the inflammation markers, C-reactive protein (CRP), fibrinogen, and white blood cell 

3 (WBC) count. Adjusting for age, sex, race/ethnicity, education, income, body mass index (BMI), physical 

4 activity, smoking status, diabetes status, inflammatory disease status, and cardiovascular disease status, 

5 the researchers found that men appeared to be more susceptible than women to Pb-associated

6 inflammation. Among women, most odds ratios for associations between quintiles of blood Pb level and 

7 tertiles of CRP, fibrinogen, and WBC count were less than 1.0 whereas corresponding odds ratios in men 

8 tended to be greater than 1.0. Additionally, compared with men with blood Pb levels less than 1.16 μg/dL, 

9 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 

10 significant increases in CRP (ORs: 2.22, 2.12, 2.85, respectively). For all inflammation markers, although 

11 the highest quintile of blood Pb level was associated with the largest odds ratio, a blood Pb dose12 dependent association was not observed. Consistent with these findings, among men in Incheon, Korea 

13 without occupational Pb exposures, Kim et al. (2007) reported positive associations of blood Pb level 

14 with WBC as well as with IL-6. These findings of associations between blood Pb levels and inflammatory 

15 mediators are consistent with Pb effects on promoting a Th2 phenotype. Th2 cells produce IL-6 which is 

16 the primary stimulus for expression of CRP and fibrinogen (Fuller & Zhang, 2001; Hage & Szalai, 2007).

17 In a genome-wide association study that included 37 autistic and 15 nonautistic children (ages 2-5 

18 years; blood Pb level range: 0.37 to 5.2 μg/dL) in California, in models that included age, sex, and autism 

19 diagnosis, blood Pb level was associated with the expression of several genes related to immune function 

20 and inflammation, including human leukocyte antigen genes (HLA-DRB) and MHC Class II-associated 

21 invariant chain CD74 (involved in antigen presentation) (Y. Tian et al., 2011). Although blood Pb levels 

22 were similar between autistic and nonautistic children and correlations were observed in both groups, they 

23 were in opposite directions (positive among autistic and negative among nonautistic). These results are 

24 consistent with findings that Pb increases MHC molecule surface expression in mouse and human HLA 

25 antigen presenting cells (Guo et al., 1996a; M. J. McCabe & Lawrence, 1991) and also suggest that Pb26 associated changes in the expression of immune genes may be modified by underlying autism.

5.6.5.2. Increased Prostaglandin E2 and Decreased Nitric Oxide 

27 Consistent with the findings presented in the 2006 Pb AQCD (U.S. EPA, 2006), recent studies 

28 continue to show that Pb exposure alters the levels of signaling molecules. These signaling molecules are 

29 involved in mediating inflammation and host resistance (Figure 5-47). For example, Pb exposure 

30 increases arachidonic acid metabolism, elevating the production of prostaglandin E2 (PGE2) (Chetty et al., 

31 2005). Additionally, production of nitric oxide (NO) by macrophages is decreased at low-moderate 

32 exposure levels (Farrer et al., 2008; Pineda-Zavaleta et al., 2004). Decreases in NO can impact not only 

33 innate host defenses, but also, acquired immunity. This is proposed to occur via the Pb-induced release of 
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1 myeloid cell (CD11b+)-mediated suppression of CD4+ T cell proliferation (Farrer et al., 2008). The result 

2 is that Pb exposure leads to an increased production of alloreactive T and B cells. Farrer et al. (2008)

3 found that Pb decreases inducible nitric oxide synthease function in myeloid cells without decreasing its 

4 abundance. The resulting loss of NO production also leads to a reduction in the production of the 

5 microbicidal metabolite peroxynitrite, thus weakening host defenses against bacteria. When this is 

6 combined with the observation that Pb can alter antigen processing (Farrer et al., 2005) and, hence, the 

7 quality and magnitude of the acquired immune response signal against pathogenic challenge, multiple 

8 arms of the host defense against infectious challenge can be compromised. The loss of NO production in 

9 innate immune cells such as macrophages would be expected to affect other physiological systems (e.g., 

10 neurological, cardiovascular, endocrine) that require NO signaling cascades. 

11 Relative to studies in animal and in vitro models, fewer epidemiologic studies have examined the 

12 effects of Pb on signaling molecules; however the limited data support associations of blood Pb level with 

13 suppressed NO production (Barbosa et al., 2006; Mishra et al., 2006; Pineda-Zavaleta et al., 2004; 

14 Valentino et al., 2007) and increased ROS production (Pineda-Zavaleta et al., 2004). Among children, in 

15 Pineda-Zavaleta et al. (2004), with increasing residential proximity to the Pb smelter, mean blood Pb 

16 levels increased (7.02 to 20.6 to 30.38 μg/dL) as did superoxide anion release from macrophages (directly 

17 activated by IFN-γ/LPS) isolated from children. NO release from macrophages (indirectly activated by 

18 phytohemagglutinin, PHA) decreased with increasing blood Pb level. After adjusting for age and sex, a 1 

19 μg/dL increase in blood Pb level was associated with a decrease in NO of 0.00089 (95% CI: -0.0017, -

20 0.00005) nmol/μg protein and an increase in superoxide anion of 0.00389 (95% CI: 0.00031, 0.00748) 

21 μmol/mg protein. Because PHA activates macrophages indirectly through the activation of lymphocytes 

22 and IFN-γ directly activates macrophages, these results suggest that Pb suppressed T cell-mediated 

23 macrophage activation and stimulated cytokine-induced macrophage activation. Results also 

24 demonstrated a larger magnitude of association between blood Pb levels and superoxide anion release in 

25 males. Although not described in detail, the association between blood Pb level and NO was reported to 

26 be not negative in girls. Barbosa et al. (2006) also observed negative associations between blood Pb level 

27 and plasma NO (quantitative results not provided) in a group of adults in Sao Paolo, Brazil residing near a 

28 battery plant, although it was not possible to identify immune cells as the specific sources of NO. 

29 In contrast to studies of populations without occupational Pb exposures, studies of occupationally30 exposed groups provided less clear indication of association of blood Pb level with NO and ROS. Among 

31 30 male Pb recycling plant workers and 27 unexposed controls, despite large differences in blood Pb 

32 levels, levels of ROS released from neutrophils (indicators of respiratory burst) were similar between 

33 workers and controls (Mishra et al., 2006). NO production of neutrophils after stimulation with zymosan34 A was lower in controls, but the difference was not statistically significant. In a study of male foundry 

35 workers (mean blood Pb level: 21.7 μg/dL), pottery workers (mean blood Pb level: 9.7 μg/dL), and 

36 unexposed workers (mean blood Pb level: 3.9 μg/dL), Valentino et al. (2007) also found lower plasma NO 
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1 levels in controls compared with Pb-exposed workers. The means (ranges) of plasma NO levels in 

2 controls, pottery workers, and foundry workers were 23.73 μM (11.21 to 55.71 μM), 28.44 μM (15.23 to 

3 57.65 μM), and 25.30 μM (15.03 to 61.98 μM), respectively. Further, although quantitative results were 

4 not reported, blood Pb level was reported to be not correlated with NO.

5.6.5.3. Cellular Death (Apoptosis, Necrosis)

5 In a study in mice, Bishayi and Sengupta (2006) found that Pb exposure elevated DNA 

6 fragmentation in splenic macrophages. Using mouse resident peritoneal macrophages, Gargioni et al. 

7 (2006) found that inorganic Pb induced both necrosis and apoptosis in vitro. While the exact pathways 

8 involved were not determined, the authors concluded that activation of the Bax pro-apoptotic protein was 

9 not the key effect of Pb on inducing macrophage apoptosis. In an in vivo study in mice, Xu et al. (2008)

10 found that a 4-week administration of Pb acetate (50-100 mg/kg, oral) significantly elevated both ROS 

11 and malondialdehyde (an indicator of ROS-induced peroxidation) levels in peripheral blood lymphocytes. 

12 The Pb-induced DNA damage, determined by the comet assay, was accompanied by elevations in p53 and 

13 Bax expression with no change in Bcl-2 expression (creating a Bax/Bcl-2 imbalance). The authors 

14 propose that this is a likely route to Pb-induced apoptosis and tumorigenesis.

5.6.5.4. Cytokine Production

15 Toxicological studies have indicated that Pb affects immune cytokine production via action on T 

16 cells, macrophages, and dendritic cells (Section 5.6.2). The combination of these three pathways of 

17 cytokine changes induced by Pb creates a hyperinflammatory state among innate immune cells, and 

18 acquired immunity is skewed toward Th2 responses. As illustrated in Figure 5-47, downstream effects 

19 include altered IgE production, ROS production, and inflammation. Cheng et al. (2006) found that Pb 

20 exposure affected TNF-α production in macrophages by affecting signaling pathways. This can result in 

21 local tissue damage during the course of immune responses affecting such targets as the liver. In a study 

22 involving macrophage-mediated liver injury in mice (A/J), substimulatory levels of Pb (10 μM) 

23 coadministered with LPS stimulated the phosphorylation of p42/44 mitogen-activated protein kinase 

24 (MAPK) and TNF-α expression (Y.-J. Cheng et al., 2006). Blocking protein kinase C or MAPK reduced 

25 TNF-α production of macrophages in vitro, which in turn, protected against Pb + LPS-induced liver 

26 injury in vivo. These findings are consistent with those of Gao et al. (2007), which showed that treatment 

27 of mouse dendritic cells with Pb produced an increased phosphorylation of the Erk/MAPK signaling 

28 molecule. 

29 The most consistent immunomodulatory effect of Pb is the skewing of immune responses away 

30 from Th1 and toward Th2. Pb was observed to skew toward Th2 cytokine production in both dendritic 

31 cells (5.6.2.6) and T cells. Lynes et al. (2006) observed that Pb suppressed the production of Th1 cytokine 
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1 IFN-γ. Gao et al. (2007) observed that Pb exposure elevated production of Th2 cytokines such as IL-4, 

2 IL-5 and IL-6. This shift to a Th2 phenotype also was demonstrated in cultures of human blood 

3 monocytes activated with Salmonella enteritidis or with monoclonal antibodies of CD3, CD28, and CD40 

4 and exposed to inorganic Pb. Pb exposure suppressed expression of Th1 cytokines, IFN-γ, IL-1β, and 

5 TNF-α, and increased secretion of Th2 cytokines, IL-5, IL-6, and IL-10 (Hemdan et al., 2005).

6 In a recent study conducted across a lifetime (developmental through adulthood) using a broad 

7 range of dietary Pb concentrations in Swiss mice (females and males), Iavicoli et al. (2006) suggested a 

8 nonlinear hierarchical cytokine response. At the lowest dietary Pb concentration (0.8 μg/dL), IL-2 and 

9 IFN-γ were elevated over the controls, indicating an enhanced Th1 response. However, as dietary Pb 

10 exposure increased (resulting in blood Pb levels 12-61 μg/dL), a Th2 phenotype was observed with 

11 suppressed IFN-γ and IL-2 and elevated IL-4 production. These findings support the notion that the 

12 immune system is remarkably sensitive to low Pb exposures and that compensatory mechanisms may be 

13 stimulated at low Pb exposures. Other studies have found variable Pb-induced changes in IL-2, with no 

14 change or elevated production, depending upon the protocol used. Recently, Gao et al. (2007) found that 

15 Pb-treated dendritic cells promoted a slight but statistically significant increase in IL-2 production among 

16 lymphocytes. A recent study on bone (in vivo and in vitro) chondrogenesis found Pb-induced increases in 

17 production of TGF-β (Zuscik et al., 2007).

18 Consistent with toxicological studies, epidemiologic studies also find higher blood Pb levels in 

19 humans to be associated with a shift towards production of Th2 cytokines relative to Th1 cytokines. One 

20 consequence of an increase in the Th2 cytokine IL-4 is the activation of B cells to induce B cell class 

21 switching to IgE. In particular, Lutz et al. (1999) provided evidence for Pb affecting this pathway by 

22 finding that children with blood Pb levels greater than 10 μg/dL had both higher levels of IL-4 and IgE 

23 (Section 5.6.3). Among adults in Incheon, Korea without occupational Pb exposures with blood Pb levels 

24 ranging from 0.337 to 10.47 μg/dL, Kim et al. (2007) found associations of blood Pb level with serum 

25 levels of TNF-α and IL-6 that were larger among men with blood Pb levels ≥ 2.51 μg/dL (median). In 

26 models that adjusted for age, sex, BMI, and smoking status, a 1 μg/dL increase in blood Pb level was 

27 associated with a 23% increase (95% CI: 4, 55%) in log of TNF-α and a 26% increase in log of IL-6 (95% 

28 CI: 0, 55%). The associations between levels of blood Pb and plasma TNF-α were greater among men 

29 who were GSTM1 null or had the TNF-α GG genotype. For the association between blood Pb level and 

30 plasma IL-6, the effect estimate was slightly elevated in TNF-α GG genotype but not elevated in the 

31 GSTM1 positive group. The effects of Pb on several physiological systems have been hypothesized to be 

32 mediated by the generation of ROS (Daggett et al., 1998). Thus, the null variant of GSTM1, which is 

33 associated with reduced metabolism of ROS, may confer increased susceptibility to Pb-associated 

34 immune effects. The results for the TNF-α polymorphism were difficult to interpret. The GG genotype is 

35 associated with lower expression of TNF-α, and the literature is mixed with respect to which variant 

36 increases risk of inflammation-related conditions.
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1 Results from studies of occupationally-exposed adults also suggested that Pb exposure may be 

2 associated with decreases in Th1 cytokines and increases in Th2 cytokines; however, analysis were 

3 mostly limited to comparisons of mean cytokine levels among different blood Pb groups or Pb-exposure 

4 groups (Di Lorenzo et al., 2007; Valentino et al., 2007; Yucesoy et al., 1997a). The exception was the 

5 study of male foundry workers, pottery workers, and unexposed workers by Valentino et al. (2007). 

6 Multiple regression analyses were performed with age, BMI, smoking, and alcohol consumption included 

7 as covariates. Although effect estimates were not reported, statistically significant associations were 

8 observed between blood Pb level and IL-10 and TNF-α. Levels of IL-2, IL-6, and IL-10 showed an 

9 increasing trend from the lowest to highest blood Pb group. In contrast with most other studies, both 

10 exposed worker groups had lower IL-4 levels compared with controls. In a similar analysis, DiLorenzo et 

11 al. (2007) separated exposed workers into intermediate (9.1-29.4 μg/dL) and high (29.4-81.1 μg/dL) 

12 blood Pb level groups, with unexposed workers comprising the low exposure group (blood Pb levels 1-11 

13 μg/dL). Excluded from this study were exposed workers from the highest end of the distribution of blood 

14 Pb levels in DiLorenzo et al. (2006). Mean TNF-α levels showed a monotonic increase from the low to 

15 high blood Pb level group. A synergistic effect was observed between blood Pb levels and smoking. 

16 Among current smokers, a 12- to 16-fold difference in TNF-α levels was observed among blood Pb 

17 groups, whereas a less than twofold difference was observed among nonsmokers. In Yucesoy et al. 

18 (1997a), levels of the Th1 cytokines, IL-1β and IFN-γ, were lower in workers than in controls; however, 

19 differences were not observed in levels of the Th2 cytokines, IL-2 and TNF-α.

5.6.6. Immune Effects of Lead within Mixtures

20 One of the most striking observations regarding Pb effects on the immune system since the 2006 Pb 

21 AQCD concerns the effects of metal mixtures. Recent studies indicate that immune effects may be 

22 observed with lower levels of Pb exposure when they occur in conjunction with other metals. Information 

23 on interactions among metal mixtures may improve risk assessment methods because most human 

24 exposures to chemicals involve mixtures at low environmental levels. In a study of mice exposed to Pb 

25 acetate (10 mg/kg by weight), As (0.5 mg/kg by weight), or both, Bishayi and Senguta (2006) reported a 

26 greater than additive effect of coadministered Pb and As on macrophages in producing a decrease in 

27 bacterial resistance, myeloperoxidase (MPO) release, and NO production. Investigators assessed the Pb28 As interaction using the multivariate ANOVA for the experimental results of MPO release and 

29 constructing an isobologram by running an ordinary least squares regression between effects (% MPO 

30 release) and dose levels of metals (single and multimetal) in log-linear form.

31 Institoris et al. (2006) reached a similar conclusion after observing that lymph node weight 

32 decreased with exposure to 20 mg/kg Pb plus a second metal (Cd or Hg) but not with 20 mg/kg of Pb 

33 alone. Another study conducted in rats (Jadhav et al., 2007) found that mixtures of Pb, Hg, and other 
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1 metals at 10 to 100 times the concentrations of the individual components typically measured in drinking 

2 water in India suppressed lymphocyte counts and antibody titers and increased neutrophil counts. In 

3 contrast with these aforementioned studies, Fortier et al. (2008) found that PbCl2-exposed (7.5-20.7 

4 μg/dL) human leukocytes did not have alterations in lymphocyte proliferation, monocytic phagocytic 

5 activity, or NK cell activity. Although the combination of 20.7 μg/dL PbCl2 plus 12.0 μg/dL 

6 methylmercuric chloride (MeHgCl) decreased lymphocyte proliferation, these effects were attributed to 

7 MeHgCl, which had a stronger suppressive effect individually. The majority of evidence indicating 

8 synergism between Pb and metals such as As, Cd, and Hg suggests that a threshold for producing Pb9 induced immune effects may be lower if additional metals are present. 

10 In a study designed to mimic exposure to particles associated with urban traffic, Wei et al. exposed 

11 human endothelial cells in culture (EA.hy926) to urban fine particles (PM2.5 enriched with Pb and other 

12 metals). Investigators found that the particle mixture increased ROS production and mitochondrial13 mediated apoptosis; however, they did not test metals individually and could not attribute findings to Pb 

14 individually or interactions between Pb and other metals within the mixture. Similarly, in two studies of 

15 mice exposed to Pb and Cd in drinking water (1 ppm of each metal in drinking water for 28 days), Pb and 

16 Cd altered antibody titres (Massadeh et al., 2007). The main aims of these studies were to demonstrate 

17 that the effects of Pb and Cd could be reversed with administration of Nigella sativa L (black cumin) or 

18 garlic extract. However, investigators did not test each metal individually to assess interactions between 

19 metals. 

20 Epidemiologic studies have not widely examined interactions between Pb and other metals. 

21 However, consistent with Bishayi and Sengupta (2006), Pineda-Zavaleta et al. (2004) (Section 5.6.5.2) 

22 reported interactions between Pb and As. In addition to Pb, As contamination of drinking water was a 

23 concern in the study area; however, urinary As levels were higher in children who had lower blood Pb 

24 levels. In multiple regression analyses, urinary As was negatively associated with NO (similar to Pb), and 

25 a statistically significant negative interaction was observed between Pb and As, indicating that high 

26 internal doses of both metals were associated with a larger decrease in NO than that associated with either 

27 metal alone. Urinary As was negatively associated with superoxide anion (opposite direction of Pb), and 

28 the Pb by As interaction was positive. Thus, although higher urinary As was associated with a lower 

29 superoxide anion level, higher internal doses both Pb and As were associated with a larger increase in 

30 superoxide anion than that associated with blood Pb level alone.

5.6.7. Summary and Causal Determination

31 The collective body of evidence integrated across epidemiologic and toxicological studies 

32 consistently demonstrates that the immune system is a major target of Pb. Rather than resulting in overt 

33 cytotoxicity to lymphoid tissues, Pb exposure has been associated predominantly with more subtle 
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1 changes in a spectrum of immune mediators and functions. The importance of Pb as an immunomodulator 

2 is particularly evident when one considers: (1) the relative sensitivity of the immune system to Pb3 induced modulation; (2) the lifetime health ramifications of exposure to Pb in early development, 

4 particularly given the recognized sensitivity of the developing immune system to environmentally5 induced programming; and (3) the consequent broad spectrum of diseases and illnesses in multiple 

6 physiological systems potentially related to Pb-associated immune dysfunction. The majority of results 

7 from animal studies indicates that immune changes are observable at blood Pb levels of <8.0 μg/dL, with 

8 new evidence in mice demonstrating that nonmonotonic cytokine changes occur at blood Pb levels of 2-3 

9 μg/dL. Consistent with the animal studies, in the newly expanded body of epidemiologic studies in 

10 children and adults without occupational Pb exposures, changes in immune parameters are commonly 

11 demonstrated in association with mean blood Pb levels in the range of <2 μg/dL to 10 μg/dL.

12 The strength of evidence for Pb-associated immune effects is derived not only from the consistency 

13 of associations but also from the coherence of findings between toxicological and epidemiologic studies 

14 and coherence of findings among the spectrum of immune changes operating within particular pathways. 

15 A large body of toxicological evidence demonstrates Pb-induced suppression of T cell proliferation, and 

16 epidemiologic evidence in children consistently links elevated blood Pb levels (as low as 2.2-3.4 μg/dL) 

17 with decreases in T cell abundance. These changes can affect cell-to-cell interactions that mediate 

18 acquired immunity required in subsequent memory responses to antigen exposures. Despite the 

19 consistency of evidence for some T-cell subtypes (e.g., CD3+, CD4+), it is unclear what effect the 

20 observed magnitudes of decrease may have in attenuating acquired immunity. 

21 The prominent effect of Pb exposure on T cells, in terms of coherence with effects on other 

22 immune endpoints and implications for developing immune-based diseases, is the skewing of immune 

23 function away from a Th1 phenotype towards a Th2 phenotype. In toxicological studies, this shift is well24 established by suppressed production of Th1 cytokines (e.g., IFN-γ) and increased production of Th2 

25 cytokines (e.g., IL-4). A recent toxicological study builds on this extant evidence by indicating that Pb 

26 may promote Th2 responses by acting directly on dendritic cells, the major effector in antigen response. 

27 Findings from recent epidemiologic studies strengthen the evidence with observations of similarly 

28 skewed cytokine profiles in association with blood Pb levels in humans in the range of 2.5-10 μg/dL. 

29 Further, toxicological and epidemiologic studies link the Pb-associated predominance of Th2 cytokines 

30 with downstream effects on humoral immunity by demonstrating Pb-associated changes in B cell 

31 abundance and changes in circulating antibody levels. An increase in IL-4 from activated Th2 cells

32 induces differentiation of B cells into antibody-producing cells, thereby amplifying B cell expansion to 

33 secrete IgE, IgA, and IgG. IgE is the primary mediator for type 1 hypersensitivity resulting in various 

34 allergic conditions and asthma. In support of this well-established mechanism, toxicological studies 

35 describe Pb-induced (blood Pb levels 10-30 μg/dL) changes in IgA, IgG, and IgM. Additionally, 

36 epidemiologic studies in children consistently demonstrate that blood Pb levels are positively associated 
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1 with B cell abundance (blood Pb levels in the range of 5-10 μg/dL) and increases in IgE (blood Pb levels 

2 as low as 2.8-3.4 μg/dL). Observations of Pb-associated increases in Th2 responses and circulating IgE 

3 levels provide biological plausibility for epidemiologic observations in children of associations of blood 

4 Pb levels (in the range of 1.16-10 μg/dL) with asthma and allergic conditions. Such epidemiologic data 

5 are limited, and additional studies with more rigorous methodology (e.g., longitudinal design to establish 

6 temporality, improved assessment of Pb exposures, adjustment for potential confounders such as 

7 smoking, SES, and exposures to other metals) are needed to substantiate the findings. 

8 Suppression of Th1 function by Pb places individuals at greater risk of certain infectious diseases 

9 and cancer. Compared with the relationships between Pb-suppressed Th1-dependent antitumor processes 

10 and effects on tumor formation, Pb effects on decreasing host resistance are relatively well-established. 

11 Pb exposure of animals (resulting in blood Pb levels <2-5 μg/dL) suppresses the DTH response, and a 

12 recent in vitro study indicates such effects may be mediated by dendritic cells. Further evidence of Pb13 associated suppressed Th1 activity is provided by toxicological and epidemiologic observations that Pb 

14 exposure and blood Pb levels, respectively, are associated impaired phagocytic and chemotactic activity 

15 of macrophages and neutrophils (blood Pb levels >25 μg/dL in humans). Th1-dependent IFN-γ normally 

16 enhances the killing capacity of macrophages. Epidemiologic studies additionally demonstrate that higher 

17 blood Pb levels are associated with lower neutrophil respiratory burst (superoxide anion release), 

18 indicative of diminished degradation of phagocytosed particles. Toxicological and epidemiologic 

19 evidence for suppressed Th1 activity and effects on macrophage and neutrophil functional activities 

20 provide biological plausibility for observations in animals and humans for associations between blood Pb 

21 levels (in the range of 3-10 μg/dL in humans) and increased risk of infection.

22 Toxicological studies demonstrate that Pb induces macrophages into a hyperinflammatory state as 

23 characterized by enhanced production of ROS, suppressed production of NO, enhanced production of 

24 TNF-α, and excessive metabolism of arachidonic acid into immunosuppressive metabolites (e.g., PGE2). 

25 Although examined in fewer studies, epidemiologic studies find higher blood Pb levels (in the range of 6-

26 20 μg/dL) to be associated with higher serum levels of ROS, TNF- α and lower serum levels of NO. 

27 These proinflammatory effects of Pb exposure on macrophages provide support for associations of Pb 

28 with elevated risk of disease in multiple physiological systems. The strongest evidence comprises Pb29 induced changes in specialized macrophages such as alveolar macrophages, testicular macrophages, and 

30 microglia, whose altered homeostasis or function may contribute to documented associations of blood Pb 

31 levels with asthma and bronchial reactivity, poor reproductive performance, and neurodegeneration, 

32 respectively. Although limited mostly to toxicological studies, Pb has been shown to induce the 

33 generation of autoantibodies, suggesting that Pb exposure may increase the risk of developing 

34 autoimmune conditions. These findings are supported by Pb-induced inflammation and tissue damage and 

35 Pb-induced T cell activation in response to new antigens.
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1 In summary, recent toxicological and epidemiologic studies support the strong body of evidence 

2 presented in the 2006 Pb AQCD that Pb exposure is associated with changes in immune cell abundance 

3 and function that subsequently lead to a broad spectrum of changes in both cell-mediated and humoral 

4 immunity to promote a Th2 phenotype and hyperinflammatory state. Toxicological and epidemiologic 

5 findings of Pb-associated decreases in T cells, inhibition of Th1-type responses, and impaired phagocytic 

6 activity of macrophages and neutrophils provide biological plausibility for evidence linking Pb exposure 

7 with increased risk of bacterial and viral infection. Additionally, toxicological and epidemiologic evidence 

8 of a Pb-associated promotion of the Th2 phenotype, increased B cell abundance, increased synthesis of 

9 IgE, and increased inflammation support observations in children for associations of blood Pb levels with 

10 asthma and allergic conditions. Although not widely examined in humans, toxicological findings indicate 

11 that Pb-induced immunomodulation may have broader implications for autoimmunity and mediating Pb 

12 effects in other physiological systems such as the nervous, cardiovascular, and reproductive systems. 

13 Animal studies and to a limited extent, epidemiologic studies, demonstrate increased susceptibility from 

14 prenatal exposures and enhanced responses with co-exposures to other metals. The consistency of 

15 findings among toxicological and epidemiologic studies and the coherence of findings between these 

16 disciplines and across the continuum of related immune responses are sufficient to conclude that there is a

17 causal relationship between Pb exposures and immune system effects.

5.7. Effects on Heme Synthesis and Red Blood 

Cell Function

5.7.1. Summary of Findings from 2006 Pb AQCD

18 The 2006 Pb AQCD reported that Pb affects developing RBCs (red blood cells [RBC]) as noted by 

19 anemia observed with blood Pb >40 μg/dL. Pb-induced anemia is thought to occur due to decreased RBC 

20 life span and effects on hemoglobin (Hb) synthesis. The exact mechanism for these effects was not 

21 known, although Pbinduced changes on iron uptake or inhibition of enzymes in the heme synthetic 

22 pathway may be responsible. 

23 The 2006 Pb AQCD indicated that Pb crosses RBC membranes through passive (i.e., energyindependent) carrier-mediated mechanisms including a vanadate-sensitive Ca2+ 24 pump. Once Pb enters the 

25 cells, it is predominantly found in protein-bound form, with Hb and aminolevulinic acid dehydratase 

26 (ALAD) both identified as targets. Pb poisoning decreases RBC survival, as well as alters RBC mobility 

27 and morphology, although the precise mechanisms by which it does so are not known. Pb exposure has 

28 been found to significantly decrease several hematological parameters including Hb, hematocrit (Hct), 

29 mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular 
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1 hemoglobin concentration (MCHC). Pb has also been observed to exert multiple effects on RBC 

2 membranes, including altered microviscosity and fluidity, decreased sialic acid content, decreased 

3 lamellar organization, decreased lipid resistance to oxidation (possibly mediated by perturbations in RBC 

4 membrane lipid profiles), and increased permeability. These alterations to RBC membranes potentially 

5 lead to RBC fragility, abnormal cellular function, RBC destruction, and ultimately anemic conditions. Pb 

6 exposure also results in increased activation of RBC scramblase, an enzyme responsible for the 

7 expression of phosphotidylserine (PS) on RBC membranes. This expression of PS decreases the life span 

8 of RBCs via phagocytosis by macrophages. Pb exposure has been observed to alter the phosphorylation 

9 profiles of membrane proteins, which may influence the activity of membrane enzymes and the 

10 functioning of receptors and channels located on the membrane. 

11 The 2006 Pb AQCD reported that Pb affects heme synthesis through the inhibition of multiple key 

12 enzymes, most notably ALAD, the enzyme that catalyzes the second, rate-limiting step in heme 

13 biosynthesis (See Figure 5-53 for a schematic representation of the heme biosynthetic pathway). The 

14 2006 Pb AQCD further reported that decreased RBC ALAD activity is the most sensitive measure of Pb 

15 exposure with a concentration-response change in the ratio of activated/nonactivated ALAD activity that 

16 is not dependent on the method of Pb administration. The inhibition of the ALAD enzyme was observed 

17 in RBCs from multiple species, including birds, Cynomolgous monkeys, and humans. Pb was also 

18 observed to inhibit other enzymes responsible for heme biosynthesis, including ferrochelatase, 

19 porphobilinogen (PBG) deaminase, and coproporphyrinogen oxidase. Pb also potentially alters heme 

20 biosynthesis through inhibition of transferrin (TF) endocytosis and iron transport. 

21 Pb has been found to alter RBC energy metabolism through inhibition of enzymes involved in 

22 anaerobic glycolysis and the pentose phosphate pathway. Pb was also found to inhibit pyrimidine 5’-

23 nucleotidase (P5N) activity and the 2006 Pb AQCD indicated that this might be another possible 

24 biomarker of Pb exposure. Inhibition of P5N results in an intracellular increase in pyrimidine nucleotides 

25 leading to hemolysis. The 2006 Pb AQCD indicated that perturbations in RBC energy metabolism may be 

26 related to significant decreases in levels of nucleotide pools, including nicotinamide adenine nucleotide 

27 (NAD), possibly due to decreased NAD synthase activity, and nicotinamide adenine nucleotide phosphate 

28 (NADP) accompanying significant increases in purine degradation products.

Pb was found to alter the activity of membrane-bound ion pumps. Potassium (K+ 29 ) permeability was 

found to be increased by Pb due to altered sensitivity of the membrane calcium (Ca2+ 30 )-binding site that 

caused selective efflux of K+ ions from the RBC membrane. Inhibition of RBC sodium (Na+)-K+ 31

32 adenosine triphosphate synthase (ATPase), acetylcholinesterase (ACh), and NADH dehydrogenase was 

also observed. In human RBCs, Na+-K+ ATPase activity was more sensitive to Pb exposure than Ca2+ 33 or 

magnesium (Mg2+ 34 ) ATPases.
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1 The 2006 Pb AQCD document identified oxidative stress as an important potential mechanism of 

2 action resulting from Pb exposure. Increased lipid peroxidation and inhibition of antioxidant enzymes 

3 (e.g., superoxide dismutase [SOD], catalase [CAT]) was observed following exposure to Pb. 

5.7.2. Effects on Red Blood Cell Functions

4 As stated in the 2006 Pb AQCD, Pb poisoning is associated with anemia resulting from shortened 

5 RBC life span and Pb effects on Hb synthesis. As of 2006, the mechanism for this was not clear, but it 

6 was determined not to be due to iron deficiency, which can be found to occur independently of Pb 

7 exposure. However, Zimmerman et al. (2006) found that blood Pb levels were statistically significantly 

8 reduced in non- or mildly anemic, iron-deficient children in India fed an iron-fortified diet for 30 weeks 

9 (p <0.02); blood Pb levels were not reduced in the children not receiving the iron-fortified diet. Although 

10 a number of studies find decreases in RBCs and/or Hct levels associated with Pb, it is not known whether 

11 this is due to reduced cell survival or a decrease in RBC cell production. However, decreased RBC 

12 survival and hematopoiesis can be expected to occur simultaneously, and any effect on RBC numbers is 

13 likely a combination of the two modes of action.

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

14 The 2006 Pb AQCD reports that Pb uptake into human RBCs occurs via passive anion transport 

15 mechanisms. Although Pb can passively cross the membrane in both directions, little of the Pb leaves the 

cell after entry. Simons (1993b) found that in vitro uptake of 203 16 Pb (1-10 μM) occurred via an anion 

17 exchanger while the efflux was via a vanadate-sensitive pathway. After entry into the RBC, radioactive Pb 

18 was found to partition with Hb at a ratio estimated to be about 6000:1 bound to unbound (Simons, 1986). 

19 However, Bergdahl et al. (1997) suggested that ALAD was the primary Pb binding protein and not Hb. 

20 The 2006 Pb AQCD also reports that the majority (approximately 98%) of Pb accumulates in RBC 

21 cytoplasm bound to protein and only about 2% is found in the membrane. This is related to the high ratio 

22 of Pb in RBCs compared to plasma Pb. Further Information on Pb binding and transport in blood can be 

23 found in the kinetics section of Chapter 4 (Section 4.2).

24 Although no studies were indentified that examined transport of Pb into RBCs, Lind et al. (2009)

25 recently observed that several zinc (Zn) ionophores (8-hydroxyquinoline derivatives and Zn and Na 

26 pyrithione) were able to effectively transport Pb out of RBCs into the extracellular space. 

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

27 Although it is known that Pb exposure shortens the RBC life span and alters RBC mobility, as of 

28 the 2006 Pb AQCD the mechanism of this was not well understood. While the mechanism is still not fully 

understood, there has been some indication for a role in free Ca2+ 29 . There are also newer studies that 
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1 examine the relationship between Pb and RBC survival, mobility, and membrane integrity. Pb-exposed 

2 workers in a recycled automobile battery factory in Mexico had 7.5 times as much blood Pb as was found 

3 in unexposed workers (74.4 ± 21.9 μg/dL in 15 exposed workers versus. 9.9 ± 2 μg/dL in 15 unexposed 

workers, p <0.01) (Quintanar-Escorza et al., 2007). Intracellular RBC free calcium levels [Ca2+ 4 ]i were 

5 significantly higher in the Pb-exposed workers than in the unexposed workers (79 ± 13 versus. 30 ± 9 nM, 

p <0.05). The level of [Ca2+]i was highly correlated (R2 6 = 0.754) with blood Pb levels in exposed workers; 

[Ca2+ 7 ]i was also elevated in the unexposed workers who had low blood Pb levels ranging from 7.9 μg/dL 

to 11.9 μg/dL. The observed elevation in [Ca2+ 8 ]i was related to an increased uptake and a decreased efflux 

due to reduced Ca2+-Mg2+-ATPase activity. In the RBCs of Pb-exposed workers, the activity of Ca2+ 9 -

Mg2+ 10 -ATPase was 28 ± 8 nmol Pi/mg protein/min versus. 40 ± 9 in unexposed workers (p <0.05). These 

11 changes were associated with increased fragility of the RBCs and dramatic morphological alterations, 

12 including the increased presence of ecinocytes (cells without normal biconcave shape) and crenocytes 

13 (speculated cells) in Pb-exposed workers. Similar dose-dependent effects were observed when RBCs 

14 from healthy subjects were incubated with Pb at concentrations from 0.2 to 6.0 μM (Quintanar-Escorza et 

al., 2010). Abam et al. (2008) also observed a decrease in the activity of RBC membrane bound Ca2+ 15 -

Mg2+ 16 -ATPase in workers exposed to Pb in a number of occupations. While the authors did not observe an 

increase in [Ca2+]i or Mg in RBCs from exposed workers, the RBC membrane [Ca2+]i and Mg2+ 17

18 concentration were increased. Abam et al. (2008) did not report on morphological changes in erythrocytes 

19 from exposed workers. Ciubar et al. (2007) found that RBC morphology was disrupted with 50% or more 

20 of RBCs exposed to Pb concentrations of 0.5 μM or higher for 24 hours at 37°C having lost the typical 

21 discocytic morphology and displaying moderate to severe echinocytosis. Exposure of RBCs to higher 

22 concentrations of Pb nitrate resulted in cell shrinkage. Ademuyiwa et al. (2009) observed that the 

23 cholesterol content of RBC plasma membranes, but not the phospholipid content, was statistically 

24 significantly higher in rats exposed to 200 ppm Pb acetate (blood Pb = 40.63 ± 9.21 μg/dL) through 

25 drinking water compared to controls. Further, the cholesterol/phospholipid ratio was increased in the rats 

26 with increased cholesterol, indicating that RBC membrane fluidity was decreased. 

27 A number of studies have investigated the effect of occupational exposure to Pb on various 

28 hematological parameters. (Ukaejiofo et al., 2009) studied hematological effects of Pb in 81 male subjects 

29 exposed to Pb at three different manufacturing companies in Nigeria. Two control groups were used for 

30 comparison (30 individuals from the same industries not involved in handling Pb and 20 individuals from 

31 the same locality but not involved in Pb handling). The exposed individuals had an average blood Pb level 

32 of 7.00 μg/dL compared to 3 μg/dL in controls drawn from industries not involved in Pb handling (control 

33 group I) and 2 μg/dL in controls drawn from the general population (control group II) (p <0.05). Exposed 

34 subjects had significantly reduced Hb and packed cell volume (PCV) levels and increased percentage of 

35 reticulocytes. Although the differences were significant between the exposed and control subjects, the 

36 study authors state that the results in the exposed subjects were at the lower range of normal for 
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1 Nigerians. The percent cell lysis did not differ between controls and exposed workers; however, when 

2 workers and controls were stratified by age, there was a significant increase in cell lysis in workers under 

3 age 30 compared to similarly aged control group II (p <0.01). Patil et al. (2006) examined hematology 

4 effects in jewelry workers in India occupationally exposed to Pb. Blood Pb was significantly higher in the 

5 jewelry workers (48.56 ± 7.39 μg/dL) compared to individuals not occupationally exposed (12.52 ± 4.08 

6 μg/dL). There was no significant difference in the Hb, MCV, or MCH; however, jewelry workers had a 

7 significant decrease in PCV and total RBCs, accompanied by a significant increase in MCHC. In addition, 

8 the exposed workers had a significant increase in total leukocyte levels. In battery manufacturing workers 

9 in India, significant decreases in Hb, PCV, MCV, MCH, MCHC, and total RBCs were observed (Patil, 

10 Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 2006) with similar blood Pb levels (53.63 ± 16.98 

11 μg/dL). Industrial workers in Pakistan occupationally exposed to Pb (blood Pb = 29.1 (range 9.0-61.1) 

12 μg/dL) had a significant increase (3.5-fold higher median) in blood Pb levels compared to age and gender 

13 matched controls (blood Pb = 8.3 (range 1.0-21.7) μg/dL) (D. A. Khan et al., 2008). The industrial 

14 workers had a significantly lower Hb, but not a significant difference in the number of RBCs. There were 

15 no significant differences in the number of white blood cells (WBCs) or in platelet counts. Karita et al. 

16 (2005) examined the relationship between hematological parameters in Pb-exposed workers (blood Pb = 

17 26.9 μg/dL) in a variety of occupational settings. Blood Pb was found to be negatively correlated with 

18 Hb, Hct, and total RBC count (p <0.01). Fonte et al. (2007) described a case-report in which a 47-year old 

19 male exposed to Pb fumes and vapors at a recycling plant (blood Pb = 148 μg/dL) experienced 

20 normocytic, normochromic anemia, along with reticulocytosis and RBC basophilic stipplings. Following 

21 chelation therapy, the hematological symptoms improved. Taken together, these studies provide consistent 

22 evidence that occupational exposure to Pb reduces the number of RBCs in circulation. Although this 

23 decrease in RBCs may be explained by both decreased cell survival and/or disruption of hematopoiesis, 

24 the observation of increased reticulocytes in Ukaejiofo et al. (2009) seems to represent compensation for 

25 decreased RBC survival due to Pb exposure. 

26 Studies in children were more equivocal than those in adults. Riddell et al. (2007) found that 21% 

27 of children 6 months to 5 years of age living in rural Philippines had blood Pb levels greater than 10 

28 μg/dL (mean = 6.9 μg/dL). Hb levels were inversely related to blood Pb, with a decrease of 3% blood Pb 

29 associated with every 1 g/dL increase in Hb. However, Huo et al. (2007) found that children living near 

30 an area where electronic waste was recycled in China had significantly higher blood Pb levels than 

31 children in the neighboring town with no waste recycling (15.3 versus 9.94 μg/dL), but no difference in 

32 the Hb levels in the children in the two towns was detected (127.55 g/L in children with the higher blood 

33 Pb levels versus 123.46 g/L). Ahamed et al. (2006) studied male urban adolescents in India. The 39 

34 adolescents were separated into groups according to their blood Pb level (group 1: <10 μg/dL, group 2: 

35 >10 μg/dL). Although the groups were similar in their age, height, weight, and body mass index, group 2 

36 had a significantly lower PCV compared to group 1. The equivocal findings in studies investigating 
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1 hematological effects in children may be due to the comparatively shorter time period and magnitude of 

2 exposure versus. those seen in occupational studies.

3 Baranowska-Bosiacka et al. (2009) examined the effects of Pb on RBC hemolysis both in vitro 

4 measuring lysate in human RBCs incubated with Pb at concentrations ranging from 100 nM to 100 μM 

5 for 5-30 minutes, and in vivo using a rat RBC lysate from rats exposed to Pb acetate (0.1 %) in drinking 

6 water for 9 months. Rats exposed to Pb in the in vivo portion of the study achieved a blood Pb 

7 concentration of 7.1 μg/dL. Both studies demonstrated that Pb exposure resulted in increased hemolysis, 

8 observed as a significant increase in extracellular Hb. Pb-induced hemolysis in these experiments may be 

9 due to inhibition of RBC phosphoribosyltransferases (Section 5.7.5.1). The in vitro studies indicated a 

10 concentration-dependent increase in the amount of hemolysis, with a significant (threefold) increase even 

11 at the lowest concentration tested (i.e., 100 nM). Lee et al. (2005) observed that rats orally administered 

12 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 

13 controls, p <0.05), and had significant decreases in Hct, Hb, and RBCs (p <0.05) (M. K. Lee et al., 2005). 

14 Male mice administered 50 mg/kg Pb nitrate in distilled water via gavage for 40 days had significantly 

15 reduced total RBC counts, total leukocyte counts, Hb, lymphocytes, and monocytes (Sharma et al., 2010). 

16 Rats exposed to 2 g/L Pb acetate in drinking water for 30 days had significantly decreased RBCs, Hb, 

17 PCV, MCH, and MCHC compared to controls (p <0.05) (Simsek et al., 2009). Microcytic and basophilic 

18 erythrocytic granulations were commonly seen in Pb-exposed animals. An indication that the decrease in 

19 RBC count was related to decreased survival, and not a disruption of hematopoiesis, was the observation 

20 of significantly increased reticulocyte density and total count compared to controls (p <0.05). Mice 

21 exposed to 1 g/L Pb acetate in drinking water for 90 days, but not those exposed for 15 or 45 days, had 

22 significantly decreased RBC counts and Hct compared to controls (p <0.05) (C. C. Marques et al., 2006). 

23 Spleen weights were also observed to be increased relative to body weight in animals exposed to Pb for 

24 45 days. Male rats administered Pb acetate in the drinking water for 4 weeks at concentrations ranging 

25 from 100 to 1,000 ppm had a dose-dependent increase in blood Pb (range: 6.57 to 22.39 μg/dL), but there 

26 were no significant changes in any of the hematological parameters (complete blood cell count 

27 performed) measured at the end of treatment (M. Y. Lee et al., 2006). Slight, nonsignificant, increases in 

28 PS expression on RBC membranes were also observed. In vitro experiments with rat and human blood 

29 did not demonstrate a significant increase in hemolysis after 4 hours of treatment with Pb acetate at 

30 concentrations up to 10 μM. 

31 Khaĭrullina et al. (2008) observed that the surface profiles of RBC membrane shadows incubated 

32 with 0.5-10 μM/l Pb acetate for three hours were much smoother than untreated RBC membranes when 

33 examined by atomic force microscopy. The authors postulate that the observed smoothing in treated RBC 

34 membranes may be due to clusterization of band 3 protein. Band 3 (anion exchanger 1 [AE1]), is a 

chloride/bicarbonate (Cl-1/HCO3

- 35 ) exchanger and is the most abundant protein in RBC membranes. AE1 is 

36 integral in carbon dioxide (CO2) transport and linkage of the cellular membrane to the underlying 
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1 cytoskeleton (Akel et al., 2007; Su et al., 2007). The observed smoothing of the RBC membrane may due 

2 to Pb interfering with how the membrane attaches to the cytoskeletal structure of the RBC through 

3 perturbation of AE1’s normal activity.

Eryptosis 

4 Eryptosis is the suicidal death of RBCs. It is characterized by cell shrinkage, membrane blebbing, 

5 and cell membrane phospholipid scrambling associated with PS exposure on the cell membrane that leads 

6 to cell destruction via macrophages (Föller et al., 2008; Lang et al., 2008). As previously reported in the 

7 2006 Pb AQCD, Kempe et al. (2005) found that exposing human RBCs to Pb at concentrations ranging 

from 0.3 to 3 μM caused increased activation of K+ 8 channels that lead to cell shrinkage and scramblase 

9 activation. The activation of scramblase increased the exposure to PS on the cell membrane, which causes 

10 an increase in destruction of the RBCs by macrophages. 

11 Shin et al. (2007) found that in vitro exposure of human RBCs to 1-5 μM Pb acetate increased PS 

12 expression in a time- and concentration-dependent manner. The maximum increase in expression of PS 

13 was 26.8 ± 3.15% (compared to deionized water) after incubation with 5 μM Pb for four hours. The 

expression of PS in RBCs is considered to be regulated through a Ca

2+ 14 dependent mechanism and, 

correspondingly, [Ca2+ 15 ]i was observed to increase with exposure to Pb (0.24 ± 0.21 μM in controls to 6.88 

16 ± 1.13 μM in RBCs treated with 5 μM Pb for one hour). Consistent with this finding, Shin et al (2007)

17 also observed that scramblase activity, which is important for induction of PS exposure and is activated 

by [Ca2+]i 18 , was increased in Pb-exposed RBCs. Flippase, which translates PS exposure to inner 

membranes, is inhibited by high levels of [Ca2+ 19 ]i and was observed to exhibit reduced activity following 

20 Pb exposure. The inhibition of flippase is additionally influenced by the depletion of cellular adenosine 

21 triphosphate (ATP). ATP levels were decreased in a dose-dependent manner following exposure to Pb. To 

22 confirm these findings in vivo, Shin et al. (2007) exposed male rats i.p. to 25, 50, or 100 mg/kg Pb 

23 acetate. Expression of PS was observed to increase in a concentration-dependent manner at concentrations 

24 ≥ 50 mg/kg, confirming the in vitro results. No hemolysis or microvesicle formation was observed in the 

25 in vitro and in vivo experiments. Ciubar et al. (2007) also found that exposure to Pb nitrate (0.5-2 μM) 

26 resulted an increase in PS exposure and cell shrinkage, which they stated were indicators of cell 

27 apoptosis. As reported above, Khaĭrullina et al. (2008) observed RBC membrane smoothing that may be 

28 due to alterations in AE1 activity. Disruptions in AE1 activity may also result in enhanced PS exposure 

and premature cell death. Akel et al. (2007) observed that in AE1-/- 29 mice, PS exposure was much greater 

30 than in wild type mice. Decreased RBCs and increased reticulocytes were also observed, an indication of 

31 high cell turnover.
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5.7.2.3. Red Blood Cell Hematopoiesis

1 Erythropoietin is a glycoprotein hormone excreted by the kidney to promote the development of 

2 RBCs in the bone marrow. Sakata et al. (2007) examined the relationship between blood Pb level and 

3 serum erythropoietin levels in Pb-exposed tricycle taxi drivers (n = 27) working at Kathmandu who were 

4 not anemic. The average blood Pb level in the taxi drivers was 6.4 μg/dL compared to 2.4 μg/dL in 

5 nondrivers. Drivers had a significantly lower level of serum erythropoietin (12.7 versus 18.8 mU/mL) 

6 compared to the nondrivers and there was a statistically significant inverse relationship between the level 

7 of serum erythropoietin and blood Pb (r = -0.68, p <0.001). No other hematological effects were 

8 observed. The Sakata et al. (2007) study demonstrated that serum erythropoietin levels are affected by Pb 

9 even at levels low enough not to cause anemia. While this is generally considered a measure of kidney 

10 toxicity, it can also be considered an indicator that Pb could possibly affect the level of RBCs through 

11 decreased levels of serum erythropoietin.

12 Celik et al. (2005) observed that exposure of female rats to 140, 250, or 500 mg/kg Pb acetate once 

13 per week for 10 weeks resulted in statistically significantly decreased numbers of polychromatic RBCs 

14 (PCE) and increased numbers of micronucleated PCEs, compared to controls (p <0.001). Alghazal et al. 

15 (2008) exposed male and female rats to 100 mg/L Pb acetate daily for 125 days and observed statistically 

16 significant increases in micronucleated PCEs in female rats (p = 0.02) but no significant reduction in the 

17 ratio of PCEs to normochromic RBCs (NCE). In male rats, a significant increase in micronucleated PCEs 

18 was observed (p <0.001) along with a decrease in the PCE/NCE ratio (p = 0.02). While the results from 

19 Alghazal et al. (2008) indicate that Pb is cytotoxic in male rats only, but is genotoxic in both sexes, Celik 

20 et al. (2005) indicates Pb is cytotoxic in female rats as well. Mice exposed to 1 g/L Pb acetate in drinking 

21 water for 90 days had statistically significant increases in micronucleated PCEs; a small, but not 

22 statistically significant decrease in the PCE/NCE ratio was also observed (C. C. Marques et al., 2006). 

23 Cyto- and genotoxicity in RBC precursor cells is a strong indication of altered hematopoiesis in bone 

24 marrow.

5.7.2.4. Membrane Proteins

25 There have been few studies examining the effects of Pb on membrane proteins since the 2006 Pb 

26 AQCD. According to the 2006 Pb AQCD report, Pb has been found to affect RBC membrane 

27 polypeptides in exposed workers (Fukumoto et al., 1983);(Apostoli et al., 1988). Fukumoto et al. (1983)

28 found decreased levels of polypeptides in band 3, which Apostoli et al. (1988) suggested may represent an 

29 anion channel protein, and increases in the level of polypeptides in bands 2, 4, 6, and 7. Fukumoto et al. 

30 (1983) suggested that the changes in the RBC membrane polypeptides may cause changes in membrane 

31 permeability. Apostoli et al. (1988) found that the changes in membrane polypeptides occurred at blood 

32 Pb levels greater than 50 μg/dL. Exposure to Pb acetate at concentrations above 100 nM for 60 minute 
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1 has also been found to increase the phosphorylation of proteins in human RBC membranes in vitro 

2 (Belloni-Olivi et al., 1996). Phosphorylation did not occur in cells depleted of protein kinase C (PKC), 

3 indicating a PKC-dependent mechanism. 

4 Huel et al. (2008) found that newborn hair and cord blood Pb levels (1.22 ± 1.41 μg/g and 3.54 ± 

5 1.72 μg/dL) were negatively associated with Ca-ATPase activity in plasma membranes of RBCs isolated 

6 from cord blood; newborn hair Pb levels were more strongly associated with cord Ca pump activity than 

7 cord blood Pb (p <0.0001 versus. p <0.05). Maternal Pb levels were not correlated with Ca pump activity 

8 in maternal or cord blood. Pb-induced disruptions in Ca homeostasis in RBCs can lead to cytotoxicity and 

9 necrosis, and these effects may be representative of cellular dysfunction in other organ systems. 

5.7.3. Effects on Red Blood Cell Heme Metabolism

10 Pb has been found to inhibit several enzymes involved in heme synthesis, namely ALAD 

11 (cytoplasmic enzyme catalyzing the second, rate-limiting, step of the heme biosynthesis pathway), 

12 coporphyrinogen oxidase (catalyses the sixth step in heme biosynthesis converting coporphyrinogen III 

13 into protoporphyrinogen IX), and ferrochelatase (catalyses the terminal step in heme synthesis converting 

14 protoporphyrin IX into heme) (Figure 5-53). The observation of decreased Hb (measured as total Hb, 

15 MCH, or MCHC) in occupationally exposed adults (Karita et al., 2005; D. A. Khan et al., 2008; Patil, 

16 Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 2006; Ukaejiofo et al., 2009), children (Riddell et al., 

17 2007), and experimental animal models (M. K. Lee et al., 2005; C. C. Marques et al., 2006; Sharma et al., 

18 2010; Simsek et al., 2009) is a direct measure of decreased heme synthesis due to Pb intoxication.
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Note: Steps in the pathway potentially affected by Pb are indicated with curved arrows pointing to 

the affected enzyme, and effects are represented 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 

1 Decreases in RBC 5-aminolevulinic acid dehydratase (ALAD) levels are strongly associated with 

2 Pb exposure in humans to such an extent that RBC ALAD activity has been used to assess Pb toxicity. 

3 Several epidemiologic studies published since the 2006 Pb AQCD evaluated the relationship between Pb 

4 exposure, blood Pb levels and ALAD activity. 

5 Patil et al. (2006) examined jewelry workers (blood Pb = 48.46 ± 7.39 μg/dL) in India 

6 occupationally exposed to Pb. In the study both the activated and nonactivated ALAD activities were 

7 measured. The study authors state that this is because decreases in ALAD activity reached a plateau and 

8 anemia can result in increased ALAD levels. Therefore, they considered the ratio of 

9 activated/nonactivated ALAD to be a good indicator of Pb toxicity. As described in the 2006 Pb AQCD, 

10 Scheuhammer (1987) studied the usefulness of this ratio in avian RBCs and found it to be a sensitive, 

11 dose-dependent measure of Pb exposure regardless of the route of exposure. They found that the 

12 activated/nonactivated ALAD ratio could be used to determine the oral exposure due to the highly 

13 positive correlation between Pb exposure in the 5–100 ppm range and ALAD activity ratio. Patil et al. 

14 (2006) observed a statistically significant decrease in nonactivated ALAD and an increase in the ratio of

15 activated to nonactivated ALAD, compared to nonexposed controls (p <0.05 and 0.001, respectively) . 
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1 The study authors state that this indicates the inhibition of heme synthesis. Urinary excretion of both ALA 

2 and PBG was statistically significantly increased in jewelry workers, a further indication of RBC ALAD 

3 inhibition. Similar results were seen in battery manufacturing workers in India (Patil, Bhagwat, Patil, 

4 Dongre, Ambekar, Jailkhani, et al., 2006). Pb-exposed workers (blood Pb = 74.4 ± 21.9 μg/dL) in a 

5 recycled automobile battery factory in Mexico had a significant decrease (~90%) in RBC ALAD activity 

6 compared to unexposed workers (9.9 ± 2 μg/dL) (Quintanar-Escorza et al., 2007). Painters in India with 

7 an average blood Pb level of 21.92 μg/dL had a significant decrease in ALAD levels compared to controls 

8 with an average blood Pb level of 3.06 μg/dL (p <0.01) (Mohammad et al., 2008). Stoleski et al. (2008)

9 observed that workers in a Pb smelter in Macedonia (blood Pb = 16.4 ± 8.5 μg/dL) had significantly 

10 decreased ALAD activity (p <0.001) and increased ALA levels (p <0.0005) compared to workers with no 

11 exposure to (blood Pb = 7.0 ± 5.4 μg/dL). Lastly, Ademuyiwa et al. (2005) observed that workers in 

12 mechanic workshops (blood Pb = 27.0 ± 1.1 - 48.9 ± 19.1 μg/dL μg/dL) had significant decreases in 

13 ALAD activity compared to controls (15.8 ± 2.8 μg/dL, p <0.001). Petrol station workers had the highest 

14 degree of ALAD inhibition relative to controls (77%), whereas welders only had a 36% decrease in 

15 ALAD activity compared to controls. A case report by Fonte et al. (2007) described a worker 

16 occupationally exposed to Pb vapors (blood Pb = 148 μg/dL) with ALAD levels substantially decreased 

17 compared to normal levels (3 versus >25 U/L). Zinc protoporphyrin (ZPP) levels were also described to 

18 be greatly increased. Following chelation therapy, the patient’s clinical picture improved.

19 Wang et al. (2010) found that even with low to moderate blood Pb levels, there was a 

20 concentration-dependent decrease in ALAD activity in both children and adults (blood Pb = 7.1 and 6.4 

21 μg/dL, respectively) in rural southwest China. Further, Wang et al. (2010) observed that the relationship 

22 between blood Pb and ALAD activity was nonlinear and exponential, with more significant decreases in 

23 ALAD activity occurring with blood Pb levels greater than 10 μg/dL. No correlation was observed 

24 between urinary ALA levels and blood Pb. Ahamed et al. (Ahamed et al., 2006) studied male urban 

25 adolescents in India. The 39 adolescents were separated into groups according to their blood Pb levels 

26 (group 1: <10 μg/dL, group 2: >10 μg/dL). Although the groups were similar in their age, height, weight, 

27 and body mass index, group 2 had a significantly lower ALAD activity than group 1 (p <0.001). When all 

28 39 adolescents were examined together, an inverse relationship was found between blood Pb and ALAD 

29 activity. Ahamed et al. (2005) also observed decreased ALAD activity in Indian children (aged 4-12) with 

30 a mean blood Pb level of 11.39 ± 1.39 μg/dL compared to children with mean blood Pb levels of 3.93 ± 

31 0.61 μg/dL. Similar decreases were also observed in children 3-6 years of age with >10 μg/dL, compared 

32 to children <10 μg/dL (Y. P. Jin et al., 2006). 

33 Rats administered 500 ppm Pb acetate in drinking water for 15 or 30 days had decreased blood 

34 ALAD activity that was related to duration of exposure and blood Pb (Rendón-Ramirez et al., 2007). 

35 Administration of Pb (25 mg/kg) to rats once a week for 4 weeks achieved a blood Pb level of 6.5 μg/dL, 
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1 which were associated with significant decreases (approximately 50% lower than control levels) in RBC 

2 ALAD activity (M. K. Lee et al., 2005).

5.7.4. Other Heme Metabolism Enzymes

3 The 2006 Pb AQCD report indicates Pb affects RBC PBG synthase (Farant & Wigfield, 1987); 

4 (Farant & Wigfield, 1990; Simons, 1995), PBG deaminase (Tomokuni & Ichiba, 1990), and TF 

5 endocytosis and iron transport across membranes (Z. M. Qian & Morgan, 1990), all of which are directly 

6 or indirectly involved in heme synthesis. Although there are no new studies that measure the effect Pb has 

7 on other heme metabolism enzymes’ activities, a number of studies investigated the effect blood Pb had 

8 on concentrations of various intermediate products in the heme biosynthetic pathway. 

9 Pb intoxication is known to inhibit the function of ferrochelatase, the enzyme that catalyzes the last 

10 step in the heme biosynthetic pathway. Under normal conditions, ferrochelatase incorporates ferrous iron 

(Fe2+ 11 ) into protoporphyrin IX, converting it into a heme molecule. However, Pb inhibits this insertion of 

Fe2+ 12 into the protoporphyrin ring and instead, Zn is inserted into the right creating ZPP. A number of 

13 recent studies have shown that blood Pb is statistically significantly associated with increased RBC ZPP 

14 levels in humans (Ademuyiwa, Ugbaja, Ojo, et al., 2005; Counter et al., 2007; Mohammad et al., 2008; 

15 Patil, Bhagwat, Patil, Dongre, Ambekar, Jailkhani, et al., 2006) and animals (Rendón-Ramirez et al., 

16 2007). Patil et al. (2006) observed a small, but not statistically significant increase in RBC ZPP levels 

17 among silver jewelry workers exposed to Pb, compared to nonexposed controls. Interestingly, Q. Wang et 

18 al. (2010) found that in children and adults living in a rural area of Southwest China, ZPP levels were 

19 decreased at the low blood levels of Pb and were only increased with higher blood Pb levels. The authors 

20 suggest that this may be representative of ALAD activities at low Pb levels, which contributes to lower 

21 ZPP levels. Scinicariello et al. (2007) performed a meta-analysis and observed that Pb-exposed 

22 individuals that carried the ALAD2 allele had slightly, but not statistically significant, lower 

23 concentrations of blood ZPP levels compared to carriers of the ALAD1 allele.

5.7.5. Effects on Other Hematological Parameters

5.7.5.1. Energy Metabolism

24 RBCs use high energy purine nucleotides (i.e., ATP and guanine triphosphate [GTP]) to support 

25 basic metabolic functions. In mature RBCs, these nucleotides are synthesized via salvage reactions via 

26 either an adenine pathway, which requires adenine phosphoribosyltransferase (APRT), or an adenosine 

27 pathway, which requires adenosine kinase. The 2006 Pb AQCD reports that Pb significantly reduces the 

28 nucleotide pool including NAD and NADP, as well as increases purine degradation products resulting in 

29 altered RBC energetics. Since the 2006 report, there have been few studies examining Pb effects on 
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1 energy metabolism. Baranowska-Bosiacka et al. (2009) examined the effects of Pb on RBC APRT and 

2 hypoxanthine-guanine phosphoribosyltransferase (HPRT) due to in vitro and in vivo exposures. For the in 

3 vitro exposure, APRT and HPRT were measured in lysate of human RBCs after exposure to Pb at 

4 concentration range from 100 nM to 100 μM for 5-30 minutes of exposure. In vivo tests measured APRT 

5 and HPRT in rat RBC lysate from rats exposed to Pb acetate (0.1 %) in drinking water for 9 months. Both 

6 the in vivo and vitro studies found a significant decrease in both HPRT and APRT levels. The levels were 

7 significantly decreased in vitro after only 5 minutes of exposure to the 100 nM concentration, but the 

8 decrease was also dose-dependent. However, the study authors considered the inhibition moderate 

9 (30-35%) even with the highest levels used in vitro. Shin et al. (2007) found a dose-dependent decrease in 

10 intracellular ATP in human RBCs in vitro with significant decreases found even with the lowest 

11 concentration (i.e., 1 μM).

5.7.5.2. Other Enzymes

The 2006 Pb AQCD reports that K+ 12 permeability was increased by Pb due to altered sensitivity of 

the membrane Ca2+-binding site that caused selective efflux of K+ 13 ions from the RBC membrane. 

However, inhibition of the RBC Na+-K+ 14 ATPase is more sensitive to Pb exposure than the inhibition of 

Ca2+-Mg2+ 15 ATPase. Only two studies since the 2006 report were found that examined the effects of Pb 

16 exposure on other enzymes. Ekinci et al. (2007) tested the effects of Pb on two carbonic anhydrase 

17 isozymes (I and II) isolated from human RBCs. Carbonic anhydrases are metalloprotein that use Zn to 

18 catalyze the equilibrium between carbon dioxide and bicarbonate in the cells of higher invertebrates. 

19 Although they found Pb nitrate inhibited both carbonic anhydrase isozymes in a concentration-dependent 

manner, the concentrations used (i.e., 2 × 10-4 to 1 × 10-3 20 M) were above those that would be 

21 physiologically relevant. Inhibition of isozyme I was noncompetitive, while the inhibition for isozyme II 

22 was uncompetitive. Bitto et al. (2006) examined the mechanisms of action of Pb-induced inhibition of 

23 P5N, an enzyme important in the pyrimidine salvage pathway that requires manganese for normal 

24 activity. Pb was observed to bind directly to the enzyme’s active site in a different position than the 

25 manganese, thus possibly resulting in improper protein folding and inhibition of activity. 

5.7.6. Red Blood Cell Oxidative Stress

26 It has been suggested that the Pb-associated decreases in ALAD activity result in increased 

27 oxidative stress, owing to the buildup of ALA. ALA can act as an electron donor in the formation of 

28 reactive oxygen species (ROS) (Ahamed & Siddiqui, 2007; Nemsadze et al., 2009). Many studies have 

29 found an association between the level of blood Pb and lipid peroxidation, antioxidant levels, or 

30 indicators of ROS production.
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5.7.6.1. Oxidative Stress, Lipid Peroxidation, and Antioxidant Enzymes

1 Malondialdehyde (MDA) is an end product of lipid peroxidation and is commonly used as an 

2 indicator of lipid peroxidation. Patil et al. (2006) found significantly higher blood Pb in the jewelry 

3 workers (48.56 ± 7.39 μg/dL) compared to individuals not occupationally exposed (12.52 ± 4.08 μg/dL) 

4 to Pb. These workers had significantly higher plasma MDA, along with significantly lower levels of RBC 

5 SOD, catalyses, and plasma ceruloplasm, all of which are indicators of oxidative stress in the RBCs. Patil 

6 et al. (2006) found similar effects in a group of battery manufacturing workers in India. Levels of MDA 

7 were significantly positively correlated with blood Pb in workers occupationally exposed to Pb, but no 

8 correlation was observed in controls. These effects were also demonstrated in vitro by Ciubar et al. 

9 (2007), but only with concentrations of 2 μM (highest concentration tested). In this study, RBCs from 

10 nine volunteers were incubated with Pb at concentrations ranging from 0.1-2 μM for 24 hours. Evidence 

11 of lipid peroxidation was also observed in auto repair apprentices in Turkey with blood Pb levels as low 

12 as 7.9 μg/dL (compared to 2.6 μg/dL in controls) (Ergurhan-Ilhan et al., 2008), including increases in 

13 glutathione peroxidase (GPx) and MDA, as well as decreases in α-tocopherol and β-carotene. Decreases 

14 were observed in SOD and CAT, but the results did not achieve statistical significance. Decreased 

15 glutathione reductase (GR) activity was observed in human RBCs incubated with 5-18 μM Pb in vitro 

16 (Coban et al., 2007). Industrial workers in Pakistan occupationally exposed to Pb had a significant 

17 increase in blood Pb levels (mean: 29.1 μg/dL, range: 9.0 to 61.1 μg/dL) compared to age and gender 

18 matched controls (mean: 8.3 μg/dL, range: 1.0 to 21.7 μg/dL) (D. A. Khan et al., 2008). The industrial 

19 workers also had increased oxidative stress as measured by increased levels of serum MDA and 

20 C-reactive protein (CRP). In painters in India with an average blood Pb level of 21.92 μg/dL (compared 

21 to 3.06 μg/dL in controls), there was a significant decrease in SOD, glutathione (GSH), and CAT 

22 accompanied by a significant increase in oxidized GSH (i.e., GSSG) and thiobarbituric acid reactive 

23 species (TBARS, expressed in terms of MDA) measured in plasma and RBC lysate (Mohammad et al., 

24 2008). Quintanar-Escorza et al. (2007) found elevated RBC lipid peroxidation measured as increased 

25 MDA levels in Pb-exposed workers in a recycled automobile battery factory in Mexico. There was a 

26 correlation in the MDA levels and blood Pb even in the unexposed workers who had low (i.e., <12 μg/dL) 

27 blood Pb levels, although the observed correlation in exposed workers was greater. Similar effects were 

28 seen when RBCs from healthy volunteers with no Pb exposure were incubated with 0.4 μM Pb for 24 

29 hours (Quintanar-Escorza et al., 2010). MDA concentrations, SOD and GPx activities were observed to be 

30 elevated in normotensive exposed workers compared to controls (S. Kasperczyk et al., 2009). The 

31 concentration of MDA was statistically significantly greater in workers with hypertension compared to 

32 both controls and Pb-exposed normotensive workers, whereas the activity of GR in hypertensive workers 

33 decreased to levels comparable to those seen in the control group. Exposure related increases in lipid 

34 peroxidation were also observed in occupationally exposed workers in Poland (S. Kasperczyk et al., 
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1 2005). The concentrations of MDA and 7-ketocholesterol (oxidized cholesterol) were significantly greater 

2 in exposed hypertensive workers compared to normotensive workers. 

3 Ahamed et al. (2005) investigated the relationship between blood Pb levels and antioxidant enzyme 

4 levels and lipid peroxidation in children aged 4-12 years in Lucknow, India. A total of 62 children, with a 

5 mean blood Pb level of 7.47 ± 3.06 μg/dL, were included in the study; children were separated into three 

6 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 

7 III, 11.39 ± 1.39 μg/dL. Lipid peroxidation, measured as blood MDA, was statistically significant greater 

8 in group III, compared to group II and I, whereas GSH was decreased in group III relative to groups II 

9 and I. Catalase activity was the only measure of oxidative stress that was statistically significantly 

10 elevated in group II compared to group I. Additionally, blood Pb levels were found to be statistically 

11 significantly positively correlated with MDA and CAT and negatively correlated with GSH. Ahamed et al. 

12 (2006) additionally studied male urban adolescents in India. The 39 adolescents were separated into 

13 groups according to their blood Pb level (group 1: <10 μg/dL, group 2: >10 μg/dL). Although the groups 

14 were similar in their age, height, weight, and body mass index, group 2 had significantly higher levels of 

15 CAT and MDA compared to group 1. There were no significant differences in blood GSH levels. 

16 Examining all the study subjects together, there was a correlation between the blood Pb level and blood 

17 MDA and RBC CAT levels, as well as an inverse relationship between ALAD activity and MDA and CAT 

18 levels. In a similar study, Ahamed et al. (2008) examined oxidative stress in Indian children with 

19 neurological disorders. There was a significantly higher blood Pb level in the study population compared 

20 to the control population (18.60 versus 10.37 μg/dL). In addition, the following indicators of oxidative 

21 stress were observed in the study population: increased blood MDA, RBC SOD, and CAT levels and 

22 decreased blood GSH levels. GPx levels were similar between the two groups. Typical indicators of Pb 

23 exposure (active/nonactive ALAD ratio and) were found to be correlated with lipid peroxidation and 

24 oxidative stress. Children aged 3-6 years old living near a steel refinery in China with blood Pb levels ≥ 

25 10 μg/dL also demonstrated a significant increase in plasma MDA compared to the children with blood 

26 Pb levels<10 μg/dL. However, levels of RBC SOD, GSH, and GPx were not different from controls (Y. P. 

27 Jin et al., 2006). 

28 Administration of Pb (25 mg/kg) to rats once a week for 4 weeks, which was related to a blood Pb 

29 level of about 6.5 μg/dL, caused a significant increase in RBC MDA levels (M. K. Lee et al., 2005). Other 

30 indications of oxidative stress included significant increases in RBC SOD and CAT levels accompanied 

31 by significant decreases in GSH and GPx. Exposure of rats to 750 mg/kg Pb acetate in drinking water for 

32 11 weeks resulted in decreased concentrations of plasma vitamin C, vitamin E, nonprotein thiol, and 

33 RBC-reduced glutathione, while simultaneously increasing the activity of SOD and GPx (Kharoubi, 

34 Slimani, Krouf, et al., 2008). CAT activity was also slightly elevated in Pb-exposed rats, but the increase 

35 failed to reach statistical significance. SOD activity was significantly decreased in rats injected with 15 

36 mg/kg Pb i.p. for seven days, but not rats exposed to 5 mg/kg Pb (Berrahal et al., 2007). GPx activity and 
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1 MDA concentrations were slightly elevated in the exposure groups, but failed to reach statistical 

2 significance.

5.7.6.2. Antioxidant Defense

3 In addition to the studies listed above that examine lipid peroxidation and oxidative stress, there 

4 have been studies that indicate that the use of antioxidants and free radical reactions were protective 

5 against Pb-induced RBC oxidative stress. Rats treated with 500 ppm Pb acetate in drinking water for 15 

6 or 30 days had an increase in free RBC protoporphyrin and TBARS that was related to length of exposure 

7 and blood Pb (Rendón-Ramirez et al., 2007). Vitamin E administration after exposure to Pb significantly 

8 reduced the TBARS levels and increased ALAD activity, compared to exposure to Pb alone. Co-exposure 

9 to vitamin E and Pb simultaneously and exposure to vitamin E before Pb exposure also prevented Pb10 induced oxidative stress. In vitro studies by Casado et al. (2006), found that hemolysis and RBC 

11 membrane damage was mediated via oxidative stress. The in vitro studies demonstrated a concentration12 and time-dependent formation in lipid peroxide that was inhibited with a number of antioxidants, 

13 including desferrioxamine (iron chelator), trolox (chain breaking antioxidant), and mannitol and Na 

formate (• 14 OH scavengers). Results suggested the role of singlet oxygen in Pb-mediated membrane 

15 damage and hemolysis of exposed RBCs. In rats exposed to 2000 ppm Pb in drinking water for 5 weeks, 

16 MDA levels were significantly increased, whereas vitamin E concentrations were significantly decreased 

17 (Caylak et al., 2008). In the case of MDA, co-exposure to Pb and a number of sulfur-containing 

18 antioxidants (e.g., L-methionine, N-acetylcysteine, and L-homocysteine) reduced concentrations to a level 

19 not statistically significantly different from controls, but statistically smaller than concentrations observed 

20 with Pb alone. Exposure to L-methionine an N-acetylcysteine also reduced Pb-induced depletion of 

21 vitamin E. 

5.7.7. Summary and Causal Determination

22 There is consistent toxicological and epidemiologic evidence that exposure to Pb induces adverse 

23 effects on hematological endpoints, including altered heme synthesis, decreased RBC survival and 

24 function, and increased RBC oxidative stress. Pb preferentially partitions into RBCs following exposure, 

25 with RBC concentrations approximately 100-fold greater than those observed in the plasma (C. Jin et al., 

26 2008; Timchalk et al., 2006). 

27 Multiple occupational epidemiologic studies have observed that Pb affects several hematological 

28 parameters such as Hb, PCV, MCV, MCH, and MCHC (Karita et al., 2005; D. A. Khan et al., 2008; Patil, 

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

30 2006; Ukaejiofo et al., 2009). Although the majority of occupationally exposed adults had blood Pb levels

31 in excess of 20 μg/dL, decreases in Hb and PCV were observed in adults with blood Pb levels of 7 μg/dL. 
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In addition, Pb exposure was shown to reduce Ca2+- and Ca2+-Mg2+ 1 -ATPase activity in RBC membranes, 

which leads to an increase in RBC [Ca2+]i 2 , increased membrane fragility, and abnormal morphological 

3 changes (Ciubar et al., 2007; Huel et al., 2008; Quintanar-Escorza et al., 2010; Quintanar-Escorza et al., 

2007). Heul observed a reduction of RBC Ca2+-Mg2+ 4 -ATPase activity at a cord blood Pb level of 3.54 

5 μg/dL. Studies in children are more equivocal than those investigating occupationally exposed adults; this 

6 may due to the comparatively shorter duration of and magnitude of exposure experienced by children. 

7 Toxicological studies have also observed decreases in Hct and Hb and increases in hemolysis and 

8 reticulocyte density in rats and mice with blood Pb levels as low as 6.6-7.1 μg/dL (Baranowska-Bosiacka 

9 et al., 2009; M. K. Lee et al., 2005; Sharma et al., 2010; Simsek et al., 2009). Pb exposure has also been 

10 observed to increase PS expression on RBC membranes, leading to cell shrinkage, eyrptosis, and 

11 destruction of the RBCs by macrophages (Ciubar et al., 2007; Shin et al., 2007). Suggestive evidence of 

12 disrupted hematopoiesis evidenced by decreased serum erythropoietin was observed in occupationally 

13 exposed adults with a blood Pb level of 6.4 μg/dL; toxicological studies in rats also indicate that Pb is 

14 cytotoxic to RBC progenitor cells. Taken together, these studies provide consistent evidence that exposure 

15 to Pb adversely effects RBC function and survival, and leads to the reduction of RBCs in circulation. 

16 Although this decrease in RBCs may be explained by both decreased cell survival and/or disruption of 

17 hematopoiesis, the observation of increased reticulocytes seems to represent compensation for decreased 

18 RBC survival due to Pb exposure. 

19 Recently, numerous epidemiologic studies have confirmed that decreases in RBC ALAD levels and 

20 activity are strongly associated with Pb exposure in adults and children at blood Pb levels as low as 6.4 

21 and 7.1 μg/dL, respectively (Ademuyiwa, Ugbaja, Ojo, et al., 2005; Mohammad et al., 2008; Patil, 

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

23 2006; Quintanar-Escorza et al., 2007). Decreases in blood ALAD activity were also seen in rats with 

24 blood Pb levels of 6.5 μg/dL (M. K. Lee et al., 2005). In addition to ALAD, recent studies have shown 

25 that Pb exposure inhibits the activity of ferrochelatase, leading to increased RBC ZPP in humans 

26 (Ademuyiwa, Ugbaja, Ojo, et al., 2005; Counter et al., 2007; Mohammad et al., 2008; Patil, Bhagwat, 

27 Patil, Dongre, Ambekar, Jailkhani, et al., 2006) and animals (Rendón-Ramirez et al., 2007). Pb has also 

28 been shown to inhibit the activities of other enzymes in RBCs, including those involved in nucleotide 

29 scavenging, energy metabolism, and acid-base homeostasis (Baranowska-Bosiacka et al., 2009; Ekinci et 

30 al., 2007).

31 Lastly, Pb exposure induces lipid peroxidation and oxidative stress in RBCs. Epidemiologic studies 

32 have observed increases in MDA in occupationally-exposed adults with blood Pb levels as low as 7.9 

33 μg/dL (Ergurhan-Ilhan et al., 2008; D. A. Khan et al., 2008; Mohammad et al., 2008; Patil, Bhagwat, 

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

35 Quintanar-Escorza et al., 2007). Other measures of oxidative stress observed included lowered activities 

36 of SOD, GR, and CAT, and increased CRP. Indices of RBC oxidative stress were also seen in adolescents 
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1 and children exposed to Pb (Ahamed et al., 2008; Ahamed et al., 2006; Y. P. Jin et al., 2006). In vitro and 

2 vivo studies have also demonstrated that prior, concurrent, or subsequent treatment with various 

3 antioxidants has been shown to at least partially ameliorate Pb-induced oxidative stress in RBCs (Casadoa 

4 et al., 2006; Cecil et al., 2008; Rendón-Ramirez et al., 2007).

5 Similar to the epidemiologic and toxicological studies that demonstrate an association between Pb 

6 exposure and hematological effects in humans and laboratory animals, the ecological literature has 

7 consistently reported on hematological responses in aquatic and terrestrial invertebrates and vertebrates 

8 (Section 7.4.5). The most consistently observed effect in metal impacted environments is decreased RBC 

9 ALAD activity. This effect has been observed across a wide range of taxa, including bivalves, fish, 

10 amphibians, birds, and mammals. More limited evidence exists regarding deleterious effects of Pb on 

11 serum enzyme levels and white blood cell counts in birds and mammals.

12 In conclusion, the recent epidemiologic and toxicological literature provides strong evidence that 

13 exposure to Pb is associated with numerous deleterious effects on the hematological system, including 

14 altered heme synthesis mediated through decreased ALAD and ferrochelatase activities, decreased RBC 

15 survival and function, decreased hematopoiesis, and increased oxidative stress and lipid peroxidation. The 

16 consistency of findings in the epidemiologic and toxicological literature and coherence across the 

17 disciplines is sufficient to conclude that there is a causal relationship between Pb exposures and effects 

18 on heme synthesis and red blood cell function.

5.8. Reproductive Effects and Birth Outcomes

19 The effect of Pb on reproductive outcomes has been of interest for years, starting in cohorts of 

20 occupationally exposed individuals. More recently, researchers have begun to focus on reproductive 

21 effects in people with environmentally relevant Pb exposure. In the toxicological and epidemiologic 

22 literature, research on reproductive effects of Pb include female and male reproductive function (hormone 

23 levels, fertility, puberty, and effects on reproductive organs and estrus), birth defects, spontaneous 

24 abortions, infant mortality, preterm birth, low birth weight/fetal growth, and other developmental effects. 

25 In epidemiologic studies, various biological measures of Pb are used including Pb measured in blood and 

26 bone; toxicological studies only report exposure using blood Pb. Bone Pb is indicative of cumulative Pb 

27 exposure. Blood Pb can represent more recent exposure, although it can also represent remobilized Pb 

28 occurring during times of bone remodeling. More detailed discussion of these measures and Pb transfer 

29 via umbilical cord blood Pb, across the placenta, and via lactation is given in Section 4.3.5 on Pb 

30 Toxicokinetics. A few studies of pregnancy-induced hypertension and eclampsia have been conducted and 

31 are reported on in the section on hypertension (Section 5.4.2.1). Briefly, the relatively small number of 
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1 studies found consistently positive associations between recent Pb exposure and pregnancy-induced 

2 hypertension. 

3 Overall, the recent reproductive literature continues to support associations reported in earlier 

4 AQCDs between Pb exposure and adverse outcomes on various parameters of sperm (function, motility, 

5 count, integrity, histology). The toxicological and epidemiologic literature also support the finding that Pb 

6 exposure is consistently associated with delayed onset of puberty in both males and females. The new 

7 information from epidemiologic and toxicological studies and conclusions from previous AQCDs are 

8 summarized below. 

5.8.1. Effects on Female Reproductive Function

9 The epidemiologic studies presented on Pb and female reproductive function in the 2006 AQCD 

10 (U.S. EPA, 2006) provided little evidence on the possible associations between Pb exposure and female 

11 reproduction and fertility. However, the 1986 and 2006 Pb AQCDs (U.S. EPA, 1986, 2006) reported

12 toxicological findings that Pb exposure is associated with effects on female reproductive function that can 

13 be classified as alterations in female sexual maturation, effects on fertility and menstrual cycle, endocrine 

14 disruption, and changes in morphology or histology of female reproductive organs including the placenta. 

15 Since the 2006 AQCD, many epidemiologic studies have been published regarding Pb levels in women 

16 and their effects on reproduction. In addition, recent toxicological studies add further knowledge of Pb17 related effects on the female reproductive system. 

5.8.1.1. Effects on Female Sex Endocrine System and Estrus Cycle

18 Multiple studies have examined the association between Pb and its effects on hormones and the 

19 estrus cyclce. Epidemiologic studies support the toxicological findings, which have the majority of the 

20 evidence. 

21 An epidemiologic study using the NHANES III database and including women aged 35-60 years 

22 old examined the relationship between blood Pb levels (mean 2.8 μg/dL) and serum follicle stimulating 

23 hormone (FSH) and luteinizing hormone (LH) (E. F. Krieg, Jr., 2007). Deviation from normal FSH and 

24 LH levels may indicate endocrine disruption related to ovary functioning. Researchers determined that as 

25 blood Pb levels increased, serum FSH and LH increased among both post-menopausal women and 

26 women with both ovaries removed. There was also an increasing trend for pre-menopausal women who 

27 were not menstruating or pregnant, although the association was not statistically significant for LH. 

28 Increasing blood Pb levels were associated with decreasing levels of serum FSH among women taking 

29 birth control pills. The inverse association was also present for LH, but it was not statistically significant. 

30 No associations between blood Pb and FSH or LH were apparent for women who were menstruating at 

31 the time of the exam or were pregnant. Further analysis found that the lowest level of blood Pb for which 
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1 a statistically significant association could be observed between blood Pb and FSH was 1.7 μg/dL among 

2 women with their ovaries removed. A limitation of the study is that FSH and LH were measured without 

3 attention to day of a woman’s menstrual cycle and LH and FSH are known to vary throughout the cycle. 

4 Another epidemiologic study was performed in Kaohsiung City, Taiwan among two groups of women 

5 aged 23-44: those who were seeking help at a fertility clinic after one year of trying to conceive, and those 

6 who had previously delivered an infant and were identified from medical records of a postpartum care 

7 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 

8 study reported a positive association between increased blood Pb levels and serum estradiol 

9 concentrations, which reflects ovary activity.

10 The effect of Pb exposure on the female endocrine system has been demonstrated in toxicological 

11 studies in the 1986 and 2006 Pb AQCD (U.S. EPA, 1986, 2006). However, the mechanism by which Pb 

12 affects the endocrine system has not been fully elucidated. Several recent articles continue to demonstrate 

13 that Pb alters the concentration of circulating hormones in female experimental animals. As mentioned 

14 previously, Pine et al. (2006) observed that maternal Pb exposure causes a decrease in basal LH levels in 

15 pre-pubertal female Fisher 344 rat pups when compared to non-Pb exposed pups during gestation and 

16 lactation. Dumitrescu et al. (2008) observed alteration of hormone levels in female Wister rats after 

17 ingesting Pb acetate (50, 100, 150 ppb) in drinking water for six months. The authors reported decreases 

18 in FSH, estradiol, and progesterone levels with increases in LH and testosterone levels. Nampoothiri and 

19 Gupta (2008) administered Pb acetate at a concentration that did not affect reproductive performance, 

20 implantation or pregnancy outcome (0.05 mg/kg body weight) to Charles Foster female rats 5 days before 

21 mating and during the gestational period. They observed a decrease in steroidogenic enzymes, 3β-HSD 

22 and 17β-HSD, activity in reproductive organs, as well as a decrease in steroid hormones (progesterone 

23 and estradiol), suggesting that chronic exposure to low levels of Pb may affect reproductive function of 

24 mothers and their offspring.

25 Kolesarova et al. (2010) conducted an in vitro study to examine the secretory activity of porcine 

26 ovarian granulose cells after Pb administration. The results of the study showed that Pb acetate 

27 concentrations of 0.046 mg/mL and 0.063 mg/mL statistically significantly inhibited IGF-1 release, but 

28 concentrations of 0.25 mg/mL and 0.5 mg/mL did not influence IGF-1 release. Progesterone release was 

29 not affected by Pb treatment; however, Pb caused a reduction in LH and FSH binding in granulose cells 

30 and increased apoptosis as evidenced by increased expression of caspase-3 and cyclin B1, suggesting a 

31 Pb-induced alteration in the pathways of proliferation and apoptosis of porcine ovarian granulose cells. 

32 Decreased gonadotropin binding was also observed in rats after Pb exposure (Nampoothiri & Gupta, 

33 2006).

34 No recent toxicological studies were found that addressed Pb-induced effects on the estrus cycle.

35 Overall, toxicological studies report alterations in hormone levels related to Pb concentration. This 

36 was also observed in epidemiologic studies. Although these changes are observed, there are discrepancies 
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1 about the direction of the hormone changes related to Pb. One explanation is that the direction of effect 

2 could vary based on current hormonal and reproductive status.

5.8.1.2. Effects on Fertility

3 Previous studies indicated that Pb exposure does not produce total sterility, but it can disrupt female 

4 fertility (U.S. EPA, 2006). Recent epidemiologic studies and studies in experimental animals support this 

5 finding. The epidemiologic studies are summarized in Table 5-27. 

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

6 Epidemiologic studies examined women having difficulty conceiving by performing studies 

7 among patients of fertility clinics or undergoing in vitro fertilization (IVF). The first of these was 

8 performed in Kaohsiung City, Taiwan among women aged 23-44 (S. H. Chang et al., 2006). A difference 

9 in blood Pb was reported between women who were seeking help at a fertility clinic after one year of 

10 trying to conceive and women who had previously delivered an infant and were identified from medical 
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1 records of a postpartum care unit at a medical center. Higher odds of infertility were observed when

2 comparing women with blood Pb levels >2.5 μg/dL to those with blood Pb levels ≤ 2.5 μg/dL. Another

3 study examining fertility reported on women in Saudi Arabia aged 19-50 years who were undergoing IVF 

4 treatment (Al-Saleh et al., 2008). Women were categorized as having achieved a pregnancy versus not 

5 achieved a pregnancy and achieved fertilization versus not achieving fertilization. The majority of women 

6 had follicular Pb levels that were below the level of detection, whereas less than 2% of women had blood 

7 Pb levels below the limit of detection. In addition, less than 2% of women had blood Pb levels that were 

8 above 10 μg/dL. Follicular Pb levels were not correlated with the blood Pb. No association was observed 

9 between blood or follicular Pb and pregnancy outcomes in either crude or adjusted models. An association 

10 was not detected between follicular Pb and fertilization but an inverse association was detected between 

11 blood Pb and fertilization. Finally, a study that included nine women undergoing IVF treatment in Rhode 

12 Island (Silberstein et al., 2006) found that median follicular Pb levels in women who achieved pregnancy 

13 were lower than the follicular Pb levels among non-pregnant women. One limitation present in these 

14 studies is that the participants, especially in the later two studies, are women who are seeking help for 

15 fertility problems. The studies are not a sample of the general population and therefore cannot be 

16 generalized to all women of childbearing age.

17 Several studies observed a decrease in litter size when females were exposed to Pb before mating 

18 or during pregnancy (Dumitrescu, Alexandra, et al., 2008; Iavicoli, Carelli, Stanek, Castellino, Li, et al., 

19 2006; Teijon et al., 2006). Pups in Teijon et al.’s study receiving 400 ppm Pb acetate in drinking water had 

20 blood Pb of 97 μg Pb/dL blood at 1 wk post-weaning and 18.2 μg Pb/dL blood at 2 wk post-weaning. 

21 Dumitrescu et al. observed a modification in sex ratio of pups born to dams exposed to Pb before mating 

22 and during pregnancy. As the dose of Pb increased, the number of females per litter also increased (i.e., 1 

23 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 

24 2.25 females in 100 ppb group; and 1 male to 2.5 females in 150 ppb group). These results are not 

25 consistent with earlier results of Ronis et al. (1998), who did not observe differences in sex ratio if liters 

26 from females exposed only during pregnancy. Thus, Pb exposure in animal studies during or before 

27 pregnancy have shown effects on litter size and mixed effects on sex ratio.

28 Nandi et al. (2010) demonstrated a dose-dependent decline in viability rate, maturation, 

29 fertilization, and cleavage rates of buffalo oocytes cultured in medium containing 1-10 μg/mL Pb acetate. 

30 Karaca and Şimşek (2007) observed an increase in the number of mast cells in ovary tissue after Pb 

31 exposure (2 g/L in drinking water) suggesting that Pb may stimulate an inflammatory response in the 

32 ovaries which may contribute to Pb-induced female infertility.

33 In contrast, Nampoothiri and Gupta (2008) did not observe any statistically significant change in 

34 fertility rate or litter size in female rats subcutaneously administered 0.05 mg/kg body weight daily before 

35 mating and during pregnancy with a resulting blood Pb of 2.49 μg/mL. Although reproductive 
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1 performance was not affected in this study, the authors did report an alteration in implantation enzymes. 

2 Cathepsin-D activity decreased and alkaline phosphatase activity increased after Pb exposure.

3 Epidemiologic and toxicological studies on the effect of Pb on fertility outcomes have generated 

4 inconsistent results. However, there is some indication that increased Pb exposure may decrease fertility.

5.8.1.3. Effects on Puberty

5 Recent toxicological studies of rodents have examined the effects of Pb on pubertal and 

6 reproductive organ development and on biomarker development. There have also been recent 

7 epidemiologic studies examining Pb levels and onset of puberty, which are summarized in Table 5-28 and 

8 in the text below. 

Table 5-28. Summary of recent epidemiologic studies of effects on puberty for females

Reference Study Location 

and Years

Outcome Study 

Population

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 centered at the mean:

log blood Pb -1.29 (p-value 

0.01)

log blood Pb -squared: -

1.01 (p-value 0.08)

Non-linear relationship 

observed and Pb below the 

mean did not appear to 

affect the odds of 

menarche. Increasing 

blood Pb from 0.49 to 0.98 

μg/dL decreased the odds 

of menarche attainment by 

72%

Gollenberg et 

al. (2010)

U.S.A.

1988-1994

Luteinizing 

hormone (LH) 

and inhibin B

Girls ages 6-11 from 

the NHANES III study

Blood Pb Median 2.5 

(range 0.07, 

29.4)

blood Pb

>10 μg/dL: 5%

OR (95% CI) for exceeding 

pubertal inhibin 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 attenuated
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Reference Study Location 

and Years

Outcome Study 

Population

Exposure 

Measurement

Mean Pb 

(SD) in 

μg/dL

Adjusted Effect 

Estimates

Naicker et al. 

(2010)

Johannesburg/Soweto, 

South Africa

Born in 1990

Self-reported 

Tanner 

staging at age 

13 and age at 

menarche 

Girls of black or mixed 

ancestry who were 

enrolled in the Birth to 

Twenty (Bt20) cohort 

(born in 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 breast 

development at age 13

<5 μg/dl: 1.00 (Ref)

≥ 5μg/dl: 2.34 (1.45, 3.79)

Delay in pubic hair 

development at age 13

<5 μg/dl: 1.00 (Ref)

≥ 5μg/dl: 1.81 (1.15, 2.84)

Delay in attainment 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 

staging and 

age at 

menarche

Girls ages 8-18 from 

the NHANES III study 

Blood Pb Geometric 

mean 

NHWhites: 1.4

NHBlacks: 2.1

MexicanAmericans: 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%

MexicanAmericans: 

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, assume results are 

per 1 μg/dL)

Breast stage: 1.01 (0.79, 

1.30)

Pubic hair stage: 1.25 

(0.83, 1.88)
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Reference Study Location 

and Years

Outcome Study 

Population

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 ages 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)

Menarche

0.7-2.0 μg/dl: 1.00 (Ref)

2.1-4.9 μg/dl: 0.42 (0.18, 

0.97)

5.0-21.7 μg/dl: 0.19 (0.08, 

0.43)

1 Several epidemiologic studies investigated the association between blood Pb and indicators of 

2 puberty onset. A study performed in NYC among 9 year old girls reported no association between Pb 

3 levels and pubertal development (Wolff et al., 2008). However, a study among girls aged 10-13 (median 

4 age 12) reported decreased levels of FSH and LH levels in the group with blood Pb of at least 10 μg/dL

5 compared to the group with blood Pb less than 10 μg/dL (Tomoum et al., 2010). In addition, there were 

6 some indications of lower Tanner stages of breast development associated with Pb levels of at least 10 

7 μg/dL, but this relationship was not present for stages of pubic hair development. A study of girls aged 

8 10-16.9 years of age in the Akwesasne Mohawk Nation reported a non-linear positive association between 

9 blood Pb and age at menarche (Denham et al., 2005). No association was observed below blood Pb of 

10 0.49 μg/dL. A study conducted in South Africa reported a positive association between blood Pb levels 

11 and age at menarche and pubertal development (Naicker et al., 2010). Blood Pb levels were associated 

12 with delayed pubertal development and later age at menarche. This study illustrates not only the 

13 association between Pb and pubertal development, but that delays can occur at low Pb levels. Multiple

14 studies have been performed examining blood Pb levels and puberty using the NHANES III database 

15 (Gollenberg et al., 2010; Selevan et al., 2003; T. Wu et al., 2003). One study included girls aged 8-16 and 

16 reported an positive association for delayed attainment of menarche and pubic hair development, but not

17 for breast development (T. Wu et al., 2003). The associations were observed even at low levels of blood 

18 Pb. Another NHANES III study included girls 8-18 years of age and reported the results stratified by race 

19 (Selevan et al., 2003). Blood Pb levels were inversely associated with Tanner stage of breast and pubic 

20 hair development and age at menarche among African Americans and with breast and pubic hair 

21 development among Mexican Americans. The associations were in the same directions for whites, but 

22 none of the associations reached statistical significance. A third study using the NHANES III database 

23 examined the association between Pb and reproductive hormones among girls 6-11 years old (Gollenberg 

24 et al., 2010). Blood Pb levels were inversely associated with inhibin B, a protein that inhibits FSH
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1 production, but no association was observed for LH. The inverse association between blood Pb and 

2 inhibin B was greater among girls with iron deficiency compared to those with high Pb but sufficient iron 

3 levels. Inhibin B and LH were chosen for this study because these hormones are, “believed to be relevant 

4 for younger girls... near the onset of puberty and...serve as markers for hypothalamic-pituitary-gonadal 

5 functioning.” 

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. 
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Table 5-29. Toxicological Exposure-Response Array Summaries for Reproductive Effects 

of Pb presented in Figure 5-54

Reference Blood Pb level 

with Effect (μg/dl) Altered 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

1 Earlier studies showed that prenatal and lactational exposures to Pb can cause a delay in the onset 

2 of female puberty in rodents. Recent studies confirm these findings and show that puberty onset is one of 

3 the more sensitive markers of Pb exposure as is demonstrated in the exposure response array (Table 5-29 

4 and Figure 5-54). Dumitrescu et al. (2008) exposed adult Wister female rats to varying doses of Pb acetate 

5 (50-150 ppb) in drinking water for 3 months before mating and during pregnancy. Vaginal opening, an 

6 indicator of sexual maturation, was statistically significantly delayed in pups from all Pb treated groups 

7 when compared to pups from non-treated dams. The age at vaginal opening in female pups from the Pb 

8 treated groups increased, in a dose-dependent manner, from 39 days to 43-47 days. The authors also 

9 observed a correlation between body weight and age at vaginal opening meaning that as body weight 

10 decreased the age at vaginal opening increased. This effect also exhibited a dose-dependent relationship. 

11 In another recent study, Iavicoli et al. (2006) reported a statistically significant delay in several 

12 biomarkers of sexual maturity in offspring (F1 generation) born to dams that ingested 3.5-40 ppm in their 

13 daily diet. Maternal ingestion of Pb at the various doses resulted in female pup blood Pb levels of 3.5-13 

14 μg/dL. For all diet groups, there was a delay in age at vaginal opening, age of first estrus, age of vaginal 

15 plug formation, and age of first parturition. A novel finding in the Iavicoli study was that very low dose 

16 Pb (blood Pb of 0.7 μg/dL, food concentration of 0.02 ppm) induced statistically significant acceleration 

17 of markers of sexual maturation in female offspring versus background Pb level animals (blood Pb of 2 

18 μg/dL) animals. There were statistically significant increases in time of vaginal opening (30% increased), 

19 first estrous, first vaginal plug formation, and first parturition at the very low Pb exposure versus 2 μg/dL

20 animals. Thus, the timing of puberty is delayed in a dose-dependent fashion with very low dose Pb having 
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1 a statistically significant earlier onset of puberty than the background Pb animals. Also, the animals 

2 exposed to the higher dose of Pb (blood Pb up to 13 μg/dL) had statistically significant delays in onset of 

3 puberty when compared to the other dose groups. 

4 In addition, Pb-induced shifts in sexual maturity were observed in the subsequent generation (F2 

5 generation) across that dose range. These animals continued to be exposed to same concentrations of Pb 

6 over multiple generations through the diet. Data results of the F2 generation closely resembled those of 

7 the F1 generation, as both generations received Pb exposure. The authors concluded that a modest 

8 elevation in blood Pb level (13 μg/dL) over background (2-3 μg/dL) can result in a profound delay in the 

9 onset of puberty (15-20%). In the F2 generation, reduction in blood Pb (0.7 μg/dL) below background (2-

10 3 μg/dL) was associated with an earlier onset of sexual maturity (30% increase) above background.

11 In the 2006 Pb AQCD (U.S. EPA, 2006), it was reported that a statistically significant reduction in 

12 the circulating levels of insulin-like growth factor 1 (IGF-1), LH, and estradiol (E2) was associated with 

13 Pb-induced delayed puberty in Fisher 344 pups. Subsequently, Pine et al. (2006) evaluated whether IGF-1 

14 replacement could reverse the effects of Pb on female puberty. The authors reported that offspring 

15 exposed to Pb during gestation and lactation (12 mg/mL; mean maternal blood Pb level 40 μg/dL) 

16 exhibited a marked increase in LH and luteinizing hormone releasing hormone (LHRH) secretion after 

IGF-1 administration (200 ng3 17 /μL) resulting in restored timing of vaginal opening such that they were the 

18 same as control. It should be noted that, IGF-1 replacement in Pb-exposed animals did not cause 

19 advanced puberty over non-Pb-exposed controls. The results of this study provide support to the theory 

20 that Pb-induced delayed onset of puberty may be due to disruption of pulsatile release of sex hormones 

21 (U.S. EPA, 2006) and not necessarily due to a direct toxic effect on the hypothalamic-pituitary-gonadal 

22 axis (Salawu et al., 2009), and IGF-1 may play a prominent role in the process.

23 In sum, epidemiologic studies consistently show a positive association between blood Pb and 

24 delayed pubertal development in girls. This association is apparent even at low blood Pb levels. New 

25 evidence from the toxicology literature continues to support Pb-induced delays in the onset of puberty. 

26 Further, the biological plausibility of delayed puberty is expanded with the toxicological literature that 

27 shows this pathway to be IGF-1-dependent. 

5.8.1.4. Summary of Effects on Female Reproductive Function

28 In summary, Pb exposure affects female reproductive function as demonstrated by both 

29 epidemiologic and toxicological studies. At low Pb levels, associations are observed with delayed puberty. 

30 Some evidence is also available regarding Pb levels and altered hormone levels as well as decreased 

31 fertility, although studies reported inconsistent findings for the later.
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5.8.2. Effects on Male Reproductive Function

1 The 2006 Pb AQCD (U.S. EPA, 2006) reported on male Pb exposure/levels and reproductive 

2 functions as measured by sperm count/motility/morphology, time to pregnancy, reproductive history, and 

3 chromosomal aberrations. Despite limitations in many of the studies, most of the studies found slight

4 associations between high Pb levels (i.e. ≥ 45 μg/dL) and reduced male fecundity or fertility (U.S. EPA, 

5 2006). Evidence provided in the 1986 Pb AQCD (U.S. EPA, 1986) also demonstrated that Pb exposure 

6 affects male reproductive function in humans and experimental animals. Recently published research has 

7 continued to support an association between Pb and reproductive function in males. These studies are 

8 described in the sections below.

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

9 Multiple epidemiologic and toxicological studies have examined the relationship between Pb and 

10 sperm and semen production, quality, and function. These studies are summarized in the text below. In 

11 addition, recent epidemiologic studies are included in Table 5-30. 

Table 5-30. Summary of recent epidemiologic studies of effects on sperm and semen

Reference

Study 

Location 

and 

Years

Study 

Population

Exposure 

Measurement

Mean Pb 

(SD) in 

μg/dL

Adjusted Effect Estimates

Hsu et al. (2009) Taiwan

NS

Men working at a 

battery plant

Blood Pb

Categorized into 3 groups: 

<25 μg/dl, 25-45 μg/dl, >45 

μg/dl 

40.2 p-values for difference across the three 

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)
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Reference

Study 

Location 

and 

Years

Study 

Population

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

Categorized as high 

exposure

workers (blood Pb 40-81 

μg/dl), low exposed workers 

(blood Pb 25-40 μg/dl), and 

controls (office workers with 

no history of occupational Pb 

exposure)

Blood Pb

High exposure 

workers: 53.1 

(2.05)

Low exposure 

workers: 34.7 

(0.83)

Controls: 8.47 

(0.54)

Seminal plasma 

Pb

High exposure workers: 2.02 

(0.23)

Low exposure workers: 2.06 

(0.40)

Controls: 1.73 

(0.16)

Mean (SE)

Sperm volume (mL) 

Controls: 2.94 (0.32)

Low exposure: 2.89 (0.22) 

High exposure: 2.98 (0.22) 

(p-value for ANOVA: 0.993)

Sperm cell count (mln/mL) 

Controls: 43.1 (7.0)

Low exposure: 44.6 (10.1)

High exposure: 42.2 (5.86)

(p-value for ANOVA: 0.400)

Normal morphology (%) 

Controls: 63.3 (2.7)

Low exposure: 57.3 (2.5)

High exposure: 58.4 (2.1)

(p-value for ANOVA: 0.266)

Progressively motile sperm after 1 h (%) 

Controls: 16.4 (3.2)

Low exposure: 14.8 (2.6)

High exposure: 10.5 (1.9)

(p-value for ANOVA: 0.217)

Motile sperm after 24 h (%) 

Controls: 4.4 (1.8)

Low exposure: 7.3 (1.7)

High exposure: 3.1 (0.8)

(p-value for ANOVA: 0.188)

p-value for correlation between blood Pb 

and sperm cell motility after 1 h: 0.011

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

1st quartile: 1.00 (ref)

2nd quartile: 0.88 (0.32, 2.44)

3rd quartile: 2.58 (0.86, 7.73)

4th quartile: 1.16 (0.37, 3.60)

Motility

1st quartile: 1.00 (ref)

2nd quartile:1.04 (0.43, 2.53)

3rd quartile: 1.95 (0.70, 5.46)

4th quartile: 1.66 (0.64, 4.29)

Morphology

1st quartile: 1.00 (ref)

2nd quartile: 0.83 (0.37, 1.87)

3rd quartile: 1.41 (0.54, 3.67)

4th quartile: 1.18 (0.50, 2.79)

Models with adjustment for multiple metals

Concentration

1st quartile: 1.00 (ref)

2nd quartile: 0.89 (1.57, 2.89)

3rd quartile: 3.94 (1.15, 13.6)

4th quartile: 2.48 (0.59, 10.4)
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Reference

Study 

Location 

and 

Years

Study 

Population

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 controls 

and 

occupationally 

exposed workers)

Categorized by work history 

as controls, low exposure (7- 10 yr of exposure for 8 h/day) 

and high exposure (>10 yr of 

exposure for 8 h/day) 

Blood Pb 

measurement

Controls 13.62 

(2.45)

Low exposure 

48.29 (4.91)

High exposure 

77.22 (1.25)

Semen Pb 

measurement

Controls 3.99 

(1.36)

Low exposure 

10.85 (0.75)

High exposure 

18.30 (2.08)

p-values for difference across the three 

groups for mean values of semen profiles 

were <0.01 for: sperm count, sperm 

protein, sperm DNA hyploidy, sperm DNA, 

sperm RNA, sperm viability, sperm 

membrane lipid peroxidation, seminal 

plasma total ascorbate, seminal plasma 

DHAA, sperm ATPase activity, sperm 

motility, sperm velocity, seminal plasma 

fructose

Naha and Manna 

(2007)

Bangalore, 

India

NS

Nonoccupationally 

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)

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 cholesterol 

Slivkova et al. 

(2009)

NS Men aged 22-48 

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 pathological 

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 infertility 

examination or for 

semen donation 

to be used for 

artificial 

insemination

Blood Pb Median: 4.92 

(range 1.13-

14.91)

Standardized regression coefficients for 

log blood Pb (units not given)

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

1 International epidemiologic studies of men occupationally exposed to Pb have reported on 

2 associations between Pb levels and semen/sperm count and quality. Most of these studies included 

3 individuals occupationally exposed to Pb and have reported blood Pb levels over 40 μg/dL. For example, 

4 two studies performed in India (Naha & Chowdhury, 2006; Naha & Manna, 2007) reported that men in 

5 the highest exposure group (men working in battery or paint manufacturing plants for 10-15 years for 8 

6 hours/day) had mean blood Pb levels of 77.22 μg/dL in one study (Naha & Chowdhury, 2006) and 68.26

7 in the other study (Naha & Manna, 2007). Control groups in these studies (those without occupational Pb 
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1 exposure) had blood Pb levels below 15 μg/dL. Increases in levels of Pb in semen were also noted across 

2 exposure groups. Both studies report decreases in sperm count and in sperm velocity and motility with 

3 increasing Pb exposure. Higher Pb exposure was also associated with increased hyploidy of sperm DNA 

4 and morphologic abnormalities (Naha & Chowdhury, 2006; Naha & Manna, 2007). Decreased viability 

5 and increased lipid peroxidation were detected (Naha & Chowdhury, 2006). A study performed in Taiwan 

6 reported that men with greater blood Pb levels had increased sperm head abnormalities, increased sperm 

7 DNA denaturation, and increased sensitivity to denaturation compared to men with lower blood Pb levels

8 (P. C. Hsu et al., 2009). No difference was detected between three Pb exposure groups and semen volume, 

9 sperm count, motility, velocity, and reactive oxygen species production. A similar study in Poland 

10 included employees exposed to Pb and compared them with a group of male office workers (A. 

11 Kasperczyk et al., 2008). Pb levels measured in seminal fluid were slightly higher among those in the 

12 exposed groups although they were not statistically different from the levels in the control group. No 

13 difference was observed for semen volume, sperm count, or sperm morphology among the groups. Sperm 

14 motility was lower in the highest exposure group compared to both the control and moderate exposure 

15 groups. Lipid peroxidation, which induces tissue damage in sperm via reactive oxygen species, was 

16 greater in the highest exposure group compared to the controls. 

17 One study performed in Croatia recruited men who had never been occupationally exposed to 

18 metals (Telisman et al., 2007). Increased blood Pb was associated with increased percent of pathologic 

19 sperm, wide sperm, and round sperm. There was also a slight increase in immature sperm although it was 

20 not statistically significant. Similar results were seen when biomarkers for Pb (erythrocyte protoporphyrin 

21 and δ-aminolevulinic acid dehydratase [ALAD]) were used instead.

22 Two studies examined Pb levels and semen quality of men at infertility clinics (Meeker et al., 2008; 

23 Slivkova et al., 2009). Meeker et al. (2008) detected no associations between increases in blood Pb and 

24 semen concentration, morphology, or motility (although a slight positive trend was observed between 

25 increasing Pb levels and motility in unadjusted models). In models that include multiple metals, blood Pb

26 was associated with being below the WHO’s limit of sperm concentration levels (less than 20 million 

27 sperm/mL), although the 95% CI was wide for the 4th quartile of Pb levels and included the null. 

28 Slivkova et al. (2009) reported a negative correlation between semen Pb and pathological changes in 

29 sperm (specifically, flagellum ball), but no correlations were observed for other alterations in the sperm.

30 An abundance of evidence in the toxicological literature demonstrates that Pb exposure is 

31 detrimental to the quality and overall health of testicular germ cells. Earlier studies showed that chronic 

32 Pb exposure (15 weeks) in adult male rabbits induced statistically significant effects on semen quality and 

33 testicular pathology at blood Pb of 16-24 μg/dL (Moorman et al., 1998). Recent studies confirm earlier 

34 studies that Pb alters sperm parameters such as sperm count, viability, motility, and morphology. Oliveira 

35 et al. (2009) observed a negative correlation between Pb dose and intact acrosomes. Rubio et al (2006), 

36 Biswas and Ghosh (2006), and Salawu et al. (2009) observed a decrease in absolute testicular weight after 
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1 Pb exposure. Rubio et al (2006) and Biswas and Ghosh (2006) also observed a Pb-induced decrease in 

2 seminal vesicle and ventral prostate weights and Rubio et al. (2006) reported that Pb acetate, in a dose3 dependent fashion (8-24 mg/kg body weight), reduced the length of certain stages of the spermatogenic 

4 cycle of rat seminiferous tubules and thus affected spermatogenesis. Reshma Anjum et al. reported 

5 decreased testicular and epididymal weights, sperm count, and viable sperm of male rats exposed to Pb 

6 acetate (273 mg/L or 819 mg/L in drinking water). Pb induced morphological abnormalities in sperm in a 

7 dose-dependent manner (Allouche et al., 2009; Massanyi et al., 2007; Oliveira et al., 2009; Salawu et al., 

8 2009; Shan et al., 2009; Tapisso et al., 2009; C. H. Wang et al., 2006). Sperm abnormalities reported after 

9 Pb exposures are amorphous sperm head, abnormal tail, and abnormal neck. Dong et al. (2009) reported 

10 decreased epididymis and body weights in mice after exposure to 0.6% Pb acetate in drinking water. 

11 However, the majority of studies did not observe a statistically significant difference in body weight or 

12 reproductive organ weights after Pb exposure at the doses used in the studies. Not all the above studies 

13 observed changes in every parameter. This may be due to the use of different strains or species, chemical 

14 form of the Pb compound administered, dosage schedule, duration of exposure, and age of animals at the 

15 time of the study (Oliveira et al., 2009).

16 Data from recent studies suggest that a component of Pb-induced toxicity is the generation of 

17 reactive oxygen species (ROS) which can then affect antioxidant defense systems of cells (Pandya et al., 

18 2010). Salawu et al. (2009) observed a statistically significant increase in malondialdehyde (MDA, 

19 oxidative stress marker) and a significant decrease in the activity of antioxidant enzymes superoxide 

20 dismutase (SOD) and catalse (CAT) in plasma and testes of adult male Sprague Dawley rats after 

21 administration of 1% Pb acetate in drinking water for 8 weeks. Supplementation with tomato paste (used 

22 as a source of antioxidants) reduced ROS production and prevented the increase in MDA formation and 

23 decrease in SOD and CAT activity. Furthermore, co-treatment of Pb with substances that are known to 

24 have antioxidant properties (i.e. tomato paste, Maca (Lepidium meyenii), and ascorbic acid) prevented the 

25 reduction in sperm count, sperm motility, and sperm viability (Madhavi et al., 2007; Rubio et al., 2006; 

26 Salawu et al., 2009; Shan et al., 2009; C. H. Wang et al., 2006). 

27 Recent studies also demonstrate that Pb may be directly toxic to mature spermatozoa (Hernandez28 Ochoa et al., 2006; Tapisso et al., 2009) as well as primary spermatocytes (Nava-Hernandez et al., 2009; 

29 Rafique et al., 2009). Nava-Hernandez et al. had two Pb exposure groups via drinking water. In their 

30 study, all Pb-treated animals had blood Pb levels statistically significantly higher than controls (L1 = 

31 19.54 μg/dL and L2 = 21.90 μg/dL); no statistically significant difference in blood Pb levels existed 

32 between the two Pb exposure groups because the L2 group drank less water than the L1 group. Piao et al. 

33 (2007) report Pb exposure caused DNA damage to sperm; the Pb exposure group had blood Pb of 67 μg/l. 

34 Piao et al. (2007) looked at the effect of Zn supplementation on Pb-induced sperm aberrations and found 

35 that the proportion of abnormal sperm was statistically significantly higher in the Pb group and the Pb+Zn 

36 group than in controls. However, the proportion of abnormal sperm in Pb+Zn group was statistically
May 2011 5-309 DRAFT – DO NOT CITE OR QUOTE

1 significantly lower than in Pb alone group. Hernandez-Ochoa et al. (2006) report that Pb reaches the 

2 sperm nucleus in the epididymis of mice chronically exposed to Pb (mean blood Pb 75.6 μg/dL) by 

3 binding to nuclear sulfhydryl groups from the DNA-protamine complex, increasing sperm chromatin 

4 condensation, and thereby interfering with the sperm maturation process without altering sperm quality 

5 parameters. Tapisso et al. (2009) observed a statistically significant increase in the number of micronuclei 

6 and frequency of sister chromatid exchange with increasing treatment duration in adult male mice 

7 administered 21.5 mg/kg body weight Pb acetate by intraperitoneal injection. Nava-Hernandez (2009)

8 reported a dose-dependent increase in DNA damage in rat primary spermatocytes after a 13-week 

9 exposure period to Pb acetate in drinking water (mean blood Pb levels between 19.5 and 21.9 μg/dL). 

10 Rafique et al. (2009) reported degenerative changes from pyknosis to apoptosis in primary spermatocytes. 

11 Pb-induced apoptosis in germ cells within the seminiferous tubules is another suggested 

12 mechanism by which Pb exerts its toxic effects on sperm production and function (C. H. Wang et al., 

13 2006). Dong et al. (2009) reported a dose-related increase in apoptosis in spermatogonia and 

14 spermatocytes of Kunming mice after exposure to 0.15-0.6% Pb acetate in drinking water. Pb-induced 

15 testicular germ cell apoptosis was associated with up-regulation of genes involved in the signal pathway 

16 of MAPK and death receptor signaling pathway of FAS. For instance, up-regulation of K-ras and Fas 

17 expressions was concomitant with activation of c-fos and active caspase-3 proteins. Wang et al. (2006)

18 observed a dose-dependent increase in the expression of apoptotic markers TGFβ1 and caspase-3 in 

19 spermatogenic cells, Sertoli cells, and Leydig cells. Shan et al. (2009) also reported a statistically

20 significant increase in mRNA expression and protein levels of Fas, Fas-L and caspase-3 after Pb 

21 exposure. Supplementation with ascorbic acid inhibited or reduced the Pb-induced apoptosis in germ cells 

22 and protected testicular structure and function (Shan et al., 2009; C. H. Wang et al., 2006) suggesting 

23 ROS generation is a major contributing factor in decreased male fertility observed after chronic Pb 

24 exposure.

25 Similar to the results summarized in previous AQCDs, recent epidemiologic and toxicological 

26 studies report negative effects of high levels of Pb on sperm and semen. Future studies will aid in 

27 determining whether this association is observed at lower Pb levels.

5.8.2.2. Hormone Levels

28 The 2006 Pb AQCD (U.S. EPA, 2006) provided evidence that Pb acts as an endocrine disruptor in 

29 males at various points along the hypothalamic-pituitary-gonadal axis. The 2006 document also reported 

30 inconsistencies in the reported effects of Pb exposure on circulating testosterone levels. Recent 

31 epidemiologic and toxicological studies are reported below. Epidemiologic studies are summarized in 

32 Table 5-31.
May 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 Population Exposure 

Measurement

Mean Pb 

(SD) in μg/dL

Adjusted Effect 

Estimates

Meeker et al. 

(2008)

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

1st quartile: 1.00 (ref)

2nd quartile: 0.13 (-0.10, 

0.37)

3rd quartile: 0.10 (-0.15, 

0.35)

4th quartile: 0.07 (-0.18, 

0.31)

LH

1st quartile: 1.00 (ref)

2nd quartile:0.004 (-0.20, 

0.21)

3rd quartile: 0.13 (-0.09, 

0.35)

4th quartile: 0.88 (-0.14, 

0.29)

Inhibin B

1st quartile: 1.00 (ref)

2nd quartile: -6.45 (-27.2, 

14.3)

3rd quartile: -4.62 (-26.6, 

17.4)

4th quartile: -7.79 (-29.0, 

13.4)

Testosterone

1st quartile: 1.00 (ref)

2nd quartile: 28.6 (-6.82, 

64.1)

3rd quartile: 15.8 (-21.8, 

53.3)

4th quartile: 39.9 (3.32, 

76.4)

SHBG

1st quartile: 1.00 (ref)

2nd quartile:-0.01 (-0.16, 

0.15)

3rd quartile: 0.04 (-0.12, 

0.21)

4th quartile: 0.07 (-0.10, 

0.23)

FAI

1st quartile: 1.00 (ref)

2nd quartile: 0.8 (-0.04, 

0.20)

3rd quartile: 0.03 (-0.10, 

0.17)

4th quartile: 0.08 (-0.05, 

0.21)

Testosterone/LH

1st quartile: 1.00 (ref)

2nd quartile: 0.07 (-0.16, 

0.30)

3rd quartile: -0.05 (-0.29, 

0.19)

4th quartile: 0.07 (-0.17, 

0.31)
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Reference

Study 

Location 

and Years

Outcome Study Population 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 difference 

>0.05 

Mean LH (SD)

Control: 5.14 (2.35)

Low exposure: 4.27 (2.52)

High exposure: 3.9 (1.69)

p-values for difference 

>0.05

Mean testosterone (SD)

Control: 5.24 (2.40)

Low exposure: 4.83 (1.21)

High exposure: 4.59 (1.27)

p-values for difference 

>0.05

Telisman et al. 

(2007)

Croatia

2002-2005

FSH, LH, 

testosterone, 

estradiol, 

prolactin

Men aged 19-55, never 

occupationally exposed to 

metals and going to a clinic 

for infertility examination or for 

semen donation to be used 

for artificial insemination

Blood Pb Median: 4.92 

(range 1.13-

14.91)

Standardized regression 

coefficients for log blood 

Pb (units not given)

Testosterone: 0.21 (pvalue <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)

1 Hormone levels were measured in a few recent epidemiologic studies. In a study of men non2 occupationally exposed to Pb in Croatia, increased blood Pb was associated with increasing serum 

3 testosterone and estradiol but decreasing serum prolactin (Telisman et al., 2007). In addition, the 

4 interaction term of blood Pb and blood cadmium levels demonstrated a synergistic effect on increasing 

5 serum testosterone levels. No association was observed between blood Pb and FSH or LH. Another study 

6 of men with high blood Pb levels reported no difference in serum FSH, LH, and testosterone among the 

7 three groups (Naha & Manna, 2007). Among men recruited from infertility clinics in Michigan, median 

8 blood Pb levels were much lower than those observed in the other studies of Pb and hormone levels 

9 among men (Meeker et al., 2010). No association was detected between blood Pb and levels of FSH, LH, 

10 InhibinB, sex hormone-binding globulin (SHBG), free androgen index (FAI) or a measure of Leydig cell 

11 function (T/LH). A positive association between the highest quartile of blood Pb and testosterone was 

12 present, but this association did not persist when other metals were included in the model. 

13 In a recent toxicological study, Rubio et al. (2006) observed a decrease in testosterone levels in Pb 

14 acetate-treated rats in a dose-related fashion (8-24 mg/kg body weight), and this decrease correlated with 

15 reduced lengths of spermatogenic cycle stages VII-VIII (spermiation) and IX-XI (onset of 

16 spermatogenesis). Pandya et al. (2010) reported altered hepatic sterodogenic enzyme activity. Biswas and 

17 Ghosh (2006) reported a Pb-induced decrease in serum testosterone and gonadotropins (FSH, LH) with 

18 inhibition of spermatogenesis, however, there was a statistically significant increase in adrenal 
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1 steroidogenic enzyme, Δ5-3β-HSD activity and serum corticosterone levels indicating disruption of the 

2 adrenocortical process. Dose-dependent decreases in serum testosterone were reported in Pb-exposed 

3 male rats (Reshma Anjum et al.). In contrast, Salawu et al. (2009) did not observe a decrease in serum 

4 testosterone between control animals and animals administered 1% Pb acetate in drinking water for 8 

5 weeks. Allouche et al. (2009) not only did not observe any statistically significant changes in serum FSH 

6 or LH, but reported an increase in serum testosterone levels after 0.05-0.3% Pb acetate treatment in 

7 drinking water (only statistically significant in animals administered 0.05% Pb acetate). The results of 

8 these recent studies further support the theory that compensatory mechanisms in the hypothalamic9 pituitary-gonadal axis may allow for the adaptation of exposed animals to the toxic endocrine effects of 

10 Pb (Rubio et al., 2006; U.S. EPA, 2006).

11 Overall, recent epidemiologic and toxicological studies report mixed outcomes regarding hormone 

12 aberrations associated with Pb exposure. These results are similar to those from the 2006 Pb AQCD on 

13 the effects of Pb exposure on circulating testosterone levels. 

5.8.2.3. Fertility

14 Epidemiologic studies have been performed comparing Pb and infertility in men. A study 

15 conducted in Turkey reported blood and seminal plasma Pb levels were different in fertile and infertile 

16 men [mean blood Pb 23.16 μg/dL (SD 5.59 μg/dL) for fertile men and 36.82 μg/dL (SD 12.30 μg/dL) for 

17 infertile men (p-values for ANOVA <0.001)] (Kiziler et al., 2007). Another study examined occupational 

18 Pb exposure (determined by self-report of occupational exposure) and detected no difference in reported 

19 exposure for infertile versus fertile men [OR 0.95 (95% CI: 0.6, 1.6)] (Gracia et al., 2005). Blood Pb was 

20 not measured but approximately 5.0% of infertile men and 5.3% fertile men reported occupational 

21 exposure to Pb. As with the fertility studies among women, a limitation present in these studies is that the 

22 cases included are men who are seeking help at fertility clinics; the studies are not a sample of the general 

23 population regarding fertility.

5.8.2.4. Puberty 

24 Research has also been published examining the association between blood Pb and onset of puberty 

25 in males. These studies are summarized in Table 5-32.
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Table 5-32. Summary of recent epidemiologic studies of effects on puberty for males.

Reference Study 

Location Outcome Study Population

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 provided
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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 

μg/dl: 3%

HR (95% CI)

Pubertal onset based on testicular 

volume

<5 μg/dl: 1.00 (Ref)

≥ 5 μg/dl: 0.73 (0.55, 0.97)

Genital development

<5 μg/dl: 1.00 (Ref)

≥ 5 μg/dl: 0.76 (0.59, 0.98)

Pubic hair development

<5 μg/dl: 1.00 (Ref)

≥ 5 μg/dl: 0.69 (0.44, 1.07)

1 Studies were performed among a cohort of Russian boys enrolled between ages 8-9 (Hauser et al., 

2 2008; P. L. Williams et al., 2010). Both the cross-sectional study (Hauser et al., 2008) and the prospective 

3 study with annual follow-ups (P. L. Williams et al., 2010) demonstrated an association between increased 

4 blood Pb levels and later onset of puberty. In addition, in a study of boys and girls in Egypt boys with 

5 higher blood Pb had delayed pubertal development compared to those with lower levels (median age in 

6 the high blood Pb group was 12.5 years compared to 13.0 years in the low blood Pb group) (Tomoum et 

7 al., 2010). In addition, compared to the low Pb group, those boys with higher blood Pb had lower 

8 testosterone, FSH, and LH levels.

9 Thus, recent studies have demonstrated a negative effect of Pb on pubertal development among 

10 boys that exists even at low blood Pb levels. No recent toxicological studies were found that addressed the 

11 effect of Pb on male sexual development and maturation; however, the 2006 Pb AQCD (U.S. EPA, 2006)

12 supported earlier findings that Pb exposure may result in delayed onset of male puberty and altered 

13 reproductive function later in life in experimental animals.

5.8.2.5. Effects on Morphology and Histology of Male Sex Organs

14 Recent toxicological studies further support historical findings that showed an 

15 association between Pb exposure and histological changes in the testes as well as germ cells. 

16 Histological changes of testes in Pb-treated animals included seminiferous tubule atrophy, 

17 Sertoli cell and Leydig cell shrinkage with pyknotic nuclei (Shan et al., 2009; C. H. Wang et 

18 al., 2006), dilatation of blood capillaries in the interstitium, undulation of basal membrane, 

19 and occurrence of empty spaces in seminiferous epithelium (Massanyi et al., 2007). 

5.8.2.6. Summary of Effects on Male Reproductive Function

20 Associations between Pb exposure and male reproductive function vary by outcome. The strongest 

21 evidence of an association is the relationship observed between high levels of Pb and negative effects on 

22 sperm and semen in both recent epidemiologic and toxicological studies and previous AQCDs. Recent 

23 toxicological studies also reported an association between Pb exposure and histological changes in the 

24 testes and germ cells. In addition, recent epidemiologic studies found Pb exposure to be associated with 
May 2011 5-315 DRAFT – DO NOT CITE OR QUOTE

1 delayed pubertal development at low blood Pb levels. This is supported by earlier toxicological studies. 

2 Similar to the 2006 Pb AQCD, recent epidemiologic and toxicological studies reported inconsistent 

3 results regarding hormone aberrations associated with Pb exposure. Mixed findings were also apparent 

4 among epidemiologic studies of fertility among men.

5.8.3. Effects on Ovaries, Embryo Development, Placental 

function, and Spontaneous Abortions

5 The 2006 Pb AQCD (U.S. EPA, 2006) included studies of Pb exposure among men and women and 

6 their associations with spontaneous abortions. The 2006 AQCD concluded that overall there was little 

7 evidence to support an association between Pb levels among women and spontaneous abortion (U.S. EPA, 

8 2006). Most of the studies examined in the 2006 AQCD assigned exposure based on living near a smelter 

9 or working in occupations that often result in Pb exposure and the results of these studies were 

10 inconsistent. Little evidence was available in the 2006 AQCD to suggest an association with paternal Pb 

11 levels (U.S. EPA, 2006), and no recent studies have been performed to examine paternal Pb levels and 

12 spontaneous abortion. Since the 2006 AQCD, multiple epidemiologic studies have been published that 

13 examine Pb levels in women and their possible association with spontaneous abortion. Additionally, 

14 toxicological studies have studied the effects of Pb on fetal loss and the contribution of the ovaries and 

15 placenta to fetal loss.

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

Women recruited 

during the second 

trimester of 

pregnancy

Whole placentas 

shortly after birth

Median (IQR):

25.8 (21.0, 36.8) 

Median Placenta Pb:

Women who had not 

previously miscarried: 27 

μg/kg

Women who had previously

miscarried: 39 μg/kg

(p-value for difference: 

0.039)

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)

Controls: 

Blood Pb: 6.5 

(4.9)

Plasma Pb: 

0.013 (0.013)

Categorized Plasma Blood 

Pb ratio:

1st tertile: 1.00 (Ref)

2nd tertile: 1.16 (p-value 

0.61)

3rd tertile: 1.90 (p-value 

0.015)

IRR (95%CI) Per 1 SD 

increase: Plasma Pb 1.12 (pvalue 0.22)

Blood Pb 0.93 (p-value 0.56)

Plasma/Blood Pb ratio 1.18 

(p-value 0.02)

Patella Pb 1.15 (p-value 

0.39)

Tibia Pb 1.07 (p-value 0.56)
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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 

cases and at study 

enrollment for 

controls

Cases: 5.3 (95% 

CI: 5.2, 5.9)

Controls: 4.5 

(95% CI: 3.7, 

5.0)

Comparisons between logtransformed blood Pb levels 

of cases and controls 

performed via Student’s ttest had a p-value of 0.03

1 Table 5-33, above, provides a summary of the recent epidemiologic studies examining the 

2 association between Pb levels and past and current spontaneous abortion. Yin et al. (2008) performed a 

3 study in the Shanxi Province of China to examine if plasma Pb levels were associated with anembryonic 

4 pregnancies (spontaneous abortions during the first trimester, which account for 15% of all spontaneous 

5 abortions). Women were enrolled at 8-12 weeks of gestation. Women who delivered a term pregnancy had

6 mean plasma Pb levels that were lower than those of women who had an anembryonic pregnancy. Of 

7 note, among cases Pb was inversely correlated with folate and vitamin B12, but this correlation was not 

8 observed among those who delivered at term; no models examining Pb levels were controlled for nutrient 

9 status. A study in Turkey reported on groups of women who either had a spontaneous abortion before the 

10 20th week of gestation or who had a viable pregnancy (Faikoglu et al., 2006). No difference was detected 

11 between the blood Pb levels of the two groups (Pb levels not reported here due to calculation errors 

12 discovered in the paper; errors do not appear to affect conclusions). A study in Mexico City examined a 

13 group of pregnant women (maximum gestational period at enrollment was 14 weeks) who had previously 

14 been pregnant and either given birth or had a spontaneous abortion (Lamadrid-Figueroa et al., 2007). 

15 Women in the highest tertile of plasma/blood Pb ratio had higher rates of previous spontaneous abortions 

16 than women in the lowest tertile. The authors state that the plasma/whole blood ratio represents the 

17 availability of Pb capable of crossing the placental barrier for a given blood concentration. No association 

18 was observed when examining the relationship between Pb and spontaneous abortions using whole blood, 

19 plasma, or bone Pb alone. Similarly, a study of placental Pb levels among pregnant women in Austria 

20 observed higher placenta Pb levels among women who had miscarried a previous pregnancy compared to 

21 women who had not miscarried a previous pregnancy although the number of women included in the 

22 study was small (only 8 women reported previously having a miscarriage) (Gundacker et al., 2010). 

23 Isolated embryo cultures are often used to understand the mechanisms responsible for aberrant 

24 embryo development as it may contribute to teratogenesis, fetal loss or negative postnatal pup outcomes. 

25 Nandi et al. (2010) demonstrated a dose-dependent decline in embryo development of fertilized buffalo 

26 oocytes cultured in medium containing 0.05-10 μg/mL Pb acetate as evidenced by reduced 

27 morula/blastocyst yield and increased four-to eight-cell arrest, embryo degeneration, and asynchronous 

28 division. This study provides evidence of the negative effect of Pb on embryo development and 

29 contributes mechanistic understand to Pb-dependent pregnancy loss. 
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1 A possible explanation for reduced fertility and impaired female reproductive success as a result of 

2 Pb exposure is changes in morphology or histology in female sex organs and the placenta (Dumitrescu et 

3 al., 2007; U.S. EPA, 2006). Wang et al. (2009) observed that elevated maternal blood Pbs (0.6-1.74 μM) 

4 compared to control (0.04 μM) were associated with decreased fetal body weight, pup body length, and 

5 placental weight in Wistar rats. The authors reported that placentae from Pb-exposed groups showed 

6 dose-dependent increasing pathology of cytoarchitecture and cytoplasmic organelles. The authors also 

7 reported a positive expression of NF-κB, a transcription factor that controls the expression of genes 

8 involved in immune responses, apoptosis, and cell cycle, in the cytotrophoblasts, decidual cells, and small 

9 vascular endothelial cells in rat placenta under a low Pb level exposure condition which correlated to 

10 blood levels. 

11 Pb-exposed (273 mg/L or 819 mg/L in drinking water, 0.05 or 0.15% Pb Acetate, respectively)

12 male rats from Reshma Anjum et al. that had dose-dependent decreases in serum testosterone, decreased 

13 male reproductive organ weight and decreased sperm were mated to untreated females. These untreated 

14 dams had dose-dependent decreased implantation rate and higher pre- and post-implantation loss, 

15 indicating paternally mediated fetal loss. 

16 As observed in sperm cells, Pb stimulates changes in antioxidant enzyme activity in rat ovaries 

17 indicating that a contributing factor in Pb-induced ovarian toxicity may be oxidative stress. Nampoothiri 

18 et al. (2007) observed a reduction in SOD activity and an increase in CAT activity along with a decrease 

19 in glutathione content and an increase in lipid peroxidation in rat granulosa cells after 15 days of Pb 

20 treatment (0.05 mg/kg body weight).

21 Previous studies demonstrated that Pb accumulates in the ovaries and causes histological changes, 

22 thus contributing to Pb-induced effects on female fertility (U.S. EPA, 2006). In support of historical 

23 studies, recent studies demonstrate histological changes in ovarian cells of pigs (Kolesarova et al., 2010)

24 and rats (Nampoothiri et al., 2007; Nampoothiri & Gupta, 2006). Kolesarova et al. (2010) observed a 

25 reduction of the monolayer of granulosa cells after Pb addition (0.5 mg/mL). Nampoothiri and Gupta 

26 (2006) reported that Pb exposure caused a decrease in cholesterol and total phospholipid content in the 

27 membranes of granulosa cells which resulted in increased membrane fluidity. A possible explanation for 

28 reduced fertility and impaired female reproductive success as a result of Pb exposure is changes in 

29 morphology or histology in female sex organs and the placenta (Dumitrescu et al., 2007; U.S. EPA, 

30 2006).

31 Overall, the recent studies support the conclusions of the last Pb AQCD that there is insufficient 

32 evidence among epidemiologic studies to suggest an association between Pb and spontaneous abortions. 

33 In addition, studies of spontaneous abortions are difficult to conduct. The majority of spontaneous 

34 abortions are during the first trimester, which makes them difficult to capture. Women may miscarry 

35 before being enrolled in a study and many women may not have known they were pregnant when they 

36 miscarried. This limits the ability to detect subtle effects, especially if higher Pb levels do lead to 
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1 increased risk of early spontaneous abortions. Toxicological data provide mechanistic understanding of 

2 the contribution of Pb to spontaneous abortions. These laboratory data show that Pb exposure impaired 

3 placental function, induced oxidative stress and histological changes in the ovaries, and affected embryo 

4 development. The toxicological and epidemiologic data up to the current date provide mixed effects on 

5 the role of Pb in spontaneous abortions. 

5.8.4. Infant Mortality and Embryogenesis

6 The 2006 AQCD (U.S. EPA, 2006) concluded that Pb exposure can increase fetal mortality and 

7 produce sublethal effects (disrupt growth and development) in offspring of Pb exposed dams at 

8 concentrations that do not result in clinical toxicity to the dams. There is substantial evidence to show that 

9 there is no apparent maternal-fetal barrier to Pb and it can easily cross the placenta and accumulate in 

10 fetal tissue during gestation (Pillai et al., 2009; Uzbekov et al., 2007; Y.-Y. Wang et al., 2009). No recent 

11 epidemiologic studies have reported on the relationship between Pb levels and infant mortality. 

5.8.5. Birth Defects

12 The 2006 Pb AQCD (U.S. EPA, 2006) reported the possibility of small associations between high 

13 Pb exposure and birth defects, but many of the studies used occupational histories instead of actual 

14 measures of blood Pb levels. Among the studies included in the 2006 AQCD, two studies reported 

15 possible associations between parental exposure to Pb and neural tube defects (Bound et al., 1997; Irgens 

16 et al., 1998). Recent studies also examined Pb levels and neural tube defects (Table 5-34). No other recent 

17 studies of Pb levels/exposure and birth defects were identified in the literature. No recent toxicological 

18 studies were found that investigated Pb-induced changes in morphology, teratology effects, or skeletal 

19 malformations of developing fetuses as a result of maternal Pb exposure; however, in the 2006 AQCD 

20 toxicological studies demonstrated associations between exposure to high doses of Pb and increased 

21 incidences of teratogenic effect in experimental animals. 

Table 5-34. Summary of recent epidemiologic studies of effects on neural tube defects.

Reference Study 

Location

Study 

Population

Exposure 

Measurement

Mean Pb (SD) in 

μg/dL

Adjusted Effect 

Estimates

Brender et al. 

(2006)

Texas

1995-2000

Infants of MexicanAmerican 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)

Blood Pb≥6.0 μg/dL: 1.5 (95% 

CI: 0.6, 4.3)
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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 MannWhitney U test (dependent on 

distribution) were 0.35 for 

maternal blood Pb and 0.63 for 

umbilical cord blood Pb

1 Among the recent epidemiologic studies (described in Table 5-34), a study of women in Turkey 

2 detected no difference between the blood Pb of mothers or the umbilical cord blood Pb of the newborns 

3 for healthy infants compared with infants with neural tube defects (cases of spina bifida occulta were 

4 excluded, but other forms of spina bifida were included) (Zeyrek et al., 2009). Brender et al. (2006)

5 performed a study of Mexican-American women living in Texas. These measurements were taken 5-6 

6 weeks postpartum, which is a limitation of this study because the levels may be different than during the 

7 developmental period of gestation. The OR comparing those with at least 6 μg/dL Pb to those with less 

8 than 6 μg/dL Pb was 1.5 (95% CI: 0.6, 4.3). This increased after adjusting for breast feeding, although 

9 this variable was not a confounder due to its inability to be associated with neural tube defects. For these 

10 women neither occupational exposure to Pb or proximity of residence to a facility with Pb air emissions at 

11 the time of conception were associated with increased odds of neural tube defects. 

12 Overall, in contrast to studies from the 2006 AQCD, recent studies of Pb and neural tube defects 

13 observed no associations. 

5.8.6. Preterm Birth

14 Research on preterm birth included in the 2006 Pb AQCD (U.S. EPA, 2006) reported inconsistent 

15 findings regarding the relationship between Pb and gestational age. Recent studies have examined this 

16 potential association and again mixed results were reported (Table 5-35). Of these studies, the ones that 

17 categorized births as preterm or term all defined preterm birth as less than 37 weeks of gestation. One 

18 limitation to note for these studies is that if Pb affects spontaneous abortion and length of gestation via a 

19 similar pathway, then the studies that only collect data at delivery and not at earlier stages of pregnancy 

20 would be biased towards the null. 
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Table 5-35. Summary of recent epidemiologic studies of effects on preterm birth.

Reference Study 

Location Outcome Study Population

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 

air 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 

Maximum maternal blood 

Pb during pregnancy

≥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 County 

area for at least 5 

mo during 

pregnancy 

Umbilical cord blood Pb 2.4 (4.3)

Geometric 

mean: 1.3

Geometric Mean:

Preterm birth: 1.4

Term birth: 1.2 

Post-term birth: 1.3

p-value for difference: >0.10

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)

1 One study of preterm birth included women living in two different residential areas over three 

2 different time periods (Berkowitz et al., 2006). One residential area was consistently unexposed but the 

3 other had a period of high Pb emissions due to damage at a local factory. The three time periods examined 

4 preterm birth rates before, during, and after the time of high exposure. No association was observed 

5 between women living in the high exposure area compared to those in the low exposure area during any 

6 of the time periods, but the number of preterm infants born during the period of high exposure was small. 

7 In another study, measurements of umbilical cord blood were taken after birth at a hospital in Nagpur, 
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1 India (Patel & Prabhu, 2009). A sample of women had their blood Pb measured and among this sample, 

2 maternal blood Pb was correlated with the umbilical cord Pb levels. Mean gestational age varied between

3 infants with >5 μg/dL Pb and infants with ≤ 5 μg/dL Pb. In a linear regression model, gestational age was 

4 found to decrease with increasing umbilical cord Pb levels. A study of women in Tennessee consisted 

5 primarily of African American women living in an urban setting (E. A. Jones et al., 2010). Mean levels of 

6 umbilical cord blood Pb were slightly higher among infants born preterm but the difference was not 

7 statistically significant. In a study taking place in California, women with information on blood Pb levels

8 based on their participation in a surveillance program (reason for participation in the surveillance program 

9 was unknown but the authors speculate it was likely because of potential Pb exposure) were matched with 

10 the birth certificates of their infants (Jelliffe-Pawlowski et al., 2006). Almost 70% of women had 

11 maximum blood Pb measurements <10 μg/dL with the majority being <5 μg/dL. Preterm birth was 

12 associated with increased blood Pb when comparing women with maximum blood Pb levels ≥ 10 μg/dL 

13 to women with blood Pb levels <10 μg/dL in adjusted analyses. In analyses of maximum Pb levels refined 

14 into further categories, when compared to maximum blood Pb levels ≤ 5 μg/dL the positive association 

15 between maximum blood Pb measurement and preterm birth was present at 20 μg/dL and higher. Finally,

16 a study in Iran reported higher maternal blood Pb for preterm births than term births (Vigeh et al., 2011). 

17 A positive association between maternal blood Pb levels and preterm birth was observed.

18 In sum, as in the 2006 Pb AQCD, recent epidemiologic studies report inconsistent findings for the 

19 relationship between Pb and preterm birth. 

5.8.7. Low Birth Weight/Fetal Growth

20 The 2006 Pb AQCD reported inconsistent study results examining the associations between Pb and 

21 birth weight/fetal growth and concluded that there could be a small effect of Pb exposure on birth weight 

22 and fetal growth (U.S. EPA, 2006). Since then, multiple studies on the relationship between Pb exposure 

23 and birth weight and fetal growth have been published using various measures of exposure, such as air 

24 levels, umbilical cord blood, and maternal blood and bone. These studies are summarized in Table 5-36 

25 below. Additionally, there have been a few recent toxicological studies evaluating the effect of Pb 

26 exposure during gestation on birth weight.
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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

1970-1981

Low birth weight

(<2,500 g and ≥37 

wk)

Small 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 

exposed/near smelter): 

pre-fire, “high-exposure 

period” (when a fire 

happened at the smelter 

and resulted in damages 

leading to high air Pb 

concentrations for 6 mo), 

and “post-fire”

Not specified Term Low birth weight:

OR (90% CI) (unexposed 

location is referent group):

Pre-fire 0.81 (0.55, 1.20) 

High exposure 2.39 (1.57, 

3.64)

Post-fire 1.28 (0.95, 1.74)

Small for gestational age: OR (90% CI) (unexposed 

location is referent group):

Pre-fire 0.98 (0.73, 1.32) 

High exposure 1.92 (1.33, 

2.76)

Post-fire 1.32 (1.05, 1.67)

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: 11.3 

(1.9)

P-values for t-tests:

Maternal blood Pb 0.07

Umbilical cord blood Pb:

0.20

P-values for correlations: 

Maternal blood Pb and 

Birth weight: Low birth weight: 0.17

Normal birth weight: 0.3

P-values for correlations: 

Umbilical cord blood Pb 

and birth weight:

Low birth weight: 0.84

normal birth weight: 0.26

Janjua et al. 

(2009)

Karachi, 

Pakistan

2005

Low birth weight 

(≤ 2,500g)

Infants of 

randomly 

selected women 

who planned to 

deliver between 

37-42 wk

Umbilical cord blood Pb Umbilical cord 

blood Pb: 10.8 

(0.2)

Prevalence ratio: 

<10 μg/dl: 1.00 (Ref)

≥ 10μg/dl: 0.82 (0.57, 

1.17)
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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 raceand gender- specific norms

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 and 

matched to birth 

records

Maximum maternal 

blood Pb during 

pregnancy

≥10 μg/dl: 

30.9%

Odd Ratios:

Low birth weight ≤5 μg/dl: 

1.00 (Ref)

6-9 μg/dl: -- 10-19 μg/dl: 2.7 (0.5, 14.8)

20-39 μg/dl: 1.5 (0.3, 7.7)

≥ 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 County 

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 Mann-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/m3 

(0.04 μg/m3)

p-value for multilevel 

regression of Pb with birth 

weight: 0.002

Increase of Pb from 0 to 

0.04 relates to a 38g 

decrease in birth weight

Increase of Pb from 0 to 

0.13 (maximum) relates to 

a 124g decrease in birth 

weight
May 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

Singleton births 

with maternal 

residence within 

5 km of Pb 

smelter

Umbilical cord blood Pb 

from delivery

Umbilical cord 

blood Pb: 3.9 

(3.6)

Linear regression 

coefficient with umbilical 

cord blood Pb as the 

dependent variable in 

model with only length and 

weight (unit not given, 

assume per 1 μg/dL): 

Length -0.46 (p-value 

0.003 ) and Weight -0.275 

(0.048)

1 Women residing in two different towns in Yugoslavia (one with a Pb smelter and one without a 

2 Pb smelter) were recruited during their first prenatal visit (Lamb et al., 2008) (study based on previous 

3 work by Factor-Litvak et al. (1991)). The blood Pb levels were greater in the town with a Pb smelter. No 

4 association was reported between maternal blood Pb and height or BMI at birth for the infants of these 

5 women. Despite the differences in maternal blood Pb between the two towns, no differences in the 

6 associations were detected. Another study using maternal blood Pb was conducted in California (Jelliffe7 Pawlowski et al., 2006). Women’s blood Pb measurements during pregnancy were matched with the 

8 corresponding birth certificates. The adjusted OR for low birth weight that compared women with blood 

9 Pb levels ≥ 10 μg/dL to women with levels <10 μg/dL was elevated. However, it was difficult to draw 

10 conclusions about the relationship between blood Pb and birth weight due to small numbers (n = 9 for low 

11 birth weight) and the subsequently large 95% CI. An association was detected for high blood Pb and 

12 having an infant who was small of his/her gestational age (SGA; defined as birth weight <10th percentile 

13 of normal weight for population-based singleton race and gender specific infants of the same gestational 

14 age). A study of term births in Iran reported no difference in blood Pb of women giving birth to a normal

15 weight infant and women giving birth to an infant with low birth weight (Iranpour et al., 2007). Finally, a 

16 study in Vienna, Austria reported an inverse association between maternal blood Pb levels and birth 

17 weight but no associations for birth length or head circumference (Gundacker et al., 2010). 

18 One study examining the association between Pb levels and birth weight used tibia bone 

19 measurements from the mothers living in Mexico City (Kordas et al., 2009). Pb tibia levels were inversely 

20 associated with birth weight but not with birth length. This association between Pb and birth weight was 

21 not modified by maternal folate consumption or maternal or infant MTHFR genotype, although the 

22 association between tibia Pb levels and birth weight was increased among women with certain genotypes 

23 (statistical tests not reported). 

24 Multiple studies examined the relationship using Pb measured from the placenta or umbilical cord. 

25 First, the study by Iranpour et al. (2007) discussed above investigated the association between umbilical 

26 cord blood Pb levels in addition to their examination of whole blood. They again report no difference in 

27 levels between term infants of normal and low birth weight. Researchers in Chile collected the placentas 

28 from term births and compared the Pb levels for those born with normal birth weights to those with low 
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1 birth weights (Llanos & Ronco, 2009). Pb levels were greater in the placentas of infants with low birth 

2 weights. In addition, the authors note that 3 low birth weight infants had extremely high Pb levels in the 

3 placentas (>1.5 μg/g) and were excluded from these analyses. A study in Brazil examined Pb levels in 

4 umbilical cord blood from term births of women residing within 5 km of a Pb smelter (Zentner et al., 

5 2006). The Pb level was found to be inversely correlated with length and weight of the infants. A third 

6 study recruited women in Pakistan (Janjua et al., 2009). Umbilical cord blood Pb levels were not 

7 associated with low birth weight. A study comparing geometric mean umbilical cord blood Pb levels 

8 reported no difference in the levels for normal and low birth weight infants among infants born to women 

9 living primarily in urban areas of Memphis, TN (E. A. Jones et al., 2010). Finally, a study in Vienna 

10 measured Pb in the placenta (Gundacker et al., 2010). A positive correlation was observed between 

11 placenta Pb and birth length and weight, however, in the same study, maternal blood Pb was inversely 

12 related to birth weight. 

13 Two studies examined air exposures and reported inverse associations between air Pb 

14 concentrations and birth weight. Williams et al. (2007) examined Pb concentrations in the air during the 

15 first trimester. The purpose of their study was to demonstrate the use of hierarchical linear models and 

16 they used the example of air pollution and birth weight in Tennessee. The model results showed an 

17 association between ambient Pb concentration and birth weight, with an estimated decrease in birth 

weight of 38 grams for every 0.04 μg/m3 18 (i.e., one standard deviation) increase in Pb concentration. The 

19 other study of air Pb levels was conducted in Idaho and included two areas over three time periods. One 

20 study area was affected by damage to a local factory that lead to high Pb emissions during one of the time 

21 periods under study (Berkowitz et al., 2006). No levels of Pb are provided. Mean birth weight for term 

22 births was decreased among women living in high exposure areas during the period of high exposure 

23 compared to those living in unexposed areas. The difference in birth weight of term births remained, but 

24 was reduced, between the two areas during the time period after the exposure ended. During the period of 

25 exposure, the odds of low birth weight among term births was increased among those living in the 

26 exposed area compared to those in the unexposed area but the odds were not different between the two 

27 study areas during the time periods before or after the high level of exposure. An increase in SGA infants

28 (defined as infants with weights less than or equal to the lowest fifth percentile of birth weight for their 

29 sex and age) was also associated with living in the exposed area during the time period of exposure. The 

30 odds of SGA infants decreased during the time period after the exposure but the odds were still elevated 

31 compared to those residing in the unexposed area. 

32 Evidence from previous toxicological studies has shown an association between gestational Pb 

33 exposure and reduced birth weight and impaired postnatal growth (U.S. EPA, 2006). More recent studies 

34 have reported conflicting results. Wang et al. (2009) demonstrated a statistically significant decrease in 

35 fetal body weight and body length of Wistar rats after maternal exposure to 0.025% Pb acetate during 

36 gestation days 1-10, 11-20, or 1-20. The greatest decrease in fetal body weight and length was observed in 
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1 the group exposed to Pb during gestation days 1-20 followed by the group exposed to Pb during gestation 

2 days 11-20. Teijón et al. (2006) observed that when pregnant dams were administered 200 ppm or 400 

3 ppm Pb acetate in drinking water, female pups had a decreased birth weight when compared to male pups 

4 of the same litter (only statistically significant at the 400 ppm dose). This effect did not persist in the 

5 postnatal growth of the rats. The results of these studies indicate that as Pb exposure increases, the body 

6 weight of exposed offspring decreases. Massó-González and Antonia-García (2009) also observed an 8-

7 20% decrease in body weight of pups from rat dams given 300 mg/L Pb acetate in drinking water (mean 

8 blood Pb level 22.8 μg/100mL), but no changes in body length were reported. In contrast, Leasure et al. 

9 (2008) reported a statistically significant inverse relationship between Pb exposure and body weight for 

10 male mice exposed to low-and high-levels of Pb during gestation. Male mice exposed to the low and high 

11 Pb concentrations during gestation were 26% and 13% heavier than controls at 1 year of age, respectively. 

12 In this study, dams were administered 27 ppm (low), 55 ppm (moderate), and 109 ppm (high) Pb in 

13 drinking water beginning 2 weeks before mating and continuing until PND10. Resulting blood Pb levels 

14 ranged from 10 μg/dL or less in the low-exposure offspring to 42 μg/dL in the high-exposure offspring at 

15 PND10. The authors also reported that when dams received low or moderate levels of Pb in drinking 

16 water from birth to weaning neither male nor female offspring exposed to Pb postnatally exhibited a 

17 difference in body weight when compared to control offspring.

18 In summary, associations were observed between Pb and low birth weight in a study of maternal 

19 bone Pb and studies of Pb air exposures and birth weight. However, the associations were less consistent 

20 when using maternal blood Pb or umbilical cord and placenta Pb as the exposure measurement. Previous 

21 toxicological studies observed an association between gestational Pb exposure and reduced birth weight

22 with moderate to high dose Pb. More recent findings using low dose Pb exposure reported increased 

23 offspring body weight after developmental Pb exposure. 

5.8.8. Toxicological Studies of Developmental Effects

5.8.8.1. Developmental Effects on Blood and Liver

24 The 1986 and 2006 AQCD reported studies that suggest Pb may alter hematopoietic and hepatic 

25 function during development. Some recent studies provide evidence that support these findings; however 

26 recent results are not consistent among the studies.

27 Massó et al. (2007) reported a decrease in liver weights of pups born to dams that consumed 300 

28 mg/L Pb in drinking water during gestation and lactation. They also report an increase in the number of 

29 erythrocytes; however their size was diminished by 62%. Pb produced microcitic anemia as evidenced by 

30 decreased hemoglobin content and hematocrit values without changes in mean corpuscular hemoglobin 

31 (MCH) concentration. No changes were observed in alkaline phosphatase (ALP) activity, CAT activity, or 
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1 thiobarbituric acid reactive substances (TBARS) production in pups at postnatal 0, but increased 

2 statistically significantly by PND21 indicating reactive oxygen generation. No change in acid phosphatase 

3 (ACP) activity was observed in the livers of pups at PND0 or 21.

4 Massó-González and Antonia-García (2009) reported normochromic and microcytic anemia and a 

5 significant decrease in hematocrit values and blood δ-aminolevulinic acid dehydratase (ALAD) activity 

6 (90% reduction) in pups from dams administered 300 mg/L Pb acetate in drinking water during gestation. 

7 The authors also reported that erythrocyte osmotic fragility was four times greater in Pb-exposed pups 

8 than in control pups. Massó-González and Antonia-García reported increases in TBARS and CAT activity

9 in the liver after Pb exposure. Intoxication with Pb also resulted in decreased liver protein concentrations 

10 and manganese-dependent SOD activity. Abnormalities in liver function were further exemplified by 

11 increases in liver concentrations of ALP and ACP.

12 Teijón et al. (2006) observed that gestational exposure to Pb caused a decrease in erythrocytes, 

13 hemoglobin, and MCH at weaning; however, by 1 and 3 months postweaning, these parameters had 

14 returned to normal values. The authors observed a slight increase in serum ALP, alanine aminotransferase 

15 (ALT), and aspartate aminotransferase (AST) levels after Pb exposure in the absence of liver histological 

16 changes. 

17 Pb-induced effects on SOD activity in the liver of fetuses after Pb intoxication was supported in a 

18 study by Uzbekov et al. (2007). The authors reported an initial increase in SOD activity in livers of pups 

19 exposed to 0.3 mg/L and 3.0 mg/L Pb nitrate during gestation for 1 month (mean daily consumption 27 

20 μg/kg). In contrast, long-term exposure (5 months) to the same concentrations of Pb nitrate concentration 

21 during gestation resulted in decreased hepatic SOD activity.

22 Effects on hepatic Phase I and Phase II enzymes after early developmental exposure of offspring to 

23 Pb during gestation and lactation was evaluated by Pillai et al. (2009). In the study, pregnant Charles 

24 Foster rats were administered 0.05 mg/kg body weight Pb subcutaneously throughout gestation until 

25 PND21. Pups were evaluated on PND56. Results of the study show that Phase I xenobiotic-metabolizing 

26 enzymes (NADPH- and NADH cytochrome c reductase) and Phase II xenobiotic- and steroid27 metabolizing enzymes (δ-glutamyl transpeptidase, UDPGT, glutathione-s-transferase, and 17β28 hydroxysteroid oxidoreductase) were reduced in both male and female pups by PND56. Only inhibition in 

29 glutathione-s-transferase and 17β-hydroxysteroid oxidoreductase activities demonstrated a sex-specific 

30 pattern (glutathione-s-transferase inhibition in males; 17β-hydroxysteroid oxidoreductase inhibition 

31 greater in females). Pb-induced histological changes observed include massive fatty degeneration in 

32 hepatocytes, large vacuoles in cytoplasm, appearance of pycnotic nuclei, and infiltration of lymphocytes 

33 in the liver. Antioxidant enzymes (SOD, CAT, glutathione peroxidase, and glutathione reductase) were 

34 also reduced after Pb intoxication. Alterations in biochemical parameters included decreased DNA, RNA, 

35 and cholesterol content.
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5.8.8.2. Developmental Effects on Skin

1 The 2006 Pb AQCD (U.S. EPA, 2006) reported one study that demonstrated Pb-induced 

2 abnormalities in skin development. No current studies were identified that addressed Pb-induced skin 

3 alterations.

5.8.8.3. Developmental Effects on the Retina

4 The 2006 AQCD concluded that Pb exposure during early postnatal development (blood Pb ~20

5 μg/dL) impaired retinal development in female Long-Evans hooded rats. A more recent study (Fox et al., 

6 2008) exposed female Long-Evans hooded rats to low (27 ppm), moderate (55 ppm), and high (109 ppm) 

7 levels of Pb acetate in drinking water beginning 2 weeks before mating, throughout gestation, and until 

8 PND10. blood Pb levels in pups on postnatal days 0-10 exposed to Pb during gestation were 10-12 μg/dL 

9 (low), 21-24 μg/dL (moderate), and 40-46 μg/dL (high). Results of the study demonstrated supernormal 

10 persistent rod photoreceptor-mediated (scotopic) electroretinograms (ERG) in adult rats similar to ERG 

11 findings in male and female children after gestational exposure to low- and moderate-levels of Pb. Low12 and moderate-levels of Pb increased neurogenesis of rod photoreceptors and rod bipolar cells without 

13 affecting Müller glial cells and statistically significantly increased the number of rods in central and 

14 peripheral retina. High-level Pb exposure (109 ppm) statistically significantly decreased the number of 

15 rods in central and peripheral retina Pb-exposure induced dose-dependent decreases in adult rat retinal 

16 dopamine synthesis and utilization/release.

5.8.8.4. Developmental Effects on Teeth 

17 Pb has been associated with multiple health effects including dental caries, however, there is very 

18 limited information available on the temporal and spatial incorporation of Pb in dental tissue (Arora et al., 

19 2005). Arora et al. (2005) demonstrated that Wistar rat pups exposed to Pb during gestation and lactation 

20 (40 mg/L of Pb nitrate in drinking water of pregnant dams) had higher concentrations of Pb on the surface 

21 of enamel and in the dentine immediately adjacent to the pulp. The authors concluded that additional 

22 research is needed on the intracellular uptake of Pb during tooth development to fully understand the 

23 spatial distribution of Pb in teeth. 

5.8.9. Summary and Causal Determination

24 Many epidemiologic and toxicological studies of the effects of Pb on reproductive outcomes have 

25 been performed since the 2006 AQCD. These studies covered outcomes such as female and male 

26 reproductive function, birth defects spontaneous abortions, infant mortality, preterm birth, low birth 

27 weight, and developmental effects. There is an abundance of evidence in the literature demonstrating that 
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1 Pb induces reproductive and developmental effects in laboratory animals exposed to Pb during gestation 

2 and/or lactation. Many of the Pb-induced effects occur in a dose-dependent manner and have been 

3 observed at maternal blood Pb levels that do not result in clinical toxicity in the dams. Additionally, 

4 epidemiologic studies have demonstrated strong evidence of an association between Pb and delayed 

5 puberty as well as decrements to sperm/semen quality and function.

6 Many of the animal toxicology studies included in the 2006 AQCD explored the effect of Pb on 

7 reproduction and development at blood Pb levels greater than 40 μg/dL, a dose where maternal toxicity 

8 can develop during pregnancy. Data from the 2006 AQCD on male fertility showed perturbed semen 

9 quality. Recent studies have shown the effects of Pb exposure during early development to include 

10 disruption of endocrine function; delay in the onset of puberty and alteration in reproductive function later 

11 in life; and changes in morphology or histology in sex organs and placenta. Additionally, epidemiologic 

12 studies of reproductive factors among males and females investigated whether Pb levels were associated 

13 with hormone levels, fertility, and onset of puberty. Epidemiologic studies showed associations between 

14 blood Pb and hormone levels for females. Studies of Pb and fertility are limited and inconsistent for 

15 females and males. Strong and consistent associations were observed between Pb levels in males in 

16 occupational settings with blood Pb levels as low as 20-45 μg/dL and sperm count and quality. Multiple 

17 studies of Pb and puberty have shown associations between blood Pb levels and delayed pubertal 

18 development for girls and boys. These associations are consistently observed in multiple epidemiologic 

19 studies and demonstrate effects on pubertal development at blood Pb levels <10ug/dL. 

20 Pb-mediated changes in levels or function of reproductive and growth hormones have been 

21 demonstrated in past and more recent toxicological studies; however the findings are inconsistent. More 

22 data are needed to determine whether Pb exerts its toxic effects on the reproductive system by affecting 

23 the responsiveness of the hypothalamic-pituitary-gonad axis or by suppressing circulating hormone levels. 

24 More recent toxicological studies suggest that oxidative stress is a major contributor to the toxic effects of 

25 Pb on male and female reproductive systems. The effects of ROS may involve interference with cellular 

26 defense systems leading to increased lipid peroxidation and free radical attack on lipids, proteins, and 

27 DNA. Several recent studies showed an association between increased generation of ROS and germ cell 

28 injury as evidenced by destruction of germ cell structure and function. Co-administration of Pb with 

29 various antioxidant compounds either eliminated Pb-induced injury or greatly attenuated its effects. In 

30 addition, many studies that observed increased oxidative stress also observed increased apoptosis which is 

31 likely a critical underlying mechanism in Pb-induced germ cell DNA damage and dysfunction. 

32 Overall, results of pregnancy outcomes were similar to those of the 2006 AQCD; inconsistent 

33 evidence of a relationship with Pb was available for preterm birth and little evidence was available to 

34 study the associations with spontaneous abortions. The previous AQCD included a few studies that 

35 reported possible associations between Pb and neural tube defects, but the recent epidemiologic studies 

36 found no association. Possible associations were observed between Pb and low birth weight when 
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1 epidemiologic studies used measures of maternal bone Pb or air exposures, but the associations were less 

2 consistent when using maternal blood Pb or umbilical cord and placenta Pb. Effects of Pb exposure 

3 during early development on toxicological studies included reduction in litter size, implantation, birth 

4 weight and postnatal growth.

5 Toxicological studies demonstrated that the effects of Pb exposure during early development 

6 include impairment of retinal development and alterations in the developing hematopoietic and hepatic 

7 systems. Negative developmental outcomes were also noted including effects on the skin and teeth.

8 Similar to toxicological and epidemiologic studies that observed Pb to be associated with delayed 

9 puberty, delays of dynamic changes in the HPT axis are seen in the ecological literature, i.e., delayed 

10 metamorphosis in Pb exposed frogs. Additionally, Pb exposure has been shown to have detrimental 

11 effects on sperm, albeit often at higher blood Pb levels in epidemiology studies but in lower doses in the 

12 toxicology literature. Again, these findings agree with the ecological literature where Pb-dependent sperm 

13 effects are seen in rotifers, earthworms, and trout. 

14 In conclusion, the recent toxicological and epidemiologic literature provides strong evidence that 

15 Pb exposure is related to delayed onset of puberty in both males and females. Additionally, Pb exposure 

16 has been shown to have detrimental effects on sperm (at high blood Pb levels in epidemiologic studies 

17 and in low doses in the toxicological literature). The data on preterm birth, low birth weight, spontaneous 

18 abortions, birth defects, hormonal influences, and fecundity are a bit more mixed and less consistent 

19 between the toxicological and epidemiologic literature. The collective body of evidence integrated across 

20 epidemiologic and toxicological studies with a focus on the strong relationship observed with negative 

21 effects on sperm and delayed pubertal onset is sufficient to conclude that there is a causal relationship 

22 between Pb exposures and reproductive effects and birth outcomes.

5.9. Effects on Other Organ Systems

5.9.1. Effects on the Hepatic System

23 Hepatotoxic effects of Pb in various animal and human models include alterations in hepatic 

24 metabolism, hepatic cell proliferation, changes in cholesterol metabolism, as well as oxidative stress25 related injury. Animal studies have also shown that exposure to Pb causes a decrease in Phase I along with 

26 a simultaneous increase in Phase II enzymes following exposure to various forms of Pb. Induction of 

27 oxidative stress is well supported by an increase in lipid peroxidation along with a decrease in glutathione

28 (GSH) levels and catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx)

29 activities
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5.9.1.1. Summary of Key Findings of the Effects on the Hepatic System from the 

2006 Lead AQCD

1 The 2006 Pb AQCD stated that some of the frequent toxicological effects in the liver following 

2 exposure to Pb included increased hepatic cell proliferation, cholesterol synthesis, DNA synthesis and 

3 glucose-6-phosphotase dehydrogenase (G6DP) activity resulting in Pb-induced hyperplasia. The AQCD 

4 concluded that cytochrome (CYP) P450 levels decreased following single exposures to Pb, primarily Pb 

5 nitrate. Inhibition of induced and constitutive expression of microsomal CYP 1A1 and 1A2 was observed 

6 among various P450 isozymes. Inhibition of Phase I enzymes was accompanied by an increase in Phase II 

7 enzymes following exposure to Pb nitrate and other Pb compounds, suggesting that Pb is capable of 

8 causing a biochemical phenotype similar to hepatic nodules. Studies relating to Pb-induced hepatic 

9 hyperplasia suggested alterations in the gluconeogenic mechanism, DNA hypomethylation along with 

10 changes in proto-oncogene expression as well as cholesterol synthesis. Cholesterol metabolism changes 

11 following exposure to Pb were reportedly mediated as a result of induction of several enzymes related to 

12 cholesterol metabolism as well as a decrease in the cholesterol catabolizing enzyme, 7 α-hydroxylase. 

13 Tumor necrosis factor α (TNF-α) was reported to be one of the major mitogenic signals that mediated Pb 

14 nitrate-induced hepatic hyperplasia in studies using inhibitors to block TNF-α activity. Other Pb-related 

15 effects presented in the 2006 Pb AQCD include liver cell apoptosis mediated by Kupffer cell derived 

16 signals and Pb-induced oxidative stress in vitro cell cultures. More recent Pb exposure experiments 

17 suggested that alterations in liver heme metabolism may involve changes in 5-aminolevulinic acid 

18 dehydrogenase (ALAD) activity, porphyrin metabolism, Transferrin (TF) gene expression and changes in 

19 iron metabolism. 

20 In humans, the 2006 Pb AQCD stated that nonspecific liver injury generally observed as increases 

21 in liver enzymes in the serum was reported in occupational studies. In addition, similar to effects noted in 

22 animal studies, cytochrome P450 activity was also suppressed in humans following exposure to Pb under 

23 various conditions. The 2006 Pb AQCD concluded that hepatic effects occurred only at high Pb exposure 

24 levels.

5.9.1.2. New Epidemiologic Studies

25 A few studies examined liver biochemical parameters effects on antioxidant status and oxidative 

26 stress resulting from occupational exposures. Patil et al. (2007) examined the effect of occupational Pb 

27 exposure to liver and kidney function in silver jewelry workers (SJW), battery manufacturing workers 

28 (BMW) and spray painters (SP) in western Maharashtra, India. Blood Pb was statistically significantly 

29 increased in all three groups: 53.63 ± 16.98 (BMW), 48.56 ± 7.39 (SJW), and 22.32 ± 8.87 μg/dL (SP), 

30 compared to controls (12.52 ± 4.08). Liver function enzymes including serum glutamic oxaloacetic 

31 transaminase (SGOT)/AST, and serum glutamic pyruvic transaminase (SGPT)/ALT levels were only 
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1 increased in the SP group compared to the concurrent control group but not in the SJW and BMW groups. 

2 Total serum protein levels were decreased in all three groups, while serum albumin levels were decreased 

3 in SJW and SP groups and increased in the BMW group. Serum globulin levels were decreased and the 

4 albumin/globulin levels were increased in the BMW and SJW groups compared to controls. In addition, 

5 bilirubin levels were increased only in the BMW group. In a study examining the impact of Pb exposure 

6 in an occupational setting, Khan et al. (2008) reported that workers in Pakistan occupationally exposed to 

7 Pb (blood Pb = 29.1 [range 9.0 to 61.1] μg/dL) had a significant increase (3.5-fold higher median) in 

8 blood levels compared to age and gender matched controls (blood Pb = 8.3 [range 1.0 to 21.7] μg/dL). 

9 Oxidative stress markers such as MDA, and gamma-glutamyl transpeptidase (GGT) were significantly 

10 increased in workers as were ALT levels. Serum albumin and total protein levels were significantly 

11 decreased in the examined workers, compared to controls. Based on these results, study authors 

12 concluded that Pb exposure causes oxidative stress and changes in liver enzymes that may lead to hepatic 

13 toxicity in exposed workers. Can et al. (2008) also reported changes in liver function enzymes among 

14 battery workers and muffler repair workers exposed to Pb in an occupational setting. Blood Pb was 

15 elevated in both worker groups (36.83 ± 8.13 and 26.99 ± 9.42 μg/dL for battery workers and muffler 

16 repair workers, respectively) versus. controls (14.81 ± 3.01 μg/dL). The study authors reported that total 

17 protein, globulin, and lactate dehydrogenase (LDH) levels were within or very close to the normal range, 

18 but were statistically significantly higher in both worker groups compared to controls. Additionally, 

19 increases in cholesterol and ALP were increased only in battery workers and muffler repair workers, 

20 respectively. Though an increase in LDH levels among the workers was observed, the study authors stated 

21 that this increase was not related to liver injury. Total protein, globulin, ALP, and LDH were also observed 

22 to be significantly correlated to blood Pb levels in workers. Though liver enzyme function changes were 

23 nominal, the study authors concluded that in an occupational setting, exposure to Pb may lead to liver 

24 injury. While Can et al. (2008) did consider the impact of smoking in their analysis, it is not clear whether 

25 Patil et al. (2007) and Khan et al. (2008) considered the impact on these factors in their analysis. In a 

26 single case study report of a 40-year old Iranian male accustomed to using opium as a pain reliever, 

27 Verheij et al. (2009) reported that a liver biopsy taken following elevated liver function enzymes exhibited 

28 bile intracytoplasmic pigmentation in the hepatocytes. The study authors reported that the blood Pb levels 

29 were highly elevated in the patient (86.0 μg/dL), and attributed exposure to Pb from Pb-contaminated 

30 opium consumption. The liver parenchyma also revealed disrupted architecture along with regenerated 

31 nodules. Pathomorphological changes, rarely seen in humans, were also reported in the form of active 

32 hepatitis along with microvesicular and macrovesicular steatosis, hemosiderosis, and cholestatis as well as 

33 lymphocytic cholangitis. The study authors stated that following chelation therapy, liver enzymes returned 

34 to normal suggesting reversal of the histological findings. However, the reversibility was not confirmed 

35 with another liver biopsy. A case report by Fonte et al. (2007) described a worker occupationally exposed 

36 to Pb vapors (blood Pb = 148 μg/dL) with hypersideremia, mixed bilirubinemia, and elevated levels of 
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1 ALT and AST. Following chelation therapy, the patient’s clinical symptoms resolved, indicating the 

2 reversibility of the Pb-induced effects on the liver.

5.9.1.3. New Toxicological Studies

Hepatic Metabolism

3 As stated in the AQCD 2006, acute exposures to Pb nitrate and other Pb compounds causes a 

4 decrease in Phase I enzymes accompanied by a simultaneous increase in Phase II enzymes. The 

5 conclusions presented in the AQCD 2006 were also reviewed and corroborated by Mudipalli (2007). 

6 Changes in biochemical parameters, suggestive of liver damage, in male Wistar rats treated with 

7 500 ppm Pb acetate in drinking water over a 10 month period included decreases in serum protein and 

8 albumin levels as well as an increase in aspartate aminotransferase (AST), alanine aminotransferase 

9 (ALT), serum alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (GGT) levels (S. et al., 

10 2009). In treated animals, the blood Pb levels steadily increased throughout the study period, reaching a 

11 maximum of approximately 110 μg/dL. The study authors reported that similar biochemical changes were 

12 not observed in animals treated with Pb acetate as well a mineral rich diet and concluded that nutritional 

13 management is important in managing Pb-related poisoning. Swarup et al. (2007) investigated serum 

14 biochemical changes in cows living in Pb-contaminated environments. Serum levels of ALT, AST, 

15 alkaline phosphatase, total protein, albumin, globulin, and A/G ratio were statistically significantly altered 

16 in cows living near Pb-Zn smelters (blood Pb = 86 ± 6) compared to control cows (blood Pb = 7 ± 1 

17 μg/dL). Significant positive correlations were found between blood Pb and ALT and AST, whereas a 

18 negative correlation was observed between blood Pb and total lipids, protein, and albumin. Upadhyay et 

19 al. (2009) investigated the effects of Pb exposure on biochemical alterations in Sprague-Dawley rats 

20 exposed to 35 mg/kg via i.p. injection for 3 days. The activities of ALT, AST, serum ALP, and acid 

21 phosphatase were all significantly increased over control in exposed animals, whereas alkaline 

22 phosphatase activity was decreased in exposed animals. Concomitant treatment with zinc and varying 

23 levels of ascorbic acid were observed to ameliorate the toxic effects of Pb.

24 Pillai et al. (2009) investigated gestational and lactational exposure to Pb on hepatic phase I and II 

25 enzymes in male and female rats. Pregnant rats were exposed to 50 μg/kg Pb acetate via subcutaneous 

26 injection daily throughout gestation, and continuing until PND21. The female and male pups were then 

27 allowed to reach sexual maturity (PND55-56) to assess continuing exposure to bioaccumulated Pb. The 

28 activities of hepatic phase I enzymes NADPH- and NADH-cytochrome c reductase were statistically 

29 significantly reduced in Pb-exposed male and female rats on PND56, compared to controls. In rats treated 

30 with 25 μg/kg Pb and Cd, the effect on phase I enzymes was increased. Pb exposure additionally 

31 decreased the activities of phase II enzymes uridine diphosphate-glucoronyl transferase and GST in males 
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1 and females, but no effect was observed on GGT or 17β-hydroxysteroid oxidoreductase. Additionally, no 

2 effect was observed in Pb-exposed rats regarding serum glutamate pyruvate dehydrogenase or ALP 

3 activities in males or females. Histological observations in both male and female rats demonstrated fatty 

4 degeneration, vacuolization, and pycnotic nuclei, indicating general hepatotoxicity following Pb 

5 exposure. In a similar study, Teijon et al. (2006) exposed Wistar rats to 200 or 400 ppm throughout 

6 gestation, lactation, and 3 months postweaning, or only 1 month postweaning. In the animals exposed 

7 continuously throughout gestation and lactation, the concentrations of Pb in the liver were elevated in the 

8 200- and 400-ppm groups 1 and 3 months postweaning. Liver concentrations of Pb were greater in the 

9 200 ppm animals compared to the 400 ppm animals at one month postweaning, but were similar between 

10 the 2 dosing regimens at 3 months postweaning. ALP activity was increased at 2 weeks postweaning in 

11 animals continuously exposed to Pb throughout gestation and lactation, whereas ALT activity was 

12 decreased only at 2 and 3 months postweaning. In animals exposed only for 1 month postweaning, serum 

13 ALP activity was significantly increased, although in a non-dose dependent manner. ALT and AST 

14 activities were not affected.

15 Cheng et al. (2006) studied the mechanism of Pb effects on bacterial lipopolysaccharide (LPS)-

16 induced TNF-α expression. A/J mice were injected via i.p with 100 μmol/kg Pb, with or without 5 mg/kg 

17 LPS. Pb alone did not affect liver function (measured as AST or ALT activity) or the level of TNF-α in the 

18 serum. In comparison, exposing the mice to low doses of Pb and LPS together caused a statistically 

19 significant increase in TNF-α induction as well as enhanced liver injury, suggesting that Pb potentiated 

20 LPS-induced inflammation. In an in vitro study, the authors reported that co-exposure of Pb and LPS 

21 stimulated the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and increased TNF-α 

22 expression in mouse whole blood cells, peritoneal macrophages, and RAW264.7 cells (a macrophage cell 

23 line) and concluded that monocytes/macrophages (rather than hepatocytes) were primarily responsible for 

24 Pb increased LPS- induced TNF-α levels via the protein kinase C (PKC)/MAPK pathway. 

Lipid Metabolism

25 In a lipid metabolism study, Ademuyiwa et al. (2009) reported that male albino Sprague Dawley 

26 rats exposed to 200, 300 and 400 ppm Pb in drinking water had blood Pb levels of 40.63 ± 9.21, 61.44 ± 

27 4.63, and 39.00 ± 7.90 μg/dL, respectively. Animals exposed to 200 ppm had liver Pb concentrations of 

28 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. 

29 Animals exposed to Pb exhibited increased hepatic cholesterogenesis at all doses tested compared to 

30 controls. Additionally, a decrease in triglyceride was observed at 300 and 400 ppm, a decrease in 

31 phospholipid levels was observed at 400 ppm. The authors also reported a positive correlation between 

32 tissue cholesterol and phospholipids compared to Pb accumulation in liver across all doses. In contrast, 

33 the association between tissue triglyceride levels and Pb accumulation was negative. In a related study, 
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1 Khotimchenko and Kolenchenko (2007) reported that adult male albino rats treated with 100 mg/kg Pb 

2 acetate for as little as 14 days exhibited disorders in lipid metabolism that were supported by increased 

3 levels of total cholesterol and triglyceride levels in the liver tissue. Pillai et al. (2009) observed decreases 

4 in total liver cholesterol in PND56 male and female rats that had been exposed to 50 μg/kg Pb acetate 

5 continuously throughout gestation and lactation. These results suggest that induction of cholesterogenesis 

6 and phospholipidosis in the liver may cause subtle effects at the cellular level that may lead to 

7 hepatotoxicity. Kojima and Degawa (2006) examined the gender-related differences in the hepatic sterol 

8 regulatory element binding protein-2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase 

9 (HMGR) gene expressions in male and female Sprague Dawley rats injected with 100 μmol/kg body 

10 weight of Pb nitrate intravenously. The SREBP-2 expression, which is a transcription factor for the 

11 HMGR gene, was significantly increased in males and females with the increase occurring earlier in male 

12 rats (6-12 hours, compared to 24-36 hours in females). In contrast, expression of the HMGR gene, a rate 

13 limiting enzyme in cholesterol biosynthesis, was significantly increased in both males and females at 

14 earlier time frames (3-48 hours in males; 12-48 hours in females) compared to the SREBP-2 gene 

15 expression. Significant increases in total liver cholesterol were also observed in males and females at 3-48 

16 and 24-48 hours, respectively. These results suggest that the SREBP-2 and HMGR gene expressions and 

17 increase in total cholesterol levels in the liver occur earlier in males compared to females and also suggest 

18 that the HMGR gene expression and increase in total cholesterol levels in the liver occur before an 

19 increase in the SREBP-2 gene expression in either sex. 

Hepatic Oxidative Stress

20 A number of studies pertaining to hepatic oxidative stress as a result of exposure to various Pb 

21 compounds were identified. Adegbesan and Adenuga (2007) reported that protein undernourished male 

22 Wistar rats injected with 100 μmol/kg Pb nitrate exhibited increased lipid peroxidation, increased CAT 

23 activity, decreased SOD activity, and increased GSH levels, compared to undernourished rats not exposed 

24 to Pb. Increased lipid peroxidation and decreased CAT and SOD activity were also observed when 

25 comparing undernourished Pb-exposed rats to wellnourished control rats. Study authors concluded that 

26 malnutrition exacerbated Pb exposure effects on liver lipid peroxidation and the involvement of free 

27 radicals in Pb toxicity. Male Foster rats exposed to 0.025 mg/kg Pb via i.p. injection also exhibited 

28 statistically significant increases in lipid peroxidation levels and decreases in SOD, CAT, and glucose-6-

29 phosphatase dehydrogenase (G6PD) levels in liver mitochondrial and postmitochondrial fraction (Pandya 

30 et al., 2010). Nonstatistically significant decreases were also observed in GSH levels and GPx and GR 

31 activities in exposed animals. Yu et al. (2008) reported similar dose-dependent increases in lipid peroxide 

32 levels and decreases in GSH levels and CAT, SOD and GPx activities in castrated boars that received a 

33 supplemental diet with 0, 5, 10, or 20 mg/kg Pb. The level of hepatic CuZnSOD mRNA was also reduced 
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1 in treated animals. The study authors suggested that this decrease in SOD mRNA expression and activity 

2 of antioxidant enzymes may lead to a reduction free radical scavenging capability, along with increased 

3 lipid peroxidation, potentially causing serious damage to hepatic function and structure. Khotimchenko 

4 and Kolinchenko (2007) also reported an increase in lipid peroxidation and development of hepatitis in 

5 male albino rat liver parenchyma following treatment with 100 mg/kg Pb acetate for as little as 14 days. 

6 Lipid peroxidation was confirmed by increases in malondialdehyde (MDA) levels along with decreases in 

7 GSH and thiol groups indicating injury in the liver antioxidant system. In another experiment, Jurczuk et 

8 al. (2007) reported that male Wistar rats treated with 500 mg/L Pb in drinking water exhibited decreases 

9 in liver vitamin E and GSH levels along with an increase in lipid peroxidation. The study authors 

10 hypothesized that vitamin E is involved in the mechanism of peroxidative action of Pb in the liver, and 

11 concluded that the suggested protective role of vitamin E in the potential toxicity by Pb may be related to 

12 scavenging of free radicals that are generated either directly or indirectly by Pb. In a study examining the 

13 effects of Pb exposure to fetuses, Massó et al. (2007) exposed pregnant Wistar rats with 300 mg/L Pb in 

14 drinking water starting at day 1 of pregnancy to parturition or until weaning to determine the effects of Pb 

15 exposure in the fetal liver. Pups exhibited liver damage that was supported by an increase in thiobarbituric 

16 acid-reactive species (TBARS) production and increased CAT activity compared to controls. In addition, 

17 increased ALP and acid phosphatase activity was also observed. Uzbekov et al. (2007) exposed female 

18 Wistar rats to 0.3 and 3.0 mg/L Pb nitrate for 1 and 5 months prior to, and continuing during pregnancy, 

19 and measured fetal hepatic SOD activity on GD20. In the fetuses from dams exposed for 1 month prior to 

20 pregnancy, an dose-dependent increase in liver SOD activity was observed, whereas SOD activity was 

21 decreased in the fetuses from dams exposed for 5 months prior to pregnancy. The increase in SOD 

22 activity in the livers of fetuses from dams exposed to 0.3 or 3.0 mg/L Pb nitrate for one month suggests 

23 that activation of SOD in response to increased free radical production, while the decrease in SOD 

24 production in fetal livers from dams exposed to the same concentrations for 5 months suggests that longer 

25 durations of Pb exposure impairs the antioxidant defense mechanism. In a study examining the role of 

26 low molecular weight thiols on peroxidative mechanism, Jurczuk et al. (2006) stated that male Wistar rats 

27 treated with 500 mg/L Pb acetate in drinking water exhibited a decrease in blood ALAD as well as 

28 decreases in GSH and nonprotein sulfhydryl (NPSH) levels in the liver. Metalothionine levels were also 

29 reported to be higher in the liver following exposure to Pb. Levels of hepatic lipid peroxidation were 

30 observed to be significantly increased in rats exposed to 35 mg/kg Pb via i.p. injection, whereas hepatic 

31 GSH was significantly decreased (Upadhyay et al., 2009). No effects on GSH or MDA levels were 

32 observed in PND56 male and female rats following continuous exposure to 50 μg/kg Pb acetate 

33 throughout gestation and lactation (Pillai et al., 2009).

34 The studies presented above all confirm the possible oxidative stress impacts following exposure to 

35 various doses of Pb administered in various forms and the potential for hepatotoxicity as a result of 

36 oxidative stress. 
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5.9.2. Effects on the Gastrointestinal System

1 Gastrointestinal effects of Pb exposure primarily include abdominal pain, constipation, and internal 

2 paralysis in humans. In animals, degeneration of the intestinal epithelial mucosa and a decrease in 

3 duodenal motility has been reported.

5.9.2.1. Summary of Key Findings on the Effects on the Gastrointestinal System 

from the 2006 Lead AQCD

4 The 2006 Pb AQCD states that a number of factors influence the gastrointestinal absorption of Pb 

5 including the chemical and physical form of Pb, the age at Pb intake, as well as various nutritional factors. 

6 Potential malabsorption of Pb as a result of degeneration of the intestinal epithelial mucosa has been 

7 observed in rats exposed to Pb. Casein micelles incidences were reported as a result of Pb present in 

8 bovine and rat milk and in infant milk formula. Pb ingestion through water was more toxic compared to 

9 Pb ingestion via milk. Pb ingested in milk was reported to be taken up by the ileal tissue, whereas Pb 

10 administered intragastrically as a soluble salt was primarily accumulated in the duodenum irrespective of 

11 vehicle used for administration. Decreases in duodenal motility and the amplitude of contractility in the 

12 intestinal tract were observed in rats following Pb exposure. Nutritional studies examining different 

13 dietary levels of Pb, calcium, and vitamin D indicated competition in absorption between Pb and calcium. 

14 Diet supplement with vitamin D Pb to an increase in intestinal absorption of Pb and calcium. In instances 

15 where severe calcium deficiency was noted, ingestion of Pb caused a clear decrease in 1,25-dihydroxy 

16 vitamin D (1,25-(OH)2D3) levels. Overall, the 2006 Pb AQCD states that studies in rat intestine have 

17 shown that the largest amount of absorption occurs in the duodenum with the mechanisms of absorption 

18 involving active transport and diffusion via the intestinal epithelial cells. Absorption has been reported to 

19 occur, through both saturable and nonsaturable pathways based on results from various animal studies. 

20 The AQCD also states that the 1986 Pb AQCD reported that common gastrointestinal effects in humans 

21 following exposure to Pb include early symptoms of abdominal pain, constipation, and internal paralysis 

22 with these symptoms generally observed at blood Pb range of 30–80 μg/dL.

5.9.2.2. New Epidemiologic Studies

23 The 2006 Pb AQCD reported that in humans, gastrointestinal effects generally include abdominal 

24 pain, constipation, and internal paralysis. In a case study, Cabb et al. (2008) reported similar symptoms in 

25 a 3-year-old child diagnosed with elevated blood Pb (19 μg/dL). The child was reported to be 

26 complaining of nonlocalized abdominal pain along with vomiting, nausea, constipation, lack of appetite, 

27 joint pains, fatigue, irritability as well as headaches. Based on detailed nutritional information obtained 

28 from the mother, the study authors inferred that the child was regularly ingesting candy contaminated 

29 with Pb. Following this the child was treated by a folk healer with “greta” an orange powder that 
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1 worsened the abdominal symptoms. When analyzed, it was determined that “greta” contained 14,000 

2 mg/kg Pb monoxide. When the child was taken off “greta” his GI symptoms began to resolve. Based on 

3 this study report, it may be concluded that Pb is capable of causing severe GI trauma and the GI effects 

4 may be reversible once exposure to Pb is ceased. In a similar case study, Fonte et al. (2007) reported that 

5 a 47-year-old male exposed to Pb fumes and vapors at a recycling plant experienced similar symptoms 

6 reported by Cabb et al. (2008) in the 3-year-old child. The male patients’ Pb levels were elevated with a 

7 blood Pb level of 148 μg/dL. Once exposure to Pb had ceased and the patient was treated with EDTA, his 

8 condition improved and the blood Pb dropped to 16 μg/dL. Kuruvilla et al. (2006) also reported 

9 gastrointestinal effects including stomach pain and gastritis along with other Pb-related clinical 

10 manifestations in battery workers and painters (blood Pb = 42.40 ± 25.53 and 8.04 ± 5.04 μg/dL, 

11 respectively) occupationally exposed to Pb in India. 

5.9.2.3. New Toxicological Studies

12 Two studies pertaining to gastrointestinal effects of Pb exposure were identified. Santos et al. 

13 (2006) examined the impact of Pb exposure on nonadrenergic noncholinergic (NANC) relaxations in rat 

14 gastric fundus. Male Wistar rats treated with 0.008% Pb acetate via drinking water for 15, 30, and 120 

15 days exhibited significant difference in NANC relaxations in the gastric fundus following electrical field 

16 stimulus (EFS). While frequency-dependent relaxations were observed in all groups, including the control 

17 group, the relaxations were significantly inhibited in rats treated with Pb acetate at all three durations. 

18 When gastric fundus strips from rats were incubated with L-nitroarginine (L-NOARG), a nitric oxide 

19 synthase (NOS) inhibiter, no additional inhibition in relaxations was observed. In contrast, incubation 

20 with sodium nitroprusside and 8-Br-GMPc (a Cyclic guanosine monophosphate [cGMP] analog), 

21 exhibited at dose-dependent relaxation in strips in the control group and group exposed to Pb acetate for 

22 120 days. Study authors concluded that chronic exposure to Pb causes inhibition in NANC relaxation 

23 probably due to the modulated release of NO from the NANC nerves or due to interaction with the 

24 intracellular transducer mechanism in the rat gastric fundus. 

25 In another study examining Pb-induced oxidative stress in the gastric mucosa, Olaleye et al. (2007)

26 treated Albino Wistar rats with 100 or 5,000 mg/L of Pb acetate for 15 weeks. Exposure to Pb acetate 

27 caused a significant increase in gastric mucosal damage caused by pretreatment with acidified ethanol. 

28 Study authors reported that though the basal gastric acid secretory rate was not altered, stomach response 

29 to histamine was significantly higher in animals treated with Pb acetate compared to the controls. 

30 Additionally, there was a significant increase in gastric lipid peroxidation in both the 100 and 5,000 mg/L 

31 dose levels. In contrast, CAT, and SOD activities and nitrite levels were significantly decreased in the 

32 gastric mucosa. Study authors concluded that exposure to Pb may increase the formation of gastric ulcers 

33 as a result of changes in the oxidative metabolism in the stomach.
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5.9.3. Effects on the Endocrine System

1 Endocrine processes that are most commonly impacted by Pb exposure include changes in the 

2 thyroid, such as changes in the thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine 

3 (T4). In addition changes in the male sex hormone levels have also been reported following exposure to 

4 Pb.

5.9.3.1. Summary of Key Findings of the Effects on the Endocrine System from 

the 2006 Lead AQCD

5 Endocrine processes that may be impacted by Pb include thyroid hormone levels, changes in male 

6 sex hormone levels, as well as changes in the production of 1,25-(OH)2D3 levels. However, these effects 

7 were reported to be observed only at blood Pb levels exceeding 30–40 μg/dL. Summary of key findings 

8 pertaining to reproductive hormones in females is presented in the section on reproductive effects and 

9 birth outcomes (Section 5.8.1). 

5.9.3.2. New Epidemiologic Studies

10 Thyroid hormone levels were reported to be impacted following exposure to various environmental 

11 contaminates, including Pb, by Abdelouahab et al. (2008), Croes et al. (2009), and Dundar et al. (2006). 

12 Abdelouahab et al. (2008) performed a cross-sectional study in a Canadian population exposed to various 

13 environmental contaminants, including Pb, following consumption of freshwater fish. The median blood 

14 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 

15 important to note that the median blood Pb level for women is lower than the limit of detection for Pb in 

16 the blood (2.1 μg/dL), effectively meaning that greater than 50% of women in the study had 

17 nondetectable levels of Pb in their blood. The study authors conducted a stratified analysis and concluded 

18 that TSH levels were negatively correlated with blood Pb in women that consumed fish contaminated 

19 with Pb and other environmental pollutants. No impacts in T3 and T4 levels were reported in women. 

20 TSH, T3 and T4 levels were not observed to be correlated with blood Pb in males. However, study 

21 authors stated that occupational exposure to Pb in men can affect pituitary thyroid axis homeostasis and 

22 the relation between low level Pb exposure thyroid hormone homeostasis in men and women needs to be 

23 investigated further. Dundar et al. (2006) examined the effects of blood Pb on thyroid function in 42 male 

24 adolescent auto repair workers exposed long term to Pb. A control group comprised of 55 healthy subjects 

25 was also used for comparison purpose. Mean blood Pb levels were reported to be higher in the auto repair 

26 workers compared to the control subjects (7.3 ± 2.92 versus. 2.08 ± 1.24 μg/dL). Free T4 (FT4) levels 

27 were significantly lower in the study group compared to the control group, which had no abnormal FT4 

28 levels reported. In contrast, free T3 (FT3) and TSH levels were comparable between the study and control 

29 group. Blood Pb level was reported to be negatively correlated with FT4 levels. Based on the study 
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1 outcome, the study authors reported that long-term exposures that result in the studied blood Pb levels 

2 may lead to lower FT4 levels without impact on T3 and TSH levels in adolescents. The study authors 

3 stated that this effect is likely secondary to the toxic effects of Pb on the pituitary-thyroid axis and to the 

4 earlier findings of primary hypothyroidism as a result of impaired production of peripheral thyroid 

5 hormones. Similar findings were reported by Croes et al. (2009) in a study conducted in Belgium. Croes 

6 et al. (2009) examined the hormone levels and degree of sexual maturity in 1679 adolescents residing in 

7 nine study areas with varying exposures to multiple industrial pollutants including Pb. The median blood 

8 Pb of the participants from the nine different regions ranged from 1.6 to 2.8 μg/dL. The study authors 

9 reported that, after correction for confounding, significant interregional differences were observed in the 

10 levels of sex hormones including total and free testosterone, estradiol, aromatase, and luteinizing hormone 

11 as well as FT3 hormone levels. When individual neighborhoods were analyzed within the larger study 

12 areas, altered levels of testosterone, aromatase, and FT3 levels were also observed. Altered sexual 

13 maturation was also observed among boys and girls of individual study areas, compared to the sexual 

14 maturation of the entire cohort. Though varying levels of sex hormones and rates of sexual maturation 

15 was observed, the study authors reported that these changes are not wholly due to exposure to various 

16 pollutants, including Pb that were measured in the study and stated that other pollutants and 

17 environmental factors may also contribute to the effects noted. 

18 Gump et al. (2008) examined cortisol response to acute stress in children (aged 9.5 years) whose 

19 prenatal and postnatal blood Pb levels had been determined prior to the study at birth (from cord blood) 

20 and at age 2.62 ± 1.2 years, respectively. For prenatal blood Pb, the children were broken into the 

21 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 

22 1.5-2.8, 2.9-4.1, 4.2-5.4, and 5.5-13.1 μg/dL. The study authors reported that blood Pb was not associated

23 with initial salivary cortisol levels. However following an acute stressor, which was comprised of a gallon 

24 of one part ice to one part water into which a child was asked to submerge his or her dominant arm for a 

25 minute, increasing prenatal and postnatal blood Pb levels were statistically significantly associated with 

26 increases in salivary cortisol responses. Children in the 2nd, 3rd and 4th prenatal blood Pb quartiles and in 

the 4th 27 postnatal quartile appeared to have increased salivary cortisol responses compared to children in 

the 1st 28 quartile. When blood Pb was treated as a continuous variable, regression analysis showed that both 

29 prenatal and postnatal blood Pb levels were significantly correlated to salivary cortisol reactivity. Based 

30 on these results, the study authors reported that relatively low prenatal and postnatal blood Pb levels, 

31 notably those well below 10 μg/dL, can alter children’s adrenocortical responses following acute stress 

32 and the health impact and behavioral aspects of this Pb-induced HPA deregulation in children needs to be 

33 further examined. 

34 In another study on the impact of Pb in children, Kemp et al. (2007) examined the blood levels in 

35 142 young, urban African-American and Hispanic children in winter and summer to determine the 

36 seasonal increase in blood Pb and its association with vitamin D, age and race. There was a 
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1 winter/summer (W/S) increase in blood Pb levels in children aged between 1 and 3 years (winter blood Pb

2 = 4.94 ± 0.45 (SE) μg/dL, summer blood Pb = 6.54 ± 0.82 (SE) μg/dL), with a smaller W/S increase 

3 observed in children aged between 4 and 8 years (winter blood Pb = 3.68 ± 0.31 (SE) μg/dL, summer 

4 blood Pb = 4.16 ± 0.36 (SE) μg/dL). Additionally, the winter and summer blood Pb levels were highly 

5 correlated with one another. The percentage of African-American children with blood Pb levels ≥ 10 

6 μg/dL increased from 12.2% in winter to 22.5% in summer. Summer and winter concentrations of 1,25-

7 (OH)2D3 were observed to differ in children aged 4-8 years and the correlation between the serum 1,25-

8 (OH)2D3. No difference in 1,25-(OH)2D3 was observed in children 1-3 years old. There was a significant 

9 correlation between seasonal differences in blood Pb and serum 1,25-(OH)2D3 in all children and 

10 African-American children between 4 and 8 years. Based on these results, the study authors concluded 

11 that higher summertime increase in serum 1,25-(OH)2D3 levels in children between 4 and 8 years is most 

12 likely due to increased sunlight-induced vitamin D synthesis and may be a contributing factor to seasonal 

13 changes in blood Pb levels. 

5.9.3.3. New Toxicological Studies

14 A single study examining the impact of Pb exposure on the endocrine system was identified. In a 

15 study examining the effects of Pb and cadmium in adult cows reared in a polluted environment in India, 

16 Swarup et al. (2007) stated that the mean plasma T3 and T4 levels were significantly higher in cows near 

17 Pb and zinc smelters (blood Pb = 86 ± 6 μg/dL) and near closed Pb and operational zinc smelters (blood 

18 Pb = 51 ± 9 μg/dL) when compared to cows in unpolluted areas (blood Pb = 7 ± 1 μg/dL). Regression 

19 analyses from 269 cows examined in the study showed a significant positive correlation between blood 

20 Pb and plasma T3 and T4 levels, whereas the correlation between blood Pb and plasma cortisol was 

21 nonsignificant. Mean plasma estradiol level was significantly higher in cows near closed Pb and 

22 operational zinc smelter industries compared to the control group. Based on these results, the study 

23 authors concluded that endocrine profile in animals can be impacted following exposure to Pb in polluted 

24 environments. 

25 Biswas and Ghosh (2006) investigated the effect of Pb exposure on adrenal and male gonadal 

26 functions in Wistar rats exposed to 8.0 mg/kg Pb acetate via i.p. injection for 21 days. Exposure to Pb was 

27 observed to significantly increase adrenal steroidogenic enzyme activity and serum corticosterone levels. 

28 Accessory sex organ (prostate and seminal vesicle) weights were decreased in Pb-exposed animals, 

29 whereas adrenal weights were increased. Spermatogenesis was decreased and the percent of spermatid 

30 degeneration was increased in animals exposed to Pb. Lastly, serum concentrations of testosterone, FSH, 

31 and LH, were decreased in Pb-exposed animals. Supplementation with testosterone during the last 14 

32 days of exposure to Pb was observed to ameliorate these effects.
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5.9.4. Effects on Bone and Teeth

1 Primary effects on bone as a result of Pb exposure include an increase in osteoporosis, increased 

2 frequencies of falls and fractures, changes in bone cell function as a result of replacement of bone calcium 

3 with Pb and depression in early bone growth. Similar to bone, calcium in the teeth is easily substituted by 

4 Pb following Pb exposure. Exposure to high levels of Pb may result in the formation of “Pb line” and Pb 

5 can also cause a decrease in cell proliferation, procollagen type I production, intracellular protein, and 

6 osteocalcin in human dental pulp cell cultures. Accumulation of Pb was also associated with tooth loss 

7 and higher incidence of periodontitis.

5.9.4.1. Summary of Key Findings of the Effects on Bone and Teeth from the 

2006 Lead AQCD

8 The 2006 Pb AQCD reported many effects on bone and some in teeth following exposure to Pb. 

9 Calcium in bone was easily substituted by Pb and taken up by the bone causing changes in bone cell 

10 function. Exposure to Pb during gestation and immediate postnatal period was reported to significantly 

11 depress early bone growth with the effects showing a dose-dependent trend, though this effect was not 

12 manifested below certain exposure thresholds. Bone effects following short-term exposure to Pb were not 

13 reported in mature animals; however, long-term exposures to Pb along with poor nutrition had an adverse 

14 effect on bone growth as well bone density. Systemic effects of Pb exposure include disruption in bone 

15 mineralization during growth, alteration in bone cell differentiation and function due to alterations in 

16 plasma levels of growth hormones and calcitropic hormones such as 1,2-[OH]2D3 and impact on calcium 

17 binding proteins and increases in calcium and phosphorus concentrations in the blood stream. Bone cell 

18 cultures exposed to Pb were shown to impact vitamin D-stimulated production of osteocalcin 

19 accompanied by inhibition of secretion of bone-related proteins such as osteonectin and collagen. In 

20 addition, Pb exposure also caused suppression in bone cell proliferation most likely due to interference 

21 from factors such as growth hormone (GH), epidermal growth factor (EGF), transforming growth factor22 beta 1 (TGF-β1), and parathyroid hormone-related protein (PTHrP).

23 Like the bone, Pb can easily substitute for calcium in the teeth and is taken and incorporated into 

24 developing teeth in experimental animals. Since teeth do not undergo remodeling like the bone during 

25 growth, most of the Pb in the teeth remains in a state of permanent storage. High dose exposure of Pb to 

26 animals has lead to the formation of a “Pb line” that is visible in both the enamel and dentin and is 

27 localized in areas of recently formed tooth structure. Areas of mineralization are easily evident in the 

28 enamel and the dentin within these “Pb lines.” Pb has also been shown to decrease cell proliferation, 

29 procollagen type I production, intracellular protein, and osteocalcin in human dental pulp cell cultures. 

30 Adult rats exposed to Pb have exhibited an inhibition of the posteruptive enamel proteinases, delayed 

31 teeth eruption times, as well as decrease in microhardness of surface enamel. Pb was reported to be 
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1 widely dispersed and incorporated into developing apatite crystal during enamel formation process; 

2 however, post formation, Pb was reported to be capable of entering and concentrating in enamel areas that 

3 were calcium deficient. The AQCD also reported that a number of epidemiologic and animal studies have 

4 both separately suggested that Pb is a caries-promoting element. 

5.9.4.2. New Toxicological and Epidemiologic Studies

5 As reported in the 2006 Pb AQCD, Pb is capable of causing significant toxicological effects in 

6 bones of humans and animals following short-term and long-term exposure. The association between Pb 

7 exposure and osteoporosis was examined in three different epidemiologic studies. Campbell and Auigner 

8 (2007) examined subjects ≥ 50 years of age using the Third National Health and Nutrition Examination 

9 Survey (NHANES III) for association between Pb exposure and osteoporosis. The study authors used the 

10 bone mineral density (BMD) in the hip as the primary variable to examine groups comprised of non11 Hispanic white men (mean blood Pb = 4.9, range: 0.7 to 48.1 μg/dl), non-Hispanic white women (mean 

12 blood Pb = 3.6, range: 0.7 to 28.7 μg/dL), African-American men (mean blood Pb = 7.7, range: 0.7 to 

13 52.9 μg/dL) and African-American women (mean blood Pb = 4.5, range: 0.7 to 23.3 μg/dL). The results 

14 indicated that the adjusted mean total hip BMD in the non-Hispanic white males who had the lowest 

15 blood Pb levels was statistically significantly higher than the males with higher blood Pb levels. Similar 

16 associations, although not statistically significant, were reported among white females. Due to the small 

17 sample size, similar results were not observed among African-American men and women. No association 

18 was observed between blood Pb and osteoporotic fractures in any gender/race. Since the NHANES data 

19 were comprised of a cross-sectional design, no inferences could be made regarding the temporal sequence 

20 of the observed association. The study authors concluded that further inquiry was needed to study the 

21 possible causal association between Pb exposure and osteoporosis. In a similar study, Sun et al. (2008)

22 examined the association between Pb exposure and osteoporosis in 192 (155 males; 37 females) Chinese 

23 individuals occupationally-exposed to Pb (blood Pb = 20.22 and 15.5 μg/dL, respectively). BMD was 

24 reported to be statistically significantly lower in exposed females compared to exposed males. When all 

25 participants (including 36 male and 21 female unexposed controls) were divided into groups according to 

26 blood Pb and urinary Pb levels, the study authors reported that there were significant decreases in BMD in 

27 groups that had high urinary Pb levels (≥ 5 μg/g creatinine) compared to groups with low urinary Pb in 

28 both genders. In contrast a significant difference was only observed between blood Pb and BMD in males 

29 with blood Pb >30 μg/dL. Prevalence of osteoporosis was reported to increase significantly with 

30 increasing blood Pb in a linear manner. Khalil et al. (2008) reported similar associations between Pb 

31 exposure and osteoporosis in older women. The study authors conducted a prospective study using 533 

32 women aged 65-78 years with a mean blood Pb of 5.3 ± 2.3 μg/dL to determine the association between 

33 blood Pb and recurring fractures. The BMD was 7% lower in the total hip (p <0.02) and 5% lower in the 
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1 femoral neck (p <0.03) in the highest blood Pb group (≥ 8 μg/dL) compared to the lowest blood Pb group 

2 (≤ 3 μg/dL). The trend across all dose groups was also observed to be statistically significant for hip and 

3 femoral neck BMD. In addition, hip, femoral neck, and calcaneus bone loss was observed to be greater in 

4 the medium (blood Pb = 4-7 μg/dL) and high Pb groups compared to the low Pb group, but the observed 

5 trend was only significant for calcaneus bone loss. Multivariate analysis indicated that women with high 

6 blood Pb levels had an increased risk of nonspine fracture and women with medium or high blood Pb 

7 levels had a higher risk of falls compared to the low blood Pb level group. Based on these results, the 

8 study authors concluded that blood Pb is associated with an increased risk of falls and fractures leading to 

9 negative health consequences including osteoporosis-related fractures. 

10 The effect of Pb exposure on bone development in younger children has been studied before and an 

11 association has been found between Pb exposure and bone development. Ignasiak et al. (2006) conducted 

12 a study on school children aged 7-15 years (463 males, 436 females) living close to copper smelters and 

13 refineries in Poland to assess the impact of Pb exposure on their growth status. The mean blood Pb for all 

14 participants was 7.7 ± 3.5 μg/dL (range: 2.0 to 33.9). Blood Pb levels were similar between males and 

15 females except at age 14.8 years, when females had lower blood Pb than males. Study results indicated 

16 that there was a statistically significant and linear relationship between blood Pb and reduced weight, 

17 height, and trunk, leg and arm lengths. Regression analysis revealed that, per every 10 μg/dL increase in 

18 blood Pb levels, height decreased 2.1 and 2.9 cm for males and females, respectively. This decrease in 

19 height was more influenced by decreases in leg length than trunk length. Based on these results, the study 

20 authors concluded that linear skeletal growth was reduced with increases in blood Pb levels and these 

21 effects were seen even at levels below 10 μg/dL. These results also indicated that there was attenuation in 

22 osteoblast activity as a result of Pb exposure. 

23 To understand the significance of bone as a target tissue of Pb toxicity, Jang et al. (2008) studied 

24 the effect of Pb on calcium release activated calcium influx (CRACI) using primary cultures of human 

25 osteoblast-like cells (OLC). When cells were incubated with 1 or 3 mM Pb, a dose-dependent impact on 

26 the CRACI observed, as was a dose-dependent increase in the influx of Pb into human OLC. These 

27 results suggest that Pb interferes with CARCI in human OLCs by initiating the CRACI (i.e. the 

28 measurable influx of calcium upon re-addition of calcium is partially inhibited by Pb) and the influx of Pb 

29 was enhanced after CRACI had been induced. Since skeletal growth in Pb-exposed children is stunted, 

30 Zuscik et al. (2007) conducted a study using murine limb bud mesenchymal cells (MSCs) to test the 

31 hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and also assessed the effects of 

32 Pb on various signaling pathways. Exposure to 1 μM Pb caused increased basal and TGF-β/BMP 

33 induction of chondrogenesis in MSCs which was supported by nodule formation and upregulation of Sox34 9, type 2 collagen, and aggrecan which are all key markers of chondrogenesis. The study authors also 

35 observed enhanced chondrogenesis during ectopic bone formation in mice that had been pre-exposed to 

36 Pb in drinking water (55 or 233 ppm, corresponding to 14 or 40 μg/dL blood Pb). MSCs exposed to Pb 
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1 exhibited an increase in TGF-β, but BMP-2 signaling was inhibited. Pb was also reported to induce NFκB

2 and inhibit AP-1 signaling. Based on these results, the study authors concluded that chondrogenesis 

3 following exposure to Pb most likely involved modulation and integration of multiple signaling pathways 

4 including TGF-β, BMP, AP-1, and NFκB.

5 Effects of Pb exposure on teeth were examined in three epidemiologic studies. Since individuals 

6 previously exposed to Pb may be impacted due to the endogenous release of Pb stored in their skeletal 

7 compartments, Arora et al. (2009) examined the association between bone Pb concentrations and loss of 

8 natural teeth in 333 male participants of the Normative Aging Study. Tooth loss in men was categorized as 

9 0, 1-8 or ≥ 9. Individuals with ≥ 9 teeth missing had significantly higher bone Pb concentrations 

10 compared to those with no tooth loss; no significant difference in blood Pb levels was observed between 

11 the categories of teeth loss. Following adjustment for different variables (age, education, smoking status, 

12 pack-years of smoking, and diabetes), men with the highest tibia Pb concentrations (>23μg/g) had higher 

13 odds of tooth loss (OR: 3.03 [95% CI: 1.60,5.75]) compared to men with tibia Pb ≤ 15 μg/g. Men with the 

14 highest patellar Pb (>36 μg/g) also had higher odds of tooth loss (OR: 2.41 [95% CI: 1.30,4.49]) 

15 compared to men with patellar Pb ≤ 22.0 μg/g. Odds of tooth loss were not statistically associated with 

16 blood Pb levels. Based on these results, the study authors concluded that long-term cumulative exposure 

17 to Pb is associated with increased odds of tooth loss. In a study examining the effects of Pb exposure on 

18 periodontitis in the United States (US), Saraiva et al. (2007) used data from NHANESIII by analyzing 

19 data for 2,500 men and 2,399 women aged between 30 and 56 years. The analysis took into account 

20 various covariates including age, NHANESIII phase, cotinine levels, poverty ration, race/ethnicity, 

21 education, BMD, diabetes, calcium intake, dental visits, and menopause in women. After adjusting for 

22 these covariates and comparing individuals with a blood Pb level of >7 μg/dL to those with a blood Pb 

23 level of <3 μg/dL, the prevalence ratios of periodontitis was 1.70 (95% CI: 1.02,2.85) for men and 3.80 

24 (95% CI: 1.66,8.73) for women. Based on these results, the study authors concluded that there was a 

25 positive and statistical association between periodontitis and Pb levels for both men and women. In a 

26 similar study, Yetkin et al. (2007) recruited 60 male subjects (30 apprentices with Pb exposure, 30 

27 controls), to examine the impact of occupational exposure to Pb on periodontal status and association 

28 between periodontitis and blood Pb or oxidative stress. The results of their analysis indicated that blood 

29 Pb was significantly higher in apprentices exposed to Pb compared to controls (7.38 ± 4.41 versus. 2.27 ± 

30 1.49 μg/dL). No clinical periodontal or oxidative stress parameters were significantly difference between 

31 apprentices and controls. While the correlation between blood Pb and periodontal parameters was not 

32 reported, significant correlations between plaque index and CAT, probing depth and SOD, clinical 

33 attachment level and SOD, and clinical attachment level and malondialdehyde in Pb-exposed apprentices 

34 was observed. These results demonstrate that there is significant association between clinical periodontal 

35 parameters and oxidative stress/damage indices in Pb-exposed apprentices. Following multiple regression 
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1 analysis, a statistically significant association between gingival index and working status, family income 

2 and either probing depth or clinical attachment level was noted. 

5.9.5. Effects on Ocular Health

3 Ocular effects most commonly observed following exposure to Pb include formation of cataract, 

4 impaired vision, edema and retinal stippling.

5.9.5.1. Summary of Key Findings of the Effects on Ocular Health from the 2006 

Lead AQCD

5 The 2006 Pb AQCD stated that various changes in the visual system were observed with Pb 

6 poisoning including retinal stippling and edema, cataract, ocular muscle paralysis and impaired vision. 

7 The AQCD reported that retinal responses were observed in children of mothers with a blood Pb range of 

8 10.5 to 32.5 μg/dL during pregnancy, while cataracts were noted in middle-aged male with tibia bone Pb 

9 levels of 31-126 μg/g.

5.9.5.2. New Toxicology and Epidemiology Studies

10 New animal studies pertaining to the ocular effects of Pb have investigated endpoints such as 

11 retinal progenitor cell proliferation and neurogenesis, and have observed effects at exposures that resulted 

12 in blood Pb levels as low as <10 μg/dL (Section 5.3.4.3). One human study pertaining to ocular effects of 

13 Pb was identified. Mosad et al. (2010) studied the association between subcapsular cataract and Pb, 

14 cadmium, vitamin C, vitamin E, and beta carotene blood levels in middle-aged male smokers compared to 

15 nonsmokers. Blood Pb was statistically significantly elevated in light (14.5 ± 0.41 μg/dL), moderate (14.5 

16 ± 0.41 μg/dL), and heavy smokers (18.7 ± 1.24 μg/dL) compared to nonsmokers (12.2 ± 0.21 μg/dL). Pb 

17 concentrations were also observed to be statistically higher in the cataracts of smokers versus. 

18 nonsmokers. Similar associations were also observed for cadmium blood and lens levels, while vitamins 

19 C, E, and beta carotene levels were significantly decreased in smokers. Based on these results, the study 

20 authors concluded that the Pb and cadmium present in high concentration in smokers were associated 

21 with cataracts due to oxidative stress which was indicated by reduced levels of antioxidants such as 

22 vitamins C, and E and beta carotene. 

5.9.6. Effects on the Respiratory System

23 The collective body of toxicological and epidemiologic studies demonstrates Pb-induced effects on 

24 multiple immunological pathways, including a shift from a Th1 to a Th2 phenotype, increased IgE 

25 antibody production, and increased inflammatory responses (Sections 5.2.5.1. and 5.6). These are well 
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1 recognized pathways that contribute to increased susceptibility to infections and also to the development 

2 of respiratory diseases such as asthma. Recent investigation of the respiratory effects of Pb exposure has 

3 been limited; however, cross-sectional studies have indicated an association of increasing blood Pb level 

4 with increased prevalence of respiratory tract illnesses (Section 5.6.4.1) and asthma in children (Section 

5 5.6.4.2). As described in Section 5.2.4, Pb has been shown to induce the generation of ROS. ROS are 

6 implicated in mediating increases in bronchial responsiveness and activating neural reflexes leading to 

7 decrements in lung function. Studies investigating these airway responses also are limited in number and 

8 collectively do not provide strong evidence of an association with blood Pb (Section 5.6.4.3). 

9 Collectively, panel and time-series studies demonstrate associations between Pb measured in PM2.5 or 

10 PM10 air samples and decreases in lung function and increases in respiratory symptoms, hospitalizations, 

11 and mortality (Section 5.6.4.3). However, common limitations of these air-Pb studies are the variable size 

12 distribution of Pb-bearing PM (Section 3.5.3) and its relationship with blood Pb levels as well as the lack 

13 of adjustment for other correlated PM chemical components.

5.9.7. Summary

14 There is evidence from epidemiologic and toxicological studies that exposure to Pb results in 

15 altered liver function and hepatic toxicity. Biochemical changes indicative of liver injury, including 

16 decreases in serum protein and albumin levels and increased AST, ALT, ALP, and GGT activities, have 

17 been observed in humans (Can et al., 2008; D. A. Khan et al., 2008; Patil et al., 2007) and animals (Y.-J. 

18 Cheng et al., 2006; S. et al., 2009). Increased hepatic cholesterogenesis, altered triglyceride and 

19 phospholipid levels, and disorders in lipid metabolism supported by increased levels of total cholesterol 

20 and triglycerides has been reported in the animal literature (Ademuyiwa et al., 2009; Khotimchenko & 

21 Kolenchenko, 2007). These results suggest that induction of cholesterogenesis and phospholipids in the 

22 liver may cause subtle effects at the cellular level, leading to hepatic injury. Multiple studies in humans 

23 and animals have observed hepatic oxidative stress, generally indicated by an increase in lipid 

24 peroxidation along with a decrease in GSH levels and CAT, SOD, and GPx activities following exposure 

25 to Pb (Adegbesan & Adenuga, 2007; D. A. Khan et al., 2008; Khotimchenko & Kolenchenko, 2007; D. Y. 

26 Yu et al., 2008). Indices of oxidative stress were additionally observed in the livers of fetuses exposed 

27 throughout gestation (Uzbekov et al., 2007).

28 Relatively few human studies have been conducted on the gastrointestinal toxicity of Pb since the 

29 completion of the 2006 Pb AQCD. Two cases studies reporting on GI symptoms in a child and adult 

30 report that elevated blood Pb was associated with nonlocalized abdominal pain, vomiting, nausea, 

31 constipation, lack of appetite, fatigue, and headaches (Cabb et al., 2008; Fonte et al., 2007). The adult’s 

32 blood Pb level was reported as 148 μg/dL. Both subjects’symptoms were reported to diminish following 

33 cessation of exposure or chelation therapy. Similar GI symptoms (stomach pain and gastritis) were 
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1 observed in battery works and painters exposed to Pb in India with blood Pb levels ranging from 0.4-

2 116.6 μg/dL (Kuruvilla et al., 2006). Toxicological evidence for Pb-induced GI health effects in rats 

3 includes inhibition of NANC relaxations in the gastric fundus and the observation of oxidative stress 

4 (lipid peroxidation, decreased SOD and CAT) in the gastric mucosa (Olaleye et al., 2007; M. R. V. Santos 

5 et al., 2006). The observation of oxidative stress was accompanied gastric mucosal damage.

6 The endocrine processes most impacted by exposure to Pb include changes in thyroid function, as 

7 well as alteration in sex and stress hormone profiles. TSH was negatively associated with blood Pb in 

8 women that ate contaminated fish (median blood Pb = 1.7 μg/dL) but not men, and FT4, but not FT3, was 

9 decreased in adolescent male auto repair workers (Abdelouahab et al., 2008; Dundar et al., 2006). 

10 Alterations in the levels of sex hormones, including total and free testosterone, estradiol, aromatase, and 

11 luteinizing hormone, were observed in Belgian adolescents environmentally exposed to Pb (Croes et al., 

12 2009). Toxicological evidence for similar effects was observed in adults cows reared in a contaminated 

13 environment and exposed to Pb. A positive correlation was reported between blood Pb and plasma T3 and 

14 T4 levels (Swarup et al., 2007). Mean plasma estradiol levels were also significantly higher in Pb-exposed 

15 cows. In children challenged with an acute stressor, increasing blood Pb was associated with significant 

16 increases in salivary cortisol responses, even at blood Pb levels less than 10 μg/dL (Gump et al., 2008).

17 Numerous epidemiologic studies investigated the association between Pb exposure and 

18 osteoporosis in adults. High blood Pb has been observed to be associated with decreased BMD in non19 Hispanic white males (Campbell & Auinger, 2007), whereas urinary Pb, but not blood Pb, was seen to be 

20 associated with decreased BMD in Chinese individuals occupationally exposed to Pb (Y. Sun, Sun, Zhou, 

21 Zhu, Zhang, et al., 2008). In elderly women, blood Pb levels were associated with an increased risk of 

22 falls and fractures, including osteoporosis-related falls (Khalil et al., 2008). Linear skeletal growth in 

23 children exposed to Pb was reduced with increased blood Pb; these effects were seen at blood levels <10 

24 μg/dL (Ignasiak et al., 2006). In vitro studies indicate that Pb interferes with CARCI in human OLCs and 

25 that Pb perturbs multiple signaling pathways during murine limb bud growth, potentially resulted in 

26 altered skeletal development (Jang et al., 2008; Zuscik et al., 2007). Epidemiologic studies investigating 

27 Pb exposure and tooth loss report that long-term, cumulative exposure to Pb is associated with increased 

28 odds of tooth loss, periodontitis in men and women, and that periodontitis is associated with oxidative 

29 stress/damage in individuals exposed in an occupational setting (Arora et al., 2009; Saraiva et al., 2007; 

30 Yetkin-Ay et al., 2007).

31 New toxicology studies have reported ocular effects (i.e., retinal progenitor cell proliferation) at 

32 blood Pb levels as low as <10 μg/dL (Section 5.3.4.3), and one human study reports and association 

33 between heavy smoking, increased blood Pb levels, and cataracts (Mosad et al., 2010). Investigation of 

34 the respiratory effects of Pb exposure has been limited; however, cross-sectional studies have indicated an 

35 association of increasing blood Pb with increased prevalence of respiratory tract illnesses (Section 
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1 5.6.4.1) and asthma in children (Section 5.6.4.2). Additionally, Pb-induced production of ROS is 

2 implicated in increased BR and decrements in lung function (Section 5.6.4.3).

3 In summary, recent toxicological and epidemiologic evidence regarding the effects of Pb 

4 exposure on the liver, GI tract, endocrine system, bone and teeth, eyes, and respiratory tract largely 

5 are confirmatory of those effects noted in the 2006 Pb AQCD. However, recent evidence of these 

6 effects is relatively limited in number, and therefore no causal determinations are made regarding 

7 Pb-induced effects in these organ systems.

5.10. Cancer

8 The previous epidemiologic studies included in the 2006 Pb AQCD (U.S. EPA, 2006) “provide[d] 

9 only very limited evidence suggestive of Pb exposure associations with carcinogenic or genotoxic effects 

10 in humans” and the studies were summarized as follows:

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.

11 The International Agency for Research on Cancer (IARC) has recently classified inorganic Pb 

12 compounds as probable human carcinogens (Group 2A of IARC classifications) based on stronger 

13 evidence in animal studies than human studies, and organic Pb compounds as not classifiable (Group 3 of 

14 IARC classifications) (IARC, 2006; Rousseau et al., 2005). Additionally, the National Toxicology 

15 Program has listed Pb and Pb compounds as “reasonably anticipated to be human carcinogens” (NTP, 

16 2004).

17 In the following sections, recent epidemiologic and toxicological studies published since the 2006 

18 Pb AQCD regarding Pb and cancer mortality and incidence are examined. In addition, recent studies of Pb 

19 and DNA and cellular damage, as well as epigenetics studies, are summarized. In epidemiologic studies, 

20 various biological measures of Pb are used including Pb measured in blood and bone. Bone Pb is 

21 indicative of cumulative Pb exposure. Blood Pb can represent more recent exposure, although it can also 

22 represent remobilized Pb occurring during times of bone remodeling. Toxicological studies only report 

23 exposure by blood Pb. More detailed discussion of these measures is given in Section 4.3.5.

5.10.1. Cancer Incidence and Mortality

24 Recent studies have included epidemiologic evaluations of the associations between Pb and both 

25 specific cancers, such as lung cancer and brain cancer, and overall cancer. Table 5-37 provides an 

26 overview of the study characteristics and results for the epidemiologic studies that reported effect 

27 estimates. This section also presents toxicological evidence on the carcinogenicity of Pb. 
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Table 5-37. Summary of epidemiologic studies of cancer incidence and mortality

Reference Study 

Location

Cancer 

Outcome

Study 

Population

Exposure 

Measurement Mean Pb (SD) Adjusted Effect Estimates

(Menke 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

Tertile 3: >=3.63 

μg/dL

Tertile 1: 1.00

Tertile 2: 0.72 (95% CI: 0.46, 1.12)

Tertile 3: 1.10 (95% CI: 0.82, 1.47)

Schober et al. 

(2006)

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: <22μg/g

Tertile 2 of patella 

Pb: 22-35μg/g 

Tertile 3 of patella 

Pb: >35 μg/g

Blood Pb Tertile 1: 1.00

Blood Pb Tertile 2: 1.03 (95% CI: 0.42, 

2.55)

Blood Pb Tertile 3: 0.53 (95% CI: 0.20, 

1.39)

Patella Pb Tertile 1: 1.00

Patella Pb Tertile 2: 0.82 (95% CI: 0.26, 

2.59)

Patella Pb Tertile 3: 0.32 (95% CI: 0.08, 

1.35)

Khalil et al. 

(2009)

Baltimore, 

MD, and 

Monongahela 

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 cumulative 

blood Pb index: 

cases 9.0 

μmol/Pb, controls 

11.9 μmol/Pb

Median peak blood Pb Level: 1.00 

(95% CI: 0.71, 1.42)

Median number of yr with at least one 

blood sample obtained: 0.96 (95% CI: 

0.91, 1.02) per μmol/L

Median cumulative Blood Pb index: 

0.99 (95% CI: 0.96, 1.02) per μmol/L

Note: similar results were observed 

when restricted to smokers only 
May 2011 5-351 DRAFT – DO NOT CITE OR QUOTE

Reference Study 

Location

Cancer 

Outcome

Study 

Population

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 

conducted from 

1972-1991

Three exposure 

scenarios 

determined for 

working lifetime 

cumulative 

exposure – all 

have similar 

medians of 

approximately 2 

mg yr/m3

NA RR for Pb exposure weighted age and 

time since exposure: 1.54 (90% CI: 1.14, 2.08)

Note: Similar results for other exposure 

determination scenarios.

Rousseau et al. 

(2007)

Montreal, 

Canada

Lung cancer 

and other 

cancer 

incidence

Men aged 35-79 Interview of job 

history and 

exposure matrix

Ever exposed to:

Organic Pb 3.0%

Inorganic Pb 

17.0%

Pb in gasoline 

emissions 38.6%

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)

Inorganic Pb: 

Esophageal 0.6 (95% CI: 0.3, 1.2)

Stomach 0.9 (95% CI: 0.6, 1.5)

Colon 0.8 (95% CI: 0.5, 1.1)

Rectum 0.8 (95% CI: 0.5, 1.3)

Pancreas 0.9 (95% CI: 0.4, 1.8)

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 

cancer
May 2011 5-352 DRAFT – DO NOT CITE OR QUOTE

Reference Study 

Location

Cancer 

Outcome

Study 

Population

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 followup 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 Tumor Study

– included 

individuals >=18 

yr diagnosed with 

brain cancer less 

than 8 wk before 

hospitalization; 

frequencymatched controls 

were individuals 

admitted to the 

same hospitals for 

non-neoplastic 

conditions

Interviews of 

lifetime work 

history and 

exposure databases

NA Meningioma:

Ever exposure to Pb 0.8 (95% CI: 0.5, 

1.3)

Glioma: Ever exposure to Pb 0.8 (95% 

CI: 0.6, 1.1)

Note: positive associations between Pb 

exposure and meningioma incidence 

was observed among individuals with 

ALAD2 genotypes, but not individuals 

with ALAD1 genotypes; these 

associations were not observed for 

glioma incidence

Bhatti et al. 

(2009)

Phoenix, AZ, 

Boston, MA, 

and 

Pittsburgh, 

PA

Brain cancer 

incidence

NCI Brain Tumor 

Study 

– included nonHispanic whites 

18 yr diagnosed 

with brain cancer 

less than 8 wk 

before 

hospitalization; 

frequencymatched controls 

were individuals 

admitted to the 

same hospitals for 

non-neoplastic 

conditions

Interviews of 

lifetime work 

history and 

exposure databases

Glioma: 70.5 

μg/m3y (193.8 

μg/m3

y)

Glioblastoma

multiform: 97.5 

μg/m3y (233.9 

μg/m3

y)

Meningioma: 

101.1 μg/m3

y 

(408.7 μg/m3

y)

Controls: 69.7 

μg/m3

y (248.8 

μg/m3y) 

Per 100 μg/m3y 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

Overall 

cancer 

incidence

Children living in 

this province at 

least five years 

Pb-related air 

pollution 

measures

NA Reported correlations between 

changes in Pb and cancer incidence – no/low correlations observed

(correlation coefficients between -0.3 

and 0.2)

Obhodas et al. 

(2007)

Island of Krk, 

Croatia

Incidence 

rates for 

neoplasms

Individuals living 

in the Island of 

Krk from 1997-

2001

Soil and 

vegetation 

samples, 

household 

potable water 

samples, 

children’s hair 

samples

NA No association observed between Pb in 

the samples and incidence of neoplasm 

(numerical results not provided)
May 2011 5-353 DRAFT – DO NOT CITE OR QUOTE

Reference Study 

Location

Cancer 

Outcome

Study 

Population

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. Overall Cancer Mortality

1 Recent studies have been performed examining the association between biologically measured Pb 

2 levels and cancer mortality. The Third National Health and Nutrition Examination Survey (NHANES III) 

3 included a nationally representative sample of U.S. adults who had blood Pb measurements taken and 

4 were followed up for 12 years. Mean blood Pb levels were 2.58 μg/dL (individuals with blood Pb levels 

5 greater than 10 μg/dL were excluded from the study). No association was observed between blood Pb and 

6 cancer mortality (HR of highest tertile compared to lowest tertile: 1.10 [95% CI: 0.82, 1.47]) (Menke et 

7 al., 2006). Another analysis of the NHANES III population, restricted to individuals 40 years and older at 

8 the time of blood Pb collection, included individuals with all blood Pb levels (including those greater than 

9 10 μg/dL) (Schober et al., 2006). Overall, 68% of the study population had blood Pb levels less than 5 

10 μg/dL and 6% had blood Pb levels greater than 10 μg/dL. Among individuals who died of cancer during 

11 the study period, 52% had blood Pb levels less than 5 μg/dL and 12% had blood Pb levels greater than 10

12 μg/dL. In this study, median follow-up time was 8.6 years and a positive association was observed 

13 between blood Pb and cancer mortality. The RRs were 1.69 (95% CI: 1.14, 2.52) for individuals with 

14 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 

15 compared to individuals with blood Pb levels less than 5 μg/dL. When categorized by age groups, point 

16 estimates comparing blood Pb levels of 5-9 versus less than 5 μg/dL were similar across all age groups 

17 but only statistically significant among 75-84 year olds. A positive association for blood Pb levels of 10 

18 μg/dL and greater was present among those 40-74 years old and 85 years and older. A study of men from 

19 the greater Boston area enrolled in the Department of Veterans Affairs Normative Aging Study reported 

20 mean blood Pb levels of 5.6 μg/dL (SD 3.4 μg/dL) but this measure was poorly correlated with measured 

21 bone Pb (Weisskopf et al., 2009). No association was detected between either measure of Pb and cancer 
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1 mortality in adjusted analyses. As part of the Study of Osteoporotic Fractures study, White women aged 

2 65-87 were included in a sub-study of blood Pb levels and cancer mortality and were followed for 

3 approximately 12 years (Khalil, Wilson, et al., 2009). Mean blood Pb levels were 5.3 μg/dL (SD 2.3 

4 μg/dL) and no association was detected between blood Pb and cancer mortality in the study population. 

5.10.1.2. Lung Cancer

5 A study was conducted among smelter workers to examine the relationship between Pb and lung 

6 cancer (incidence and mortality combined) (Lundstrom et al., 2006). Pb exposure was measured with 

7 three different variables (peak blood Pb values, number of years Pb samples were obtained, and 

8 cumulative blood Pb index) but none showed an association with lung cancer. Median follow-up in the 

9 study was about 30 years and the peak blood Pb values during employment were 49.7 μg/dL for lung 

10 cancer cases and 55.9 μg/dL for controls. A study in the UK of tin smelters reported no association 

11 between Pb exposure and lung cancer mortality in unweighted analyses, but when the analyses were 

12 weighted by age and time since exposure, positive associations were apparent (S. R. Jones et al., 2007). 

13 Pb exposure was calculated in this study by combining historical air sampling data and personnel record 

14 cards, which specified work histories. The median Pb exposure was estimated to be approximately 2 mgyear/m3 15 and the smelters were exposed to other metals as well, such as arsenic and antimony. A 

16 population-based case-control study performed among men in Montreal, Canada assessed Pb exposure via 

17 interviews regarding job histories and calculated the likely Pb exposures associated with the job activities 

18 (Rousseau et al., 2007). No association was apparent between organic Pb, inorganic Pb, or Pb from 

19 gasoline emissions and lung cancer. 

20 Other studies of Pb and lung cancer were performed by comparing the lung tissue of individuals 

21 with lung cancer to those without lung cancer. The controls for these studies were individuals with 

22 metastases in the lung from other primary cancers (De Palma et al., 2008) and individuals with non23 cancerous lung diseases (De Palma et al., 2008; Kuo et al., 2006). One study reported increased Pb 

24 concentrations were observed in the cancerous and non-cancerous lung tissue of individuals with non25 small cell lung cancer compared to control groups (although the authors report these results may be 

26 confounded by smoking) (De Palma et al., 2008), but no statistical difference in Pb levels was reported 

27 for lung tissue of individuals with lung cancer compared to controls in the other study (Kuo et al., 2006). 

5.10.1.3. Brain Cancer

28 A few studies of brain cancer examined the association between cancer and Pb using exposures 

29 determined via exposure databases and patient interviews about past jobs and known exposures. The 

30 National Longitudinal Mortality Study, a study that included a national sample of the U.S. population, 

31 estimated Pb exposure based on current/most recent employment among individuals (van Wijngaarden & 
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1 Dosemeci, 2006). Although not all estimates are statistically significant, a pattern of increased 

2 associations between Pb exposure and brain cancer mortality was observed in the study population. In a 

3 case-control study of brain tumors, glioma was reported to have no association with any Pb exposure 

4 metric; however, positive associations were observed between high cumulative Pb exposure and 

5 meningioma among individuals with ALAD2 genotypes (Rajaraman et al., 2006). The association was not 

6 present among individuals with ALAD1 genotypes. A third study of the association between Pb exposure 

7 and brain tumors reported none or slight overall associations with types of brain tumors; however, 

8 positive associations were observed among individuals with certain single nucleotide polymorphisms 

9 (SNPs) (Bhatti et al., 2009). After control for multiple comparisons, individuals with GPX1 variants had 

10 positive associations between cumulative Pb exposure and glioblastoma multiforme and meningioma,

11 whereas individuals without RAC2 variants showed a positive association between Pb and glioblastoma 

12 multiforme and individuals without XDH variants displayed a positive association between Pb and 

13 meningioma. 

5.10.1.4. Breast Cancer

14 A few studies examined Pb levels and breast tumors among individuals with and without breast 

15 tumor and/or cancer present. One study of newly diagnosed breast cancer patients and controls examined 

16 Pb levels in blood and hair samples and reported higher levels in both for cancer cases, although the 

17 difference in the hair samples was not statistically significant (Alatise & Schrauzer, 2010). Siddiqui et al. 

18 (2006) observed higher blood Pb levels in women with benign and malignant tumors compared to 

19 controls. Additionally, although blood Pb levels were higher among those with malignant breast tumors 

20 compared to those with benign tumors, both had similar levels of Pb detected in breast tissues. Another 

21 study of Pb levels present in breast tissue also reported no statistical difference in Pb levels (Pasha, Malik, 

22 et al., 2008b). However, one study of breast tissue did observe a statistically significant difference 

23 between Pb levels in the breast tissue of cancer cases and controls (Ionescu et al., 2007). Finally, a study 

24 of Pb levels in urine reported a positive association between urine Pb and breast cancer but this 

25 association became null when women taking nonsteroidal aromatase inhibitors but not taking 

26 bisphostphonates (a combination responsible for bone loss) were excluded from the analysis (McElroy et 

27 al., 2008). 

28 Overall, these studies demonstrate the possibility that women with breast cancer may have 

29 increased Pb levels in blood measurement, whereas the results for actual breast tissue are mixed. 

30 However, these studies are limited by their study design. The samples are taken after cancer is already 

31 present in the cases, leading to issues of temporality for the Pb levels. Additionally, the sample sizes are 

32 often small and the studies may be underpowered. 
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5.10.1.5. Other Cancers

1 Studies of multiple cancers or cancers not listed above have also been performed. An ecologic 

2 analysis compared levels of Pb in the air from 1990 to 2005 with incidence rates of cancer (cancer sites 

3 not specified) among children during this time period (Absalon & Slesak, 2010). The highest Pb levels 

were measured in 1990 when over 50% of the study area exceeded the limit of 1 μg/m2 4 ·year. No 

5 correlation was observed both overall and in sex-specific analyses. A similar study examined correlations 

6 between Pb concentrations in soil, water, vegetation, and hair samples with incidence of neoplasms 

7 (Obhodas, 2007). No correlations were reported.

8 A study performed among men evaluated multiple cancer outcomes and determined exposures to 

9 organic Pb, inorganic Pb, and Pb in gasoline emissions via interviews regarding job histories and then 

10 subsequent exposure approximations by chemists and hygenists (Rousseau et al., 2007). Organic Pb

11 exposure was positively associated with stomach cancer. A positive association was also observed for 

12 rectal cancer when population-based controls were used but was null when the control population was 

13 limited to individuals with other types of cancers. No association was detected for cancers of the 

14 esophageous, colon, pancreas, prostate, bladder, kidney, melanoma, or non-Hodgkin’s lymphoma. None 

15 of the cancers were associated with exposure to inorganic Pb. When occupational exposure to Pb in 

16 gasoline was categorized as unexposed, nonsubstantial level, and substantial level, a positive association 

17 with stomach cancer was observed when cancer controls were used (association not present when 

18 population controls were employed as the control group). Another case-control study using participant 

19 interviews and a job exposure matrix, including only men, reported no association between Pb exposure 

20 and esophageal squamous cell carcinomas, but an association was present between high Pb exposure and 

21 adenocarcinmoa of the esophagus (Santibanez et al., 2008). 

22 Several studies compared Pb levels in blood, tissue, and urine of individuals who have cancer with 

23 individuals who are cancer-free. Compared to control groups, increased Pb levels were observed in the 

24 blood and bladder tissue of individuals with bladder cancer (Golabek et al., 2009), the kidney tissue of 

25 individuals with renal cell carcinoma (with highest levels among those with the highest stage tumors) 

26 (Calvo et al., 2009), the tissue (but not serum) of individuals with laryngeal cancer (Olszewski et al., 

27 2006), the blood of individuals with gastric cancer (Khorasani et al., 2008), the plasma and hair of 

28 individuals with gastrointestinal cancer (Pasha et al., 2010), the blood and hair of individuals with non29 specified types of cancer (Pasha et al., 2007; Pasha, Malik, & Shah, 2008), and the hair of individuals 

30 with benign tumors (Pasha, Malik, et al., 2008a). No statistical difference in Pb levels was reported for 

31 colon tissue of individuals with colorectal polyps (Alimonti et al., 2008) or urine of individuals with 

32 bladder cancer (C. N. Lin et al., 2009) compared to control groups. A study examining Pb levels in kidney 

33 tissue reported the highest levels of Pb in normal kidney tissue samples that were adjacent to neoplastic 

34 tumors. The Pb levels reported in the kidney tissue of neoplastic tumors were elevated compared to those 
May 2011 5-357 DRAFT – DO NOT CITE OR QUOTE

1 detected in corpses without neoplastic tumors of the kidney (Cerulli et al., 2006). All of these comparison 

2 studies are limited by the inability to examine temporality; the presence of Pb may be due to changes that 

3 result from having cancer, not changes that result in cancer. Many of these studies attempted to control for 

4 this by including only cases who have not undergone certain treatments. Additionally, studies are limited 

5 by their small sample size and the selection of the control populations. Control populations are supposed 

6 to represent the general population from which the cases are drawn; some of the control subjects in these 

7 studies are individuals with diseases/conditions warranting tissue resections, which are not prevalent in 

8 the general population. 

5.10.1.6. Toxicological Models of Carcinogenicity

Carcinogenicity in Animal Models

9 Inorganic Pb has been shown to act as a carcinogen in animal toxicology models. Most commonly 

10 the kidneys (Azar et al., 1973; Kasprzak et al., 1985; Koller et al., 1985; Van Esch & Kroes, 1969) are 

11 targeted but the testes, brain, adrenals, prostate, pituitary, and mammary gland have also been affected

12 (IARC, 2006). More recently it has been shown that early life transplacental and lactational exposure of 

13 laboratory rodents to inorganic Pb induces carcinogenicity in adulthood (Waalkes et al., 1995). Chronic, 

14 lifetime exposure to Pb is also associated with carcinogenicity in laboratory rodents (Koller et al., 1985). 

15 One recent study considered Pb-dependent carcinogenesis in laboratory animals. Tokar et al. (2010)

16 considered tumorigenesis in rodents. Homozygous metallothionein I/II knockout mice and their 

17 corresponding wild type controls (groups of ten mice each) were exposed by drinking water to 2,000 or 

18 4,000 ppm Pb(Ac)2 and compared to untreated controls. Study animals were exposed in utero, through 

19 birth and lactation, and then directly to drinking water until 8 weeks old. The metallothionein I/II 

20 knockout mice had increased testicular teratomas and renal and urinary bladder preneoplasia. Pb exposed 

21 wild-type mice were not statistically significantly different than controls. The data suggest 

22 metallothionein can protect against Pb-induced tumorigenesis. Concerns with the study are that the doses 

23 are at levels of Pb humans would not likely be exposed to and there is no metallothionein null condition 

24 in humans though there is variability in the expression of metallothionein. The data do not address 

25 whether this variability has any impact on Pb carcinogenesis.

Neoplastic Transformation Studies, Human Cell Cultures

26 Three studies considered Pb-dependent carcinogenesis in human cells. Xie et al. (2007) treated 

27 BEP2D cells (human papilloma virus- immortalized human bronchial cells) with 0, 1, 5, or 10 μg/cm2 

28 PbCrO4 for 120 h. PbCrO4 induced foci formation in a concentration-dependent manner. Xie et al. (2008)

29 treated BJhTERT cells (hTERT-immortalized human skin fibroblasts) and ATLD-2 cells (hTERT-
May 2011 5-358 DRAFT – DO NOT CITE OR QUOTE

immortalized human skin fibroblasts deficient in Mre11) with 0, 0.1, 0.5, and 1 μg/cm2 1 PbCrO4 for 120 h. 

2 PbCrO4 induced foci formation in a concentration-dependent manner in the Mre11 deficient cells. Xie et 

3 al., (2008) treated BJhTERT cells (hTERT-immortalized human skin fibroblasts) and ATLD-2 cells 

(hTERT-immortalized human skin fibroblasts deficient in Mre11) with 0, 0.1, 0.5, and 1 μg/cm2 4 PbCrO4

5 for 24 or 120 h. Mre11 was required to prevent PbCrO4-induced neoplastic transformation.

Immune Modulation of Tumorogenesis by Pb

6 As described in the prior AQCD for Pb (U.S. EPA, 2006), Pb-induced immunotoxicity affecting 

7 tumors results from a combination of suppressed Th1 responses and misregulated inflammation. The 

8 intersection of these two general Pb-induced alterations that elevate the risk of cancer. First, Pb-induced 

9 misregulation of inflammation involving innate immune cells results in chronic insult to tissues. Decades 

10 of excessive lipid and DNA oxidation production by overproduction of ROS and weakened anti-oxidant 

11 defenses increase the likelihood of mutagenesis, cellular instability, and tumor cell formation. In support 

12 of this, Xu et al. (J. Xu et al., 2008) found evidence that supports the association with Pb exposure and 

13 DNA damage and concluded that it is a route to increased Pb-induced tumorigenesis. The second 

14 component of increased risk of cancer involves Pb-induced suppression of Th1-dependent anti-tumor 

15 immunity as acquired immunity shifts statistically significantly toward Th2 responses. With cytotoxic T 

16 lymphocytes and other cell-mediated defenses dramatically lessened, the capacity to resist cancer may be 

17 compromised.

5.10.2. Cancer Biomarkers

18 A study of men aged 21-40 years never occupationally exposed to metals examined prostate 

19 specific antigen (PSA), a biomarker for prostate cancer. This study reported a positive association 

20 between blood Pb and PSA levels in adjusted analyses (Pizent et al., 2009). The median blood Pb level

21 was 2.6 μg/dL (range 1.0-10.8 μg/dL). The authors note that the study population was young and at lower 

22 risk of prostate cancer than older men.

5.10.3. DNA and Cellular Damage

23 Multiple studies have been performed examining the relationship between Pb and DNA and 

24 cellular damage. Details of the recent epidemiologic and toxicological studies follow.

5.10.3.1. Epidemiologic Evidence for DNA and Cellular Damage

25 Multiple studies examined the relationship between Pb and sister chromatid exchange (SCE). A 

26 study of male policeman reported mean blood Pb levels for the study population of 43.5 μg/dL 
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1 (Wiwanitkit et al., 2008). When categorized as having high or low blood Pb levels (cut-off at 49.7 μg/dL), 

2 the higher blood Pb group was observed to have higher mean SCE. Another study of adult males 

3 compared the SCE of storage battery manufacturing workers (mean blood Pb levels of 40.14 μg/dL) and 

4 office workers (mean blood Pb levels of 9.77 μg/dL) (Duydu et al., 2005). The exposed workers had 

5 higher SCE levels and also a greater number of cells with the SCEs per cell higher than the 95th

6 percentile of the population. Finally, a study of children aged 5-14 years old (mean blood Pb levels of 

7 7.69 μg/dL [SD 4.29 μg/dL]) reported no correlation with SCE (Mielzyńska et al., 2006). However, the 

8 study did report a positive association between blood Pb and micronuclei levels. 

9 Other studies of DNA damage have reported mixed results. A study of children ages 6-11 years old 

10 and environmentally-exposed to Pb reported no association between blood Pb and DNA basal damage or 

11 repair ability after a peroxide challenge (Méndez-Gómez et al., 2008). Another study included adult 

12 participants aged 50-65 years and reported an association between blood Pb and carcinoembryonic 

13 antigen (CEA) but not with DNA-strand breaks or oxidative DNA damage (De Coster et al., 2008). A 

14 study conducted among workers exposed to Pb (mean blood Pb levels of 30.3 μg/dL) and unexposed 

15 controls (mean blood Pb levels of 3.2 μg/dL) reported positive associations between the exposed group 

16 and cytogenetic damage (measured by micronuclei frequency), chromosomal aberrations, and DNA 

17 damage (although this was not statistically significant in linear regression models controlling for age) 

18 (Grover et al., 2010). A study of painters in India, where Pb-concentrations in paint are high, reported 

19 mean blood Pb levels of 21.56 μg/dL (SD 6.43 μg/dL) among painters who reported painting houses for 

20 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 

21 0.96 μg/dL) for healthy workers who had not been occupationally exposed to Pb. Cytogenetic damage 

22 was increased among the painters compared to the healthy controls. Another study compared the blood Pb 

23 of metal workers and office workers and reported greater blood Pb levels (both current and 2 year 

24 average) among the metal workers (blood Pb levels at least 20 μg/dL for metal workers compared to 

25 blood Pb levels less than 10 μg/dL for the office workers) (Olewińska et al., 2010). Overall, the workers 

26 had increased DNA strand breaks versus the office workers (this held true at various blood Pb levels). 

27 Finally, a study of Pb battery workers with symptoms of Pb toxicity and a group of controls were 

28 examined (Shaik & Jamil, 2009). Higher chromosomal aberrations, micronuclei frequency, and DNA 

29 damage were reported for the battery workers as compared to the controls. 

5.10.3.2. Toxicological Evidence for DNA and Cellular Damage

Sister Chromatid Exchanges

30 One study, Tapisso et al. (2009), considered sister chromatid exchanges (SCE) in rodents. Algerian 

31 mice (groups of six mice each) were exposed by intraperitoneal injection to 5 or 10 doses of 0.46 mg/kg 
May 2011 5-360 DRAFT – DO NOT CITE OR QUOTE

1 Pb(Ac)2 and compared to untreated controls. The SCE in bone marrow were elevated after Pb exposure 

2 alone, which increased with time. Co-exposure with cadmium or zinc further increased SCE levels. 

3 Two studies considered SCE in cultured human cells. In one study, Ustundag and Duydu (2007), 

4 considered the ability of N-acetylcysteine and melatonin to reduce led nitrate-induced SCE in a single 

5 human donor. Cells were treated with 0, 1, 5, 10, or 50 μM Pb(NO3)2. SCE statistically significantly 

6 increased at every Pb concentration in a concentration dependent manner. Both 1 and 2 mM N7 acetylcysteine and melatonin were able to statistically significantly reduce SCE levels. Exposure times 

8 were not provided. The full interpretation of these data is limited by the limited number of donors and the 

9 absence of an exposure time for the SCE assay. In the other study, Turkez et al. considered the ability of 

10 boron compounds to prevent Pb chloride-induced SCE in human lymphocytes. Cells were obtained from 

11 4 non-smoking donors. Both 3 and 5 ppm Pb chloride induced a statistically significant increase in SCE 

12 levels over controls. Boron was able to statistically significantly diminish these levels. Exposure times 

13 were not provided. The full interpretation of these data is limited by the limited number of donors and the 

14 absence of an exposure time for the SCE assay.

Micronuclei Formation

15 Two studies considered MN in rodents. Alghazal et al. (2008), considered the ability of Pb(Ac)2

16 trihydrate to induce MN in bone marrow of Wistar rats. Animals were given a daily dose of 100 mg/l in 

17 their drinking water for 125 days. The mean number of MN in male and female rats was statistically 

18 significantly higher than unexposed controls. The second study, Tapisso et al., (2009), considered Pb

19 alone, Pb plus zinc and Pb plus cadmium-induced MN in rodents. Algerian mice were exposed by 

20 intraperitoneal injection to 5 or 10 doses of 0.46 mg/kg Pb(Ac)2 and compared to untreated controls. The 

21 MN in bone marrow were elevated after Pb exposure, which increased with time. Co-exposure with 

22 cadmium or zinc did not further increase MN levels.

23 Three studies considered MN in cultured human cells. In one study, Ustundag and Duydu (2007)

24 considered the ability of N-acetylcysteine and melatonin to reduce led nitrate-induced MN in a single 

25 human donor. Cells were treated with 0, 1, 5, 10, or 50 μM Pb(NO3)2. MN statistically significantly 

26 increased at the two highest Pb concentrations in a concentration dependent manner. Both 1 and 2 mM N27 acetylcysteine and melatonin were not able to statistically significantly reduce MN levels. Exposure times 

28 were not provided. The full interpretation of these data is limited by the limited number of donors and the 

29 absence of an exposure time for the MN assay. The second study, Turkez et al., considered the ability of 

30 boron compounds to prevent Pb chloride-induced MN in human lymphocytes. Cells were obtained from 4 

31 non-smoking donors. Both 3 and 5 ppm Pb chloride induced a statistically significant increase in MN 

32 levels over controls. Boron induced a statistically significantly attenuation of these levels. Exposure times 

33 were not provided. The full interpretation of these data is limited by the limited number of donors and the 
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1 absence of an exposure time for the MN assay. The third study, Gastaldo et al. (2007), evaluated the 

2 ability of Pb to induce MN. Human endothelial HMEC cell line was treated with 1–1000 μM Pb(NO3)2

3 for 24 h. MN increased in a statistically significant, concentration-dependent manner.

HPRT Mutations

4 Two studies evaluated HPRT mutations in human cell cultures. Li et al., (2008), evaluated Pb(Ac)2-

5 induced HPRT in the non-small-cell lung carcinoma tumor cell line, CL3 and normal human diploid 

6 fibroblasts (specific tissue source not provided). All cells were exposed to 0, 100, 300 or 500 μM Pb(Ac)2

7 for 24 hours in serum-free medium ± a 1-hour pretreatment with a MKK1/2 inhibitor or a PKC-alpha 

8 inhibitor. Pb alone did not induce HPRT mutations. Inhibiting the ERK pathway via either inhibitor 

9 statistically significantly increased Pb-induced mutagenesis. A second study, Wang et al. (2008), 

10 investigated Pb(Ac)2-induced HPRT mutations in CL3 cells. All cells were exposed to 0, 100, 300 or 500 

11 μM Pb(Ac)2 for 24 hours in serum-free medium ± a 1-hour pretreatment with a PKC-alpha inhibitor or 

12 siRNA fpr PKC-alpha. Pb alone did not induce HPRT mutations. Inhibiting the PKC-alpha via either 

13 inhibitor statistically significantly increased Pb-induced mutagenesis.

14 One study considered HPRT in animal cell culture. McNeill et al. (2007) considered Pb(Ac)2

15 induced HPRT mutations in Chinese hamster ovary AA8 cells and AA8 cells overexpressing human 

16 Ape1. Cells were treated with 5 μM Pb(Ac)2 for 6 hours. No increases in HPRT mutations were observed 

17 after Pb exposure in either cell line.

Chromosomal Aberrations

18 Only one study (El-Ashmawy et al., 2006) considered Pb in laboratory rodents. The study focused 

19 on dietary exposure to Pb(Ac)2 administered as a single dose of 0.5% w/w. Male Swiss albino mice, 30 

20 per group, were studied. In addition, the authors considered the protective effects of turmeric and myrrh 

21 powder. The study reported statistically significant levels of chromosomal aberrations in the Pb treatment 

22 alone group, particularly with respect to fragments, deletions, ring chromosomes, gaps, and end-to-end 

23 associations. The turmeric and myrrh powders were protective. Concerns with the study include the use of 

24 only a single dose of Pb(Ac)2 along with the high levels of unusual aberrations such as ring chromosomes 

25 and end-to-end associations. Typically, these aberrations are rare after metal exposure, but were the most 

26 common in this study raising questions about the quality of the metaphase preparations. One additional 

27 concern was that only 50 metaphases per dose were analyzed instead of the more common 100 

28 metaphases per dose. The authors did not explain why their spectrum of aberrations was so different or 

29 why they only used one dose or analyzed fewer metaphases per dose. 

30 Seven studies considered the ability of Pb to induce chromosomal aberrations in cultured human 

31 cells. One study (Pasha Shaik et al., 2006) considered the ability of Pb(NO3)2 to induce chromosomal 
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1 aberrations in primary human peripheral blood lymphocytes obtained from healthy, non-smoking donors. 

2 Cells were treated with 0, 1.2 or 2 mM Pb(NO3)2 for 2 h. No increase in chromosomal aberrations was 

3 reported. Some aneuploidy was observed. Concerns with the study are that only a 2 hour exposure was 

4 used, which may not be long enough for DNA damage to be expressed as a chromosomal aberration. It 

5 also appears from the data presentation that only three subjects were used; one for a control, one for the 

6 low dose and one for the high dose. Experiments were not repeated, thus given the small number of 

7 subjects, this study may not have had sufficient power to detect any effects. Holmes et al. (2006), treated 

WHTBF-6 cells (hTERT-immortalized human lung cells) with 0, 0.1, 0.5, or 1 μg/cm2 8 PbCrO4 for 24-120

hours or with 0, 0.1, 0.5, 1, 5 or 10 μg/cm2 9 PbO for 24 or 120 hours. PbCrO4 induced statistically 

10 significant, concentration-dependent increases in centrosome abnormalities and aneuploidy. Wise et al. 

(2006) treated BEP2D cells with 0, 0.5, 1, 5, or 10 μg/cm2 11 PbCrO4 for 24 hours. PbCrO4 induced 

12 statistically significant concentration-dependent increases in chromosomal aberrations. Holmes et al.

(2006), treated WHTBF-6 cells with 0, 0.1, 0.5, or 1 μg/cm2 13 PbCrO4 for 24-72 hours. PbCrO4 induced 

14 statistically significant, concentration-dependent increases in chromosomal aberrations. The effects were 

attributed to the chromate anion. Wise et al. (2006), treated WHTBF-6 cells with 0, 0.1, 0.5, or 1 μg/cm2 15

16 PbCrO4 for 24-120 hours. PbCrO4 induced statistically significant, concentration-dependent increases in 

17 spindle assembly checkpoint disruption, effects of mitosis and aneuploidy. By contrast, chromate-free 

18 PbO did not induce centrosome amplification. The effects were attributed to the chromate anion. Xie et al. 

(2007) treated BEP2D cells with 0, 1, 5, or 10 μg/cm2 19 PbCrO4 for 24 hours. PbCrO4 induced statistically 

20 significant, concentration-dependent increases in chromosomal aberrations and aneuploidy.

Wise et al. (2010) treated WHTBF-6 cells with 0, 0.1, 0.5, or 1 μg/cm2 21 PbCrO4 for 24 hours in a 

22 study comparing 4 chromate compounds. PbCrO4 induced statistically significant, concentration23 dependent increases in chromosomal aberrations, but was the least potent chromate based on administered 

24 concentration. 

25 Five studies considered the ability of PbCrO4 to induce chromosome aberrations in rodent cell 

26 cultures. All focused on PbCrO4. Duzevik et al. (2006) treated Chinese hamster ovary (CHO) cells with 0, 

0.1, 0.5, or 1 μg/cm2 27 PbCrO4 for 24 h. Specific CHO lines used included AA8 (wildtype) EM9 (XRCC1-

28 deficient), and H9T3 (EM9 complemented with human XRCC1 gene). PbCrO4 induced statistically 

29 significant, concentration-dependent increases in chromosomal aberrations that were statistically 

30 significantly increased by XRCC1 deficiency. Nestmann and Zhang (2007) treated Chinese hamster ovary 

cells (clone WB(L)) with 0, 0.1, 0.5, 1, 5, or 10 μg/cm2 31 PbCrO4 (as pigment yellow) for 18 h. No 

32 increases in chromosomal aberrations were observed. Savery et al. (2007) treated CHO cells with 0, 0.1, 

0.5, 1, or 5 μg/cm2 33 PbCrO4 for 24 h. Specific CHO lines used included AA8 (wildtype) KO40 (Fancg34 deficient), and 40BP6 (Fancg complemented). PbCrO4 induced statistically significant, concentration35 dependent increases in chromosomal aberrations that were increased by Fancg deficiency. Camrye et al., 

(2007) treated CHO cells with 0, 0.1, 0.5, 1, 5, or 10 μg/cm2 36 PbCrO4 for 24 hours. Specific CHO lines 
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1 used included CHO-K1 (parental), xrs-6 (Ku80 deficient), and 2E (xrs-6 complemented with Ku80 gene). 

2 PbCrO4induced statistically significant, concentration-dependent increases in chromosomal aberrations 

3 that were not affected by Ku80 deficiency. Stackpole et al. (2007) treated CHO and Chinese hamster lung 

(CHL) cells with 0, 0.1, 0.5, or 1 μg/cm2 4 PbCrO4 for 24 hours. Specific CHO lines used included AA8 

5 (wildtype), irs1SF (XRCC3-deficient), and 1SFwt8 (XRCC3 complemented). CHL lines used included 

6 V79 (wildtype), irs3 (Rad51C deficient) and irs3#6 (Rad51C complemented). PbCrO4 induced 

7 statistically significant, concentration-dependent increases in chromosomal aberrations that were 

8 statistically significantly increased by both XRC3 and Rad51C deficiency.

9 Three studies considered the ability of PbCrO4 to induce chromosome aberrations in marine 

10 mammal cell cultures. All focused on PbCrO4. Li Chen et al. (2009) treated primary North Atlantic right 

whale lung and skin fibroblasts with 0, 0.5, 1.0, 2.0, and 4.0 μg/cm2 11 PbCrO4 for 24 hours. Wise et al.

(2009) treated primary Steller sea lion lung fibroblasts with 0, 0.1, 0.5, 1 and 5 μg/cm2 12 PbCrO4 for 24 

hours. Wise et al. (2011) treated primary sperm whale skin fibroblasts with 0, 0.5, 1, 3, 5, and 10 μg/cm2 13

14 PbCrO4 for 24 hours. In all three studies, PbCrO4 induced statistically significant, concentration15 dependent increases in chromosomal aberrations.

COMET Assay

16 Three studies considered the ability of Pb to induce DNA single strand breaks measured by the 

17 comet assays in laboratory animals. Xu et al. (2008) considered the ability of Pb(Ac)2 to induce DNA 

18 damage measured by the comet assay in lymphocytes in male ICR mice. Animals (5 per group) were 

19 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

20 exposure statistically significantly increased both tail length and tail moment in a dose-dependent manner.

21 Nava-Hernandez et al. (2009) considered the ability of Pb(Ac)2 to induce DNA damage measured by the 

22 comet assay in primary spermatocyte DNA of male Wistar rats. Animals (3 per group) were treated for 13 

23 weeks with 0, 250 or 500 mg/l Pb in their drinking water. There was statistically significantly less DNA 

24 damage in the controls compared to the two treatment groups. Narayana and Al-Bader (2011) considered 

25 the ability of Pb(NO3)2 to induce DNA damage measured by the comet assay in liver tissue of adult male 

26 Wistar rats. Animals (8 per group) were treated for 60 days with doses of 0, 0.5 or 1% Pb(NO3)2 in their 

27 drinking water. There were no statistical differences between treated groups and controls.

28 Two studies considered the ability of Pb to induce DNA strand breaks measured by the comet assay 

29 in cultured human cells. Only, one study (Pasha Shaik et al., 2006) considered the ability of Pb to induce 

30 DNA single strand breaks using the comet assay in primary human peripheral blood lymphocytes 

31 obtained from healthy, non-smoking donors. Cells were treated with 0, 2.1, 2.4, 2.7, 3.0, 3.3 Pb(NO3)2 for 

32 2 hours. Increased comet tail length with increased dose was reported.
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1 Concerns with the study are that apparently no untreated control was used. It also appears from the 

2 data presentation that only five subjects were used; one for each dose. Experiments were not repeated, 

3 thus given the small number of subjects and the absence of a negative control, this study may only be 

4 detecting background levels. Xie et al. (2008) treated BJhTERT cells (hTERT-immortalized human skin 

5 fibroblasts) and ATLD-2 cells (hTERT-immortalized human skin fibroblasts deficient in Mre11) with 0, 

0.1, 0.5, and 1 μg/cm2 6 PbCrO4 for 24 hours. PbCrO4 induced a concentration-dependent increase in DNA 

7 double strand breaks measured by the comet assay.

8 Two studies considered Pb-induced DNA single strand breaks in rodent cell cultures using the 

9 comet assay. Xu et al. (2006), treated PC12 cells with 0, 0.1, 1 or 10 μM Pb(Ac)2. Both tail length and tail 

10 moment statistically significantly increased in a concentration-dependent manner. Kermani et al. (2008)

11 exposed mouse bone marrow-mesenchymal stem cells to 60 μM Pb(Ac)2 for 48 hours. There was an 

12 increase in several comet assay measurements including tail length.

Other Assays

13 Three studies considered the ability of Pb to induce DNA double strand breaks measured by 

14 measuring gamma-H2A.X foci formation in cultured human cells. Xie et al. (2008) treated BJhTERT 

15 cells (hTERT-immortalized human skin fibroblasts) and ATLD-2 cells (hTERT-immortalized human skin 

fibroblasts deficient in Mre11) with 0, 0.1, 0.5, and 1 μg/cm2 16 PbCrO4 for 24 hours. PbCrO4 induced a 

17 concentration-dependent increase in DNA double strand breaks measured by gamma-H2A.X foci 

18 formation. Gastaldo et al. (2007) evaluated the ability of Pb to induce DNA double strand breaks measure 

19 with gamma-H2A.X foci formation and by pulse-field gel electrophoresis in cultured human cells. Human 

20 endothelial HMEC cell line was treated with 1 to 1,000 μM Pb(NO3)2 for 24 hours. DNA double strand 

21 breaks increased in a concentration-dependent manner. Wise et al. (2010) treated WHTBF-6 cells with 0, 

0.1, 0.5, or 1 μg/cm2 22 PbCrO4 for 24 hours in a study comparing 4 chromate compounds. PbCrO4 induced 

23 statistically significant, concentration-dependent increases in DNA double strand breaks measured by 

24 gamma-H2A.X foci formation, at a similar level to the other 3 compounds. 

25 Four studies considered Pb and DNA repair. All were done in cultured cells. Li et al., (2008), 

26 evaluated Pb(Ac)2-induced effects on nucleotide excision repair efficiency in CL3 cells. All cells were 

27 exposed to 0, 100, 300 or 500 μM Pb(Ac)2 for 24 hours in serum-free medium. Pb increased nucleotide 

28 excision repair efficiency. Gastaldo et al. (2007) evaluated the ability of Pb to affect DNA repair in 

29 cultured human cells. Human endothelial HMEC cell line was treated with 100 μM Pb(NO3)2 for 24 

30 hours. Pb inhibited non-homologous end joining (NHEJ) repair, over activates MRE11-dependent repair 

31 and increased Rad51-related repair. Xie et al. (2008) treated BJhTERT cells (hTERT-immortalized human 

32 skin fibroblasts) and ATLD-2 cells (hTERT-immortalized human skin fibroblasts deficient in Mre11) with 

0, 0.1, 0.5, and 1 μg/cm2 33 PbCrO4 for 24 or 120 hours. Mre11 was required to prevent PbCrO4-induced 
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1 DNA double strand breaks. McNeill et al, (2007) considered Pb(Ac)2 effects on Ape1. Chinese hamster 

2 ovary cells (AA8) were treated with 0, 0.5, 5, 50, or 500 μM Pb(Ac)2 and then whole cell extracts were 

3 used to determine AP site incision activity. The data show that Pb reduced AP endonuclease function. 

4 Finally, two studies considered Pb-induced cellular proliferation in laboratory animals. Fortoul et al. 

5 (2005) exposed adult male CD1 mice (24 animals per group) to 0.01 M Pb(Ac)2, 0.006 M ca.dmium 

6 chloride or a mixture of the two chemicals for 1 h twice a week for 4 weeks by inhalation. The lungs were 

7 then examined by electron microscopy for changes. Pb induced cellular proliferation in the lungs.

8 Kermani et al. (2008) exposed mouse bone marrow-mesenchymal stem cells to 0-100 μM Pb(Ac)2 for 48 

9 hours. As measured by the MTT assay, Pb decrease cell proliferation at all concentrations tested.

5.10.3.3. Mechanisms of Action

10 The carcinogenic mechanism of action of Pb is poorly understood. Three well-accepted general 

11 paradigms of carcinogenesis include multistage carcinogenesis (including initiation, promotion, and 

12 progression), genomic instability, and epigenetic modification. Of the aforementioned paradigms, it is 

13 unclear which of these best fit Pb. For example, multistage carcinogenesis involves a series of cellular and 

14 molecular changes that result from the progressive accumulation of mutations that induce alterations in 

15 cancer-related genes. Pb does not appear to follow this paradigm and the literature suggests it is weakly 

16 mutagenic. Pb does appear to have some ability to induce chromosomal mutation and DNA damage, i.e. 

17 clastogenicity. However, the ability of Pb to alter gene expression (epigenetic effects) and to interact with 

18 proteins may be a means by which Pb induces its carcinogenicity. It is known that Pb can replace zinc in 

19 zinc-binding (zinc-finger) proteins, which include hormone receptors, cell-cycle regulatory proteins, the 

20 Ah receptor, estrogen receptor, p53, DNA repair proteins, protamines, and histones. These zinc-finger 

21 proteins all bind to specific recognition elements in DNA. Thus, Pb may act at a post-translational stage 

22 to alter protein structure of Zn-finger proteins, which can in turn alter gene expression, DNA repair and 

23 other cellular functions. To recapitulate, cancer develops from one or a combination of multiple 

24 mechanisms including modification of DNA via epigenetics or enzyme dysfunction and genetic instability 

25 or mutation(s). These modifications then provide the cancer cells with a selective growth advantage. In 

26 this schematic, Pb appears to contribute to epigenetic changes, and chromosomal aberrations.

27 The genomic instability paradigm requires a cascade of genome-wide changes caused by 

28 interfering with DNA repair, kinetochore assembly, cellular checkpoints, centrosome duplication, 

29 microtubule dynamics or a number of cell maintenance processes. There are some data that suggest Pb

30 may interfere with some of these processes, but the data are few as these areas are rarely studied for Pb. 

31 Furthermore, the bulk of the literature in this area involves PbCrO4 and it is unclear if the effects are due 

32 to Pb or chromate. Epigenetic modifications lead to cancer by altering cellular functions without altering 

33 the genetic material. The most commonly studied epigenetic change is methylation alterations. Data show 
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1 Pb can induce epigenetic changes, but studies are still missing to clearly tie these effects to Pb-induced 

2 carcinogenesis and genotoxicity. Thus, the mechanism is difficult to define but, if Pb is a human 

3 carcinogen, the mechanism likely involves either genomic instability or epigenetic modification 

4 paradigms or some combination of the two, but it not likely to occur by a multistage carcinogenesis 

5 paradigm. More work is needed to determine the mechanism. 

6 No recent studies of the protective role of calcium or zinc in Pb-dependent carcinogenesis or 

7 genotoxicity were found. There were some data suggesting that metallothionein protects rodents from Pb8 induced cancers. There were some data suggesting that boron, melatonin, N-acetylcysteine, turmeric and 

9 myrrh protecting cells against Pb-induced genotoxicity. There were some data suggesting that Pb mimics

10 or antagonizes selenium in rodents. These data are discussed in more detail elsewhere in the cancer 

11 section.

5.10.3.4. Effects of Lead within Mixtures

12 Three studies considered the impact of mixtures with Pb. All considered genotoxicity. Mendez13 Gomez et al., (2008), considered 65 children from Mexico exposed to both arsenic and Pb. DNA damage 

14 and decreased DNA repair were seen using the comet assay and other assays, but did not correlate with 

15 arsenic or Pb levels. Tapisso et al., (2009), considered Pb alone, Pb plus zinc and Pb plus cadmium16 induced MN in rodents. Algerian mice (groups of six mice each) were exposed by intraperitoneal to 5 or 

17 10 doses of 0.46 mg/kg Pb(Ac)2 compared to untreated controls. The MN in bone marrow were elevated 

18 after Pb alone exposure, which increased with time. Co-exposure with cadmium or zinc increased SCE 

19 levels, but did not further increase MN levels. Glahn et al., (2008) performed a gene array study in 

20 primary normal human bronchial epithelial cells from 4 donors treated with 550 μg/l Pb chloride, 15 μg/l 

21 cadmium sulphate, 25 μg/l cobalt chloride or all three combined for 72 hours. There was a clear 

22 interaction of all three metals impacting RNA expression.

23 One new publication details the interaction of Pb and selenium in virus-dependent mammary tumor 

24 formation. No recent studies of the protective role of calcium in Pb-dependent carcinogenesis or 

25 genotoxicity were found. There were some data suggesting that boron, melatonin, N-acetylcysteine, 

26 turmeric and myrrh protect cells against Pb-induced genotoxicity (Sections 5.10.3.5, 5.10.3.7 and 

27 5.10.3.10.).

28 One study considered Pb and selenium interactions in carcinogenesis in laboratory animals. 

29 Schrauzer (2008) considered the impact of selenium on carcinogensis by studying 4 groups of weanling 

30 virgin female C3H/St mice infected with murine mammary tumor virus (groups of 20-30 mice). One set 

31 of two groups were fed a diet containing 0.15 ppm selenium and then were exposed via drinking water to 

32 acetic acid (control group) or 0.5 ppm Pb(Ac)2 (treated group). The second set of two groups were fed a 

33 diet containing 0.65 ppm selenium and then similarly exposed to acetic acid or 0.5 ppm Pb(Ac)2. The 
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1 effects of selenium and Pb on the tumors caused by the virus were studied. The study is primarily focused 

2 on the general effects of a low selenium diet. The data suggest that selenium is anticarcinogenic as in the 

3 control groups the animals exposed to the higher selenium levels had fewer mammary tumors and these 

4 tumors had a delayed onset. Pb exposure with low selenium caused the same delayed onset as the higher 

5 dose of selenium and also caused some reduction in the tumor frequency. Pb exposure with higher 

6 selenium increased the tumor frequency and the onset of the tumors. Pb also induced weight loss at 14 

7 months in both exposed groups. The data suggest that there may be interactions of Pb and selenium, but 

8 they suggest that Pb mimics or antagonizes selenium. They do not suggest that selenium is protective of 

9 Pb-induced toxicity or carcinogenesis.

5.10.4. Epigenetics

10 Epigenetic studies have been conducted to examine the associations between Pb levels and global 

11 DNA methylation markers [Alu and long interspersed nuclear element-1 (LINE-1)] (Pilsner et al., 2009; 

12 R. O. Wright et al., 2010). Wright et al. (2010) utilized a sample of participants from the Normative Aging 

13 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 

14 measures, and 4.1 μg/dL (SD 2.4 μg/dL) for blood measures. In both crude and adjusted analyses, patella 

15 Pb levels were inversely associated with LINE-1 methylation but not with Alu. When examining the 

16 relationship between patella Pb and LINE-1 more closely, a non-linear trend was observed with leveling 

17 off at higher Pb levels. No associations were observed for tibia or blood Pb and either LINE-1 or Alu. The 

18 second study included maternal-infant pairs from the Early Life Exposures in Mexico to Environmental 

19 Toxicants (ELEMENT) study and measured LINE-1 and Alu methylation in umbilical cord blood samples 

20 (Pilsner et al., 2009). In unadjusted models, maternal tibia Pb levels [mean 10.5 μg/g (SD 8.4 μg/g)] were 

21 inversely associated with Alu methylation and maternal patella Pb levels [12.9 μg/g (SD 14.3 μg/g)] were 

22 inversely associated with LINE-1 methylation. The associations persisted in adjusted models although the 

23 association between patella Pb and LINE-1 was only apparent when the adjusted models also included 

24 umbilical cord blood Pb levels. No association was detected between umbilical cord Pb levels and the 

25 DNA methylation markers. Overall, the studies consistently demonstrate an association between patella 

26 Pb levels and LINE-1 methylation. 

27 Toxicological studies have been performed examining Pb-dependent epigenetic changes and gene 

28 expression, DNA repair, and mitogenesis. Glahn et al., (2008) performed a gene array study in primary 

29 normal human bronchial epithelial cells from 4 donors after in vitro treatment of the cells with 550 μg/l 

30 Pb chloride, 15 μg/l cadmium sulphate, 25 μg/l cobalt chloride or all three combined for 72 hours. The 

31 authors describe a pattern of RNA expression changes indicating “ ...coordinated stress-response and cell32 survival signaling, deregulation of cell proliferation, increased steroid metabolism, and increased 

33 expression of xenobiotic metabolizing enzymes”. These are all known targets of possible epigentic 
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1 changes, but full interpretations of the data as epigenetic changes are complicated by the absence of a 

2 measure to determine if these changes were a result of genotoxic effects.

5.10.5. Summary and Causal Determination

3 In summary, the toxicology literature on the genotoxic, mutagenic, and carcinogenic potential of Pb 

4 have strong evidence of effects in laboratory animals. In laboratory studies, high-dose Pb has been 

5 demonstrated to be an animal carcinogen. There are data to suggest Pb is a human carcinogen among 

6 toxicological studies, but they are not definitive. The three toxicological studies of neoplastic 

7 transformation in cultured cells both were positive, but both focused on PbCrO4 and attributed the 

8 positive response to the CrO4 and not the Pb. Mechanistic understanding of the carcinogenicity of Pb is 

9 expanding with work on the antioxidant selenium and metallothionein, a protein that binds Pb and 

10 reduces its bioavailability. In separate studies, selenium and metallothionein are protective against the 

11 effect of Pb on carcinogenicity. Pb is clastogenic and mutagenic in some but not all models. 

12 Clastogenicity and mutagenicity may be possible mechanisms contributing to cancer but are not 

13 absolutely associated with the induction of cancer. Because Pb has a higher atomic weight than zinc, Pb 

14 replaces zinc at many zinc binding or zinc finger proteins. This substitution has the potential to induce 

15 indirect effects that can contribute to carcinogenicity via interactions at hormone receptors, at cell-cycle 

16 regulatory proteins, with tumor suppressor genes like p53, with DNA repair enzymes, with histones, etc. 

17 These indirect effects may act at a post-translational level to adversely alter protein structure and DNA 

18 repair. Also, epigenetic changes associated with Pb exposure are beginning to appear in the literature. 

19 These modifications may further alter DNA repair or change the expression of a tumor suppressor gene or 

20 oncogene in an adverse fashion. Thus, the animal toxicology literature provides a strong base for 

21 understanding the potential contribution of Pb exposure to cancer in laboratory animals. 

22 Multiple epidemiologic studies have been performed examining cancer incidence and mortality, 

23 determined with biological measures and exposure databases. Mixed results have been reported for cancer 

24 mortality studies; one strong epidemiologic study demonstrated a positive association between blood Pb 

25 and cancer mortality, but the other studies reported null results. Although the previous Pb AQCD reported 

26 some studies demonstrating an association between Pb exposure and lung cancer, current studies mostly 

27 include studies of occupational exposure and observed no associations. Most studies of Pb and brain 

28 cancer were null among the overall study population, but positive associations were observed among 

29 individuals with certain genotypes. A limited amount of research on other types of cancer has been 

30 performed. The previous AQCD reported evidence that suggested an association between Pb exposure 

31 and stomach cancer, but recent studies of this association are lacking. One study examining Pb and 

32 stomach cancer has been performed since the last AQCD and the results of the study are mixed. 
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1 Among epidemiologic studies, positive associations were observed between high Pb levels and 

2 SCE among adults but not children. Other epidemiologic studies of DNA damage reported inconsistent 

3 results. Consistent with previous toxicological findings, Pb does appear to have genotoxic activity 

4 inducing SCE, MN and DNA strand breaks, but continues to not produce chromosomal aberrations except 

5 for PbCrO4; this again is likely due to the chromate. Pb does not appear to be very mutagenic as the HPRT 

6 assays were typically negative unless a cell signaling pathway was disturbed. 

7 Epigenetic effects, particularly with respect to methylation and effects on DNA repair were also 

8 consistently seen. Epigenetic studies examining Pb and LINE-1 and Alu consistently demonstrated an 

9 inverse association between patella Pb and LINE-1 methylation. Toxicological studies do show that Pb

10 can activate or interfere with a number of signaling and repair pathways, though it is unclear whether 

11 these are in response to epigenetics or to genotoxicity. Thus, an underlying mechanism is still uncertain, 

12 but likely involves either genomic instability or epigenetic modifications or both.

13 Overall, there is some epidemiologic evidence supporting associations between Pb and cancer. 

14 Strong evidence from toxicological studies demonstrates an association between Pb and cancer, 

15 genotoxicity/clastogenicity or epigenetic modification. The collective body of evidence integrated across 

16 epidemiologic and toxicological studies is sufficient to conclude that there is a likely causal relationship 

17 between Pb exposures and cancer.

5.11. Overall Summary

18 The evidence reviewed in this chapter describes the recent findings regarding the health effects of 

19 Pb. Table 5-38 provides an overview of the causal determinations for each of the health categories 

20 evaluated.

Table 5-38. Summary of causal determinations for Pb.

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 relationship
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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

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