United States
Environmental Protection
Agency
Environmental Criteria and
Assessment Office
Research Triangle Park NC 27711
EPA-600/8-83-028A
October 1983
External Review Draft
Research and Development
xvEPA
Air Quality
Criteria for Lead
Volume IV of IV
Review
Draft
(Do Not
Cite or Quote)
NOTICE
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
-------
EPA-600/8-83-028A
October 1983
Draft External Review Draft
Do Not Quote or Cite
Air Quality Criteria
for Lead
Volume IV
NOTICE
This document is a preliminary draft. It has not been formally released by EPA and should not at this stage
be construed to represent Agency policy. It is being circulated for comment on its technical accuracy and
policy implications.
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
-------
NOTICE
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
il
-------
ABSTRACT
The document evaluates and assesses scientific information on the health
and welfare effects associated with exposure to various concentrations of lead
in ambient air. The literature through 1983 has been reviewed thoroughly for
information relevant to air quality criteria, although the document is not
intended as a complete and detailed review of all literature pertaining to
lead. An attempt has been made to identify the major discrepancies in our
current knowledge and understanding of the effects of these pollutants.
Although this document is principally concerned with the health and
welfare effects of lead, other scientific data are presented and evaluated in
order to provide a better understanding of this pollutant in the environment.
To this end, the document includes chapters that discuss the chemistry and
physics of the pollutant; analytical techniques; sources, and types of
emissions; environmental concentrations and exposure levels; atmospheric
chemistry and dispersion modeling; effects on vegetation; and respiratory,
physiological, toxicological, clinical, and epidemiological aspects of human
exposure.
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PRELIMINARY DRAFT
CONTENTS
Page
VOLUME I
Chapter 1.
VOLUME II
Chapter 2.
Chapter 3.
Chapter 4.
Chapter 5.
Chapter 6.
Chapter 7.
Chapter 8.
VOLUME III
Chapter 9.
Chapter 10.
Chapter 11.
Executive Summary and Conclusions
Introduction
Chemical and Physical Properties
Sampling and Analytical Methods for Environmental Lead
Sources and Emissions
Transport and Transformation
Environmental Concentrations and Potential Pathways to Human Exposure
Effects of Lead on Ecosystems
Quantitative Evaluation of Lead and Biochemical Indices of Lead
Exposure in Physiological Media
Metabolism of Lead
Assessment of Lead Exposures and Absorption in Human Populations
Volume IV
Chapter 12. Biological Effects of Lead Exposure
Chapter 13. Evaluation of Human Health Risk Associated with Exposure to Lead
and Its Compounds
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
12-1
13-1
iv
-------
PRELIMINARY DRAFT
TABLE OF CONTENTS
LIST OF FIGURES ix
LIST OF TABLES ix
12. BIOLOGICAL EFFECTS OF LEAD EXPOSURE 12-1
12.1 INTRODUCTION 12-1
12.2 SUBCELLULAR EFFECTS OF LEAD IN HUMANS AND EXPERIMENTAL ANIMALS 12-3
12.2.1 Effects of Lead on the Mitochondrion 12-4
12.2.1.1 Effects of Lead on Mitochondria! Structure 12-4
12.2.1.2 Effects of Lead on Mitochondrial Function 12-4
12.2.1.3 In Vivo Studies 12-4
12.2.1.4 Iji Vrtro Studies 12-6
12.2.2 Effects of Lead on the Nucleus 12-7
12.2.3 Effects of Lead on Membranes 12-8
12.2.4 Other Organellar Effects of Lead 12-9
12.2.5 Summary of Subcellular Effects of Lead 12-9
12.3 EFFECTS OF LEAD ON HEME BIOSYNTHESIS AND ERYTHROPOIESIS/ERYTHROCYTE
PHYSIOLOGY IN HUMANS AND ANIMALS 12-12
12.3.1 Effects of Lead on Heme Biosynthesis 12-12
12.3.1.1 Effects of Lead on 6-Aminolevulinic Acid Synthetase 12-13
12.3.1.2 Effects of Lead on 6-Aminolevulinic Acid Dehydrase and
ALA Accumulation/Excretion 12-14
12.3.1.3 Effects of Lead on Heme Formation from Protoporphyrin ... 12-19
12.3.1.4 Other Heme-Related Effects of Lead 12-24
12.3.2 Effects of Lead on Erythropoiesis and Erythrocyte Physiology 12-25
12.3.2.1 Effects of Lead on Hemoglobin Production 12-25
12.3.2.2 Effects of Lead on Erythrocyte Morphology and Survival .. 12-26
12.3.2.3 Effects of Lead on Pyrimidine-5'-Nucleotidase Activity
and Erythropoietic Pyrimidine Metabolism 12-28
12.3.3 Effects of Alkyl Lead on Heme Synthesis and Erythopoiesis 12-30
12.3.4 The Interrelationship of Lead Effects on Heme Synthesis and
the Nervous System 12-30
12.3.5 Summary and Overview 12-33
12.3.5.1 Lead Effects on Heme Biosynthesis 12-33
12.3.5.2 Lead Effects on Erythropoiesis and
Erythrocyte Physiology 12-37
12.3.5.3 Effects of Alkyl Lead Compounds on Heme Biosynthesis
and Erythropoiesis 12-38
12.3.5.4 Relationships of Lead Effects on
Heme Synthesis and Neurotoxicity 12-38
12.4 NEUROTOXIC EFFECTS OF LEAD 12-40
12.4.1 Introduction 12-40
12.4.2 Human Studies 12-41
12.4.2.1 Neurotoxic Effects of Lead Exposure in Adults 12-44
12.4.2.2 Neurotoxic Effects of Lead Exposure in Children . 12-50
12.4.3 Animal Studies 12-76
12.4.3.1 Behavioral Toxicity: Critical Periods for Exposure and
Expression of Effects 12-77
23PB13/D 9/20/83
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PRELIMINARY DRAFT
TABLE OF CONTENTS (continued).
12.4.3.2 Morphological Effects 12-100
12.4.3.3 Electrophysiological Effects 12-102
12.4.3.4 Biochemical Alterations 12-105
12.4.3.5 Accumulation and Retention of Lead in the Brain 12-110
12.4.4 Integrative Summary of Human and Animal Studies of Neurotoxicity .. 12-110
12.4.4.1 Internal Exposure Levels at Which Adverse
Neurobehavioral Effects Occur 12-114
12.4.4.2 The Question of Irreversibility 12-115
12.4.4.3 Early Development and the Susceptibility to
Neural Damage 12-116
12.4.4.4 Utility of Animal Studies in Drawing Parallels
to the Human Condition 12-116
12.5 EFFECTS OF LEAD ON THE KIDNEY 12-121
12.5.1 Historical Aspects 12-121
12.5.2 Lead Nephropathy in Childhood 12-121
12.5.3 Lead Nephropathy in Adults 12-122
12.5,3.1 Lead Nephropathy Following Childhood Lead Poisoning 12-122
12.5.3.2 "Moonshine" Lead Nephropathy 12-124
12.5.3.3 Occupational Lead Nephropathy 12-124
12.5.3.4 Lead and Gouty Nephropathy 12-129
12.5.3.5 Lead and Hypertensive Nephrosclerosis 12-132
12.5.3.6 General Population Studies 12-133
12.5.4 Mortality Data 12-134
12.5.5 Experimental Animal Studies of the Pathophysiology of
Lead Nephropathy 12-135
12.5.5.1 Lead Uptake By the Kidney 12-135
12.5.5.2 Intracellular Binding of Lead in the Kidney 12-137
12.5.5.3 Pathological. Features of Lead Nephropathy 12-137
12.5.5.4 Functional Studies 12-139
12.5.6 Experimental Studies of the Biochemical Aspects of
Lead Nephrotoxicity 12-140
12.5.6.1 Membrane Marker Enzymes and Transport Functions 12-140
12.5.6.2 Mitochondrial Respiration/Energy-Linked
Transformation 12-140
12.5.6.3 Renal Heme Biosynthesis 12-141
12.5.6.4 Lead Alteration of Renal Nucleic Acid/Protein
Synthesis 12-142
12.5.6.5 Lead Effects on the Renin-Angiotension System 12-144
12.5.6.6 Effects of Lead on Uric Acid Metabolism 12-145
12.5.6.7 Effects of Lead on Vitamin 0 Metabolism in the Kidney ... 12-145
12.5.7 General Summary and Comparison of Lead Effects in Kidneys of
Humans and Animal Models 12-146
12.6 EFFECTS OF LEAD ON REPRODUCTION AND DEVELOPMENT 12-147
12.6.1 Human Studies 12-147
12.6.1.1 Historical Evidence 12-147
12.6.1.2 Effects of Lead Exposure on Reproduction 12-148
12.6.1.3 Placental Transfer of Lead 12-152
12.6.1.4 Effects of Lead on the Developing Human 12-152
12.6.1.5 Summary of the Human Data 12-156
12.6.2 Animal Studies 12-156
12.6.2.1 Effects of Lead on Reproduction 12-156
12.6.2.2 Effects of Lead on the Offspring 12-160
vi
23PB13/D 9/20/83
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PRELIMINARY DRAFT
TABLE OF CONTENTS (continued).
12.6.2.3 Effects of Lead on Avian Species 12-171
12.6.3 Summary 12-171
12.7 GENETOXIC AND CARCINOGENIC EFFECTS OF LEAD 12-173
12.7.1 Introduction 12-173
12.7.2 Carcinogenesis Studies with Lead and its Compounds 12-176
12.7.2.1 Human Epidemiological Studies 12-176
12.7.2.2 Induction of Tumors in Experimental Animals 12-180
12.7.2.3 Cell Transformation 12-185
12.7.3 Genotoxicity of Lead 12-188
12.7.3.1 Chromosomal Aberrations 12-188
12.7.3.2 Effect of Lead on Bacterial and Mammalian
Mutagenesis Systems 12-193
12.7.3.3 Effect of Lead on Parameters of DNA
Structure and Function 12-194
12.7.4 Summary and Conclusions 12-195
12.8 EFFECTS OF LEAD ON THE IMMUNE SYSTEM 12-196
12.8.1 Development and Organization of the Immune System 12-196
12.8.2 Host Resistance 12-197
12.8.2.1 Infectivity Models 12-198
12.8.2.2 Tumor Models and Neoplasia 12-200
12.8.3 Humoral Immunity 12-200
12.8.3.1 Antibody Titers 12-200
12.8.3.2 Enumeration of Antibody Producing Cells
(Plaque-Forming Cells) 12-202
12.8.4 Cell-Mediated Immunity 12-204
12.8.4.1 Delayed-Type Hypersensitivity 12-204
12.8.4.3 Interferon 12-206
12.8.5 Lymphocyte Activation by Mitogens 12-206
12.8.5.1 In Vivo Exposure 12-206
12.8.5.2 In Vitro Exposure 12-208
12.8.6 Macrophage Function 12-209
12.8.7 Mechanisms of Lead Immunomodulation 12-211
12.8.8 Summary 12-211
12.9 EFFECTS OF LEAD ON OTHER ORGAN SYSTEMS 12-212
12.9.1 The Cardiovascular System 12-212
12.9.2 The Hepatic System 12-214
12.9.3 The Endocrine System 12-216
12.9.4 The Gastrointestinal System 12-218
12.10 CHAPTER SUMMARY 12-218
12.10.1 Introduction 12-218
12.10.2 Subcellular Effects of Lead 12-218
12.10.3 Effects of Lead on Heme Biosynthesis, Erythropoiesis, and
Erythrocyte Physiology in Humans and Animals 12-221
12.10.4 Neurotoxic Effects of Lead 12-227
12.10.4.1 Internal Exposure Levels at Which Adverse
Neurobehavioral Effects Occur 12-228
12.10.4.2 The Question of Irreversibility 12-229
12.10.4.3 Early Development and the Susceptibility to Neural
Damage 12-230
12.10.4.4 Utility of Animal Studies in Drawing Parallels to the
Human Condition 12-230
vii
23PB13/D 9/20/83
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PRELIMINARY DRAFT
TABLE OF CONTENTS (continued).
12.10.5 Effects of Lead on- the Kidney 12-232
12.10.6 Effects of Lead on Reproduction and Development 12-233
12.10.7 Genotoxic and Carcinogenic Effects of Lead 12-234
12.10.8 Effects of Lead on the Immune System 12-234
12.10.9 Effects of Lead on Other Organ Systems 12-235
12.11 REFERENCES 12-236
APPENDIX 12-A 12A-1
APPENDIX 12-B 12B-1
APPENDIX 12-C TftA
APPENDIX 12-D 12D-1
13.1 INTRODUCTION 13-1
13.2 EXPOSURE ASPECTS 13-2
13.2.1 Sources of Lead Emission in the United States 13-2
13.2.2 Environmental Cycling of Lead 13-4
13.2.3 Levels of Lead in Various Media of Relevance to Human Exposure 13-5
13.2.3.1 Ambient Air Lead Levels 13-6
13.2.3.2 Level s of Lead in Dust 13-6
13.2.3.3 Levels of Lead in Food 13-7
13.2.3.4 Lead Levels in Drinking Water 13-7
13.2.3.5 Lead in Other Media 13-11
13.2.3.6 Cumulative Human Lead Intake From Various Sources 13-11
13.3 LEAD METABOLISM: KEY ISSUES FOR HUMAN HEALTH RISK EVALUATION 13-11
13.3.1 Differential Internal Lead Exposure Within Population Groups 13-12
13.3.2 Indices of Internal Lead Exposure and Their Relationship to External
Lead Levels and Ti ssue Burdens/Effects 13-13
13.4 DEMOGRAPHIC CORRELATES OF HUMAN LEAD EXPOSURE AND RELATIONSHIPS BETWEEN
EXTERNAL AND INTERNAL LEAD EXPOSURE INDICES 13-16
13.4.1 Demographic Correlates of Lead Exposure 13-16
13.4.2 Relationships Between External and Internal Lead Exposure Indices 13-18
13.4.3 Proportional Contributions of Lead in Various Media to Blood Lead in
Human Populations 13-23
13.5 BIOLOGICAL EFFECTS OF LEAD RELEVANT TO THE GENERAL HUMAN POPULATION 13-27
13.5.1 Introduction • 13-27
13.5.2 Dose-Effect Relationship for Lead-Induced Health Effects 13-29
13.5.2.1 Human Adults 13-29
13.5.2.2 Children 13-31
13.6 DOSE-RESPONSE RELATIONSHIPS FOR LEAD IN HUMAN POPULATIONS 13-36
13.7 POPULATIONS AT RISK 13-40
13.7.1 Children as a Population at Risk 13-40
13.7.1.1 Inherent Susceptibility of the Young 13-40
13.7.1.2 Exposure Consideration 13-41
13.7.2 Pregnant Women and the Conceptus as a Population at Risk 13-41
13.7.3 Description of the United States Population in Relation to Potential
Lead Exposure Risk 13-42
13.8 SUMMARY AND CONCLUSIONS 13-44
13-9 REFERENCES 13-46
viii
23PB13/0 9/20/83
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PRELIMINARY DRAFT
LIST OF FIGURES
Figure Page
12-1 Lead effects on heme biosynthesis 12-13
12-2 Maximal motor nerve conduction velocity (NCV) of the median nerve plotted
against the actual Pb-B level (ug/100 ml) for 78 workers occupationally
exposed to lead and for 34 control subjects 12-49
12-3 (a) Predicted SW voltage and 95% confidence bounds in 13- and 75-month-old
children as a function of blood lead level, (b) Scatter plots of SW data
from children aged 13-47 months with predicted regression^lines for ages
18, 30, and 42 months, (c) Scatter plots for children aged 48-75 months
with predicted regression lines for ages 54 and 66 months. These graphs
depict the linear interaction of blood lead level and age 12-73
12-4 Peroneal nerve conduction velocity versus blood lead level, Idaho, 1974 12-75
12-5 Probit plot of incidence of renal tumors in male rats 12-186
13-1 Pathways of lead from the environment to man 13-3
13-2 Geometric mean blood lead levels by race and age for younger children in the
NHANES II study, and the Kellogg/Silver Valley and New York childhood
screening studies 13-17
13-3 Dose-response for elevation of EP as a function of blood lead level using
probit analysis 13-38
13-4 Dose-response curve for FEP as a function of blood lead level:
in subpopulations 13-38
13-5 EPA calculated dose-response curve for ALA-U 13-39
LIST OF TABLES
Table Page
12-1 Summary of results of human studies on neurobehavioral effects 12-55
12-2 Effects of lead on activity in rats and mice 12-81
12-3 Recent animal toxicology studies of lead effects on learning in rodent
species 12-83
12-4 Recent animal toxicology studies of lead effects on learning in primates 12-89
12-5 Summary of key studies of morphological effects of iji vivo lead exposure 12-101
12-6 Summary of key studies of electrophysiological effects of jji vivo
lead exposure 12-103
12-7 Summary of key studies on biochemical effects of .in vivo lead exposure 12-106
12-8 Index of blood lead and brain lead levels following exposure 12-111
12-9 Summary of key studies of in vitro lead exposure 12-119
12-10 Morphological features of lead nephropathy in various species 12-138
12-11 Effects of lead exposure on renal heme biosynthesis 12-143
12-12 Statistics on the effect of lead on pregnancy 12-148
12-13 Effects of prenatal exposure to lead pn the offspring of laboratory and
domestic animals 12-161
12-14 Effects of prenatal lead exposure on offspring of laboratory animals 12-163
12-15 Reproductive performance of Fx lead-intoxicated rats 12-166
ix
23PB13/D 9/20/83
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PRELIMINARY DRAFT
LIST OF TABLES (continued).
Table Page
12-16 Expected and observed deaths for malignant neoplasms Jan. 1, 1947 -
Dec. 31, 1979 for lead smelter and battery plant workers 12-177
12-17 Expected and observed deaths resulting from specified malignant neoplasms
for lead smelter and battery plant workers and levels of significance by
type of statistical analysis according to one-tailed tests 12-178
12-18 Examples of studies on the incidence of tumors in experimental animals
exposed to lead compounds 12-181
12-19 Mortality and kidney tumors in rats fed lead acetate for two years 12-185
12-20 Cytogenetic investigations of cells from individuals exposed to lead:
10 positive studies 12-189
12-21 Cytogenetic investigations of cells from individuals exposed to lead:
6 negative studies 12-190
12-22 Effect of lead on host resistance to infectious agents 12-197
12-23 Effect of lead on antibody titers 12-200
12-24 Effect of lead on the development of antibody-producing cells (RFC) 12-202
12-25 Effect of lead on cell-mediated immunity 12-204
12-26 Effect of lead exposure on mitogen activation of lymphocytes 12-206
12-27 Effect of lead on macrophage and reticuloendothelial system function 12-209
12-B Tests commonly used in a psycho-educational battery for children 12-B2
13-1 Summary of baseline human exposures to lead 13-8
13-2 Relative baseline human lead exposures expressed per kilogram body weight 13-9
13-3 Summary of potential additive exposures to lead 13-11
13-4 Summary of blood inhalation slopes, (P) 13-19
13-5 Estimated contribution of leaded gasoline to blood lead by inhalation and
non-inhalation pathways 13-22
13-6 Direct contributions of air lead to blood lead in adults at fixed inputs of
water and food lead 13-24
13-7 Direct contributions of air lead to blood lead in children at fixed inputs
of food and water 1 ead 13-25
13-8 Contributions of dust/soil lead to blood lead in children at fixed inputs
of air, food, and water lead 13-26
13-9 Summary of lowest observed effect levels for key lead-induced health effects
i n adul ts 13-30
13-10 Summary of lowest observed effect levels for key lead-induced health effects
in children 13-32
13-11 EPA-estimated percentage of subjects with ALA-U exceeding limits for various
blood lead levels 13-39
13-12 Provisional estimate of the number of individuals in urban and rural
population segments at greatest potential risk to lead exposure 13-43
23PB13/D 9/20/83
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PRELIMINARY DRAFT
LIST OF ABBREVIATIONS
AAS
Ach
ACTH
ADCC
AOP/0 ratio
AIDS
AIHA
All
ALA
ALA-D
ALA-S
ALA-U
APDC
APHA
ASTM
ASV
ATP
B-cells
Ba
BAL
BAP
BSA
BUN
BW
C.V.
CaBP
CaEDTA
CBD
Cd
CDC
CEC
CEH
CFR
CMP
CNS
CO
COHb
CP-U
cBah
D.F.
DA
DCMU
DDP
DNA
DTH
EEC
EEC
EMC
EP
EPA
Atomic absorption spectrometry
Acetylcholine
Adrenocoticotrophic hormone
Antibody-dependent cell-mediated cytotoxicity
Adenosine diphosphate/oxygen ratio
Acquired immune deficiency syndrome
American Industrial Hygiene Association
Angiotensin II
Aminolevulinic acid
Aminolevulinic acid dehydrase
Aminolevulinic acid synthetase
Aminolevulim'c acid in urine
Ammonium pyrrolidine-dithiocarbamate
American Public Health Association
Amercian Society for Testing and Materials
Anodic stripping voltammetry
Adenosine triphosphate
Bone marrow-derived lymphocytes
Barium
British anti-Lewisite (AKA dimercaprol)
benzo(a)pyrene
Bovine serum albumin
Blood urea nitrogen
Body weight
Coefficient of variation
Calcium binding protein
Calcium ethylenediaminetetraacetate
Central business district
Cadmium
Centers for Disease Control
Cation exchange capacity
Center for Environmental Health
reference method
Cytidine monophosphate
Central nervous system
Carbon monoxide
Carboxyhemoglobin
Urinary coproporphyrin
plasma clearance of p-aminohippuric acid
Copper
Degrees of freedom
Dopamine
[3-(3,4-dichlorophenyl)-l,l-dimethy1urea
Differential pulse polarography
Deoxyribonucleic acid
Delayed-type hypersensitivity
European Economic Community
Electroencephalogram
Encephalomyocarditi s
Erythrocyte protoporphyrin
U.S. Environmental Protection Agency
TCPBA/D
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9/20/83
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PRELIMINARY DRAFT
LIST OF ABBREVIATIONS (continued).
FA Fulvic acid
FDA Food and Drug Administration
Fe Iron
FEP Free erythrocyte protoporphyrin
FY Fiscal year
G.M. Grand mean
G-6-PD Glucose-6-phosphate dehydrogenase
GABA Gamma-aminobutyric acid
GALT Gut-associated lymphoid tissue
GC Gas chromatography
GFR Glomerular filtration rate
HA Humic acid
Hg Mercury
hi-vol High-volume air sampler
HPLC High-performance liquid chromatography
i-iti. Intramuscular (method of injection)
i.p. Intraperitoneally (method of injection)
i-v. Intravenously (method of injection)
IAA Indol-3-ylacetic acid
IARC International Agency for Research on Cancer
ICD International classification of diseases
ICP Inductively coupled plasma
IDMS Isotope dilution mass spectrometry
IF Interferon
ILE Isotopic Lead Experiment (Italy)
IRPC International Radiological Protection Commission
K Potassium
LAI Leaf area index
LDH-X Lactate dehydrogenase isoenzyme x
LCj-f. Lethyl concentration (50 percent)
LDj" Lethal dose (50 percent)
LH Luteinizing hormone
LIPO Laboratory Improvement Program Office
In National logarithm
LPS Lipopolysaccharide
LRT Long range transport
mRNA Messenger ribonucleic acid
ME Mercaptoethanol
MEPP Miniature end-plate potential
MES Maximal electroshock seizure
MeV Mega-electron volts
MLC Mixed lymphocyte culture
MMD Mass median diameter
MMED Mass median equivalent diameter
Mn Manganese
MND Motor neuron disease
MSV Moloney sarcoma virus
MTD Maximum tolerated dose
n Number of subjects
N/A Not Available
TCPBA/0 X11 9/20/83
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PRELIMINARY DRAFT
LIST OF ABBREVIATIONS
NA
NAAQS
NADB
NAMS
NAS
NASN
NBS
NE
NFAN
NFR-82
NHANES II
Ni
OSHA
P
P
PAH
Pb
PBA
Pb(Ac)_
PbB *
PbBrCl
PBG
PFC
PH
PHA
PHZ
PIXE
PMN
PND
PNS
ppm
PRA
PRS
PWM
Py-5-N
RBC
RBF
RCR
redox
RES
RLV
RNA
S-HT
SA-7
sent
S.D.
SOS
S.E.M.
SES
SGOT
Not Applicable
National ambient air quality standards
National Aerometric Data Bank
National Air Monitoring Station
National Academy of Sciences
National Air Surveillance Network
National Bureau of Standards
Norepinephrine
National Filter Analysis Network
Nutrition Foundation Report of 1982
National Health Assessment and Nutritional Evaluation Survey II
Nickel
Occupational Safety and Health Administration
Potassium
Significance symbol
Para-aminohippuric acid
Lead
Air lead
Lead acetate
concentration of lead in blood
Lead (II) bromochloride
PorphobiUnogen
Plaque-forming cells
Measure of acidity
Phytohemagglutinin
Polyacrylamide-hydrous-zirconia
Proton-induced X-ray emissions
Polymorphonuclear leukocytes
Post-natal day
Peripheral nervous system
Parts per million
Plasma renin activity
Plasma renin substrate
Pokeweed mitogen
Pyrimide-5'-nucleotidase
Red blood cell; erythrocyte
Renal blood flow
Respiratory control ratios/rates
Oxidation-reduction potential
Reticuloendothelial system
Rauscher leukemia virus
Ribonucleic acid
Serotonin
Simian adenovirus
Standard cubic meter
Standard deviation
Sodium dodecyl sulfate
Standard error of the mean
Socioeconomic status
Serum glutamic oxaloacetic transaminase
TCPBA/D
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9/20/83
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PRELIMINARY DRAFT
LIST OF ABBREVIATIONS (continued).
slg
SLAMS
SMR
Sr
SRBC
SRMs
STEL
SW voltage
T-cells
t- tests
TBL
TEA
TEL
TIBC
TML
TMLC
TSH
TSP
U.K.
UMP
USPHS
VA
WHO
XSF
XZ
Zn
2PP
Surface immunoglobulin
State and local air monitoring stations
Standardized mortality ratio
Strontium
Sheep red blood cells
Standard reference materials
Short-term exposure limit
Slow-wave voltage
Thymus-derived lymphocytes
Tests of significance
Tri-n-butyl lead
Tetraethyl-ammonium
Tetraethyllead
Total iron binding capacity
Tetramethyllead
Tetramethyllead chloride
Thyroid-stimulating hormone
Total suspended particulate
United Kingdom
Uridine monophosphate
U.S. Public Health Service
Veterans Administration
Deposition velocity
Visual evoked response
World Health Organization
X-Ray fluorescence
Chi squared
Zinc
Erythrocyte zinc protoporphyrin
MEASUREMENT ABBREVIATIONS
dl
ft
9
g/gal
g/ha-mo
km/hr
1/min
mg/km
ug/m3
mm
pmol
ng/cm2
nm
nM
sec
deciliter
feet
gram
gram/gallon
gram/hectare-month
kilometer/hour
liter/minute
mi 11igram/ki1ometer
microgram/cubic meter
millimeter
micrometer
nanograms/square centimeter
namometer
nanomole
second
TCPBA/D
xiv
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Chapter 12: Biological Effects of Lead Exposure
Contributing Authors
Dr. Max Costa
Department of Pharmacology
University of Texas Medical School
Houston, TX 77025
Dr. J. Michael Davis
Environmental Criteria and Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Or. Jack Dean
Immunobiology Program and Immunotoxicology/
Cell Biology Program
CUT
P.O. Box 12137
Research Triangle Park, NC 27709
Dr. Bruce Fowler
Laboratory of Pharmacology
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27709
Dr. Lester Grant
Director, Environmental Criteria and
Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Ronald D. Hood
Department of Biology
The University of Alabama
P.O. Box 1927
University, AL 35486
Dr. Loren Koller
School of Veterinary Medicine
University of Idaho
Moscow, ID 83843
Dr. David Lawrence
Microbiology and Immunology
Department
Albany Medical College of Union
University
Albany, NY 12208
Dr. Paul Mushak
Department of Pathology
UNC School of Medicine
Chapel Hill, NC 27514
Dr. Dr. David Otto
Clinical Studies Division
MD-58
U.S. Environmental Protection
Agency
Research Triangle Park, NC 27711
Dr. Magnus Piscator
Department of Environmental Hygiene
The Karolinska Institute 104 01
Stockholm
Sweden
Dr. Stephen R. Schroeder
Division for Disorders of
Development and Learning
Biological Sciences Research Center
University of North Carolina
Chapel Hill, NC 27514
Dr. Richard P. Wedeen
V.A. Medical Center
Tremont Avenue
East Orange, NJ 07019
Dr. David Weil
Environmental Criteria and
Assessment Office
MD-52
U.S. Environmental Protection
Agency
Research Triangle Park, NC 27711
xv
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The following persons reviewed this chapter at EPA's request. The evaluations
and conclusions contained herein, however, are not necessarily those of the
reviewers.
Dr. Carol Angle
Department of Pediatrics
University of Nebraska
College of Medicine
Omaha, NE 68105
Dr. Lee Annest
Division of Health Examin. Statistics
National Center for Health Statistics
3700 East-West Highway
Hyattsville, MD 20782
Dr. Donald Barltrop
Department of Child Health
Westminister Children's Hospital
London SW1P 2NS
England
Dr. Irv Billick
Gas Research Institute
8600 West Bryn Mawr Avenue
Chicago, IL 60631
Dr. Joe Boone
Clinical Chemistry and
Toxicology Section
Center for Disease Control
Atlanta, GA 30333
Dr. Robert Bornschein
University of Cincinnati
Kettering Laboratory
Cincinnati, OH 45267
Dr. A. C. Chamberlain
Environmental and Medical Sciences Division
Atomic Energy Research Establishment
Harwell 0X11
England
Dr. Neil Chernoff
Division of Developmental Biology
MD-67
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Julian Chisolm
Baltimore City Hospital
4940 Eastern Avenue
Baltimore, MD 21224
Dr. Jerry Cole
International Lead-Zinc Research
Organization
292 Madison Avenue
New York, NY 10017
Dr. Anita Curran
Commissioner of Health
Westchester County
White Plains, NY 10607
Dr. Cliff Davidson
Department of Civil Engineering
Carnegie-Mellon University
Schenley Park
Pittsburgh, PA 15213
Dr. H. T. Delves
Chemical Pathology and Human
Metabolism
Southampton General Hospital
Southampton S09 4XY
England
Dr. Fred deSerres
Associate Director for Genetics
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27709
Dr. Joseph A. DiPaolo
Laboratory of Biology, Division
of Cancer Cause and Prevention
National Cancer Institute
Bethesda, MD 20205
Dr. Robert Dixon
Laboratory of Reproductive and
Developmental Toxicology
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27711
xv i
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Dr. Clair Ernhart
Department of Psychiatry
Cleveland Metropolitan General Hospital
3395 Scranton Road
Cleveland, OH 44109
Dr. Sergio Fachetti
Section Head - Isotope Analysis
Chemistry Division
Joint Research Center
121020 Ispra
Varese, Italy
Dr. Virgil Perm
Department of Anatomy and Cytology
Dartmouth Medical School
Hanover, NH 03755
Dr. Alf Fischbein
Environmental Sciences Laboratory
Mt. Sinai School of Medicine
New York, NY 10029
Dr. Jack Fowle
Reproductive Effects Assessment Group
U.S. Environmental Protection Agency
RD-689
Washington, DC 20460
Dr. Bruce Fowler
Laboratory of Pharmocology
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27709
Dr. Warren Galke
Department of Biostatistics and Epidemiology
School of Allied Health
East Carolina University
Greenville, NC 27834
Mr. Eric Goldstein
Natural Resources Defense Council, Inc.
122 E. 42nd Street
New York, NY 10168
Dr. Harvey Gonick
1033 Gayley Avenue
Suite 116
Los Angeles, CA 90024
Dr. Robert Goyer
Deputy Director
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27711
Dr. Philippe Grandjear
Department of Environmental Medicine
Institute of Community Health
Odense University
Denmark
Dr. Stanley Gross
Hazard Evaluation Division
Toxicology Branch
U.S. Environmental Protection
Agency
Washington, DC 20460
Dr. Paul Hammond
University of Cincinnati
Kettering Laboratory
Cincinnati, OH 45267
Dr. Kari Hemminki
Institute of Occupational Health
Tyoterveyslaitos-Haartmaninkatu
1 SF-00290 Helsinki 29
Finland
Dr. V. Houk
Center for Disease Control
1600 Clifton Road, NE
Atlanta, GA 30333
Dr. Carole A. Kimmel
Perinatal and Postnatal Evaluation
Branch
National Center for Toxicological
Research
Jefferson, AR 72079
Or. Kristal Kostial
Institute for Medical Research
and Occupational Health
YU-4100 Zagreb
Yugoslavia
Dr. Lawrence Kupper
Department of Biostatistics
UNC School of Public Health
Chapel Hill, NC 27514
xvi i
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Dr. Phillip Landrigan
Division of Surveillance,
Hazard Evaluation and Field Studies
Taft Laboratories - NIOSH
Cincinnati, OH 45226
Dr. Alais-Yves Leonard
Centre Betude De L'Energie Nucleaire
B-1040 Brussels
Belgium
Dr. Jane Lin-Fu
Office of Maternal and Child Health
Department of Health and Human Services
Rockville, MD 20857
Dr. Don Lynam
Air Conservation
Ethyl Corporation
451 Florida Boulevard
Baton Rouge, LA 70801
Dr. Kathryn Mahaffey
Division of Nutrition
Food and Drug Administration
1090 Tusculuni Avenue
Cincinnati, OH 45226
Dr. Ed McCabe
Department of Pediatrics
University of Wisconsin
Madison, WI 53706
Dr. Chuck Nauman
Exposure Assessment Group
U.S. Environmental Protection Agnecy
Washington, DC 20460
Dr. Herbert L. Needleman
Children's Hospital of Pittsburgh
Pittsburgh, PA 15213
Or. Forrest H. Nielsen
Grand Forks Human Nutrition Research Center
USDA
Grand Forks, ND 58202
Dr. Stephen Overman
Toxicology Institute
New York State Department of
Health
Empire State Plaza
Albany, NY 12001
Dr. H. Mitchell Perry
V.A. Medical Center
St. Louis, MO 63131
Dr. Jack Pierrard
E.I. duPont de Nemours and
Company, Inc.
Petroleum Laboratory
Wilmington, DE 19898
Dr. Sergio Piomelli
Columbia University Medical School
Division of Pediatric Hematology
and Oncology
New York, NY 10032
Dr. Robert Putnam
International Lead-Zinc
Research Organization
292 Madison Avenue
New York, NY 10017
Dr. Rabinowitz
Children's Hospital Medical Center
300 Longwood Avenue
Boston, MA 02115
Dr. Dr. Larry Reiter
Neurotoxicology Division
MD-74B
U.S. Environmental Protection
Agency
Research Triangle Park, NC 27711
Dr. Cecil R. Reynolds
Department of Educational Psychology
Texas A & M University
College Station, TX 77843
Dr. Patricia Rodier
Department of Anatomy
University of Rochester Medical
Center
Rochester, NY 14642
xviii
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Dr. Harry Roels
Unite de Toxicologie Industrie!le et Medicale
Universite de Louvain
Brussels, Belgium
Dr. John Rosen
Head, Division of Pediatric Metabolism
Montefiore Hospital and Medical Center
111 East 210 Street
Bronx, NY 10467
Dr. Michael Rutter
Department of Psychology
Institute of Psychiatry
DeCrespigny Park
London SE5 SAL
England
Dr. Anna-Maria Seppalainen
Institutes of Occupational Health
Tyoterveyslaitos
Haartmanikatu 1
00290 Helsinki 29
Finland
Dr. Ellen Silbergeld
Environmental Defense Fund
1525 18th Street, NW
Washington, DC 20036
Dr. Ron Snee
E.I. duPont de Nemours and
Company, Inc.
Engineering Department L3167
Wilmington, DE 19898
Dr. J. William Sunderman, Jr.
Department of Pharmacology
University of Connecticut
School of Medicine
Farmington, CT 06032
Dr. Gary Ter Haar
Toxicology and Industrial
Hygiene
Ethyl Corporation
451 Florida Boulevard
Baton Rouge, LA 70801
Dr. Hugh A. Til son
Laboratory of Behavioral and
Neurological Toxicology
NIEHS
Research Triangle Park, NC 27709
Mr. Ian von Lindern
Department of Chemical Engineering
University of Idaho
Moscow, ID 83843
xix
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Chapter 13: Risk Assessment
Principal Authors
Dr. Lester Grant
Director, Environmental Criteria and
Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Contributing Authors
Dr. Robert Eli as
Environmental Criteria and Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Vic Hasselblad
Biometry Division
MD-55
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Dennis Kotchmar
Environmental Criteria and Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Paul Mushak
Department of Pathology
UNC School of Medicine
Chapel Hill, NC 27514
Dr. Alan Marcus
Department of Mathematics
Washington State University
Pullman, Washington 99164-2930
Dr. David Weil
Environmental Criteria and
Assessment Office
U.S Environmental Protection
Agency
Research Triangle Park, NC 27711
xx
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PRELIMINARY DRAFT
12. BIOLOGICAL EFFECTS OF LEAD EXPOSURE
12.1 INTRODUCTION
As noted in Chapter 2, air quality criteria documents evaluate scientific knowledge of
relationships between pollutant concentrations and their effects on the environment and public
health. Early chapters of this document (Chapters 3-7) discuss: physical and chemical pro-
perties of lead; measurement methods for lead in environmental media; sources of emissions;
transport, transformation, and fate; and ambient concentrations and other aspects of the ex-
posure of the U.S. population to lead in the environment. Chapter 8 evaluates the projected
impact of lead on ecosystems. Chapters 9-11, immediately proceeding this one, discuss:
measurement techniques for lead in biologic media; aspects related to the uptake, distribu-
tion, toxicokinetics, and excretion of lead; and the relationship of various external and
internal lead exposure indices to each other. This chapter assesses information regarding
biological effects of lead exposure, with emphasis on (1) the qualitative characterization of
various lead-induced effects and (2) the delineation of dose-effect relationships for key
effects most likely of health concern at ambient exposure levels presently encountered by the
general population of the United States.
In discussing biological effects of lead, one should note at the outset that, to date,
lead has not been demonstrated to have any beneficial biological effect in humans. Some in-
vestigators have hypothesized that lead may serve as an essential element in certain mammalian
species (e.g., the rat) and have reported experimental data interpreted as supporting such an
hypothesis. However, a critical evaluation of these studies presented in Appendix 12-A of
this chapter raises serious questions regarding interpretation of the reported findings; and
the subject studies are currently undergoing intensive evaluation by an expert committee con-
vened by EPA. Therefore, pending the final report from that expert committee, the present
chapter does not address the issue of potential essentiality of lead.
It is clear from the evidence evaluated in this chapter that there exists a continuum of
biological effects associated with lead across a broad range of exposure. At rather low
levels of lead exposure, biochemical changes, e.g., disruption of certain enzymatic activities
involved in heme biosynthesis and erythropoietic pyrimidine metabolism, are detectable. With
increasing lead exposure, there are sequentially more pronounced effects on heme synthesis and
a broadening-of lead effects to additional biochemical and physiological mechanisms in various
tissues, such that increasingly more severe disruption of the normal functioning of many dif-
ferent organ systems becomes apparent. In addition to impairment of heme biosynthesis, signs
of disruption of normal functioning of the erythropoietic and nervous systems are among the
earliest effects observed in response to increasing lead exposure. At increasingly higher
exposure levels, more severe disruption of the erythropoietic and nervous systems occurs; and
APB12/A 12-1 9/20/83
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PRELIMINARY DRAFT
other organ systems are also affected so as to result in the manifestation of renal effects,
disruption of reproductive functions, impairment of immunological functions, and many other
biological effects. At sufficiently high levels of exposure, the damage to the nervous system
and other effects can be severe enough to result in death or, in some cases of non-fatal lead
poisoning, long-lasting sequelae such as permanent mental retardation.
The etiologies of many of the different types of functional disruption of various mamma-
lian organ systems derive (at least in their earliest stages) from lead effects on certain
subcellular organelles, which result in biochemical derangements (e.g., disruption of heme
synthesis processes) common to and affecting many tissues and organ systems. Some major
effects of lead on subcellular organelles common to numerous organ systems in mammalian spe-
cies are discussed below in Section 12.2, with particular emphasis on lead effects on mito-
chondrial functions. Subsequent sections of the chapter discuss biological effects of lead in
terms of various organ systems affected by that element and its compounds (except for Section
12.7, which assesses genotoxic and carcinogenic effects of lead). Additional cellular and
subcellular aspects of the biological effects of lead are discussed within respective sections
on particular organ systems.
Sections 12.3 to 12.9 have been sequenced generally according to the degree of known vul-
nerability of each organ system to lead. Major emphasis is placed first on discusssion of the
three systems classically considered to be most sensitive to lead (i.e., the hematopoietic,
the nervous, and the renal systems). The next sections then discuss the effects of lead on
reproduction and development (in view of the impact of lead on the fetus and pregnant women),
as well as gametotoxic effects of lead; Genotoxic effects of lead and evidence for possible
carcinogenic effects of lead are then reviewed, followed by discussion of lead effects on the
immune system and, lastly, other organ systems.
This chapter is subdivided mainly according to organ systems to facilitate presentation
of information. It must be noted, however, that, in reality, all systems function in delicate
concert to preserve the physiological integrity of the whole organism and all systems are in-
terdependent. Thus, not only do effects in a critical organ often exert impacts on other
organ systems, but low-level effects that might be construed as unimportant in a single speci-
fic system may be of concern in terms of their cumulative or aggregate impact.
Special emphasis is placed on the discussion of lead exposure effects in children. They
are particularly at risk due to sources of exposure, mode of entry, rate of absorption and re-
tention, and partitioning of lead in soft and hard tissues. The greater sensitivity of
children to lead toxicity, their inability to recognize symptoms, and their dependence on par-
ents and health care professionals all make them an especially vulnerable population requiring
special consideration in developing criteria and standards for lead.
APB12/A 12-2 9/20/83
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PRELIMINARY DRAFT
12.2 SUBCELLULAR EFFECTS OF LEAD IN HUMANS AND EXPERIMENTAL ANIMALS
The biochemical or molecular basis for lead toxicity is the ability of the toxicant, as a
metallic cation, to bind to ligating groups in biomolecular substances crucial to normal phy-
siological functions, thereby interfering with these functions via such mechanisms as competi-
tion with native essential metals for binding sites, inhibition of enzyme activity, and
inhibition or other alterations of essential ion transport. The relationship of this basis
for lead toxicity to organ- and organelle-specific effects is modulated by: (1) the inherent
stability of such binding sites for lead; (2) the compartmentalization kinetics governing lead
distribution among body compartments, among tissues, and within cells; and (3) differences in
biochemical and physiological organization across tissues and cells due to their specific
function. Given complexities introduced by factors 2 and 3, it is not surprising that no
single, unifying mechanism of lead toxicity has been demonstrated to apply across all tissues
and organ systems.
In the 1977 Air Quality Criteria Document for Lead, cellular and subcellular effects of
lead were discussed, including effects on various classes of enzymes. Much of the literature
detailing the effects of lead on enzymes per se has entailed study of relatively pure enzymes
vn vitro in the presence of added lead. This was the case for data discussed in the earlier
document and such information continues to appear in the literature. Much of this material is
of questionable relevance for effects of lead iji vivo. On the other hand, lead effects on
certain enzymes or enzyme systems are recognized as integral mechanisms of action underlying
the impact of lead on tissues HI vivo and are logically discussed in later sections below on
effects at the organ system level.
This subsection is mainly concerned with organellar effects of lead, especially those
that provide some rationale for lead toxicity at higher levels of biological organization.
While a common mechanism at the subcellular level that would account for all aspects of lead
toxicity has not been identified, one fairly common cellular response to lead is the impair-
ment of mitochondrial structure and function; thus, mitochondrion effects are accorded major
attention here. Lead effects on other organelles have not been as extensively studied as
mitochondrion effects; and, in some cases, it is not clear how the available information,
e.g., that on lead-containing nuclear inclusion bodies, relates to organellar dysfunction.
12.2.1 Effects of Lead on the Mitochondrion
The mitochondrion is clearly the target organelle for toxic effects of lead on many tis-
sues, the characteristics of vulnerability varying somewhat with cell type. Given early re-
cognition of this sensitivity, it is not surprising that an extensive body of jjn vivo and jn
vitro data has accumulated, which can be characterized as evidence of: (1) structural injury
to the mitochondrion; (2) impairment of basic cellular energetics and other mitochondrial
functions; and (3) uptake of lead by mitochondria jn vivo and in vitro.
APB12/A 12-3 9/20/83
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PRELIMINARY DRAFT
12.2.1.1 Lead Effects on Mitochondria"! Structure. Changes in mitochondria! morphology with
lead exposure have been well documented in humans and experimental animals and, in the case of
the kidney, are a rather early response to such exposure. Earlier studies have been reviewed
by Goyer and Rhyne (1973), followed by later updates by Fowler (1978) and Bull (1980).
Chronic oral exposure of adult rats to lead (1 percent lead acetate in diet) results in
structural changes in renal tubule mitochondria, including swelling with distortion or loss of
cristae (Goyer, 1968). Such changes have also been documented in renal biopsy tissue of lead
workers (Wedeen et al., 1975; Biagini et al., 1977) and in tissues other than kidney, i.e.,
heart (Malpass et al., 1971; Moore et al., 1975b), liver (Hoffman et al., 1972), and the cen-
tral (Press, 1977) and peripheral (Brashear et al., 1978) nervous systems.
While it appears that relatively high level lead exposures are necessary to detect struc-
tural changes in mitochondria in some" animal models (Goyer, 1968; Hoffman et al., 1972),
changes in rat heart mitochondria have been seen at blood lead levels as low as 42 ug/dl.
Also, in the study of Fowler et al. (1980), swollen mitochondria or renal tubule cells were
seen in rats chronically exposed to lead from gestation to 9 months of age at a dietary lead
dosing level as low as 50 ppm and a median blood lead level of 26 ug/dl (range 15-41 ug/dl).
Taken collectively, these differences point out relative tissue sensitivity, dosing protocol,
and the possible effect of developmental status (Fowler et al., 1980) as important factors in
determining lead exposure levels at which mitochondria are affected in various tissues.
12.2.1.2 Lead Effects on Mitochondria! Function. Both jji vivo and jjn vitro studies dealing
with such effects of lead as the impact on energy metabolism, intermediary metabolism, and
intracellular ion transport have been carried out in various experimental animal models. Of
particular interest for this section are such effects of lead in the developing versus the
adult animal, given the greater sensitivity of the young organism to lead.
12.2.1.3 In Vivo Studies. Uncoupled energy metabolism, inhibited cellular respiration using
succinate and NAD-linked substrates, and altered kinetics of intracellular calcium have all
been documented for animals exposed to lead jn vivo, as reviewed by Bull (1980).
Adult rat kidney mitochondria, following chronic oral feeding of lead in the diet (1 per-
cent lead acetate, 10 or more weeks) showed a marked sensitivity of the pyruvate-NAD reductase
system (Goyer, 1971), as indicated by impairment of pyruvate-dependent respiration indexed by
ADP/0 ratio and respiratory control rates (RCRs). Succinate-mediated respiration in these
animals, however, was not different from controls. In contrast, Fowler et al. (1980) found in
rats exposed HI utero (dams fed 50 or 250 ppm lead) and for 9 months postnatal ly (50 or 250
ppm lead in their diet) renal tubule mitochondria that exhibited decreased state 3 respiration
and RCRs for both succinate and pyruvate/malate substrates. This may have been due to longer
exposure to lead or a differential effect of lead exposure during early development.
APB12/A 12-4 9/20/83
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PRELIMINARY DRAFT
Intraperitoneal administration of lead to adult rats at doses as low as 12 mg/kg over 14
days was associated with inhibition of potassium-stimulated respiration in cerebral cortex
slices with impairment of NAD(P)H oxidation using glucose but not pyruvate as substrate (Bull
et al., 1975). This effect occurred at a corresponding blood lead of 72 ug/dl and a brain
lead content of 0.4 ug/g, values below those associated with overt neurotoxicity. Bull
(1977), in a later study, demonstrated that the respiratory response of cerebral cortical tis-
sue from lead-dosed rats receiving a total of 60 mg Pb/kg (10 mg/kg x 6 dosings) over 14 days
was associated with a marked decrease in turnover of intracellular calcium in a cellular com-
partment that appears to be the mitochondrion. This is consistent with the observation of
Bouldin et al. (1975) that lead treatment leads to increased retention of calcium in guinea
pig brain.
Numerous studies have evaluated relative effects of lead on mitochonodria of developing
vs. adult animals, particularly in the nervous system. Holtzman and Shen Hsu (1976) exposed
rat pups at 14 days of age to lead via milk of mothers fed lead in the diet (4 percent lead
carbonate) with exposure lasting for 14 days. A 40 percent increase in state 4 respiratory
rate of cerebellar mitochonodria was seen within one day of treatment and was lost at the end
of the exposure period. However, at this later time (28 days of age), a substantial inhibi-
tion of state 3 respiration was observed. This early effect of lead on uncoupling oxidative
phosphorylation is consistent with the results of Bull et al. (1979) and McCauley et al.
(1979). In these investigations, female rats received lead in drinking water (200 ppm) from
14 days before breeding through weaning of the pups. At 15 days of age, cerebral cortical
slices showed alteration of potassium-stimulated respiratory response and glucose uptake.
Holtzman et al. (1980a) compared mitochondrial respiration in cerebellum and cerebrum in
rat pups exposed to lead beginning at 14 days of age (via milk of mothers fed 4 percent lead
carbonate) and in adult rats maintained on the same diet. Cerebellar mitochondria showed a
very early loss (by 2 days of exposure) of respiratory control in the pups with inhibition of
phosphorylation-coupled respiration for NAD-linked substrates but not for succinate. Such
changes were less pronounced in mitochondria of the cerebrum and were not seen for either
brain region in the adult rat. This regional and age dependency of mitochondrial impairment
parallels features of lead encephalopathy.
In a second study addressing this issue, Holtzman et al. (1981) measured the cytochrome
contents of cerebral and cerebellar mitochondria from rat pups exposed either from birth or at
14 days of age via the same dosing protocol noted above. These were compared to adult animals
exposed in like fashion. Pups exposed to lead from birth showed statistically significant
reductions of cytochrome b, cytochromes c + GI, and cytochromes a + a3 in cerebellum by 4
weeks of exposure. Changes in cerebral cytochromes, in contrast, were marginal. When lead
exposure began at 14 days of age, little effect was observed, and adult rats showed little
APB12/A 12-5 9/20/83
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PRELIMINARY DRAFT
change. This study indicates that the most vulnerable period for lead effects on developing
brain oxidative metabolism is the same period where a major burst in such activity begins.
Related to effects of lead on energy metabolism in the developing animal mitochondrion is
the effect on brain development. In the study of Bull et al. (1979) noted earlier, cerebral
cytochrome c + GI levels between 10 and 15 days of age decreased in a dose-dependent fashion
at all maternal dosing levels (5-100 mg Pb/liter drinking water) and corresponding blood lead
values for the rat pups (11.7-35.7 ug/dl). Delays in synaptic development in these pups also
occurred, as indexed by synaptic counts taken in the parietal cortex. As the authors con-
cluded, uncoupling of energy metabolism appears to be causally related to delays in cerebral
cortical development.
Consistent with the effects of lead on mitochondria! structure and function are ui vivo
data demonstrating the selective accumulation of lead in mitochondria. Studies in rats using
radioisotopic tracers *luPb (Castallino and Aloj, 1969) and *0!«Pb (Barltrop et al., 1971)
demonstrate that mitochondria will accumulate lead in significant relative amounts, the nature
of the accumulation seeming to vary with the dosing protocol. Sabbioni and Marafante (1976)
as well as Murakami and Hurosawa (1973) also found that lead is selectively lodged in mito-
chondria. Of interest in regard to the effects of lead on brain mitochondria are the data of
Moore et al. (1975a) showing uptake of lead by guinea pig cerebral mitochondria, and the
results of Krigman et al. (1974c) demonstrating that mitochondria in brain from 6-month-old
rats exposed chronically to lead since birth showed the highest uptake of lead (34 percent),
followed by the nuclear fraction (31 percent). While the possibility of translocation of lead
during subcellular fractionation can be raised, the distribution pattern seen in the reports
of Barltrop et al. (1971) and Castallino and Aloj (1969) over multiple time points make this
unlikely. Also, findings of i_n vivo uptake of lead in brain mitochondria are supported by ui
vitro data discussed below.
12.2.1.4 In Vitro Studies. In vitro studies of both the response of mitochondrial function
to lead and its accumulation by the organelle have been reported, using both isolated mito-
chondria and tissues. Bull (1980) reviewed such data published up to 1979.
Significant reductions in mitochondrial respiration, using both NAD-linked and succinate
substrates have been reported for isolated heart and brain mitochondria. The lowest levels of
lead associated with such effects appear to be 5 uM or, in some cases, less. Available evi-
dence suggests that the sensitive site for lead in isolated mitochondria is before cytochrome
b in the oxidative chain and involves either tricarboxylic acid enzymes or non-heme protein/
ubiquinone steps. If substrate specificity is compared, e.g., succinate vs. glutamate/malate
oxidation, there appear to be organ-specific differences. As Bull (1980) noted, tissue-
specific effects of lead on cellular energetics may be one bases for differences in toxicity
across organs. Also, several enzymes involved in intermediary metabolism in isolated mito-
APB12/A 12-6 9/20/83
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PRELIMINARY DRAFT
chondria have been observed to undergo significant inhibition in activity in the presence of
lead, and these have been tabulated by Bull (1980).
One focus of studies dealing with lead effects on isolated mitochondria has been ion
transport—particularly that of calcium. Scott et al. (1971) have shown that lead movement
into rat heart mitochondria involves active transport, with characteristics similar to those
of calcium, thereby establishing a competitive relationship. Similarly, lead uptake into
brain mitochondria is also energy dependent (Holtzman et al., 1977; Goldstein et al., 1977).
The recent elegant studies of Pounds and coworkers (Pounds et al., 1982a,b), using labeled
calcium or lead and desaturation kinetic studies of these labels in isolated rat hepatocytes,
have elucidated the intracellular relationship of lead to calcium in terms of cellular com-
partmentalization. In the presence of graded amounts of lead (10, 50, or 100 uM), the kinetic
analysis of desaturation curves of calcium-45 label showed a lead dose-dependent increase in
the size of all three calcium compartments within the hepatocyte, particularly that deep com-
partment associated with the mitochondrion (Pounds et al., 1982a). Such changes were seen to
be relatively independent of serum calcium or endogenous regulators of systemic calcium meta-
bolism. Similarly, the use of lead-210 label and analogous kinetic analysis (Pounds et al.,
1982b) showed the same three compartments of intracellular distribution as noted for calcium,
including the deep component (which has the mitochondrion). Hence, there is striking overlap
in the cellular metabolism of calcium and lead. These studies not only further confirm easy
entry of lead into cells and cellular compartments, but also provide a basis for perturbation
by lead of intracellular ion transport, particularly in neural cell mitochondria, where there
is a high capability for calcium transport. Such capability is approximately 20-fold higher
than in heart mitochondria (Nicholls, 1978).
Given the above observations, it is not surprising that a number of investigators have
noted the in vitro uptake of lead into isolated mitochondria. Walton (1973) noted that lead
is accumulated within isolated rat liver mitochondria over the range of 0.2-100 uM lead; and
Walton and Buckley (1977) extended this observation to mitochondria in rat kidney cells in
culture. Electron nricroprobe analyses of isolated rat synaptosomes (Silbergeld et al., 1977)
and capillaries (Silbergeld et al., 1980b) incubated with lead ion have established that sig-
nificant accumulation of lead, along with calcium, occurs in the mitochondrion. These obser-
vations are consistent with the kinetic studies of Pounds et al. (1982a,b), and the in vitro
data for isolated capillaries are in accord with the observations of Toews et al. (1978), who
found significant lead accumulation in brain capillaries of the suckling rat.
12.2.2 Effects of Lead on the Nucleus
With lead exposure, a cellular reaction typical of many species (including humans) is the
formation of intranuclear lead-containing inclusion bodies, early data for which have been sum-
APB12/A 12-7 9/20/83
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PRELIMINARY DRAFT
man'zed by Goyer and Moore (1974). In brief, these lead-bearing inclusion bodies: (1) have
been verified as to lead content by X-ray microanalysis (Carroll et al., 1970); (2) consist
of a rather dense core encapsulated by a fibrillary envelope; (3) are a complex of lead and
the acid fractions of nuclear protein; (4) can be disaggregated jn v itro by EDTA; (5) can
appear very rapidly after lead exposure (Choie et al., 1975); (6) consist of a protein moiety
in the complex which is synthesized de novo; and (7) have been postulated to serve a protec-
tive role in the cell, given the relative amounts of lead accumulated and presumably rendered
toxicologically inert.
Based on renal biopsy studies, Cramer et al. (1974) concluded that such inclusion body
formation in renal tubule cells in lead workers is an early response to lead entering the kid-
ney, in view of decreased presence as a function of increased period of employment. Schumann
et al. (1980), however, observed a continued exfoliation of inclusion-bearing tubule cells
into urine of workers having a variable employment history.
Any protective role played by the lead inclusion body appears to be an imperfect one, to
the extent that both subcellular organelle injury and lead uptake occur simultaneously with
such inclusion formation, often in association with severe toxicity at the organ system level.
For example, Osheroff et al. (1982), observed lead inclusion bodies in the anterior horn cells
of the cervical spinal cord and neurons of the substantia nigra (as well as in renal tubule
cells) in the adult rhesus monkey, along with damage to the vascular walls and glisl processes
and ependymal cell degeneration. At the light- and electron-microscope level, there were no
signs of neuronal damage or altered morphology except for the inclusions. As noted by the
authors, these inclusions in the large neurons of the substantia nigra show that the neuron
will take up and accumulate lead. In the study of Fowler et al. (1980), renal tubule inclu-
sions were observed simultaneously with evidence of structural and functional damage to the
mitochondrion, all at relatively low levels of lead. Hence, it appears that a limited pro-
tective role for these inclusions may extend across a range of lead exposure.
Chromosomal effects and other indices of genotoxicity in humans and animals are discussed
in Section 12.7 of this chapter.
12.2.3 Effects of Lead on Membranes
In theory, the cell membrane is the first organelle to encounter lead, and it is not sur-
prising that cellular effects can be ascribed to interactions at cellular and intracellular
membranes, mainly appearing to be associated with ion transport processes across membranes.
In Section 12.3 a more detailed discussion is accorded the effects of lead on the membrane as
they relate to the erythrocyte in terms of increased cell fragility and increased osmotic
resistance. These effects can be rationalized, in large part, by the documented inhibition by
lead of erythrocyte membrane (Na*. K+)-ATPase.
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Lead also appears to interfere with the normal processes of calcium transport across mem-
branes of various tissue types. Silbergeld and Adler (1978) have described lead-induced
retardation of the release of the neurotransmitter, acetylcholine, in peripheral cholinergic
synaptosomes, due to a blockade of calcium binding to the synaptosomal membrane reducing
calcium-dependent choline uptake and subsequent release of acetylcholine from the nerve ter-
minal. Calcium efflux from neurons is mediated by the membrane (Na , K )-ATPase via an ex-
change process with sodium. Inhibition of the enzyme by lead, as also occurs with the
erythroctye (see above), increases the concentration of calcium within nerve endings (Goddard
and Robinson, 1976). As seen from the data of Pounds et al. (1982a), lead can also elicit
retention of calcium in neural cells by easy entry into the cell and by directly affecting the
deep calcium compartment within the cell, of which the mitochondrion is a major component.
12.2.4 Other Organellar Effects of Lead
Studies of morphological alterations of renal tubule cells in the rat (Chang et al.,
1981) and rabbit (Spit et al., 1981) with varying lead treatments have demonstrated lead-
induced lysosomal changes. In the rabbit, with relatively modest levels of lead exposure
(0.25 or 0.5 mg Pb/kg, 3 times weekly over 14 weeks) and corresponding blood lead values of 50
and 60 ug/dl, there was a dose-dependent increase in the amount of lysosomes in proximal con-
voluted uubule cells, as well as increased numbers of lysosomal inclusions. In the rat, expo-
sure to 10 mg Pb/kg i.v. (daily over 4 weeks) resulted in the accumulation of lysosomes, some
gigantic, in the pars recta segment of renal tubules. These animal data are consistent with
observations made in lead workers (Cramer et al., 1974; Wedeen et al., 1975) and appear to
represent a disturbance in normal lysosomal function, with the accumulation of lysosomes being
due to enhanced degradation of proteins arising from effects of lead elsewhere within the
cell.
12.2.5 Summary of Subcellular Effects of Lead
The biological basis of lead toxicity is closely linked to the ability of lead to bind to
ligating groups in biomolecular substances crucial to normal physiological functions. This
binding interferes with physiological processes by such mechanisms as: competition with
native essential metals for binding sites; inhibition of enzyme activity; and inhibition or
other changes in essential ion transport.
The main target organelle for lead toxicity in a variety of cell and tissue types clearly
is the mitochondrion, followed probably by cellular and intracellular membranes. Mitochon-
drial effects take the form of structural changes and marked disturbances in mitochondrial
function within the cell, especially energy metabolism and ion transport. These effects are
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associated, in turn, with demonstrable accumulation of lead in mitochondria, both iji vivo and
J_n vitro. Structural changes include mitochondrial swelling in many cell types, as well as
distortion and loss of cristae, which occur at relatively moderate levels of lead exposure.
Similar changes have been documented irt lead workers across a wide range of exposure levels.
Uncoupled energy metabolism, inhibited cellular respiration using both succinate and
nicotinamide adenine dinucleotide (NAD)-linked substrates, and altered kinetics of intracellu-
lar calcium have been demonstrated jn vivo using mitochondria of brain and non-neural tissue.
In some cases, relatively moderate lead exposure levels have been associated with such
changes, and several studies have documented the relatively greater sensitivity of this organ-
elle in young versus adult animals in terms of mitochondrial respiration. The cerebellum
appears to be particularly sensitive, providing a connection between mitochondrial impairment
and lead encephalopathy. Impairment by lead of mitochondrial function in the developing brain
has also been associated with delayed brain development, as indexed by content of various
cytochromes. In the rat pup, ongoing lead exposure from birth is required for this effect to
be expressed, indicating that such exposure must occur before, and is inhibitory to, the burst
of oxidative metabolism activity that normally occurs in the young rat during 10 to 21 days
postnatally.
In vivo lead exposure of adult rats has also been observed to markedly inhibit cerebral
cortex intracellular calcium turnover (in a cellular compartment that appears to be the mito-
chondrion) at a brain lead level of 0.4 ppm. These results are consistent with a separate
study showing increased retention of calcium in the brain of lead-dosed guinea pigs. A number
of reports have described the jm vivo accumulation of lead in mitochondria of kidney, liver,
spleen, and brain tissue, with one study showing that such uptake was slightly more than
occurred in the nucleus. These data are not only consistent with the various deleterious ef-
fects of lead on mitochondria but are also supported by other, vn vitro findings.
Significant decreases in mitochondrial respiration jn vitro using both NAO-1inked and
succinate substrates have been observed for brain and non-neural tissue mitochondria in the
presence of lead at micromolar levels. There appears to be substrate specificity in the inhi-
bition of respiration across different tissues, which may be a factor in differential organ
toxicity. Also, a number of enzymes involved in intermediary metabolism in isolated mitochon-
dria have been observed to undergo significant inhibition of activity with lead.
A major focus of research on lead effects on isolated mitochondria has concerned ion
(especially calcuim) transport. Lead movement into brain and other tissue mitochondria, as
does calcium movement, involves active transport. Recent sophisticated kinetic analyses of
desaturation curves for radiolabeled lead or calcium indicate that there is striking overlap
in the cellular metabolism of calcium and lead. These studies not only establish a basis for
easy entry of lead into cells and cell compartments, but also provide a basis for impairment
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by lead of intracellular ion transport, particularly in neural cell mitochondria, where the
capacity for calcium transport is 20-fold higher than even in heart mitochondria.
Lead is also selectively taken up in isolated mitochondria jji vitro, including the mito-
chondria of synaptosoines and brain capillaries. Given the diverse and extensive evidence of
lead's impairment of mitochondria! structure and function as viewed from a subcellular level,
it is not surprising that these derangements are logically held to be the basis of dysfunction
of heme biosynthesis, erythropoiesis, and the central nervous system. Several key enzymes in
the heme biosynthetic pathway are intramitochondrial, particularly ferrochelatase. Hence, it
is to be expected that entry of lead into mitochondria will impair overall heme biosynthesis,
and in fact this appears to be the case in the developing cerebellum. Furthermore, the levels
of lead exposure associated with entry of lead into mitochondria and expression of mitochon-
drial injury can be relatively moderate.
Lead exposure provokes a typical cellular reaction in human and other species that has
been morphologically characterized as a lead-containing nuclear inclusion body. Although it
has been postulated that such inclusions constitute a cellular protection mechanism, such a
mechanism is an imperfect one. Other organelles, e.g., the mitochondrion, also take up lead
and sustain injury in the presence of nuclear inclusion bodies. Chromosomal effects and other
indices of genotoxicity in humans and animals are considered later, in Section 12.7.
In theory, the cell membrane is the first organelle to encounter lead and it is not sur-
prising that cellular effects of lead can be ascribed to interactions at cellular and intra-
cellular membranes in the form of distrubed ion transport. The inhibition of membrane
(Na ,K )-ATPase of erythrocytes as a factor in lead-impaired erythropoiesis is noted else-
where. Lead also appears to interfere with the normal processes of calcium transport across
membranes of different tissues. In peripheral cholinergic synaptosomes, lead is associated
with retarded release of acetylcholine owing to a blockade of calcium binding to the membrane,
while calcium accumulation within nerve endings can be ascribed to inhibition of membrane
(Na+,K+)-ATPase.
Lysosomes accumulate in renal proximal convoluted tubule cells of rats and rabbits given
lead over a wide range of dosing. This also appears to occur in the kidneys of lead workers
and seems to represent a disturbance in normal lysosomal function, with the accumulation of
lysosomes being due to enhanced degradation of proteins because of the effects of lead else-
where within the cell.
In so far as effects of lead on the activity of various enzymes are concerned, many of
the available studies concern in vitro behavior of relatively pure enzymes with marginal rele-
vance to various effects in vivo. On the other hand, certain enzymes are basic to the effects
of lead at the organ or organ system level, and discussion is best reserved for such effects
in ensuing sections of the document dealing with these systems.
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12.3 EFFECTS OF LEAD ON HEME BIOSYNTHESIS AND ERYTHROPOIESIS/ERYTHROCYTE PHYSIOLOGY IN HUMANS
AND ANIMALS
Lead has well-recognized effects not only on heme biosynthesis, a crucial process common
to many organ systems, but also on erythropoiesis and erythrocyte physiology. This section is
therefore divided for purposes of discussion into: (1) effects of lead on heme biosynthesis
and (2) effects of lead on erythropoiesis and erythrocyte physiology. Discussion of the
latter is further subdivided into effects of lead on hemoglobin production, cell morphology
and survival, and erythropoietic nucleotide metabolism. The interrelationship of effects of
lead on heme biosynthesis and neurotoxic effects of lead are discussed in a final subsection.
Attention is accorded to discussion of effects of both inorganic lead and alkyl lead compounds
used as gasoline additives.
12.3.1 Effects of Lead on Heme Biosynthesis
The effects of lead on heme biosynthesis are very well known because of both their prom-
inence and the large number of studies of these effects in humans and experimental animals.
In addition to being a constituent of hemoglobin, heme is a prosthetic group of a number of
tissue hemoproteins having diverse functions, such as myoglobin, the P-450 component of the
mixed function oxidase system, and the cytochromes of cellular energetics. Hence, any effects
of lead on heme biosynthesis will, perforce, pose the potential for multi-organ toxicity.
At present, much of the available information concerning the effects of lead on heme bio-
synthesis have been obtained by measurements in blood, due in large part to the relative ease
of assessing such effects via measurements in blood and in part to the fact that blood is the
vehicle for movement of metabolites from other organ systems. On the other hand, a number of
reports have been concerned with lead effects on heme biosynthesis in tissues such as kidney,
liver, and brain. In the discussion below, various steps in the heme biosynthetic pathway
affected by lead are discussed separately, with information describing erythropoietic effects
usually appearing first, followed by studies involving other tissues.
The process of heme biosynthesis results in formation of the porphyrin, protoporphyrin
IX, starting with glycine and succinyl-coenzyme A. It culminates with the insertion of iron
at the center of the porphyrin ring. As may be noted in Figure 12-1, lead interferes with
heme biosynthesis by disturbing the activity of three major enzymes: (1) it indirectly stim-
ulates, by feedback derepression, the mitochondrial enzyme delta-aminolevulinic acid synthe-
tase (ALA-S), which mediates the condensation of glycine and succinyl-coenzyme A to form
delta-aminolevulinic acid (6-ALA); (2) it directly inhibits the cytosolic enzyme delta-amino-
levulinic acid dehydrase (ALA-D), which catalyzes the.cyclocondensation of two units of ALA to
porphobilinogen; (3) it disturbs the mitochondrial enzyme ferrochelatase, found in liver, bone
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MITOCHONDRAIL MEMBRANE
MITOCHONDRION
HEME
GLYCINE FERRO f ^
+ CHELATASE X 7
SUCCINYL-CoA
\
^^^* Pb
^^
^ ?
Fe + PROTOPORPHYRIN < ||
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PRELIMINARY DRAFT
J_n vitro and ni vivo experimental data have provided mixed results in terms of the direc-
tion of the effect of lead on ALA-S activity. Silbergeld et al. (1982) observed that ALA-S
activity was increased in kidney with acute lead exposure in rats, while chronic treatment was
associated with increased activity of the enzyme in spleen. In liver, however, ALA-S activity
was reduced under both acute and chronic dosing. Fowler et al. (1980) reported that renal
ALA-S activity was significantly reduced in rats continuously exposed to lead jm utero,
through development, and up to 9 months of age. Meredith and Moore (1979) noted a steady in-
crease in hepatic ALA-S activity when rats were given lead parenterally over an extended
period of time. Maxwell and Meyer (1976) and Goldberg et al. (1978) also noted increased
ALA-S activity in rats given lead parenterally. It appears that the type and time-frame of
dosing influences the observed effect of lead on the enzyme activity. Using a rat liver cell
line (RLC-GAI) in culture, Kusell et al. (1978) demonstrated that lead could produce a time-
dependent increase in ALA-S activity. Stimulation of activity was observed at lead levels as
low as 5 x 10" M, with maximum stimulation at 10" M. The authors report that the activity
increase was associated with biosynthesis of more enzyme, rather than stimulation of the pre-
existing enzyme. Lead-stimulated ALA-S formation was also not limited to liver cells; rat
gliomas and mouse neuroblastomas showed similar results.
12.3.1.2 Effects of Lead on 6-Aminolevulinic Acid Dehydrase and ALA Accumulation/Excretion.
Delta-aminolevulinic acid dehydrase (5-aminolevulinate hydrolase; porphobilinogen synthetase;
E.G. 4.2.1.24; ALA-0) is a sulfhydryl, zinc-requiring allosteric enzyme in the heme biosynthe-
tic pathway which catalyzes the conversion of two units of ALA to porphobilinogen. The
enzyme's activity is very sensitive to inhibition by lead, the inhibition being reversed by
reactivation of the sulfhydryl group with agents such as dithiothreitol (Granick et al.,
1973), zinc (Finelli et al., 1975), or zinc plus glutathione (Mitchell et al., 1977).
The activity of ALA-D appears to be inhibited at virtually all blood lead levels studied
so far, and any threshold for this effect remains to be identified (see discussion below).
Dresner et al. (1982) found that ALA-D activity in rat bone marrow suspensions was signifi-
cantly inhibited to 35 percent of control levels in the presence of 5 x 10 M (0.5 pM) lead.
This potency, on a comparative molar basis, was unmatched by any other metals tested.
Recently, Fujita et al. (1981) showed evidence of an increase in the amount of ALA-D in ery-
throcytes in lead-exposed rats, ascribed to an increased rate of ALA-D synthesis in bone
marrow cells. Hence, the commonly observed net inhibition of activity occurs even in the face
of an increase in ALA-D synthesis.
Hernberg and Nikkanen (1970) found that enzyme activity was correlated inversely with
(logarithmic) blood lead values in a group of urban, non-exposed subjects. Enzyme activity
inhibition was 50 percent at a blood lead level of 16 pg/dl. Other reports have confirmed
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PRELIMINARY DRAFT
these observations across age groups and exposure categories (Alessio et al., 1976b; Roels et
al., 1975b; Nieberg et al., 1974; Wada et al., 1973). A ratio of activated to inhibited en-
zyme activity (versus a single activity measurement, which does not accommodate intersubject
genetic variability) measured against blood lead in children with values between 20 and 90
pg/dl was employed by Granick et al. (1973) to obtain an estimated threshold of 15 ug/dl for
an effect of lead. On the other hand, Hernberg and Nikkanen (1970) observed no threshold in
their subjects, all of whom were at or below 16 pg/dl. The lowest blood lead actually mea-
sured by Granick et al. (1973) was higher than the values measured by Hernberg and Nikkanen
(1970).
Kuhnert et al. (1977) reported that ALA-D activity measures in erythrocytes from both
pregnant women and cord blood of infants at delivery are correlated with the corresponding
blood lead values, using the activated/inhibited activity ratio method of Granick et al.
(1973). The correlation coefficient of activity with lead level was higher in fetal erythro-
cytes (r = -0.58, p <0.01) than in the mothers (r = -0.43, p <0.01). The mean inhibition
level was 28 percent in mothers vs. 12 percent in the newborn. A study by Lauwerys et al.
(1978) in 100 pairs of pregnant women and infant cord blood samples confirms this observation,
i.e., for fetal blood r = 0.67 (p <0.001) and for maternal blood r = -0.56 (p <0.001).
While several factors other than lead may affect the activity of erythrocyte ALA-D, much
of the available information suggests that most of these factors do not materially compromise
the interpretation of the relationship between enzyme activity and lead or the use of this
relationship for screening purposes. Border et al. (1976) questioned the reliability of ALA-D
activity measurement in subjects concurrently exposed to both lead and zinc, since zinc also
affects the activity of the enzyme. The data of Meredith and Moore (1980) refute this objec-
tion. In subjects without exposure, having serum zinc values of 80-120 uM, there was only a
minor activating effect with increasing zinc when contrasted to the correlation of activity
and blood lead in these same subjects. In workers exposed to both lead and zinc, serum zinc
values were greater than in subjects with just lead exposure, but the mean level of enzyme ac-
tivity was still much lower than in controls (p <0.001).
The preceding discussion indicates that neither differences within the normal range of
physiological zinc in humans nor combined excessive zinc and lead exposure in workers materi-
ally affects ALA-D activity. The obverse of this, lead exposure in the presence of zinc defi-
ciency, is probably the more significant issue, but one that has not been well studied. Since
ALA-D is a zinc-requiring enzyme, one would expect that optimal activity would be governed by
jji vivo zinc availability. Furthermore, zinc deficiency could potentially have a dual dele-
terious effect on ALA-D activity, first by reduced activity with reduced zinc availability and
second, by enhanced lead absorption in the presence of zinc deficiency (see Section 10.5), the
increased lead burden further inhibiting ALA-D activity.
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The recent study of Roth and Kirchgessner (1981) indicates that ALA-D activity is signi-
ficantly decreased in the presence of zinc deficiency. In zinc-deficient rats showing reduced
serum and urinary zinc levels, the level of erythrocyte ALA-D activity was only 50 percent
that of pair-fed controls, while urinary ALA was significantly elevated. Although these in-
vestigators did not measure blood lead in deficient and control animal groups, it would appear
that the level of inhibition is more than could be accounted for just on the basis of in-
creased lead absorption from diet. Given the available information documenting zinc defi-
ciency in children (Section 10.5) as well as the animal study of Roth and Kirchgessner (1981),
the relationship of lead, zinc deficiency, and ALA-D activity in young children merits fur-
ther, careful study.
Moore and Meredith (1979) noted the effects of carbon monoxide on the activity of ALA-D,
comparing moderate or heavy smokers with non-smokers. At the highest level of carboxyhemoglo-
bin measured in their smoker groups, the depression of ALA-D activity was 2.1 percent. In
these subjects, a significant inverse correlation of ALA-D activity and blood lead existed,
but there was no significant correlation of such activity and blood carboxyhemoglobin levels.
While blood ethanol has been reported to affect ALA-D activity (Moore et al., 1971;
Abdulla et al., 1976), its effect is significant only with intake corresponding to acute alco-
hol intoxication. Hence, relevance of this observation to screening is limited, particularly
in children. The effect is reversible, declining with clearing of alcohol from the blood
stream.
The inhibition of ALA-D activity in erythrocytes by lead apparently reflects a similar
effect in other tissues. Secchi et al. (1974) observed that there was a clear correlation in
26 lead workers between hepatic and erythrocyte ALA-D activity as well as the expected inverse
correlation between such activity and blood lead in the range 12-56 ug/dl. In suckling rats,
Millar et al. (1970) noted decreased enzyme activity in brain and liver as well as red cells
when lead was administered orally. In the study of Roels et al. (1977), tissue ALA-D changes
were not observed when dams were administered 1, 10, or 100 ppm lead in drinking water. How-
ever, the recent report of Silbergeld et al. (1982) described moderate inhibition of ALA-D
activity in brain and significant inhibition in kidney, liver, and spleen when adult rats were
acutely exposed to lead given intraperitoneally; chronic exposure was associated with reduced
activity in kidney, liver, and spleen. Gerber et al. (1978) found that neonatal mice exposed
to lead from birth through 17 days of age at a level of 1.0 mg/ral in water showed significant
decreases in brain ALA-D activity (p <0.01) at all time points studied. These results support
the data of Millar et al. (1970) for the suckling rat. In this study by Millar et al., rats
exposed from birth through adulthood only showed significant decreases of brain ALA-D activity
at 15 and 30 days, which also supports other data for the developing rodent. It would appear,
therefore, that brain ALA-D activity is more sensitive to lead in the developing animal than
in the adult.
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The study of Dieter and Finley (1979) sheds light on the relative sensitivity of ALA-D
activity in several regions of the brain and permits comparison of blood vs. brain ALA-D acti-
vity as a function of lead level. Mallard ducks given a single pellet of lead showed, by 4
weeks, 1 ppm lead in blood, 2.5 ppm lead in liver, and 0.5 ppm lead in brain. Cerebellar
ALA-D activity was reduced by 50 percent at a lead level below 0.5 ppm; erythrocyte enzyme
activity was lowered by 75 percent. Hepatic ALA-D activity was comparable to cerebellar acti-
vity or somewhat less, although the lead level in the liver was 5-fold higher. Cerebellar
ALA-D activity was significantly below that for cerebrum. In an avian species, then, at
blood lead levels where erythrocyte ALA-D activity is significantly depressed, activity of the
enzyme in cerebellum was even more affected relative to lead concentration. The Roels et al.
(1977) data may reflect a lower effective dose delivered to the rat pups in maternal milk as
well as the dose taken in by the dams themselves, since they similarly showed no tissue enzyme
activity changes.
The inhibition of ALA-D is reflected by increased levels of its substrate, ALA, in urine
(Haeger, 1957) as well as in whole blood or plasma (Meredith et al., 1978; MacGee et al.,
1977; Chisolm, 1968; Haeger-Aronsen, 1960). The detailed study of Meredith et al. (1978),
which involved both control subjects and lead workers, indicated that in elevated lead expo-
sure the increase in urinary ALA is preceded by a significant rise in circulating levels of
ALA. The overall relationship of plasma ALA to blood lead was exponential and showed a per-
ceptible continuation of an ALA-blood lead correlation into the control group to include the
lowest value, 18 ug/dl. The relationship of plasma ALA to urinary levels of the precursor was
found to be exponential, indicating that as plasma ALA increases, a greater proportion under-
goes excretion into urine. Inspection of the plot of urinary vs. plasma ALA in these subjects
shows that the correlation persists down to the plasma ALA concentration corresponding to the
lowest blood lead level, 18 ug/dl. Cramer et al. (1974) have demonstrated that ALA clearance
into urine parallels glomerular filtration rate across a range of lead exposures, suggesting
that increased urinary output with increasing circulating ALA is associated with decreased tu-
bular reabsorption (Moore et al., 1980). This study employed the method of Haeger-Aronsen
(1960), which does not account for the presence of ami no-acetone. If ami no-acetone were in-
terfering at low blood lead levels, however, one might expect an obliteration of the associa-
tion, since this metabolite is not affected by lead exposure and its concentration should be
randomly distributed in plasma and urine of the subjects.
Urinary ALA has been employed extensively as an indicator of excessive lead exposure,
particularly in occupational settings (e.g., Davis et al., 1968; Selander and Cramer, 1970;
Alessio et al., 1976a). The reliability of this test in initial screening of children for
lead exposure has been questioned by Specter et al. (1971) and Blanksma et al. (1969), who
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pointed out the failure of urinary ALA analysis to detect lead exposure when compared with
blood lead values. This is due to the fact that an individual subject will show a wide vari-
ation in urinary ALA with random sampling. Chisolm et al. (1976) showed that reliable levels
could only be obtained with 24-hour collections. In children with blood lead levels above 40
ug/dl the relationship of ALA in urine to blood lead becomes similar to that observed in lead
workers (see below).
A correlation exists between blood lead and the logarithm of urinary ALA in workers
(Meredith et al., 1978; Alessio et al., 1976a; Roels et al., 1975a; Wada et al., 1973;
Selander and Cramer, 1970) and in children (National Academy of Sciences, 1972). Selander and
Cramer (1970) reported that two different correlation curves were obtained, one for individ-
uals below 40 ug/dl blood lead, and a different one for values above this, although the degree
of correlation was less than with the entire group. A similar observation has been reported
by Lauwerys et al. (1974) from a study of 167 workers (10-75 ug/dl). Meredith et al. (1978)
found that the correlation curve for blood ALA vs. urinary ALA was linear below a blood lead
of 40 |jg/dl, as was the relationship of blood ALA to blood lead. Hence, there was also a
linear relationship between blood lead and urinary ALA below 40 ug/dl, i.e., a continuation of
the correlation below the commonly accepted threshold blood lead value of 40 ug/dl (see
below). Tsuchiya et al. (1978) have questioned the relevance of using single correlation
curves to describe the blood lead-urinary ALA relationship across a broad range of exposure,
because they found that this relationship in workers showing moderate, intermediate, and high
lead exposure could be described by three correlation curves of differing slope. This finding
is consistent with the observations of Selander and Cramer (1970) as well as the results of
Meredith et al. (1978) and Lauwerys et al. (1974). Chisolm et al. (1976) described an expo-
nential correlation between blood lead and urinary ALA in children 5 years old or younger,
with blood lead ranging from 25 to 75 pg/dl. In adolescents with blood lead below 40 ug/dl,
no clear correlation was observed.
It is apparent from the above reports (Tsuchiya et al., 1978; Meredith et al., 1978;
Selander and Cramer, 1970) that circulating ALA and urinary ALA are elevated and correlated at
blood lead values below 40 ug/dl in humans. This is consistent, as in the Meredith et al.
study, with the significant and steady increase in ALA-D inhibition concomitant with rising
blood levels of ALA, even at blood lead values considerably below 40 ug/dl. Increases of ALA
in tissues of experimental animals exposed to lead have also been documented. In the study of
Silbergeld et al. (1982), acute administration of lead to adult rats was associated with an
elevation in spleen and kidney ALA vs. that of controls, while in chronic exposure there was a
moderate increase in ALA in the brain and a large increase (9-15 fold) in kidney and spleen.
Liver levels with either form of exposure were not materially affected, although there was
inhibition of liver ALA-D, particularly in the acute dose group.
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12.3.1.3 Effects of Lead on Heme Formation from Protoporphyrin. The accumulation of proto-
porphyrin in the erythrocytes of individuals with lead intoxication has been recognized since
the 1930s (Van den Bergh and Grotepass, 1933), but it has only recently been possible to study
this effect through the development of sensitive and specific analytical techniques that per-
mit quantitative measurement. In particular, the development of laboratory microtechniques
and the hematofluorometer have allowed the determination of dose-effect relationships as well
as the use of such measurements to screen for lead exposure.
In humans under normal circumstances, about 95 percent of the protoporphyrin in cir-
culating erythrocytes is zinc protoporphyrin (ZPP) with the remaining 5 percent present as
"free" protoporphyrin (Chisolm and Brown, 1979). Accumulation of protoporphyrin IX in the
erythrocytes is the result of impaired iron (II) placement in the porphyrin moiety to form
heme, an intramitochondrial process. In lead exposure, the porphyrin acquires a zinc ion, in
lieu of the native iron, with the resulting ZPP tightly bound in the available heme pockets
for the life of the erythrocyte, about 120 days (Lamola et al., 1975a,b).
In lead poisoning, the accumulation of protoporphyrin differs from that seen in the con-
genital disorder, erythropoietic protoporphyria. In the latter case, there is a defect in
ferrochelatase function, leading to loose attachment of the porphyrin, accumulated without up-
take of zinc, on the surface of the hemoglobin. Loose attachment permits diffusion into
plasma and ultimately into the skin, where photosensitivity is induced. This behavior is ab-
sent in lead-associated porphyrin accumulation. The two forms of porphyrin, free and zinc-
containing, differ sufficiently in fluorescence spectra to permit a laboratory distinction.
With iron deficiency, there is also accumulation of protoporphyrin in the heme pocket as the
zinc complex, resembling in large measure the characteristics of lead intoxication.
The elevation of erythrocyte ZPP has been extensively documented as being exponentially
correlated with blood lead in children (Piomelli et al., 1973; Kammholz et al., 1972; Sassa et
al., 1973; Lamola et al., 1975a,b; Roels et al., 1976) and in adult workers (Valentine et al.,
1982; Lilis et al., 1978; Grandjean and Lintrup, 1978; Alessio et al., 1976b; Roels et al.,
1975a, 1979; Lamola et al., 1975a,b). Reigart and Graber (1976) and Levi et al. (1976) have
demonstrated that ZPP elevation can predict which children tend to increase their blood lead
levels, a circumstance which probably rests on the nature of chronic lead exposure in certain
groups of young children where a pulsatile blood lead curve is superimposed on some level of
ongoing intake of lead which continues to elevate the ZPP values.
Accumulation of ZPP only occurs in erythrocytes formed during lead's presence in erythro-
poietic tissue, resulting in a lag of several weeks before the fraction of new ZPP-rich cells
is large enough to influence total cell ZPP level. On the other hand, elevated ZPP in ery-
throcytes long after significant lead exposure has ceased appears to be a better indicator of
APB12/B 12-19 9/20/83
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PRELIMINARY DRAFT
resorption of stored lead in bone than other measurements. Alessio et al. (1976b) reported
that former lead workers, removed from exposure at the workplace for more than 12 months in
all cases, still showed the typical logarithmic correlation with blood or urinary lead. How-
ever, the best correlation was observed between ZPP and chelatable lead, that fraction of
total body burden considered toxicologically active (see Chapter 10). This post-exposure re-
lationship for adults clearly indicates that significant levels of hematologically toxic lead
continue to circulate long after exposure to lead has ceased.
In a report relevant to the problem of multi-indicator measurement to assess the degree
of lead exposure, Hesley and Wimbish (1981) studied changes in blood lead and ZPP in two
groups, newly exposed lead workers or those removed from significant exposure. In new
workers, blood lead achieved a plateau at 9-10 weeks, while ZPP continued to rise over the
entire study interval of 24 weeks. Among workers removed from exposure, both blood lead and
ZPP values remained elevated up to the end of this study period (33 weeks), but the decline in
ZPP concentration lagged behind blood lead in reaching a plateau. These investigators logi-
cally concluded that the difficulty in demonstrating reliable blood lead-ZPP relationships may
reflect differences in when the two measures reach plateau. Similarly, more reliance should
be placed on ZPP vs. blood lead levels before permitting re-entry into areas of elevated lead
exposure.
The threshold for the effect of lead on ZPP accumulation is affected by the relative
spread of blood lead values and the corresponding concentrations of ZPP. In many cases these
range from "normal" levels in non-exposed subjects up to values reflecting considerable expo-
sure. Furthermore, iron deficiency is also associated with ZPP elevation, particularly in
children 2-3 years or younger.
In adults, Roels et al. (1975b) found that a cutoff for the relationship of erythrocyte
protoporphyrin (EP) elevation to blood lead was 25-30 ug/dl, confirmed by the log-transformed
data of Joselow and Flores (1977), Grandjean and Lintrup (1978), Odone et al. (1979), and
Herber (1980).
In older children, 10-15 years of age, the data of Roels et al. (1976) indicate a thres-
hold for effect of 15.5 ug/dl. The population dose-response relationship between EP and blood
lead in these children Indicated that EP levels were significantly higher (>2 SDs) than the
reference mean in 50 percent of the children at a blood lead level of 25 ug/dl. In the age
range of children studied here, iron deficiency is uncommon and these investigators did not
note any significant hematocrit change in the exposure group. In fact, it was lower in the
control group, although these subjects had lower ZPP levels. In this study, then, iron defi-
ciency was unlikely to be a confounding factor in the primary relationship. Piomelli et al.
(1977) obtained a comparable threshold value (15.5 ug/dl) for lead's effect on ZPP elevation
APB12/B 12-20 9/20/83
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PRELIMINARY DRAFT
in children who were older than 4 years as well as those who were 2-4 years old. Were iron
deficiency a factor in the results for this large study population (1816 children), one would
expect a greater impact in the younger group, where the deficiency is more common.
Within the blood lead range considered "normal," i.e., below 30-40 ug/dl, assessment of
any ZPP-blood lead relationship is strongly influenced by the relative analytical proficiency
of the laboratory carrying out both measurements, particularly for blood lead at lower values.
The type of statistical treatment of the data is also a factor, as are some biological sources
of variability. With respect to subject variability, Grandjean (1979) has documented that ZPP
increases throughout adulthood while hemoglobin remains relatively constant. Hence, age
matching is a prerequisite. Similarly, the relative degree of ZPP response is sexually dicho-
tomous, being greater in females for a given blood lead level (see discussion below).
Suga et al. (1981) claimed no apparent correlation between blood lead levels below
40 pg/dl and blood ZPP content in an adult population of 395 male and female subjects. The
values for males and females were combined, based on no measured differences in ZPP response,
which is at odds with the studies of Stuik (1974), Roels et al. (1975b), Zielhuis et al.
(1978), Odone et al. (1978), and Toriumi and Kawai (1981). Also, EP was found to increase
with increasing age, despite the fact that the finding of no correlation between blood lead
and ZPP was based on a study population with all age groups combined.
Piomelli et al. (1982) investigated both the threshold for the effect of lead on ZPP ac-
cumulation and a dose-response relationship in 2004 children, 1852 of whom had blood lead
values below 30 ug/dl. In this study, blood lead and EP measurements were done in facilities
with a high proficiency for both blood lead and ZPP analyses. The study employed two statis-
tical approaches (segmental line techniques and probit analysis), both of which revealed an
average threshold blood lead level of 16.5 ug/dl in either the full group or the children with
blood values below 30 ug/dl. In this report, the effect of iron deficiency and other non-lead
factors was tested and removed using the Abbott formula (Abbott, 1925). With respect to popu-
lation dose-response relationships, it was found that blood lead values corresponding to sig-
nificant EP elevation at more than 1 or 2 standard deviations above a reference mean in 50
percent of the subjects were 28.6 or 35.6 ug/dl blood lead, respectively. At a blood lead
level of 30 ug/dl, furthermore, it was determined that 27 percent of children would have an EP
greater than 53 ug/dl.
Comparison of ZPP elevation among adult males and females and children at a given blood
lead level generally indicates that children and adult females are more sensitive to this ef-
fect of lead. Lamola et al. (1975a,b) demonstrated that the slope of ZPP vs. blood lead was
steeper in children than in adults. Roels et al. (1976) found that women and children were
equally more sensitive in response than adult males, a finding also observed in the population
APB12/B 12-21 9/20/83
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PRELIMINARY DRAFT
studied by Odone et al. (1979). Other comparisons between adults, either as groups studied at
random or in a voluntary lead exposure study, also document the sensitivity of females vs.
males to this effect of lead (Stuik, 1974; Roels et al., 1975b, 1976, 1979; Toriumi and Kawai,
1981). The heightened response of females to lead-associated EPP elevation was investigated
in rats (Roels et al., 1978a) and shown to relate to hormonal interactions with lead, con-
firming the human data of Roels et al. (1975b, 1976, 1979) that iron status is not a factor in
the phenomenon.
The effect of lead on iron incorporation into protoporphyrin in the heme biosynthetic
pathway is not restricted to the erythropoietic system. Evidence of a generalized effect of
lead on tissue heme synthesis at low levels of lead exposure comes from the recent studies of
Rosen and coworkers (Rosen et al., 1980, 1981; Mahaffey et al., 1982). Children with blood
lead levels in the range 12-120 M9/dl showed a strong negative correlation (r = -0.88) with
serum 1,25-dihydroxy vitamin D (1,25-(OH)*D). The slopes of the regression lines for subjects
having blood lead below 30 (jg/dl were not materially different from those over this level.
Furthermore, when lead-intoxicated children were subjected to chelation therapy, it was
observed that the depressed levels of serum 1,25-(OH);,D returned to normal, while values of
serum 25-hydroxy vitamin D (the precursor to 1,25-(OH)^D) remained the same. This indicates
that lead has an inhibitory effect on renal 1-hydroxylase, a cytochrome P-450 mediated mito-
chondrial enzyme system that converts 25-(OH)D to 1,25-(OH);;D. The low end of the blood lead
range associated with lowered 1,25-(OH)20 levels and accompanying 1-hydroxylase activity inhi-
bition corresponds to the lead level associated with the onset of EP accumulation in erythro-
poietic tissue (see above). Sensitivity of renal mitochondrial 1-hydroxylase activity to lead
is consistent with a large body of information showing the susceptibility of renal tubule cell
mitochondria to injury by lead and with the chronic lead exposure animal model of Fowler et
al. (1980), discussed in more detail below.
Formation of the heme-containing protein cytochrome P-450, which is an integral part of
the hepatic mixed function oxygenase system, has been documented as being affected by lead
exposure, particularly acute lead intoxication, in animals (Alvares et al., 1972; Scappa et
al., 1973; Chow and Cornish, 1978; Goldberg et al., 1978; Meredith and Moore, 1979) and humans
(Alvares et al., 1975; Meredith et al., 1977; Fischbein et al., 1977). Many of these studies
used altered drug detoxification rates as a functional measure of such effects. In the work
of Goldberg et al. (1978), increasing level of lead exposure in rats was correlated with both
steadily decreasing P-450 content of hepatic microsomes and decreased activity in the detoxi-
fying enzymes aniline hydroxylase and aminopyrine demethylase, while the data of Meredith and
Moore (1979) showed that continued dosing of rats with lead results in steadily decreased
microsomal P-450 content, decreased total heme content of microsomes, and increased ALA-S
activity.
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PRELIMINARY DRAFT
Of interest in this regard are data relating to neural tissue. Studies of organotypic
chick dorsal root ganglion in culture document that the nervous system has heme biosynthetic
capability (Whetsell et al., 1978) and that this cell system, in the presence of lead, elabo-
rates increased porphyrinic material (Sassa et al., 1979). Chronic administration of lead to
neonatal rats indicates that at low levels of exposure, with modest elevations of blood lead,
there is a retarded growth in the respiratory chain hemoprotein cytochrome C and disturbed
electron transport function in the developing rat cerebral cortex (Holtzman and Shen Hsu,
1976; Bull et al., 1979). These effects on the developing organism are accentuated by in-
creased whole body lead retention in both developing children and experimental animals, as
well as by higher retention of lead in brain of suckling rats compared with adults (see
Chapter 10).
Heme oxygenase activity is elevated in lead-intoxicated animals (Maines and Kappas, 1976;
Meredith and Moore, 1979) in which relatively high dosing is employed, indicating that normal
repression of this enzyme's activity is lost, further adding to heme reduction and loss of
regulatory control on the heme biosynthetic pathway.
The mechanism(s) underlying derangement of heme biosynthesis leading to ZPP accumulaton
in lead intoxication rests with either ferrochelatase inhibition, impaired mitochondrial
transport of iron, or a combination of both. Inferentially, the resemblance of lead-associ-
ated ZPP accumulation to a similar effect of iron deficiency is consistent with the unavaila-
bility of iron to ferrochelatase rather than direct enzyme inhibition, while the porphyrin
pattern seen in the congenital disorder, erythropoietic porphyria, where ferrochelatase itself
is affected, is different from that seen in lead intoxication. Similarly, lead-induced
effects on mitochondrial morphology and function are well known (Goyer and Rhyne, 1973;
Fowler, 1978), and such disturbances may include impaired iron transport (Borova et al.,
1973).
Several animal studies indicate that the effects of lead on heme formation may involve
both ferrochelatase inhibition and impaired mitochondrial transport of iron. Hart et al.
(1980) observed that acute lead exposure in rabbits is associated with a two-stage hemato-
poietic response, the earlier one resulting in significant formation of free vs. zinc proto-
porphyrin with considerable hemolysis, and a later phase (where ZPP is formed) which otherwise
resembles the common features of lead intoxication. Subacute exposure shows more of the typi-
cal porphyrin response reported with lead. These data may suggest that acute lead poisoning
is quite different from chronic exposure in terms of the nature of hematological derangement.
Fowler et al. (1980) maintained rats on a regimen of oral lead, starting with exposure of
their dams to lead in water and continuing through 9 months after birth at levels up to 250
ppm lead. The authors observed that the activity of kidney mitochondrial ALA-S and ferro-
APB12/B 12-23 9/20/83
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PRELIMINARY DRAFT
chelatase, but not that of the cytosolic enzyme ALA-D, was inhibited. Ferrochelatase activity
was inhibited at 25, 50, and 250 ppm exposure levels, being 63 percent of the control values
at the 250 ppm level. Depression of state-3 respiration control ratios was observed for both
succinate and pyruvate. Ultrastructurally, the mitochondria were swollen and lysosomes were
rich in iron. In this study, reduced ferrochelatase activity was observed while there was
concomitant mitochondrial injury and disturbance of function. The accumulation of iron may be
the result of phagocytized dead mitochondria or it may represent intracellular
accumulation of iron owing to the inability of mitochondria to use tha element. Ibrahim et
al. (1979) have shown that excess intracellular iron under conditions of iron overload is
stored in cytoplasmic lysosomes. The observation of disturbed mitochondrial respiration sug-
gests, as do the mitochondrial function data of Holtzman and Shen Hsu (1976) and Bull et al.
(1979) for the developing nervous system, that intramitochondrial transport of iron would be
impaired. Flatmark and Romslo (1975) demonstrated that iron transport in mitochondria is
energy linked and requires an intact respiration chain at the level of cytochrome C, whereby
iron (III) on the C-side of the mitochondrial inner membrane is reduced before transport to
the M-side and utilization in heme formation.
The above results are particularly interesting in terms of relative tissue response.
While the kidney was affected, there was no change in blood indices of hematological derange-
ment in terms of inhibited ALA-D activity or accumulation of ZPP. This suggests that there is
a difference in dose-effect functions among different tissues, particularly with lead exposure
during development of the organism. It appears that while indices of erythropoietic effects
of lead may be more accessible, they may not be the most sensitive as indicators of heme bio-
synthesis derangement in other organs.
12.3.1.4 Other Heme-Related Effects of Lead. An increased excretion of coproporphyrin in the
urine of lead workers and children with lead poisoning has long been recognized, and urinary
coproporphyrin measurement has been used as an indicator of lead poisoning. The mechanism of
such accumulation is not understood in terms of differentiating among direct enzyme inhibi-
tion, accumulation of substrate secondary to inhibition of heme formation, or impaired move-
ment of the coproporphyrin intranritochondnally. Excess coproporphyrin excretion differs as
an indicator of lead exposure from EP accumulation in that the former is a measure of ongoing
lead intoxication without the lag in response seen with EP (Piomelli and Graziano, 1980).
In lead intoxication, there is an accumulation of porphobilinogen with elevated excretion
in urine, owing to inhibition by lead of the enzyme uroporphyrinogen URO-I-synthetase (Piper
and Tephly, 1974). In vitro studies of Piper and Tephly (1974) using rat and human erythro-
cyte and liver preparations indicate that it is the erythrocyte URO-I-synthetase in both rats
and humans that is sensitive to the inhibitory effect of lead; activity of the hepatic enzyme
APB12/B 12-24 9/20/83
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PRELIMINARY DRAFT
is relatively insensitive. Significant inhibition of the enzyme's activity occurs at 5 uM
-4
lead with virtually total inhibition of activity in human red cell hemolysates at 10 M. Ac-
cording to Piper and van Lier (1977), the lower sensitivity of hepatic URO-I-synthetase activ-
ity to lead is due to a protective effect afforded by a pteridine derivative, pteroylpolyglu-
tamate. It appears that the protection does not occur through lead chelation, since hepatic
ALA-D activity was reduced in the presence of lead. The studies of Piper and Tephly (1974)
indicate that it is inhibition of URO-I-synthetase in erythroid tissue or erythrocytes that
accounts for the accumulation of its substrate, porphobilinogen.
12.3.2 Effects of Lead on Erythropoiesis and Erythrocyte Physiology
12.3.2.1 Effects of Lead on Hemoglobin Production. Anemia is a manifestation (sometimes an
early one) of chronic lead intoxication. Typically, the anemia is mildly hypochromic and usu-
ally normocytic. It is associated with reticulocytosis, owing to shortened cell survival, and
the irregular presence of basophilic stippling. Its genesis lies in both decreased hemoglobin
production and increased rate of erythrocyte destruction. Not only is anemia commonly seen in
children with lead poisoning, but it appears to be more severe and frequent among those with
severe lead intoxication (World Health Organization, 1977; National Academy of Sciences, 1972;
Lin-Fu, 1S73; Betts et al., 1973).
While the anemia associated with lead intoxication in children shows features of iron-
deficiency anemia, there are differences in cases of severe intoxication. These differences
include reticulocytosis, basophilic stippling, and a significantly lower total iron binding
capacity (TIBC). These latter features suggest that iron-deficiency anemia in young children
is exacerbated by lead. The reverse is also true.
In young children, iron deficiency occurs at a significant rate, based on national
(Mahaffey and Michaelson, 1980) and regional (Owen and Lippman, 1977) surveys and is known to
be correlated with increased lead absorption in humans (Yip et al., 1981; Chisolm, 1981;
Watson et al., 1980; Szold, 1974; Watson et al., 1958) and animals (Hamilton, 1978; Barton et
al., 1978; Mahaffey-Six and Goyer, 1972). Hence, prevalent iron deficiency can be seen to
potentiate the effects of lead in reduction of hemoglobin by both increasing lead absorption
and exacerbating the degree of anemia.
Also in young children, there is a negative correlation between hemoglobin level and
blood lead levels (Adebonojo, 1974; Rosen et al., 1974; Betts et al., 1973; Pueschel et al.,
1972). These studies generally involved children under 6 years of age where iron deficiency
may have been a factor. In adults, a negative correlation at blood lead values usually below
80 ug/dl has been observed (Grandjean, 1979; Lilis et al., 1978; Roels et al., 1975a; Wada,
1976), while several studies did not report any relationship below 80 ug/dl (Valentine et al.,
APB12/B 12-25 9/20/83
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PRELIMINARY DRAFT
1982; Reels et al., 1979; Ramirez-Cervantes et al., 1978). In adults, iron deficiency would
be expected to play less of a role in this relationship; Lilis et al. (1978) reported that the
significant correlation between lead in blood and hemoglobin level was observed in workers
where serum iron and TIBC were indistinguishable from controls.
The blood lead threshold for effects on hemogloblin has not been conclusively estab-
lished. In children, this value appears to be about 40 ug/dl (World Health Organization,
1977), which is somewhat lower than in adults (Adebonojo, 1974; Rosen et al., 1974; Betts et
al., 1973; Pueschel et al., 1972). Tola et al. (1973) observed no effect of lead on new work-
ers until the blood lead had risen to a value of 50 |jg/dl after about 100 days. The regres-
sion analysis data of Grandjean (1979), Lilis et al. (1978), and Wada (1976) show persistence
of the negative correlation of blood lead and hemoglobin below 50 ug/dl. Human population
dose-response data for the lead-hemoglobin relationship are limited. For lead workers, Baker
et al. (1979) have calculated the corresponding dose-response (<14.0 g Hb/dl): 5 percent at
blood lead of 40-59 ug/dl; 14 percent at blood lead of 60-79 ug/dl; and 36 percent at values
above 80 ug/dl. In 202 lead workers, Grandjean (1979) noted the following percentage of
workers having a hemoglobin level below 14.4 g/dl as a function of blood lead: <25 ug/dl, 17
percent; 25-60 ug/dl, 26 percent; >60 ug/dl, 45 percent.
The underlying mechanisms of lead-associated anemia appear to be a combination of reduced
hemoglobin production and shortened erythrocyte survival because of direct cell damage. Ef-
fects of lead on hemoglobin production, furthermore, rest with disturbances of both heme and
globin biosynthesis.
Biosynthesis of globin, the protein moiety of hemoglobin, also appears to be inhibited in
lead exposure (Dresner et al., 1982; Wada et al., 1972; White and Harvey, 1972; Kassenaar et
al., 1957). White and Harvey (1972) reported a decrease of globin synthesis in reticulocytes
ID vitro in the presence of lead at levels as low as 1.0 uM, corresponding to a blood lead
level of 20 ug/dl. These data are in accord with the observation of Dresner et al. (1982),
who noted a reduced globin synthesis (76 percent of controls) in rat bone marrow suspensions
exposed to 1.0 uM lead. White and Harvey (1972) also noted that there was a decreased synthe-
sis of alpha chains vs. beta chains.
Disturbance of globin biosynthesis is a consequence of lead's effects on heme formation
since cellular heme regulates protein synthesis in erythroid cells (Levere and Granick, 1967)
and regulates the translation of globin messenger RNA (Freedman and Rosman, 1976). The dis-
turbance in the translation of mRNA in erythroid tissue may also reflect the effect of lead on
pyrimidine metabolism.
12.3.2.2 Effects of Lead on Ervthrocyte Morphology and Survival. It is clear that there is a
hemolytic component to lead-induced anemia in humans owing to shortened erythrocyte survival,
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PRELIMINARY DRAFT
and the various aspects of this effect have been reviewed by Waldron (1966), Goldberg (1968),
Moore et al. (1980), Valentine and Paglia (1980), and Angle and Mclntire (1982).
The relevant studies of shortened cell life with lead intoxication include observations
of the behavior of red cells to mechanical and osmotic stress under HI vivo and i_n vitro con-
ditions. Waldron (1966) has discussed the frequent reports of increased mechanical fragility
of erythrocytes from lead-poisoned workers, beginning with the work of Aub et al. (1926). In-
creased osmotic resistance of erythrocytes from subjects with lead intoxication is a parallel
finding, both jri vivo (Aub and Reznikoff, 1924; Harris and Greenberg, 1954; Horiguchi et al.,
1974) and in vitro (Qazi et al., 1972; Waldron, 1964; Clarkson and Kench, 1956). Using an ap-
paratus called a coil planet centrifuge, Karai et al. (1981) studied erythrocytes of lead
workers and found significant increases in osmotic resistance; at the same time mean corpuscu-
lar volume and reticulocyte counts were not different from controls. Karai et al. suggest
that one mechanism of increased resistance involves impairment of hepatic lecithin-cholesterol
acyltransferase, leading to a build-up of cholesterol in the cell membrane. This resembles
the increased osmotic resistance seen in obstructive jaundice where increased membrane choles-
terol has been observed (Cooper et al., 1975). Karai et al. (1981) also reported an increased
cholesterol-phospholipid ratio in lead workers' erythrocytes.
Erythrokinetic data in lead workers and children with lead-associated anemia have been
reported. Shortening of erythrocyte survival has been demonstrated by Hernberg et al. (1967a)
using tritium-labeled difluorophosphonate. Berk et al. (1970) used detailed isotope studies
of a subject with severe lead intoxication to determine shorter erythrocyte life span, while
Lei ken and Eng (1963) observed shortened cell survival in three of seven children. These
studies, as well as the reports of Landaw et al. (1973), White and Harvey (1972), Albaharry
(1972), and Dagg et al. (1965), indicate that hemolysis is not the exclusive mechanism of ane-
mia and that diminished erythrocyte production also plays a role.
The molecular basis for increased cell destruction with lead exposure includes the inhi-
bition by lead of the activities of the enzymes (Na+, K+)-ATPase and pyrimidine-5'-nucleoti-
dase. Erythrocyte membrane (Na+, K+)-ATPase is a sulfhydryl enzyme and inhibition of its ac-
tivity by lead has been well documented (Raghavan et al., 1981; Secchi et al., 1968; Hasan et
al., 1967; Hernberg et al., 1967b). In the study of Raghavan et al. (1981), enzyme activity
was inversely correlated with membrane lead content (p <0.001) in lead workers with or without
symptoms of overt lead toxicity, while correlation with whole blood lead was poor. With en-
zyme inhibition, there is irreversible loss of potassium ion from the cell with undisturbed
input of sodium into the cell, resulting in a relative increase in sodium. Since the cells
"shrink," there is a net increase in sodium concentration, which likely results in increased
mechanical fragility and cell lysis (Moore et al., 1980).
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Both with lead exposure and in subjects with a genetic deficiency of the enzyme pyrimi-
dine-5'-nucleotidase, activity is reduced, leading to impaired phosphorolysis of the nucleo-
tides cytidine and uridine phosphate, which are then retained in the cell, causing interfer-
ence with the conservation of the purine nucleotides necessary for cellular energetics (Angle
and Mclntire, 1982; Valentine and Paglia, 1980). A more detailed discussion of lead's inter-
action with this enzyme is presented in Section 12.3.2.3.
In a series of studies dealing with the hemolytic relationship of lead and vitamin E
deficiency in animals, Levander et al. (1980) observed that lead exposure exacerbates the
experimental hemolytic anemia associated with vitamin E deficiency by enhancing mechanical
fragility, i.e., retarded cell deformability. These workers note that vitamin E deficiency is
seen with children having elevated blood lead levels, especially subjects having glucose-6-
phosphate dehydrogenase (G-6-PD) deficiency, indicating that the synergistic relationship seen
in animals may exist in humans.
Glutathione is a necessary factor in erythrocyte function and structure. In workers ex-
posed to lead, Roels et al. (1975a) found that there is a moderate but significant decrease in
red cell glutathione compared with controls. This appears to reflect lead-induced impairment
of glutathione synthesis.
12.3.2.3 Effects of Lead on Pyrimidine-S'-Nucleotidase Activity and Erythropoietic Pyrimidine
Metabolism. The presence in lead intoxication of basophilic stippling and an anemia of hemo-
lytic nature is similar to what is seen in subjects having a congenital deficiency of
pyrimidine-B'-nucleotidase (Py-5-N), an enzyme mediating the phosphorolysis of the pyrimidine
nucleotides, cytidine and uridine phosphates. With inhibition these nucleotides accumulate in
the red cell or reticulocyte, there is a retardation of ribonuclease-mediated ribosomal RNA
catabolism in maturing cells, and the resulting accumulation of aggregates of incompletely de-
graded ribosomal fragments accounts for the phenomenon of basophilic stippling.
In characterizing the enzyme Py-5-N, Paglia and Valentine (1975) .observed that its
activity was particularly sensitive to inhibition by certain metals, particularly lead,
prompting further investigation of the interplay between lead intoxication and disturbances of
erythropoietic pyrimidine metabolism. Paglia et al. (1975) observed that in subjects occupa-
tional^ exposed to lead but having no evidence of basophilic stippling or significant fre-
quency of anemia, the activity of Py-5-N was reduced to about 50 percent of control subjects
and was most impaired in one worker with anemia, about 11 percent of normal. There was a
general inverse correlation between enzyme activity and blood lead level. In this report,
normal erythrocytes incubated with varying levels of lead showed detectable inhibition at
levels as low as 0.1-1.0 uM, with consistent 50 percent inhibition at about 10 uM. Subse-
quently, these investigators (Valentine et al., 1976) observed that an individual with severe
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PRELIMINARY DRAFT
lead intoxication had an 85 percent decrease in Py-5-N activity, basophilic stippling, and
accumulation of pyrimidine nucleotides, mainly cytidine triphosphate. Since these parameters
approached values seen in the congenital deficiency of Py-5-N, the data suggest a common eti-
ology for the hemolytic anemia and stippling in both lead poisoning and the congenital dis-
order.
Several other reports of investigations of Py-5-N activity and pyrimidine nucleotide
levels in lead workers have been published (Paglia et al., 1977; Buc and Kaplan, 1978). In
nine workers having overt lead intoxication and blood lead values of 80-160 ug/dl, Py-5-N
activity was significantly inhibited while the pyrimidine nucleotides comprised 70-80 percent
of the total nucleotide pool, in contrast to trace levels in unexposed individuals (Paglia
et al., 1977). In the study of Buc and Kaplan (1978), lead workers with or without overt lead
intoxication all showed reduced activity of Py-5-N, which was inversely correlated with blood
lead when the activity was expressed as a ratio with G-6-PD activity to accommodate an
enhanced population of young cells due to hemolytic anemia. Enzyme inhibition was observed
even when other indicators of lead exposure were negative.
Angle and Mclntire (1978) observed that in 21 children 2-5 years old, with blood lead
levels of 7-80 ug/dl, there was a negative linear correlation between Py-5-N activity and
blood lead (r = -0.60, p <0.01). Basophilic stippling was only seen in the child with the
highest blood lead value and only two subjects had reticulocytosis. While adults tended to
show a threshold for inhibition of Py-5-N at a blood lead level of 44 ug/dl or higher,
there was no clear response threshold in these children. In a related investigation with 42
children 1-5 years old having blood lead levels of <10 to 72 ug/dl, Angle et al. (1982) noted
that there was: (1) an inverse correlation (r = -0.64, p <0.001) between the logarithm of
Py-5-N activity and blood lead; (2) a positive log-log correlation between cytidine phosphates
and blood lead in 15 of these children (r = 0.89, p <0.001); and (3) an inverse relationship
in 12 subjects between log of enzyme activity and cytidine phosphates (r = -0.796, p <0.001).
Study of the various relationships at low levels was made possible by the use of anion-
exchange high performance liquid chromatography. In these studies, there was no threshold of
effects of lead on either enzyme activity or cell nucleotide content even below 10 ug/dl.
Finally, there was a significant positive correlation of pyrimidine nucleotide accumulation
and the accumulation of ZPP.
In subjects undergoing therapeutic chelation with EOTA, Py-5-N activity increased, while
there was no effect on pyrimidine nucleotides (Swanson et al., 1982), indicating that the py-
rimidine accumulation is associated with the reticulocyte.
The metabolic significance of Py-5-N activity inhibition and nucleotide accumulation with
lead exposure is derived from its effects on red cell membrane stability and survival by al-
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teration of cellular energetics (Angle and Mclntire, 1982), leading to cell lysis. A further
consequence may be feedback inhibition of mRNA and protein synthesis, in that denatured mRNA
may alter globin mRNA or globin chain synthesis. It was noted earlier that disturbances in
heme production also affect the translation of globin mRNA (Freedman and Rosman, 1976).
Hence, these two lead-associated disturbances of erythroid tissue function potentiate the ef-
fects of each other.
12.3.3 Effects of Alkyl Lead on Heme Synthesis and Erythropoiesis
In the Section 10.7 discussion of alkyl lead metabolism, it was noted that transforma-
tions of tetraethyl and tetramethyl lead jji vivo result in generation not only of neurotoxic
trialkyl lead metabolites but also of products of further dealkylation, including inorganic
lead. One would therefore expect alkyl lead exposure to be associated with, in addition to
other effects, some of those effects classically related to inorganic lead exposure.
Chronic gasoline sniffing has been recognized as a problem habit among children in rural
or remote areas (Boeckx et al., 1977; Kaufman, 1973). When such practice involves leaded gas-
oline, the potential exists for lead intoxication. Boeckx et al. (1977) conducted surveys of
children in remote Canadian communities in regard to the prevalence of gasoline sniffing and
indications of chronic lead exposure. In one group of 43 children, all of whom sniffed gaso-
line, mean ALA-D activity was only 30 percent that of control subjects, with a significant
correlation between the decrease in enzyme activity and the frequency of sniffing. A second
survey of 50 children revealed similar results. Two children having acute lead intoxication
associated with gasoline sniffing showed markedly lowered hemoglobin, elevated urinary ALA,
and elevated urinary coproporphyrin. The authors estimated that more than half of disadvan-
taged children residing in rural or remote areas of Canada may have chronic lead exposure via
this habit, consistent with the estimate of Kaufman (1973) of 62 percent for children in rural
American Indian communities in the Southwest.
Robinson (1978) described two cases of pediatric lead poisoning due to habitual gasoline
sniffing, one of which showed basophilic stippling. Hansen and Sharp (1978) reported that a
young adult with acute lead poisioning due to chronic gasoline sniffing not only had basophi-
lic stippling, but a 6-fold increase in urinary ALA, elevated urinary coproporphyrin, and an
EP level about 4-fold above normal. In the reports of Boeckx et al. (1977) and Robinson
(1978), increased lead levels were measured in urine in the course of chelation therapy, indi-
cating that significant amounts of inorganic lead were present.
12.3.4 The Interrelationship of Lead Effects on Heme Synthesis and the Nervous System
Lead-associated disturbances in heme biosynthesis as a possible factor in the neurologi-
cal effects of lead have been studied because of (1) the recognized similarity between
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classic signs of lead neurotoxicity and many, but not all, of the neurological components of
the congenital disorder, acute intermittent porphyria, and (2) some unusual aspects of lead
neurotoxicity. Both acute attack porphyria and lead intoxication with neurological symptoms
are variably accompanied by abdominal pain, constipation, vomiting, paralysis or paresis,
demyelination, and psychiatric disturbances (Dagg et al. , 1965; Moore et a!., 1980; Silbergeld
and Lamon, 1980). According to Angle and Mclntire (1982), some of the unusual features of
lead neurotoxicity are consistent with deranged hematopoiesis: (1) a lag in production of
neurological symptoms; (2) the incongruity of early deficits in affective and cognitive func-
tion with the regional distribution of lead in the brain; and (3) a better correlation of neu-
robehavioral deficits with erythrocyte protoporphyrin than with blood lead. Item 3, it should
be noted, is not universally the case (Hammond et al., 1980; Spivey et al., 1979).
While the nature and pattern of the derangements in heme biosynthesis in acute attack
porphyria and lead intoxication differ in many respects, both involve excessive systemic ac-
cumulation and excretion of ALA, and this common feature has stimulated numerous studies of a
connection between hemato- and neurotoxicity. In vitro data (Whetsell et al., 1978) have
shown that the nervous system is capable of heme biosynthesis in the chick dorsal root gan-
glion. Sassa et al. (1979) found that the presence of lead in these preparations increases
production of porphyrinic material, i.e., there is disturbed heme biosynthesis with accumula-
tion of one or more porphyrins and, presumably, ALA. Millar et al. (1970) reported inhibited
brain ALA-D activity in suckling rats exposed to lead, while Silbergeld et al. (1982) observed
similar inhibition in brains of adult rats acutely exposed to lead. In the latter study,
chronic lead exposure was also associated with a moderate increase in brain ALA without inhi-
bition of ALA-D, suggesting an extra-neural source of the heme precursor. Moore and Meredith
(1976) administered ALA to rats and observed that exogenous ALA can penetrate the blood-brain
barrier. These reports suggest that ALA can either be generated iji situ in the nervous system
or can enter the nervous system from elsewhere.
Neurochemical investigations of ALA action in the nervous system have evaluated interac-
tions with the neurotransmitter gamma-aminobutyric acid (GABA). Interference with GABAergic
function by exposure to lead is compatible with such clinical and experimental signs of lead
neurotoxicity as excitability, hyperactivity, hyperreactivity, and, in severe lead intoxica-
tion, convulsions (Silbergeld and Laroon, 1980). Of particular interest is the similarity in
chemical structure between ALA and GABA, which differ only in that ALA has a carbonyl group on
the alpha carbons, and GABA has a carbonyl group on the beta carbon.
While chronic lead exposure appears to alter neural pathways involving GABA function
(Piepho et al., 1976; Silbergeld et al., 1979), this effect cannot be duplicated iji vitro
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using various levels of lead (Silbergeld et a!., 1980). This suggests that lead does not
impart the effect by direct interaction or an intact multi-pathway system is required. In
vitro studies (Silbergeld et al., 1980a; Nicoll, 1976) demonstrate that ALA can displace GABA
from synaptosomal membranes associated with synaptic function of the neurotransmitter on the
GABA receptor, but that it is less potent than GABA by a factor of 10a-104, suggesting that
levels of ALA achieved even with severe intoxication may not be effectively competitive.
A more significant role for ALA in lead neurotoxicity may well be related to the observa-
tion that GABA release is subject to negative feedback control through presynaptic receptors
on GABAergic terminals (Snodgrass, 1978; Mitchell and Martin, 1978). Brennan and Cantrill
(1979) found that ALA inhibits K+-stimulated release of GABA from pre-loaded synaptosomes by
functioning as an agonist at the presynaptic receptors. The effect is evident at 21.0 uM ALA,
while the inhibiting effect is abolished by the GABA antagonists bicuculline and picrotoxin.
Of interest also is the demonstration (Silbergeld et al., 1980a) that synaptosomal release of
preloaded aH-GABA, both resting and K -stimulated, is also inhibited in animals chronically
treated with lead, paralleling the in vitro data of Brennan and Cantrill (1979) using ALA.
Silbergeld et al. (1982) described the comparative effects of lead and the agent succi-
nylacetone, given acutely or chronically to adult rats, in terms of disturbances in heme syn-
thesis and neurochemical indices. Succinylacetone, a metabolite that can be isolated from the
urine of patients with hereditary tyrosinemia (Lindblad et al., 1977) is a potent inhibitor of
heme synthesis, exerting its effect by ALA-D inhibition and derepression of ALA synthetase
(Tschudy et al., 1980, 1983). Both agents, ui vivo, showed significant inhibition of high
affinity Na -dependent uptake of 14C-GABA by cortex, caudate, and substantia nigra. However,
neither agent affected GABA uptake in vitro. Similarly, both chronic or acute lead treatment
and chronically administered succinylacetone reduced the seizure threshold to the GABA antago-
nist, picrotoxin. While these agents may involve entirely different mechanisms of toxicity to
the GABAergic pathway, the fact remains that two distinct potent inhibitors of the heme bio-
synthetic pathway and ALA-D, which do not impart a common neurochemical effect by direct
action on a neurotransmitter function, have a common neurochemical action iji vivo.
Human data relating the hemato- and neurotoxicity of lead to each other are limited.
Hammond et al. (1980) reported that the best correlates of the frequency of neurological symp-
toms in 28 lead workers were urinary and plasma ALA, which showed a higher correlation than
EP. These data support a connection between heme biosynthesis impairment and neurological
effects of ALA. Of interest here is the clinical report of Lamon et al. (1979) describing the
effect of hematin [Fe(III)-heme] given parenterally to a subject with lead intoxication. Over
the course of treatment (16 days), urinary coproporphyrin and ALA significantly dropped
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and such neurological symptoms as lower extremity numbness and aching diminished. Blood lead
levels were not altered during the treatment. Although remission of symptoms in this subject
may have been spontaneous, the outcome parallels that observed in hematin treatment of sub-
jects with acute porphyria in terms of similar reduction of heme indicators and relief of
symptoms (Lamon et al., 1979).
Taken collectively, all of the available data strongly suggest that ALA, formed in situ
or entering the brain, is neurotoxic to GABAergic function in particular. It inhibits
K+-stimulated GABA release by interaction with presynaptic receptors, where ALA appears to be
particularly potent at very low levels, based on vn vitro results. As described in the sec-
tion on heme biosynthesis, lead can affect both cellular respiration and cytochrome C levels
in the nervous system of the developing rat, which may contribute to manisfestation of some
symptoms of lead neurotoxicity. Hence, more than the issue of ALA neurotoxicity should be
considered in assessing connections between lead-induced hemato- and neurotoxicity.
12.3.5 Summary and Overview
12.3.5.1 Lead Effects on Heme Biosynthesis. Lead effects on heme biosynthesis are well known
because of both their prominence and numerous studies of such effects in humans and experimen-
tal animals. The process of heme biosynthesis starts with glycine and succinyl-coenzyme A,
proceeds through formation of protoporphyrin IX, and culminates with the insertion of divalent
iron into the porphyrin ring, thus forming heme. In addition to being a constituent of hemo-
globin, heme is the prosthetic group of many tissue hemoproteins having variable functions,
such as myoglobin, the P-450 component of the mixed function oxygenase system, and the cyto-
chromes of cellular energetics. Hence, disturbance of heme biosynthesis by lead poses the
potential for multi-organ toxicity.
At present, steps in the heme synthesis pathway that have been best studied in regard to
lead effects involve three enzymes: (1) stimulation of mitochondrial delta-aminolevulinic
acid synthetase (ALA-S), which mediates formation of delta-aminolevulinic acid (ALA); (2) di-
rect inhibition of the cytosolic enzyme, delta-aminolevulinic acid dehydrase (ALA-D), which
catalyzes formation of porphobilinogen from two units of ALA; and (3) inhibition of insertion
of iron (II) into protoporphyrin IX to form heme, a process mediated by ferrochelatase.
Increased ALA-S activity has been found in lead workers as well as lead-exposed animals,
although the converse, an actual decrease in enzyme activity, has also been observed in
several experimental studies using different exposure methods. It appears, then, that enzyme
activity increase via feedback derepression or activity inhibition may depend on the nature of
the exposure. Using rat liver cells in culture, ALA-S activity was stimulated w vitro at
levels as low as 5.0 pM or 1.0 ug Pb/g preparation. The increased activity was seen to be due
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to biosynthesis of more enzyme. The threshold for lead stimulation of ALA-S activity in
humans, based on a study using leukocytes from lead workers, appears to be about 40 ug Pb/dl.
The generality of this apparent threshold to other tissues depends on how well the sensitivity
of leukocyte mitochondria mirrors that in other systems. The relative impact of ALA-S activi-
ty stimulation on ALA accumulation at lower lead exposure levels appears to be much less than
the effect of ALA-D activity inhibition, to the extent that at ALA-D activity is significantly
depressed at 40 pg/dl blood lead, where ALA-S activity only begins to be affected.
Erythrocyte ALA-D activity is very sensitive to lead inhibition, which is reversed by re-
activation of the sulfhydryl group with agents such as dithiothreitol, zinc, or zinc plus glu-
tathione. Zinc levels that achieve reactivation, however, are well above physiological
levels. Although zinc appears to offset inhibitory effects of lead observed in human eryth-
rocytes vn vitro and in animal studies, lead workers exposed to both zinc and lead do not show
significant changes in the relationship of ALA-D activity to blood lead compared with just
lead exposure; nor does the range of physiological zinc in non-exposed subjects affect the
activity. In contrast zinc deficiency in animals significantly inhibits activity, with con-
comitant accumulation of ALA in urine. Since zinc deficiency has also been demonstrated to
increase lead absorption, the possibility exists for dual effects of such deficiency on ALA-D
activity: (1) a direct effect on activity due to reduced zinc availability; and (2) increased
lead absorption leading to further inhibition of activity.
Erythrocyte ALA-D activity appears to be inhibited at virtually all blood lead levels
measured so far, and any threshold for this effect in either adults or children remains to be
determined. A further measure of this enzyme's sensitivity to lead is a report that rat bone
marrow suspensions show inhibition of ALA-D activity by lead at a level of 0.1 pg/g suspen-
sion. Inhibition of ALA-D activity in erythrocytes apparently reflects a similar effect in
other tissues. Hepatic ALA-D activity was inversely correlated in lead workers with both
erythrocyte activity as well as blood lead levels. Of significance are experimental animal
data showing that (1) brain ALA-D activity is inhibited with lead exposure and (2) this inhi-
bition appears to occur to a greater extent in developing vs. adult animals, presumably re-
flecting greater retention of lead in developing animals. In the avian brain, cerebellar
ALA-D activity is affected to a greater extent than that of the cerebrum and, relative to lead
concentration, shows inhibition approaching that occurring in erythrocytes.
Lead inhibition of ALA-D activity is reflected by elevated levels of its substrate, ALA,
in blood, urine, and soft tissues. In one study, increases in urinary ALA were preceded by a
rise in circulating levels of the metabolite. Blood ALA was elevated at all corresponding
blood lead values down to the lowest determined (18 ug/dl), while urinary ALA increased expo-
nentially with blood ALA.
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Urinary ALA is employed extensively as an indicator of excessive lead exposure in lead
workers. The value of this measurement in pediatric screening, however, is diagnostically
limited if only spot urine collection is done, more satisfactory data being obtainable with
24-hour collections. Numerous independent studies document a direct correlation between blood
lead and the logarithm of urinary ALA in human adults and children; the threshold for urinary
ALA increases is commonly accepted as being 40 jjg/dl. However, several studies of lead
workers indicate that the correlation of urinary ALA with blood lead continues below this
value, and one study found that the slope of the dose-effect curve in lead workers is depen-
dent upon level of exposure.
The health significance of lead-inhibited ALA-D activity and accumulation of ALA at lower
lead exposure levels is controversial, to the extent that the "reserve capacity" of ALA-D
activity is such that only the level of inhibition associated with marked accumulation of the
enzyme's substrate, ALA, in accessible indicator media may be significant. However, it is not
possible to quantify, at lower levels of lead exposure, the relationship of urinary ALA to
target tissue levels nor to relate the potential neurotoxicity of ALA at any accumulation
level to levels in indicator media; i.e., the blood lead threshold for neurotoxicity of ALA
may be different from that associated with increased urinary excretion of ALA.
Accumulation of protoporphyrin in erythrocytes of lead-intoxicated individuals has been
recognized since the 1930s, but it has only recently been possible to quantitatively assess
the nature of this effect via development of sensitive, specific microanalysis methods. Accu-
mulation of protoporphyrin IX in erythrocytes results from impaired placement of iron (II) in
the porphyrin moiety to form heme, an intramitochondrial process mediated by ferrochelatase.
In lead exposure, the porphyrin acquires a zinc ion in lieu of native iron, thus forming zinc
protoporphyrin (ZPP), and is tightly bound in available heme pockets for the life of the ery-
throcytes. This tight sequestration contrasts with the relatively mobile non-metal, or free,
protoporphyrin (FEP) accumulated in the congenital disorder ierythropoietic protoporphyria.
Elevation of erythrocyte ZPP has been extensively documented as being exponentially cor-
related with blood lead in children and adult lead workers and is presently considered one of
the best indicators of undue lead exposure. Accumulation of ZPP only occurs in erythrocytes
formed during lead's presence in erythroid tissue, resulting in a lag of at least several
weeks before such build-up can be measured. The level of such accumulation in erythrocytes of
newly employed lead workers continues to increase when blood lead has already reached a pla-
teau. This influences the relative correlation of ZPP and blood lead in workers with short
exposure histories. In individuals removed from occupational exposure, the ZPP level in blood
declines much more slowly than blood lead, even years after removal from exposure or after a
drop in blood lead. Hence, ZPP level appears to be a more reliable indicator of continuing
intoxication from lead resorbed from bone.
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The threshold for detection of lead-induced ZPP accumulation is affected by the relative
spread of blood lead and corresponding ZPP values measured. . In young children (< 4 yr old),
the ZPH elevation associated with iron-deficiency anemia must also be considered. In adults,
numerous studies indicate that the blood lead threshold for ZPP elevation is about 25-30
|jg/dl. In children 10-15 years old, the threshold is about 16 ug/dl; in this age group, iron
deficiency is not a factor. In one study, children over 4 years old showed the same thresh-
old, 15.5 |jg/dl, as a second group under 4 years old, indicating that iron deficiency was not
a factor in the study. Fifty percent of the children had significantly elevated EP levels (2
standard deviations above reference mean EP) at 25 ug/dl blood lead.
At blood lead levels below 30-40 ug/dl, any assessment of the ZPP-blood lead relationship
is strongly influenced by the relative analytical proficiency for measurement of both blood
lead and EP. The types of statistical analyses used are also important. In a recent detailed
statistical study involving 2004 children, 1852 of whom had blood lead values below 30 ug/dl,
segmental line and probit analysis techniques were employed to assess the dose-effect thres-
hold and dose-response relationship. An average blood lead threshold for the effect using
both statistical techniques yielded a value of 16.5 ug/dl for either the full group or those
subjects with blood lead below 30 ug/dl. The effect of iron deficiency was tested for and
removed. Of particular interest was the finding that the blood lead values corresponding to
EP elevations more than 1 or 2 standard deviations above the reference mean in 50 percent of
the children were 28.6 or 35.7 ug Pb/dl, respectively. Hence, fully half of the children had
significant elevations of EP at blood lead levels around 30 ug/dl, the currently accepted cut-
off value for undue lead exposure. From various reports, children and adult females appear to
be more sensitive to lead effects on EP accumulation at any given blood lead level, with
children being somewhat more sensitive than adult females.
Lead effects on heme formation are not restricted to the erythropoietic system. Recent
studies show that the reduction of serum 1,25-(OH);/D seen with even low level lead exposure is
apparently the result of lead inhibition of the activity of renal 1-hydroxylase, a cytochrome
P-450 mediated enzyme. This heme-containing protein, cytochrome P-450 (an integral part of
the hepatic mixed function oxygenase system), is affected in humans and animals by lead expo-
sure, especially acute intoxication. Reduced P-450 content correlates with impaired activity
of detoxifying enzyme systems such as aniline hydroxylase and aminopyrine demethylase.
Studies of organotypic chick dorsal root ganglion in culture show that the nervous system
not only has heme biosynthetic capability but such preparations elaborate porphyrim'c material
in the presence of lead. In the neonatal rat, chronic lead exposure, resulting in moderately
elevated blood lead, is associated with retarded increases in the hemoprotein, cytochrome C,
and disturbed electron transport in the developing cerebral cortex. These data parallel ef-
fects of lead on ALA-D activity and ALA accumulation in neural tissue. When both these
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effects are viewed in the toxicokinetic context of increased retention of lead in both devel-
oping animals and children, there is an obvious, serious potential for impaired heme-based
metabolic function in the nervous system of lead-exposed children.
As can be concluded from the above discussion, the health significance of ZPP accumula-
tion rests with the fact that it is evidence of impaired heme and hemoprotein formation in
many tissues, arising from entry of lead into mitochondria. Such evidence for reduced heme
synthesis is consistent with much data documenting lead-associated effects on mitochondria.
The relative value of the lead-ZPP relationship in erythropoietic tissue as an index of this
effect in other tissues hinges on the relative sensitivity of the erythropoietic system com-
pared with other organ systems. One study of rats exposed to low levels of lead over their
lifetime demonstrated that protoporphyrin accumulation in renal tissue was already significant
at levels of lead exposure where little change was seen in erythrocyte porphyrin levels.
Other steps in the heme biosynthesis pathway are also known to be affected by lead, al-
though these have not been as well studied on a biochemical or molecular level. Coproporphy-
rin levels are increased in urine, reflecting active lead intoxication. Lead also affects the
activity of the enzyme uroporphyrinogen-I-synthetase, resulting in an accumulation of its sub-
strate, porphobilinogen. The erythrocyte enzyme has been reported to be much more sensitive
to lead than the hepatic species, presumably accounting for much of the accumulated substrate.
Ferrochelatase is an intramitochondrial enzyme, and impairment of its activity, either di-
rectly by lead or via impairment of iron transport to the enzyme, is evidence of the presence
of lead in mitochondria.
12.3.5.2 Lead Effects on Erythropoiesis and Erythrocyte Physiology. Anemia is a manifesta-
tion of chronic lead intoxication, being characterized as mildly hypochromic and usually nor-
mocytic. It is associated with reticulocytosis, owing to shortened cell survival, and the
variable presence of basophilic stippling. Its occurrence is due to both decreased production
and increased rate of destruction of erythrocytes. In young children (< 4 yr old) iron
deficiency anemia is exacerbated by lead effects, and vice versa. Hemoglobin production is
negatively correlated with blood lead in young children, where iron deficiency may be a con-
founding factor, as well as in lead workers. In one study, blood lead values that were
usually below 80 ug/dl were inversely correlated with hemoglobin content. In these subjects,
no iron deficiency was found. The blood lead threshold for reduced hemoglobin content is
about 50 ug/dl in adult lead workers and somewhat lower (40 ug/dl) in children.
The mechanism of lead-associated anemia appears to be a combination of reduced hemoglobin
production and shortened erythrocyte survival because of direct cell injury. Lead effects on
hemoglobin production involve disturbances of both heme and globin biosynthesis. The hemoly-
tic component to lead-induced anemia appears to be due to increased cell fragility and in-
creased osmotic resistance. In one study using rats, the hemolysis associated with vitamin E
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deficiency, via reduced cell deformability, was exacerbated by lead exposure. The molecular
basis for increased cell destruction rests with inhibition of (Na , K )-ATPase and pyrimidine-
5'-nucleotidase. Inhibition of the former enzyme leads to cell "shrinkage" and inhibition of
the latter results in impaired pyrimidine nucleotide phosphorolysis and disturbance of the
activity of the purine nucleotides necessary for cellular energetics.
12.3.5.3 Effects of Alkyl Lead Compounds on Heme Biosynthesis and Erythropoiesis. Tetraethyl
lead and tetramethyl lead, components of leaded gasoline, undergo transformation jn vivo to
neurotoxic trialkyl metabolites as well as further conversion to inorganic lead. Hence, one
might anticipate that exposure to such agents may show effects commonly associated with inor-
ganic lead in terms of heme synthesis and erythropoiesis. Various surveys and case reports
show that the habit of sniffing leaded gasoline is associated with chronic lead intoxication
in children from socially deprived backgrounds in rural or remote areas. Notable in these
subjects is evidence of impaired heme biosynthesis as indexed by significantly reduced ALA-D
activity. In several case reports of frank lead toxicity from habitual leaded gasoline
sniffing, effects such as basophilic stippling in erythrocytes and significantly reduced hemo-
globin have also been noted.
12.3.5.4 Relationships of Lead Effects on Heme Synthesis to Neurotoxicity. The role of lead-
associated disturbances of heme biosynthesis as a possible factor in neurological effects of
lead is of considerable interest because of: (1) similarities between classical signs of lead
neurotoxicity and several neurological components of the congenital disorder, acute intermit-
tent porphyria; and (2) some of the unusual aspects of lead neurotoxicity. There are two
possible points of connection between lead effects on heme biosynthesis and the nervous
system. Associated with both lead neurotoxicity and acute intermittent porphyria is the
common feature of excessive systemic accumulation and excretion of ALA. Secondly, lead neuro-
toxicity reflects, to some degree, impaired synthesis of heme and hemoproteins involved in
crucial cellular functions. Available information indicates that ALA levels are elevated in
the brain of lead-exposed animals, arising via jn situ inhibition of brain ALA-D activity or
via transport to the brain after formation in other tissues. ALA is known to traverse the
blood-brain barrier. Hence, ALA is accessible to, or formed within, the brain during lead
exposure and may express its neurotoxic potential.
Based on various in vitro and HI vivo data obtained in the context of neurochemical
studies of lead neurotoxicity, it appears that ALA can readily play a role in GABAergic func-
tion, particularly inhibiting release of the neurotransmitter GABA from presynaptic receptors,
where ALA appears to be very potent even at low levels. In an jn vitro study, agonist behav-
ior by ALA was demonstrated at levels as low as 1.0 uM ALA. This in vitro observation sup-
ports results of a study using lead-exposed rats in which there was reported inhibition of
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both resting and K+-stiraulated release of preloaded 3H-GABA from nerve terminals. Further
evidence for an effect of some agent other than lead acting directly is the observation that
i_n vivo effects of lead on neurotransmitter function cannot be duplicated with in vitro pre-
parations to which lead is added. Human data on lead-induced associations between disturbed
heme synthesis and neurotoxicity, while limited, also suggest that ALA may function as a neu-
rotoxicant.
The connection of impaired heme and hemoprotein synthesis in the neonatal rat brain was
noted earlier, in terms of reduced cytochrome C production and impaired operation of the cyto-
chrome C respiratory chain. Hence, one might expect that such impairment would be most promi-
nent in areas of relatively greater cellularization, such as the hippocampus. As noted in
Chapter 10, these are also regions where selective lead accumulation occurs.
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12.4 NEUROTOXIC EFFECTS OF LEAD
12.4.1 Introduction
Historically, neurotoxic effects have long been recognized as being among the more severe
consequences of human lead exposure (Tanqueral Des Planches, 1839; Stewart, 1895; Prendergast,
1910; Oliver, 1911; Blackfan, 1917). Since the early 1900's, extensive research has focused on
the elucidation of lead exposure levels associated with the induction of various types of neu-
rotoxic effects and related issues, e.g. critical exposure periods for their induction and
their persistence or reversibility. Such research, spanning more than 50 years, has provided
expanding evidence indicating that progressively lower lead exposure levels, previously accep-
ted as "safe," are actually sufficient to cause notable neurotoxic effects of lead.
The neurotoxic effects of extremely high exposures resulting in blood lead levels in ex-
cess of 80-100 |jg/dl, have been well documented—especially in regard to increased risk for
fulminant lead encephalopathy (a well-known clinical syndrome characterized by overt symptoms
such as gross ataxia, persistent vomiting, lethargy, stupor, convulsions, and coma of such
severity that immediate medical attention is required). The persistence of neurological
sequelae in cases of non-fatal lead encephalopathy has also been well established. The neuro-
toxic effects of subencephalopathic lead exposures in both human adults and children, however,
continues to represent a major area of controversy and interest. Reflecting this, much
research during the past 10-15 years has focussed on the delineation of exposure-effect rela-
tionships for: (1) the occurrence of overt signs and symptoms of neurotoxicity in relation to
other indicators of subencephalopathic overt lead intoxication; and (2) the manifestation of
more subtle, often difficult-to-detect indications of altered neurological functions in appar-
ently asymptomatic (i.e., not overtly lead-poisoned) individuals.
The present assessment critically reviews the available scientific literature on the neu-
rotoxic effects of lead, first evaluating the results of human studies bearing on the subject
and then focusing on pertinent animal toxicology studies. The discussion .of human studies is
divided into two major subsections focusing on neurotoxic effects of lead exposure in (1)
adults and (2) children. Both lead effects on the central nervous system (CMS) and the peri-
pheral nervous system (PNS) are discussed in each case. In general, only relatively brief
overview summaries are provided in regard to findings bearing on the effects of extremely high
level exposures resulting in encephalopathy or other frank signs or symptoms of overt lead in-
toxication. Studies concerning the effects of lower level lead exposures are assessed in
more detail, especially those dealing with non-overtly lead intoxicated children. As for the
animal toxicology studies, particular emphasis is placed on the review of studies that help to
address certain important issuas raised by the human research findings, rather than attempting
an exhaustive review of all animal toxicology studies concerning the neurotoxic effects of
lead.
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12.4.2 Human Studies
Defining exposure-effect or dose-response relationships between lead and particular
neurotoxic responses in humans involves two basic steps. First, there must be an assessment
of the internal lead burden resulting from external doses of lead received via various routes
of exposure (such as air, water, food, occupational hazards, house dust, etc.). Internal lead
burdens may be indexed by lead concentrations in blood, teeth, or other tissue, or by other
biological indicators. The second step involves an assessment of the relationship of internal
exposure indices to behavioral or other types of neurophysiological responses. The difficulty
of this task is reflected by current controversies over existing data. Studies vary greatly
in the quality of design, precision of assessment instruments, care in data collection, and
appropriateness of statistical analyses employed. Many of these methodological problems are
broadly common to research on toxic agents in general and not just to lead alone.
Although epidemiological studies of lead effects have immediate environmental relevance
at the human level, difficult problems are often associated with the interpretation of the
findings, as noted in several reviews (Bornschein etal., 1980; Cowan and Leviton, 1980;
Rutter, 1980; Valciukas and Lilis, 1980; Neddleman and Landrigan, 1981. The main problems
are: (1) inadequate markers of exposure to lead; (2) insensitive measures of performance; (3)
bias in selection of subjects; (4) inadequate handling of confounding covariates; (5) inappro-
priate statistical analyses; (6) inappropriate generalization and interpretation of results;
and (7) the need for "blind" evaluations by experimenters and technicians. Each of these pro-
blems are briefly discussed below.
Each major exposure route—food, water, air, dust, and soil—contributes to a person's
total daily intake of lead (see Chapters 7 and 11 of this document). The relative contribu-
tion of each exposure route, however, is difficult to ascertain; neurotoxic endpoint measure-
ments, therefore, are most typically evaluated in relation to one or another indicator of
overall internal lead body burden. Subjects in epidemiological studies may be misclassified
as to exposure level unless careful choices of exposure indices are made based upon the hypo-
theses to be tested, the accuracy and precision of the biological media assays, and the
collection and assay procedures employed. Chapter 9 of this document evaluates different
measures of internal exposure to lead and their respective advantages and disadvantages. The
most commonly used measure of internal dose is blood lead concentration, which varies as a
function of age, sex, race, geographic location, and exposure. The blood lead level is a use-
ful marker of current exposure but generally does not reflect cumulative body lead burdens as
well as lead levels in teeth. Hair lead levels, measured in some human studies, are not
viewed as reliable indicators of internal body burdens at this time. Future research may
identify a more standard exposure index, but it appears that a risk classification similar to
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that of the U.S. Centers for Disease Control (1978) in terms of blood lead and FEP levels will
continue in the foreseeable future to be the standard approach most often used for lead expo-
sure screening and evaluation. Much of the discussion below is, therefore, focused, on
defining dose-effect relationships for human neurotoxic effects in terms of blood lead levels;
some ancillary information on pertinent teeth lead levels is also discussed.
The frequency and timing of sampling for internal lead burdens represent another impor-
tant factor in evaluating studies of lead effects on neurological and behavioral functions.
For example, epidemiological studies often rely on blood lead and/or erythrocyte protoporphy-
rin (EP) levels determined at a single point in time to retrospectively estimate or character-
ize internal exposure histories of study populations that may have been exposed in the past to
higher levels of lead than those indicated by a single current blood sample. Relatively few
prospective studies exist that provide highly reliable estimates of critical lead exposure
levels associated with observed neurotoxic effects in human adults or children, especially in
regard to the effects of subencephalopathic lead exposures. Some prospective longitudinal
studies on the effects of lead on early development of infants and young children (e.g.,
Bornschein, 1983) are currently in progress, but the results of these studies are not yet
available. The present assessment of the neurotoxic effects of lead in humans must, there-
fore, rely heavily on published epidemiological studies which typically provide exposure
history information of only limited value in defining exposure-effect relationships.
Key variables that have emerged in determining effects of lead on the nervous system in-
clude (1) duration and intensity of exposure and (2) age at exposure. Evidence suggest that
young organisms with developing nervous systems are more vulnerable than adults with fully
matured nervous systems. Particular attention is, therefore, accorded below to discussion of
neurotoxic effects of lead in children as a special group at risk.
Precision of measurement is a critical methodological issue, especially when research on
neurotoxicity leaves the laboratory setting. Neurotoxicity is often measured indirectly with
psychometric or neurometric techniques in epidemiological studies (Valciukas and Lilis, 1980).
The accuracy with which these tests reflect what they purport to measure (validity) and the
degree to which they are reproducible (reliability) are issues central to the science of mea-
surement theory. Many cross-sectional population studies make use of instruments that are
only brief samples of behavior thought to be representative of some relatively constant
underlying traits, such as intelligence. Standardization of tests is the subject of much
research in psychometrics. The quality and precision of specific test batteries have been
particularly controversial issues in evaluating possible effect levels for neurotoxic effects
of lead exposure in children. Table 12B (Appendix 12B) lists some of the major tests used,
together with their advantages and weaknesses. The following review places most weight on
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results obtained with age-normed, standardized psychometric test instruments and well-
controlled, standardized nerve conduction velocity (NCV) tests. Other measures, such as reac-
tion time, finger tapping, and certain electrophysiological measures (e.g., cortical evoked
and slow-wave potentials) are potentially more sensitive indices, but are still experimental
measures whose clinical utility and psychometric properties with respect to the neuro-
behavioral toxicity of lead remain to be more fully explored.
Selection bias is a critical issue in epidemiological studies in which attempts are made
to generalize from a small sample to a large population. Volunteering to participate in a
study and attendance at special clinics or schools are common forms of selection bias that
often limit how far the results of such studies can be generalized. These factors may need to
be balanced in lead neurotoxicity research since reference groups are often difficult to find
because of the pervasiveness of lead in the environment and the many non-lead covariates that
also affect performance. Selection bias and the effects of confounding can be reduced by
choosing a more homogeneous stratified sample, but the generalizability of the results of such
cohort studies is thereby limited.
Perhaps the greatest methodological concern in epidemiological studies is controlling for
confounding covariates, so that residual effects can be more confidently attributed to lead.
Among adults, the most important covariates are age, sex, race, educational level, exposure
history, alcohol intake, total food intake, dietary calcium and iron intake, and urban vs.
rural styles of living (Valciukas and Lilis, 1980). Among children, a number of developmental
covariates are additionally important: parental socioeconomic status (Needleman etal.,
1979); maternal IQ (Perino and Ernhart, 1974); pica (Barltrop, 1966); quality of the care-
giving environment (Hunt et al., 1982; Milar et al., 1980); dietary iron and calcium intake,
vitamin D levels, body fat and nutrition (Mahaffey and Michael son, 1980; Mahaffey, 1981); and
age at exposure. Preschool children below the age of 3-5 years appear to be particularly vul-
nerable, in that the rate of accumulation of aven a low body-lead burden is higher for them
than for adults (National Academy of Sciences, Committee on Lead in the Human Environment,
1980). Potential confounding effects of covariates become particularly important when trying
to interpret threshold effects of lead exposure. Each covariate alone may not be significant,
but, when combined, may interact to pose a cumulative risk which could result in under- or
overestimation of a small effect of lead
Statistical considerations important not only to lead but to all epidemiological studies
include adequate sample size (Hill, 1966), the use of multiple comparisons (Cohen and Cohen,
1975), and the use of multivariate analyses (Cooley and Lohnes, 1971). Regarding sample size,
false negative conclusions are at times drawn from small studies with low statistical power.
.t, is often difficult and expensive to use large sample sizes in complex research such as that
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on lead neurotoxicity. This fact makes it all the more important to use sensitive assessment
instruments which have a high level of discriminating power and can be combined into factors
for multivariate analysis. Multiple statistical comparisons can then be made while reducing
the likelihood of finding a certain number of significant differences by chance alone. This
is a serious problem, because near-threshold effects are often small and variable.
A final crucial issue in this and other research revolves around the care taken to assure
that investigators are isolated from information that might identify subjects in terms of
their lead exposure levels at the time of assessment and data recording. Unconscious biases,
nonrandom errors, and arbitrary data correction and exclusion can be ruled out only if a study
is performed under blind conditions or, preferably, double-blind conditions.
With the above methodological considerations in mind, the following sections evaluate
pertinent human studies, including an overview of lead exposure effects in adults, followed by
a more detailed assessment of neurotoxic effects of lead exposures in children.
12.4.2.1 Neurotoxic Effects of Lead Exposures in Adults.
12.4.2.1.1 Overt lead intoxication in adults. Severe neurotoxic effects of extreme exposures
to high levels of lead, especially for prolonged periods that produce overt signs of acute
lead intoxication, are well documented in regard to both adults and children. The most pro-
found (CNS) effects in adults have been referred to for many years as the clinical syndrome of
lead encephalopathy, described in detail by Aub et al. (1926), Cantarow and Trumper (1944),
Cumings (1959), and Teisinger and Styblova (1961). Early features of the syndrome that may
develop within weeks of initial exposure include dullness, restlessness, irritability, poor
attention span, headaches, muscular tremor, hallucinations, and loss of memory. These symp-
toms may progress to delirium, mania, convulsions, paralysis, coma, and death. The onset of
such symptoms can often be quite abrupt, with convulsions, coma, and even death occurring very
rapidly in patients who shortly before appeared to exhibit much less severe or no symptoms of
acute lead intoxication (Cumings, 1959; Smith et al., 1938). Symptoms of lead encephalopathy
indicative of severe CNS damage and posing a threat to life are generally not seen in adults
except at blood lead levels well in excess of 120 pg/dl (Kehoe, 1961a,b,c). Other data
(Smith et al., 1938) suggest that acute lead intoxication, including severe gastrointestinal
symptoms and/or signs of encephalopathy can occur in some adults at blood lead levels around
100 ug/dl, but ambiguities make this data difficult to interpret.
In addition to the above CNS effects, lead also clearly damages peripheral nerves at tox-
ic, high exposure levels that predominantly affect large myelinated nerve fibers (Vasilescu,
1973; Feldman et al., 1977; Englert, 1980). Pathologic changes in peripheral nerves, as shown
in animal studies, can include both segmental demyelination and, in some fibers, axonal degen-
eration (Fullerton, 1966). The former types of changes appear to reflect lead effects on
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Schwann cells, with concomitant endoneurial edema and disruption of myelin membranes
(Windebank and Dyck, 1981). Apparently lead induces a breakdown in the blood-nerve barrier
which allows lead-rich edema fluid to enter the endoneurium (Dyck etal., 1980; Windebank
etal., 1980). Remyelination observed in animal studies suggests either that such lead
effects may be reversible or that not all Schwann cells are affected equally (Lampert and
Schochet, 1968; Ohnishi and Dyck, 1981). Reports of plantar arch deformities due to old per-
ipheral neuropathies (Emmerson, 1968), however, suggest that lead-induced neuropathies of
sufficient severity in human adults could result in permanent peripheral nerve damage. Mor-
phologically, peripheral neuropathies are usually detectable only after prolonged high expo-
sure to lead, with distinctly different sensitivities and histological differences existing
among mammalian species. In regard to man, as an example, Buchthal and Behse (1979, 1981),
using nerve biopsies from a worker with frank lead neuropathy (blood lead = 150 ug/dl), found
histological changes indicative of axonal degeneration in association in NCV reductions that
corresponded to loss of large fibers and decreased amplitude of sensory potentials.
Data from certain studies provide a basis by which to estimate lead exposure levels at
which adults exhibit overt signs or symptoms of neurotoxicity and to compare such levels with
those associated with other types of signs and symptoms indicative of overt lead intoxication
(Lilis et al., 1977; Irwig et al., 1978; Dahlgren et al., 1978; Baker et al., 1979; Hanninen
etal., 1979; Spivey etal., 1979; Fischbein etal., 1980; Hammond etal., 1980). These
studies evaluated the incidence of various clinical signs and symptoms of lead intoxication
across a wide range of lead exposures among occupationally exposed smelter and battery plant
workers. The reported incidences of particular types of signs and symptoms, both neurological
and otherwise, and associated lead exposure levels varied considerably from study to study,
but they collectively provide evidence indicating that overt neurological, gastrointestinal,
and other lead-related symptoms can occur among adults starting at blood lead levels as low as
40-60 ug/dl. Considerable individual biological variability is apparent, however, among vari-
ous study populations and individual workers in terms of observed lead levels associated with
overt signs and symptoms of lead intoxication, based on comparisons of exposure-effect and
dose-response data from the above studies. Irwig et al (1978), for example, report data for
black South African lead workers indicative of clearly increased prevalence of both neurologi-
cal and gastrointestinal symptoms at blood lead levels over 80 ug/dl. Analogously, Hammond
et al. (1980) reported significant increases in neurological (both CNS and PNS) and gastro-
intestinal symptoms among American smelter workers with blood lead levels often exceeding
80 ug/dl, but not among workers whose exposure histories did not include levels above 80
ug/dl—findings in contrast to the results of several other studies. Lilis et al. (1977), for
instance, found that CNS symptoms (tiredness, sleeplessness, irritability, headaches) were
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reported by 55 percent and muscle or joint pain by 39 percent of a group of lead smelter
workers whose blood lead levels had never been found to exceed 80 ug/dl. Low hemoglobin
levels (<14g/dl) were found in more than 33 percent of these workers. Also, Spivey et al.
(1977) reported significantly increased neurological (mainly CNS, but some PNS) symptoms and
joint pain among a group of 69 lead workers with mean ± standard deviation blood lead levels
of 61.3 ± 12.8 ug/dl in comparison to a control group with 22.0 ±5.9 ug/dl blood lead values.
Hanninen et al. (1979) similarly reported finding significantly increased neurological (both
CNS and PNS) and gastrointestinal symptoms among 25 lead workers with maximum observed blood
lead levels of 50-69 |jg/dl and significantly increased CNS symptoms among 20 lower exposure
workers with maximum blood lead values below 50 ug/dl, compared in each case against a refer-
ent control group (N = 23) with blood lead values of 11.9 ±4.3 M9/dl (mean ± standard devia-
tion).
Additional studies provide evidence of overt signs or symptoms of neurotoxicity occurring
at still lower lead exposure levels than those indicated above. Baker et al. (1979) studied
dose-response relationships between clinical signs and symptoms of lead intoxication among
lead workers in two smelters. No toxicity was observed at blood lead levels below 40 ug/dl.
However, 13 percent of those workers with blood lead values in the range 40-79 ug/dl had
extensor muscle weakness or gastrointestinal symptoms; and anemia occurred in 5 percent of the
workers with 40-59 ug/dl blood lead levels, in 14 percent with levels of 60-79 ug/dl, and in
36 percent with blood lead levels exceeding 80 ug/dl. Also, Fischbein et al. (1980), in a
study of 90 cable splicers intermittently exposed to lead, found higher zinc protoporphyrin
levels (an indicator of impaired heme synthesis associated with lead exposure) among workers
reporting CNS or gastrointestinal symptoms than among other cable splicers not reporting such
symptoms. Only 5 percent of these workers had blood lead levels in excess of 40 pg/dl, and
the mean ± standard deviation blood lead levels for the 26 reporting CNS symptoms were 28.4
±7.6 ug/dl and 30 ±9.4 ug/dl for the 19 reporting gastrointestinal symptoms. Caution must be
exercised in accepting these blood levels as being representative of average or maximum lead
exposures of this worker population, however, in view of the highly intermittent nature of
their exposure and probable much higher resulting peaks in their blood lead levels than those
coincidentally measured at the time of their blood sampling.
Overall, the above results appear to support the following conclusions: (1) overt signs
and symptoms of neurotoxicity in adults are manifested at roughly comparable lead exposure
levels as other types of overt signs and symptoms of lead intoxication, such as gastrointesti-
nal complaints; (2) the neurological signs and symptoms are indicative of both central and
peripheral nervous system effects; (3) such overt signs and symptoms, both neurological and
otherwise, occur at markedly lower blood lead levels than the 60 or 80 ug/dl criteria levels
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previously established or recently discussed as being "safe" for occupationally exposed
adults; and (4) lowest observed effect levels for the neurological signs and symptoms can most
credibly be stated to be in the 40 to 60 (jg/dl range. Insufficient information exists pre-
sently by which to estimate with confidence to what extent or for how long such overt signs
and symptoms persist in adults after termination of precipitating external lead exposures, but
at least one study (Dahlgren, 1978) reports evidence of abdominal pain persisting for as long
as 29 months after exposure termination among 15 smelter workers, including four whose blood
lead levels were between 40 and 60 ug/dl while working.
12.4.2.1.2 Non-Overt lead intoxication in adults. Of special importance for establishing
standards for exposure to lead is the question of whether exposures lower than those producing
overt signs or symptoms of lead intoxication result in less obvious neurotoxic effects in
otherwise apparently healthy individuals. Attention has focused in particular on whether ex-
posures leading to blood lead levels below 80-100 ug/dl may lead to behavioral deficits or
other neurotoxic effects in the absence of classical signs of overt lead intoxication.
In adults, if such neurobehavioral deficits occurred with great frequency, one might ex-
pect this to be reflected by performance measures in the workplace, such as higher rates of
absences or reduced psychomotor performances among occupationally exposed lead workers. Some
epidemiological studies have investigated possible relationships between elevated blood lead
and general health as indexed by records of sick absences certified by physicians (Araki et
al., 1982; Robinson, 1976; Shannon et a!., 1976; Tola and Nordman, 1977). However, sickness
absence rates are generally poor epidemiologic outcome measures that may be confounded by many
variables and are difficult to relate specifically to lead exposure levels. Much more useful
are studies discussed below which evaluate lead exposures in relation to direct measurements
of CMS or peripheral neurological functions.
Only a few studies have employed sensitive psychometric and/or neurological testing
procedures in an effort to demonstrate specific lead-induced neurobehavioral effects in
adults. For example, Morgan and Repko (1974) reported deficits in hand-eye coordination and
reaction time in an extensive study of behavioral functions in 190 lead-exposed workers (mean
blood lead level = 60.5 ± 17.0 ug/dl). The majority of the subjects were exposed between 5
and 20 years. In a similar study, Milburn et al. (1976) found no differences between control
and lead-exposed workers on numerous psychometric and other performance tests. On the other
hand, several recent studies (Arnvig et al., 1980; Grandjean et al., 1978; HSnninen et al.,
1978; Mantere et al. , 1982; Valciukas et al., 1978) have found disturbances in visual motor
performance, IQ test performance, hand dexterity, mood, nervousness, and coping in lead
workers with blood lead levels of 50-80 ug/dl. A graded dose-effect relationship for non-
overt CMS lead effects in otherwise apparently asymptomatic adults is indicated by such
studies.
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In addition to the above studies indicative of CNS dysfunctions in non-overtly lead
intoxicated adults, numerous investigations have provided electrophysiological data indicating
that peripheral nerve dysfunction in apparently asymptomatic adults can be associated with
blood lead values below 80 |jg/dl. Such peripheral nerve deficits, i.e. slowed nerve conduc-
tion veolocity (NCV), were established by Seppalainen et al. (1975) for lead workers whose
blood lead levels were as low as 50 ug/dl and had never exceeded 70 pg/dl during their entire
exposure period (mean = 4.6 years), as determined by regular monitoring. Similar results were
obtained in a study by Melgaard et al. (1976) on automobile mechanics exposed to TEL and other
lead compounds in lubricating and high-pressure oils. Results of an analysis of the workers'
blood for lead, chromium, copper, nickel, and manganese indicated a clear association between
lead exposure and peripheral nerve dysfunction. Half of the workers (10 to 20) had elevated
blood lead levels (60-120 ug/dl) and showed definite electromyographic deficits. The mean
blood lead level for the control group was 18.6 (jg/dl. Melgaard et al. (1976) reported addi-
tional results on associating lead exposures with polyneuropathy of unknown etiology in 10
cases from the general population. Another study reported by Araki and Honma (1976) provided
further confirmation of the Seppalainen et al. (1975) and Melgaard et al. (1976) findings in
that evidence for peripheral neuropathy effects were reported for lead industry workers with
blood lead values of 29 to 70 ug/dl.
More recent studies by Araki et al (1980), Ashby (1980), Bordo et al. (1982), Johnson
et al. (1980), Seppalainen et al. (1979), and Seppalainen and Hernberg (1980, 1982) have con-
firmed a dose-dependent slowing of NCV in lead workers with blood lead levels below 70 to 80
Hg/dl. Seppalainen et al. (1979) observed NCV slowing in workers with blood lead levels
across a range of 29 to 70 (jg/dl (Figure 12-2); and Seppalainen and Hernberg (1980, 1982)
found NCV slowing in workers with maximum blood lead levels of 30 to 48 ug/dl, but not among
workers with levels below 30 ug/dl. Buchthal and Behse (1979), Lilis et al. (1977), and
Paulev et al. (1979), in contrast, found no signs of neuropathy below 80 ug/dl. Reports of
low blood lead levels (below 50 ug/dl) in some of the above studies should be viewed with cau-
tion until further confirmatory data are reported for larger samples using well verified blood
assay results. Nonetheless, these studies are consistent with a continuous dose-response
relationship between blood lead concentration and extent and degree of peripheral nerve dys-
function in non-overtly lead intoxicated adults.
The above studies on nerve conduction velocity provide convergent evidence for peripheral
nerve dysfunctions occurring in adults with blood lead levels in the 30-70 ug/dl range but not
exhibiting overt signs of lead intoxication. Furthermore, although it might be argued that
peak levels of lead may have been significant and that substantially higher lead body burdens
existing before the time of some of the studies were actually responsible for producing the
dysfunctions, it appears that in several cases (Seppalainen et al., 1975; SeppSlMinen and
2BPB12/B 12-48 9/20/83
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o
«
I
III
UJ
111
u.
O
U
80
70
60
50
40
I =1
y = -0.141x + 63.5
r=-0.377
p < 0.001
_] I
10 20 30 40
ACTUAL BLOOD LEAD CONCENTRATION,
50
60
Figure 12-2. Maximal motor nerve conduction velocity (NCV) of the
median nerve plotted against the actual blood lead level for 78
workers occupatibnally exposed to lead and for 34 control subjects.
Source: Seppalainen et al. (1979).
12-49
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PRELIMINARY DRAFT
Hernberg, 1980) blood levels that had never exceeded 70 pg/dl were related to increased peri-
pheral nerve dysfunction; and, in the Seppalainen and Hernberg (1982) study, NCV slowing was
associated with maximum levels of 30-48 (jg/dl. The studies by Seppalainen and her co-workers
are generally methodologically sound, having been well controlled for the possible effects of
extraneous factors such as history, length, and type of exposure, multiple assessments of dif-
ferent nerves, temperature differences at the NCV assessment sites, plus relevant confounding
covariates. Thus, when the Seppalainen et al. (1975) results are viewed collectively with
the data from other studies reviewed here, substantial evidence can be stated to exist for
peripheral nerve dysfunctions occurring in adults at blood lead levels of as low as 30 to 50
ug/dl. The question as to whether these reflect mild, reversible effects (Buchthal and Behse,
1981) or are true early warning signals of progressively more serious peripheral neuropathies
important in the diagnosis of otherwise unrecognized toxic effects of lead (Feldman et al.,
1977; Seppalainen and Hernberg, 1980) is still a matter of some dispute. Nevertheless, it is
clear that these effects represent departures from normal neurologic functioning and their
potential relationship to other extremely serious effects (see, for example, the next para-
graph) argues for prudence in interpreting their potential health significance.
There are several reports of previous overexposure to heavy metals in amyotrophic lateral
sclerosis (ALS) patients and patients dying of motor neuron disease (MND). Conradi et al.
(1976, 1978a,b, 1980) found elevated cerebrospinal fluid lead levels in ALS patients as com-
pared with controls. Thus, the possible pathogenic significance of lead in ALS needs to be
further explored. In addition, Kurlander and Patten (1978) found that lead levels in spinal
cord anterior horn cells of MND patients were nearly three times that of control subjects and
that lead levels correlated with illness durations. Despite chelation therapy for about a
year, high lead levels remained in their tissue.
12.4.2.2 Neurotoxic Effects of Lead Exposure in Children.
12.4.2.2.1 Overt lead intoxication in children. Symptoms of encephalopathy similar to those
that occur in adults have been reported to occur in infants and young children (Prendergast,
1910; Oliver and Vogt, 1911; Blackfan, 1917; McKahann and Vogt, 1926; Giannattasio et al.,
1952; Cumings, 1959; Tepper, 1963; Chisolm, 1968), with a markedly higher incidence of severe
encephalopathic symptoms and deaths occurring among them than in adults. This may reflect the
greater difficulty in recognizing early symptoms in young children, thereby allowing intoxica-
tion to proceed to a more severe level before treatment is initiated (Lin-Fu, 1973). In
regard to the risk of death in children, the mortality rate for encephalopathy cases was
approximately 65 percent prior to the introduction of chelation therapy as standard medical
practice (Greengard et al., 1965; National Academy of Sciences, 1972; Niklowitz, 1975;
Niklowitz and Mandybur, 1975). The following mortality rates have been reported for children
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experiencing lead encephalopathy since the inception of chelation therapy as the standard
treatment approach: 39 percent (Ennis and Harrison, 1950); 20 to 30 percent (Agerty, 1952);
24 percent (Mellins and Jenkins, 1955); 18 percent (Tanis, 1955); and 5 percent (Lewis et al.,
1955). These data, and those tabulated more recently (National Academy of Sciences, 1972),
indicate that once lead poisoning has progressed to the point of encephalopathy, a life-
threatening situation clearly exists and, even with medical intervention, is apt to result in
a fatal outcome. Historically there have been three stages of chelation therapy. Between
1946 and 1950, dimercaprol (BAL) was used. From 1950 to 1960, calcium disodium ethylenedia-
minetetraacetate (CaEDTA) completely replaced BAL. Beginning in 1960, combined therapy with
BAL and CaEDTA (Chisolm, 1968) resulted in a very substantial reduction in mortality.
Determining precise values for lead exposures necessary to produce acute symptoms, such
as lethargy, vomiting, irritability, loss of appetite, dizziness, etc., or later neurotoxic
sequelae in humans is difficult in view of the usual sparsity of data on environmental lead
exposure levels, period(s) of exposure, or body burdens of lead existing prior to manifesta-
tion of symptoms. Nevertheless, enough information is available to permit reasonable esti-
mates to be made regarding the range of blood lead levels associated with acute encephalo-
pathic symptoms or death. Available data indicate that lower blood lead levels among children
than among adults are associated with acute encephalopathy symptoms. The most extensive
compilation of information on a pediatric population is a summarization (National Academy of
Sciences, 1972) of data from Chisolm (1962, 1965) and Chisolm and Harrison (1956). This data
compilation relates occurrence of acute encephalopathy and death in children in Baltimore to
blood lead levels determined by the Baltimore City Health Department (using the dithizone
method) between 1930 and 1970. Blood lead levels formerly regarded as "asymptomatic" and
other signs of acute lead poisoning were also tabulated. Increased lead absorption in the
absence of detected symptoms was observed at blood lead levels ranging from 60 to 300 ug/dl
(mean = 105 pg/dl). Acute lead poisoning symptoms other than signs of encephalopathy were
observed from approximately 60 to 450 ng/dl (mean = 178 (jg/dl). Signs of encephalopathy
(hyperirritability, ataxi a, convulsions, stupor, and coma) were associated with blood lead
levels of approximately 90 to 700 or 800 ug/dl (mean = 330 ug/dl). The distribution of blood
lead levels associated with death (mean = 327 ug/dl) was essentially the same as for levels
yielding encephalopathy. These data suggest that blood lead levels capable of producing death
in children are essentially identical to those associated with acute encephalopathy and that
such effects are usually manifested in children starting at blood lead levels of approximately
100 ug/dl. Certain other evidence from scattered medical reports (Gant, 1938; Smith et al.,
1938; Bradley et al., 1956; Bradley and Baumgartner, 1958; Comings, 1959; Runwno et al., 1979),
however, suggests that acute encephalopathy in the most highly susceptible children may be
associated with blood lead levels in the range of 80-100 pg/dl. These latter reports are
evaluated in detail in the 1977 EPA document Air Quality Criteria for Lead (U.S. EPA, 1977).
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From the preceding discussion, it can be seen that severity of symptoms varies widely for
different adults or children as a function of increasing blood lead levels. Some show irre-
versible CNS damage or death at blood lead levels around 100 MQ/dl, whereas others may not
show any of the usual clinical signs of lead intoxication even at blood lead levels in the 100
to 200 ug/dl or higher range. This diversity of response may be due to: (1) individual bio-
logical variation in lead uptake or susceptibility to lead effects; (2) changes in blood lead
values from the time of initial damaging intoxication; (3) greater tolerance for a gradually
accumulating lead burden; (4) other interacting or confounding factors, such as nutritional
state or inaccurate determinations of blood lead; or (5) lack of use of blind evaluation pro-
cedures on the part of the evaluators. It should also be noted that a continuous gradation of
frequency and severity of neurotoxic symptoms extends into the lower ranges of lead exposure.
Morphological findings vary in cases of fatal lead encephalopathy among children
(Blackman, 1937; Pentschew, 1965; Popoff et al., 1963). Reported neuropathologic findings are
essentially the same for adults and children. On macroscopic examination the brains are often
edematous and congested. Microscopically, cerebral edema, altered capillaries (endothelial
hypertrophy and hyperplasia), and peri vascular glial proliferation often occur. Neuronal
damage is variable and may be caused by anoxia. However, in some cases gross and microscopic
changes are minimal (Pentschew, 1965). Pentschew (1965) described neuropathology findings for
20 cases of acute lead encephalopathy in infants and young children. The most common finding
was activation of intracerebral capillaries characterized by dilation of the capillaries, with
swelling of endothelial cells. Diffuse astrocytic proliferation, an early morphological
response to increased permeability of the blood-brain barrier, was often present. Concurrent
with such alterations, especially evident in the cerebellum, were changes that Pentschew
(1965) attributed to hemodynamic disorders, i.e., ischemic changes manifested as cell
necrosis, perineuronal incrustations, and loss of neurons, especially in isocortex and basal
ganglia.
Attempts have been made to understand better brain changes associated with encephalopathy
by studying animal models. Studies of lead intoxication in the CNS of developing rats have
shown vasculopathic changes (Pentschew and Garro, 1966), reduced cerebral cortical thickness
and reduced number of synapses per neuron (Krigman et al., 1974a), and reduced cerebral axonal
size (Krigman et al., 1974b). Biochemical changes in the CNS of lead-treated neonatal rats
have also demonstrated reduced lipid brain content but no alterations of neural lipid composi-
tion (Krigman et al., 1974a) and a reduced cerebellar DNA content (Michaelson, 1973). In
cases of lower level lead exposure, subjectively recognizable neuropathologic features may not
occur (Krigman, 1978). Instead there may be subtle changes at the level of the synapse
(Silbergeld etal., 1980a) or dendritic field, myelin-axon relations, and organization of
2BPB12/B 12-52 9/20/83
-------
PRELIMINARY DRAFT
synaptic patterns (Krigman, 1978). Since the nervous system is a dynamic structure rather
than a static one, it undergoes compensatory changes (Norton and Culver, 1977), maturation and
aging (Sotelo and Palay, 1971), and structural changes in response to environmental stimuli
(Coss and Glohus, 1978). Thus, whereas massive structural damage in many cases of acute
encephalopathy would be expected to lead to lasting neurotoxic sequalae, some other CNS
effects due to severe early lead insult might be reversible or compensated for, depending upon
age and duration of toxic exposure. This raises the question of whether effects of early
overt lead intoxication are reversible beyond the initial intoxication or continue to persist.
In cases of severe or prolonged nonfatal episodes of lead encephalopathy, there occur
neurological sequelae qualitatively similar to those often seen following traumatic or infec-
tious cerebral injury, with permanent sequelae being more common in children than in adults
(Mel 1 ins and Jenkins, 1955; Chi solm, 1962, 1968). The most severe sequelae in children are
cortical atrophy, hydrocephalus, convulsive seizures, and severe mental retardation (Mel 1 ins
and Jenkins, 1955; Perlstein and Attala, 1966; Chisolm, 1968). Children who recover from
acute lead encephalopathy but are re-exposed to lead almost invariably show evidence of per-
manent central nervous system damage (Chisolm and Harrison, 1956). Even if further lead expo-
sure is minimized, 25 to 50 percent show severe permanent sequelae, such as seizure disorders,
blindness, and hemiparesis (Chisolm and Barltrop, 1979).
Lasting neurotoxic sequelae of overt lead intoxication in children in the absence of
acute encephalopathy have also been reported. Byers and Lord (1943), for example, reported
that 19 out of 20 children with previous lead poisoning later made unsatisfactory progress in
school, presumably due to sensorimotor deficits, short attention span, and behavioral dis-
orders. These latter types of effects have since been confirmed in children with known high
exposures to lead, but without a history of life-threatening forms of acute encephalopathy
(Chisolm and Harrison, 1956; Cohen and Ahrens, 1959; Kline, 1960). Perlstein and Attala
(1966) also reported neurological sequelae in 140 of 386 children (37 percent) following lead
poisoning without encephalopathy. Such sequelae included mental retardation, seizures, cere-
bral palsy, optic atrophy, and visual-perceptual problems in some children with minimal intel-
lectual impairment. The severity of sequelae was related to severity of earlier observed
symptoms. For 9 percent of those children who appeared to be without severe symptoms at the
time of diagnosis of overt lead poisoning, mental retardation was observed upon later follow-
up. The conclusion of the neurological effects observed by Perlstein and Attala (1966) being
persisting effects of earlier overt lead intoxication without encephalopathy might be ques-
tioned in view of no control group having been included in the study; however, it is extremely
unlikely that 37 percent of any randomly selected control group from the general pediatric
population would exhibit the types of neurological problems observed in that proportion of the
cohort of children with earlier lead intoxication studied by Perlstein and Attala (1966).
2BPB12/B 12-53 9/20/83
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PRELIMINARY DRAFT
Numerous studies (Cohen et al., 1976; Fejerman et al., 1973; Pueschel et al., 1972; Sachs
et al., 1978, 1979, 1982) suggest that, in the absence of encephalopathy, chelation therapy
may ameliorate the persistence of neurotoxic effects of overt lead poisoning (especially cog-
nitive, perceptual, and behavioral deficits). On the other hand, one recent study found a
residual effect on fine motor performance even after chelation (Kirkconnell and Hicks, 1980).
In summary, pertinent literature definitively demonstrates that lead poisoning with
encephalopathy results in a greatly increased incidence of permanent neurological and cogni-
tive impairments. Also, several studies further indicate that children with symptomatic lead
poisoning in the absence of encephalopathy also show a later increased incidence of neurologi-
cal and behavioral impairments.
12.4.2.2.2 Non-Overt lead intoxication in children
In addition to neurotoxic effects associated with overt lead intoxication in children,
growing evidence indicates that lead exposures not leading to overt lead intoxication in
children can induce neurological dysfunctions. This issue has attracted much attention and
generated considerable controversy during the past 10 to 15 years. However, the evidence for
and against the occurrence of significant neurotoxic deficits at relatively low levels of lead
exposure is quite mixed and largely interpretable only after a thorough critical evaluation of
methods employed in the various important studies on the subject. Based on the five criteria
listed earlier (i.e., adequate markers of exposure to lead, sensitive measures, appropriate
subject selection, control of confounding covariates, and appropriate statistical analysis),
the 20 population studies summarized in Table 12-1 were conducted rigorously enough to warrant
at least some consideration here. Even so, no epidemiological study is completely flawless
and, therefore, overall interpretation of such findings must be based on evaluation of:
(1) the internal consistency and quality of each study; (2) the consistency of results ob-
tained across independently conducted studies; and (3) the plausibility of results in view of
other available information.
Rutter (1980) has classified studies evaluating neurobehavioral effects of lead exposure
in non-overtly lead intoxicated children according to several types, including four categories
reviewed below: (1) clinic-type studies of children thought to be at risk because of high lead
levels; (2) other studies of children drawn from general (typically urban or suburban) pedia-
tric populations; (3) samples of children living more specifically in close proximity to lead
emitting smelters; and (4) studies of mentally retarded or behaviorally deviant children.
Major attention is accorded here to studies falling under the first three categories. As
will be seen, quite mixed results have emerged from the studies reviewed.
12.4.2.2.2.1 Clinic-type studies of children with high lead levels. The clinic-type
studies are typified by evaluation of children with relatively high lead body burdens as
identified through lead screening programs or other large-scale programs focussing on mother-
infant health relationships and early childhood development.
2BPB12/B 12-54 9/20/83
-------
TABLE 12-1.
SUMMARY OF PUBLISHED RESULTS FROM STUDIES OF LEAD EFFECTS ON KEUROBEHAVIORAL FUNCTIONS
OF NON-OVERTLY LEAD INTOXICATED CHILDREN
Reference
Clinic-type Studies
De la Burde and
Choate (1972)
Oe la Burde and
Choate (1975)
RUMO et al.
(1974. 1979)
_,
PO
Ul
w Kotok (1972)
Kotok et al.
(1977)
Perl no and
Ernhart (1974)
Ernhart et al.
(1981)
Population
studied N/group
of Children with High Lead Levels
Inner city Control = 72
(Richmond, VA) Lead = 70
Follow-up Control = 67
same subjects Lead = 70
Inner city Control Ss = 45
(Providence, RI)
Short Pb Ss = 15
Long Pb S_s = 20
Post enceph Pb = 10
Inner city Control = 25
(New Haven, CT) Lead = 24
Inner city Control = 36
(Rochester, NY) Lead = 31
Inner city Control = 50
(New York, NY)
Lead = 30
Follow-up sa»e Control = 31
subjects Lead = 32
Age at
testing, yr
4
4
7
7
4-8 (x = 5.8)
4-8 (x = 5.6)
4-8 (x = 5.6)
4-8 (x = 5.3)
1.1 -5 .5 (x =
1.0 - 5.8 (x =
1.9 - 5.6 (x =
1.7 - 5.4 (- =
3-6
3-6
8-13
Blood lead,
ug/dl
Not assayed0
40-100
See above e
See above
x = 23 ± 8
x = 61 ± 7
x = 68 ± 13
x = 88 ± 40
2.7) 20-55
2.8) 58-137
3.6) 11-40
3.6) 61-200
10-30
40-70
21.3+3.71
32.4±5.3
Psychometri c
tests employed
Stanford-Binet IQ
Other measures
WISC Full Scale IQ
Neurologic exam
Other neasures
McCarthy General
Cognitive
McCarthy Subscales
Neurologic exam
rating
Objective neurologic
tests
Denver Developmental
Scale
IQ Equivalent for
six ability classes:
Social maturity;
Spatial relations;
Spoken vocab;
Info, comprehension;
Visual attention;
Auditory neaory
McCarthy General
Cognitive
McCarthy Subscales
McCarthy General Cog-
nitive Index
McCarthy Subscales
Reading Tests
Exploratory Tests
(Bender Gestalt,
Oraw-A-Child)
Conners Teachers Rating
Scale
Summary of results
(C=control; Pb=lead)
C = 94 Pb = 89
C > Pb on 3/4 tests
C = 90 Pb = 87
C better than Pb
C > Pb on 9/10 tests
C = 93; S = 94;
L = 88; P = 77
C+S > L > P on 5/5
tests
C+S > L > P on ratings
C+S > L > P on 3/12
tests
C > Pb on 1/3 Subscales
IQ Equivalent for
each:
C = 126 Pb = 124
C = 101 Pb = 92;
C = 93 Pb = 92;
C = 96 Pb = 95;
C = 93 Pb = 90
C = 100 Pb = 93
C = 90 Pb = 80
C > Pb on 5/5 scales
Shared Variance =7.7
(2/5) 8.0, 7.4
1.3
•>
i
Levels of .
significance
p <0.05
N.S.-p <0.01
p <0.01
p <0.01
N.S.-p <0.001
N.S.-p <0.01
(P vs C)
N.S.-p <0.01
(P vs C)
N.S.
N.S.-p <0.01
(P vs C)
N.S.
p <0.10 for
spatial
p >0.10 for all
other ability
classes
p <0.01
N.S.-p <0.01
p <0.05
f, <0.05
M.S.
N.S.
N.S.
-D
73
rn
r—
i — t
3
i — i
?
•K
-<
0
TO
J.
-n
~H
-------
TABLE 12-1. (continued)
Population Age at
Reference studied H/group testing, yr
General Population Studies
Needleman et al. General population Control = 100 7
(1979) - (Boston, MA area) Lead =58 7
ro HcBride et al. Urban and suburban Moderate = C. 100 4,5
i, (1982) (Sydney, Australia)
o» Low = C.100 4,5
Yule et al . Urban Group 1 = 20 9
(1981) (London, England) Group 2 = 29 9
Group 3 = 29 8
Group 4 = 21 8
Yule et al. Same subjects Same Same
(1983)
Blood lead. Psychometric Sumary of results
ug/dl tests employed (C=contro); Pb=lead)
PbT < 10 ppwh WISC Full scale IQ
PbT > 20 ppm WISC Verbal IQ
WISC Performance IQ
Seashore Rhythm Test
Token Test
Sentence Repetition Test
Delayed Reaction Tine
Teacher's Behavior
Rating
19-30 ug/dl Peabody Picture Voc.
Test
0.5-9 Mfl/dl Fine Motor Tracking
Pegboard
Tapping Test
Beam Walk
Standing Balance
Rutter Activity Scale
&.&* WISC-R Full Scale IQ
11.6 Verbal IQ
14.5 Performance IQ k
19.6 Vernon Spelling Test .
Vernon Arithmetic lest
Neale Reading Test
Same Needleman Teacher's
Behavior Ratings
Conners Teachers
C = 106.6 Pb = 102.1
C = 103.9 Pb = 99.3
C = 108.7 Pb = 104.9
C = 21.6 Pb = 19.4
C = 24.8 Pb = 23.6
C = 12.6 !>b= 11.3
C > Pb on 3/4 blocks
C = 9.5 Pb = 8.2
C = 105 Pb = 104
C > Pb 1/4 comparisons
C = 20 Pb = 20
C = 30 Pb = 31
C = 5 Pb = 4
C > Pb 1/4 comparisons
C = 1.9 Pb - 2.1
Gpl < Gp2 > Gp3 > Gp4
Gpl < Gp2 > Gp3 > Gp4
Gpl < Gp2 > Gp3 > Gp4
Gpl > Gp2 > Gp3 > Gp4
Gpl < Gp2 > Gp3 > Gp4
Gpl < Gp2 > Gp3 > Gp4
Linear Trend 3/4 itens
Gpl < Gps2-4
Levels of .
significance
p < 0.03
p <0.03
N.S.
p <0.002
N.S.
p <0.04
p <0.01
p <0.02
N.S.
p <0.05
N.S.
N.S.
N.S.
p <0.05
N.S.
p <0.029
p <0.04
N.S.
p <0. 001
N.S.
p <0.001
p <0.05
p <0.05
Questionnaire
Factors 1,2,4,5
Rutter Teacher Rating
Scale for Activity
73
Linear Trend 25/36 items N.S.
-------
Table 12-1. (continued)
Population Age at Blood lead. Psychometric Summary of results
Reference studied N/group testing, yr ug/d? tests employed (Decontrol; Pb=lead)
Smith et al. Urban Hi = 155 6,7 PbT £8.0 WISC-R Full Scale
(1983) (London, UK) Ned = 103 6,7 PbT =5-5. 5 Verbal IQ
Low = 145 6,7 PbT < 2.5 Performance IQ
(AH in ug/g)
x PbB = 13.1
(jg/dl Word Reading Test
Seashore Rhythm Test
Visual Sequential Kemory
Sentence Memory
Shape Copying
<•— ' Mathematics
i Mean Visual RT (sees)
JJJ Conners Teachers Ratings
rule and Lansdown Urban 80 9 7-12 WISC-R Full Scale
(1983) (London, UK) 82 9 13-24 Verbal IQ
Performance IQ
Neale Reading Ace.
Neale Reading Coup.
Vernon Spelling
Vernon Hath
Harvey et al. Urban 189 2.5 15.5 British Ability Scales
(1983) (Birmingham, UK) Naming
Recal 1
Comprehension
Recognition
IQ
Stanford-Binet Items
Shapes
Blocks
Beads
Playroom Activity
HIGH
105
103
106
40
20
20
9
14
15
.39
13
Low
107
104
108
114
113
101
100
Levels of .
significance
NED LOW
105
103
106
42
20
19
9
14
15
.37
11
Regression F
<1
1.26
<1
<1
<1
<1
2.34
2.46
?
107
105
108
45
21
20
9
14
16
.37
11
Hi
105
103
106
111
109
99
99
Ratio
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
z
TO
O
TO
-------
Table 12-1. (continued)
ro
i
oo
Reference
Population
studied
Age at Blood lead, Psychometric Summary of results
N/group testing, yr yg/dl tests employed (C=control; Pb=lead)
Levels of .
significance
Smelter Area Studies
Landrigan et al.
(1975)
McNeil and Ptasnik
(1975)
Ratcliffe (1977)
Winneke et al.
(19B2a)
Winneke et al.
(1982b)
Smelter area
(El Paso, TX)
Smelter area
(El Paso, TX)
Smelter area
Manchester, Eng.
Smelter area
(Ouisburg. FRG)
Smelter area
(Stolburg, FRG)
Control = 46 3-15 (x = 9.3) <40 WISC Full Scale IQfn C = 93 Pb = 87
Lead = 78 3-15 (x = 8.3) 40-68 WPPSI Full Scale IQ9 C = 91 Pb = 86
WISC + WPPSI Combined C = 93 Pb = 88
WISC + WPPSI Subscales C > Pb on 13/14 scales
Neurologic testing C > Pb on 4/4 tests
Control = 61-152 1.5 - 18(Mdn = 9) <40 McCarthy General
Lead = 23-161 1.5 - 18(Mdn = 9) >40 Cognitive C = 82 Pb = 81
WISC- WAI S Full Scale
IQ C = 89 Pb = 87
Oseretsky Motor Level C = 101 Pb = 97
California Person-
ality C > Pb, 6/10 items
Frostig Perceptual
Quotient C = 100 Pb = 103
Finger-Thumb
Apposition C = 27 Pb = 29
Control = 23 4-7 28.2 Griffiths Mental Oev. C = 101-111 Pb = 97-107
Lead = 24 4-8 44.4 Frostig Visual
Perception C = 14. 3 Pb = 11.8
Pegboard Test C = 17.5 Pb = 17.3
C = 19.5 Pb = 19.8
Control =26 8 PbT =2.4 pprah German WISC Full Scale C = 122 Pb = 117
Lead = 26 8 PbT = 9.2 ppm Verbal IQ C = 130 Pb = 124
No PbB Performance IQ C = 130 Pb = 123
Bender Gesta It Test C = 17.2 Pb=19.6
Standard Neurological C = 2.7 Pb = 7.2
Tests
Conners Teachers Rating C = ? Pb = ?
Scale
89 9.4 PbT = £.16 pp«h German WISC Full Scale Prop, of Variance=-O.Q
PbB =14.3 ug/dl IQ
Verbal 0 -0.5
Performance IQ +0.6
Bender Gestalt Test +2.1
Standard Neurological +1.2
Tests
Conners Teachers Rating 0.4-1.3
Scale
Wiener Reaction Performance +2.0
N.S.
N.S.
p <0.01
N.S.-p <0.01
N.S.-p <0.001
N.S.
N.S,
N.S.
p <0.05
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
p <0.05
N.S.
N.S.
N.S.
N.S.
N.S.
p <0.05
N.S.
N.S.
N.S.
-o
m
r~
3
^
^
TO
-C
o
3>
— 1
rtean test scores for control children indicated by C = x; mean scores for respective lead-exposed groups indicated by Pb = x, except for Ryronno (1979)
study where C = control, S = short-term lead-exposed subjects, L = long-term lead-exposed group, and P = post-encephalopathy lead group. N.S. = non-
significant, I.e. p >0.05. Note exception of p <0.10 listed for spacial ability results in Kotok.et al. (1977) study. Significance levels are those found
after partial ing out confounding covariates. Urinary coproporphyrin levels were not elevated. Or £30 pg/dl with positive radiologic findings, suggesting
earlier exposure in excess of 50-60Jig/dl. Assays for lead in teeth showed the Pb-exposed group to be approximately twice as high as controls (202
ug/g vs..112 M9/g. respectively). Used for children over 5 years of age. 9Used for children under 5 years of age. Main measure was dentine lead
(PbT). Dentine levels not reported for statistical reasons. JBJood lead levels taken 9-12 months prior to testing; none above 33 ug/dl. Data not
corrected for age.
-------
PRELIMINARY DRAFT
Of the several pediatric studies presenting evidence for CMS deficits being associated
with lead exposure in asymptomatic children, most all are either retrospective or cross-
sectional studies except the work of De la Burde and Choate (1972, 1975). De la Burde and
Choate (1972) observed neurological dysfunctions, fine motor dysfunction, impaired concept
formation, and altered behavioral profiles in 70 preschool children exhibiting pica and ele-
vated blood lead levels (in all cases above 30 |jg/dl; mean = 59 ug/dl) in comparison with
matched control subjects not engaging in pica. Subjects were drawn from the Collaborative
Study of Cerebral Palsy, Mental Retardation, and Other Neurologic Disorders of Infancy and
Childhood (Broman et al. , 1975), which was conducted in Richmond, Virginia, and had a total
population of 3400 mothers. The De la Burde and Choate study population was drawn from this
group, in which all mothers were followed throughout pregnancy and all children were post-
natal^ evaluated by regular pediatric neurologic examinations, psychological testing, and
medical interviews. All children subject to prenatal, perinatal, and early postnatal insults
were excluded from the study, and all had to have normal neurologic examinations and Bayley
tests at eight to nine months of age. These are important points which add value to the study.
It is unfortunate that blood lead data were not regularly obtained; however, at the time of
the study in the late 1960s, 10 to 20 ml of venous blood was required for a blood lead deter-
mination and such samples usually had to be obtained by either jugular or femoral puncture.
The other control features (housing location and repeated urinary coproporphyrin tests) would
be considered the state of the art for such a study at the time that it was carried out.
In a follow-up study on the same children (at 7 to 8 years old), De la Burde and Choate
(1975) reported continuing CNS impairment in the lead-exposed group as assessed by a variety
of psychological and neurological tests. In addition, seven times as many lead-exposed
children were repeating grades in school or being referred to the school psychologist, despite
many of their blood lead levels having by then dropped significantly from the initial study.
In general, the De la Burde and Choate (1972, 1975) studies appear to be methodologically
sound, having many features that strengthen the case for the validity of their findings. For
example, there were appreciable numbers of children (67 lead-exposed and 70 controls) whose
blood lead values were obtained in preschool years and who were old enough (7 years) during
the follow-up study to cooperate adequately for reliable psychological testing. The psycho-
metric tests employed were well standardized and acceptable as sensitive indicators of neuro-
behavioral dysfunction, and the testing was carried out in a blind fashion (i.e., without the
evaluators knowing which were control or lead-exposed subjects).
2BPB12/B 12-59 9/20/83
-------
PRELIMINARY DRAFT
The De la Burde and Choate (1972, 1975) studies might be criticized on several points,
but none provide sufficient grounds for rejecting their results. One difficulty is that blood
lead values were not determined for control subjects in the initial study; but the lack of
history of pica, as well as tooth lead analyses done later for the follow-up study, render it
improbable that appreciable numbers of lead-exposed subjects might have been wrongly assigned
to the control group. Subjects in the control group did have a history of pica, but not for
paint. Also, results indicating no measurable coproporphyrins in the urine of control sub-
jects at the time of initial testing further confirm proper assignment of those children to
the nonexposed control group. A second point of criticism is the use of multiple chi-square
statistical analyses, but the fact that the control subjects did significantly better on vir-
tually every measure makes it unlikely that all of the observed effects were due to chance
alone. One last problem concerns ambiguities in subject selection which complicate interpre-
tation of the results obtained. Because the lead-exposed group included children with blood
lead levels of 40 to 100 pg/dl, or of at least 30 ^g/dl with "positive radiographic findings
of lead lines in the long bones, metallic deposits in the intestines, or both," observed defi-
cits might be attributed to blood lead levels as low as 30 (jg/dl. Other evidence (Betts et
al., 1973), however, suggests that such a simple interpretation is probably not accurate.
That is, the Betts et al. (1973) study indicates that lead lines are usually seen only if
blood levels exceed 60 (jg/dl for most children at some time during exposure, although some
(about 25 percent) may show lead lines at blood lead levels of 40 to 60 |jg/dl. In view of
this, the de la Burde and Choate results can probably be most reasonably interpreted as
showing persisting neurobehavioral deficits at blood lead levels of 40 to 60 ug/dl or higher.
In another clinic-type child study, Rummo et al. (1974, 1979), found significant neuro-
behavioral deficits (hyperactivity, lower scores on McCarthy scales of cognitive function,
etc.) among Providence, Rhode Island, inner-city children who had previously experienced high
levels of lead exposure that had produced acute lead encephalopathy. Mean maximum blood lead
levels recorded for those children at the time of encephalopathy were 88 ± 40 pg/dl. However,
children with moderate blood lead elevation but not manifesting symptoms of encephalopathy
were not significantly different (at p <0.05) from controls on any measure of cognitive func-
tioning, psychomotor performance, or hyperactivity. Still, when the data from the Rummo et
al. (1979) study for performance on the McCarthy General Cognitive Index or several McCarthy
Subscales are compared (see Table 12-1), the scores for long-term moderate-exposure subjects
consistently fall below those for control subjects and lie between the latter and the encepha-
lopathy group scores. Thus, it appears that long-term moderate lead exposure may have, in
fact, exerted dose-related neurobehavioral effects. The overall dose-response trend might
have been shown to be statistically significant if other types of analyses were used or if
2BPB12/B 12-60 9/20/83
-------
PRELIMINARY DRAFT
larger samples were assessed. However, control for confounding variables in the different
exposure groups would also have to be considered. Note that (1) the maximum blood lead levels
for the short-term and long-term exposure subjects were all greater than 40 ug/dl (means =
61 ± 7 and 68 ± 13 ug/dl, respectively), whereas control subjects all had blood lead levels
below 40 pg/dl (mean = 23 ± 8 ug/dl), and (2) the control and lead-exposed subjects were
inner-city children well matched for socioeconomic background, parental education levels,
incidence of pica, and other pertinent factors, but not parental IQ.
A somewhat similar pattern of results emerged from a study by Kotok et al. (1977) in
which 36 Rochester, New York, control-group children with blood lead levels less than 40 ug/dl
were compared with 31 children having distinctly elevated blood lead levels (61 to 200 ug/dl)
but no classical lead intoxication symptoms. Both groups were well matched on important back-
ground factors, notably including their propensity to exhibit pica. Again, no clearly statis-
tically significant differences between the two groups were found on numerous tests of cogni-
tive and sensory functions. However, mean scores of control-group children were consistently
higher than those of the lead-exposed group for all six of the ability classes listed. Kotok
(1972) had reported earlier that developmental deficiencies (using the comparatively insen-
sitive Denver Development Screening test) in a group of children having elevated lead levels
(58 to 137 ug/dl) were identical to those in a control group similar in age, sex, race,
environment, neonatal condition, and presence of pica, but whose blood lead levels were lower
(20 to 55 ug/dl). Children in the lead-exposed group, however, had blood lead levels as high
as 137 ug/dl, whereas some control children had blood lead levels as high as 55 ug/dl. Thus,
the study essentially compared two groups with different degrees of markedly elevated lead
exposure rather than one of lead-exposed vs. nonexposed control children.
Peri no and Ernhart (1974) reported a relationship between neurobehavioral deficits and
blood lead levels ranging from 40 to 70 ug/dl in a group of 80 inner-city preschool black
children, based on the results of a cross-sectional study including children detected as
having elevated lead levels via the New York City lead screening program. One key result
reported was that the high-lead children had McCarthy Scale IQ scores markedly lowe than those
of the low-lead group (mean IQ = 90 vs 80, respectively). Also, the normal correlation of
0.52 between parents' intelligence and that of their offspring was found to be reduced to only
0.10 in the lead-exposed group, presumably because of the influence of another factor (lead)
that interfered with the normal intellectual development of the lead-exposed children. One
obvious possible alternative explanation for the reported results, however, might be differ-
ences in the educational backgrounds of parents of the control subjects when compared with
lead-exposed subjects, because parental education level was found to be significantly nega-
tively related to blood lead levels of the children participating in the Perino and Ernhart
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(1974) study. The importance of this point lies in the fact that several other studies
(McCall et al., 1972; Elardo et al., 1975; Ivanans, 1975) have demonstrated that higher paren-
tal education levels are associated with more rapid development and higher intelligence quo-
tients (IQs) for their children.
Ernhart et al. (1981) were able to follow up 63 of the 80 preschool children of the
Peri no and Ernhart (1974) study once they reached school age, using the McCarthy IQ scales,
various reading achievement tests, the Bender-Gestalt test, the Draw-A-Child test, and the
Conners Teacher's Questionnaire for hyperactivity. The children's blood lead levels cor-
related significantly with FEP (r = 0.51) and dentine lead levels (r = 0.43), but mean blood
lead levels of the moderately elevated group had decreased after five years. When control
variables of sex and parent IQ were extracted by multivariate analyses, the observed differ-
ences were reported to be greatly reduced but remained statistically significant for three of
seven tests on the McCarthy scales in relation to concurrently measured blood lead levels but
not in relation to the earlier blood lead levels for the same children. This led Ernhart et
al. (1981) to reinterpret their 1974 (Perino and Ernhart, 1974) IQ results (in which they
had not controlled for parental education) as either not likely being due to lead or, if due
to lead, then representing only minimal effects on intelligence.
The Perino and Ernhart (1974) and Ernhart et al. (1981) studies were intensively reviewed
by an expert committee convened by EPA in March, 1983 (see Appendix 12-C). The committee
found that blood lead measurements used in the Perino and Ernhart (1974) study were of accep-
table reliability and the psychometric measures for children were acceptable. However, the
IQ measure used for their parents was of questionable utility, other confounding variables
may not have been adequately measured, and the statistical analyses did not deal adequately
with confounding variables. As for the Ernhart et al. (1981) follow-up study, the committee
found the psychometric measures to be acceptable, but the blood lead sampling method raised
questions about the reliability of the reported blood lead levels and the statistical analyses
did not adequately control for confounding factors. The committee concluded, therefore, that
the Perino and Ernhart (1974) and Ernhart et al. (1981) study results, as published, neither
confirm nor refute the hypothesis of associations between neuropsychologic deficits and low-
level lead exposures in children. It was also recommended that the entire Ernhart data set be
reanalyzed, using statistical analyses that better control for confounding factors and includ-
ing longitudinal analyses of data for subjects that were evaluated in both the Perino and
Ernhart (1974) and the Ernhart et al. (1981) studies. A sample longitudinal analysis provided
by one committee member, using uncorrected blood lead values and unadjusted psychometric
scores from such subjects, suggested that an association may exist between changes in blood
lead levels and changes in IQ scores from the first to the second sampling point.
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Two recent reports of a study of 193 children from the Philadelphia cohort of the Collab-
orative Perinatal Project at age seven years examined the persistence of lead-related neuro-
psychological deficits using circumpulpal rather than primary dentine lead assays at ages
10-14 years (Shapiro and Marecek, 1983, Marecek et al., 1983). Performance differences on
several subtests of the Wechsler Intelligence Scale for Children (WISC) and Bender-Gestalt
Test were found to persist after four years, these effects being more evident when related
to circumpulpal than to primary dentine lead levels. Methodologically, this study suffers
from sampling bias, subject ascertainment bias, poor control of covarying social factors, and
use of different testers at different testing periods, with no notation as to their blindess.
Odenbro et al. (1983) studied psychological development of children (aged 3-6 yr) seen
in Chicago Department of Health Clinics (August, 1976 - February, 1977), evaluating Denver
Development Screening test (DDST) and Wechsler IQ scales (WPPSI) scores in relation to blood
lead levels obtained by repeated sampling during the three previous years. A significant cor-
relation (r = -0.435, p <0.001) was reported between perceptual-visual-motor ability and mean
blood levels measured. Statistically significant (p <0.005) deficits in verbal productivity
and perceptual visual motor performance (measured by the WPPSI) were found for children with
mean blood lead levels of 30-40 ug/dl versus control children with mean blood lead levels
<25 ug/dl, using two-tailed Student's t-tests. These results are highly suggestive of neuro-
psychologic deficits being associated with blood lead levels of 30-60 ug/dl in preschool
children. However, questions can be raised regarding the adequacy of the statistical analyses
employed, especially in regard to sufficient control for confounding covariates, e.g., parental
IQ, education, and socioeconomic status.
The above studies generally found higher lead-exposure groups to do more poorly on IQ or
other types of psychometric tests. However, many studies did not control for importtant con-
founding variables or, when such were taken into account, differences between lead exposed and
control subjects were often no longer statistically significant. Still, the consistency of
finding lower IQ values among at-risk higher lead children across the studies lends credence
to cognitive deficits occurring in apparently asymptomatic children with relatively high blood
lead levels. The De la Burde studies in particular point to 40-60 ug/dl as likely lowest
observed effect levels among such children.
12.4.2.2.2.2 General population studies. These studies evaluated samples of non-overtly
lead intoxicated children drawn from and thought to be representative of the general pediatric
population. They generally aimed to evaluate asymptomatic children with lower lead body bur-
dens than those of children in most of the above clinic-type studies.
A pioneering, general population study was reported by Needleman et al. (1979), who used
shed deciduous teeth to index lead exposure. Teeth were donated from 70 percent of a total
population of 3329 first and second grade children from two towns near Boston. Almost all
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children who donated teeth (2146) were rated by their teachers on an eleven-item classroom
behavior scale devised by the authors to assess attention disorders. An apparent dose-response
function was reported for ratings on the behavior scale not taking potentially confounding
variables into account. After excluding various subjects for control reasons, two groups
(<10th and >90th percentiles of primary dentine lead levels) were provisionally selected for
further in-depth neuropsychologic testing. Later, some provisionally eligible children were
also excluded for various reasons, leaving 100 low-lead (<10 ppm dentine lead) children for
comparison with 58 high-lead (>20 ppm dentine lead) children in statistical analyses reported
by Needleman et al. (1979). A preliminary analysis on 39 non-lead variables showed significant
differences between the low- high-lead groups for age, maternal IQ and education, maternal
age at time of birth, paternal SES, and paternal education. Some of these variables were
entered as covariates into an analysis of covariance along with lead. Significant effects
(p <0.05) were reported for full-scale WISC-R IQ scores, WISC-R verbal scales scores, for 9 of
11 classroom behavior scale items, and several experimental measures of perceptual-motor
behavior.
Additional papers published by Needleman and coworkers report on results of the same or
further analyses of the data discussed in the initial paper by Needleman et al. (1979). For
example, a paper by Needleman (1982) provided a summary overview of findings from the Needleman
et al. (1979) study and findings reported by Burchfiel et al. (1980) that are discussed later
i,n this section concerning EEC patterns for a subset of children included in the 1979 study.
Needleman (1982) summarized results of an additional analysis of the 1979 data set reported
elsewhere by Needleman, Levitan and Bellinger (1982). More specifically, cumulative frequency
distributions of verbal IQ scores for low- and high-lead subjects from the 1979 study were
reported by Needleman et al. (1982), and the key point made was that the average IQ deficit of
four points demonstrated by the 1979 study did not just reflect children with already low IQs
having their cognitive abilities further impaired. Rather the entire distribution of IQ scores
across all IQ levels was shifted downward in the high-lead group, with none of the children in
that group having verbal IQs over 125. Another paper, by Bellinger and Needleman (1983), pro-
vided still further follow-up analyses of the 1979 N. Eng. J. Med. data set, focusing mainly
on comparison of the low- and high-lead children's observed versus expected IQs based on their
mother's IQ. Bellinger and Needleman reported that regression analyses showed that IQs of
children with elevated levels of dentine-lead (>20 ppm) fell below those expected based on
their mothers' IQs and the amount by which a child's IQ falls below the expected value in-
creases with increasing dentine-lead levels in a nonlinear fashion. Scatter plots of IQ
residuals by dentine-lead levels, as illustrated and discussed by Bellinger and Needleman
(1983), indicated that regressions for children with 20-29.9 ppm dentine lead in the high-lead
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group did not reveal significant associations between increasing lead levels in that range and
IQ residuals, in contrast to statistically significant (p <0.05) correlations between IQ resi-
duals and dentine-lead for high-lead group children with 30-39.9 ppm dentine lead levels.
The Needleman et al. (1979) study and spin-off analyses published later by Needleman and
coworkers were critically evaluated by the same expert committee noted above that was convened
by EPA in March, 1983, and which evaluated the Perino and Ernhart (1974) and Ernhart et al.
(1981) studies (see Appendix 12-C). In regard to the original study reported by Needleman
et al. (1979), the expert committee found that dentine-lead was adequately determined as a
measure of cumulative lead exposure and the psychometric data for the subject children gener-
ally appeared to be adequately collected and of acceptable reliability. However, the commit-
tee concluded that the reported dose-response relationship between dentine-lead levels and
teachers' ratings of classroom behavior cannot be accepted as valid, due to: (1) serious res-
ervations regarding the adequacy of classification of subjects into lead exposure categories
using only the first dentine-lead level obtained for each child and (2) failure to control for
effects of confounding variables. The committee also found that the reported statistically
significant effects of lead on IQ and other behavioral neuropsychologic abilities measured for
the low- and high-lead groups could not be accepted as valid, due to: (1) errors made in cal-
culations of certain parental IQ scores entered as a control variable in analyses of covari-
ance; (2) failure to take age and father's education into account adequately in the analyses
of covariance; (3) use of a forward elimination approach rather than a backwards elimination
strategy in statistical analyses; (4) concerns regarding the basis for classification of
children in terms of dentine-lead levels; and (5) questions about possible bias due to exclu-
sion of data for large numbers of provisionally eligible subjects from statistical analyses.
The committee concluded, therefore, that the study results, as published by Needleman et al.
(1979), neither confirm nor refute the hypothesis of associations between neuropsychologic
deficits and low-level lead exposure in non-overtly lead intoxicated children. In regard to
the publications by Needleman (1982), Needleman et al. (1982), and Bellinger and Needleman
(1983) describing further analyses of the same data set reported on by Needleman et al.
(1979), the committee concluded that the findings reported in these later papers also cannot
be accepted as valid, in view of the above reservations regarding the basic analyses reported
by Needleman et al. (1979) and additional problems with the later "spin-off" analyses. The
committee also recommended that the entire Needleman data set be reanalyzed, correcting for
errors in data calculation and entry, using better Pb exposure classification, and appropri-
ately adjusting for confounding factors.
A recent study of urban children in Sydney, Australia (McBride et al. , 1982) involved 454
preschoolers (aged 4-5 yr) with blood lead levels of 2 to 29 (jg/dl. Children born at the
Women's Hospital in Sydney were recruited via personal letter. No blood lead measures were
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available on non-participants. Blood levels were evaluated after neurobehavioral testing, but
earlier exposure history was apparently not assessed. Using a multiple statistical comparison
procedure and Bonferroni correction to protect against study-wise error, no statistically
significant differences were found between two groups with blood lead levels more than one
standard deviation above and below the mean (>19 ug/dl vs. <9 (jg/dl) on the Peabody Picture
Vocabulary IQ Test, on a parent rating scale of hyperactivity devised by Rutter, or on three
tests of motor ability (pegboard, standing balance, and finger tapping). In one test of fine
motor coordination (tracking), five-year old boys in the higher lead group performed worse
than boys in the lower lead group. In one test of gross motor skill (walking balance), results
for the two age groups were conflicting. This study suffers from many methodological weak-
nesses and cannot be regarded as providing evidence for or against an effect of low-level lead
exposures in non-overtly lead intoxicated children. For example, a comparison of socioeconomic
status (father's occupation and mother's education) of the study sample with the general popu-
lation showed that it was higher than Bureau of Census statistics for the Australian work force
as a whole. There was apparently some self-selection bias due to a high proportion of profes-
sionals living near the hospital. Also, other demographic variables such as mother's IQ, pica,
and caregiving environment were not evaluated.
Another recent large scale study (Smith et al., 1983) of tooth lead, behavior, intelli-
gence and a variety of other psychological skills was carried out in a general population sam-
ple of over 4000 children aged 6 to 7 years in three London boroughs, 2663 of whom donated
shed teeth for analysis. Of these, 403 children were selected to form six groups, one each of
high (8 ug/g or more), intermediate (5-5,5 ug/g), and low (2.5 ug/g or less) tooth lead levels
for two socioeconomic groups (manual vs. non-manual workers). Parents were intensively inter-
viewed at home regarding parental interest and attitudes toward education and family charac-
teristics and relationships. The early history of the child was then studied in school using
tests of intelligence (WISC-R), educational attainment, attention, and other cognitive tasks.
Teachers and parents completed the Conners behavior questionnaires. Results showed that in-
telligence and other psychological measures were strongly related to social factors, especi-
ally social grouping. Lead level was linked to a variety of factors in the home, especially
the level of cleanliness, and to a lesser extent, maternal smoking. There was no statisti-
cally significant link between lead level and IQ or academic performance. However, when rated
by teachers (but not by parents), there were small, reasonably consistent (but not statisti-
cally significant) tendencies for high-lead children to show more behavioral problems after
the different social covariables were taken into account statistically. The Smith et al.
(1983) study has much to recommend it: (1) a well-drawn sample of adequate size; (2) three
tooth lead groupings based on well-defined classifications minimizing possible overlaps of
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exposure groupings using whole tooth lead values, including quality-controlled replicate ana-
lyses comparisons for the same tooth and duplicate analyses comparisons across multiple teeth
from the same child; (3) blood lead levels on a subset of 92 children (averaging 13.1 ug/dl),
which correlated reasonably well with tooth lead levels (r = 0.45); (4) cross-stratified
design of social groups; (5) extensive information on social covariates and exposure sources;
and (6) statistical control for potentially confounding covariates in the analsyes of study
results. However, one possible source of selection bias was that tooth donors had a signifi-
cantly higher social status than non-donors. Thus, the reported results may be less generali-
zable to the lower socioeconomic working classes, where one might expect the effects of lead
exposure to be greater (Yule and Lansdown, 1983).
Harvey et al. (1983) also recently reported that blood lead made no significant contri-
bution to IQ decrements after appropriate allowance had been made for social factors. This
study involved 189 children, average age 2.5 years and 15.5 ug/dl blood lead, of middle class
workers from the inner city of Birmingham, England. The investigators utilized a wide range
of behavioral measures of activity level and psychomotor performance. Strengths of this study
are: (1) a well-drawn sample, (2) extensive evaluation of 15 confounding social factors, (3)
a wide range of abilities evaluated, and (4) blind evaluations. However, evaluation of lead
burden was based on only a single venous blood sample, so that exposure history was not docu-
mented as well as in the study by Smith et al. (1983). Nevertheless, a stronger correlation
between IQ and blood lead levels was found in children of manual workers (r = -0.32) than in
children of non-manual workers (r = -0.06), consistent with findings from the Yule and
Lansdown (1983) study discussed below.
Yule et al. (1981) carried out a pilot study on the effects of low-level lead exposure on
85 percent of a population of 195 children aged 6-12 years, whose blood lead concentrations
had been determined some nine months earlier as part of a European Economic Community survey.
The blood lead concentrations ranged from 7 to 32 ug/dl, and the children were assigned to
four quartiles encompassing the following values: 7 to 10 ug/dl; 11 to 12 ug/dl; 13 to 16
ug/dl; and 17 to 32 ug/dl. The tests of achievement and intelligence were similar to those
used in the Lansdown et al. (1974) and Needleman et al. (1979) studies. There were signifi-
cant associations between blood lead levels and scores on tests of reading, spelling, and in-
telligence, but not on mathematics (Yule et al., 1981). These differences in performance
largely remained after age, sex, and father's occupation were taken into account. However,
other potentially confounding social factors were not controlled in this study. Another paper
by Yule et al. (1983) dealt with the results pertinent to attention deficits. While there
were few differences between groups on the Rutter Scale, the summed scores on the Needleman
questionnaire across the blood lead groupings approached significance (p = 0.096). Three of
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the questionnaire items showed a significant dose-response function ("Day Dreamer," "Does not
Follow Sequence of Direction," "Low Overall Functioning"). Nine of 11 items were highly cor-
related with children's IQ. Therefore, the Needleman questionnaire may be tapping IQ-related
attention deficits as opposed to measures of conduct disorder and socially maladaptive behav-
ior (Yule et al. , 1983). The hyperactivity factors on the Conners and Rutter scales were
reported to be related to blood lead levels (7-12 vs. 13-32 ug/dl), but the authors noted that
caution is necessary in interpreting their findings in view of the crude measures of social
factors available and differences between countries in diagnosing attention deficit disorders.
Moreover, since the blood lead values reported were determined only once (nine months before
psychological testing), earlier lead exposures may not be fully reflected and the reported
blood lead levels cannot be accepted confidently as those with which any behavioral effects
might be associated. Also, home environment and parental IQ and education were not evaluated.
Yule and Lansdown (1983) reported a second, better designed study with similar methods
and procedures using 194 children living in a predominantly lower-middle-class area of London
near a busy roadway. In this study, a lengthy structured interview yielded data on sources of
exposure, medical history, and many potentially confounding variables. Parental IQ was also
examined. In contrast to the first pilot study, no statistically significant relationships
were found even before social class was controlled for in the statistical analyses. Still,
the authors stated that there was some evidence of weak associations between lead level and
intelligence in working-class groups but whether these are of a causal nature in either direc-
tion is unclear.
Two studies by Winneke and colleagues, the first a pilot study (Winneke et al., 1982a)
and the second an extended study (Winneke et al., 1982b) discussed later, employed teeth lead
analyses analogous to some of the above studies. In the pilot study, incisor teeth were
donated by 458 children aged 7 to 10 years in Duisburg, Germany, an industrial city with air-
borne lead concentrations between 1.5 and 2.0 ug/V. Two extreme exposure groups were formed,
a Tow-lead group with 2.4 ug/g mean tooth lead level (n = 26) and another, high-lead group
with 7 ug/g mean tooth lead level (n = 16), and matched for age, sex, and father's occupa-
tional status. The two groups did not differ significantly on confounding covariates, except
that the high-lead group showed more perinatal risk factors. Parental IQ and quality of the
home environment were not among the 52 covariables examined. The authors found a marginally
significant decrease (p <0.10) of 5-7 IQ points and a significant decrease in perceptual-
motor integration (p <0.05), but no significant differences in hyperactivity as measured by
the Conners Teachers' Questionnaire administered during testing. As with the Yule et al.
(1981) study, the inadequacy of the background social measures (e.g., parental IQ, caregiving
environment, and pica), and group differences in perinatal factors weaken this study.
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None of the general population studies reviewed provide strong evidence for neuropsycho-
logic deficits being associated with relatively low body lead burdens in non-overtly lead
intoxicated children representative of general pediatric populations. All of the studies
reporting statistically significant associations between cognitive (IQ) or other behavior
(e.g., attentional) deficits have methodological weaknesses, especially inadequate control for
confounding covariates such as parental IQ or socioeconomic status. On the other hand, in
view of the consistent pattern of results from such studies showing relationships between lead
and neuropsychologic deficits before major confounding variables are controlled for, one
cannot completely rule out the possibility that lead may be contributing to the observed
deficits, especially given the cross-sectional design used in such studies (see Appendix 12-C
introduction). The findings of no significant associations between lead and cognitive/behav-
ioral deficits in several recently reported studies (generally controlling better for con-
founding variables) may not be incompatible with this possibility, in view of the latter
studies apparently having evaluated children with lead body burdens likely generally lower
than the former studies reporting at least suggestive evidence for lead effects on cognitive
and behavioral functions.
12.4.2.2.2.3 Smelter area studies. These studies evaluated children with elevated lead
exposures associated with residence in close proximity to lead emitting smelters.
For example, Lansdown et al. (1974) reported a relationship between blood lead level In
children and the distance they lived from lead-processing facilities, but no relationship
between blood lead level and mental functioning. However, only a minority of the lead-exposed
cohort had blood lead levels over 40 ug/dl. Furthermore, this study failed to consider ade-
quately social factors such as socioeconomic status.
In another study, Landrigan et al. (1975) found that lead-exposed children living near an
El Paso, Texas, smelter scored significantly lower than matched controls on measures of per-
formance IQ and finger-wrist tapping. The control children in this study were, however, not
well matched by age or sex to the lead-exposed group, although the results remained statisti-
cally significant after adjustments were attempted for age differences. McNeil and Ptasnik
(1975) found negative results in another sample of children living near the same lead smelter
in El Paso who were generally comparable medically and psychologically to matched controls
living elsewhere in the same city, except for the direct effects of lead (blood lead level,
free erythrocyte protoporphyrin levels, and X-ray findings). An extensive critique of these
two studies made by another expert committee (see Appendix 12-D) found that no reliable con-
clusions could be based on either of the two El Paso smelter studies in view of various metho-
dological and other problems affecting the conduct of the studies.
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A later study by Ratcliffe (1977) of children living near a battery factory in Manchester,
England, found no relation between their blood levels taken at two years of age (28 ug/dl vs.
44 ug/dl in low- vs. high-lead groups) and testing done at age five on the Griffiths Mental
Development Scales, the Frostig Developmental Test of Visual Perception, a pegboard test, or a
behavioral questionnaire. The differences in scores, although small, favored the low-lead
exposure children, i.d., they had somewhat better scores than the higher exposure group. The
failure to repeat blood lead assays at age five weakens this otherwise adequate study; poten-
tially higher blood lead levels occurring after age two among control children may have less-
ened exposure differences between the low- and high-lead groups.
Winneke et al. (1982b) carried out a study which involved 115 children aged 9.4 years
living in the lead smelter town of Stolberg. Tooth lead (X = 6.16 ppm, range = 2.0-38.5 ppm)
and blood lead levels (X = 13.4 ug/dl; range = 6.8-33.8 ug/dl were significantly correlated
(r = 0.47; p <0.001) for the children studied. Using stepwise multiple regression analysis,
the authors found significant (p <0.05) or marginally significant (p <0.10) associations be-
tween tooth lead levels and measures of perceptual-motor integration, reaction time perfor-
mance, and four behavioral rating dimensions, including distractibility. This was true even
after taking into account age, sex, duration of labor at birth, and socio-heredity background
as covariates. However, the proportion of explained variance due to lead never exceeded
6 percent for any of these outcomes, and no significant association was found between tooth
lead and WISC verbal-IQ after the effects of socio-hereditary background were eliminated.
The above smelter area studies, again, do not provide strong evidence for cognitivie or
behavior deficits being associated with, lead exposure in nonovertly lead exposed children.
At the same time, the possibility of such deficits being associated with lead exposure in
apparently asymptomatic children cannot be ruled out, either, given the overall pattern of
results obtained with the cross sectional study design typically employed (see Appendix 12-C
introduction).
Several studies have also reported significant associations between hair lead levels and
behavioral or cognitive testing endpoints (Pihl and Parkes, 1977; Hole et al., 1979; Hansen
et al., 1980; Capel et al., 1981; Ely et al., 1981; Thatcher et al., 1982a,b; Marlowe et al.,
1982, 1983; Marlowe and Errera, 1982). Measures of hair lead are easily contaminated by ex-
ternal exposure and are generally questionable in terms of accurately reflecting internal body
burdens (see Chapter 9). Such data, therefore, cannot be credibly used to evaluate relation-
ships between absorbed lead and nervous system effects and are not discussed further.
12.4.2.2.2.4 Studies of mentally retarded or behavioral1y abnormal children. Other stu-
dies, of mentally retarded or autistic individuals and infants, have shown such abnormal popu-
lations to have somewhat higher lead levels than the control groups (Beattie et al. , 1975;
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David et al. , 1972, 1976, 1979a,b, 1982a,b, 1983; Moore et al., 1977). However, whether dis-
orders such as mental retardation, hyperactivity, autism, etc. are the causes or the effects
of lead exposure is a difficult issue to resolve, and most of the studies cited employed
study designs not capable of achieving such resolution. Still, results of at least one study
(David et al. , 1983) indicate that chelation therapy leading to reduced lead levels resulted
in some improvement in behavior among a group of retarded individuals, suggesting that lead
may contribute to deviant behavior patterns among such behaviorally abnormal populations, even
if lead was not the key etiological factor originally causing the retardation or other behav-
ioral abnormalities.
12.4.2.2.2.5 Electrophysiological studies of lead effects in children. In addition to
studies using psychometric and behavioral testing approaches, electrophysiological studies of
CMS lead neurotoxicity in non-overtly lead-intoxicated children have been conducted.
Burchfiel et al. (1980) used computer-assisted spectral analysis of a standard EEG exam-
ination on 41 children from the Needleman et al. (1979) study and reported significant EEG
spectrum differences in percentages of low-frequency delta activity and in alpha activity in
spontaneous EEGs of the high-lead children. Percentages of alpha and delta frequency EEG
activity and results for several psychometric and behavioral testing variables (e.g., WISC-R
full-scale IQ and verbal IQ, reaction time under varying delay, etc.) for the same children
were then employed as input variables (or "features") in direct and stepwise discriminant anal-
yses. The separation determined by these analyses for combined psychological and EEG variables
(p <0.005) was reported to be strikingly better than the separation of low-lead from high-lead
children using either psychological (p <0.041) or EEG (p <0.079) variables alone. Unfortun-
ately, no dentine lead or blood lead values were reported for the specific children from the
Needleman et al. (1979) study who underwent the EEG evaluations reported by Burchfiel et al.
(1980), and making it impossible to estimate lead-exposure levels associated with observed EEG
effects. (See also Appendix 12-C).
The relationship between low-level lead exposure and neurobehavioral function (including
electrophysiological responses) in children aged 13-75 months was extensively explored in
another study, conducted at the University of North Carolina in collaboration with the U.S.
Environmental Protection Agency. Psychometric evaluation (Milar et al., 1980, 1981a) revealed
lower IQ scores for children with elevated blood lead levels of 30 ug/dl or higher compared
with children with levels under 30 ug/dl, but the observed IQ deficits were confounded by
poorer home caregiver environment scores in children with elevated blood lead levels (Milar
et al., 1980); and no relationship between blood lead and hyperactive behavior (as indexed by
standardized playroom measures and parent-teacher rating scales) was observed in these child-
ren (Milar et al., 1981a). On the other hand, electrophysiological assessments, including
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analyses of slow cortical potentials during sensory conditioning (Otto et al. , 1981) and EEC
spectra (Benignus et al., 1981), did provide evidence of CMS effects of lead in the same
children. In contrast to psychometric and behavioral findings, a significant linear relation-
ship between blood lead (ranging from 6 to 59 pg/dl) and slow wave voltage (SW) was observed
(Otto et al., 1981) as depicted in Figure l?-3. Analyses of quadratic and cubic trends in SW
voltage, moreover, did not reveal any evidence of a threshold for this effect. The slope of
the blood lead x SW voltage function, however, varied systematically with age. No effect of
blood lead on EEC power spectra or coherence measures was observed, but the relative amplitude
of synchronized EEC between left and right hemispheres (gain spectra) increased relative to
blood lead levels (Benignus et al., 1981). A significant cubic trend for gain between the
left and right parietal lobes was found with a major inflection point at 15 ug/dl. This
finding suggests that EEC gain is altered at blood lead levels as low as 15 (jg/dl, although
the clinical and functional significance of this measure has not been established. A follow-
up study of slow cortical potentials and EEC spectra in a subset (28 children aged 35 to 93
months) of the original sample was carried out two years later (Otto et al., 1982). Slow wave
voltage during sensory conditioning again varied as a linear function of blood lead, even
though the mean lead level had declined by 11 ug/dl (from 32.5 ug/dl to 21.1 pg/dl). The
similarity of SW results obtained at initial and follow-up assessments suggests that the ob-
served alterations in this parameter of CNS function arc persistent, despite a significant
decrease in the mean blood lead level during the two-year interval.
Results of the neurobehavioral study and two-year follow-up described above are important
for several reasons. First, no significant relationship between child IQ and EEC measures was
found in the initial (Benignus et al., 1981; Otto et al., 1981) or follow-up study. SW volt-
age and EEG gain thus appear to provide CNS indices of lead exposure effects that may be both
more sensitive than and independent of standardized psychometric measures used in other stud-
ies. Electrophysiologica^ measures such as these hold considerable promise as indicators of
CNS function that are free of cultural bias and other linguistic and motor constraints atten-
dant to traditional paper-and-pencil or behavioral tests. Observation of a linear relation-
ship between SW voltage and blood lead within a range of 6 to 59 ug/dl, without evidence of
any threshold effect level, is also provocative, particularly in view of the apparent persis-
tence of the effect over a two-year interval. The inflection point in the EEG gain function
at 15 ug/dl provides additional evidence of the effect of lead exposure of CNS function in
young children at levels considerably below those previously considered to be safe (30 ug/dl).
Interpretation of these electrophysiological data, however, must be carefully tempered in view
of: (1) SW voltage and EEG gain are both experimental measures, the clinical and functional
significance of which is presently unknown; (2) estimated effective blood lead levels associ-
ated with the EEG effects are somewhat probalematic because the effects might have resulted
2BPB12/B 12-72 9/20/83
-------
<
I
I
30
20
10
0
-10
-20
-30
-40
20
10
0
-10
-20
-30
20
10
0
-10
-20
J I
I
I I I
1 I
(c)'
o 48 59
D 60 75
I I I
1
I
I
I
I
I
I
I
5 10 15 20 25 30 35 40 45 50 55
PbB LEVEL, ^g/dl
Figure 12-3. (a) Predictad SW voltage and 95% confidence
bounds in 13- and 75-month old children as a function of
blood lead level, (b) Scatter plots of SW data from children
aged 13-47 months with predicted regression lines for
ages 18, 30, and 42 months, (c) Scatter plots for children
aged 48-75 months with predicted regression lines for
ages 54 and 66 months. These graphs depict the linear in-
teraction of blood lead and age.
Source: Otto et al. (1981).
12-73
-------
PRELIMINARY DRAFT
from higher blood lead levels prior to the reported studies; and (3) the study sample was rela-
tively small (n = 43 for the original and 28 for the follow-up SW analyses). In view of these
caveats, these findings need to be replicated in an independent sample. Nevertheless, they
provide clear evidence of altered CNS functioning being associated with relatively low level
lead exposure of non-overtly lead intoxicated children and at least lead levels likely well
below 30 ug/dl.
The adverse effects of lead on peripheral nerve function in children remain to be consi-
dered. Lead-induced peripheral neuropathies, although often seen in adults after prolonged
exposures, are rare in children. Several articles (Anku and Harris, 1974; Erenberg et al.,
1974; Seto and Freeman, 1964), however, describe case histories of children with lead-induced
peripheral neuropathies, as indexed by electromyography, assessment of nerve conduction ve-
locity, and observation of other overt neurological signs, such as tremor and wrist or foot
drop. Frank neuropathic effects have been observed at blood lead levels of 60 to 80 ug/dl
(Erenberg et al. , 1974). In other cases, signs indicative of peripheral neuropathy have been
reported to be associated with blood lead values of 30 |jg/dl. In these latter cases, however,
lead lines in long bones suggest probable past exposures leading to peak blood lead levels at
least as high as 40 to 60 ug/dl and probably in excess of 60 ug/dl (based on the data of Betts
et al., 1973). In each of these case studies, some, if not complete, recovery of affected
motor functions was reported after treatment for lead poisoning. A tentative association has
also been hypothesized between sickle cell disease and increased risk of peripheral neuropathy
as a consequence of childhood lead exposure. Half of the cases reported (10 out of 20) in-
volved inner-city black children, several with sickle cell anemia (Anku and Harris, 1974;
Feldman et al., 1973; Lampert and Schochet, 1968; Seto and Freeman, 1964; Imbus et al., 1978).
In summary, (1) evidence exists for frank peripheral neuropathy in children, and (2) such
neuropathy can be associated with blood lead levels at least as low as 60 ug/dl and, possibly,
as low as 40-60 ug/dl.
Further evidence for lead-induced peripheral nerve dysfunction in children is provided by
the data from two studies by Feldman et al. (1972, 1977) of inner city children and from a
study by Landrigan et al. (1976) of children living in close proximity to a smelter in Idaho.
The nerve conduction velocity results from this latter study are presented in Figure 12-4 in
the form of a scatter diagram relating peroneal nerve conduction velocities to blood lead
levels. No clearly abnormal conduction velocities were observed, although a statistically
significant negative correlation was found between peroneal NCV and blood lead levels
(r = -0.38, p <0.02 by one-tailed t-test). These results, therefore, provide evidence for
significant slowing of nerve conduction velocity (and, presumably, for advancing peripheral
neuropathy as a function of increased blood lead levels), but do not allow clear statements to
be made regarding a threshold for pathologic slowing of NCV.
2BPB12/B 12-74 9/20/83
-------
u
-SC
E
1-
u
o
^
LU
>
z
O
IDUCTIi
2
O
u
oo.uu
82.90
77.60
72.40
67.20
62.00
56.80
51.60
46.40
41.20
36 00
I I I I I I I I I
YICONDUCTION VELOCITY) = 54.8 - .045 x (BLOOD LEAD)
_ (r --0.38) (n - 202) -
~ • —
•
, •
0 —
• •
• • _
, •
* « • .
••
__••••• *•
• ^ •* • . •
(— * •/ • ^* • • —
• •»_•• *^*» •• • •
^^T^jt*?. . * •
~ .^f^^^^T^^-----^ ~
- \-^l . "' •"•• •*
' . . l'*. '
• •
• * *
ii r i i i i i i
0 15 30 45 60 75 90 105 120 135 1E
BLOOD LEAD, M9/dl
Figure 12-4. Peroneal nerve conduction velocity versus blood lead level, Idaho,
1974.
Source: Landrigan et al. (1976).
12-75
-------
PRELIMINARY DRAFT
12.4.3 Animal Studies
The following sections focus on recent experimental studies of lead effects on behavioral,
morphological, physiological, and biochemical parameters of nervous system development and
function in laboratory animals. Several basic areas or issues are addressed: (1) behaviorial
toxicity, including the question of critical exposure periods for concurrent induction or
later expression of behavioral dysfunction in motor development, learning performance, and
social interactions; (2) alterations in morphology, including synaptogenesis, dendritic deve-
lopment, myelination, and fiber tract formation; (3) perturbations in various electrophysiolo-
gical parameters, e.g., ionic mechanisms of neurotransmission or conduction velocities in
various tracts; (4) disruptions of biochemical processes such as energy metabolism and
chemicaT neurotransmission; (5) the persistence or reversibility of the above types of effects
beyond the cessation of external lead exposure; and (6) the issue of "threshold" for neuro-
toxic effects of lead.
Since the initial description of lead encephalopathy in the developing rat (Pentschew and
Garro, 1966), considerable effort has been made to define more closely the extent of nervous
system involvement at subencephalopathic levels of lead exposure. This experimental effort
has focused primarily on exposure of the developing organism. The interpretation of a large
number of studies dealing with early irj vivo exposure to lead has, however, been made diffi-
cult by variations in certain important experimental design factors across available studies.
One of the more notable of the experimental shortcomings of some studies has been the
occurrence of undernutrition in experimental animals (U.S. Environmental Protection Agency,
1977). Conversely, certain other studies of lead neurotoxicity in experimental animals have
been confounded by the use of nutritionally fortified diets, i.e., most commercial rodent
feeds (Michaelson, 1980). In general, deficiencies of certain minerals result in increased
absorption of lead, whereas excesses of these minerals result in decreased uptake (see Chapter
10). Commercial feeds may also be contaminated by variable amounts of heavy.metals, including
as much as 1.7 ppm of lead (Michaelson, 1980). Questions have also been raised about possible
nutritional confounding due to the acetate radical in lead acetate solutions, which are often
used as the source of lead exposure in experimental animal studies ( Barrett and Livesey,
1982).
Another important factor that varies among many studies is the route of exposure to lead.
Exposure of the suckling animal via the dam would appear to be the most "natural" method, yet
may be confounded by lead-induced chemical changes in milk composition. On the other hand,
intragastric gavage allows one to determine precisely the dose and chemical form of admini-
stered lead, but the procedure is quite stressful to the animal and does not necessarily
reflect the actual amount of lead absorbed by the gut. Injections of lead salts (usually
BPB12/A 12-76 9/20/83
-------
PRELIMINARY DRAFT
performed intraperitoneally) do not mimic natural exposure routes and can be complicated by
local tissue calcinosis at the site of repeated injections.
Another variable in experimental animal studies that merits attention concerns species
and strains of experimental subjects used. Reports by Mykkanen et al. (1980) and Overmann et
al. (1981) have suggested that hooded rats and albino rats may differ in their sensitivity to
the toxic effects of lead, possibly because of differences in their rates of maturation and/or
rates of lead absorption. Such differences may account for variability of lead effects
and exposure-response relationships between different species as well.
Each of the above factors may lead to widely variable internal lead burdens in the same
or different species exposed to roughly comparable amounts of lead, making comparison and
interpretation of results across studies difficult. The force of this discussion, then, is to
emphasize the importance of measurements of blood and tissue concentrations of lead in experi-
mental studies. Without such measures, attempts to formulate dose-response relationships are
futile. This problem is particularly evident in later sections dealing with the morpholo-
gical, biochemical, and electrophysiological aspects of lead neurotoxicity. In vitro studies
accorded attention in those sections, in contrast to vn vivo studies, are of limited relevance
in dose-response terms. The ui vitro studies, however, provide valuable information on basic
)
mechanisms underlying the neurotoxic effects of lead.
The following sections discuss and evaluate the most recent studies of nervous system in-
volvement at subencephalopathic exposures to lead. Older studies reviewed in the previous
Air Quality Criteria Document for Lead (U.S. Environmental Protection Agency, 1977) are cited
as needed to illustrate particular points but, in general, the discussion below focuses on
more recent work.
12.4.3.1 Behavioral Toxicity: Critical Periods for Induction and Expression of Effects. The
1977 EPA review (U.S. Environmental Protection Agency, 1977) of animal behavioral studies and
a number of articles since then (e.g., Shigeta et al. , 1977; Zenick et al., 1979; Crofton et
al., 1980; Kimmel, 1983) have pointed to the perinatal period of ontogeny as a particularly
critical time for the induction of behavioral effects due to lead exposure. Such findings are
consistent with the general pattern of development of the nervous system in the experimental
animals that have been investigated (see Reiter, 1982) and are reviewed in some detail in the
ensuing sections of this chapter.
Alterations in the behavior of rats exposed after weaning or after maturation have also
been reported (Angell and Weiss, 1982; Cory-Slechta and Thompson, 1979; Cory-Slechta et al.,
1981; Donald et al., 1981; Geist and Mattes, 1979; Lanthorn and Isaacson, 1978; Nation et al.,
1982; Shapiro et al., 1973). These findings stand in contrast to results from other studies
showing some effects in rats as being produced only by early perinatal exposure (e.g., Brown,
BPB12/A 12-77 9/20/83
-------
PRELIMINARY DRAFT
1975; Brown et al., 1971; Padich and Zenick, 1977; Shigeta et al., 1977; Snowdon, 1973).
Nevertheless, behavioral effects of relatively low-level exposure to lead have also been noted
in adult subjects of other species, including pigeons (Barthalmus et al., 1977; Dietz et al.,
1979) and fish (Weir and Mine, 1970), and the effects of lead exposure during adulthood are
not to be dismissed as inconsequential, although the present evaluation focuses mainly on the
effects of lead exposure early in development.
12.4.3.1.1 Development of motor function and reflexes. A variety of methods have been used
to assess the effect of lead on the ability of experimental animals to respond appropriately,
either by well defined motor responses or gross movements, to a defined stimulus. Such
responses have been variously described as reflexes, kineses, taxes, and "species-specific"
behavior patterns. The air righting reflex, which refers to the ability to orient properly
with respect to gravity while falling through the air and to land on one's feet, is only one
of several commonly used developmental markers of neurobehavioral function (Tilson and Harry,
1982). Overmann et al. (1979) found that development of this particular reflex was slowed in
rat pups exposed to lead via their dams (0.02 or 0.1 percent lead as lead acetate in the dams'
drinking water). However, neither the auditory startle reflex nor the ability to hang
suspended by the front paws was affected.
Grant et al. (1980) exposed rats indirectly to lead j_n utero and during lactation through
the mothers' milk and, after weaning, directly through drinking water containing the same
lead concentrations their respective dams had been given. In addition to morphological and
physical effects [see Sections 12.5, 12.6, and 12.11 for discussions of this work as reported
by Kimmel et al. (1980), Fowler et al. (1980), Faith et al. (1979), and Luster et al. (1978)],
there were delays in the development of surface righting and air righting reflexes in subjects
exposed under the 0.005 and 0.025 percent lead conditions; other reflexive patterns showed no
effect. The median blood lead concentration for the 0.005-percent subjects at postnatal day
(PND) 11 was 35 pg/dl; the median brain lead concentration was 0.07 ug/g. Locomotor develop-
ment generally showed no significant alteration due to lead exposure. Body weight was signif-
icantly depressed for the most part in the 0.005- and 0.025-percent pups.
The ontogeny of motor function was also investigated by Overmann et al. (1981). Exposure
of pups to lead was limited to the period from parturition to weaning and occurred through
adulteration of the dams' drinking water with lead acetate (0.01 or 0.1 percent lead acetate).
The development of swimming performance was assessed on alternate days from PND 6 to 24. No
alterations in swimming ability were found. Rotorod performance was also tested at PND 21,
30, 60, 90, 150, and 440. Overall, the ability to remain on a rotating rod was significantly
impaired (p <0.01) at 0.1 percent and tended to be impaired (0.10 > p > 0.05) at 0.1 percent
(blood lead values were not reported). However, data for individual days were statistically
BPB12/A 12-78 9/20/83
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PRELIMINARY DRAFT
significant only on PND-60 and 150. An adverse effect of lead exposure on rotorod performance
at PND 30-70 was also found in an earlier study by Overmann (1977) at a higher exposure level
of 30 mg/kg lead acetate by intubation (average PbB value at PND 21 was 173.5 ± 32.0 ug/dl).
At blood lead concentrations averaging 33.2 ± 1.4 ug/dl, however, performance was not
impaired. Moreover, other studies using rotorods at average blood lead concentrations of
approximately 61 ug/dl (Zenick et al. , 1979) and 30 to 48 ug/dl (Grant et al., 1980) have not
found significant effects of lead on such performance when tested at PND 21 and 45, respec-
tively. Comparisons between studies are confounded by differences in body weight and age at
time of testing and by differences in speed and size of the rotorod apparatus (Zenick et al.,
1979).
Delays in the development of gross activity in rat pups have been reported by Crofton et
al. (1980) and by Jason and Kellogg (1981). It should be noted that very few studies have
been designed to measure the rate of development of activity. Ideally, subjects should be
assessed daily over the entire period of development in order to detect any changes in the
rate at which a behavior pattern occurs and matures. In the study by Crofton et al. (1980),
photocell interruptions by pups as they moved through small passageways into an "exploratory
cage" adjacent to the home cage were automatically counted on PND 5 to 21. Pups exposed jji
utero through the dams' drinking water (0.01 percent solution of lead as lead chloride) lagged
controls by approximately one day in regard to characteristic changes in daily activity count
levels starting at PND 16. (Blood lead concentrations at PND 21 averaged 14.5 ± 6.8 ug/dl for
representative pups exposed to lead iji utero and 4.8 ± 1.5 ug/dl for controls.) Another form
of developmental lag in gross activity around PND 15-18, as measured in an automated activity
chamber, was reported by Jason and Kellogg (1981). Rats were intubated on PND 2-14 with lead
at 25 mg/kg (PbB = 50.07 ± 5.33 ug/dl) and 75 mg/kg (PbB = 98.64 ± 9.89 ug/dl). In this case,
the observed developmental lag was in the characteristic decrease in activity that normally
occurs in pups at that age (Campbell et al. , 1969; Melberg et al., 1976); thus, lead-exposed
pups were significantly more active than control subjects at PND 18.
One question that arises when ontogenetic effects are discovered concerns the possible
contribution of the lead-exposed dam to her offsprings' slowed development through, for exam-
ple, reduced or impaired maternal care giving behavior. A detailed assessment of various
aspects of maternal behavior in chronically lead-exposed rat dams by Zenick et al. (1979),
discussed more fully in Section 12.4.3.1.4, and other studies using cross-fostering techniques
(Crofton et al., 1980; Mykkanen et al., 1980) suggest that the deleterious effects observed in
young rats exposed to lead via their mothers' milk are not ascribable to alterations in the
dams' behavior toward their offspring. Chronically lead-exposed dams may, if anything, tend
to respond adaptively to their developmentally retarded pups by, for example, more quickly
retrieving them to the nest (Davis, 1982).
BPB12/A 12-79 9/20/83
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PRELIMINARY DRAFT
12.4.3.1.2 Locomotor activity. The spontaneous activity of laboratory animals has been meas-
ured frequently and in various ways as a behavioral assay in pharmacology and toxicology
(Reiter and MacPhail, 1982). Such activity is sometimes described as gross motor activity or
exploratory behavior, and is distinguished from the motor function tests noted in the previous
section by the lack of a defined eliciting stimulus for the activity. With reports of hyper-
activity in lead-exposed children (see Section 12.4.2), there has naturally been considerable
interest in the spontaneous activity of laboratory animals as a model for human neurotoxic
effects of lead (see Table 12-2). As the 1977 review (U.S. Environmental Protection Agency,
1977) of this material demonstrated, however, and as other reviews (e.g., Jason and Kellogg,
1980; Michaelson, 1980; Mullenix, 1980) have since confirmed, the use of activity measures as
an index of the neurotoxic effects of lead has been fraught with difficulties.
First, there is no unitary behavioral endpoint that can be labeled "activity." Activity
is, quite obviously, a composite of many different motor actions and can comprise diverse be-
havior patterns including (in rodents) ambulation, rearing, sniffing, grooming, and, depending
on one's operational definition, almost anything an animal does. These various behavior pat-
terns may vary independently, so that any gross measure of activity which fails to differen-
tiate these components will be susceptible to confounding. Thus, different investigators'
definitions of activity are critical to interpreting and comparing these findings. When these
definitions are sufficiently explicit operationally (e.g., activity as measured by rotations
of an "activity wheel"), they are frequently not comparable with other operational definitions
of activity (e.g., movement in an open field as detected by photocell interruptions). More-
over, empirical comparisons show that different measures of activity do not necessarily cor-
relate with one another quantitatively (e.g., Copobianco and Hamilton, 1976; Tapp, 1969).
In addition to these rather basic difficulties, activity levels are influenced greatly by
numerous variables such as age, sex, estrous cycle, time of day, novelty of environment, and
food deprivation. If not controlled properly, any of these variables can confound measure-
ments of activity levels. Also, nutritional status has been a frequent confounding variable
in experiments examining the neurotoxic effects of lead on activity (see the review by U.S.
Environmental Protection Agency, 1977; Jason and Kellogg, 1980; Michaelson, 1980). In
general, it appears that rodents exposed neonatally to sufficient concentrations of lead
experience undernutrition and subsequent retardation in growth; and, as Loch et al. (1978)
have shown, retarded growth per se can induce increased activity of the same types that were
attributed to lead alone in some earlier studies.
In view of the various problems associated with the use of activity measures as a behav-
ioral assay of the neurotoxic effects of lead, the discrepant findings summarized in Table
12-2 should come as no surprise. Until the measurement of "activity" can be better
BPB12/A 12-80 9/20/83
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PRELIMINARY DRAFT
TABLE 12-2. EFFECTS OF LEAD ON ACTIVITY IN RATS AND MICE
Increased
Decreased
Age-dependent,
qualitative, mixed or
no change
Driscoll and Stegner
(1978)
Goiter and Michaelson
(1975)
Kostas et al. (1976)
Overmann (1977)
Petit and Alfano (1979)
Sauerhoff and Michaelson
(1973)
Silbergeld and Goldberg
(1973, 1974a,b)
Weinreich et al. (1977)
Winneke et al. (1977)
Driscoll and Stegner
(1976)
Flynn et al. (1979)
Gray and Reiter (1977)
Reiter et al. (1975)
Verlangieri (1979)
Barrett and Livesey (1982)
Brown (1975)
Crofton et al. (1980)
Cutler (1977)
Dolinsky et al. (1981)
Dubas and Hrdina (1978)
Geist and Balko (1980)
Geist and Praed (1982)
Grant et al. (1980)
Gross-Selbeck and
Gross-Selbeck (1981)
Hastings et al. (1977)
Jason and Kellogg (1981)
Kostas et al. (1978)
Krehbiel et al. (1976)
Loch et al. (1978)
Minsker et al. (1982)
Mullenix (1980)
Ogilvie and Martin (1982)
Rafales et al. (1979)
Schlipkoter and Winneke (1980)
Sobotka and Cook (1974)
Sobotka et al. (1975)
Zimering et al. (1982)
BPB12/A
12-81
9/20/83
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PRELIMINARY DRAFT
standardized, there appears to be little basis for comparing or utility in further discussing
the results of studies listed in Table 12-2.
12.4.3.1.3 Learning ability. When animal learning studies related to the neurotoxic effects
of lead were reviewed in 1977 (U.S. Environmental Protection Agency, 1977), a number of criti-
cisms of existing studies were noted. A major limitation of early work in this field was the
lack of adequate information on the exposure regimen (dosage of lead, how precisely adminis-
tered, timing of exposure) and the resulting body burdens of lead in experimental subjects
(concentrations of lead in blood, brain, or other tissue; time course of blood or tissue lead
values, etc.). A review of studies appearing since 1977 reveals a notable improvement in this
regard. A number of more recent studies have also attempted to control for the confounding
factors of litter effects and undernutrition—variables that were generally not controlled in
earlier studies.
Unfortunately, other criticisms are still valid today. The reliability and validity of
behavioral assays remain to be established adequately, although progress is being made. The
reliability of a number of common behavioral assays for neurotoxicity is currently being de-
termined by several independent U.S. laboratories (Kimmel et al. , 1982). The results of this
program should heli- standardize some behavioral testing procedures and perhaps create some
reference methods in behavioral toxicology. Also, as well-described studies are replicated
within and between laboratories, the reliability of certain experimental paradigms for demon-
strating neurotoxic effects is effectively established.
Some progress is also being made in dealing with the issue of the validity of animal be-
havioral assays. As the neurological and biochemical mechanisms underlying reliable behavi-
oral effects become better understood, the basis for extrapolating from one species to another
becomes stronger and more meaningful. An awareness of different species' phylogenetic, evo-
lutionary, and ecological relationships can also help elucidate the basis for comparing
behavioral effects in one species with those observed in another (Davis, 1982).
Tables 12-3 and 12-4 summarize exposure conditions, testing conditions, and results of a
number of recent studies of animal learning (see U.S. Environmental Protection Agency, 1977,
for a summary of earlier studies). Some general issues emerge from an examination of these
studies. One point of obvious interest is the lowest level of exposure at which behavioral
effects are clearly evident. Such a determination is best done on a species-by-species basis.
Rats seem to be the species of choice for the great majority of the behavioral studies,
despite the concerns that have repeatedly been expressed concerning the appropriateness of
this species as a subject for behavioral investigation (e.g., Lockard, 1968, 1971; Zeigler,
1973). Of the studies not obviously confounded by nutritional or litter effects, those by
Winneke et al. (1977, 1982c) and by Cory-Slechta and Thompson (1979) report alterations in
BPB12/A 12-82 9/20/83
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TABLE 12-3. RECENT ANIMAL TOXICOLOGY STUDIES OF LEAD EFFECTS ON LEARNING IN RODENT SPECIES
PO
i
CO
tO
Experimental
animal
(species
Reference or strain)
Hastings Rat
et al. (L-E)
(1977)
Overmann Rat
(1977) (L-E)
Padich Rat
and Zenick (CD)
(1977)
Winneke Rat
et al. (W)
(1977)
Lead exposure Treatment
Pb cone.
(medium)
0.01
or
0.05X
(water)
10,
30, or
90 mg/kg
(gavage)
375
mg/kg
(water)
372
mg/kg
(food)
period groups
(route) (n)
PND 0- C (12)
21 Pb, (12)
(dam's Pb2 (12)
milk)
PND 3-21 C
(direct) Pb, 12-
Pb2 25
Pb3 ea.
Preconception 0-0 (10)
to 0-Pb (10)
a) Weaning Pb-0 (10)
(via dam) Pb-Pb (10)
or
b) termination
(via dan
and direct);
or
Weaning to
termination
(direct only)
Preconception C (20)
- Testing Pb (20)
(via dam
and
direct)
Litters
per
group
Random
selection
fron 9
litters
j
5
5
5
5
,
(random
selection
from
110 male
pups)
Tissue lead
(age measured)
PbB
(20 d):
C: 11 pg/dl
Pb,: 29
Pb2: 42
(60 d):
C: 4
Pb,: 5
Pb2: 9
PbB (21 d)
C: 15 |jg/dl
Pbj: 33.2
Pb2: 173.5
Pb3: 226.1
»
PbB
(-16 d):
C: 1.7 M9A11
Pb: 26.6
(-190 d)
Pb: 28.5
Testing
Age at paradigm
testing (task)
~90- Operant
186 d (successive
brightness
discrin. )
26-29 d Aversive
conditioning
(1) active
2) passive)
67-89 d Operant
(inhibit
response)
E-maze
(discrin. :
79-101 d 1) spatial
83-105 d 2) tactile
95-117 d 3) visual)
42- Operant
? d (FR 20)
100- Lashley
200 d jumping
stand
(visual
discrim.
of stimulus:
1) orientation
2) size)
Non-
behavioral
effects
None
None
Body wt.
of Pb-Ss
< 0-Ss from
birth to
weaning.
Body wt. of
Pb-Ss > C-Ss;
however, sTze
of Pb-Ss
litters
< C-S
litters.
Behavioral
results
No sig. differences
between Pb-Ss
and C-Ss in
learning original
or reversed
discrim. task.
Pb3-Ss sig. slower
in acquisition and
extinction of active
avoidance response;
no sig. diffs. for
passive avoidance.
All Pb groups failed
to inhibit responses
as well as C-Ss.
No sig. diffs. on E-
maze tasks, except
Pbz>3-Ss sig. worse
than C-Ss when on
tactile discrira.
Pb-Pb group
had sig. fewer
rewarded responses
even though
responding at
sig. higher rate.
Pb-Ss sig. slower
to learn size
discrimination;
no diff. between
Pb and C groups
on orientation
discrim. (a rela-
tively easy task).
-------
TABLE 12-3. (continued)
Experimental
animal
(species
Reference or strain)
Dietz Rat
et al. Expt. l(L-E)
(1978)
Expt. 2(CD)
Lanthorn Rat
& Isaacson (L-E)
r? (1978)
r\j \ Aft **j
1
CO
Cory- Rat
Schlecta (S-0)
&
Thompson
(1979)
Cory- Rat
Schlecta (S-D)
et al.
(1981)
Lead exposure Treatment
Pb cone.
(medium)
200
»g/kg
(gavage)
0.01X
(water)
0.27X
(water)
(1) 0.005,
(2) 0.03,
or
(3) 0.1X
(water)
(1) 0.01
or
(2) 0.03%
(water)
period groups
(route) (n)
PNO C (6)
3-30 Pb (7)
(direct)
Preconcep- C (4)*
tion to Pb (4)
termination
(via dan
until weaning,
then direct)
Adult C (4)
(direct) Pb (6)
PNO la:
20- C (4)*
(a) 70 Pb (5)
or Ib:
(b) 150 C (4)*
(direct) Pb (6)
2-
C (3)*
Pb (4)
3:
C (4)*
Pb (5)
PND C (4)
21-? Pbt (5)
Pb2 (5)
Litters
per Tissue lead Age at
group (age measured) testing
? 3 mo
2, split or
21 mo
? ? 8 mo
? ? Adul t
random PbB (150 d): 55-
assign- C: -6 ug/dl 140 d
ment la: -3
Ib: -7
2: -27
3: -42
random Brain-Pb 55-
assign- (past-test): ? d
Bent C: 14-26 ng/g
Pb,: 40-142
Pb2: 320-1080
Testing
paradigm
(task)
Operant
(minimum
20- sec pd.
between
bar-presses)
T-raaze
(1) spontan.
alternation
2) light
di scrim.
3) spatial
di scrim. )
Operant
(FI-
30 sec)
Operant
(minimum
duration
bar- press)
Non-
behavioral
effects
None
Pb-body
wt. lower
1 wk. prior
to test; C
reduced to
same wt. at
test.
C-Ss
pair- fed
to control
for loss
of body
wt.
None
None
Behavioral
results
Short IRTs (£4 sec)
more prevalent in
Pb-Ss than in
C-Ss, but did not
result in differsnt
reward rates; Pb-Ss
showed higher varia-
bility in response-
rate under d-amphet-
amine treatment.
•o
Pb-Ss had sig. ^
lower rate of i—
spontaneous alterna- ^
tion; Pb-Ss sig. •— i
slower than C-Ss ^
only on 1st spatial 'X
discrim. task. ~<
o
30
Increased response — i
rate and inter-S
variability in groups
Pb! , and Pb2; de-
creased response rate
in group Pb3; effects
in Pbt reversed after
exposure terminated.
Pb groups impaired:
decreased response
durations; increased
response latencies;
failure to improve
performance by
external stimulus
control.
"Weight-matched controls
-------
TABLE 12-3. (continued)
i
00
01
Experimental
animal Lead exposure Treatment Litters
(species Pb cone.
Reference or strain) (medium)
Geist fiat
& Mattes (S-D)
(1979)
F>ynn fiat
et al. (L-E)
(1979) Expt. 1
Expt. 2
Expt. 3
Petit Rat
& Alfano (L-E)
(1979)
0.001
or
0.0025X
(water)
0.25X
(water)
0.1X
(water) ,
225 mg/kg
(gavage),
0.25X
(water)
same
as above
except 90
•gAg
(gavage)
0.2 or
2%
(food)
period
(route)
PNO 23-
termi nation
(direct)
Preconception
- PND 22
(via dam)
Preconception
- Birth
(via dam),
Birth -
Weaning
(direct),
Weaning
- termination
(direct)
same as
above except
stopped at
PNO 33
PND
1-25
groups per Tissue Lead Age at
(n) group (age measured) testing
C (7) ? ? 58-
Pbt (7) ? d
Pb4 (7)
C (8) 8 Brain-Pb (3 d): ?
Pb (10) 10
C: ~0
Pb: 0.174 ug/g
(30-34 d):
no sig. diffs.
C (12) 6 (75-76 d): 49-
Pb (12) 6 C: 0.13 ug/g 58 d
Pb: 1.85
C (10) . see above 58-
Pb (10) * 60 d
C, (22) ~7 PbB 66-
C' (22) each; (25 d): 115 d
nlt (22) split C: 2 ug/dl
Pb/ (22) for "i" Pb^ 331
Pb2? (22) (isola- Pba: 1297
Pb2 (22) tion and
6 "e" (en-
richment)
conditions
Testing Non-
paradigm behavioral
(task) effects
Hebb-
Willians
maze
(find way
to goal
box)
Radial
arm maze
(spontaneous
alternation)
Passive
avoidance
(remain
in 1 of 2
compartments
to avoid
elect, shock)
Shuttle-box
signalled
avoidance
(move from one
compartment to
other to avoid
elect, shock)
Hebb-
Williams
maze
(find way
to goal
box)
Passive
avoidance
(remain
in compart-
ment to
avoid shock)
None
Brain wts.
of Pb-Ss
< C-SsT
no other
differences.
None
None
Body wts.
of Pb2-Ss
< C-Ss,
Pbj-Ss
> C-Ss;
gross
toxicity
in Pbz-Ss;
lower
brain
wts. in
Pb-Ss
Behavioral
results
Pbj- and Pb2-Ss
made sig. nore
errors than C-Ss;
Pb2-Ss slower
than~C-Ss to
traverse maze.
No sig. difference
between Pb-Ss
and C-Ss.
No sig. difference
in trials to criterion,
but Pb-Ss made
sig. fewer partial
excursions from
"safe" compartment.
No sig. difference
between Pb-Ss
and C-Ss.
No sig. diff.
between Pb- and C-
Ss in naze learning;
Tsol ati on-reared
Pb-Ss less success-
ful than C.-Ss
on passive-avoidance
task; enrichment-
reared Pbj-Ss = C -Ss
but Pb2 -Ss~sig.
worse on passive
avoidance.
-D
30
m
i— i
^
'Z.
TO
O
70
— 1
-------
TABLE 12-3 (continued;
Experimental
animal Lead exposure Treatment Litters
(species Pb cone.
Reference or strain) (nedium)
Zenick Rat 500
et al. (CO) ngAg
(1978) (water)
Zenick Rat 375
et al. (CO) mg/kg
(1979) (water)
Hastings Rat 0.01
et al. (L-E) or
(1979) 0.1X
(water)
,
ro
CO
cr,
Schlipkbter Rat 0.23X
& Winneke (?) (food)
(1980) Expt. 1
Expt. 2 0.07SX
(food)
Expt. 4 0.025
or
0.075X
(food)
period groups per
(route) (n) group
Preconception C (10) 5
- Weaning Pb (10) 5
(via dan)
Preconception 0-0 (?) 5
to Pb-0 (?) 5
a) Weaning Pb-Pb (?) 5
(via dan)
or
b) teroi nation
(via dan and direct)
PND 0 C (23) Random
- 21 Pb, (11) selection
(daw's Pbj (13) from
•ilk) 15
litters
Preconception C (?) ?
- PND 120 Pbj (18)
(via dam
and direct)
a) Prenatal- C (10) ?
7 «o Pb2 (10)
(via dan Pb2* (10)
and direct) °
b) Prenatal -
Weaning
(via dam)
Expt. 2 C (14) ?
ft>3 (14)
Pb3* (14)
Prenatal C (10) ?
- 7 mo Pb., (10)
(via dam pb«" (10)
and direct) D
Tissue lead Age at
(age measured) testing
? 30-
40 d
55-
63 d
? 42-
? d
PbB (20 d): 120 d
C: 11 ug/dl
Pbj: 29
Pb2: 65
270 d
Brain-Pb
(20 d):
C: 12.5 ug%
Pba: 29 330 d
Pb2: 65
PbB 7 mo
all C: <5 ug/dl
Pb! (120 d):
39.5
(8 mo):
12.0
Pb, :
(21ad) 29.2
(7 mo) 27.0
(21bd) 29.2
(7 mo) 5.2
Pb, :
(21ad) 29.9
(7 mo) 30.8
(21bd) 29.9
(7 mo) 1.8
(120 d)
Pb« : 17.8
Pb«*: 28.6
Testing Non-
paradigm behavioral
(task) effects
Water T-maze Body wt. of
1) black-white Pb-Ss <
discrim. C-Ss from
2) shape birth to
discrim. 50 d.
Operant Body wt.
(FI-1 min) of Pb-Ss
< 0-Ss
from birth
to weaning.
(1) Operant None
(simult. vis.
discrim. )
(2) T-maze
(success, vis.
discrim. )
(3) Operant
(go/no-go task)
Lashley ?
jumping
stand
(cue -
size
discrim. )
" ?
it ^
Water ?
maze
(spatial
discrim. )
Behavioral
results
On both discrim.
tasks, Pb-Ss
made sig. more
errors with sig.
shorter response
Pb-Pb group had sig.
fewer rewarded
responses across
sessions than Pb-0
or 0-0 groups.
Pb2-Ss sig. slower
to reach criterion
than C-Ss on
simultaneous visual
discrimination task;
no sig. differences
on successive and
go/no-go discrim.
tasks.
Sig. increase in
error repetition
by Pbi-$s.
Non-sig. (p <0.10)
increase in error
repetition by Pb2-Ss.
No sig. diffs.
between Pb3-Ss
and C-Ss.
35% of Pb«-|s failed
to reach criterion
(vs. 10% C-Ss); 35%
also failed retest
after 1 wk (vs. 0%
C-Ss).
TO
m
t —
|5
•z.
70
•<
a
70
-r\
—1
-------
PRELIMINARY GRAFT
TABLE 12-3. (continued)
Experimental
animal
(species
Reference or strain)
Gross-
Selbeck
& Gross-
CA 1 t%AV>lr
be 1 beck
(1981)
Angel 1
& Weiss
(1982)
Milar
-; et al.
7s (1981b)
CO
Nation
et al.
(1982)
Winneke
et al.
(1982c)
Rat Ft
(W)
Lead exposure
Pb cone.
(medium)
0.5 g/kg
(food)
period
(route)
Treatment
groups
(n)
Litters
per
group
Postweaning C (6) ?
- termination Pb (6)
(direct)
F2 " Preconception C (6) ?
- Weaning Pb (6)
Rat
(L-E)
Rat
(L-E)
Rat
(S-D)
Rat
(W)
Expt. 1
Expt. 2
0.1X
(water)
25, 100,
or 200
•tg/kg
10 mg/kg
(food)
0.004,
0.012,
or
0.037X
(food)
(via dam)
PNO 3-21
(dam's
milk)
and/or
21-130
(direct)
PND 4-31
(direct)
PND 100-
termi na-
tion
(direct)
0-0 (20)
0-Pb (20)
Pb-0 (24)
Pb-Pb (24)
C (10)
Pbi (5)
Pb* (4)
C (8)
Pb (8)
Preconception C(16)
- Testing
(via dam
and
direct)
-Continuation of Expt.
Pbi (16)
Pb4 (16)
Pb3 (16)
1- C (10)
Pb2 (10)
Pb3 (10)
5, split
6, split
3
4
4
?
Random
selection
from 5-6
litters
per condi-
tion
(females
dropped;
Tissue lead
(age measured)
PbB
(-180 d):
C: 6.2 ug/dl
Pb: 22.7
(-110 d):
C: 3.7
Pb: 4.6
PbB(130d):
0-0: 2 ug/dl
0-Pb: 66
Pb-0: 9
Pb-Pb: 64
PbB (32 d)
C: 5 ug/dl
Pb!: 26
PbK: 123
7
7
?
no Pb! group
for Expt.
2)
Testing
Age at paradigm
testing (task)
Adult Operant
(DRH)
3-4
mo
58- Operant
130 d (Mult
FI-TO-
FR-TO)
50 d Operant
(spatial
alternation
levers)
156 d Operant
(conditioned
suppression
of respond-
ing on mult.
VI schedule)
70- Shuttle-box
100 d signalled
avoidance
(move from
one compart-
ment to avoid
elect, shock)
190- Lashley
250 d jumping stand
(size discrim
Non-
behavioral
effects
None
11
Pb-Pb Ss
sig. lower
body wt.
postweaning
Pb2-Ss
sig.
slower
rate
None
ALA-D at
90 d:
C: 7.05 U/l
Pbj: 4.26
Pb2: 1.92
Pb3: 1.18
. )
Behavioral
results
Both F, and F2
(especially F2)
Pb-Ss had greater
% rewarded responses
than C-Ss, i.e. ,
Pb-Ss bar-pressed
at higher rate
than C-Ss.
Groups exposed post-
weaning (0-Pb, Pb-
Pb) had longer
Inter- Response
Times; group ex-
posed preweaning
(Pb-0) had
shorter IRTs.
No sig. differences
between C-Ss
and Pb-Ss.
Presentation of tone
associated with
electrical shock
disrupted steady-
state responding
more in PB-Ss than
in C-Ss.
Expt. 1 Pb-Ss sig.
faster than~C-Ss to
learn avoidance
response.
Expt. 2 Pb-Ss
sig. slower~than
C-Ss to learn
size discrim.
-o
m
»— i
t — i
-c.
0
3>
— 1
-------
TABLE 12-3 (continued)
_l
PO
1
oo
00
Reference
Taylor
et al.
(1982)
Kowalski
et al.
(1982)
McLean
et al.
(1982)
"Inferred
Experimental
animal Lead exposure
(species Pb cone. period
or strain) (medium) (route)
Treatment Litters
groups per
(n) group
Rat 0.01 Preconception C (12) 6*
(CO) or - Weaning Pbj (16) 8*
0.02X (via dam) C2 (4) 2*
(water) Pb* (4) 2*
Mouse 0.0002% Adult
(Wistar) (water) (direct)
Mouse 0.002 or Adult
(Swiss) 0.2X (direct)
(water)
from information in report.
C (16) ?
Pb (16)
C (16) ?
Pbj (16)
Pb2 (16)
Tissue lead
(age measured)
PbB (21 d)
C: 3.7 |jg/dl
Pb,: 38.2
Pb2: 49.9
7
?
Testing Non-
Age at paradigm behavioral
testing (task) effects
11 d Runway None
(traverse
alley to
reach dan
and dry
suckle)
(13 d Water T-maze None
after (spatial
start of discrim. )
exposure)
(10 d Water T-maze None
after (spatial
start of di scrim.)
expos . )
Behavioral
results
No sig. diffs.
in acquisition of
response, but
both Pb groups
sig. slower to
extinguish when
response no longer
rewarded.
Pb-Ss made more
errors than C-Ss;
food deprivation
exacerbated effect.
Pb-Ss showed no
improvement in
performance com-
pared to C-Ss.
;o
m
i
i— i
^
2
>
-c
o
-n
-H
Abbreviations:
?
ALA-D
C
CD
DRH
Fj
F2
FI
FR
IRT
L-E
N/A
information not given in report
delta Aminolevulinic Acid Dehydrase
Control group
substrain of Sprague Dawley
Differential Reinforcement of High
1st Filial generation
2nd Filial generation
Fixed Interval
Fixed Ratio
Inter Response Time
Long Evans
Not Applicable
response rates
NaAc
Pb
Pb(Ac)2
PbB
PND
S
s-o
TO
U/l
VI
W
WGTA
X
sodium acetate
lead-exposed group
lead acetate
blood lead
Post-Natal Day
Subject
Sprague Dawley
Tine Out
pmol ALAO/nin x liter erythrocytes
Variable Interval
Wistar
Wisconsin General Testing Apparatus
experimental group
-------
TABLE 12-4. RECENT ANIMAL TOXICOLOGY STUDIES OF LEAD EFFECTS ON LEARNING IN PRIMATES
ro
i
00
to
Experimental
animal
(species
Reference or strain)
Bushnel 1 Monkey
& Bowman (Macaca
(1979a> mulatto)
Expt. 1
Expt. 2
Test 1
TAF *• t
lest £
T»^ + ^
lest a
Bushnel 1 Monkey
& Bowman (Macaca
(1979b) mulatta)
Lead exposure
Pb cone. period
(medium) (route)
-0.07 or Daily for
0.16X 1st yr
(•ilk) (direct,}
adjusted
to main-
tain tar-
get PbB
—same as Expt. 1—
Treatment Litters
groups per
(n) group
C (4) N/A
Pb, (3)
Pbj. (3)
C (4) N/A
Pb, (4)
Pbz (4)
Continuation of Expt. 2 —
_* f A. <*
after exposure terminated at 12 mo
--Continuation of Bushnell & Bowman (1979a)--
Tissue lead
(age measured)
PbB (1st yr):
C: ~5 pg/dl*
Pb,: 37*
Pb2: 58*
PbB (1st yr):
C: ~4 ug/dl*
Pb,: 32*
Pb2: 65*
r\L_ n / 1 C
PbB (16
BO):
C: ~5 pg/dl
Pbj: 19
Pb2: 46
PbB (56
mo):
C: 4 ug/dl
Pb * * 5
Pbz: 6
Testing Non-
Age at paradigm behavioral
testing (task) effects
5- WGTA (form None
10 mo di scrim.
reversal
learning)
1.5- 2-choice None
4.5 «o maze
(discr.
reversal
learning)
non-food
reward
5- WGTA None
12 mo (series
of 4
reversal
discr.
problems)
^ C U/*TA UnAa
is wti I A None
mo (discr.
reversal
learning,
more
49- WGTA None
55 mo (spatial
discr.
reversal
learning)
Behavioral
results
Both Pb-exposed
groups retarded
in reversal learn-
ing; Pb2-Ss
especially impaired
on 1st reversal
following over-
training.
Pb2-Ss sig.
retarded on 1st
reversal (confirms
Expt. 1 using different
task and reward to
control for possible
confounding by motiva-
tional or motor
factors).
Both Pb groups
retarded in
reversal learning;
Pb2-Ss
impaired on 1st
reversals regard-
less of prior over-
training.
DK ~Cc viat ^«*/4aH
Pu2-bs retaraeo
on 1st reversal.
Both Pb-exposed
groups retarded
in reversal learn-
ing; 3 Pbj-Ss
failed to retain
-o
TO
m
•z.
TO
O
5
^
motor pattern for
operating WGTA
from 2 yrs
earlier.
"Corrected annual averages obtained fron Bushnell (1978)
-------
TABLE 12-4 (continued)
Reference
Rice
& Willes
(1979)
Rice
et al.
(1979)
Experimental
animal
(species
or strain)
Monkey
(Macaca
fascicu-
laris)
Lead exposure
Pb cone.
(medium)
500
ugA9
(•ilk)
Continuation
period
(route)
Daily
for 1st
year
(direct)
of Rice &
Treatment Litters
groups per
(n) group
C (4) N/A
Pb (4)
Tissue lead
(age measured)
PbB
(200 d):
C: <5 ug/dl
Pb: 35-70
(400 d):
Pb: 20-50
PbB (400+ d):
20-30 ug/dl
Age at
testing
421-
714 d
2.5-
3 yr
Testing
paradigm
(task)
WGTA
(form
di scrim.
reversal )
Operant
(mult. FI-
TO)
Non-
behavioral
effects
None
None
Behavioral
results
Pb-Ss slower
to learn successive
reversals.
Pb-Ss responded
at higher rates, had
shorter IRTs,
PO
o
TO
and tended to
respond more during S
time-out (unrewarded) i— i
CD
70
Abbreviations:
? information not given in report
ALA-D delta Aminolevulinic Acid Dehydrase
C Control group
CD substrain of Sprague Dawley
DRH Differential Reinforcement of High response rates
Ft 1st Filial generation
F2 2nd Filial generation
FI Fixed Interval
FR Fixed Ratio
IRT Inter Response lime
L-E Long Evans
N/A Not Applicable
NaAc
Pb
Pb(Ac)2
PbB
PND
S
S-D
TO
U/l
VI
W
WGTA
X
sodium acetate
lead-exposed group
lead acetate
blood lead
Post-Natal Day
Subject
Sprague Dawley
Time Out
umol ALAD/min x liter erythrocytes
Variable Interval
Wistar
Wisconsin General Testing Apparatus
experimental group
-------
PRELIMINARY DRAFT
learning task performances by rats with blood lead levels below 30 ug/dl. Winneke et al.
(1977) exposed Wistar rats jm utero and postnatally to a diet containing 0.07 percent lead as
lead acetate. Between PND 100 and 200 the subjects were tested on two types of visual discri-
mination learning tasks using either "easy" stimuli (vertical vs. horizontal stripes) or
"difficult" stimuli (white circles or differing diameters). Blood lead concentrations were
measured at about PND 16 (26.6 ug/dl) and PND 190 (28.5 ug/dl). Although there were no
significant differences between lead-exposed and control subjects on the easy discrimination
task, the lead-exposed subjects performed significantly (p <0.01) worse than controls on the
size discrimination task. The performance of the lead group continued around change level
(50 percent correct) essentially throughout the 4-week training period; control subjects began
to improve after about 2 weeks of training and reached an error rate of about 15 percent by 3
to 4 weeks. Stated differently, 8 out of 10 control animals reached criterion performance
levels within 27 days, whereas only one of the lead-exposed subjects did (p <0.01).
More recently, Winneke et al. (1982c) repeated the size discrimination experiment and
added another test involving shock avoidance. As in the earlier study, exposure started jr\
utero and continued through behavioral testing. Different concentrations of lead acetate in
the diet were used to yield average blood lead levels of 18.3 and 31.2 pg/dl after 130 days of
feeding, compared to 5 ug/dl for control subjects. These values were not determined directly
from the subjects in this study but were based on separate work by Schlipkbter and Winneke
(1980). However, ALA-D activity was measured directly in selected female subjects at
PND-90 and was found to be inhibited 73 percent and 83 percent, respectively, for the
different levels of lead exposure. Consistent with their earlier findings, Winneke et al.
(1982c) found that lead-exposed subjects were significantly slower to reach criterion perfor-
mance levels on the size discrimination task. However, on the shock avoidance task, the lead-
exposed subjects were significantly quicker than control subjects to reach the criterion of
successful performance. Although seemingly incongruous with the impairment found in the
discrimination task, the latter finding is consistent with results obtained by Driscoll and
Stegner (1976), who found performance on a shock avoidance task enhanced by lead exposure at a
level high enough (-0.15 percent lead in dams' drinking water) to cause a 20 percent weight
reduction in the subjects prior to weaning. Both the size discrimination deficits and the
enhanced avoidance performance are indicative of alterations in normal neural functioning
consequent to lead exposure.
Cory-Slechta and Thompson (1979) exposed Sprague-Dawley rats to 0.0025, 0.015, or 0.05
percent drinking water solutions of lead as lead acetate starting at PND 20-22. Operant con-
ditioning on a fixed-interval 30-second schedule of reinforcement (food pellet delivered upon
the first bar-press occurring at least 30 sec after preceding pellet delivery) began at PND
BPB12/A 12-91 9/20/83
-------
PRELIMINARY DRAFT
55-60. Blood lead concentrations measured at approximately PND 150 were reported in graphical
form roughly as follows: 0.0025-percent solution group, 5 to 10 ^ig/dl PbB; 0.015-percent
group, 25 to 30 pg/dl PbB; 0.05-percent group, 40 to 45 (jg/dl PbB. Subjects exposed to 0.0025
or 0.015 percent lead solutions showed a "significantly" (no probability value reported)
higher median response rate than matched controls during the first 30 sessions of training;
response rates continued to be significantly higher over the next 60 sessions for the 0.0025-
percent group and over the next 30 sessions for the 0.015-percent group (at which points
training terminated for each group). Moreover, latencies to the first response in the 30-sec
interval (the beginning of the typical "fixed-interval scallop" cumulative response pattern)
were significantly shorter in the 0.0025- and 0.015-percent groups. However, response rates
for the group exposed to the 0.05 percent solution were significantly lower than the control
group's rates for the first 40 sessions; correspondingly, response latencies were longer for
the highest exposure group.
Other work by Cory-Slechta et al. (1981) repeated the earlier study's exposure regimen
(using 0.005 and 0.015 percent solutions) and examined the effects on another aspect of oper-
ant performance. In this study the subjects were required to depress a bar for a specified
minimum duration (0.5 to 3.0 sec) before a food pellet could be delivered. Intersubject vari-
ability increased greatly in the lead-exposed groups (see also, e.g., Cory-Slechta and
Thompson, 1979; Dietz et al., 1978; Hastings et al. , 1979). In general, though, treated sub-
jects tended to shorten their response durations (p = 0.04 for the 0.005-percent group;
p = 0.03 for the 0.015-percent group). This tendency would contribute toward a reduced rate
of reinforcement, which is associated with (and perhaps accounts for) an observed tendency
toward increased response latencies in the lead-exposed subjects (p = 0.04 in the 0.015-
percent group). Although blood lead values were not reported by Cory-Slechta et al. (1981),
brain lead concentrations at approximately PND 200 ranged from 40 to 142 ng/g for the 0.005-
percent group and 320 to 1080 ng/g for the 0.015-percent group. Given the same exposure regi-
mens in the two studies, blood lead values should be comparable.
The Gross-Selbeck and Gross-Selbeck (1981) study (partly described below in Section
12.4.3.1.5) also tested Wistar rats exposed post-weaning to a diet containing 0.05 percent
lead daily until completion of behavioral testing at ~180 days of age, at which time the
average blood lead level was 22.7 ug/dl. Although no differences were apparent in preliminary
operant barpress training, differences between lead-treated and control groups did appear when
the subjects were required to bar-press at a very high rate (e.g., 2 presses per second). The
lead-treated subjects outperformed, i.e., bar-pressed more rapidly than, the control subjects.
Except for monkeys, few other species have recently been studied in sufficient detail to
warrant discussion here. One of the primate studies, that by Bushnell and Bowman (1979b),
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is discussed under Section 12.4.3.1.5 because it examined learning ability some time after
neonatal exposure to lead had terminated. In brief, that study showed impaired discrimination
reversal learning performance at 40 months of age, even though lead exposure was limited to
the first 12 months and the mean blood lead level was about 32 ug/dl for the "low-lead" group
during that period. When measured following behavioral testing, average blood lead concentra-
tions were similar to control levels, i.e., 5-6 ug/dl. v
Other studies of nonhuman primates, however, have examined learning ability while lead
exposure was ongoing. In a more comprehensive report, to which the above-described study
(Bushnell and Bowman, 1979b) was a follow-up, Bushnell and Bowman (1979a) ran a series of
tests on discrimination reversal learning in rhesus monkeys over the second through sixteenth
months of life. Lead acetate was fed to the subjects during the first 12 months so as to
maintain nominal blood lead levels of 50 and 80 ug/dl in the low-lead and high-lead groups
(actual blood lead concentrations varied considerably during the first year, particularly for
the high-lead groups). Although lead dosing was terminated at 12 months, blood lead levels
were still somewhat elevated over control levels at the completion of behavioral testing
(18.75 ± 2.87 ug/dl, low-lead group; 46.25 ± 6.74 ug/dl, high-lead group). The basic finding
that appeared consistently throughout this series of tests, including two separate experiments
involving different groups of subjects (see Table 12-4), was that young rhesus monkeys with
blood lead levels on the order of 30 to 50 ug/dl, compared to control groups with levels of
approximately 5 ug/dl, were significantly retarded in their ability to learn a visual discri-
mination task in which the cues were reversed from time to time according to specified
criteria. In addition, the higher exposure subjects were especially slow in mastering the
first reversal problem, following extended training on the original discrimination task.
Rice and Willes (1979) attempted to replicate the Bushnell and Bowman (1979a) findings.
They fed Rhesus monkeys lead acetate from day one of life and obtained blood lead concentra-
tions in their four experimental subjects between 35 and 70 ug/dl around PND 200, which
dropped to 20-50 ug/dl by PND 400; the four control subjects' levels were generally 5 ug/dl or
lower. At 2-3 years of age, while lead exposure continued, the subjects were trained on a
WGTA form-discrimination task similar to that used by Bushnell and Bowman (1979a). Consistent
with the latter study, Rice and Willes (1979) used a reversal-learning paradigm in which the
correct discriminative cue was reversed once the task was mastered. Although initially the
lead-treated monkeys performed better than controls (fewer trials to criterion and fewer
errors), over successive reversals (4 through 12) the control subjects made fewer errors and
required fewer trials to reach criterion performance in each daily session. This difference
disappeared following session 12, which was extended 500 trials beyond the criterion level
("overtraining"). Overall, the lead-treated subjects appeared to make more errors in per-
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forming the reversal tasks; analysis of variance yielded a significant main effect (p = 0.05),
but this applied only to sessions 6 through 12, which would seem to be a somewhat arbitrary
selection of data for analysis. The authors did note, however, that the success of the lead-
treated monkeys in the first few trials appeared to result from the treated subjects' reluc-
tance to manipulate the novel negative stimulus after 100 pretraining trials in which only the
positive stimulus was presented. Thus, the unexpected initial success of the lead-exposed
subjects may have been an artifact of the pretraining procedure. By this interpretation, the
lead-treated monkeys in Rice and Willes' (1979) study and the high-lead group of monkeys in
Bushnell and Bowman's (1979a) study were both showing perturbed behavior, that is, refractori-
ness to alter their behavior under changed conditions.
Rice and her coworkers studied the same two groups of subjects at 2-3 years of age on an
operant conditioning task involving a multiple fixed-interval/time-out schedule of reinforce-
ment (Rice et al., 1979). This schedule alternated a 10- to 90-sec time-out period, during
which responses were unrewarded, with an 8-min fixed interval, at the end of which a push on a
lighted disk was rewarded. The lead-treated monkeys, whose blood-lead levels had by then sta-
bilized at 20-30 (jg/dl, showed a higher response rate than controls during the fixed interval
and shorter pauses between responses (lower median interresponse times). The treated monkeys
also tended to respond more during the time-out period, even though responses were not reward-
ed.
In conclusion, it appears that alterations in behavior in rats and monkeys occur as a
consequence of chronic exposure to dietary lead resulting in blood lead levels on the order of
30-50 ug/dl. These alterations in behavior are clearly indicative of altered neural function-
ing, especially in the CMS in view of certain of the tasks employed. Another question that
arises, however, is whether such alterations represent impairment in overall functioning of
the lead-exposed subjects. As some studies indicate, lead-treated subjects may actually per-
form better than non-treated control subjects on certain learned tasks. For example, in the
Winneke et al. (1982c) study, the task on which lead-exposed rats excelled required the sub-
jects to move from one compartment to the other in a two-compartment shuttle box in order to
avoid receiving an electrical shock to the feet. A successful avoidance response had to occur
within 5 seconds after the onset of a warning stimulus. Similar findings have been reported
by Oriscoll and Stegner (1976) for shock-avoidance performance. As previously described, a
study by Gross-Selbeck and Gross-Setback (1981) required rats to press a bar for food under an
operant conditioning schedule that rewarded only high rates of responding. By responding more
rapidly, the lead-treated subjects were more successful than untreated control subjects in
maximizing their rewards.
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Because of the contingencies of reinforcement specified in the just-cited experiments, a
tendency to respond with greater alacrity or less hesitation was properly adaptive for the
subjects. Other conditions, however, could make the same tendency unadaptive, as, for exam-
ple, in the study by Cory-Slechta et al. (1981), which required rats to press a bar and hold
it down longer than rats are normally inclined to do. In that case the lead-treated subjects
were less successful than untreated controls. Thus, success or failure (or enhancement or
impairment of performance) may be misleading designations for the behavioral alterations mea-
sured under arbitrary experimental conditions (cf. Penzien et al. , 1982). Of greater impor-
tance may be the underlying tendency to respond more rapidly or "excessively," regardless of
whether or not such responding is appropriate for the reinforcement contingencies of an
experiment. Such a tendency may be inferred from results of other studies of the neurotoxic
effects of lead (e.g., Angell and Weiss, 1982; Overmann, 1977; Rice et al., 1979). Taken
together, these reports might be interpreted as suggesting a possible "hyper-reactivity"
(cf. Winneke et al., 1982c) in lead-treated animals. They and others (e.g., Petit and Alfano,
1979) have noted the commonality of such types of behavioral deficits with experimental
studies of lesions to the hippocampus (see also Sections 12.4.3.2.1 and 12.4.3.5.).
12.4.3.1.4 Effects of lead on social behavior. The social behavior and organization of even
phylogenetically closely related species may be widely divergent. For this and other reasons,
there is little or no basis to assume that, for example, aggressiveness in a lead-treated
Rhesus monkey provides a model of aggressiveness in a lead-exposed human child. However,
there are other compelling grounds for including animal social behavior in the present review.
As in the case of nonsocial behavior patterns, characteristics of an animal's interactions
with conspecifics may reflect neurological (especially CNS) impairment due to toxic exposure.
Also, certain aspects of animal social behavior have evolved for the very purpose (in a non-
teleological sense) of indicating an individual's physiological state or condition (Davis,
1982). Such behavior could potentially provide a sensitive and convenient indicator of toxi-
cological impairment.
Two early reports (Silbergeld and Goldberg, 1973; Sauerhoff and Michaelson, 1973) sug-
gested that lead exposure produced increased aggressiveness in rodents. Neither report, how-
ever, attempted to quantify these observations of increased aggression. Later, Hastings et
al. (1977) examined aggressive behavior in rats that had been exposed to lead via their dams'
milk. Solutions containing 0, 0.01, or 0.05 percent lead as lead acetate constituted the dams'
drinking water from parturition to weaning at PND 21, at which time exposure was terminated.
This lead treatment produced no change in growth of the pups. Individual pairs of male off-
spring (from the same treatment groups) were tested at PND 60 for shock-elicited aggression.
Both lead-exposed groups (average blood lead levels of 5 and 9 ug/dl and brain lead levels of
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8 and 14 |jg/100g) showed significantly less aggressive behavior than the control group. There
were no significant differences among the groups in the flinch/jump thresholds to shock, which
suggests that the differences seen in shock-elicited aggression were not caused by differences
in sensitivity to shock.
A study by Drew et al. (1979) utilized apomorphine to induce aggressive behavior in 90-
day-old rats and found that earlier lead exposure attenuated the drug-induced aggressiveness.
Lead exposure occurred between birth and weaning primarily through the dams' milk or through
food containing 0.05 percent lead as lead acetate. No blood or tissue concentrations of lead
were measured. There were no significant differences in the weights of the lead-treated and
control animals at PND 10, 20, 30, or 90.
Using laboratory mice exposed as adults, Ogilvie and Martin (1982) also observed reduced
levels of aggressive behavior. Since the same subjects showed no differences in vitality or
open field activity measures, the reduction in aggressiveness did not appear to be due to a
general effect of lead on motor activity. Blood lead levels were estimated from similarly
treated groups as being approximately 160 ug/dl after 2 weeks of exposure and as 101 pg/dl
after 4 weeks of exposure.
Cutler (1977) used ethological methods to assess the effects of lead exposure on social
behavior in laboratory mice. Subjects were exposed from birth (via their dams' milk) and
post-weaning to a 0.05 percent solution of lead as lead acetate (average brain lead concentra-
tions were 2.45 nmol/g for controls and 4.38 nmol/g for experimental subjects). At 8 weeks of
age social encounters between subjects from the same treatment group were analyzed in terms of
numerous specified, identifiable behavioral and postural elements. The frequency and dura-
tion of certain social and sexual investigative behavior patterns were significantly lower in
lead-treated mice of both sexes than in controls. Lead-exposed males also showed significant-
ly reduced agonistic behavior compared with controls. Overall activity levels (nonsocial as
well as social behavior) were not affected by the lead treatment. Average body weights did
not differ for the experimental and control subjects at weaning or at the time of testing.
A more recent study by Cutler and coworkers (Donald et al., 1981) used a similar
paradigm of exposure and behavioral evaluation, except that exposure occurred either only pre-
natal ly or postnatally and testing occurred at two times, 3-4 and 14-16 weeks of age. Sta-
tistically significant effects were found only for the postnatal exposure group. Although
total activity in postnatally exposed mice did not differ from that of controls at either age
of testing, the incidence of various social activities did differ significantly. As juveniles
(3-4 weeks old), lead-treated males (and to some extent, females) showed decreased social in-
vestigation of a same-sex conspecific. This finding seems to be consistent with Cutler's
(1977) earlier observations made at 8 weeks of age. Aggressive behavior, however, was almost
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nonexistent in both control and lead-treated subjects in the later study, and so could not be
compared meaningfully. Although the authors do not comment on this aspect of their study, it
seems likely that differences in the strains of laboratory mice used as subjects could well
have been responsible for the lack of aggressive behavior in the Donald et al.'s (1981) study
(cf., e.g., Adams and Boice, 1981). Later testing at 14 to 16 weeks revealed that lead-exposed
female subjects engaged in significantly more investigative behavior of a social or sexual
nature than did control subjects, while males still showed significant reductions in such
behavior when encountering another mouse of the same sex. This apparent disparity between
male and female mice is one of relatively few reports of gender differences in sensitivity to
lead's effects on the nervous system (cf. Cutler, 1977; Verlangieri, 1979). In this case,
Donald et al. (1981) hypothesized that the disparity might have been due to differences in
brain lead concentrations: 74.7 (jmol/kg in males versus 191.6 (jmol/kg in females (blood lead
concentrations were not measured). The Donald et al. (1981) study, along with the above-
mentioned study of Ogilvie and Martin (1982), point out the importance of not focusing exclu-
sively on perinatal exposure in assessing neurotoxic effects of chronic lead exposure.
The social behavior of rhesus monkeys has also been evaluated as a function of early lead
exposure. A study by Allen et al. (1974) reported persistent perturbations in various aspects
of the social behavior of lead-exposed infant and juvenile monkeys, including increased cling-
ing, reduced social interaction, and increased vocalization. However, exposure conditions
varied considerably in the course of this study, with overt toxicity being evident as blood
lead levels at times ranged higher than 500 ug/dl.
A more recent study consisting of four experiments (Bushnell and Bowman, 1979c) also
examined social behavior in infant Rhesus monkeys, but under more systematically varied expo-
sure conditions. In experiments 1 and 2, daily ingestion of lead acetate during the first
year of life resulted in blood lead levels of 30-100 M9/d1» Wltn consequent suppression of
play activity, increased clinging, and greater disruption of social behavior when the play
environment was altered. Experiment 3, a comparison of chronic and acute lead exposure (the
latter resulting in a peak blood lead concentration of 250-300 ug/dl during weeks 6-7 of
life), revealed little effect of acute exposure except in the disruption that occurred when
the play environment was altered. Otherwise, only the chronically exposed subjects differed
significantly from controls in various categories of social behavior. Experiment 4 of the
study showed that prenatal exposure alone, with blood lead concentrations of exposed infants
ranging between 33 and 98 ug/dl at birth, produced no detectable behavioral effects under the
same procedures of evaluation. Overall, neither aggressiveness nor dominance was clearly
affected by lead exposure.
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Another aspect of social behavior--interact!on between mothers and their offspring—was
examined in lead-exposed rats by Zenick et al. (1979). Dams chronically received up to 400
mg/kg lead acetate in their drinking water on a restricted daily schedule (blood lead concen-
trations averaged 96.14 ± 16.54 ug/dl in the high-exposure group at day 1 of gestation). Dams
and their litters were videotaped on PND 1-11, and the occurrence of certain behavior patterns
(e.g., lying with majority of pups, lying away from pups, feeding) was tabulated by the exper-
imenters. In addition, dams were tested for their propensity to retrieve pups removed from
the nest. Neither analysis revealed significant effects of lead exposure on the behavior of
the dams. However, restricted access to drinking water (whether lead-treated or not) appeared
to confound the measures of maternal behavior.
The above studies suggest that aggressive behavior in particular is, if anything, reduced
in laboratory animals as a result of exposure to lead. Certain other aspects of social behav-
ior in laboratory mice, namely components of sexual interaction and social investigation, also
appear to be reduced in lead-treated subjects, although there may be gender differences in
this regard following chronic post-maturational exposure. Young rhesus monkeys also appear to
be sensitive to the disruptive effects of lead on various aspects of social behavior. All of
these alterations in social behavior are indicative of altered neural functioning as a conse-
quence of lead exposure in several mammalian species.
12.4.3.1.5 Persistence of neonatal exposure effects. The specific question of persisting,
long-term consequences of lead effects on the developing organism has been addressed in a
number of studies by carrying out behavioral testing some time after the termination of lead
exposure. Such evidence of long-term effects has been reported for rhesus monkeys by Bushnell
and Bowman (1979b). Their subjects were fed lead acetate so as to maintain blood lead (PbB)
levels of either 50 ± 10 (low-lead) or 80 ± 10 ug/dl (high-lead) throughout the first year of
life (actual means and standard errors for the year were reported as 31.71 ± 2.75 and 65.17 ±
6.28 ug/dl). Lead treatment was terminated at 12 months of age, after which blood lead levels
declined to around 5-6 ug/dl at 56 months. At 49 months of age the subjects were re-
introduced to a discrimination reversal training procedure using new discriminative stimuli.
Despite their extensive experience with the apparatus (Wisconsin General Test Apparatus)
during the first two years of life, most of the high-lead subjects failed to retain the simple
motor pattern (pushing aside a small wooden block) required to operate the apparatus.
Remedial training largely corrected this deficit. However, both high- and low-lead groups
required significantly more trials than the control group (p <0.05) to reach criterion per-
formance levels. This difference was found only on the first discrimination task and nine
reversals of it. Successive discrimination problems showed no differential performance
effects, which indicates that with continued training the lead-treated subjects were able to
achieve the same level of performance as controls.
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Studies using rats have also suggested that behavioral perturbations may be evident some
time after the termination of exposure to lead. Hastings et al. (1979) exposed rat pups to
lead through their mothers' milk by providing the dams 0.01 or 0.1 percent solutions of lead
as lead acetate for drinking water. Exposure was stopped at weaning, at which time average
blood-lead values were 29 (± 5) and 65 (± 25) ug/dl, respectively. At 120 days of age the
subjects were placed on an operant conditioning simultaneous visual discrimination task.
Although Hastings et al. (1979) did not actually measure blood lead levels in adult subjects
at the time of behavioral testing, they presumed that the levels for control and experimental
groups were by then probably quite similar, i.e., on the order of 10 ug/dl, based on prior
work (Hastings et al., 1977.) Forty-six percent of the high-lead group and 37 percent of the
low-lead group failed to learn the task within 60 days; only 4 percent of the control group
failed to reach criterion. In terms of time to reach criterion, controls required a mean of
23 days while the low-lead subjects required 32 days and the high-lead rats 39 days (high-lead
vs. controls, p <0.01). Additional testing on a successive discrimination task at 270 days of
age and a go/no-go discrimination task at 330 days revealed no significant differences between
controls and lead-treated subjects. Since the three tests were not counter-balanced in pres-
entation, there is no way to determine whether the lack of effects in the two latter tests may
have been a function of the order of testing or age at the time of testing or, more simply, a
function of the latter tests' lack of sensitivity to neurotoxic effects.
Gross-Selbeck and Gross-Selbeck (1981) also found alterations in the operant behavior of
adult rats after perinatal exposure to lead via mothers whose blood lead levels averaged 20.5
ug/dl. At the time of testing (3 to 4 months postnatally) the lead-exposed subjects' blood
lead levels averaged 4.55 ug/dl, compared to 3.68 ug/dl in control subjects. Although the two
groups appeared qualitatively similar in their behavior in an open-field test and in prelimi-
nary bar-press training, the lead-exposed subjects tended to respond at a much higher rate
than did control subjects when rewarded for responding quickly. Since the schedule differen-
tially reinforced high response rates, the lead-exposed subjects performed more successfully
than did the control subjects. This was true for three different variations on the basic
schedule examined by the authors. As noted earlier, in this case, the heigtened response rate
was adaptive within the context of the particular task used but may not have been under other
contingencies. Most importantly here, it is indicative of altered CNS function persisting for
months beyond the cessation of lead exposure early in development.
Results from the above studies indicate that behavioral effects may exist as sequelae to
past lead exposure early in development of mammalian species, even though blood lead levels at
the time of later behavioral assessment are essentially "normal."
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12.4.3.2 Morphological Effects
12.4.3.2.1 In vivo studies. Recent key findings on the morphological effects of i_n vivo lead
exposure on the nervous system are summarized in Table 12-5. It would appear that certain
types of glial cells are sensitive to lead exposure, as Reyners et al. (1979) found a
decreased density of oligodendrocytes in cerebral cortex of young rats exposed from birth to
0.1 percent lead in their food. Higher exposure concentrations (0.2-0.4 percent lead salts),
especially during the prenatal period (Bull et al., 1983), can reduce synaptogenesis and
retard dendritic development in the cerebral cortex (McCauley and Bull, 1978; McCauley et al.,
1979, 1982) and the hippocampus of developing rats (Campbell et al., 1982, and Alfano and
Petit, 1982). Some of these effects, e.g. on cerebral cortex appear to be transient (McCauley
et al. , 1979, 1982). Suckling rats subjected to increasing exposures of lead exhibit more
pronounced effects, such as reduction in the number and average diameter of axons in the optic
nerve at 0.5 percent lead acetate exposure (Tennekoon et al., 1979), a general retardation of
cortical synaptogenesis at 1.0 percent lead carbonate exposure (Averill and Needleman, 1980),
or a reduction in cortical thickness at 4.0 percent lead carbonate exposure (Petit and
LeBoutillier, 1979). This latter exposure concentration also causes a delay in the onset and
peak of Schwann cell division and axonal regrowth in regenerating peripheral nerves in chroni-
cally exposed adult rats (Ohnishi and Dyck, 1981). In short, both neuronal and glial compo-
nents of the nervous system appear to be affected by neonatal or chronic lead exposure.
Organolead compounds have also been demonstrated to have a deleterious effect on the mor-
phological development of the nervous system. Seawright et al. (1980) administered triethyl
lead acetate (EtgPb) by gavage to weanling (40-50 g) and "young adult" (120-150 g) rats.
Single doses of 20 mg EtgPb/kg caused impaired balance, convulsions, paralysis, and coma in
both groups of treated animals. Peak levels in blood and brain were noted two days after ex-
posure, with extensive neuronal necrosis evident in several brain regions by three days post-
treatment. Weekly exposures to 10 mg EtsPb/kg for 19 weeks resulted in less severe overt
signs of intoxication (from which the animals recovered) and moderate to severe loss of
neurons in the hippocampal region only.
12.4.3.2.2 In vitro studies. Bjb'rklund et al. (1980) placed tissue grafts of developing ner-
vous tissue in the anterior eye chambers of adult rats. When the host animals were given 1 or
2 percent lead acetate in their drinking water, the growths of substantia nigral and hippocam-
pal, but not cerebellar, grafts were retarded. Grafts of the developing cerebral cortex in
host animals receiving 2 percent lead exhibited a permanent 50 percent reduction in size
(volume), whereas 1 percent lead produced a slight increase in size in this tissue type. The
authors felt that this anomalous result might be explained by a hyperplasia of one particular
cell type at lower concentrations of lead exposure.
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TABLE 12-5. SUMMARY OF KEY STUDIES OF MORPHOLOGICAL EFFECTS OF IN VIVO LEAD EXPOSURE
Species
Exposure protocol
Peak blood
lead level
Observed
effect
Reference
Young rats
Adult rats
0.1X Pb
PND 0-90
in chow
0.1% Pb(Ac)2 in
dams' drinking
water PNO 0-32
0.2% PbCl2 in dams'
drinking water from
gestation thru PNDO
80 ug/dl
at birth
0.2% Pb(Ac)2 in
dams' drinking
water PNO 0-25
0.4% PbC03 in
dams' drinking
water PND 0-30
0.5% Pb(Ac)2 in
dams' drinking
water PND 0-21
IX PbC03 in chow
PND 0-60
4% PbC03 in dams'
chow PND 0-28
4% PbC03 in dams'
chow PNO 0-25
4% PbCOj in chow
for 3 mos.
4% PbCOj in chow
PND 0-150
385 pg/dl
(PND 21)
258 ug/dl
(PND 28)
300
(PND 150)
Decreased density of
oligodendrocytes in cerebral
cortex
Significant inhibition in
myelin deposition and
maturation in whole brain
Less mature synoptic profile
in cerebral cortex at PND-
15
30% reduction in synoptic
density in cerebral cortex
at PND15 (returned to normal
at PN021)
15-30% reduction in
synaptic profiles in
hippocampus
Retardation in temporal
sequence of hippocampal
dendritic development
10-15% reduction in number
of axons in optic nerve;
skewing of fiber diameters
to smaller sizes
Retardation of cortical
synaptogenesis over and
above any nutritional
effects
13% reduction in
coctical thickness
and total brain weight;
reduction in synaptic
density
Reduction in hippocampal
length and width; similar
reduction in afferent
projection to hippocampus
Delay in onset and peak
of Schwann cell division
and axonal regrowth in
regenerating nerves
Demyelination of peri-
pheral nerves beginning
PND 20-35
Reyners et al. (1979)
Stephens and
Gerber (1981)
McCauley and Bull
(1978)
McCauley et al. (1979)
McCauley et al. (1982)
Campbell et al. (1982)
Alfano and Petit
(1982)
Tennekoon et al.
(1979)
Averill and Needleman
(1980)
Petit and
LeBoutillier (1979)
Alfano et al. (1982)
Ohnishi and Dyck
(1981)
Windebank et al.
1980
PND: post-natal day
Pb(Ac)2: lead acetate
PbC03: lead carbonate
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Organolead compounds have also been demonstrated to affect neuronal growth (Grundt et
al. , 1981). Cultured cells from embryonic chick brain were exposed to 3.16 uM triethyllead
chloride in the incubation medium for 48 hr, resulting in a 50 percent reduction in the number
of cells exhibiting processes. There was no observed effect on glial morphology.
Other investigations have focused on morphological aspects of the blood-brain barrier and
its possible disruption by lead intoxication (Kolber et al., 1980). Capillary endothelial
cells isolated from rat cerebral cortex and exposed to 100 uM lead acetate J_n vitro
(Silbergeld et al., 1980b) were examined by electron microscopy and X-ray microprobe analysis.
Lead deposits were found to be sequestered preferentially in the mitochondria of these cells
in much the same manner as calcium. This affinity may be the basis for lead-induced disrup-
tion of transepithelial transport of Ca* and other ions.
12.4.3.3 Electrophysiological Effects.
12.4.3.3.1 In vivo studies. Recent key findings on the electrophysiological effects of jm
vivo lead exposure are summarized below in Table 12-6. The visual system appears to be par-
ticularly susceptible to perturbation by neonatal lead exposure. Suckling rats whose dams
were given drinking water containing 0.2 percent lead acetate had significant alterations in
their visual evoked responses (VER) and decreased visual acuity at PND 21, at which time their
blood lead levels were 65 ug/dl (Cooper et al., 1980; Fox et al., 1977; Impelman et al., 1982;
Fox and Wright, 1982; Winneke, 1980). Both of these observations are indicative of depressed
conduction velocities in the visual pathways. These same exposure levels also increased the
severity of the maximal electroshock seizure (MES) response in weanling rats who exhibited
blood lead levels of 90 ug/dl (Fox et al., 1978, 1979). The authors speculated that neonatal
lead exposure acts to increase the ratio of excitatory to inhibitory systems in the developing
cerebrospinal axis. Such exposure can also lead to lasting effects on the adult nervous
system, as indicated by persistent decreases in visual acuity and spatial resolution in 90-day
old rats exposed only from birth to weaning to 0.2 percent lead acetate (Fox et al., 1982).
The adult nervous system is also vulnerable to lead-induced perturbation at low levels of
exposure. Hietanen et al. (1980) found that chronic exposure of adult rabbits to 0.2 percent
lead acetate in drinking water resulted in an 85 percent inhibition of motor conduction velo-
city in the sciatic nerve.
12.4.3.3.2 In vitro studies. Palmer et al. (1981) and Olson et al. (1981) looked at intrao-
cular grafts of cerebellar tissue from 14- to 15-day-old rats in host animals treated for 2
months with drinking water containing 1 percent lead acetate, followed by plain water for 4-5
months. They found no alterations in total growth or morphology of grafts in treated vs.
control hosts, yet the Purkinje neurons in the lead-exposed grafts had almost no spontaneous
activity. Host cerebellar neurons, on the other hand, and both host and graft neurons in
BPB12/A 12-102 9/20/83
-------
TABLE 12-6. SUMMARY OF KEY STUDIES OF ELECTROPHYSIOLOGICAL
EFFECTS OF IN VIVO LEAD EXPOSURE
Species
Suckling rat
Exposure protocol
0.2% Pb(Ac)z in
dams' drinking water
PND 0-20
0.2% Pb(Ac)z in
dams' drinking water
Peak blood
lead level
90 ug/dl
(PND 20)
65 ug/dl
(PND 21)
Observed
effect
More rapid appearance
and increased severity of
MES response
1) Increased latencies and
decreased amplitudes of
Reference
Fox et al .
(1978, 1979)
Fox et al.
(1977);
PND 0-21
o
CO
Young rhesus
monkeys
Adult rabbit
Pb(Ac)a solutions
in food
PND 0-365
0.2% Pb(Ac)s in
drinking water for
4 weeks
300 ug/dl
(PND 60)
85 Mg/cil
primary and secondary
components of VER;
2) decreased conduction
velocities in visual
pathways;
3) 25-50% decrease in
scotopic visual acuity
4) persistent decreases
in visual acuity and
spatial resolution at
PND 90
Severe impairment
of discrimination
accuracy; loss of
scotopic function
85% reduction in motor
conduction velocity of
sciatic nerve
Impel man et al.
(1982);
Cooper et al.
(1980);
Winneke (1980);
Fox and Wright
(1982)
Fox et al. (1982)
Bushnell et al.
(1977)
Hietanen et al.
(1980)
PND: post-natal day
Pb(Ac)^: lead acetate
MES: maximal electroshock seizure
VER: visual evoked response
-------
PRELIMINARY DRAFT
control animals, all exhibited significant levels of spontaneous activity. Taylor et al.
(1978) recorded extracellularly from cerebellar Purkinje cells in adult rats both j_n situ and
in intraocular grafts in an effort to determine what effect lead had on the norepinephrine
(NE)-induced inhibition of Purkinje cell spontaneous discharge. Application of exogenous NE
to both i_n s'itu and j_n oculo cerebellum produced 61 and 49 percent inhibitions of spontaneous
activity, respectively. The presence of 5-10 |jM lead reduced this inhibition to 28 and 13
percent, respectively. This "disinhibition" was specific for NE, as responses to both cho-
linergic and parallel fiber stimulation in the same tissue remained the same. Furthermore,
application of lead itself did not affect spontaneous activity, but did inhibit adenylate
cyclase activity in cerebellar homogenates at the same concentration required to disinhibit
the NE-induced reduction of spontaneous activity (3 to 5 uM).
Fox and Sillman (1979) looked at receptor potentials in the isolated, perfused bullfrog
retina and found that additions of lead chloride caused a reversible, concentration-dependent
depression of rod (but not cone) receptor potentials. Concentrations of 5 uM produced an
average 16 percent depression, while 12.5 uM produced an average 23 percent depression.
Evidence that lead does indeed resemble other divalent cations, in that it appears to
interfere with chemically mediated synaptic transmission, has been obtained in studies of
peripheral nerve function. For example, lead is capable of blocking neural transmission at
peripheral adrenergic synapses (Cooper and Steinberg, 1977). Measurements of the contraction
force of the rabbit saphenous artery following stimulation of the sympathetic nerve endings
indicated that lead blocks muscle contraction by an effect on the nerve terminals rather than
by an effect on the muscle. Since the response recovered when the Ca2 concentration was in-
creased in the bathing solution, it was concluded that lead does not deplete transmitter
stores in the nerve terminals, but more likely blocks NE release.
It has also been demonstrated that lead depresses synaptic transmission at the peripheral
neuromuscular junction by impairing acetylcholine (ACh) release from presynaptic terminals
(Kostial and Vouk, 1957; Manalis and Cooper, 1973; Cooper and Manalis, 1974). This depression
of neurotransmitter release evoked by nerve stimulation is accompanied by an increase in the
spontaneous release of ACh, as evidenced by the increased frequency of spontaneous miniature
endplate potentials (MEPPs). Kolton and Yaari (1982) found that this increase in MEPPs in the
frog nerve/muscle preparation could be induced by lead concentrations as low as 5 uM.
The effects of lead on neurotransmission within the central nervous system have also been
studied. For example, Kim et al. (1980) fed adult rabbits 165 mg lead carbonate per day for
five days and looked at Ca2 retention in brain slices. Treated animals showed a 75 percent
increase in Ca2 retention time, indicating that lead inhibited the mediated efflux of Ca2*
from the incubated brain slice. Investigation of the iri vitro effects of lead on Ca2 binding
BPB12/A 12-104 9/20/83
-------
PRELIMINARY DRAFT
was carried out by Silbergeld and Adler (1978) on caudate synaptosomes. They determined that
50 uM lead caused an 8-fold increase in 45Ca2+ binding and that in both control and lead-
treated preparations the addition of ATP increased binding, while ruthenium red and Ca2
decreased it. Further findings in this series of experiments demonstrated that lead inhibits
the Na -stimulated loss of Ca2 by mitochondria and that blockade of dopamine (DA) uptake by 5
uM benztropine reversed the lead-stimulated increase in Ca2 uptake by synaptosomes. The
authors concluded that lead affects the normal mechanisms of Ca2 binding and uptake, perhaps
by chelating with DA in order to enter the nerve terminal. By inhibiting the release of Ca2
bound to mitochondria there, lead essentially causes an increase in the Ca2 concentration
gradient across the nerve terminal membrane. As a result, more Ca2 would be expected to
enter the nerve terminal during depolarization, thus effectively increasing synaptic neuro-
transmission at dopaminergic terminals without altering neuronal firing rates.
12.4.3.4 Biochemical Alterations. The majority of previous investigations of biochemical
alterations in the nervous system following exposure to lead have focused on perturbations of
various neurotransmitter systems, probably because of the documentation extant on the neuro-
physiological and behavioral roles played by these transmitters. Recently, however, somewhat
more attention has been centered on the impact of lead exposure on energy metabolism and other
cellular homeostatic mechanisms such as protein synthesis and glucose transport. A signifi-
cant portion of this work has, however, been conducted i_n vitro.
12.4.3.4.1 In vivo studies. Recent key findings on the biochemical effects of iji vivo expo-
sure are summarized in Table 12-7. Although the majority of recent work has continued to
focus on neurotransmitter function, it appears that the mechanisms of energy metabolism are
also particularly vulnerable to perturbation by lead exposure. McCauley, Bull, and coworkers
have demonstrated that exposure of prenatal rats to 0.02 percent lead chloride in their dams'
drinking water leads to a marked reduction in cytochrome content in cerebral cortex, as well
as a possible uncoupling of energy metabolism. Although the reduction in cytochrome content
is transient and disappears by PND 30, it occurs at blood lead levels as low as 36 ug/dl
(McCauley and Bull, 1978; Bull et al., 1979); delays in the development of energy metabolism
may be seen as late as PND 50 (Bull, 1983).
There does not appear to be a selective vulnerability of any particular neurotransmitter
system to the effects of lead exposure. Pathways utilizing dopamine (DA), norepinephrine
(NE), serotonin (5-HT), and yaminobutyric acid (GABA) are all affected in neonatal animals at
lead-exposure concentrations of 0.2-2.0 percent lead salts in dams' drinking water. Although
the blood lead values reported following exposure to the lower lead concentrations (0.2-0.25
percent lead acetate or lead chloride) range from 47 pg/dl (Goldman et al. , 1980) to 87 ug/dl
(Govoni et al., 1980), a few general observations can be made:
BPB12/A 12-105 9/20/83
-------
PRELIMINARY DRAFT
TABLE 12-7. SUMMARY OF KEY STUDIES ON BIOCHEMICAL
EFFECTS OF IN VIVO LEAD EXPOSURE
Species
Exposure protocol
Peak blood
lead level
Observed
effect
Reference
Suckling rat 0.004% Pb(Ac)2 in
dams' drinking water
PND 0-35
0.02% PbCl2 in dams' 80 ug/dl
drinking water from (at birth)
gestation thru PND 36 \ig/d'\
0-21 (PND 21)
0.2% Pb(Ac)2 in 47 pg/dl
dams' drinking water (PND 21)
PND 0-21
0.25% Pb(Ac)2 in
dams' drinking water
PND 0-35
0.25% Pb(Ac)2 in
dams' drinking water
PND 0-35
0.25% Pb(Ac)$> in
dams' drinking water
PND 0-35
0.25% Pb(Ac)2 in 87 pg/dl
dams' drinking water (PND 42)
PND 0-42
Decline in synthesis and
turnover of striatal DA
1) Transient 30% reduction
in cytochrome content of
cerebral cortex;
2) possible uncoupling of
energy metabolism
3) delays in development of
energy metabolism
1) 23% decrease in NE levels
of hypotholamus and
striatum;
2) increased turnover of
NE in brainstem
Decline in synthesis
and turnover of striatal
DA
Increase in DA synthesis
in frontal cortex and
nuc. accumbens(10-30%
and 35-45%, respectively)
1) 50% increase in DA
binding to striatal
D2 receptors;
2) 33% decrease in DA binding
to nuc. accumbens D2 receptors
1) 31% increase in GABA
specific binding in
cerebellum; 53% increase
in GMP activity;
2) 36% decrease in GABA-
specific binding in striatum;
47% decrease in GMP activity
Govoni et al.
(1979, 1980);
Memo et al.
(1980a, 1981)
McCauley and
Bull (1978);
McCauley et
al. (1979);
Bull et al.
(1979)
Bull (1983)
Goldman et al
(1980)
Govoni et al.
(1978a)
Govoni et al.
(1979, 1980);
Memo et al.
(1980a, 1981)
Lucchi et al.
(1981)
Govoni et al.
(1978b, 1980)
BPB12/A
12-106
9/20/83
-------
PRELIMINARY DRAFT
TABLE 12-7. (continued)
Species
Exposure protocol
Peak blood
lead level
Observed
effect
Reference
Young rat
0.25% Pb(Ac)2 in
dam's drinking water
PND 0-21; 0.004% or
0.25% until PND 42
0.5-1% Pb(Ac)2 in
drinking water
PND 0-60
0.25-1% Pb(Ac)j in
drinking water
PND 0-60
75 mg Pb(Ac)2/kg
b.w./day via
gastric intubation
PND 2-14
72-91 g/dl
(PND 21)
98 ug/dl
(PND 15)
2% Pb(Ac)2 in dam's
drinking water PND 0-21
then 0.002-0.008% until
PND 56
1) 12 and 34% elevation of Memo et al.
GABA binding in cerebellum (1980b)
for 0.004% and 0.25%, respec-
tively;
2) 20 and 45% decreases in GABA
binding in striatum for 0.004%
and 0.25%, respectively
1) Increased sensitivity
to seizures induced
by GABA blockers;
2) increase in GABA synthesis
in cortex and striatum;
3) inhibition of GABA uptake
and release by synaptosomes
from cerebellum and basal
ganglia;
4) 70% increase in GABA-
specific binding in
cerebellum
1) 40-50% reduction of
whole-brain ACh by PND 21;
2) 36% reduction by PND 30
(return to normal values
by PND 60)
1) 20% decline in striatal
DA levels at PND 35;
2) 35% decline in striatal DA
turnover by PND 35;
3) Transient depression of DA
uptake at PND 15;
4) Possible decreased DA
terminal density
1) non-dose-dependent
elevations of NE in
midbrain (60-90%) and
DA and 5-HT in midbrain,
striatum and hypothalamus
(15-30%);
2) non-dose-dependent depression
of NE in hypothalamus and
striatum (20-30%).
SiIbergeld
et al.
(1979, 1980a)
Modak et al.
(1978)
Jason and
Kellogg (1981)
Dubas et al.
(1978)
PND: post-natal day
Pb(Ac)j: lead acetate
PbCls: lead chloride
NE: norepinephrine
BPB12/A
DA: dopamine
GABA: Y'aroinobutyric acid
GMP: guanosine monophosphate
5-HT: serotonin
12-107
9/20/83
-------
PRELIMINARY DRAFT
(1). Synthesis and turnover of DA and NE are depressed in the striatum, and elevated in mid-
brain, frontal cortex, and nucleus accumbens. This seems to be paralleled by concomitant
increases in DA-specific binding in striatum and decreases in DA-specific binding In
nucleus accumbens, possibly involving a specific subset (D2) of DA receptors (Lucchi
et al., 1981). These findings are probably reflective of sensitization phenomena result-
ing from changes in the availability of neurotransmitter at the synapse.
(2). The findings for pathways utilizing GABA show similar parallels. Increases in GABA syn-
thesis in striatum are coupled with decreases in GABA-specific binding in that region,
while the converse holds true for the cerebellum. In these cases, cyclic GMP activity
mirrors the apparent changes in receptor function. This increased sensitivity of cere-
be liar postsynaptic receptors (probably a response to the lead-induced depression of pre-
synaptic function) is likely the basis for the finding that lead-treated animals are more
susceptible to seizures induced by GABA-blocking agents such as picrotoxin or strychnine
(Silbergeld et al., 1979).
12.4.3.4.2 In vitro studies. Any alterations in the integrity of the blood-brain barrier can
have serious consequences for the nervous system, especially in the developing organism.
Kolber et al. (1980) examined glucose transport in isolated microvessels prepared from the
brains of suckling rats given 25, 100, 200, or 1000 mg lead/kg body weight daily by intra-
gastric gavage. On PND 25, they found that evan the lowest dose blocked specific transport
sites for sugars and damaged the capillary endothelium. In vitro treatment of the preparation
with concentrations of lead as low as 0.1 jjM produced the same effects.
Purdy et al. (1981) examined the effects in rats of varying concentrations of lead ace-
tate on the whole-brain synthesis of tetrahydrobiopterin (BH4), a cofactor for many important
enzymes, including those regulating catecholamine synthesis. Concentrations of lead as low as
0.01 uM produced a 35 percent inhibition of BH4 synthesis, while 100 uM inhibited the BH4 sal-
vage enzyme, dihydropteridine reductase, by 40 percent. This would result in a decreased con-
version of phenylalanine to tyrosine and thence to DOPA (the initial steps in dopamine synthe-
sis), as well as decreases in the conversion of trytophan to its 5-hydroxy form (the initial
step in serotonin synthesis). These decrements, if occurring HI vivo, could not be ameliora-
ted by increased dietary intake of BH4, as it does not cross the blood-brain barrier.
Lead has also been found to have an inhibitory effect on mitochondrial respiration in the
cerebrum and cerebellum of immature or adult rats at concentrations greater than 50 pM
(Holtzman et al., 1978b). This effect, which was equivalent in both brain regions at both
ages studied, is apparently due to an inhibition of nicotinamide adenine dinucleotide (NAD)-
1 inked dehydrogenases within the mitochondrial matrix. These same authors found that this
lead-induced effect, which is an energy-dependent process, could be blocked _in vitro by
BPB12/A 12-108 9/20/83
-------
PRELIMINARY DRAFT
addition of ruthenium red to the incubation medium (Holtzman et al., 1980b). In view of the
fact that Ca2 uptake and entry into the mitochondria! matrix is also blocked by ruthenium
red, it is possible that both lead and Ca2 share the same binding site/carrier in brain mito-
chondria. These findings are supported by the work of Gmerek et al. (1981) on adult rat
cerebral mitochondria, with the exception that they observed respiratory inhibition at 5 uM
lead acetate, which is a full order of magnitude lower than the Holtzman et al. (1978b, 1980b)
studies. Gmerek and co-workers offer the possibility that this discrepancy may have been due
to the inadvertent presence of EDTA in the incubation medium used by Holtzman et al.
Organolead compounds have also been demonstrated to have a deleterious effect on cellular
metabolism in the nervous system. For example, Grundt and Neskovic (1980) found that concen-
trations of triethyl lead chloride as low as 5-7 uM caused a 40 percent decrease in the incor-
poration of S04 or serine into myelin galacto-lipids in cerebellar slices from 2-week-old
rats. Similarly, Konat and coworkers (Konat and Clausen, 1978, 1980; Konat et al., 1979) ob-
served that 3 uM triethyl lead chloride preferentially inhibited the incorporation of leucine
into myelin proteins in brain stem and forebrain slices from 22-day-old rats. This apparent
inhibition of myelin protein synthesis was two-fold greater than that observed for total pro-
tein synthesis (approximately 10 vs. 20 percent, respectively). In addition, acute intoxica-
tion of these animals by i.p. injection of triethyl lead chloride at 8 mg/kg produced equiva-
lent results accompanied by a 30 percent reduction in total forebrain myelin content.
Interestingly, while a suspension of cells from the forebrain of these animals (Konat et
al., 1978) exhibited a 30 percent inhibition of total protein synthesis at 20 uM triethyl lead
chloride (the lowest concentration examined), a cell-free system prepared from the same tissue
was not affected by triethyl lead chloride concentrations as high as 200 uM. This result,
coupled with a similar, although not as severe, inhibitory effect of triethyl lead chloride on
oxygen consumption in the cell suspension (20 percent inhibition at 20 uM) would tend to indi-
cate that the inhibition of rat forebrain protein synthesis is related to an inhibition of
cellular energy-generating systems.
The effects of organolead compounds on various neurotransmitter systems have been inves-
tigated in adult mouse brain homogenates. Bondy et al. (1979a,b) demonstrated that micromolar
concentrations (5 uM) of tri-n-butyl lead (TBL) acetate were sufficient not only to cause a 50
percent decline in the high affinity uptake of GABA and DA in such homogenates, but also to
stimulate a 25 percent increase in GABA and DA release. These effects were apparently selec-
tive for DA neurons at lower concentrations, as only DA uptake or release was affected at 0.1
(jM, albeit mildly so. The effect of TBL acetate on DA uptake appears to be specific, as there
is a clear dose-response relationship down to 1 uM TBL (Bondy and Agarwal, 1980) for inhibi-
tion (0-60 percent) of spiroperidol binding to rat striatal DA receptors. A concomitant
BPB12/A 12-109 9/20/83
-------
PRELIMINARY DRAFT
inhibition of adenyl cyclase in this dose range (50 percent) suggests that TBL may affect the
entire postsynaptic binding site for DA.
12.4.3.5 Accumulation and Retention of Lead in the Brain. All too infrequently, experimen-
tal studies of the neurotoxic effects of lead exposure do not report the blood-lead levels
achieved by the exposure protocols used. Even less frequently reported are the concomitant
tissue levels found in brain or other tissues. From the recent information that is available,
however, it is possible to draw some limited conclusions about the relationship of exposure
concentrations to blood and brain lead concentrations. Table 12-8 calculates the blood lead/
brain lead ratios found in recent studies where such information was available. It can be
seen that, at exposure concentrations greater than 0.2 percent and for exposure periods longer
than birth until weaning (21 days in rats), the ratio generally falls below unity. This
suggests, that, even as blood lead levels reach a steady state and then fall due to excretion
or some other mechanism, lead continues to accumulate in brain.
Further evidence bearing on this was derived from a set of studies by Goldstein et al.
(1974), who reported that administration of a wide range of doses of radioactive lead nitrate
to one-month-old rats resulted in parallel linear increases in both blood and brain lead
levels during the ensuing 24 hours. This suggests that deposition of lead in brain occurs
without threshold and that, at least initially, it is proportional to blood lead concentra-
tion. However, further studies by Goldstein et al. (1974) followed changes in blood and brain
lead concentrations after cessation of lead exposure and found that, whereas blood lead levels
decreased dramatically (by an order of magnitude or more) during a 7-day period, brain lead
levels remained essentially constant over the one-week postexposure period. Thus, with even
intermittent exposures to lead, it is not unexpected that brain concentrations would tend to
remain the same or even to increase although blood lead levels may have returned to "normal"
levels. Evidence confirming this comes from findings of: (1) Hammond (1971), showing that
EDTA administration causing marked lead excretion in urine of young rats did not significantly
lower brain lead levels in the same animals; and (2) Goldstein et al. (1974), showing that
although EDTA prevented the ir\ vitro accumulation of lead into brain mitochondria, if lead was
added first then EDTA was ineffective in removing lead from the mitochondria. These results,
overall, indicate that, although lead may enter the brain in rough proportion to circulating
blood lead concentrations, it is then taken up by brain cells and tightly bound into certain
subcellular components (such as mitochondrial membranes) and retained there for quite long
after initial external exposure ceases and blood lead levels markedly decrease. This may help
to account for the persistence of neurotoxic effects of various types noted above long after
the cessation of external lead exposure.
BPB12/A 12-110 9/20/83
-------
PRELIMINARY DRAFT
TABLE 12-8. INDEX OF BLOOD LEAD AND BRAIN LEAD LEVELS FOLLOWING EXPOSURE
Species
(strain)
Suckling rat
(Charles
River-CD)
Suckling rat
(Charles
River)
Suckling rat
(Charles
River-CD)
Suckling rat
(Long-Evans)
Suckling rat
(Long-Evans)
Suckling rat
(Holtzman-
albino)
Suckling rat
Suckling rat
(Holtzman-
albino)
Suckling rat
(Long-Evans)
Suckling rat
(Long-Evans)
Suckling rat
(Long-Evans)
Suckling mice
(ICR Swiss
albino)
Suckling rat
(Wistar)
Exposure
0.0005% PbCl2
in water
PND 0-21
0.003% PbCl2
in water
PND 0-21
0.005% Pb(Ac)2
in water from
conception
0.01% Pb(Ac)2
in water from
conception
0.02% PbCl2
in water
PND 0-21
0.02% Pb(Ac)2
in water
PND 0-21
0.02% Pb(Ac)2
in water from
PND 0-21
0.05% Pb(Ac)2
in water
PND 0-21
0.1% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
0.2X Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
0.25% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 2-60
0.5% Pb(AC)2
in water
PND 2-60
Time of Blood lead
assay (ug/dl)
PND 21
PND 21
PND 11
PND 30
PND 11
PND 30
PND 21
PND 10
PNO 21
PND 21
PND 21
PND 21
PND 21
PND 21
PND 10
PND 21
PND 21
PND 21
PND 21
PND 30
PND 60
PND 30
PND 60
12
21
22
18
35
48
36
21.7
25.2
29
12
20
65
47
49.6
89.4
65.0
65.1
72
115*
35*
308*
73*
Brain lead
(ug/lOOg)
8
11
3
11
7
22
25
6.3
13
29
20
50
65
80
19
82
53
53
230
84
99
172
222
Blood: brain
lead ratio
1.5
1.9
7.0
1.6
5.0
2.2
1.4
3.4
1.9
1.0
0.6
0.4
1.0
0.6
2.6
1.1
1.2
1.2
0.3
.
-
.
-
Reference
Bull et al.
(1979)
Grant et al.
(1980)
Bull et al.
(1979)
Fox et al.
(1979)
Hastings
et al. (1979)
Goldman et al .
(1980)
Hastings et
al. (1979)
Goldman et al.
(1980)
Fox et al.
(1979)
Fox et al.
(1977)
Cooper et al.
(1980)
Modak et al.
(1978)
Shigeta et al.
(1979)
12-111
-------
PRELIMINARY DRAFT
Table 12-8. (continued)
Species
(strain)
Suckling rat
(Sprague-
Dawley)
Suckling rat
(Sprague-
Dawley)
Suckling rat
(Long-Evans)
Young mice
(ICR Swiss
albino)
Adult rat
(Charles
River-CD)
Adult rat
(Wistar)
Time of
Exposure assay
0.25% Pb(Ac)2 PND 42
in water from
gestation until
PND 42
0.5% Pb(Ac)2 PNO 21
in water
PND 0-21
IX Pb(Ac)2 PND 21
in water
PND 0-21
4% PbC03 PND 27
in water
PND 0-27
25 mg/kg Pb(Ac)2 PND 15
by gavage
PND 2-14
75 mg/kg Pb(Ac)2 PND 15
by gavage
PND 2-14
0.25% Pb(Ac)2 PND 60
in water
PND 0-60
0.5% Pb(Ac)2 PND 60
in water
PND 0-60
IX Pb(Ac)2 PHD 60
in water PND 0-60
0.0005X Pb(Ac)2
in water for 21 days
0.003% Pb(Ac)2
In water for 21 days
0.02% Pb(Ac)2
in water for 21 days
0.15X Pb(Ac)2
in water for 3 months
0.4X Pb(Ac)2
in water for 3 months
IX Pb(Ac)2
1n water for 3 months
Blood lead
(MQ/dl)
87
70
91
__.
50
98
91
194
223
9
11
29
31
69
122
Brain lead Blood: brain
(ug/lOOg) lead ratio
85
280
270
1.36
40
60
410
360
810
10
12
100
12-18
(depending
on region)
16-34
(depending
on region)
37-72
(depending
on region)
1.0
0.25
0.3
—
1.3
1.6
0.2
0.5
0.3
0.9
0.9
0.29
2.6-1.7
(depending
on region)
4.3-2.0
(depending
on region)
3.3-1.7
(depending
on region)
Reference
Govani et al.
(1980)
Wince et al.
(1980)
Jason and
Kellogg (1981)
Kodak et al .
(1978)
Bull et al.
(1979)
Ewers and
Erbe (1980)
PND: post-natal day
Pb(Ac)2: lead acetate
PbC12: lead chloride
•Expressed as pg Pb/lOOg blood.
12-112
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PRELIMINARY DRAFT
The uptake of lead into specific neural and non-neuronal elements of the brain has also
been studied and provides insight into possible morphological correlates of certain lead
effects discussed above and below as being observed in vivo or in vitro. For example, Stumpf
"girt
et al. (1980), via autoradiographic localization of Pb, found that ependymal cells, glial
cells, and endothelial cells of brain capillaries concentrate and retain lead above background
levels for several days after injections of tracer amounts of the elements. These cells are
non-neural elements of brain important in the maintenance of "blood-brain barrier" functions,
and their uptake and retention of lead, even with tracer doses, provides evidence of a mor-
phological basis by which lead effects on blood-brain barrier functions may be exerted.
Again, the retention of lead in these non-neuronal elements for at least several days after
original exposure points towards the plausibility of lead exerting effects on blood-brain
barrier functions long after external exposure ceases and blood lead levels decrease back
toward normal levels. Uptake and concentration of lead in the nuclei of some cortical neurons
210
even several days after administration of only a tracer dose of Pb was also observed by
Stumpf et al. (1980) and provide yet another plausible morphological basis by which neurotoxic
effects might be exerted by lead long after external exposure terminates and blood lead levels
return to apparently "normal" levels.
12.4.4 Integrative Summary of Human and Animal Studies of Neurotoxicity
An assessment of the impact of lead on human and animal neurobehavioral function raises a
number of issues. Among the key points addressed here are: (1) the internal exposure levels,
as indexed by blood lead levels, at which various adverse neurobehavioral effects occur; (2)
the reversibility of such deleterious effects; and (3) the populations that appear to be most
susceptible to neural damage. In addition, the question arises as to the utility of using
animal studies to draw parallels to the human condition.
12.4.4.1 Internal Exposure Levels at Which Adverse Neurobehavioral Effects Occur. Markedly
elevated blood lead levels are associated with neurotoxic effects of lead exposure (including
severe, irreversible brain damage as indexed by the occurrence of acute and/or chronic enceph-
alopathic symptoms) in both humans and and animals. For most adult humans, such damage typi-
cally does not occur until blood lead levels exceed 120 ng/dl. Evidence does exist, however,
for acute encephalopathy and death occurring in some human adults at blood lead levels below
120 ug/dl. In children, the effective blood lead level for producing encephalopathy or death
is lower, starting at approximately 100 ug/dl. Again, however, evidence exists for encepha-
lopathy occurring in some children at lower blood lead levels, i.e., at 80-100 jjg/dl.
It should be emphasized that, once encephalopathy occurs, death is not an improbable out-
come, regardless of the quality of medical treatment available at the time of acute crisis.
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In fact, certain diagnostic or treatment procedures themselves tend to exacerbate matters and
push the outcome toward fatality if the nature and severity of the problem are not fully rec-
ognized or properly diagnosed. It is also crucial to note the rapidity with which acute
encephalopathic symptoms can develop or death can occur in apparently asymptomatic individuals
or in those apparently only mildly affected by elevated body burdens of lead. It is not
unusual for rapid deterioration to occur, with convulsions or coma suddenly appearing and with
progression to death within 48 hours. This strongly suggests that, even in apparently asymp-
tomatic individuals, rather severe neural damage probably does exist at high blood lead levels
even though it is not yet overtly manifested in obvious encephalopathic symptoms. This con-
clusion is further supported by numerous studies showing that children with high blood lead
levels (over 80-100 |jg/dl), but not observed to manifest acute encephalopathic symptoms, are
permanently cognitively impaired, as are most children who survive acute episodes of frank
lead encephalopathy.
Other evidence tends to confirm that some type of neural dysfunction exists in apparently
asymptomatic children, even at much lower levels of blood lead. The body of studies on low-
or moderate-level lead effects on neurobehavioral functions, as summarized in Table 12-1, pre-
sents a rather impressive array of data pointing to that conclusion. Several well-controlled
studies have found effects that are clearly statistically significant, whereas others have
found nonsignificant but borderline effects. Even certain studies reporting generally non-
significant findings at times contain data confirming some statistically significant effects,
which the authors attribute to various extraneous factors. It should also be noted that,
given the apparent non-specific nature of some of the behavioral or neural effects probable at
low levels of lead exposure, one would not expect to find striking differences in every
instance. The lowest blood lead levels associated with significant neurobehavioral (e.g.
cognitive) deficits both in apparently asymptomatic children and in developing rats and
monkeys generally appear to be in the range of 30-50 ug/dl. Also, certain behavioral and
electrophysiological effects indicative of CNS deficits have been reported at lower levels,
supporting a continuous dose-response relationship between lead and neurotoxicity. Such
effects, when combined with adverse social factors (such as low parental IQ, low socioeconomic
status, poor nutrition, and poor quality of the caregiving environment) can place children,
especially those below the age of three years, at significant risk. However, it must be
acknowledged that nutritional covariates, as well as demographic social factors, have been
poorly controlled in many of the pediatric neurobehavioral studies reviewed above. Socio-
economic status also is a crude measure of parenting and family structure that requires fur-
ther assessment as a possible contributor to observed results of neurobehavioral studies.
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Timing, type, and duration of exposure are also important factors in both animal and
human studies. It is often uncertain whether observed blood lead levels represent the levels
that were responsible for observed behavioral deficits. Monitoring of lead exposures in human
subjects in all cases has been highly intermittent or non-existent during the period of life
preceding neurobehavioral assessment. In most human studies, only one or two blood lead
values are provided per subject. Tooth lead may be an important cumulative exposure index;
but its modest, highly variable correlation to blood lead or FEP and to external exposure
levels makes findings from various studies difficult to compare quantitatively. The com-
plexity of the many important covariates and their interaction with dependent measures of
modest validity, e.g., IQ tests, may also account for many of the discrepancies among the dif-
ferent studies.
The precise medical or health significance of the neuropsychological and electrophysio-
logical effects associated with low-level lead exposure as reported in the above studies is
difficult to state with confidence at this time. Observed IQ deficits and other behavioral
changes, although statistically significant in some studies, tend to be relatively small as
reported by the investigators, but nevertheless may still affect the intellectual development,
school performance, and social development of the affected children sufficiently to be regard-
ed as adverse. This would be especially true if such impaired intellectual development or
school performance and disrupted social development were reflective of persisting, long-term
effects of low-level lead exposure in early childhood. The issue of persistence of such lead
effects, however, remains to be more clearly resolved. Still, some study results reviewed
above suggest that significant low-level lead-induced neurobehavioral and EEG effects may, in
fact, persist at least into later childhood, and a number of animal studies demonstrate long-
term persistence into adulthood of neurologic dysfunctions induced by relatively moderate or
low level lead exposures early in postnatal development of mammalian species.
12.4.4.2 The Question of Irreversibility. Little research on humans is available on persis-
tence of effects. Some work suggests the possibility of reversing mild forms of peripheral
neuropathy in lead workers, but little is known regarding the reversibility of lead effects on
central nervous system function in humans. A recent two-year follow-up study of 28 children
of battery factory workers found a persistent relation between blood lead and altered slow
wave voltage of cortical slow wave potentials. Current human psychometric studies, however,
will have to be supplemented by prospective longitudinal studies of the effects of lead on
development in order to better elucidate persistence or reversibility of neurotoxic effects of
lead exposure early in infancy or childhood.
Various animal studies provide evidence that alterations in neurobehavioral function may
be long-lived, with such alterations being evident long after blood lead levels have returned
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to control levels. These persistent effects have been demonstrated in monkeys as well as rats
under a variety of learning performance test paradigms. Such results are also consistent with
morphological, electrophysiological, and biochemical studies on animals that suggest lasting
changes in synaptogenesis, dendritic development, myelin and fiber tract formation, ionic
mechanisms of neurotransmission, and energy metabolism.
12.4.4.3 Early Development and Susceptibility to Neural Damage. On the question of early
childhood vulnerability, the neurobehavioral data are consistent with morphological and bio-
chemical studies of the susceptibility of the heme biosynthetic pathway to perturbation by
lead. Various lines of evidence suggest that the order of susceptibility neurotoxic effects of
lead is: young > adult; female > male. Animal studies also have pointed to the perinatal
period of ontogeny as a particularly critical time for a variety of reasons: (1) it is a
period of rapid development of the nervous system; (2) it is a period where good nutrition is
particularly critical; and (3) it is a period where the caregiver environment is vital to nor-
mal development. However, the precise boundaries of a critical period for lead exposure are
not yet clear and may vary depending on the species and function or endpoint that is being
assessed. Nevertheless, there is general agreement that human infants and toddlers below the
age of three years are at special risk because of ijn utero exposure, increased opportunity for
exposure because of normal mouthing behavior of lead-containing objects, and increased rates
of lead absorption due to various factors, e.g., iron and calcium deficiencies.
12.4.4.4 Utility of Animal Studies in Drawing Parallels to the Human Condition. Animal
models are used to shed light on questions where it would be impractical or ethically unaccep-
table to use human subjects. This is particularly true in the case of exposure to environmen-
tal toxins such as lead. In the case of lead, it has been most effective and convenient to
expose developing animals via their mothers' milk or by gastric gavage, at least until
weaning. Very often, the exposure is continued in the water or food for some time beyond
weaning. This approach does succeed in simulating at least two features commonly found in
human exposure: oral intake and exposure during early development. The preweaning postnatal
period in rats and mice is of particular relevance in terms of parallels with the first two
years or so of human brain development.
However, important questions exist concerning the comparability of animal models to
humans. Given differences between humans, rats, and monkeys in heme chemistry, metabolism,
and other aspects of physiology and anatomy, it is difficult to state what constitutes an
equivalent internal exposure level (much less an equivalent external exposure level). For
example, is a blood lead level of 30 ug/dl in a suckling rat equivalent to 30 ug/dl in a
three-year-old child? Until an answer is available to this question, i.e., until the function
describing the relationship of exposure indices in different species is available, the utility
of animal models for deriving dose-response functions relevant to humans will be limited.
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Questions also exist regarding the comparability of neurobehavioral effects in animals
with human behavior and cognitive function. One difficulty in comparing behavioral endpoints
such as locomotor activity is the lack of a consistent operational definition. In addition to
the lack of standardized methodologies, behavior is notoriously difficult to "equate" or com-
pare meaningfully across species because behavioral analogies do not demonstrate behavioral
homologies. Thus, it is improper to assume, without knowing more about the responsible
underlying neurological structures and processes, that a rat's performance on an operant
conditioning schedule or a monkey's performance on a stimulus discrimination task necessarily
corresponds directly to a child's performance on a cognitive function test. Nevertheless,
deficits in performance by mammalian animals on such tasks are indicative of likely altered
CMS functions, which is reasonable to assume will likely parallel some type of altered CNS
function in humans as well.
In terms of morphological findings, there are reports of hippocampal lesions in both
lead-exposed rats and humans that are consistent with a number of independent behavioral find-
ings suggesting an impaired ability to respond appropriately to altered contingencies for
rewards. That is, subjects with hippocampal damage tend to persist in certain patterns of
behavior even when changed conditions make the behavior inappropriate; the same sort of ten-
dency seems to be common to a number of lead-induced behavioral effects. Other morphological
findings in animals, such as demyelination and glial cell decline, are comparable to human
neuropathologic observations only at relatively high exposure levels.
Another neurobehavioral endpoint of interest in comparing human and animal neurotoxicity
of lead is electrophysiological function. Alterations of electroencephalographic patterns and
cortical slow wave voltage have been reported for lead-exposed children, and various electro-
physiological alterations both 1_n vivo (e.g., in rat visual evoked response) and u; vitro
(e.g., in frog miniature endplate potentials) have also been noted in laboratory animals.
Thus, far, however, these lines of work have not converged sufficiently to allow for much in
the way of definitive conclusions regarding electrophysiological aspects of lead neuro-
toxicity.
Biochemical approaches to the experimental study of lead effects on the nervous system
have been basically limited to laboratory animal subjects. Although their linkage to human
neurobehavioral function is at this point somewhat speculative, such studies do provide in-
sight on possible neurochemical intermediaries of lead neurotoxicity. No single neurotrans-
mitter system has been shown to be particularly sensitive to the effects of lead exposure;
lead-induced alterations have been demonstrated in various neurotransmitters, including
dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid. In addition, lead has been
shown to have subcellular effects in the central nervous system at the level of mitochondrial
function and protein synthesis. In particular, the work of McCauley, Bull, and co-workers has
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indicated that delays seen in cortical synoptogenesis and metabolic maturation following pre-
natal lead exposure may well underly the delayed development of exploratory and locomoter
function seen in other studies of the neurobehavioral effects of lead.
Given the difficulties in formulating a comparative basis for internal exposure levels
among different species, the primary value of many animal studies, particularly J_n vitro
studies, may be in the information they can provide on basic mechanisms involved in lead
neurotoxicity. A number of key j_n vitro studies are summarized in Table 12-9. These stu-
dies show that significant, potentially deleterious effects on nervous system function occur
at i_n situ lead concentrations of 5 uM and possibly lower. This suggests that, at least
intracellularly or on a molecular level, there may exist essentially no threshold for certain
neurochemical effects of lead. The relationship between blood lead levels and lead concen-
trations at extra- or intracellular sites of action, however, remains to be determined.
Despite the problems in generalizing from animals to humans, both the animal and the
human studies show considerable internal consistency in that they both support a continuous
dose-response functional relationship between lead and neurotoxic biochemical, morphological,
electrophysiological, and behavioral effects.
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TABLE 12-9. SUMMARY OF KEY STUDIES OF IN VITRO LEAD EXPOSURE
INi
I
Preparation
Adult rat brain
Isolated microvessels
from rat brain
Adult mouse
brain homogenate
Exposure
concentration
0.1 uM Pb(Ac)2
0.1 uM Pb(Ac)2
0.1-5 uM tri-n-butyl
lead (TBL)
Results
35% inhibition of whole-
brain BH4 synthesis
Blockade of sugar-specific
transport sites in capi-
llary endothelial cells
1) 50% decline in high
affinity uptake of DA;
2) 25% increase in
release of DA
Reference
Purdy et al.
(1981)
Kolber et al .
(1980)
Bondy et al.
(1979a,b)
Adult rat striatum
Embryonic chick
brain cell culture
Brainstem and forebrain
slices from PND-22 rats
Adult rat
cerebellar homogenates
Adult rat
cerebellar mitochondria
Adult frog
nerve/muscle preparation
Isolated, perfused
bullfrog retina
1-5 |jM TBL
3 MM (Et3Pb)Cl2
3 uM (Et3Pb)Cl2
3-5 (jM Pb++
5 MM Pb(Ac)z
5 uH Pb++
5 (jM Pb"*"1"
0-60% inhibition of spiro-
peridal binding to DA
receptors
50% reduction in no. of
cells exhibiting processes
Inhibition of teucine in-
corporation into myelin
proteins
Inhibition of adenylate
cyclase activity
Inhibition of respiration
Increase in frequency of
MEPP's (indicative of
depression of synaptic
transmission)
Depression of rod (but not
cone) receptor potentials
Bondy and Agarwal
(1980)
Grundt et al.
(1981)
Konat and Clausen
(1978, 1980)
Konat et al.
(1979)
Taylor et al.
(1978)
Gmerek et al.
(1981)
Kolton and Yaari
(1982)
Fox and Sillman
(1979)
•33
-f.
-------
TABLE 12-9. (continued)
INJ
O
Preparation
Cerebellar slices
from PND-14 rats
In oculo culture of
cerebellar tissue
from PND-15 rats
Cell suspension from
forebrain of PND-22 rats
Adult rat cerebral
and cerebellar mitochondria
Adult rat caudate
synaptosomes
Exposure
concentration
5-7 pM (EtaPb)Cl2
5-10 pM Pb"*"1"
20 uM (EtgPb)Cl2
50 pM Pb(Ac)2
50 pM PbCl2
Results
Inhibition of incorporation
of S04 and serine into
myelin galactolipids
"Disinhibition" of NE-
induced inhibition of
spontaneous activity in
Purkinje cells
30% inhibition of total
protein synthesis
Inhibition of respiration
8-fol (^increase in binding
of Ca to mitochondria
Reference
Grundt and
Neskovic (1980)
Taylor et al .
(1978)
Konat et al.
(1978)
Holtzman et al .
(1978b, 1980b)
Silbergeld and
Adler (1978)
Capillary endothelial
cells from rat cere-
cortex
100 pM Pb(Ac)z
(effectively increases
Ca gradient across ter-
minal membrane, thus in-
creasing synatic trans-
mission without altering
firing rates)
Pb preferentially seques-
ter^d in mitochondria like
Ca . (Possible basis for
Pb-induced disruo_£ion of
transmembrane Ca
transport)
Silbergeld et al.
(1980b)
PND: post-natal day
Pb(Ac)z: lead acetate
PbClj: lead chloride
Et3Pb: triethyl lead
TBL: tri-n-butyl lead
DA: dopamine
NE: norepinephrine
BH4: tetrahydrobiopterin
MEPP's: miniature endplate potentials
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PRELIMINARY DRAFT
12.5 EFFECTS OF LEAD ON THE KIDNEY
12.5.1 Historical Aspects
The first description of renal disease due to lead was published by Lancereaux (1862).
In a painter with lead encephalopathy and gout, Lancereaux noted tubulo-interstitial disease
of the kidneys at autopsy. Distinctions between glomerular and tubulo-interstitial forms of
kidney disease were not, however, clearly defined in the mid-nineteenth century. Ollivier
(1863) reported observations in 37 cases of lead poisoning with renal disease and thus intro-
duced the idea that lead nephropathy was a proteinuric disease, a confusion with primary
glomerular disease that persisted for over a century. Under the leadership of Jean Martin
Charcot, interstitial nephritis characterized by meager proteinuria in lead poisoning was
widely publicized (Charcot, 1868; Charcot and Gombault, 1881) but not always appreciated by
contemporary physicians (Danjoy, 1864; Gepper, 1882; Lorimer, 1886).
More than ninety years ago, the English toxicologist Thomas Oliver (1885, 1891) distin-
guished acute effects of lead on the kidney from lead-induced chronic nephropathy. Acute
renal effects of lead were seen in persons dying of lead poisoning and were usually restricted
to non-specific changes in the renal proximal tubular lining cells. Oliver noted that a
"true interstitial nephritis" developed later, often with glomerular involvement.
In an extensive review of the earlier literature, Pejic (1928) emphasized that changes in
the proximal tubules, rather than the vascular changes often referred to in earlier studies
(Gull and Sutton, 1872), constitute the primary injury to the kidney in lead poisoning. Many
subsequent studies have shown pathological alterations in the renal tubule with onset during
the early or acute phase of lead intoxication. These include the formation of inclusion
bodies in nuclei of proximal tubular cells (Blackman, 1936) and the development of functional
defects as well as ultrastructural changes, particularly in renal tubular mitochondria.
12.5.2 Lead Nephropathy in Childhood
Dysfunction of the proximal tubule was first noted as glycosuria in the absence of hyper-
glycemia in childhood pica (McKhann, 1926). Later it was shown that the proximal tubule
transport defect included aminoaciduria (Wilson et al., 1953). Subsequently, Chisolm et al.
(1955) found that the full Fanconi syndrome was present: glycosuria, aminoaciduria, phos-
phaturia (with hypophosphatemia), and rickets. Proximal tubular transport defects appeared
only when blood lead levels exceeded 80 ug/dl. Generalized aminoaciduria was seen more con-
sistently in Chisolm1s (1962, 1968) studies than were other manifestations of renal dysfunc-
tion. The condition was related to the severity of clinical toxicity, with the complete
Fanconi syndrome occurring in encephalopathic children when blood lead concentrations exceeded
150 ug/dl (National Academy of Sciences, 1972). Children who were under three years of age
excreted 4 to 12.8 mg of lead chelate during the first day of therapy with CaEDTA at 50 mg/kg
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day. The aminoaciduria disappeared after treatment with chelating agents and clinical remis-
sion of other symptoms of lead toxicity (Chisolm, 1962). This is an important observation
relative to the long-term or chronic effects of lead on the kidney.
In a group of children with slight lead-related neurological signs reported by Pueschel
et al. (1972), generalized aminoaciduria was found in 8 of 43 children with blood lead levels
of 40 to 120 |jg/dl. It should be noted that the children reported to have aminoaciduria in
this study were selected because of a blood lead level of ^50 ug/dl or a provocative chelation
test of >500 ug of lead chelate per 24 hours.
Although children are considered generally to be more susceptible than adults to the
toxic effects of lead, the relatively sparse literature on childhood lead nephropathy probably
reflects a greater clinical concern with the life-threatening neurologic symptoms of lead in-
toxication than with the transient Fanconi syndrome.
12.5.3 Lead Nephropathy in Adults
There is convincing evidence in the literature that prolonged lead exposure in humans can
.result in chronic lead nephropathy in adults. This evidence is reviewed below in terms of six
major categories: (1) lead nephropathy following childhood lead poisoning; (2) "moonshine"
lead nephropathy; (3) occupational lead nephropathy; (4) lead and gouty nephropathy; (5) lead
and hypertensive nephrosclerosis; and (6) general population studies.
12.5.3.1 Lead Nephropathy Following Childhood Lead Poisoning. Reports from Queensland,
Australia (Gibson et al., 1892; Nye, 1933; Henderson, 1954; Emmerson, 1963) points to a strong
association between severe childhood lead poisoning, including central nervous system
symptoms, and chronic nephritis in early adulthood. The Australian children sustained acute
lead poisoning when confined to the enclosed, raised terraces peculiar to the houses around
Brisbane. The houses were painted with white lead, which the children ingested by direct con-
tamination of their fingers or by drinking lead-sweetened rain water as it flowed over the
weathered surfaces. Two fingers brushed against the powdery paint were shown to pick up about
2 mg of lead (Murray, 1939). Henderson (1954) followed up 401 untreated children who had been
diagnosed as having lead poisoning in Brisbane between 1915 and 1935. Of these 401 subjects,
death certificates revealed that 165 had died under the age of 40, 108 from nephritis or
hypertension. This is greatly in excess of expectation. Information was obtained from 101 of
the 187 survivors, and 17 of these had hypertension and/or albuminun'a.
In a more recent study, Emmerson (1963) presented a criterion for implicating lead as an
etiological factor in such patients: the patients should have an excessive urinary excretion
of lead following administration of CaEOTA. Leckie and Tompsett (1958) had shown that
increasing the CaEDTA dosage above 2 g/day intravenously had little effect on the amount of
lead chelate excreted by adults. They observed little difference in chelatable lead excretion
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when 1 g was compared with 2 g (i.v.). Similarly, the magnitude of lead chelated when 1 g is
given i.v. or 2 g i.m. (over 12 hr) appears to be the same (Albahary et al., 1961; Emmerson,
1963; Wedeen et al., 1975). Adult control subjects without undue lead absorption excrete less
than 650 pg lead chelate during the first post-injection day if renal function is normal, or
over 4 days if renal function is severely reduced. The level of reduction of glomerular fil-
tration rate (GFR) at which the EDTA lead-mobilization test is no longer reliable has not been
precisely defined but probably exceeds a reduction of 85 percent (serum creatinine concentra-
tions in excess of about 6 mg/dl). In Emmerson1s (1963) study 32 patients with chronic renal
disease attributable to lead poisoning had elevated excretion of lead chelate. Intranuclear
inclusions are associated with recent acute exposure but are often absent in chronic lead
nephropathy or after the administration of CaNaj-EDTA (Goyer and Wilson, 1975).
The Australian investigators established the validity of the EDTA lead-mobilization test
for the detection of excessive past lead absorption and further demonstrated that the body
lead stores were retained primarily in bone (Emmerson, 1963; Henderson, 1954; Inglis et al.,
1978). Bone lead concentration averaged 94 ug/g wet weight in the young adults dying of lead
nephropathy in Australia (Henderson and Inglis, 1957; Inglis et al., 1978), compared with mean
values ranging from 14 to 23 ug/g wet weight in bones from non-exposed individuals (Barry,
1975; Emmerson, 1963; Gross et al., 1975; Wedeen, 1982).
Attempts to confirm the relationship between childhood lead intoxication and chronic
nephropathy have not been successful in at least two studies in the United States. Tepper
(1963) found no evidence of increased chronic renal disease in 139 persons with a well-
documented history of childhood plumbism 20 to 35 years earlier at the Boston Children's
Hospital. The study population was 165 patients (after review of 524 case records) who met
any two of the following criteria: 1) a definite history of pica or use of lead nipple
shields; 2) X-ray evidence of lead-induced skeletal alterations; or 3) characteristic
symptoms. No uniform objective measure of lead absorption was reported in this study. In 42
of the 139 subjects clinical studies of renal function were performed and included urinalysis,
endogenous creatinine clearance, urine culture, urine concentrating ability, 24-hour protein
excretion, and phenolsulfonphthalein excretion. Only one patient was believed to have died of
lead nephropathy; three with creatinine clearances under 90 ml/min were said to have had
inadequate urine collections. Insufficient details concerning past lead absorption and
patient selection were provided to permit generalized conclusions from this report.
Chisolm et al. (1976) also found no evidence of renal disease (as judged by routine
urinalysis, blood urea nitrogen, serum uric acid, and creatinine clearance) in 55 adolescents
known to have been treated for lead intoxication 11 to 16 years earlier. An important dis-
tinction between the Australian group and those patients in the United States studied by
Chisolm et al. (1976) was that none of the latter subjects showed evidence of increased
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residual body lead burden by the EDTA lead-mobilization test. This U.S. study was carried out
on adolescents between 12 and 22 years of age in the late 1960s. During acute toxicity in
early childhood, blood lead levels had ranged from 100 to 650 ug/dl; all received immediate
chelation therapy. Follow-up chelation tests performed with 1 g EDTA i.m. (with procaine)
approximately a decade later resulted in 24-hour lead-chelate excretion of less than 600 |jg in
45 of 52 adolescents. The absence of renal disease in this study led Chisolm et al. to sug-
gest that lead toxicity in the Australian children may have been of a different type, with a
more protracted course than that experienced by the American children. On the other hand,
chelation therapy of the American children may have removed lead stored in bone and thus pre-
vented the development of renal failure later in life. Most children in the United States who
suffer from overt lead toxicity do so early in childhood, between the ages of 1 and 4, the
source often being oral ingestion of flecks of wall paint and plaster containing lead.
12.5.3.2 "Moonshine" Lead Nephropathy. In the United States, chronic lead nephropathy in
adults was first noted among illicit whiskey consumers in the southeastern states. The pre-
revolutionary tradition of homemade whiskey ("moonshine") was modernized during the Prohibi-
tion era for large scale production. The copper condensers traditionally used in the illegal
stills were replaced by truck radiators with lead-soldered parts. Illegally produced whiskey
might contain up to 74 mg of lead per liter (Eskew et al., 1961). The enormous variability in
moonshine lead content has recently been reiterated in a study of 12 samples from Georgia, of
which five contained less than 10 ug/1 but one contained 5.3 mg/1 (Gerhardt et al., 1980).
Renal disease often accompanied by hypertension and gout was common among moonshiners
(Eskew et al., 1961; Morgan et al., 1966; Ball and Sorenson, 1969). These patients usually
sought medical care because of symptomatic lead poisoning characterized by colic, neurological
disturbances, and anemia, although more subtle cases were sometimes detected by use of the
i.v. EDTA lead-mobilization test (Morgan, 1968; Morgan and Burch, 1972). While acute sympto-
matology, including azotemia, sometimes improved during chelation therapy, residual chronic
renal failure, gout, and hypertension frequently proved refractory, thus indicating underlying
chronic renal disease superimposed on acute renal failure due to lead (Morgan, 1975).
12.5.3.3 Occupational Lead Nephropathy. Although rarely recognized in the United States
(Brieger and Reiders, 1959; Anonymous, 1966; Greenfield and Gray, 1950; Johnstone, 1964;
Kazantzis, 1970; Lane, 1949; Malcolm, 1971; Mayers, 1947), occupational lead nephropathy,
often associated with gout and hypertension, was widely identified in Europe as a sequela to
overt lead intoxication in the industrial setting (Albahary et al., 1961, 1965; Cramer et al.,
1974; Danilovic, 1958; Galle and Morel-Maroger, 1965; Lejeune et al., 1969; Lilis et al., 1967,
1968; Radosevic" et al., 1961; Radulescu et al., 1957; Richet et al., 1964, 1966; Tara and
Francon, 1975; Vigdortchik, 1935). Some important recent studies are summarized here.
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Richet et al. (1964) reported renal findings in eight lead workers, all of whom had
repeated episodes of lead poisoning, including colic. Intravenous EDTA lead-mobilization
tests ranged from 587 to 5930 ug lead-chelate excretion per 24 hours. Four of these men had
reduced glomerular filtration rates, one had hypertension with gout, one had hypertension
alone, and one had gout alone. Proteinuria exceeded 200 mg/day in only one patient. Five of
seven renal biopsies were abnormal showing minor glomerular sclerosis but severe interstitial
nephritis and vascular sclerosis by light microscopy. The one patient with proteinuria of 1.7
gm/day showed extensive glomerular hyalinization. Electron microscopy showed intranuclear and
cytoplasmic inclusions and ballooning of mitochondria in proximal tubule cells. The presence
of intranuclear inclusion bodies is helpful in establishing a relationship between renal
lesions and lead toxicity, but inclusion bodies are not always present in persons with chronic
lead nephropathy (Cramer et al., 1974; Wedeen et al., 1975, 1979).
Richet et al. (1966) subsequently recorded renal findings in 23 symptomatic lead workers
in whom blood lead levels ranged from 30 to 87 |jg/dl. Six had diastolic pressures over 90
mm Hg, three had proteinuria exceeding 200 mg/day, and five had gout. In 5 of 21 renal biop-
sies, glomeruli showed minor hyalinization, but two cases showed major glomerular disease
(their creatinine clearances were 20 and 33 ml/min, respectively). Interstitial fibrosis and
arteriolar sclerosis were seen in all but two biopsies. Intranuclear inclusion bodies were
noted in 13 cases. Electron jnicroscopy showed loss of brush borders, iron-staining intra-
cellular vacuoles, and ballooning of mitochondria in proximal tubule epithelial cells.
Effective renal plasma flow (C ., plasma clearance of p-aminohippuric acid) by the
single injection disappearance technique was measured in 14 lead-poisoned Rumanian workers be-
fore and after chelation therapy by Lilis et al. (1967). C . increased from a pre-treatment
mean of 428 ml/min (significantly less than the control mean of 580 ml/min) to a mean of 485
ml/min after chelation therapy (p <0.02). However, no significant increase in GFR (endogenous
creatinine clearance) was found. Lilis et al. interpreted the change in effective renal
plasma flow as indicating reversal of the renal vasoconstriction that accompanied acute lead
toxicity. Although neither blood lead concentrations nor long-term follow-up studies of renal
function were provided, it seems likely that most of these patients suffered from acute,
rather than chronic, lead nephropathy.
In a subsequent set of 102 cases of occupational lead poisoning studied by Lilis et al.
(1968), seven cases of clinically verified chronic nephropathy were found. In this group, en-
dogenous creatinine clearance was less than 80 ml/min two weeks or more after the last episode
of lead colic. The mean blood lead level approximated 80 M9/dl (range 42 to 141 ug/dl.) All
patients excreted more than 10 mg lead chelate over 5 days during therapy consisting of 2 g
CaNa.^EDTA i.v. daily. Nephropathy was more common among those exposed to lead for more than
10 years than among those exposed for less than 10 years. Most of the Rumanian lead workers
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had experienced lead colic, and 13 of 17 had persistent hypertension that followed the appear-
ance of renal failure by several years. Proteinuria was absent except in two individuals who
excreted 250 and 500 mg/1. Hyperuricemia was not evident in the absence of azotemia. In both
studies by Lillis, reduced urea clearance preceded reduced creatinine clearance.
Cramer et al. (1974) examined renal biopsies from five lead workers exposed for 0.5 to 20
years in Sweden. Their blood lead levels ranged from 71 to 138 ug/dl, with GFR ranging from
65 to 128 ml/min, but C . exceeding 600 ml/min in all. Although plasma concentrations of
valine, tyrosine, and phenylalanine were reduced, excretion of these amino acids was not sig-
nificantly different from controls. A proximal tubular reabsorptive defect might, therefore,
have been present without increased amino acid excretion because of low circulating levels:
increased fractional excretion may have occurred without increased absolute amino acid ex-
cretion. Albuminuria and glycosuria were not present. Glomeruli were normal by electron
microscopy. Intranuclear inclusions in proximal tubules were found in two patients with nor-
mal GFRs, and peritubular fibrosis was present in the remaining three patients who had had the
longest occupational exposure (4 to 20 years).
Wedeen et al. (1975, 1979) reported on renal dysfunction in 140 occupationally exposed
men. These investigators used the EDTA lead-mobilization test (1 g CaEDTA with 1 ml of 2 per-
cent procaine given i.m. twice, 8 to 12 hr apart) to detect workers with excessive body lead
stores. In contrast to workers with concurrent lead exposure (Alessio et al., 1979), blood
lead measures have proven unsatisfactory for detection of past lead exposure (Baker et al.,
1979; Havelda et al., 1980; Vitale et al. , 1975). Of the 140 workers tested, 113 excreted
1000 ug or more of lead-chelate in 24 hr compared with a normal upper limit of 650 |jg/day
(Albahary et al., 1961; Emmerson, 1973; Wedeen et al., 1975). Glomerular filtration rates
measured by li;bl-iothalamate clearance in 57 men with increased mobilizable lead revealed
reduced renal function in 21 (GFR less than 90 ml/min per 1.73 m* body surface area). When
workers over age 55 or with gout, hypertension, or other possible causes of renal disease were
excluded, 15 remained who had previously unsuspected lead nephropathy. Their GFRs ranged
between 52 and 88 ml/min per 1.73 m*. Only three of the men with occult renal failure had
ever experienced symptoms attributable to lead poisoning. Of the 15 lead nephropathy
patients, one had a blood lead level over 80 ug/dl, three repeatedly had blood levels under 40
ug/dl, and eleven had blood levels between 40 and 80 ug/dl at the time of the study. Thus,
blood lead levels were poorly correlated with degree of renal dysfunction. The failure of
blood lead level to predict the presence of lead nephropathy probably stems from the indepen-
dence of blood lead from cumulative bone lead stores (Gross, 1981; Saenger et al., 1982a,b).
Percutaneous renal biopsies from 12 of the lead workers with reduced GFRs revealed focal
interstitial nephritis in six. Non-specific changes were present in proximal tubules, includ-
ing loss of brush borders, deformed mitochondria, and increased lysosomal bodies. Intra-
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nuclear inclusion bodies were not found in the renal biopsies from these men who had experi-
enced long-term occupational exposure and who had had chelation tests shortly before biopsy.
In experimental animals, chelation results in the rapid disapperance of lead-induced intra-
nuclear inclusions (Goyer and Wilson, 1975). The presence of a variety of immunoglobulin
deposits by fluorescent microscopy suggests (but does not prove) the possibility that some
stages of lead nephropathy in adults may be mediated by immune mechanisms.
Eight patients with pre-azoteraic occupational lead nephropathy were treated with 1 g
CaEDTA (with procaine) i.m. three times weekly for 6 to 50 months. In four patients, GFR rose
by 20 percent or more by the time the EDTA test had fallen to less than 850 ug Pb/day. The
rise in GFR was paralleled by increases in effective renal plasma flow (C ,) during CaEDTA
treatment. These findings indicate that chronic lead nephropathy may be reversible by chela-
tion therapy, at least during the pre-azotemic phase of the disease (Wedeen et a!., 1979).
However, much more information will have to be obtained on the value of long-term, low-dose
chelation therapy before this regimen can be widely recommended. There is, at present, no
evidence that interstitial nephritis itself is reversed by chelation therapy. It may well be
that only functional derangements are corrected and that the improvement in GFR is not accom-
panied by disappearance of tubulo-interstitial changes in kidney. Chronic volume depletion,
for example, might be caused by lead-induced depression of the renin-angiotension-aldosterone
system (McAllister et al., 1971) or by direct inhibition of (Na+, K+)ATPase-mediated sodium
transport (Nechay and Williams, 1977; Nechay and Saunders, 1978a,b,c; Raghavan et al., 1981;
Secchi et al., 1973). On the other hand, volume depletion would be expected to produce pre-
renal azotetrria, but this was not evident in these patients. The value of chelation therapy in
chronic lead nephropathy once azotemia is established is unknown.
The prevalence of azotemia among lead workers has recently been confirmed in health sur-
veys conducted at industrial sites (Baker et al., 1979; Hammond et al., 1980; Landrigan et
al., 1982; Lilis et al., 1979, 1980). Interpretation of these data is, however, hampered by
the weak correlation generally found between blood lead levels and chronic lead nephropathy in
adults, the absence of matched prospective controls, and the lack of detailed diagnostic in-
formation on the workers found to have renal dysfunction. Moreover, blood serum urea nitrogen
(BUN) is a relatively poor indicator of renal function because it is sensitive to a variety of
physiological variables other than GFR, including protein anabolism, catabolism, and hydra-
tion. Several other measures of renal function are more reliable than the BUN, including in
order of increasing clinical reliability: serum creatinine, endogenous creatinine clearance,
and 12bl-iothalamate or inulin clearance. It should be noted that none of these measures of
GFR can be considered reliable in the presence of any acute illness such as lead colic or
encephalopathy. Elevated BUN in field surveys may, therefore, sometimes represent transient
acute functional changes rather than chronic intrinsic renal disease.
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The variable susceptibility of the kidneys to the nephrotoxic effects of lead suggests
that environmental factors in addition to lead may participate in the expression of renal
damage. Industrial workers are often exposed to a variety of toxic materials, some of which,
such as cadmium (Buchet et al., 1980), are themselves nephrotoxic. In contrast to cadmium,
lead does not increase urinary excretion of beta-2-microglobulins (Batuman et al., 1981;
Buchet et al., 1980) or lysozyme (Wedeen et al., 1979). Multiple interactions between en-
vironmental toxins may enhance susceptibility to lead nephrotoxicity. Similarly, nephro-
toxicity may be modulated by reductions in 1,25-dihydroxy vitamin 03, increased 6-beta-hydro-
xycortisol production (Saenger et al., 1981, 1982a,b), or immunologic alterations
(Gudbrandsson et al., 1981; Keller and Brauner, 1977; Kristensen, 1978; Kristensen and
Andersen, 1978). Reductions in dietary intake of calcium, copper, or iron similarly appear to
increase susceptibility to lead intoxication (Mahaffey and Michaelson, 1980).
The slowly progressive chronic lead nephropathy resulting from years of relatively low-
dose lead absorption observed in adults is strikingly different from the acute lead nephro-
pathy arising from the relatively brief but intense exposure arising from childhood pica.
Typical acid-fast intranuclear inclusions are, for example, far less common in the kidneys of
adults (Cramer et al., 1974; Wedeen et al., 1975). Although aminoaciduria has been found to
be greater in groups of lead workers than in controls (Clarkson and Kench, 1956; Goyer et al.,
1972), proximal tubular dysfunction is more difficult to demonstrate in adults with chronic
lead nephropathy than in acutely exposed children (Cramer et al., 1974). It should be remem-
bered, however, that children with the Fanconi syndrome have far more severe acute lead intox-
ication than is usual for workmen on the job. In contrast to the reversible Fanconi syndrome
associated with childhood lead poisoning, proximal tubular reabsorptive defects in occupa-
tional ly exposed adults are uncommon and subtle; clearance measurements are often required to
discern impaired tubular reabsorption in chronic lead nephropathy. Hyperuricemia is frequent
among lead workers (Albahary et al., 1965; Garrod, 1859; Hong et al., 1980; Landrigan et al.,
1982), presumably a consequence of specific lead inhibition of uric acid excretion, increased
uric acid production (Emmerson et al., 1971; Granick et al., 1978; Ludwig, 1957), and pre-
renal azotemia from volume depletion. The hyperuricemia in adults contrasts with the reduced
serum uric acid levels usually associated with the Fanconi syndrome in childhood lead
poisoning. Although aminoaciduria and glycosuria are unusual in chronic lead nephropathy,
Hong et al. (1980) reported a disproportionate reduction in the maximum reabsorptive rate for
glucose compared with para-aminohippuric acid (PAH) in five of six lead workers they studied.
PAH transport has not been consistently altered beyond that expected in renal failure of any
etiology (Hong et al., 1980; Wedeen et al., 1975). Biagini et al. (1977) have, however,
reported a good negative linear correlation between the one-day EDTA lead-mobilization test
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and C ^ in 11 patients with histologic evidence of lead-induced ultrastructural abnormalities
in proximal tubules.
The differences between lead nephropathy in children and adults would not appear to be a
consequence of the route of exposure, since a case of pica in an adult (geophagic lead nephro-
pathy) studied by Wedeen et al. (1978) showed the characteristics of chronic rather than acute
lead nephropathy; intranuclear inclusions were absent and the GFR was reduced out of propor-
tion to the effective renal plasma flow.
12.5.3.4 Lead and Gouty Nephropathy. Renal disease in gout can often be attributed to well
defined pathogenetic mechanisms including urinary tract stones and acute hyperuricemic nephro-
pathy with intratubular uric acid deposition (Bluestone et al., 1977). In the absence of
intra- or extra-renal urinary tract obstruction, the frequency, mechanism, and even the exist-
ence of a renal disease peculiar to gout remains in question. While some investigators have
described "specific" uric acid-induced histopathologic changes in both glomeruli and tubules
(Gonick et al., 1965; Sommers and Churg, 1982), rigorously defined controls with comparable
degrees of renal failure were not studied simultaneously. Specific histologic changes in the
kidneys in gout have not been found by others (Pardo et al., 1968; Bluestone et al., 1977).
Glomerulonephritis, vaguely defined "pyelonephritis" (Heptinstall, 1974), or intra- and extra-
renal obstruction may have sometimes been confused with the gouty kidney, particularly in
earlier studies (Fineberg and Altschul, 1956; Gibson et al., 1980b; Mayne, 1955; McQueen,
1951; Schnitker and Richter, 1936; Talbott and Terplan, 1960; Williamson, 1920).
The histopathology of interstitial nephritis in gout appears to be non-specific and can-
not usually be differentiated from that of "pyelonephritis," nephrosclerosis, or lead nephro-
pathy on morphologic grounds alone (Barlow and Beilin, 1968; Bluestone et al., 1977; Greenbaum
et al., 1961; Heptinstall, 1974; Inglis et al., 1978). Indeed, renal histologic changes in
non-gouty hypertensive patients have been reported to be identical to those found in gout
patients (Cannon et al., 1966). In these hypertensive patients, serum uric acid levels paral-
leled the BUN.
Confusion between glomerular and interstitial nephritis can in part be explained by the
tendency of proteinuria to increase as renal failure progresses, regardless of the underlying
etiology (Batuman et al., 1981). In the absence of overt lead intoxication it may, there-
fore, be difficult to recognize surreptitious lead absorption as a factor contributing to
renal failure in gouty patients. Further, medullary urate deposits, formerly believed to be
characteristic of gout (Brown and Mallory, 1950; Mayne, 1955; McQueen, 1951; Fineberg and
Altschul, 1956; Talbott and Terplan, 1960), have more recently been reported in end-stage
renal disease patients with no history of gout (Cannon et al., 1966; Inglis et al., 1978;
Linnane et al., 1981; Ostberg, 1968; Verger et al., 1967). Whether such crystalline deposits
contribute to, or are a consequence of, renal damage cannot be determined with confidence. In
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the presence of severe hyperuricemia (serum uric acid greater than 20 |jg/dl), intraluminal
crystal deposition may produce acute renal failure because of tubular obstruction (Emmerson,
1980) associated with grossly visible medullary streaks. In chronic renal failure without
gout or massive hyperuricemia, the functional significance of such medullary deposits is un-
clear (Linnane et al., 1981). Moreover, medullary microtophi, presumably developing around
intraluminal deposits, may extend into the renal interstitium, inducing foreign body reactions
with giant cell formation. Such amorphous deposits may require alcohol fixation and
deGalantha staining for identification (Verger et al., 1967). Because of the acid milieu,
medullary deposits are usually uric acid, while microtophi developing in the neutral pH of the
renal cortex are usually monosodium urate. Both amorphous and needle-like crystals have been
demonstrated in kidneys of non-gout and hyperuricemic patients frequently in association with
arteriolonephrosclerosis (Inglis et al., 1978; Cannon et al., 1966; Ostberg, 1968). Urate
deposits therefore, are not only not diagnostic, but may be the result, rather than the
cause, of interstitial nephritis. The problem of identifying unique characteristics of the
gouty kidney has been further confounded by the coexistence of pyelonephritis, diabetes
mellitis, hypertension, and the aging process itself.
Although the outlook for gout patients with renal disease was formerly considered grim
(Talbott, 1949; Talbott and Terplan, 1960), more recent long-term follow-up studies suggest a
benign course in the absence of renovascular or other supervening disease (Fessel, 1979; Yli
and Berger, 1982; Yli, 1982). Over the past four decades the reported incidence of renal
disease has varied from greater than 25 percent (Fineberg and Altschul, 1956; Henck et al.,
1941; Talbott, 1949; Talbott and Terplan, 1960; Wyngaarden, 1958) to less than 2 percent, as
observed by Yii (1982) in 707 patients followed from 1970 to 1980. The low incidence of renal
disease in some hyperuricemic populations does not support the view that elevated serum uric
acid levels of the degree ordinarily encountered in gout patients is harmful to the kidneys
(Emmerson, 1980; Fessel, 1979; Ramsey, 1979; Reif et al., 1981). Similarly, the failure of
the xanthine oxidase inhibitor, allopurinol, to reverse the course of renal failure in gout
patients despite marked reductions in the serum uric acid (Bowie et al., 1967; Levin and
Abrahams, 1966; Ogryzlo et al., 1966; Rosenfeld, 1974; Wilson et al., 1967) suggests that
renal disease in gout may be due in part to factors other than uric acid. Some studies have,
however, suggested a possible slowing of the rate of progression of renal failure in gout by
allopurinol (Gibson et al., 1978, 1980a,b; Briney et al., 1975). While the contribution of
uric acid to the renal disease of gout remains controversial, the hypothesized deleterious
effect of hyperuricemia on the kidney has no bearing on other potential mechanisms of renal
damage in these patients.
Although hyperuricemia is universal in patients with renal failure, gout is rare in such
patients except when the renal failure is due to lead. Gout occurs in approximately half of
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the patients with lead nephropathy (Emmerson, 1963, 1973; Ball and Sorenson, 1969; Richet et
al., 1965). Moreover, among gout patients in Scotland without known lead exposure, blood lead
levels were found to be higher than in non-gouty controls (Campbell et al., 1978). The long
association of lead poisoning with gout raises the possibility that lead absorption insuffi-
cient to produce overt lead intoxication may, nevertheless, cause gout with slowly progressive
renal failure. Garrod (1859), Ball and Sorenson (1969), and Emmerson et al. , (1971) demon-
strated that lead reduces uric acid excretion, thereby creating the internal milieu in which
gout can be expected. The mechanism of hyperuricemia in lead poisoning is, however, unclear.
Serum uric acid levels would be expected to rise in association with lead induced pre-renal
azotemia; increased proximal tubule reabsorption of uric acid could result from reduced glo-
merular filtration rate due to chronic volume depletion. Increased tubular reabsorption of
uric acid in lead nephropathy was suggested by the pyrazinamide suppression test (Emmerson,
1971), but interpretation of this procedure has been questioned (Holmes and Kelly, 1974).
Lead inhibition of tubular secretion of uric acid, therefore, remains another possible mecha-
nism of reduced uric acid excretion. In addition, some investigators have found increased
uric acid excretion in saturnine gout patients, thereby raising the possibility that lead in-
creases uric acid production in addition to reducing uric acid excretion (Emmerson et al.,
1971; Ludwig, 1957; Granick et al., 1978).
Having specifically excluded patients with gout or hypertension from their study of occu-
pational lead nephropathy, Wedeen and collaborators examined the possible role of lead in the
etiology of the gouty kidney (Batuman et al., 1981). To test the hypothesis that surrepti-
tious lead absorption may sometimes contribute to renal failure in gout, 44 armed service
veterans with gout were examined by the EDTA lead-mobilization test. Individuals currently
exposed to lead (including lead workers) were specifically excluded. Collection of urine dur-
ing the EDTA lead-mobilization test was extended to three days because reduced GFR delays
excretion of the lead chelate (Emmerson, 1963). Note that the. EDTA test does not appear to be
nephrotoxic even for patients with preexisting renal failure (Wedeen et al., 1983). Half of
the gout patients had normal renal function and half had renal failure as indicated by serum
creatinines over 1.5 mg/dl (mean = 3.0; standard error = 0.4 mg/dl), reflecting approximately
70 percent reduction in renal function. The groups were comparable in regard to age, duration
of gout, incidence of hypertension, and history of past lead exposure. The mean (and standard
error) blood lead concentration was 26 (± 3) pg/dl in the patients with reduced renal function
and 24 (± 3) ug/dl in the gout patients with normal kidney function. The gout patients with
renal dysfunction, however, excreted significantly more lead chelate than did those without
renal dysfunction (806 ± 90 and 470 ± 52 pg Pb over 3 days, respectively).
Ten control patients with comparable renal failure excreted 424 ± 72 yg lead during the
3-day EDTA test (2 g i.m.). The non-gout control patients with renal failure had normal lead
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stores (Emmerson, 1973; Wedeen et al., 1975), indicating that the excessive mobilizable lead
in the gout patients with renal failure was not a consequence of reduced renal function per
se. These studies suggest that excessive lead absorption may sometimes be responsible for the
gouty kidney in contemporary patients, as appeared to be the case in the past (Wedeen, 1981).
While the EDTA lead-mobilization test cannot prove the absence of other forms of renal
disease, when other known causes are excluded by appropriate diagnostic studies, a positive
EDTA test can indicate that lead may be a contributing cause of renal failure.
The source of lead exposure in these armed service veterans could not be determined with
confidence. A history of transient occupational exposure and occasional moonshine consumption
was common among all the veterans, but the medical histories did not correlate with either the
EDTA lead-mobilization test or the presence of renal failure. The relative contributions of
airborne lead, industrial sources, and illicit whiskey to the excessive body lead stores dem-
onstrated by the EDTA lead-mobilization test could not, therefore, be determined.
12.5.3.5 Lead and Hypertensive Nephrosclerosis. Hypertension is another putative complica-
tion of excessive lead absorption that has a long and controversial history. Hypertension has
often been associated with lead poisoning, frequently together with renal failure (Beevers et
al., 1980; Dingwall-Fordyce and Lane, 1963; Emmerson, 1963; Legge, 1901; Lorimer, 1886;
Morgan, 1976; Oliver, 1891; Richet et al., 1966; Vigdortchik, 1935). However, a number of in-
vestigators have failed to find such an association (Belknap, 1936; Brieger and Rieders, 1959;
Cramer and Dahlberg, 1966; Fouts and Page, 1942; Malcolm, 1971; Mayers, 1947; Ramirez-
Cervantes et al., 1978). Because of the absence of both uniform definitions of excessive lead
exposure and prospective control populations, the true contribution of lead to hypertension at
various levels and durations of exposure is unknown. Similarly, it is not clear whether
lead-induced hypertension is mediated by renal disease, vascular effects, or mechanisms invol-
ving vasoactive hormones or sodium transport. Definitive epidemiological studies remain to be
performed, but the etiologic role of lead in hypertension is likely to remain clouded as long
as the etiology of "essential" hypertension is unknown.
Among non-occupationally exposed individuals, hypertension and serum uric acid levels
have been found to correlate with blood lead levels (Beevers et al., 1976). Moreover, the
kidneys of patients with chronic lead nephropathy may show uric acid microtophi and the vas-
cular changes of "benign essential hypertension" even in the absence of gout and hypertension
(Cramer et al., 1974; Inglis et al., 1978; Morgan, 1976; Wedeen et al., 1975). In a long-term
follow-up study of 624 patients with gout, Yli and Berger (1982) reported that while hyper-
uricemia alone had no deleterious effect on renal function, decreased renal function was more
likely to occur in gout patients with hypertension and/or ischemic heart disease than in those
with uncomplicated gout.
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Like gout, hypertension was specifically excluded from the study of occupational lead
nephropathy by Wedeen et al. (1975, 1979) in order to isolate lead-induced renal disease.
Hypertension by itself is widely accepted as a cause of renal failure. Currently, however,
the renal sequelae of moderate hypertension appear to be less dramatic than in the past
(Kincaid-Smith, 1982). In order to determine if unsuspected excessive body lead stores might
contribute to the renal disease of hypertension, 3-day EDTA (2 g i.m.) lead mobilization tests
were performed in hypertensive armed service veterans with and without renal failure (Batuman
et al., 1983). A significant increase in mobilizable lead was found in hypertensive subjects
with renal disease compared to those without renal disease. Control patients with renal fail-
ure again demonstrated normal mobilizable lead, thereby supporting the view that renal
failure is not responsible for the excess mobilizable lead in patients with hypertension and
renal failure. These findings suggest that patients who would otherwise be deemed to have
essential hypertension with nephrosclerosis can be shown to have underlying lead nephropathy
by the EDTA lead-mobilization test when other renal causes of hypertension are excluded.
The mechanism whereby lead induces hypertension remains unclear. Although renal disease,
particularly at the end-stage, is a recognized cause of hypertension, renal arteriolar histo-
logic changes may precede both hypertension and renal disease (Wedeen et al., 1975). Lead may
therefore induce hypertension by direct or indirect effects on the vascular system.
Studies of hypertension in moonshine consumers have indicated the presence of hyporenin-
emic hypoaldosteronism. A blunted plasma renin response to salt depletion has been described
in lead poisoned patients; this response can be restored to normal by chelation therapy
(McAllister et al., 1971; Gonzalez et al., 1978; Sandstead et al., 1970a). The diminished
renin-aldosterone responsiveness found in moonshine drinkers could not, however, be demon-
strated in occupationally exposed men with acute lead intoxication (Campbell et al., 1979).
Although the impairment of the renin-aldosterone system appears to be independent of renal
failure and hypertension, hyporeninemic hypoaldosteronism due to lead might contribute to the
hyperkalemia (Morgan, 1976) and the exaggerated natriuresis (Fleischer et al., 1980) of some
patients with "benign essential hypertension." Since urinary kailikrein excretion is reduced
in lead workers with hypertension, it has been suggested that the decrease in this vasodilator
may contribute to lead-induced hypertension (Boscolo et al., 1981). The specificity of kalli-
krein suppression in the renal and hypertensive manifestions of excessive lead absorption can-
not, however, be determined from available data, because lead workers without
hypertension and essential hypertensive patients without undue lead absorption also have
reduced urinary kailikrein excretion.
12.5.3.6 General Population Studies. Few studies have been performed to evaluate the possi-
ble harmful effects of lead on the kidneys in populations without suspected excessive lead
absorption from occupational or moonshine exposure.
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An epidemiological survey in Scotland of households with water lead concentrations in ex-
cess of WHO recommendations (100 H9/1) revealed a close correlation between water lead content
and blood lead and serum urea concentrations (Campbell et al., 1977). In 970 households lead
concentrations in drinking water ranged from <0.1 to >8.0 mg/1. After clinical and biochemi-
cal screening of 283 subjects from 136 of the households with water lead concentrations in
excess of 100 ug/1, a subsample of 57 persons with normal blood pressure and elevated serum
urea (40 ug/dl) was compared with a control group of 54- persons drawn from the study group
with normal blood pressure and normal serum urea. The frequency of renal dysfunction in indi-
viduals with elevated blood lead concentratons (>41 |jg/dl) was significantly greater than that
of age- and sex-matched controls.
Since 62 general practitioners took part in the screening, the subsamples may have come
from many different areas; however, it was not indicated if matching was done for place of re-
sidence. The authors found a significantly larger number of high blood lead concentrations
among the persons with elevated serum urea and claimed that elevated water lead concentration
was associated with renal insufficiency as reflected by raised serum urea concentrations.
This is difficult to accept since serum urea is not the method of choice for evaluating renal
function. Despite reservations concerning use of the BUN for assessing renal function (de-
scribed above), these findings are consistent with the view that excessive lead absorption
from household water causes renal dysfunction. However, the authors used unusual statistical
methods and could not exclude the reverse causal relationship, i.e., that renal failure had
caused elevated blood lead levels in their study group. A carefully matched control popula-
tion of azotemic individuals from low lead households would have been helpful for this
purpose. A more convincing finding in another subsample was a strong association between
hyperuricemia and blood lead level. This was also interpreted as a sign of renal insuffici-
ency, but it may have represented a direct effect of lead on uric acid production or renal
excretion.
These investigators have also found a statistically significant correlation between blood
lead concentration and hypertension. Tap-water lead did not, however, correlate with blood
lead among the hypertensive group, thus suggesting that other environmental sources of lead
may account for the presence of high blood lead concentrations among hypertensive persons in
Scotland (Beevers et al., 1976, 1980).
12.5.4 Mortality Data
Cooper and Gaffey (1975) analyzed mortality data available from 1267 death certificates
for 7032 lead workers who had been hired by 16 smelting or battery plants between 1900 and
1969. Standardized mortality ratios revealed an excess of observed over predicted deaths from
"other hypertensive disease" and "chronic nephritis and other renal sclerosis." The authors
DPB12/A 12-134 9/20/83
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PRELIMINARY DRAFT
concluded that "high levels of lead absorption such as occurred in many of the workers in this
series, can be associated with chronic renal disease." Although renal carcinomas have been
observed in lead poisoned rats, no increase in cancer rates was evident in this study of lead
workers (Cooper, 1976; see Section 12.7). Reports of renal carcinoma among lead workers are
distinctly unusual (Baker et al., 1980).
In a more limited study of 241 Australian smelter employees who were diagnosed as lead
poisoned between 1928 and 1959 by a government medical board, 140 deaths were identified
between 1930 and 1977 (McMichael and Johnson, 1982). Standard proportional mortality rates of
the lead-exposed workers compared with 695 non-lead-exposed employees revealed an overall
three-fold excess in deaths due to chronic nephritis and a two-fold excess in deaths due to
cerebral hemorrhage in the lead-exposed workers. Over the 47 years of this retrospective
study the number of deaths from chronic nephritis decreased from an initial level of 36 per-
cent to 4.6 percent among the lead-exposed workers, compared with a drop from 8.7 percent to
2.2 percent among controls. From 1965 to 1977 the age-standardized mortality rates from
chronic nephritis were the same for the lead-worker and control groups, although both rates
were higher than the proportional mortality rate for the general population of Australian
males. The latter observation indicated that the excessive deaths from chronic nephritis
among lead-poisoned workers at the smelter had declined in recent decades.
Despite substantial evidence that lead produces interstitial nephritis in adults, the im-
pact of chronic lead nephropathy on the general population is unknown. The diagnosis of lead
nephropathy is rarely made in dialysis patients in the United States. The absence of the
diagnosis does not, however, provide evidence for the absence of the disease. Advanced renal
failure is usually encountered only many years after excessive lead exposure. Moreover, acute
intoxication may never have occurred, and neither heme enzyme abnormalities nor elevated blood
lead levels may be present at the time renal failure becomes apparent. The causal relation-
ship between lead absorption and renal disease may therefore not be evident. It is likely
that such cases of lead nephropathy have previously been included among other diagnostic
categories such as pyelonephritis, interstitial nephritis, gouty nephropathy, and hypertensive
nephrosclerosis. Increasing proteinuria as lead nephropathy progresses may also cause con-
fusion with primary glomerulonephritis. It should also be noted that the End Stage Renal
Disease Program (Health Care Financing Administration, 1982) does not even include the diag-
nosis of lead nephropathy in its reporting statistics, regardless of whether the diagnosis is
recognized by the attending nephrologist.
12.5.5 Experimental Animal Studies of the Pathophysiology of Lead Nephropathy
12.5.5.1 Lead Uptake by the Kidney. Lead uptake by the kidney has been studied jn vivo and
In vitro using renal slices. Vander et al. (1977) performed renal clearance studies in dogs
DPB12/A 12-135 9/20/83
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PRELIMINARY DRAFT
two hours after a single i.v. dose of 0.1 or 0.5 mg lead acetate containing 1-3 mCi of 20aPb
or 1 hour after continous i.v. infusion of 0.1-0.15 mg/kg-hour. These investigators reported
that 43-44 percent of the plasma lead was ultrafiltrable, with kidney reabsorptian values of
89-94 percent for the ultrafiltrable fraction. A subsequent stop-flow analysis investigation
by Victery et al. (1979a), using dogs given a single i.v. dose of lead acetate at 0.2 or 10.0
mg/kg, showed both proximal and distal tubular reabsorption sites for lead. Distal reabsorp-
tion was not linked to sodium chloride or calcium transport pathways. Proximal tubule reab-
sorption was demonstrated in all animals tested during citrate or bicarbonate infusion.
Another experiment (Victery et al., 19796) examined the influence of acid-base status on renal
accumulation and excretion of lead in dogs given 0.5-50 pg/kg hr as an infusion or in rats
given access to drinking water containing 500 ppm Pb for 2-3 months. These showed that
alkalosis increased lead entry into tubule cells via both luminal and basolateral membranes,
with a resultant increase in both renal tissue accumulation and urinary excretion of lead.
Similarly, acutely induced alkalosis increased lead excretion in rats previously given access
to drinking water containing 500 ppm lead for 2-3 months. These authors also concluded that
the previously reported acute exposure experiments concerning the renal handling of lead were
at least qualitatively similar to results of the chronic exposure experiments and that rats
were an acceptable model for investigating the effects of alkalosis on the excretion of lead
following chronic exposure.
In vitro studies (Vander et al., 1979) using slices of rabbit kidney incubated with 2oaPb
acetate at lead concentrations of 0.1 or 1.0 uM over 180-minute time intervals showed that a
steady-state uptake of *oaPb by slices (ratio of slice: medium uptake in the range of 10-42)
was reached after 90 minutes and that lead could enter the slices as a free ion. Tissue slice
uptake was reduced by a number of metabolic inhibitors, thus suggesting a possible active
transport mechanism. Tin (Sn IV) was found to markedly reduce *oaPb uptake into the slices
but not to affect lead efflux or para-aminohippurate accumulation. This finding raises the
possibility that Pb and Sn (IV) compete for a common carrier.
Subsequent studies also using rabbit kidney slices (Vander and Johnson, 1981) showed that
co-transport of *U3Pb into the slices in the presence of organic anions such as cysteine,
citrate, glutathione, histidine, or serum ultrafiltrate was relatively small compared with up-
take due to ionic lead.
In summary, it is clear from the above iji vivo and ijn vitro studies on several different
animal species that renal accumulation of lead is an efficient process that occurs in both
proximal and distal portions of the nephron and at both luminal and basolateral membranes.
The transmembrane movement of lead appears to be mediated by an uptake process that is subject
to inhibition by several metabolic inhibitors and the acid-base status of the organism.
DPB12/A 12-136 9/20/83
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PRELIMINARY DRAFT
12.5.5.2 Intracellular Binding of Lead in the Kidney. The bioavailability of lead inside
renal tubule cells under low or *03Pb tracer exposure conditions is mediated in part by bind-
ing to several high affinity cytosolic binding proteins (Oskarsson et al., 1982; Mistry et
al., 1982) and, at higher exposure conditions, by the formation of cytoplasmic and intra-
nuclear inclusion bodies (Goyer et al., 1970a). These inclusion bodies have been shown by
both cell fractionation (Goyer et al., 1970a) and X-ray microanalysis (Fowler et al., 1980) to
contain the highest intracellular concentrations of lead. Saturation analysis of the renal
63,000 dalton (63K) cytosolic binding protein has shown that it possesses an approximate dis-
sociation constant (Kd) of 10" M (Mistry et al., 1982). These data quantify the high af-
finity nature of this protein for lead and explain the previously reported finding (Oskarsson
et al., 1982) that this protein constitutes a major intracellular lead-binding site in the
kidney cytosol. Biochemical studies on the protein components of isolated rat kidney intra-
nuclear inclusion bodies have shown that the main component has an approximate molecular
weight of 27K (Moore et al., 1973) or 32K (Shelton and Egle, 1982) and that it is rich in the
dicarboxylic amino acids glutamate and aspartate (Moore et al., 1973). The isoelectric point
of the main nuclear inclusion body protein has been reported to be pi = 6.3 and appeared from
two-dimensional gel analysis to be unique to nuclei of lead-injected rats (Shelton and Egle,
1982). The importance of the inclusion bodies resides with the suggestion (Goyer et al.,
1970a; Moore et al., 1973; Goyer and Rhyne, 1973) that, since these structures contain the
highest intracellular concentrations of lead in the kidney proximal tubule and hence account
for much of the total cellular lead burden, they sequester lead to some degree away from
sensitive renal organelles or metabolic (e.g., heme biosynthetic) pathways until their capac-
ity is exceeded. The same argument would apply to the high affinity cytosolic lead-binding
proteins at lead exposure levels below those that cause formation of inclusion bodies. It is
currently unclear whether lead-binding to these proteins is an initial step in the formation
of the cytoplasmic or nuclear inclusion bodies (Oskarsson et al., 1982).
12.5.5.3 Pathological Features of Lead Nephropathy. The main morphological effects of lead
in the kidney are manifested in renal proximal tubule cells and interstitial spaces between
the tubules. A summary of morphological findings from some recent studies involving a number
of animal species is given in Table 12-10. In all but one of these studies, formation of
intranuclear inclusion bodies is a common pathognomic feature for all species examined. In
addition, proximal tubule cell cytomegaly and swollen mitochondria with increased numbers of
lysosomes were also observed in two of the chronic exposure studies (Fowler et al., 1980; Spit
et al., 1981). Another feature reported in three of these studies (Mass et al., 1964; White,
1977; Fowler et al., 1980) was the primary localization of morphological changes in the
straight (Sa) segments of the proximal tubule, thereby indicating that not all cell types of
DPB12/A 12-137 9/20/83
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TABLE 12-10. MORPHOLOGICAL FEATURES OF LEAD NEPHROPATHY IN VARIOUS SPECIES
OO
Species
Rabbit
Rat
Dog
Monkey
Rat
Rabbit
Ringed
dove
Morphological findings
Increased
Nuclear mitochondrial Increased Interstitial
Pb dose regimen inclusions swelling lysosomes fibrosis
. D* rD acetate in * ~ — -------------my-- _______ ^
diet for up to 55
weeks
1% Pb in d.w. for + + ND +
9 weeks
ou pg ru/Kg Tor D weeics + NU — nu
6 days/week for 9 months
0, 0.5, 5, 25, 50, 250 + + —
pp» Pb**
0, 0.25, 0.50 pg Pb/kg*** -- — +
3 days/wk for 14 weeks
100 Mg Pb/ml** + + —
Reference
Hass et al. , 1964
Goyer, 1971
White, 1977
Colle et al. , 1980
Fowler et al., 1980
Spit et al., 1981
Kendal 1 et al . , 1981
'RELIMINARY DRAf
—i
* Dosed by oral gavage
** Drinking water ad libitum
***Subcutaneous injection
NO - Not determined
-------
PRELIMINARY DRAFT
the kidney are equally involved in the toxicity of lead to this organ. Interstitial fibrosis
has also been reported in rabbits (Hass et al., 1964) given diets containing 0.5 percent lead
acetate for up to 55 weeks and in rats (Goyer, 1971) given drinking water containing lead ace-
tate for 9 weeks.
12.5.5.4 Functional Studies.
12.5.5.4.1 Renal blood flow and glomerular filtration rate. Studies by Aviv et al. (1980)
concerning the impact of lead on renal function as assessed by renal blood flow (RBF) and
glomerular filtration rate (GFR) have reported significant (p <0.01) reductions in both of
these parameters in rats at 3 and 16 weeks after termination of exposure to 1 percent lead
acetate in drinking water. Statistically significant (p <0.05) reduction of GFR has also been
recently described (Victery et al., 1981) in dogs 2.5-4 hours after a single i.v. dose of 3.0
mg Pb/kg. In contrast, studies by others (Johnson and Kleinman, 1979; Hammond et al., 1982)
were not able to demonstrate reduction in GFR or RBF using the rat as a model. The reasons
behind these reported differences are presently unclear but may be related to differences in
experimental design, age, or other variables.
12.5.5.4.2 Tubular function. Exposure to lead has also been reported to produce tubular dys-
function (Studnitz and Haeger-Aronsen, 1962; Goyer, 1971; Mouw et al., 1978; Suketa et al.,
1979; Victery et al., 1981, 1982a,b, 1983). An early study (Studnitz and Haeger-Aronsen,
1962) reported aminoaciduria in rabbits given a single dose of lead at 125 mg/kg, with urine
collected over a 15-hour period. Goyer et al. (1970b) described aminoaciduria in rats follow-
ing exposure to 1 percent lead acetate in the diet for 10 weeks. Wapnir et al. (1979) con-
firmed a mild hyperaminoaciduria in rats injected with lead at 20 mg/kg five times a week for
six weeks but found no changes in urinary excretion of phosphate or glucose.
Other studies (Mouw et al., 1978; Suketa et al., 1979; Victery et al., 1981, 1982a,b,
1983) have focused attention on increased urinary excretion of electrolytes. Mouw et al.
(1978) reported increased urinary excretion of sodium, potassium, calcium, and water in dogs
given a single i.v. injection of lead at 0.6 or 3.0 mg/kg over a 4-hour period despite a con-
stant GFR, indicating decreased tubular reabsorption of these substances. Suketa et al.
(1979) treated rats with a single oral dose of lead at 0, 5, 50, or 200 mg/kg and killed the
animals at 0, 6, 12, or 24 hours after treatment. A dose-related increase in urinary sodium,
potassium, and water was observed over time. Victery et al. (1981, 1982a,b, 1983) studied
zinc excretion in dogs over a 4-hour period following an i.v. injection of lead at 0.3 or 3.0
mg/kg. These investigators reported maximal increases in zinc excretion of 140 ng/min at the
0.3 mg/kg dose and 300 ng/min at the 3.0 mg/kg dose at the end of the 4-hour period. In con-
trast, in studies by Mouw et al. (1978) no changes in urinary excretion of sodium or potassium
were noted. Urinary protein or magnesium excretion were also unchanged.
DPB12/A 12-139 9/20/83
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PRELIMINARY DRAFT
The results of the above studies indicate that acute or chronic lead treatment is capable
of producing tubular dysfunction in several species of animals, as manifested by increased
2+ 2+ + +
urinary excretion of ami no acid nitrogen and some ions such as Zn , Ca , Na , K , and water.
12.5.6 Experimental Studies of the Biochemical Aspects of Lead Nephrotoxicity
12.5.6.1 Membrane Marker Enzymes and Transport Functions. The biochemical effects of lead in
the kidney appear to be preferentially localized in the cell membranes and mitochondrial and
nuclear compartments following either acute or chronic lead exposure regimens.
Oral exposure of rats to lead acetate in the diet at concentrations of 1-2 percent for
10-40 weeks was found to produce no significant changes in renal slice water content or in
accumulation of paraminohippurate (PAH) or tetraethyl-ammonium (TEA). However, tissue glucose
synthesis at 40 weeks and pyruvate metabolism were both significantly (p <0.05) reduced
(Hirsch, 1973).
Wapnir et al. (1979) examined biochemical effects in kidneys of rats injected with lead
acetate (20 mg/kg) five days per week for six weeks. They observed a significant (p <0.05)
reduction in renal alkaline phosphatase activity and an increase in (Mg +)-ATPase, but no sig-
nificant changes in (Na+,K+)-ATPase, glucose-6-phosphatase, fructose 1-6 diphosphatase, tryp-
tophan hydroxylase, or succinic dehydrogenase. These findings indicated that preferential
effects occurred only in marker enzymes localized in the brush border membrane and mitochon-
drial inner membrane. Suketa et al. (1979) reported marked (50-90 percent) decreases in renal
(Na+,K+)-ATPase at 6-24 hours following a single oral administration of lead acetate at a dose
of 200 mg/kg. A later study (Suketa et al., 1981) using this regi-men showed marked decreases
in renal (Na+, K+)-ATPase but no significant changes in (Mg )-ATPase after 24 hours, thus in-
dicating inhibition of a cell membrane marker enzyme prior to changes in a mitochondrial
marker enzyme.
12.5.6.2 Mitochondria! Respiration/Energy-Linked Transformation. Effects of lead on renal
mitochondrial structure and function have been studied by a number of investigators (Goyer,
1968; Goyer and Krall, 1969a,b; Fowler et al., 1980, 1981a,b). Examination of proximal tubule
cells of rats exposed to drinking water containing 0.5-1.0 percent lead acetate for 10 weeks
(Goyer, 1968; Goyer and Krall, 1969a,b) or 250 ppm lead acetate for 9 months (Fowler et al.,
1980) has shown swollen proximal tubule cell mitochondria in situ. Common biochemical find-
ings in these studies were decreases in respiratory control ratios (RCR) and inhibition of
state-3 respiration, which.was most marked for NAD-linked substrates such as pyruvate/malate.
Goyer and Krall (1969a,b) found these respiratory effects to be associated with a decreased
capacity of mitochondria to undergo energy-linked structural transformation.
Jji vitro studies (Garcia-Cafiero et al., 1981) using 10" M lead demonstrated decreased
renal mitochondrial membrane transport of pyruvate or glutamate associated with decreased res-
DPB12/A 12-140 9/20/83
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PRELIMINARY DRAFT
piration for these two substrates. Other jm vitro studies (Fowler et al., 1981a,b) have shown
decreased renal mitochondria! membrane energization as measured by the fluorescent probes
l-anilino,-8 napthalenesulfonic acid (ANS) or ethidium bromide following exposure to lead
acetate at concentrations of 10 to 10" M lead. High amplitude mitochondrial swelling was
also observed by light scattering.
The results of the above studies indicate that lead produces mitochondrial swelling both
2r\ situ and ui vitro, associated with a decrease in respiratory function that is most marked
for RCR and state-3 respiration values. The structural and respiratory changes appear to be
linked to lead-induced alteration of mitochondrial membrane energization.
12.5.6.3 Renal Heme Biosynthesis. There are several reports concerning the effects of lead
on renal heme biosynthesis following acute or chronic exposure. Silbergeld et al. (1982) in-
jected rats with lead at 10 uM/kg per day for three days and examined effects on several tis-
sues including kidney. These investigators found an increase in 6-aminolevulinic acid syn-
thetase (ALA-S) following acute injection and no change following chronic exposure (first in-
directly via their dams' drinking water containing lead at 10 mg/ml until 30 days of age and
then directly via this drinking water to 40-60 days of age). Renal tissue content of 6-amino-
levulinic acid (ALA) was increased in both acutely and chronically exposed rats. Renal
6-aminolevulinic acid dehydrase (ALA-D) was found to be inhibited in both acute and chronic
treatment groups. Gibson and Goldberg (1970) injected rabbits s.c. with lead acetate at doses
of 0, 10, 30, 150, or 200 mg Pb/week for up to 24 weeks. The mitochondrial enzyme ALA-S in
kidney was found to show no measurable differences from control levels. Renal ALA-D, which is
found in the cytosol fraction, showed no differences from control levels when glutathione was
present but was significantly reduced (p <0.05) to 50 percent of control values for the pooled
lead-treated groups when glutathione was absent. Mitochondrial heme synthetase (ferrochel-
atase) was not significantly decreased in lead-treated versus control rabbits, but this enzyme
_4
in the kidney was inhibited by 72 and 94 percent at lead-acetate concentrations of 10 and
10 M lead, respectively. Accumulation (12-15 fold) of both ALA and porphobilinogen (PBG)
was also observed in kidney tissue of lead-treated rabbits relative to controls. Zawirska and
Medras (1972) injected rats with lead acetate at a dose of 3 mg Pb/day for up to 60 days and
noted a similar renal tissue accumulation of uroporphyrin, coproporphyrin, and protoporphyrin.
A study by Fowler et al. (1980) using rats exposed through 9 months of age to 50 or 250 ppm
lead acetate in drinking water showed significant inhibition of the mitochondrial enzymes
ALA-S and ferrochelatase but no change in the activity of the cytosolic enzyme ALA-D. Similar
findings have been reported for ALA-D following acute i.p. injection of lead acetate at doses
of 5-100 mg Pb/kg at 16 hours prior to sacrifice (Woods and Fowler, 1982). In the latter two
studies, reduced glutathione was present in the assay mixture.
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PRELIMINARY DRAFT
To summarize the above studies (also see Table 12-11), the pattern of alteration of renal
heme biosynthesis by lead is somewhat different from that usually observed with this agent in
other tissues (see Section 12.3). A general lack of lead-induced inhibition of renal ALA-D is
one frequently reported observation in this tissue except under conditions of high-level expo-
sure. Such a finding could result from the presence of the recently described high affinity
cytosolic lead-binding proteins (Oskarsson et al., 1982; Mistry et al., 1982) in the kidney
and/or the formation of lead-containing intranuclear inclusion bodies in this tissue (Goyer,
1971; Fowler et al., 1980), which would sequester most of the intracellular lead away from
other organelle compartments until the capacity of these mechanisms is exceeded. Based on the
observations of Gibson and Goldberg (1970), tissue or assay concentrations of glutathione may
also be of importance to the effects of lead on this enzyme. The observed lack of ALA-S in-
duction in kidney mitochondria reported in the above studies may have been caused by decreased
mitochondrial protein synthesis capacity or, as previously suggested (Fowler et al., 1980), by
overwhelming inhibition of this enzyme by lead, such that any inductive effects were not mea-
surable. Further research is needed to resolve these questions.
12.5.6.4 Lead Alteration of Renal Nucleic Acid/Protein Synthesis. A number of studies have
shown marked increases in renal nucleic acid or protein synthesis following acute or chronic
exposure to Pb acetate. One study (Choie and Richter, 1972a) conducted on rats given a single
intraperitoneal injection of lead acetate showed an increase in 3H-thymidine incorporation. A
subsequent study (Choie and Richter, 1972b) involved rats given intraperitoneal injections of
1-7 mg lead once per week over a 6-month period. Autoradiography of 3H-thymidine incorpora-
tion into tubule cell nuclei showed a 15-fold increase in proliferative activity in the lead-
treated rats relative to controls. The proliferative response involved cells both with and
without intranuclear inclusions. Follow-up autoradiographic studies in rats given three
intraperitoneal injections of lead acetate (0.05 mg Pb/kg) 48 hours apart showed a 40-fold
increase in 3H-thymidine incorporation 20 hours after the first lead dose and 6 hours after
the second and third doses.
Choie and Richter (1974a) also studied mice given a single intracardiac injection of lead
(5 ug Pb/g) and demonstrated a 45-fold maximal increase in DNA synthesis in proximal tubule
cells as judged by aH-thymidine autoradiography 33 hours later. This increase in DNA synthe-
sis was preceded by a general increase in both RNA and protein synthesis (Choie and Richter,
1974b). The above findings were essentially confirmed with respect to lead-induced increases
in nucleic acid synthesis by Cihlk and Seifertova (1976), who found a 13-fold increase in
aH-thymidine incorporation into kidney nuclei of mice 4 hours after an intracardiac injection
(5 ug Pb/g) of lead acetate. This finding was associated with a 34-fold increase in the
mitotic index but no change in the activities of thymidine kinase or thymide monophosphate
kinase. Stevenson et al. (1977) have also reported a 2-fold increase in 3H-thymidine or
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PRELIMINARY DRAFT
TABLE 12-11. EFFECTS OF LEAD EXPOSURE ON RENAL HEME BIOSYNTHESIS
Species
Rabbit
Rat
Rat
Rat
(dams)
(newborns)
Rat
(dams)
(suckling)
Rat
Rat
Rat
Pb dose regimen
10, 30, 150, 200
mg Pb/kg/wk (s.c.)
3 mg Pb/day
(s.c.)
10, 100, 1000,
5000 ppm Pb in
d.w. for 3 wks
10 ppm in d.w.
during:
3 wks before mating
3 wks of pregnancy
3 wks after delivery
100 ppm Pb
in d.w. for
3 wks
0.5, 5, 25, 50, 250
ppm Pb in d.w. for
9 months
5, 25, 50, 100 mg
Pb/kg (i.p.) 16 hrs.
prior to sacrifice
10 pM Pb/kg/3 day
(i.p.)
10 mg Pb/ml in d.w.
for 10-30 days
ALA-S
NC**
NM***
NM
NM
NM
NM
NM
4.
NM
t
NC
ALA-0
±4-
NM
4-
NC
NC
NC
±4-
NC
NC
4.
4-
FC*
NC
NM
NM
NM
NM
NM
NM
4-
NM
NM
NM
Renal tissue
porphyrins
t ALA, PBG
(12-15 x)
t uro- ,
copro-, proto-
porphyrins
t at 1000 and
5000 ppm
t ALA-urine
NC
t
NC
t
NM
NM
tALA
tALA
Reference
Gibson and
Goldberg, 1970
Zawirska and
Medras', 1972
Buchet et al. ,
1976
Hubermont
et al . , 1976
Roels et al. ,
1977
Fowler et al. ,
1980
Woods and
Fowler, 1982
Silbergeld
et al . , 1982
*FC - Ferro chelatase **NC - Not changed relative to controls ***NM - Not measured
DPB12/A
12-143
9/20/83
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PRELIMINARY DRAFT
14C-orotic acid incorporation into kidney DNA or RNA of rats given a single intraperitoneal
injection of lead chloride three days earlier.
The above studies clearly demonstrate that acute or chronic administration of lead by
injection stimulates renal nucleic acid and protein synthesis in kidneys of rats and mice.
The relationship between this proliferative response and formation of intranuclear inclusion
bodies is currently unknown; nor is the basic mechanism underlying this response and the
formation of renal adenomas in rats and mice following chronic lead exposure understood.
12.5.6.5 Lead Effects on the Renin-Angiotension System. A study by Mouw et al. (1978) used
dogs given a single intravenous injection of lead acetate at doses of 0.6 or 3.0 mg Pb/kg and
observed over a 4-hour period. Subjects showed a small but significant decrease in plasma
renin activity (PRA) at 1 hour, followed by a large and significant (p <0.05) increase from
2.5 to 4.0 hours. Follow-up work (Goldman et al., 1981) using dogs given a single intravenous
injection of lead acetate at 3.0 mg Pb/kg showed changes in the renin-angiotensin system over
a 3-hour period. The data demonstrated an increase in PRA, but increased renin secretion oc-
curred in only three of nine animals. Hepatic extraction of renin was virtually eliminated in
all animals, thus providing an explanation for the increased blood levels of rem'n. Despite
the large observed increases in PRA, blood levels of angiotensin II (All) did not increase
after lead treatment. This suggests that lead inhibited the All converting enzyme.
Exposure of rats to drinking water containing 0.5 mg Pb/ml for three weeks to five months
(Fleischer et al., 1980) produced an elevation of PRA after six weeks of exposure in those
rats on a sodium-free diet. No change in plasma renin substrate (PRS) was observed. At five
months, PRA was significantly higher in the lead-treated group on a 1-percent sodium chloride
diet, but the previous difference in renin levels between animals on an extremely low-sodium
(1 meq) vs. 1-percent sodium diet had disappeared. The lead-treated animals had a reduced
ability to decrease sodium excretion following removal of sodium from the diet.
Victory et al. (1982a) exposed rats to lead rn utero and to drinking water solution con-
taining 0, 100, or 500 ppm lead as lead acetate for six months. Male rats on the 100 ppm lead
dose became significantly hypertensive at 3.5 months and remained in that state until termi-
nation of the experiment at six months. All female rats remained normotensive as did males at
the 500-ppm dose level. PRA was found to be significantly reduced in the 100-ppm treatment
males and normal in the 500-ppm treatment groups of both males and females. Dose-dependent
decreases in AII/PRA ratios and renal renin content were also observed. Pulmonary All con-
verting enzyme was not significantly altered. It was concluded that, since the observed
hypertension in the 100-ppm group of males was actually associated with reduction of PRA and
All, the renin-angiotensin system was probably not directly involved in this effect.
Webb et al. (1981) examined the vascular responsiveness of helical strips of tail
arteries in rats exposed to drinking water containing 100 ppm lead for seven months. These
DPB12/A 12-144 9/20/83
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PRELIMINARY DRAFT
investigators found that the mild hypertension associated with this regimen was associated
with increased vascular responsiveness to a-adrenergic agonists.
Male rats exposed to lead in utero and prior to weaning indirectly by their dams' drink-
ing water containing 0, 5, or 25 ppm lead as lead acetate, followed by direct exposure at the
same levels for five months (Victery et al., 1982b), showed no change in systolic blood pres-
sure. Rats exposed to the 25 ppm dose showed a significant (p <0.05) decrease in basal PRA.
Stimulation of renin release by administration of polyethylene glycol showed a significant
increase in PRA but low All values. These yielded a significant (p <0.001) decrease in the
AII/PRA ratio. Basal renal renin concentrations were found to be significantly reduced in
both the 5 ppm (p <0.05) and 25 ppm (p <0.01) dose groups relative to controls.
Victery et al. (1983) exposed rats \n utero to lead by maternal administration of 0, 5,
25, 100, or 500 ppm lead as lead acetate. The animals were continued on their respective dose
levels through one month of age. All exposure groups had PRA values significantly (p <0.05)
elevated relative to controls. Renal renin concentration was found to be similar to controls
in the 5 and 25 ppm groups but significantly increased (p <0.05) in the 100 and 500 ppm
groups. The plasma AII/PRA ratio was similar to controls in the 100 ppm group but was signi-
ficantly reduced (p <0.05) in the 500 ppm group.
It appears from the above studies that lead exposure at even low dose levels is capable
of producing marked changes in the renin-angiotension system and that the direction and mag-
nitude of these changes is mediated by a number of factors, including dose level, age, and sex
of the species tested, as well as dietary sodium content. Lead also appears capable of
directly altering vascular responsiveness to a-adrenergic agents. The mild hypertension ob-
served with chronic low level lead exposure appears to stem in part from this effect and not
from changes in the renin-angiotensin system. (See also Section 12.9.1 for a discussion of
other work on the hypertensive effects of lead.)
12.5.6.6 Lead Effects on Uric Acid Metabolism. A report by Mahaffey et al. (1981) on rats
exposed concurrently to lead, cadmium, and arsenic alone or in combination found significantly
(p <0.05) increased serum concentrations of uric acid in the lead-only group. While the bio-
chemical mechanism of this effect is not clear, these data support certain observations in
humans concerning hyperuricemia as a result of lead exposure (see Section 12.5.3) and, also,
confirm an earlier report by Goyer (1971) showing increased serum uric acid concentration in
rats exposed- to 1 percent lead acetate in drinking water for 84 weeks.
12.5.6.7 Lead Effects on Kidney Vitamin D Metabolism. Smith et al. (1981) fed rats vitamin
D-deficient diets containing either low or normal calcium or phosphate for two weeks. The
animals were subsequently given the same diets supplemented with 0.82 percent lead as lead
acetate. Ingestion of lead at this dose level significantly reduced plasma levels of 1,25
dihydrocholecalciferol in cholecalciferol-treated rats and in rats fed either a low phospho-
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rous or low calcium diet while it had no effect in rats fed either a high calcium or normal
phosphorous diet. These data suggest decreased production of 1,25-dihydrocholecalciferol in
the kidney in response to lead exposure in concert with dietary deficiencies.
12.5.7 General Summary: Comparison of Lead Effects in Kidneys of Humans and Animal Models
It seems clear from the preceding review that, in general, results of experimental animal
studies have confirmed findings reported for human kidney function in individuals exposed to
lead for prolonged time periods and that these studies have helped illuminate the mechanisms
underlying such effects. Similar morphological changes are found in kidneys of humans and
animals following chronic lead exposure, including nuclear inclusion bodies, cytomegaly,
swollen mitochondria, interstitial fibrosis, and increased numbers of iron-containing lyso-
somes in proximal tubule cells. Physiological renal changes observed in humans have also been
confirmed in animal model systems in regard to increased excretion of amino acids and elevated
serum urea nitrogen and uric acid concentrations. The inhibitory effects of lead exposure on
renal blood flow and glomerular filtration rate are currently less clear in experimental model
systems; further research is needed to clarify the effects of lead on these functional para-
meters in animals. Similarly, while lead-induced perturbation of the renin-angiotensin system
has been demonstrated in experimental animal models, further research is needed to clarify the
exact relationships among lead exposure (particularly chronic low-level exposure), alteration
of the renin-angiotensin system, and hypertension in both humans and animals.
On the .biochemical level, it appears that lead exposure produces changes at a number of
sites. Inhibition of membrane marker enzymes, decreased mitochondria! respiratory function/
cellular energy production, inhibition of renal heme biosynthesis, and altered nucleic acid
synthesis are the most marked changes thus far reported. The extent to which these mito-
chondrial alterations occur is probably mediated in part by the intracellular bioavailability
of lead, which is determined by its binding to high affinity kidney cytosolic binding proteins
and deposition within intranuclear inclusion bodies.
Recent studies in humans have indicated that the EDTA lead-mobilization test is the most
reliable technique for detecting persons at risk for chronic nephropathy. Blood lead measure-
ments are a less satisfactory indicator because they may not accurately reflect cumulative
absorption some time after exposure to lead has terminated.
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12.6 EFFECTS OF LEAD ON REPRODUCTION AND DEVELOPMENT
Data from human and animal studies indicate that lead may exert gametotoxic, embryotoxic,
and (according to animal studies) teratogenic effects that could influence the survival and
development of the fetus and newborn. It appears that prenatal viability and development may
also be affected by lead indirectly, via effects on various health parameters of the expectant
mother. The vulnerability of the conceptus to such effects of lead has contributed to concern
that the unborn may constitute a group at risk for lead health effects. Also, certain infor-
mation regarding lead effects on male reproductive functions has led to concern regarding the
impact of lead on men.
12.6.1 Human Studies
12.6.1.1 Historical Evidence. Findings suggesting that lead exerts adverse effects on human
reproductive functions have existed in the literature since before the turn of the century.
For example, Paul (1860) observed that severely lead-poisoned pregnant women were likely to
abort, while those less severely intoxicated were more likely to deliver stillborn infants.
Legge (1901), in summarizing the reports of 11 English factory inspectors, found that of 212
pregnancies in 77 female lead workers, only 61 viable children were produced. Fifteen workers
never became pregnant; 21 stillbirths and 90 miscarriages occurred. Of 101 children born, 40
died in the first year. Legge also noted that when lead was fed to pregnant animals, they
typically aborted. He concluded that maternal exposure to lead resulted in a direct action of
the element on the fetus.
Four years later, Hall and Cantab (1905) discussed the increasing use of lead in nostrums
sold as abortifacients in Britain. Nine previous reports of the use of diachylon ("lead plas-
ter") in attempts to cause miscarriage were cited and 30 further cases of known or apparent
use of lead in attempts to terminate real or suspected pregnancy listed. Of 22 cases de-
scribed in detail, 12 resulted in miscarriage and all 12 exhibited marked signs of plumbism,
including a blue gum line (in eight cases the women were known to have attempted to induce
abortion). Hall's report was soon followed by those of Cadman (1905) and Eales (1905), who
described three more women who miscarried following consumption of lead-containing pills.
Oliver (1911) then published statistics on the effect of lead on pregnancy in Britain
(Table 12-12). These figures showed that the miscarriage rate was elevated among women em-
ployed in industries in which they were exposed to lead. Lead compounds were said by Taussig
(1936) to be known for their embryotoxic properties and their use to induce abortion.
In a more recent study by Lane (1949), women exposed to lead levels of 750 ug/m3 were ex-
amined for effects on reproduction. Longitudinal data on 15 pregnancies indicated an increase
in the number of stillbirths and abortions. No data were given on urinary lead in women, but
men in this sample had urinary levels of 75 to 100 ug/liter.
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TABLE 12-12. STATISTICS ON THE EFFECT OF LEAD ON PREGNANCY
Number of Number of
abortions and neonatal deaths
stillbirths per (first year) per
Sample 1000 females 1000 females
Housewives 43.2 150
Female workers (mill work) 47.6 214
Females exposed to lead premaritally 86.0 157
Females exposed to lead after marriage 133.5 271
Source: Oliver (1911).
The above studies clearly demonstrate an adverse effect of lead at high levels on human
reproductive functions, and include evidence of increased incidence of miscarriages and still-
births when women are exposed to lead during pregnancy. The mechanisms underlying these ef-
fects are unknown at this time. Many factors could contribute to such results, ranging from
lead effects on maternal nutrition or hormonal state before or during pregnancy to more direct
gametotoxic, embryotoxic, fetotoxic, or teratogenic effects that could affect parental fertil-
ity or offspring viability during gestation. Pregnancy is a stress that may place a woman at
higher risk for toxic lead exposure. Both iron deficiency and calcium deficiency increase sus-
ceptibility to lead, and women have an increased risk of both deficiencies during pregnancy
and postpartum (Rom, 1976).
Such studies as those of Legge, Hall, and Oliver suffer from methodological inadequacies.
They must be mentioned, however, because they provide evidence that effects of lead on repro-
duction occurred at times when women were exposed to high levels of lead. Nevertheless, evi-
dence for adverse reproductive outcomes in women with obvious lead poisoning is of little help
in defining the effects of lead at significantly lower exposure levels. Efforts have been
made to define more precisely the points at which lead may affect reproductive functions in
both the human female and male, as well as in animals, as reviewed below.
12.6.1.2 Effects of Lead Exposure on Reproduction.
12.6.1.2.1 Effects associated with exposure of women to lead. Since the time of the above
reports, women have been largely, though not entirely (Khera et al., 1980), excluded from oc-
cupational exposure to lead; and lead is no longer used to induce abortion. Thus, little new
information is available on reproductive effects of chronic exposure of women to lead. Vari-
ous reports (Pearl and Boxt, 1980; Qazi et al., 1980; Timpo et al., 1979; Singh et al., 1978;
Angle and Mclntire, 1964) suggest that relatively high prenatal lead exposures do not invari-
ably result in abortion or in major problems readily detectable in the first few years of life
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These findings are based on only a few case histories, however, and are obviously not an ade-
quate sample. The data are confounded by numerous variables, and longer follow-ups are
needed.
In a sample population exposed to lead and to other toxic agents (including arsenic and
sulfur dioxide) from the Ronnskar smelter, Nordstrom et al. (1978b) found an increased fre-
quency of spontaneous abortions among women living closest to the smelter. In addition to the
exposure to multiple environmental toxins, however, the study was confounded by failure to
match exposed and control populations for socioeconomic status. A further study by the same
authors (Nordstrom et al., 1979a) determined that female smelter workers at the Ronnskar
smelter had an increased frequency of spontaneous miscarriage when the mother was employed by
the smelter during pregnancy or had been so employed prior to pregnancy and still lived near
the smelter. Also, women who worked in more highly polluted areas of the smelter were more
likely to have aborted than were other employees. This report, however, suffers from the same
deficiencies as the earlier study.
In regard to potential lead effects on ovarian function in human females, Panova (1972)
reported a study of 140 women working in a printing plant for less than one year (1 to 12
months) where ambient air levels were <7 jjg Pb/m3. Using a classification of various age
groups (20-25, 26-35, and 36-40 yr) and type of ovarian cycle (normal, anovular, and disturbed
lutein phase), Panova claimed that statistically significant differences existed between the
lead-exposed and control groups in the age range 20 to 25 years. Panova1s report, however,
does not show the age distribution, the level of significance, or data on the specificity of
her method for classification. Zielhuis and Wibowo (1976), in a critical review of the above
study, concluded that the study design and presentation of data were such that it is difficult
to evaluate the author's conclusions. It should also be noted that no consideration was given
to the dust levels of lead, an important factor in print shops.
Unfortunately, little else besides the above study appears to exist in regard to assess-
ing the effects of lead on human ovarian function or other factors affecting female fertility.
Studies offering firm data on maternal variables, e.g., hormonal state, that are known to af-
fect the ability of the pregnant woman to carry the fetus full term are also lacking.
12.6.1.2.2 Effects associated with exposure of men to lead. Lead-induced effects on male re-
productive functions have been reported in several instances. Among the earliest of these was
the review of Stofen (1974), who described data from the work of Neskov in the USSR involving
66 workers exposed chiefly to lead-containing gasoline (organic lead). In 58 men there was a
decrease or disappearance of erection, in 41 there was early ejaculation, and in 44 there were
a diminished number of spermatocytes. These results were confounded, however, by the presence
of the other constituents of gasoline.
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Lancranjan et al. (1975) reported lead-related interference with male reproductive func-
tions. A group of 150 workmen who had long-term exposure to lead in varying degrees was
studied. Clinical and toxicological criteria were used to categorize the men into four
groups: lead-poisoned workmen (mean blood lead level = 74.5 ug/dl) and those showing moderate
(52.8 ug/dl), slight (41 ug/dl), or "physiologic" (23 ug/dl) exposure to lead. Moderately
increased lead absorption (52.8 ug/dl) was said to result in gonadal impairment. The effects
on the testes were believed to be direct, in that tests for impaired hypothalamopituitary
influence were negative. Also, semen analysis revealed asthenospermia and hypospermia in all
groups except those with "physiologic" absorption levels, and increased teratospermia was seen
in the two highest lead exposure groups.
An apparently exposure-related increase in erectile dysfunction was also found by
Lancranjan et al. (1975). Problems with ejaculation and libido were said to be more common in
the lead exposed groups, but their incidences did not seem to be dose-dependent. Control in-
cidences of these difficulties were invariably lower than those of the lead exposed groups,
however, so the lack of a clear cut dose-response relationship may have merely been due to in-
appropriate assignment of individuals to the high, moderate, and low exposure groups.
The Lancranjan et al. (1975) study has been criticized by Zielhuis and Wibowo (1976), who
stated that the distributions of blood lead levels appeared to be skewed and that exposure
groups overlapped in terms of lead intake. Thus, the means for each putative exposure group
may not have been representative of the individuals within a group. It is difficult to dis-
cern, however, if the men were improperly assigned to exposure level groups, as blood lead
levels may have varied considerably on a short term basis. Zielhuis and Wibowo also stated
that the measured urinary ALA levels were unrealistically high for individuals with the stated
blood lead levels. This suggests that if the ALA values were correct, the blood lead levels
may have been underestimated. Other deficiencies include failure to use matched controls and
exclusion of different proportions of individuals per exposure group for the semen analyses.
Plechaty et al. (1977) measured lead concentrations in the semen of 21 healthy men.
Semen lead levels were generally less than blood lead levels, and no correlation was found be-
tween lead content of the semen and sperm counts or blood lead levels in this small sample.
Hypothalamic-pituitary-testicular relationships were investigated by Braunstein et al.
(1978) in men occupationally exposed at a lead smelter. Six subjects had 2-11 years of expo-
sure to lead and exhibited marked symptoms of lead toxicity. All had received one or more
courses of EDTA chelation therapy. This group was referred to as "lead-poisoned" (LP). Four
men from the same smelter had no signs of lead toxicity, but had been exposed for 1-23 years
and were designated "lead-exposed" (LE). The control (C) group consisted of nine volunteers.
Mean (± standard error) blood lead levels for the LP, LE, and C groups were 38.7 (± 3),
29.0 (± 5), and 16.1 (± 1.7) ug/dl, respectively, at the time of the study. Previously, how-
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ever, the LP and LE groups had exhibited values as high as 88.2 (± 4) and 80 (± 0) ug/dl,
respectively. All three groups were chelated and 24-hour urinary lead excretion values were
999 (± 141), 332 (± 17), and 225 (± 31) pg for the LP, LE, and C groups, respectively. Fre-
quency of intercourse was significantly less in both lead-exposed groups than in controls.
Sperm concentrations in semen of the LP and LE men ranged from normal to severely oligosper-
mic, and one from the LP group was unable to ejaculate. Testicular biopsies were performed on
"the two most severely lead-poisoned men," one with aspermia and one with testicular pain.
Both men showed increased peritubular fibrosis, decreased spermatogenesis, and Sertoli cell
vacuolization. The two lead groups exhibited reduced basal serum testosterone levels, but
displayed a normal increase in serum testosterone following stimulation with human chorionic
gonadotrophin. A similar rise in serum follicle-stimulating hormone was seen following treat-
ment with clomiphene citrate or gonadotrophin releasing hormone, although the LP men exhibited
a lower than expected increase in luteinizing hormone (LH). The LE men also appeared to have
a decreased LH response, but the difference was not significant.
The results of the Braunstein et al. (1978) study suggest that lead exposure at high
levels may result in a defect in regulation of LH secretion at the hypothalamic-pituitary
level, resulting in abnormal dynamics of LH secretion. They also indicate a likely direct
effect on the testes, resulting in oligospermia and peritubular fibrosis. Nevertheless, the
possibility remains that such effects may have been precipitated by the EDTA chelation ther-
apy, and the numbers of men studied were quite small.
More recently, Wildt et al. (1983) compared two groups of men exposed to lead in a
Swedish battery factory. The 29 high-lead group men had had blood lead levels >50 |ig/dl at
least once prior to the study, while the 30 "controls" seldom exceeded 30 ug/dl. There were
two test periods eight months apart. For the first test, 15 men were in the high lead and 24
in the control groups, respectively, and 17 were in each group for the second test. Fourteen
and 15 of these men from the high lead and control groups, respectively, took part in both
tests. Blood lead values were obtained periodically over a six-month period. For the two
high lead groups, blood lead values were 46.1 and 44.6 pg/dl, respectively (range 25-75);
corresponding values for the controls were 31.1 and 21.5 ug/dl (range 8-39). The high lead
men tended to exhibit decreased function of the prostate and/or seminal vesicles, as measured
by seminal plasma constituents (fructose, acid phosphatase, Mg, and Zn); however, a signifi-
cant difference was seen only in the case of zinc. More men in the high lead than in the
control group had low semen volume values, but the numbers of individuals did not allow a
reliable statistical analysis. The heads of sperm of high lead individuals were more likely
to swell when exposed to a detergent solution, viz. sodium dodecyl sulfate (SDS), a test of
functional maturity, but the values were still in a normal range. Conversely, the leakage of
lactate dehydrogenase isoenzyme X (LDH-X) was greater in control semen samples.
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The values for live and for motile sperm were lower in the control group. The data were
skewed, however, by the presence of several of the same men with low values in the control
groups for both sampling times. Another confounding factor was the fact that the high lead
and control groups differed in a significant way: ten of the control men had present or past
urogenital tract infections versus none in the high lead group, possibly explaining the inci-
dence of control samples with lowered sperm motility and viability. The observed decrease in
SOS resistance in sperm of high lead group men may have been related to their apparent abnor-
mal prostatic function, or to an effect of lead on sperm maturation. In evaluating the above
results, it must also be noted that even the "controls" had elevated blood lead levels.
12.6.1.3 Placenta! Transfer of Lead. The transfer of lead across the human placenta and its
potential threat to the concaptus have been recognized for more than a century (Paul, 1860).
Documentation of placental transfer of lead to the fetus and data on resulting fetal blood
lead levels help to build the case for a potential, but as yet not clearly defined, threat of
subtle embryotoxicity or other deleterious health effects.
The placental transfer of lead has been established, in part, by various studies that
have disclosed measurable quantities of lead in human fetuses or newborns, as well as off-
spring of experimental animals. The relevant data on prenatal lead absorption have been re-
viewed in Chapter 10, Section 2.4 of this document, and thus work dealing only with lead
levels will not be discussed further here.
12.6.1.4 Effects of Lead on the Developing Human.
12.6.1.4.1 Effects of lead exposure on fetal metabolism. Prenatal exposure of the conceptus
to lead, even in the absence of overt teratogenicity, may be associated with other health
effects. This is suggested by studies relating fetal and cord-blood levels to changes in
fetal heme synthesis. Haas et al. (1972) examined 294 mother-infant pairs for blood lead and
urinary ALA levels. The maternal blood lead mean was 16.89 ug/dl; and the fetal blood lead
mean was 14.98 ug/dl, with a correlation coefficient of 0.54 (p <0.001). In the infants,
blood lead levels and urinary ALA were positively correlated (r = 0.19, p <0.01), although the
data were based on spot urines (which tends to limit their value) The full biological signi-
ficance of the elevated ALA levels is not clear, but the positive correlation between lead in
blood and urinary ALA for the group as a whole indicates that increased susceptibility of heme
synthesis occurs at relatively low blood lead levels in the fetus or newborn infant.
Subsequently, Kuhnert et al. (1977) measured ALA-D activity and levels of erythrocyte
lead in pregnant urban women and their newborn offspring. Cord erythrocyta lead levels ranged
from 16 to 67 ug/100 ml of cells, with a mean of 32.9. Lead levels were correlated with in-
hibition of ALA-D activity (r = -0.58, p <0.01), suggesting that typical urban lead exposures
could affect fetal enzyme activity. Note, however, that ALA-D activity is related to blood
cell age, being highest in the younger cells. Thus, results obtained with cord blood, with
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its high percentage of immature cells, are not directly comparable to those obtained with
adult blood. In a later study, Lauwerys et al. (1978) found no lead-related increase in
erythrocyte porphyrin levels in 500 mothers or their offspring. They did, however, report
negative correlations between ALA-D activity and blood lead levels in both mothers and their
newborns. Maternal blood lead levels averaged 10.2 pg/dl (range 3.1-31 pg/dl). Corresponding
values for the newborns were 8.4 ug/dl and 2.7-27.3. Such results indicate that ALA-D activ-
ity may be a more sensitive indicator of fetal lead toxicity than erythrocyte porphyrin or
urinary ALA levels.
12.6.1.4.2 Other toxic effects of intrauterine lead exposure. Fahim et al. (1976), in a
study on maternal and cord-blood lead levels, determined blood lead values in women having
preterm delivery and premature membrane rupture. Such women residing in a so-called "lead
belt" (mining and smelting area) had significantly higher blood lead levels than women from
the same area delivering at full term. Fahim et al. (1976) also noted that among 249 pregnant
women in a control group outside the lead belt area, the percentages of women having preterm
deliveries and premature rupture were 3 and 0.4, respectively, whereas corresponding values
for the lead area (n = 253) were 13.04 and 16.99, respectively. A confusing aspect of this
study, however, is the similarity of blood lead levels in women from the nonlead and lead belt
areas. In fact, no evidence was presented that women in the lead belt group had actually
received a greater degree of lead exposure during pregnancy than did control individuals.
Also, questions exist regarding analytical aspects of this study. Specifically, other workers
(e.g., see summary table in Clark, 1977) have typically found blood lead levels in mothers and
their newborn offspring to be much more similar than those of Fahim et al. (1976).
In another study, Clark (1977) detected no effects of prenatal lead exposure in newborns
with regard to birth weight, hemoglobin, or hematocrit. He compared children born of 122
mothers living near a Zambian lead mine with 31 controls from another area. Maternal and
infant blood lead levels for the mine area were 41.2 (± 14.4) and 37.9 (± 15.3) ug/dl, respec-
tively. Corresponding values for control mothers and offspring were 14.7 (± 7.5) and 11.8 (±
5.6) ug/dl.
There is also some evidence that lead levels in bone samples from stillborn children are
higher than would be expected (Khera et al., 1980; Bryce-Smith et al., 1977), but the data are
inconclusive.
Nordstrb'm et al. (1979b) examined birth weight records for offspring of female employees
of the RSnnskSr smelter and found decreased birth weights related to: (1) employment of the
mothers at the smelter during pregnancy, (2) distance that the mothers lived from the smelter,
and (3) proximity of the mother's job to the actual smelting process. Similar results were
also seen for children born to mothers merely living near the smelter (Nordstrb'm et al.,
1978a). NordstrSm et al. (1979b) also investigated birth defects in offspring of the female
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smelter workers and in populations living at various distances from the Rdnnska'r smelter.
They concluded that the frequencies of both single and multiple malformations were increased
when the mother worked at the smelter during pregnancy.
The number of smelt?*1 '.-mrkpi s wi*.!i ty^ifm-nipr! offspring was relatively small (39/1291).
The incidence of children with birth defects whose mothers worked while pregnant was 5.8 per-
cent (17 of 291). Five of the six offspring with multiple malformations were in this group,
suggesting that the observed effect may have been a real one. Nevertheless, the crucial
factor in evaluating all of the Rb'nnskar studies is the exposure of workers and the nearby
population to a number of toxic substances including not only lead, but arsenic, mercury, cad-
mium, and sulfur dioxide as well.
Alexander and Delves (1981) found that the mean blood lead concentrations of pregnant and
non-pregnant control women living in an urban area of England were approximately 4 pg/dl
higher than those for similar groups living in a rural area. The mean concentrations for the
urban and rural pregnant women were 15.9 and 11.9 ug/dl, respectively (p <0.001), but there
were no demonstrable effects of the higher maternal blood lead levels on any aspect of peri-
natal health. The rate for congenital abnormality was higher in the rural area, suggesting
that whatever the cause, it was unlikely to be related to maternal levels of lead.
Additional studies of placental lead and stillbirths have not clarified the situation.
Khera et al. (1980) measured placental and stillbirth tissue lead in occupationally exposed
women 1n the United Kingdom. Regardless of the incidence of stillbirths, placental lead con-
centrations were found to increase with duration of occupational exposure, from 0.29 ug/g at
<1 yr exposure to 0.48 ug/g at >6 Vr exposure for a group of 26 women aged 20-29 years. Pla-
cental lead concentrations also increased with age of the mother, independently of time of oc-
cupational exposure, and ranged from 0.30 (± 0.16) |jg/g for those <20 yrs old to 0.51 (± 0.44)
H9/9 for those £30 yrs old. Average placental lead concentrations for 20 occupationally ex-
posed women whose babies were stillborn were higher [0.45 (± 0.32) ug/g] than the average
level of 0.29 (± 0.09) ug/g for placentas from eight mothers who had not been occupationally
exposed for at least two years. The authors noted, however, that it was not possible to say
whether occupational exposure caused any of the stillbirths or whether the high lead levels
were merely consequential to the fetal death. It is somewhat disconcerting that the placental
lead concentrations were about three times lower than those reported earlier by this group
(Wibberley et al., 1977). These differences were attributed to methodological changes and to
changes in concentration during storage of placentas at -20°C (Khera et al., 1980).
The placental lead concentrations reported by Alexander (1982) are, however, similar to
the earlier results of Wibberley et al. (1977), with mean values of 1.34 (± 0.15) ug/g for
seven stillbirths and 1.27 (± 0.48) ug/g for seven matched healthy controls. The wide range
of concentrations reported for the controls (0.34-5.56 ug/g) and the differences in concentra-
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tion with site of sampling makes it difficult to draw any useful conclusions from the results
presented by Alexander. Clearly these analytical discrepancies in placental lead measurements
must be resolved if any interpretation of their significance is to be made.
An additional study by Reels et al. (1978b) reported placental lead values of 0.08
(± 0.05) ug/g (range = 0.01-0.40 ug/g) from a variety of locations in Belgium, but these data
indicated no correlation between lead concentration and birth weight. In contrast, placental
lead has been reported to be associated with decreased activity of a placental enzyme, steroid
sulfatase (Karp and Robertson, 1977). A similar association was found for mercury, suggesting
that either metal or both together could have affected the enzyme activity or that the authors
had merely uncovered a spurious correlation.
12.6.1.4.3 Paternally mediated effects of lead. There is increasing evidence that exposure
of male laboratory animals to toxic agents can result in adverse effects on their offspring,
including decreased litter size, birth weight, and survival. Mutagenic effects are the most
likely cause of such results, but other mechanisms have been proposed (Soyka and Joffe, 1980).
In the following cases, exposure of human males to lead has been implicated as the cause of
adverse effects on the conceptus.
According to Koinuma (1926) in a brief report, 24.7 percent of workmen exposed to lead in
a storage battery plant had childless marriages, while the value for men not so exposed was
14.8 percent. Rates for miscarriages or stillbirths in wives of lead-exposed men and controls
were 8.2 and 2.8 percent, respectively, while corresponding figures for neonatal deaths were
24.2 and 19.2 percent. These comparisons were based on 170 lead-exposed and 128 control men.
These differences in fertility and prenatal mortality, while not dramatic, are suggestive of a
male-mediated lead effect; however, the reliability of the methodology used in this study can-
not be determined, due to the brevity of the report.
In a study of the pregnancies of 104 Japanese women before and after their husbands began
lead-smelter work, miscarriages increased to 8.30 percent of pregnancies from a pre-exposure
rate of 4.70 percent (Nogaki, 1957). The miscarriage rate for 75 women whose husbands were
not occupationally exposed to lead was 5.80 percent. In addition, exposure to lead was
related to a significant increase in the ratio of male to female offspring at birth. Lead
content of paternal blood ranged from 11 to 51.7 ug/dl [mean = 25.4 (± 1.26) ug/dl], but was
not correlated with reproductive outcome, except in the case of the male to female offspring
ratio. The reported blood lead levels appear low, however, in view of the occupational expo-
sure of these men, and were similar to those given for controls [mean - 22.8 (± 1.63)
Hg/dl]. Also, maternal age and parity appear not to have been well controlled for in the
analysis of the reproductive data. Another report (Van Assen, 1958) on fatal birth defects in
children conceived during a period when their fathers were lead poisoned (but neither before
nor after) also hints at paternally-mediated effects of lead.
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In the above study by Nordstrom et al. (1979b), women employed at the Rb'nnskar smelter
were found to have higher abortion rates if their husbands were also employed at the smelter.
This was true only of their third or later pregnancies, however, suggesting that the effect
was related to long-term exposure of the male gametogenic stem cells. Whether this was a lead
effect or that of other toxins from the smelter is not clear.
12.6.1.5 Summary of the Human Data. The literature on the effects of lead on human reproduc-
tion and development leaves little doubt that lead can, at high exposure levels, exert signi-
ficant adverse health effects on reproductive functions. Most studies, however, only looked
at the effects of prolonged moderate to high exposures to lead, e.g., those encountered in
industrial situations, and many reports do not provide definite information on exposure levels
or blood lead levels at which specific effects were observed. Also the human data were
derived from studies involving relatively small numbers of individuals and therefore do not
allow for discriminating statistical analysis. These reports are often additionally con-
founded by failure to obtain appropriate controls and, in some cases, by the presence of addi-
tional toxic agents or disease states. These and other factors obviously make interpretation
of the data difficult. It appears possible that effects on sperm or on the testis may occur
due to chronic exposure resulting in blood lead values of 40-50 ug/dl, based on the Lancranjan
et al. (1975) and Wildt et al. (1983) studies, but additional data are greatly needed. Expo-
sure data related to reproductive functions in the female are so lacking that even a rough
estimate is impossible. Data on maternal exposure levels at which effects may be seen in
human fetuses or infants are also quite meager, although the results of Haas et al. (1972),
Kuhnert et al. (1977), and Lauwerys et al. (1978) suggest possible perinatal effects on heme
metabolism at maternal blood levels considerably below 30 ug/dl. The human data on actual
absorbed doses are even more lacking than those on blood lead, values, adding to the impreci-
sion of conclusions relating lead exposure to reproductive outcome.
12.6.2 Animal Studies
12.6.2.1 Effects of Lead on Reproduction.
12.6.2.1.1 Effects of lead on male reproductive functions. Among the first investigators to
report infertility in male animals due to lead exposure were Puhac et al. (1963), who exposed
rats to lead via their diet. Ability to sire offspring returned, however, 45 days after ces-
sation of treatment. More recently, Varma et al. (1974) gave a solution of lead subacetate in
drinking water to male Swiss mice for four weeks (mean total intake of lead = 1.65 g). The
fertility of treated males was reduced by 50 percent. Varma and coworkers calculated the
mutagenicity index (number of early fetal deaths/total implants) to be 10.4 for lead-treated
mice versus 2.98 for controls (p <0.05). The major differences in fecundity appeared to have
been due to differing pregnancy rates, however, rather than prenatal mortality. Impairment of
DPB12/G 12-156 9/20/83
-------
PRELIMINARY DRAFT
male fertility by lead rather than lead-induced mutagenicity was thus likely to have been the
primary toxic effect observed. Additionally, it has been suggested by Leonard et al. (1973),
that effects seen following administration of lead acetate in water may be due to resulting
acidity, rather than to lead. Also, Eyden et al. (1978) found no decrease in fertility of
male mice fed 0.1 percent lead acetate in the diet for 64 weeks.
Several animal studies have found lead-associated damage to the testes or prostate,
generally at relatively high doses. Golubovich et al. (1968) found a decrease in normal sper-
matogonia in the testes of rats gavaged for 20 days with lead (2 mg/kg/day). Desquamation of
the germinal epithelium of the seminiferous tubules was also increased, as were degenerating
spermatogonia. Hilderbrand et al. (1973) also noted testicular damage in male rats given oral
lead (100 ug/day for 30 days). Egorova (cited in Stbfen, 1974) injected lead at a dose of 2
|jg/kg six times over a ten-day period and reported testicular damage.
Ivanova-Chemishanska et al. (1980) investigated the effect of lead on male rats adminis-
tered 0.0001 or 0.01 percent solutions of lead acetate over a four-month period. The authors
reported that changes in enzymatic activity and in levels of disulfide and ATP were observed
in testicular homogenates. No histopathological changes in testicular tissue were found, but
the fertility index for treated males was decreased. Offspring of those males exhibited post-
partum "failure to thrive" and stunted growth. Such data suggest biological effects due to
chronic lead exposure of the male, but the study is difficult to evaluate due to limited in-
formation on the experimental methods, particularly the dose levels actually received.
In a more recent study of lead effects on the male reproductive tract, no histopathologi-
cal changes were seen during an examination of the testes of rabbits (Willems et al., 1982).
Five males per group were dosed subcutaneously with up to 0.5 mg/kg lead acetate three times
weekly for 14 weeks. Blood lead levels at termination of treatment were 6.6 and 61.5 ug/dl
for control and high dose rabbits, respectively.
Lead-related effects on spermatozoa have also been published. For example, Stowe et al.
(1973) reported the results of a low calcium and phosphate diet containing 100 ppm lead (as
acetate) fed to dogs from 6 to 18 weeks of age. This dose resulted in a number of signs of
toxicity, including spermatogonia with hydropic degeneration. In the Maisin et al. (1975)
study, male mice received up to 1 percent lead in the diet, and the percentage of abnormal
spermatozoa increased with increasing lead exposure. Eyden et al. (1978) also fed 1 percent
lead acetate in the diet to male mice. By the eighth week, abnormal sperm had increased;
however, the affected mice showed weight loss and other signs of general toxicity. Thus, the
spermatogenesis effect was not indicative of differential sensitivity of the gonad to lead.
Krasovskii et al. (1979) observed decreased motility, duration of motility, and osmotic
stability of sperm from rats given 0.05 mg/kg lead orally for 20-30 days. Damage to gonadal
DPB12/G 12-157 9/20/83
-------
PRELIMINARY DRAFT
blood vessels and to Leydig cells was also seen. Rats treated for 6-12 months exhibited ab-
normal sperm morphology and decreased spermatogenesis. In the report of Willems et al. (1982)
described above, however, no effects on sperm count or morphology were seen in rabbits.
Lead acetate effects on sperm morphology were also tested in mice given about one six-
teenth to one half an LD5o dose by i.p. injection on five consecutive days (Bruce and Heddle,
1979; Wyrobek and Bruce, 1978; Heddle and Bruce, 1977). The two lowest doses (apparently 100
and 250 mg/kg) resulted in only a modest increase in morphologically abnormal sperm 35 days
after treatment, but the 500 or 900 mg/kg doses resulted in up to 21 percent abnormal sperm.
That lead could directly affect developing sperm or their cellular precursors is made
more plausible by the data of Timm and Schulz (1966), who found lead in the seminiferous
tubules of rats and in their sperm. The mechanisms for lead effects on the male gonad or
gamete are unknown, however, although Golubovich et al. (1968) found altered RNA levels in the
testes of lead exposed rats. They suggested that testicular damage was related to diminished
ribosomal activity and inhibition of protein synthesis. As noted above, Ivanova-Chemishanska
et al. (1980) observed biochemical changes in testes of lead-treated mice. Nevertheless, such
.observations are only initial attempts to determine a mechanism for observed lead effects. A
more likely mechanism for such effects on the testis may be found in the work of Donovan et
al. (1980), who found that lead inhibited androgen binding by the cytosolic receptors of mouse
prostate. This could provide a mechanism for the observation of Khare et al. (1978), who
found that injection of lead acetate into the rat prostate resulted in decreased prostatic
weight; no such changes were seen in other accessory sex glands or in the testes.
Effects on hormonal production or on hormone receptors could also explain the results of
Maker et al. (1975), who observed a delay in testicular development and an increase in age of
first mating in male mice of two strains whose dams were given 0.08 percent lead (C57B1/6J) or
0.5 percent lead (Swiss-Webster albino) during pregnancy and lactation. The weanling males
were fed these same doses in their diets through 60 days of age.
Another potential mechanism underlying lead effects on sperm involves its affinity for
sulfhydryl groups. Mammalian sperm possess high concentrations of sulfhydryls believed to be
involved in the maintenance of motility and maturation via regulation of stability in sperm
heads and tails (Bedford and Calvin, 1974; Calvin and Bedford, 1971). It has also been found
that blockage of membrane thiols inhibits sperm maturation (Reyes et al., 1976).
12.6.2.1.2 Effects associated with exposure of females to lead. Numerous studies have
focused on lead exposure effects in females. For example, effects of lead on reproductive
functions of female rats were studied by Hilderbrand et al. (1973), using animals given lead
acetate orally at doses of 5 and 100 ug for 30 days. Control rats of both sexes had the same
blood lead levels. Blood lead levels of treated females were higher than those of similarly
treated males: 30 versus 19 ug/dl at the low dose, and 53 versus 30 ug/dl at the high dose.
DPB12/G 12-158 9/20/83
-------
PRELIMINARY DRAFT
The females exhibited irregular estrus cycles at both doses. When blood lead levels reached
50 ug/dl, they developed ovarian follicular cysts, with reductions in numbers of corpora
lutea.
In a subsequent study (Der et al., 1974), lead acetate (100 ug lead per day) was injected
s.c. for 40 days in weanling female rats. Treated rats received a low-protein (4 percent) or
adequate-protein (20 percent) diet; controls were given the same diets without lead. Females
on the low protein, high lead diet did not display vaginal opening during the treatment period
and their ovaries decreased in weight. No estrous cycles were observed in animals from either
low protein group; those of the adequate diet controls were normal, while those of the rats
given adequate protein plus lead were irregular in length. Endometrial proliferation was also
inhibited by lead treatment. Blood lead levels were 23 ug/dl in the two control groups, while
values for the adequate and low protein lead-treated groups were 61 and 1086 ug/dl, respec-
tively. The reports of Hilderbrand et al. (1973) and Der et al. (1974) suggest that lead
chronically administered in high doses can interfere with sexual development in rats and the
body burden of lead is greatly increased by protein deprivation.
Maker et al. (1975) noted a delay in age at first conception in female mice of two
strains exposed to 0.08 percent (C57B1/6J) or 0.5 percent lead (Swiss-Webster) indirectly via
the maternal diet (while HI utero and nursing) and directly up to 60 days of age. These
females were retarded in growth and tended to conceive only after reaching weights approxi-
mating those at which untreated mice normally first conceive. Litters from females that had
themselves been developmentally exposed to at least 0.5 percent lead had lower survival rates
and retarded development. More recently, Grant et al. (1980) reported delayed vaginal opening
in rats whose mothers were given 25, 50, or 250 ppm lead (as lead acetate) in their drinking
water during gestation and lactation followed by equivalent exposure of the offspring after
weaning. The vaginal opening delays in the 25 ppm females occurred in the absence of any
growth retardation or other developmental delays, in association with median blood lead levels
of 18-29 ug/dl.
Although most animal studies have used rodents, Vermande-Van Eck and Meigs (1960) admin-
istered lead chloride i.v. to female rhesus monkeys. The monkeys were given 10 mg/ week for
four weeks and 20 mg/week for the next seven months. Lead treatment resulted in cessation of
menstruation, loss of color of the "sex skin" (presumably due to decreased estrogen produc-
tion), and pathological changes in the ovaries. One to five months after lead treatment
ceased menstrual periods resumed, the sex skin returned to a normal color, and the ovaries
regained their normal appearance. Thus, there was an apparent reversal of lead effects on
female reproductive functions, although there were no confirmatory tests of fertility.
The above studies indicate that pre- and/or post-natal exposure of female animals to lead
can affect pubertal progression and hypothalamic-pituitary-ovarian-uterine functions. The
DPB12/G 12-159 9/20/83
-------
PRELIMINARY DRAFT
observations of delayed vaginal opening may reflect delayed ovarian estrogen secretion,
suggesting toxicity to the ovary, hypothalamus, or pituitary. One study has demonstrated
decreased levels of circulating follicle-stimulating hormone (Petrusz et al., 1979), and
others discussed previously have shown lead-induced ovarian atrophy (Stowe and Goyer, 1971;
Vermande-Van Eck and Meigs, 1960), again suggesting toxicity involving the hypothalamic-
pi tui tary-ovari an-endometri al axi s.
12.6.2.2 Effects of Lead on the Offspring. This section discusses developmental studies of
offspring whose parents (one or both) were exposed to lead. Possible male-mediated effects as
well as effects of exposure during gestation are reviewed. Results obtained for offspring of
females given lead following implantation or throughout pregnancy are summarized in Tables
12-13 and 12-14.
12.6.2.2.1 Male mediated effects. A few studies have focused on male-mediated lead effects
on the offspring, suggesting that paternally transmitted effects of lead may cause reductions
in litter size, offspring weight, and survival rate.
Cole and Bachhuber (1914), using rabbits, were the first to report paternal effects of
lead intoxication. In their study, the litters of dams sired by lead-intoxicated male rabbits
were smaller than those sired by controls. Weller (1915) similarly demonstrated reduced birth
weights and survival among offspring of lead-exposed male guinea pigs.
Offspring of lead-treated males from the Ivanova-Chemishanska et al. (1980) study de-
scribed above were affected in a variety of ways, e.g. they exhibited "failure to thrive" and
lower weights than did control progeny at one and three weeks postpartum. These results are
difficult to interpret, however, without more specific information on the experimental methods
and dosing procedures.
12.6.2.2.2 Results of lead exposure of both parents. Only a few studies have assessed the
effects of lead exposure of both parents on reproduction. Schroeder and Mitchener (1971)
found a reduction in the number of offspring of rats and mice given drinking water containing
25 ppm lead. According to the data of Schroeder et al. (1970), however, animals in the 1971
study may have been chromium deficient, and the Schroeder and Mitchener (1971) results are in
marked contrast to those of an earlier study by Morris et al. (1938), who reported no signifi-
cant reduction in weaning percentage among offspring of rats fed 512 ppm lead.
In another study, Stowe and Goyer (1971) assessed the relative paternal and maternal
effects of lead as measured by effects on the progeny of lead-intoxicated rats. Female rats
fed diets with or without 1 percent lead were mated with normal males. The pregnant rats were
continued on their respective rations with or without lead throughout gestation and lactation.
Offspring of these matings, the Ft generation, were fed the rations of their dams and were
mated in combinations as follows: control female to control male (CF-CM), control female to
DPB12/G 12-160 9/20/83
-------
TABLE 12-13. EFFECTS OF PRENATAL EXPOSURE TO LEAD ON THE OFFSPRING OF LABORATORY AND DOMESTIC ANIMALS:
STUDIES USING ORAL OR INHALATION ROUTES OF EXPOSURE
re
i
Treatment
Species
Rat
Mouse
Test agent
Lead acetate
Lead acetate
Tetraethy) lead
Tetraaethyl lead
Triaethyl lead
chloride
Lead nitrate
Lead (aerosol)
Lead acetate
Dose and aode
512 ppa in diet
10.000 ppa in diet
45.2 ag/kg/day, po
45.5 ag/kg/day, po
31.9-47.8 ag/kg/day, po
63.7 ag/kg/day, po
150 ag/kg/day, po
256-478 ag/kg/day in
water
31.9-319 ppm in water
0.32-159 ppa in water
1.6-3.2 ng/kg/day, po
0.064 ag/kg/day, po
0.64 ag/kg/day, po
6.4 ag/kg/day, po
10-28.7 ag/kg/day, po
3.6-7.2 ag/kg/day, po
1 ppe in water
10 ppa in water
1 or 3 ag/a3, inhaled
10 mg/a3, inhaled
3,185 ppe in diet
780-1,593 ppa in diet
3,185 ppa in diet
1,593-6,370 ppa in diet
1,595-3,185 ppn in diet
45.3 ag/kg/day, po
455 ng/ kg/day, po
Tiaingc
all
all
6-16
6-8
all
all
6-18
all.LAC
all
all
9-11 or 12-14
6-16
6-16
6-8
9-11 or 12-14
9-11 or 12-14
all
all
1-21
1-21
1-7
1-16,17, or 18
1-16,17, or 18
1-15,16, or 17
7-16,17, or 18
6-16
6-8
Effect on the offspring3
Mortality Fetotoxicity Malformation Reference
? Morris et al . (1938)
+ + ? Stowe and Goyer (1971)
- - Kennedy et al. (1975)
Miller et al. (1982)
+ - - Wardell et al. (1982)
? +d ? Murray et al . (1978)
±* + - Dilts and Ahokas (1979, 1980)
+ - Kiimel et al. (1980)
± + - McClain and Becker (1972)
Kennedy et al. (1975)
± + - McClain and Becker (1972)
+
-. ? Hubermont et al. (1976)
+f,9 ?
? -* ? Prigge and Greve (1977)
+ t N/A Jacquet (1977)
? S>} . ? Jacquet et al. (1977b)
? +k ? Gerber and Maes (1978)
? +1 ? Gerber et al. (1978)
Kennedy et al . (1975)
-o
no
m
r~
i — t
2
Z
no
O
•yo
— 1
-------
TABLE 12-13. (continued)
PO
i
TNJ
Species
House
Sheep
Treatment
Test agent Dose and node
0.1-1.0 g/1 in water
637-3,185 ppM in diet
1,593 ppM in diet
3,185 ppM in diet
1,250 ppM in diet
3,185 ppM in diet
1,250 ppM in diet
2,500-5,000 PDM
in diet
1,250 p»M in diet
Tetraethyl lead 0.06 ng/kg/day, po
0.64 Mg/kg/day, po
6.4 Mg/kg/day, po
Lead powder 0.5-16 itg/kg/day, in
diet1
Effect on the offspring3
Ti«ringc Mortality Fetotoxicity Malformation
all - ? ?
1-18 * ? ?
1-16,17, or 18 +
1-16,17, or 18 + +
all +
1-16,17, or 18 + +
all +
all + +
all +
6-16 - -
6-16 + +
6-8 + +
all + ? -
Reference
Leonard et al. (1973)
Maisin et al. (1975)
Jacquet et al. (1975)
Jacquet (1976)
Kennedy et al. (1975)
Sharaia and Buck (1976)
a+ = present; - = effect not seen; t = ambiguous effect; ? = effect not examined or insufficient data.
b As elemental lead.
Specific gestation days when exposed; LAC = also during lactation.
Decreased numbers of dendritic spines and Malformed spines at day 30 postpartuM.
eLitter size values for high dose group suggestive of an effect.
fALAQ activity was decreased.
9Free tissue porphyrins increased in kidneys.
Hematocrit was decreased.
'Fetal porphyrins were increased, except in the low dose fetuses assayed on gestation day 18.
^Decreased heae and fetal weight.
Incorporation of Fe into he«e decreased, and growth was retarded.
reased placental blood flow.
-------
TABLE 12-14. EFFECTS OF PRENATAL LEAD EXPOSURE ON OFFSPRING OF LABORATORY ANIMALS:
RESULTS OF STUDIES EMPLOYING ADMINISTRATION OF LEAD BY INJECTION
Treatment
Species
Rat
House
Test agent Dose and node
Lead acetate 15.9 ng/kg, ip
Lead nitrate 31.3 ng/kg, iv
31.3 ng/kg, iv
31.3 ng/kg, iv
3.13 ng/kg, iv
15.6 ng/kg, iv
15.6 ng/kg, iv
unknown, iv
31.3 ng/kg, iv
15.6 Bg/kg, iv
5 «g/kg, iv
25 ng/kg, iv
Lead chloride 7.5 ng/kg/
75 ng/kg/
Trinethyl lead 20.2 ng/kg, iv
chloride 23.8 ng/kg, iv
Lead acetate 9.56-22.3 mg/kg, ip
9.56 wg/kg, ip
22.3 mg/kg, ip
22.3 ntg/kg, ip
Lead chloride 29.8 mg/kg, iv
29.8 ng/kg, iv
Tiningc
9
8
9 or 16
10- 14,
15,17
9 or 15
9
15
8 or 9
17
17
9 or 15
9 or 15
9
9
12
9,10,13, or 15
8
9
9
10 or 12
3 or 4
6
Effect on the offspring
Mortality fetotoxicity Malformation Reference
+ +• + Zegarska et al. (1974)
+ + McClain and Becker (1975)
+d * *
+ +•
Hackett et al. (1978a,b)
+ t +
+ ? ?
+ ? + Core Antich and Arooedo Mon
(1980)
+ - Minsker et al. (1982)
+ +•
Hackett et al . (1982)
+ * *,-
± - - McLellan et al. (1974)
+ +
+
+9 +
- + + Jacquet and Gerber (1979)
4- +
t + +
_
t ? ? Wide and Nilsson (1977)
+ N/A N/A
-------
TABLE 12-14. (continued)
no
i
Species
Haaster
Test agent
Lead acetate
Lead acetate or
chloride
Lead nitrate
Treatment
Dose and Mode
31.9 -fl/ kg, iv
31.9 or 37.3 «g/kg, iv
31.3 mg/kg, iv
15.6-31.3 no/kg, iv
31.3 ng/kg, iv
31.3 mg/kg, iv
Timingc
8
8
7, 8. or 9
8 or 9
8
8
Effect on the offspring3
Mortality Fetotoxicity Malformation Reference
+ ? + Ferm (1969)
? ? + Ferrn and Carpenter (1967)
? ? + Fern and Carpenter (1967)
-» ? * Ferm and Ferm (1971)
-» + + Carpenter and Ferm (1977)
+ +h + Gale (1978)
+ = effect present; - = effect not seen; ± = ambiguous effect; ? = effect not examined or insufficient data.
b As elemental lead.
cSpecific gestation days when exposed.
''with the exception of day 17.
eNo fetuses survived to be examined for Malformation.
No dosage route specified.
''Only after day 10 treatment.
Delayed ossification (fetal weights not given).
Dosage was varied dally to Maintain a blood lead level of = 40 pg/dl (range = 30 to 70 pg/dl).
-o
•so
73
-------
PRELIMINARY DRAFT
lead-intoxicated male (CF-PbM), lead-intoxicated female to control male (PbF-CM), and lead-
intoxicated female to lead-intoxicated male (PbF-PbM). The results are shown in Table 12-15.
The paternal effects of lead included reductions of 15 percent in the number of pups per
litter, 12 percent in mean pup birth weight, and 18 percent in pup survival rate. The mater-
nal effects of lead included reductions of 26 percent in litter size, 19 percent in pup birth
weight, and 41 percent in pup survival. The combined male and female effects of lead toxicity
resulted in reductions of 35 percent in the number of pups per litter, 29 percent in pup birth
weight, and 67 percent in pup survival to weaning. Stowe and Goyer classified the effects of
lead upon reproduction as gametotoxic, intrauterine, and extrauterine. The gametotoxic ef-
fects of lead seemed to be irreversible and had additive male and female components. Intra-
uterine effects were presumed to be due to lead uptake by the conceptus, plus gametotoxic ef-
fects. The extrauterine effects were due to the passage of lead from the dam to the nursing
pups, adding to the gametotoxic and intrauterine effects.
Leonard et al. (1973), however, found no effect on the reproductive performance of groups
of 20 pairs of mice given lead in their drinking water over a nine-month period. Lead doses
ranged from 0.1 to 1.0 g/1. A total amount of 31 g/kg was ingested at the high dose, equiva-
lent to ingestion of 2.2 kg lead by a 70 kg man over the same time period.
12.6.2.2.3 Lead effects on implantation and early development. Numerous studies have been
performed to elucidate mechanisms by which lead causes prenatal death. They suggest two
mechanisms of action for lead, one on implantation and the other (mainly at higher doses) on
fetal development. The latter is discussed primarily in Section 12.6.2.2.4.5.
Maisin et al. (1975) exposed female mice to dietary lead for 18 days after mating; both
the number of pregnancies and surviving embryos decreased. Similarly, exposure of female mice
to lead via their diet (0.125-1.00 percent) from mating to 16-18 days afterward (Jacquet,
1976; Jacquet et al., 1975) resulted in decreased pregnancy incidence and number of corpora
lutea; increased number of embryos dying after implantation at the highest dosages; decreased
body weights of surviving fetuses; and treated dam fatalities at the high dose.
Jacquet and co-workers also described effects of maternal dietary lead exposure on pre-
implantation mouse embryos (Jacquet, 1976; Jacquet et al., 1976). They found lead in the diet
to be associated with retardation of cleavage in embryos, failure of trophoblastic giant cells
to differentiate, and absence of an uterine decidual reaction. Maisin et al. (1978) also
found delayed cleavage in embryos of mice fed lead acetate prior to mating and up to 7 days
afterwards.
Giavini et al. (1980) further confirmed the ability of lead to affect the preimplantation
embryo in studies of rats transplacentally exposed to lead nitrate, and Wide and Nilsson
(1977, 1979) reported that inorganic lead had similar effects on mice. Jacquet (1978) was
able to force implantation in that species by use of high doses of progesterone, while Wide
DPB12/G 12-165 9/20/83
-------
TABLE 12-15. REPRODUCTIVE PERFORMANCE OF
LEAD- INTOXICATED RATS
Parameter
Type of
CF-CM
Litters observed
Pups per litter
Pup birth weight, g
Weaned rats per litter
Survival rate, %
Litter birth weight, „
Dam breeding weight
Litter birth weight, ^
Dam whelping weight
Gestational gain,
Pups per litter 9
Nonfetal gestational
gain per fetus, g
22
11.90
6.74
9.84
89.80
28.04
19.09
11.54
3.93
± 0.403
± 0.15
± 0.50
± 3.20
±1.30
± 0.80
± 0.60
± 0.38
CF-PbM
24
10.10
5.92
7.04
73.70
22.30
15.97
11.20
4.83
± 0.
± 0.
± 0.
± 7.
± 0.
± 0.
i 0.
± Q.
50
13C
77C
90
90C
58C
74
47
mating
PbF-CM
36
8.78
5.44
5.41
52.60
19.35
14.28
11.17
4.15
± 0.
± 0.
± 0.
± 7.
± 1.
± 0.
± 0.
± 0.
30b
13C'd
74C'd
20
00C
66C
54
42
PbF-PbM
16
7.75 ±
4.80 ±
2.72 ±
30.00 ±
15.38 ±
1 1 . 58 ±
12.34 ±
3.96 ±
0.50C
0.1 9C
0.70C
8.20C
1.10C
0.78C
1.24
0.46
,d,e
,d,e
,d,f
,d,f
,d,f
*Mean ± S.E.M.
Significantly (p <0.05)
CSignificantly (p <0.01)
Significantly (p <0.01)
p
Significantly (p <0.01)
Significantly (p <0.05)
less than mean for CF-CM.
less than mean for CF-CM.
less than mean for CF-PbM.
less than mean for PbF-CM.
less than mean for PbF-CM.
•30
Source: Stowe and Goyer (1971).
-------
PRELIMINARY DRAFT
(1980) determined that administration of estradiol-170 and progesterone could reverse the ef-
fects of lead on implantation. Wide suggested that the lead-induced implantation blockage was
mediated by a decrease in endometrial responsiveness to both sex steroids. Jacquet (1976) and
Jacquet et al. (1977b) had attributed lead-induced prevention of implantation in the mouse to
a lack of endogenous progesterone alone, stating that estrogen levels were unaffected. Later,
however, Jacquet et al. (1977a) stated that estrogen levels also decreased, a finding not sup-
ported by Wide and Wide (1980). The latter authors did find a lead-induced increase in
uterine estradiol receptors, but no change in binding affinities.
In order to examine lead effects early in gestation, Wide and Nilsson (1977) examined
embryos from untreated mice and from mothers given 1 mg lead chloride on days 3, 4, or 6 of
pregnancy. Embryonic mortality was greater in lead-treated litters; in the day-6 group some
abnormal embryos were observed by day 8. In a later experiment, Wide (1978) removed blasto-
cysts from lead-treated mice. She found that they attached and grew normally during three
days of ij\ vitro culture. Other blastocysts from untreated mothers were cultured in media
containing lead, and a dose-dependent decrease in the number of normally developing embryos
was seen.
A study employing domestic sheep was reported by Sharma and Buck (1976), who fed lead
powder to pregnant ewes throughout gestation. Levels in the diet were varied from 0.5 to 16
mg/kg/day in an effort to keep blood lead levels near 40 H9/dl (actual levels ranged from 30
to 70 ug/dl). Such treatment resulted in a greatly decreased lambing percentage but no gross
malformations. However, the number of subjects was small.
12.6.2.2.4 Teratogem'city and prenatal toxlcity of lead in animals.
12.6.2.2.4.1 High dose effects on the conceptus. Teratogem'c effects refer to physical
defects (malformations) in the developing offspring. Prenatal toxicity (embryotoxicity, feto-
toxicity) includes premature birth, prenatal death, stunting, histopathological effects, and
transient biochemical or physiological changes. Behavioral teratogenicity, consisting of be-
havioral alterations or functional (e.g., motor, sensory) deficits resulting from i_n uterg
exposure, is dealt with in Section 12.4 of this chapter.
Teratogenicity of lead, at high exposure levels, has been demonstrated in rodents and
birds, with some results suggesting a species-related specificity of certain gross teratogenic
effects. Perm and Carpenter (1967), as well as Perm and Perm (1971), reported increased
embryonic resorption and malformation rates when various lead salts were administered i.v. to
pregnant hamsters. Teratogenic effects were largely restricted to the tail region, including
malformations of sacral and caudal vertebrae resulting in absent or stunted tails. Gale
(1978) found the same effects plus hydrocephalus, among six strains of hamsters and noted dif-
ferences in susceptibility, suggesting a genetic component in lead-induced teratogenicity.
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Zegarska et al. (1974) performed a study with rats injected with lead acetate at midges-
tation. They reported embryonic mortality and malformations, McClain and Becker (1975) sub-
sequently administered lead nitrate i.v. to rats on one of days 8-17 of gestation, producing
malformations and embryo- and feto-toxicity. Hackett et al. (1978, 1982a,b) also gave lead
i.v. to rats and found malformations and high incidences of prenatal mortality. Minsker et
al. (1982) gave lead i.v. to dams on day 17 of gestation and observed decreased birth weights,
as well as decreased weight and survival by postpartum day 7.
In another study, Miller et al. (1982) used oral doses of lead acetate up to 100 mg/kg
given daily to rats before breeding and throughout pregnancy and found fetal stunting at the
high dose, but no other effects. Maternal blood lead values ranged from 80 to 92 M9/dl prior
to mating and from 53 to 92 ug/dl during pregnancy. Pretreatment and control blood lead
levels averaged 6 to 10 ug/dl. Also, Wardell et al. (1982) gavaged rats daily with lead doses
of up to 150 mg/kg from gestation day 6 through day 18 and observed decreased prenatal surviv-
al at the high dose, but no malformations.
Perm (1969) reported that teratogenic effects of i.v. lead in hamsters are potentiated in
the presence of cadmium, leading to severe caudal dysplasia. This finding was duplicated by
Hi 1 be!ink (1980). In addition to caudal malformations, lead appears to influence the morphol-
ogy of the developing brain. For example, Murray et al. (1978) described a significant
decrease in number of dendritic spines and a variety of morphological abnormalities of such
spines in parietal cortex of 30-day-old rat pups exposed to lead during gestation and nursing,
during the postweaning period only, or during both periods. Morphometric analysis of rats
transplacentally exposed to lead indicated that cellular organelles were altered as a function
of dose and stage of development at exposure (Klein et al., 1978). These results indicate
that morphologically apparent effects of lead on the brain could be produced by exposure dur-
ing pregnancy alone, a question not addressed by Murray et al. (1978).
A variety of studies relating neurobehavioral effects to prenatal lead exposure have also
been published. These studies are discussed in Section 12.4.3 of this chapter.
12.6.2.2.4.2 Low dose effects on the conceptus. There is a paucity of information re-
garding the teratogenicity and developmental toxicity of prolonged low-level lead exposure.
Kimmel et al. (1980) exposed female rats chronically to lead acetate via drinking water (0.5,
5, 50, and 250 M9/9) from weaning through mating, gestation, and lactation. They observed a
decrease in fetal body length of female offspring at the high dose, and the female offspring
from the 50 and 250 ug/g groups weighed less at weaning and showed delays in physical develop-
ment. Maternal toxicity was evident in the rats given 25 ug/g or higher doses, corresponding
to blood lead levels of 20 ug/dl or higher. Reiter et al. (1975) observed delays in the
development of the nervous system in offspring exposed to 50 ug/g lead throughout gestation
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and lactation. Whether these delays in development resulted from a direct effect of lead on
the nervous system of the pups or reflect secondary changes (resulting from malnutrition, hor-
monal imbalance, etc.) is not clear. Whatever the mechanisms involved, these studies suggest
that low-level, chronic exposure to lead may induce postnatal developmental delays.
12.6.2.2.4.3 Prenatal effects of organolead compounds. In an initial study of the ef-
fects of organolead compounds in animals, McClain and Becker (1972) treated rats orally with
7.5-30 mg/kg tetraethyl lead, 40-150 mg/kg tetramethyl lead, or 15-38 mg/kg trimethyl lead
chloride, given in three divided doses on gestation days 9-11 or 12-14. The last compound was
also given i.v. at doses of 20 to 40 mg/kg on one of days 8-15 of pregnancy. The highest dose
of each agent resulted in maternal death, while lower doses caused maternal toxicity. At all
dose levels, fetuses from dams given multiple treatment weighed less than controls. Single
treatments at the highest doses tended to have similar effects. In some cases delayed ossifi-
cation was observed. In addition, direct intra-amniotic injection of trimethyl lead chloride
at levels up to 100 (jg per fetus caused increasing fetal mortality.
Kennedy et al. (1975) administered tetraethyl lead by gavage to mice and rats during the
period of organogenesis at dose levels up to 10 mg/kg. Maternal toxicity, prenatal mortality,
and developmental retardation were noted at the highest doses in both species, although mater-
nal treatment was discontinued after only three days due to excessive toxicity. In a subse-
quent study involving alkyl lead, Odenbro and Kihlstrom (1977) treated female mice orally with
triethyl lead at doses of up to 3.0 mg/kg/day on days 3 to 5 following mating. The highest
treatment levels resulted in decreased pregnancy rates, while at 1.5 mg/kg, lower implantation
rates were seen. In order to elucidate the mechanism of implantation failure in organolead-
intoxicated mice, Odenbro et al. (1982) measured plasma sex steroid levels in mice five days
after mating. Levels of both estradiol and progesterone, but not estrone, were decreased
following intraperitoneal triethyl lead chloride on days three and four of gestation. Such
results suggest a hormonal mechanism for blockage of implantation, a finding also suggested
for inorganic lead (Wide, 1980; Jacquet et al., 1977a).
12.6.2.2.4.4 Effects of lead on fetal physiology and metabolism. Biochemical indicators
of developmental toxicity have been the subject of a number of investigations, as possible in-
dicators of subtle prenatal effects. Hubermont et al. (1976) exposed female rats to lead in
drinking water before mating, during pregnancy, and after delivery. In the highest exposure
group (10 ppm), maternal and offspring blood lead values were elevated and approached 68 and
42 ug/dl, respectively. Inhibition of ALA-D and elevation of free tissue porphyrins were also
noted in the newborns. Maternal diets containing up to 0.5 percent lead were associated with
increased fetal porphyrins and decreased ALA-D activity by Jacquet et al. (1977a). Fetuses in
the high dose group had decreased weights, but no data were presented on maternal weight gain
or food consumption (which could have influenced fetal weight).
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In the only inhalation exposure study (Prigge and Greve, 1977), rats were exposed
throughout gestation to an aerosol containing 1, 3, or 10 mg Pb/m3 or to a combination of 3 mg
Pb/m3 and 500 ppm carbon monoxide CO. Both maternal and fetal ALA-D activities were strongly
inhibited by lead exposure in a dose-related manner. In the presence of lead plus CO, how-
ever, fetal (but not maternal) ALA-D activity was higher than in the group given lead alone,
possibly due to the increase in total ALA-D seen in the CO-plus-lead treated fetuses. Fetal
body weight and hematocrit were decreased in the high-dose lead group, while maternal values
were unchanged, thus suggesting that the fetuses were more sensitive to lead effects than were
the mothers. Granahan and Huber (1978) also reported decreased hematocrit, as well as reduced
hemoglobin levels, in fetal rats from lead intoxicated dams (1000 ppm in the diet throughout
gestation).
Gerber and Maes (1978) fed pregnant mice diets containing up to one percent lead from day
7 to 18 of pregnancy and determined levels of heme synthesis. Incorporation of iron into
fetal heme was inhibited, but glycine incorporation into heme and protein was unaffected.
Gerber et al. (1978) also found that dietary lead given late in gestation resulted in dimin-
ished placental blood flow but did not decrease uptake of a non-metabolizable amino acid,
alpha-amino isobutyrate. The authors could not decide whether lead-induced fetal growth
retardation was due to placental insufficiency or to the previously described reduction in
heme synthesis (Gerber and Maes, 1978). They did not mention the possibility that the treated
mothers may have reduced their food consumption, resulting in a reduced nutrient supply to the
fetus, regardless of fetal ability to absorb nutrients.
More recently, Wardell et al. (1982) exposed rat fetuses jm utero to lead by gavaging
their pregnant mothers with 150 mg/kg lead from gestation days 6 to 18. On day 19, fetal limb
cartilage was tested for ability to synthesize protein, DMA, and proteoglycans, but no adverse
effects were seen.
12.6.2.2.4.5 Possible mechanisms of lead-induced teratogenesis. The reasons for the
localization of many of the gross teratogenic effects of lead are unknown at this time. Ferm
and Ferm (1971) have suggested that the observed specificity could be explained by an inter-
ference with specific enzymatic events. Lead alters mitochondria! function and enhances or
inhibits enzymes (Vallee and Ulmer, 1972); any or all such effects could interfere with normal
development. Similarly, inhibition of ALA has been suggested as a mechanism of teratogenesis
by Cole and Cole (1976).
In an attempt to study the mechanics of lead induction of sacral-tail region malforma-
tions, Carpenter and Ferm (1977) examined hamster embryos treated at mid-gestation during the
critical stage for response to teratogens in this species. The initial effects were edema of
the tail region of embryos 30 hours after maternal exposure, followed by blisters and hema-
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tomas. These events disrupted normal caudal development, presumably by mechanical displace-
ment. The end results seen in surviving fetuses were missing, stunted, or malformed tails and
anomalies of the lower spinal cord and adjacent vertebrae.
12.6.2.2.4.6 Maternal factors in lead-induced teratogenesis and fetotoxicity. Nutri-
tional factors may also have a bearing on the prenatal toxicity of lead. Jacquet and Gerber
(1979) reported increased mortality and defects in fetuses of mice given i.p. injections of
lead while consuming a calcium deficient diet during gestation. In several treatment groups,
lead-treated calcium deficient mothers had low blood calcium levels, while controls on the
same diet had normal values. It is not certain how meaningful these data are, however, as
there was no clear dose-response relationship within diet groups. In fact, fetal weights were
said to be significantly higher in two of the lead-treated groups (on the normal diet) than in
the untreated controls. Another problem with the study was that litter numbers were small.
Another study on interactions of lead with other elements was done by Dilts and Ahokas
(1979), who exposed rats to lead in their drinking water throughout gestation. Controls were
pair-fed or fed ad libitum. Lead treatment was said to result in decreased fetal weight, and
dietary zinc supplementation was claimed to be associated with a protective effect against
fetal stunting. The data presented do not allow differentiation of effects due to maternal
stress (e.g., decreased food consumption) from direct effects on the fetus. Litter numbers
were small, and some of the data were confusing (e.g., a lead-treated and a pair-fed group
with very similar litter sizes and total litter weights, but rather dissimilar average fetal
weights; live litter weight divided by live litter size does not give the authors' values for
average fetal, weight). Also, no data were given on maternal or fetal lead or zinc levels. In
a further report on apparently the same animals as above, Dilts and Ahokas (1980) found that
lead inhibited cell division and decreased protein contents of the fetal placentas, evis-
cerated carcasses, and livers. Such lead-related effects were not influenced by maternal zinc
supplementation.
12.6.2.3 Effects of Lead on Avian Species. The effects of lead on the reproduction and
development of various avian species have been studied by a number of investigators, primarily
out of interest in the effects of lead shot ingested by wildlife or out of interest in an
avian embryo model for the experimental analysis of ontogenetic processes. The relevance of
such studies to the health effects of lead on humans is not clear. Consequently, these
studies are not discussed further here.
12.6.3 Summary
The most clear-cut data described in this section on reproduction and development are de-
rived from studies employing high lead doses in laboratory animals. There is still a need for
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more critical research to evaluate the possible subtle toxic effects of lead on the fetus,
using biochemical, ultrastructural, or behavioral endpoints. An exhaustive evaluation of
lead-associated changes in offspring will require consideration of possible additional effects
due to paternal lead burden. Neonatal lead intake via consumption of milk from lead-exposed
mothers may also be a factor at times. Also, it must be recognized that lead effects on re-
production may be exacerbated by other environmental factors (e.g., dietary influences,
maternal hyperthermia, hypoxia, and co-exposure to other toxins).
There are currently no reliable data pointing to adverse effects in human offspring fol-
lowing paternal exposure to lead, and the early studies of high dose exposure in pregnant
women indicate toxic—but not teratogenic—effects on the conceptus. Effects on reproductive
performance in women are not well documented, but industrial exposure of men to lead at levels
resulting in blood lead values of 40-50 ug/dl appear to have resulted in altered testicular
function. Unfortunately, the human data regarding lead effects during development currently
do not lend themselves to accurate estimation of no-effect levels.
The paucity of human exposure data forces an examination of the animal studies for indi-
cations of threshold levels for effects of lead on the conceptus. It must be noted that the
animal data are almost entirely derived from rodents. Based on these rodent data, it seems
likely that fetotoxic effects have occurred in animals at chronic exposures to 600-1000 ppm
lead in the diet. Subtle effects appear to have been observed at 10 ppm in the drinking water,
while effects of inhaled lead have been seen at levels of 10 mg/m3. With acute exposure by
gavage or by injection, the values are 10-16 mg/kg and 16-30 mg/kg, respectively. Since
humans are most likely to be exposed to lead in their diet, air, or water, the data from other
routes of exposure are of less value in estimating harmful exposures. Indeed, it seems likely
that teratogenic effects occur only when the maternal dose is given by injection.
Although human and animal responses may be dissimilar, the animal evidence does document
a variety of effects of lead exposure on reproduction and development. Measured or apparent
changes in production of or response to reproductive hormones, toxic effects on the gonads,
and toxic or teratogenic effects on the conceptus have all been reported. The animal data
also suggest subtle effects on such parameters as metabolism and cell structure that should be
monitored in human populations. Well-designed human epidemiological studies involving large
numbers of subjects are still needed. Such data could clarify the relationship of exposure
levels and durations to blood lead values associated with significant effects and are needed
for estimation of no-effect levels.
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12.7 GENOTOXIC AND CARCINOGENIC EFFECTS OF LEAD
12.7.1 Introduction
Potential carcinogenic, genotoxic (referring to alteration in structure or metabolism of
DNA), and mutagenic roles of lead are considered here. Epidemiological studies of occupation-
ally exposed populations are considered first. Such studies investigate possible associations
of lead with induction of human neoplasia. Epidemiological studies are important because they
assess the incidence of disease in humans under actual ambient exposure conditions. However,
such studies have many limitations that make it difficult to assess the carcinogenic activity
of any specific agent. These include general problems in accurately determining the amount
and nature of exposure to a particular chemical agent; in the absence of adequate exposure
data it is difficult to determine whether each individual in a population was equally exposed
to the agent in question. It is also often difficult to assess other factors, such as expo-
sure to carcinogens in the diet, and to control for confounding variables that may have con-
tributed to the incidence of any neoplasms. These factors tend to obscure the effect of lead
alone. Also, in an occupational setting a worker is often exposed to various chemical com-
pounds, making it more difficult to assess epidemiologically the injurious effect resulting
specifically from exposure to one, such as lead.
A second approach considered here examines the ability of specific lead compounds to in-
duce tumors in experimental animals. The advantage of these studies over epidemiological in-
vestigations is that a specific lead compound, its mode of administration, and level of expo-
sure can be well defined and controlled. Additionally, many experimental procedures can be
performed on animals that for ethical reasons cannot be performed on humans, thereby allowing
a better understanding of the course of chemically induced injury. For example, animals may
be sacrificed and necropsies performed at any desired time during the study. Factors such as
diet and exposure to other environmental conditions can be well controlled, and genetic vari-
ability can be minimized by use of well established and characterized animal lines. One
problem with animal studies is the difficulty of extrapolating such data to humans; however,
this drawback is perhaps more important in assessing the toxicity of organic chemicals than in
assessing inorganic agents. The injury induced by many organic agents is highly dependent
upon reactive intermediates formed jm vivo by the action of enzymatic systems (e.g., micro-
somal enzymes) upon the parent compound. Both qualitative and quantitative differences be-
tween the metabolic capabilities of humans and experimental animals have been documented
(Neal, 1980). With inorganic compounds of lead, however, the element of interest undergoes
little alteration iji vivo and, therefore, the ultimate toxic agent is less likely to differ
between experimental animals and humans (Costa, 1980). The carcinogenic action of most
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organic chemicals is dependent upon activation of a parent pro-carcinogen, whereas most metal-
lic carcinogens undergo little alteration i_n vivo to produce their oncogenic effects (Costa,
1980).
A third approach discussed below is HI vitro studies. Animal carcinogen bioassays are
presently the preferred means for assessing carcinogenic activity but they are extremely ex-
pensive and time consuming. As a result, much effort has been directed toward developing
suitable in vitro tests to complement HI vivo animal studies in evaluating potential oncogen-
icity of chemicals. The cell transformation assay has as its endpoint neoplastic transforma-
tion of mammalian cells and is among the most suitable J_n vitro systems because it examines
cellular events closely related to carcinogenesis (Heck and Costa, 1982a). A general problem
with this assay system, which is less troublesome with reference to metal compounds, is that
it employs fibroblastic cells in culture, which lack many HI vivo metabolic systems. Since
lead is not extensively metabolized HI vivo, addition of liver microsomal extracts (which has
been attempted in this and similar systems) is not necessary to generate ultimate carcino-
gen(s) from this metal (see above). However, if other indirect factors are involved with lead
carcinogenesis i_n vivo, then these might be absent in such culture systems (e.g., specific
lead-binding proteins that direct lead interactions i_n vivo with oncogenically relevant
sites). There are also technical problems related to the culturing of primary cells and dif-
ficulties with the final microscopic evaluation of morphological transformations, which are
prone to some subjectivity. However, if the assay is performed properly it can be very relia-
ble and reproducible. Modifications of this assay system (i.e., exposure of pregnant hamsters
to a test chemical followed by culturing and examination of embryonic cells for transplacen-
tal ly induced transformation) are available for evaluation of jji vivo metabolic influences,
provided that the test agent is transported to the fetus. Additionally, cryopreservation of
primary cultures isolated from the same litter of embryos can control for variation in cell
populations exposed to test chemicals and give more reproducible responses in replicate ex-
periments (Pienta, 1980). A potential advantage of the cell transformation assay system is
the possibility that cultured human cells can be transformed jr> vitro. Despite numerous at-
tempts, however, no reproducible human-cell transformation system has yet been sucessfully
established which has been evaluated with a number of different chemicals of defined carcino-
genic activity.
Numerous processes have been closely linked with oncogenic development, and specific
assay systems that utilize events linked mechanistically with cancer as an endpoint have been
developed to probe whether a chemical agent can affect any of these events. These systems in-
clude assays for mutations, chromosomal aberrations, development of micronuclei, enhancement
of sister chromatid exchange, effects on DMA structure, and effects on DMA and RNA polymerase.
These assay systems have been used to examine the genotoxicity of lead and facilitate the
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assessment of possible lead carcinogenicity. Chromosomal aberration studies are useful
because human lymphocytes cultured from individuals after exposure to lead allow evaluation of
genotoxic activity that occurred under the influence of an ijn vivo metabolic system. Such
studies are discussed below in relationship to genotoxic effects of lead. However, a neo-
plastic change does not necessarily result, and evaluations of some less conspicuous types of
chromosomal aberrations are somewhat subjective since microscopy is exclusively utilized in
the final analyses. The sensitivity of detection of chromosomal changes also tends to be less
than other measurable DMA effects, e.g., the induction of DMA repair. However, it is reason-
able to assume that if an agent produces chromosomal aberrations it may have potential carcin-
ogenic activity. Many carcinogens are also mutagenic and this fact, combined with the low
cost and ease with which bacterial mutation assays can be performed, has resulted in wide use
of these systems in determining potential carcinogenicity of chemicals. Mutation assays can
also be performed with eukaryotic cells and several studies are discussed below that examined
the mutagenic role of lead in these systems. However, in bacterial systems such as the Ames
test, metal compounds with known human carcinogenic activity are generally negative and,
therefore, this system is not useful for determining the potential oncogenicity of lead.
Similarly, even in eukaryotic systems, metals with known human cancer-causing activity do not
produce consistent mutagenic responses. Reasons for this lack of mutagenic effect remain un-
clear, and it appears that mutagenicity studies of lead cannot be weighed heavily in assessing
its genotoxicity.
Other test systems that probe for effects of chemical agents on DMA structure may be use-
ful in assessing the genotoxic potential of lead. Sister chromatid exchange represents the
normal movement of DNA in the genome and enhancement of this process by potentially carcino-
genic agents is a sensitive indicator of genotoxicity (Sandberg, 1982). However, these
studies usually involve tissue cultures; consequently, jjn vivo interactions related to such
effects have not been addressed with this system. Numerous recently developed techniques can
be used to assess DNA damage induced by chemical carcinogens. One of the most sensitive is
alkaline elution (Kohn et al., 1981), which may be used to study DNA lesions produced iji vivo
or in cell culture. This technique can measure DNA strand breaks or crosslinks in DNA, as
well as repair of these lesions, but lead compounds have not been studied with this technique.
Assessment of the induction of DNA repair represents one of the most sensitive techniques for
probing genotoxic effects. The reason for this is that the other procedures measure DNA
lesions that have persisted either because they were not recognized by repair enzymes or
because their number was sufficiently great to saturate DNA repair systems. Measurement of
DNA repair activation is still possible even if the DNA lesion has been repaired, but effects
of lead compounds on DNA repair have not been studied. There are a few isolated experiments
within publications that examined the ability of lead compounds to induce ONA damage, but this
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line of investigation requires further work. There are some well-conducted studies of the
effect of lead along with other water soluble metals on isolated DNA and RNA polymerases,
which suggest mutagenic mechanisms occurring in intact cells. The ability of lead to affect
the transcription of DNA and RNA merits concern in regard to its potential oncogenic and muta-
genic properties.
12.7.2 Carcinogenesis Studies with Lead and its Compounds
12.7.2.1 Human Epidemiological Studies. Epidemiological studies of industrial workers, where
the potential for lead exposure is usually greater than for a "normal population," have been
conducted to evaluate the role of lead in the induction of human neoplasia (Cooper, 1976,
1981; Cooper and Gaffey, 1975; Chrusciel, 1975; Dingwall-Fordyce and Lane, 1963; Lane, 1964;
McMichael and Johnson, 1982; Neal et al., 1941; Nelson et al., 1982). In general, these
studies made no attempt to consider types of lead compounds to which workers were exposed or
to determine probable routes of exposure. Some information on specific lead compounds encoun-
tered in the various occupational settings, along with probable exposure routes, would have
made the studies more interpretable and useful. As noted in Chapter 3, with the exception of
lead nitrate and lead acetate, many inorganic lead salts are relatively water insoluble. If
exposure occurred by ingestion, the ability of water-insoluble lead salts (e.g., lead oxide
and lead sulfide) to dissolve in the gastrointestinal tract may contribute to understanding of
their ultimate systemic effects in comparison to their local actions in the gastrointestinal
tract. Factors such as particle size are also important in the dissolution of any water in-
soluble compounds in the gastrointestinal system (Mahaffey, 1983). When considering other
routes of exposure (e.g., inhalation), the water solubility of the lead compound in question,
as well as the particle size, are extremely important, both in terms of systemic absorption
and contained injury in the immediate locus of the retained particle (see Chapter 10). A
hypothetical example is the inhalation of an aerosol of lead oxide versus a water soluble lead
salt such as lead acetate. Lead oxide particles having a diameter of <5 urn would tend to
deposit in the lung and remain in contact with cells there until they dissolved, while soluble
lead salts would dissipate systemically at a much more rapid rate. Therefore, in the case of
inhaled particulate compounds, localized exposure to lead might produce injury primarily in
respiratory tissue, whereas with soluble salts systemic (i.e., CNS, kidney, and erythropoie-
tic) effects might predominate.
The studies of Cooper and Gaffey (1975) and Cooper (1976, 1981) examined the incidence of
cancer in a large population of industrial workers exposed to lead. Two groups of individuals
were identified as the lead-exposed population under consideration: smelter workers from six
lead production facilities and battery plant workers (Cooper and Gaffey, 1975). The authors
reported (see Table 12-16) that total mortality from cancer was higher in lead smelter workers
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than in a control population in two ways: (1) the difference between observed and expected
values for the types of malignancies reported; and (2) the standardized mortality ratio, which
indicates a greater than "normal" response if it is in excess of 100 percent. These studies
report not only an excess of all forms of cancer in smelter workers but also a greater level
of cancer in the respiratory and digestive systems in both battery plant and smelter workers.
The incidence of urinary system cancer was also elevated in the smelter workers (but not in
the battery plant workers), although the number of individuals who died from this neoplasm was
very small. As the table indicates, death from neoplasm at other sites was also elevated com-
pared with a normal population, but these results were not discussed in the report. Kang et
al. (1980) examined the Cooper and Gaffey (1975) report and noted an error in the statistical
equation used to assess the significance of excess cancer mortality. Table 12-17, from Kang
et al., 1980, shows results based on a corrected form of the statistical equation used by
Cooper and Gaffy; it also employed another statistical test claimed to be more appropriate.
Statistical significance was observed in every category listed with the exception of battery
plant workers, whose deaths from all forms of neoplasia were not different from a control
population.
TABLE 12-16. EXPECTED AND OBSERVED DEATHS FOR MALIGNANT NEOPLASMS
JAN. 1, 1947 - DEC. 31, 1979 FOR LEAD SMELTER AND BATTERY PLANT WORKERS
Causes.of Death
(ICDT Code)
Obs
Smelters
Exp SMR* Obs
Battery plant
Exp SMR+
All malignant neoplasms (140-205)
Buccal cavity & pharynx (140-248)
Digestive organs peritoneum (150-159)
Respiratory system (160-164)
Genital organs (170-179)
Urinary organs (180-181)
Leukemia (204)
Lymphosarcoma lymphatic and
hematopoietic (200-203, 205)
Other sites
69
0
25
22
4
5
2
3
8
54.95
1.89
17.63
15.76
4.15
2.95
2.40
3.46
6.71
133
—
150
148
101
179
88
92
126
186
6
70
61
8
5
6
7
23
180.34
6.02
61.48
49.51
18.57
10.33
7.30
9.74
17.39
111
107
123
132
46
52
88
77
142
International Classification of Diseases.
"Correction of +5.55% applied for 18 missing death certificates.
^-Correction of +7.52% applied for 71 missing death certificates.
Source: Cooper and Gaffey (1975).
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TABLE 12-17. EXPECTED AND OBSERVED DEATHS RESULTING FROM SPECIFIED MALIGNANT NEOPLASMS
FOR LEAD SMELTER AND BATTERY PLANT WORKERS AND LEVELS OF SIGNIFICANCE BY
TYPE OF STATISTICAL ANALYSIS ACCORDING TO ONE-TAILED TESTS
Probability
Causes.of death
(ICDT code)
Number
Ob-
served
of deaths
Ex-
pected
SMR* Pois-
son**
This
anal-
ysis***
Cooper
and
Gaffey****
Lead smelter workers:
All malignant neoplasms
(140-205)
Cancer of the digestive organs
peritoneum (250-159)
Cancer of the respiratory system
(160-164)
Battery plant workers:
69
25
22
54.95 133 <0.02 <0.01 <0.02
17.63 150 <0.03 <0.02 <0.05
15.76 148 <0.05 <0.03 >0.05
All malignant neoplasms
(140-205)
Cancer of the digestive organs,
peritoneum (150-159)
Cancer of the respiratory system
(160-164)
186
70
61
180.34
61.48
49.51
111
123
132
>0.05
<0.05
<0.03
>0.05
<0.04
<0.02
>0.05
>0.05
<0.03
'International Classification of Diseases.
*SMR values were corrected by Cooper and Gaffey for missing death certificates under the
assumption that distribution of causes of death was the same in missing certificates as in
those that were obtained.
**0bserved deaths were recalculated as follows: adjusted observed deaths = (given SMR/100) x
expected deaths.
***Given z = (SMR - 100) Vexpected/100.
****Given z = (SMR - 100)A/100 x SMR/expected.
Source: Rang et al. (1980).
Cooper and Gaffey (1975) did not discuss types of lead compounds that these workers may
have been exposed to in smelting operations, but workers thus employed likely ingested or
inhaled oxides and sulfides of lead. Since these and other lead compounds produced in the in-
dustrial setting are not readily soluble in water it could be that the cancers arising in res-
piratory or gastrointestinal systems were caused by exposure to water-insoluble lead com-
pounds. Although the Cooper and Gaffey (1975) study had a large sample (7032), only 2275 of
the workers (32.4 percent) were employed when plants monitored urinary lead. Urinary lead
values were available for only 9.7 percent of the 1356 deceased employees on whom the cancer
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PRELIMINARY DRAFT
mortality data were based. Only 23 (2 percent) of the 1356 decedents had blood lead levels
measured. Cooper and Gaffey (1975) did report some average urinary and blood lead levels,
where 10 or more urine or at least three blood samples were taken (viz., battery plant
workers: urine lead = 129 ug/1, blood lead = 67 ug/dl; smelter workers: urine lead = 73
ug/1, blood lead = 79.7 ug/dl). Cooper (1976) noted that these workers were potentially
exposed to other materials, including arsenic, cadmium, and sulfur dioxide, although no data
on such exposures were reported. In these and other epidemiological studies in which selec-
tion of subjects for monitoring exposure to an agent such as lead is left to company discre-
tion, it is possible that individual subjects are selected primarily on the basis of obvious
signs of lead exposure, while other individuals who show no symptoms of lead poisoning would
not be monitored (Cooper and Gaffey, 1975). It is also not clear from these studies when the
lead levels were measured, although the timing of measurement would make little difference
since no attempt was made to match an individual's lead exposure to any disease process.
In a follow-up study of the same population of workers, Cooper (1981) concluded that lead
had no significant role in the induction of neoplasia. However, he did report standardized
mortality ratios (SMRs) of 149 percent and 125 percent for all types of malignant neoplasms in
lead battery plant workers with < 10 and > 10 years of employment, respectively. SMR is a
percentage value that is based upon comparison of an exposed population relative to a control
population. If the value exceeds 100 percent, the incidence of death is greater than normal
but not necessarily statistically significant. In battery workers employed for 10 years or
more there was an unusually high incidence of cancer listed as "other site" tumors (SMR = 229
percent; expected = 4.85, observed = 16). Respiratory cancers were elevated in the battery
plant workers employed for less than 10 years (SMR = 172 percent). Similarly, in workers in-
volved with lead production facilities for more than 10 years the SMR was 151 percent. Again,
in the absence of good lead exposure documentation, it is difficult to assess the contribution
of lead to the observed results. Cooper (1981) suggested that the excess of respiratory
cancers could have been due to a lack of correction for smoking histories.
A recent study (McMichael and Johnson, 1982) examined the historical incidence of cancers
in a population of smelter workers diagnosed as having lead poisoning. The incidence of can-
cer in a relatively small group of 241 workers was compared with 695 deceased employees from
the same company. The control group had been employed during approximately the same period
and was asserted to be free from lead exposure, although there were no data to indicate lead
levels in either the control or the experimental group. Based upon diagnoses of lead poison-
ing made in the 1920s and 1930s for a majority of the deaths, the authors concluded that there
was a considerably lower incidence of cancer in lead-poisoned workers. However, there is no
indication of how lead poisoning was diagnosed. It is difficult to draw any conclusions from
this study with regard to the role of lead in human neoplasia.
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PRELIMINARY DRAFT
Evaluation of the ability of lead to induce human neoplasia must await further epidemio-
logical studies in which other factors that may contribute to the observed effects are well
controlled for and the disease process is assessed in individuals with well documented expo-
sure histories. Little can now be reliably concluded from available epidemiological studies.
12.7.2.2 Induction of Tumors in Experimental Animals. As discussed in the preceding sections
it is difficult to obtain conclusive evidence of the carcinogenic potential of an agent using
only epidemiological studies. Experiments testing the ability of lead to cause cancer in
experimental animals are an essential aspect of understanding its oncogenicity in humans.
However, a proper lifetime animal feeding study to assess the carcinogenic potential of lead
following National Cancer Institute guidelines (Sontag et al., 1976) has not been conducted.
The cost of such studies exceed $1 million and consequently are limited only to those agents
in which sufficient evidence based upon iji vitro or epidemiological studies warrants such an
undertaking. The literature on lead carcinogenesis contains many smaller studies where only
one or two doses were employed and where toxicological monitoring of experimental animals ex-
posed to lead was generally absent. Some of these studies are summarized in Table 12-18.
Most mainly serve to illustrate that numerous different laboratories have induced rvenal tumors
in rats by feeding them diets containing 0.1 percent or 1.0 percent lead acetate. In some
cases, other lead formulations were tested, but the dosage selection was not based upon lethal
dose values. In most cases, only one dose level was used. Another problem with many of these
studies was that the actual concentrations of lead administered and internal body burdens
achieved were not measured. Some of these studies are discussed very briefly; others are
omitted entirely because they contribute little to our understanding of lead carcinogenesis.
Administration of 1.0 percent lead acetate (10,000 ppm) resulted in kidney damage and a
high incidence of mortality in most of the studies in Table 12-18. However, kidney tumors
were also evident at lower dosages (e.g., 0.1 percent lead acetate in the diet), which pro-
duced less mortality among the test animals. As discussed in Section 12.5, renal damage is
one of the primary toxic effects of lead. At 0.1 percent lead acetate (1000 ppm) in the diet,
the concentration of lead measured in the kidney was 30 ug/g while 1 percent lead acetate
resulted in 300 ug/g of lead in the kidneys of necropsied animals (Azar et al., 1973). In
most of the studies with rats fed 0.1 or 1.0 percent lead in the diet, the incidence of kidney
tumors increased between the lower and higher dosage, suggesting a relationship between the
deposition of lead in the kidney and the carcinogenic response. Renal tumors were also
induced in mice at the 0.1 percent oral dosage of lead subacetate but not in hamsters that
were similarly exposed to this agent (Table 12-18).
Other lead compounds have also been tested in experimental animals, but in these studies
only one or two dosages (generally quite high) were employed, making it difficult to assess
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PRELIMINARY DRAFT
TABLE 12-18. EXAMPLES OF STUDIES ON THE INCIDENCE OF TUMORS IN EXPERIMENTAL
ANIMALS EXPOSED TO LEAD COMPOUNDS
Species
Rat
Rat
Rat
Mouse
Rat
Rat
Rat
Mouse
Rat
Hamster
Mouse
Rat
Rat
Pb compound
Pb phosphate
Pb acetate
Pb
subacetate
Pb
naphthenate
Pb phosphate
Pb
subacetate
Pb
subacetate
Tetraethyl
lead in
tricaprylin
Pb acetate
Pb
subacetate
Pb
subacetate
Pb nitrate
Pb acetate
Dose and mode
120-680 mg
(total dose s.c. )
1% (in diet)
0.1% and
1.0% (in diet)
20% in benzene
(dermal 1-2
times weekly)
1.3 g (total
dosage s.c. )
0.5-1%
(in diet)
1% (in diet)
0.6 mg (s.c. )
4 doses between
birth and 21 days
3 mg/day for
2 months;
4 mg/day for
16 months (p.o. )
1.0% (in
0.5% diet)
0.1% and
1.0% (in diet)
25 g/1 in
drinking water
3 mg/day (p.o.)
Incidence (and type) of
neoplasms
19/29 (renal tumors)
15/16 (kidney tumors)
14/16 (renal carcinomas)
11/32 (renal tumors)
13/24 (renal tumors)
5/59 (renal neoplasms)
(no control with
benzene)
29/80 (renal tumors)
14/24 (renal tumors)
31/40 (renal tumors)
5/41 (lymphomas)
in females, 1/26 in
males, and 1/39 in
controls
72/126 (renal tumors)
23/94 males (testicular
[Leydig cell] tumors)
No significant incidence
of renal neoplasms
7/25 (renal carcinomas)
at 0.1%
Substantial death at 1.0%
No significant incidence
of tumors
89/94 (renal, pituitary,
cerebral gliomas,
adrenal, thyroid, pro-
Reference
Zollinger
(1953)
Boyland et
al. (1962)
Van Esch
et al. (1962)
Baldwin et
al. (1964)
Balo et al.
(1965)
Mass et al.
(1967)
Mao and
Molnar (1967)
Epstein and
Mantel (1968)
Zawirska and
Medras' (1968)
Van Esch and
Kroes (1969)
Van Esch and
Kroes (1969)
Schroeder et
al. (1970)
Zawirska
and
Medras, 1972
static, mammary tumors)
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PRELIMINARY DRAFT
TABLE 12-18. (continued)
Species Pb Compound Dose and mode
Incidence (and type) of
neoplasms
Reference
Rat
Pb acetate
Hamster
Rat
Pb oxide
Pb powder
0, 10, 50, 100,
1000, 2000 ppm
(in diet) for
2 yr
10 intratracheal
administrations
U mg)
10 mg orally 2 times
each month
10 mg/monthly
for 9 months;
then 3 monthly
injections of 5 mg
No tumors 0-100 ppm; Azar et al.
5/50 (renal tumors) at (1973)
500 ppm; 10/20 at 1000 ppm;
16/20 males, 7/20 females
at 2000 ppm
0/30 without benzopyrene,
12/30 with benzopyrene
(lung cancers)
5/47 (1 lymphoma,
4 leukemias)
1/50 (fibrosarcoma)
Kobayashi
and
Okamoto (1974)
Furst et al.
(1976)
the potential carcinogenic activity of lead compounds at relatively nontoxic concentrations.
It is also difficult to assess the true toxicity caused by these agents, since properly
designed toxicity studies were generally not performed in parallel with these cancer studies.
As shown in Table 12-18, lead nitrate produced no tumors in rats when given at very low
concentrations, but lead phosphate administered subcutaneously at relatively high levels in-
duced a high incidence of rsnal tumors in two studies. Lead powder administered orally
resulted in lymphomas and leukemia; when given intramuscularly only one fibrosarcoma was pro-
duced in 50 animals. Lead naphthenate applied as a 20 percent solution in benzene two times
each week for 12 months resulted in the development of four adenomas and one renal carcinoma
in a group of 50 mice (Baldwin et al., 1964). However, in this study control mice were not
painted with benzene. Tetraethyl lead at 0.6 mg given in four divided doses between birth and
21 days to female mice resulted in 5/36 surviving animals developing lymphomas while 1/26
males treated similarly and 1/39 controls developed lymphomas (Epstein and Mantel, 1968).
Lead subacetate has also been tested in the mouse lung adenoma bioassay (Stoner et al.,
1976). This assay measures the incidence of nodules forming in the lung of strain A/Strong
mice following parenteral administration of various test agents. Nodule formation in the lung
does not actually represent the induction of lung cancer but merely serves as a general meas-
ure of carcinogenic potency independent of lung tissue (Stoner et al., 1976). Lead subacetate
was administered to mice at 150, 75, and 30 mg (total dose), which represented the maximum
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PRELIMINARY DRAFT
tolerated dose (MTD), 1/2 MTD, and 1/5 MTD, respectively, over a 30-week period using 15 sepa-
rate i.p. injections (Stoner et a!., 1976). Survivals at the three doses were 15/20 (MTD),
12/20 (1/2 MTD), and 17/20 (1/5 MTD), respectively, with 11/15, 5/12, and 6/17 survivors
having lung nodules. Only at the highest doses was the incidence of nodules greater than in
the untreated 1 or 2 highest groups. However, these authors concluded that on a molar-dose
basis lead subacetate was the most potent of all the metallic compounds examined. Injection
of 0.13 mmol/kg lead subacetate was required to produce one lung tumor per mouse, indicating
that this compound was about three times more potent than urethane (at 0.5 mmol/kg) and
approximately 10 times more potent than nickelous acetate (at 1.15 mmol/kg). The mouse lung
adenoma bioassay has been one of the most utilized systems for examining carcinogenic activity
in experimental animals and is well recognized as a highly accurate test system for assessing
potential carcinogenic hazard (Stoner et al., 1976). Lead oxide combined with benzopyrene
administered intratracheally resulted in 11 adenomas and 1 adenocarcinoma in a group of 15
hamsters, while no lung neoplasias were observed in groups receiving benzopyrene or lead oxide
alone (Kobayashi and Okamoto, 1974).
Administration of lead acetate to rats has been reported to produce other types of
tumors, e.g., testicular, adrenal, thyroid, pituitary, prostate, lung (Zawirska and Medras,
1968), and cerebral gliomas (Oyasu et al., 1970). However, in other animal species, such as
dogs (Azar et al., 1973; Fouts and Page, 1942) and hamsters (Van Esch and Kroes, 1969), lead
acetate induced either no tumors or only kidney tumors (Table 12-18).
The above studies seem to implicate some lead compounds as carcinogens in experimental
animals but were not designed to address the question of lead carcinogenesis in a definitive
manner. In contrast, a study by Azar et al. (1973) examined the oncogenic potential of lead
acetate at a number of doses and in addition monitored a number of toxicological parameters in
the experimental animals. Azar et al. (1973) gave 0, 10, 50, 100, 1000 and 2000 ppm dose
levels of lead (as lead acetate) to rats during a two-year feeding study. Fifty rats of each
sex were utilized at doses of 10 to 500 ppm, while 100 animals of each sex were used as con-
trols. After the study was under way for a few months, a second 2-year feeding study was ini-
tiated using 20 animals of each sex in groups given doses of 0, 1000, or 2000 ppm. The study
also included four male and four female beagle dogs at each dosage of lead ranging from 0 to
500 ppm in a 2-year feeding study. During this study, the clinical appearance and behavior of
the animals were observed, and food consumption, growth, and mortality were recorded. Blood,
urine, fecal, and tissue lead analyses were done periodically using atomic absorption spectro-
photometry. A complete blood analysis was done periodically, including blood count, hemo-
globin, hematocrit, stippled cell count, prothrombin time, alkaline phosphatase, urea nitro-
gen, glutamic-pyruvate transaminase, and albumin-to-globulin ratio. The activity of the
enzyme alpha-ami no!evulinic acid dehydrase (ALA-D) in the blood and the excretion of its sub-
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PRELIMINARY DRAFT
strate, delta-aminolevulinic acid (6-ALA) in the urine were also determined. A thorough ne-
cropsy, including both gross and histologic examination, was performed on all animals. Repro-
duction was also assessed (see Section 12.6).
Table 12-19 depicts the mortality and incidence of kidney tumors reported by Azar et al.
(1973). At 500 ppm (0.05 percent) and above, male rats developed a significant number of re-
nal tumors. Female rats did not develop tumors except when fed 2000 ppm lead acetate. Two
out of four male dogs fed 500 ppm developed a slight degree of cytomegaly in the proximal con-
voluted tubule but did not develop any kidney tumors. The number of stippled red blood cells
increased at 10 ppm in the rats but not until 500 ppm in the dogs. ALA-D was decreased at 50
ppm in the rats but not until 100 ppm in the dogs. Hemoglobin and hematocrit, however, were
not depressed in the rats until they received a dose of 1000 ppm lead. These results illus-
trate that the induction of kidney tumors coincides with moderate to severe toxicological
doses of lead acetate, for it was at 500-1000 ppm lead in the diet that a significant
increase in mortality occurred (see Table 12-19). At 1000 and 2000 ppm lead, 21-day-old wean-
ling rats showed no tumors but did show histological changes in the kidney comparable to those
seen in adults receiving 500 ppm or more lead in their diet. Also of interest from the Azar
et al. (1973) study is the direct correlation obtained in dogs between blood lead level and
kidney lead concentrations. A dietary lead level of 500 ppm produced a blood lead concentra-
tion of 80 ug/dl, which corresponds to a level at which humans often show clinical signs of
lead poisoning (see Section 12.4.1). The kidney lead concentration corresponding to this
blood lead level was 2.5 ug/g (wet weight), while at 50 ug/dl in blood the kidney lead levels
were 1.5 ug/g. Presumably blood and kidney lead were determined at about the same time,
although this was not clear from the report. At this level of lead, kidney tumors were in-
duced in the rats but not the dogs. However, it is apparent from the above differences in
hematological parameters that dogs tolerate higher levels of lead than rats. As shown in
Figure 12-5, the induction of renal tumors by lead acetate was linearly proportional to the
dietary levels of lead fed to male rats. It may be concluded, therefore, that chronic lead
exposure of rats producing blood lead levels comparable to those at which clinical signs of
toxicity would be evident in humans results in a significant elevation in the incidence of
kidney tumors.
Animal carcinogenesis studies conducted with lead and its compounds are numerous; how-
ever, with the exception of the study by Azar et al., (1973) they do not provide much useful
information. Most of the studies shown in Table 12-18 were conducted with only one lead com-
pound in one animal species, the rat. In cases where other lead compounds were tested or where
other animal species were used, only a single high dosage level was administered, and para-
meters of toxicity such as those monitored in the Azar et al. (1973) study were not measured.
Although it is clear from these studies as a whole that lead is a carcinogen in experimental
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PRELIMINARY DRAFT
TABLE 12-19. MORTALITY AND KIDNEY TUMORS IN RATS FED LEAD ACETATE FOR TWO YEARS
Nominal (actual)3
concentration in
ppm of Pb in diet
0 (5)
10 (18)
50 (62)
100 (141)
500 (548)
0 (3)
1000 (1130)
2000 (2102)
No. of rats
of each sex
100
50
50
50
50
20
20
20
% Mortal
Male
37
36
36
36
52
50
50
80
ityb
Female
34
30
28
28
36
35
50
35
% Kidney
Male
0
0
0
0
10
0
50
80
tumors
Female
0
0
0
0
0
0
0
35
Measured concentration of lead in diet.
Includes rats that either died or were sacrificed iji extremis.
Source: Azar et al. (1973).
animals, until more investigations such as that of Azar et al. (1973) are conducted it is
difficult to determine the relative carcinogenic potency of lead. There remains a need to
test thoroughly the carcinogenic activity of lead compounds in experimental animals. These
tests should include several modes of administration, many dosages spanning non-toxic as well
as toxic levels, and several different lead compounds or at least a comparison of a relatively
water-soluble form such as lead acetate with a less soluble form such as lead oxide.
12.7.2.3 Cell Transformation. Although cell transformation is an jm vitro experimental
system, its end point is a neoplastic change. There are two types of cell transformation
assays: (1) those employing continuous cell lines, and (2) those employing cell cultures pre-
pared from embryonic tissue. Use of continuous cell lines has the advantage of ease in prepa-
ration of the cell cultures, but these cells generally have some properties of a cancer cell.
The absence of a few characteristics of a cancer cell in these continuous cell lines allows
for an assay of cell transforming activity. End points include morphological transformation
(ordered cell growth to disordered cell growth), ability to form colonies in soft agar-con-
taining medium (a property characteristic of cancer cells), and ability of cells to form
tumors when inoculated into experimental animals. Assays that utilize freshly isolated embry-
onic cells are generally preferred to those that use cell lines, because embryonic cells have
not yet acquired any of the characteristics of a transformed cell. The cell transformation
assay system has been utilized to examine the potential carcinogenic activity of a number of
chemical agents, and the results seem to agree generally with the results of carcinogenesis
CPB12/A 12-185 9/20/83
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0.1
0.2
0.5 1 2 5
DIETARY LEAD, 103 ppm
Figure 12-5. Probit plot of incidence of renal tumors in male rats.
Source: U.S. Environmental Protection Agency (1980) based on
Azar et al. (1973).
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PRELIMINARY DRAFT
tests using experimental animals. Cell transformation assays can be made quantitative by
assessing the percentage of surviving colonies exhibiting morphological transformation.
Verification of a neoplastic change can be accomplished by cloning these cells and testing
their ability to form tumors in animals.
Lead acetate has been shown to induce morphological transformation in Syrian hamster em-
bryo cells following a continuous exposure to 1 or 2.5 ug/ml of lead in culture medium for
nine days (Dipaolo et al., 1978). The incidence of transformation increased from 0 percent in
untreated cells to 2.0 and 6.0 percent of the surviving cells, respectively, following treat-
ment with lead acetate. Morphologically transformed cells were capable of forming fibrosarco-
mas when cloned and administered to "nude" mice and Syrian hamsters, while no tumor growth
resulted from similar inoculation with untreated cells (Dipaolo et al., 1978). In the same
study lead acetate was shown to enhance the incidence of simian adenovirus (SA-7) induction of
Syrian hamster embryo cell transformation. Lead acetate also caused significant enhancement
(-2-3 fold) at 100 and 200 ug/ml following three hours of exposure. In another study (Casto
et al., 1979), lead oxide also enhanced SA-7 transformation of Syrian hamster embryo cells
almost 4 fold at 50 uM following three hours of exposure (Casto et al., 1979). The signifi-
cance of enhanced virally induced carcinogenesis in relationship to the carcinogenic potential
of an agent is not well understood.
Morphological transformation induced by lead acetate was correlated with the ability of
the transformed cells to form tumors in appropriate hosts (see above), indicating that a truly
neoplastic change occurred in tissue culture. The induction of neoplastic transformation by
lead acetate suggests that this agent is potentially carcinogenic at the cellular level. How-
ever, with jjn vitro systems such as the cell transformation assay it is essential to compare
the effects of other, similar types of carcinogenic agents in order to evaluate the response
and to determine the reliability of the assay. The incidence of transformation obtained with
lead acetate was greater than the incidence following similar exposure to NiCl2, but less than
that produced by CaCr04 (Heck and Costa, 1982a). Both nickel and chromium have been impli-
cated in the etiology of human cancer (Costa, 1980). These results thus suggest that lead
acetate has effects similar to those caused by other metal carcinogens. In particular, the
ability of lead acetate to induce neoplastic transformation in cells in a concentration-depen-
dent manner is highly suggestive of potential carcinogenic activity. It should also be noted
that lead acetate induced these transformations at concentrations that decreased cell survival
by only 27 percent (Heck and Costa, 1982a). Further studies from other laboratories utilizing
the cell transformation assay and other lead compounds are needed.
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12.7.3 Genotoxicity of Lead
Since cancer is krtown to be a disease of altered gene expression, numerous studies have
evaluated changes in DMA consequent to exposure to suspected carcinogenic agents. The general
response associated with such alterations in regulation of DNA function has been called geno-
toxicity. Various assay systems developed to examine specific changes in DNA structure and
function caused by carcinogenic agents include assays that evaluate chromosomal aberrations,
sister chromatid exchange, mutagenicity, and functional and structural features of DNA meta-
bolism. Lead effects on these parameters are discussed below.
12.7.3.1 Chromosomal Aberrations. Two approaches have been used in the analysis of effects
of lead on chromosomal structure. The first approach involves culturing lymphocytes either
from humans exposed to lead or from experimental animals given a certain dosage of lead. The
second approach involves exposing cultured lymphocytes directly to lead. For present pur-
poses, emphasis will not be placed on the type of chromosomal aberration induced, since most
of the available studies do not appear to associate any specific type of chromosomal aberra-
tion with lead exposure. It should be noted, however, that moderate aberrations include gaps
and fragments, whereas severe aberrations include dicentric rings, trans locations, and ex-
changes. Little is known of the significance of chromosomal aberrations in relationship to
cancer, except that in a number of instances genetic diseases associated with chromosomal
aberrations often enhance the probability of neoplastic disease. However, implicit in a mor-
phologically distinct change in genetic structure is the possibility of an alteration in gene
expression that represents a salient feature of neoplastic disease.
Contradictory reports exist regarding lead effects in inducing chromosomal aberrations
(Tables 12-20 and 12-21). These studies have been grouped in two separate tables based upon
their conclusions. Those studies reporting a positive effect of lead on chromosomal aberra-
tions are indexed in Table 12-20, whereas studies reporting no association between lead expo-
sure and chromosomal aberrations are indexed in Table 12-21. Unfortunately, these studies are
difficult to evaluate fully because of many unknown variables (e.g., absence of sufficient
evidence of lead intoxication, no dose-response relationship, and absence of information
regarding lymphocyte culture time). To illustrate, in a number of the studies where lead ex-
posure correlated with an increased incidence of chromosomal aberrations (Table 12-20), lym-
phocytes were cultured for 72 hours. Most cytogenetic studies have been conducted with a
maximum culture time of 48 hours to avoid high background levels of chromosomal aberrations
due to multiple cell divisions during culture. Therefore, it is possible that the positive
effects of lead on chromosomal aberrations may have been due to the longer culture period.
Nonetheless, it is evident that in the negative studies the blood lead concentration was gen-
erally lower than In the studies reporting a positive effect of lead on chromosomal aberra-
tions, although in many of the latter instances blood lead levels indicated severe exposure.
CPB12/A 12-188 9/20/83
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TABLE 12-20. CYTOGENETIC INVESTIGATIONS OF CELLS FROM INDIVIDUALS EXPOSED TO LEAD: POSITIVE STUDIES
Number of
exposed Number of
subjects control s
8 14
10 10
14 5
105
—
NJ
j
CO
^ 11 (before
and after ex-
posure)
44 15
23 20
20 20
26 (4 low, not
16 medium, given
6 high ex-
posure)
12 18
Cell Blood (ug/dl)
culture or urine
time (hrs.) (ug/1) level
? 62. -89.
(blood)
72 60. -100.
(blood)
48 155-720
(urine)
72 11.6-97.4
mean, 37.7
(blood)
6S-70 34. -64.
(blood)
72 30. -75.
(blood)
48 44. -95.
(not given)
46-48 53. -100.
(blood)
72 22.5-65.
(blood)
48-72 24-49
(blood)
Exposed
subjects
Workers in a lead
oxide factory
Workers In a chem-
ical factory
Workers in a zinc
plant, exposed to
fumes & dust of
cadmium, zinc &
lead
Blast-furnace work-
ers, metal grin-
ders, scrap metal
processers
Workers in a
lead- acid battery
plant and a lead
foundry
Individuals in a
lead oxide fac-
tory
Lead-acid battery
Belters, tin workers
Ceramic, lead &
Battery workers
Smelter workers
Electrical storage
battery workers
Type of
damage
Chromatid and
chromosome
Chromatid gaps,
breaks
Gaps, fragments.
exchanges, dicen-
trics, rings
"Structural ab-
normalities,"
gaps , breaks ,
hyperploidy
Gaps, breaks.
fragments
Chromatid and
chromosome
aberrations
Dicentrics,
rings, fragments
Breaks, frag-
ments
Gaps, chroma-
tid and chro-
mosome aberra-
tions
Chromatid and
chromosome aberra-
tions
Remarks
Increase in
chromosomal damage
correlated with
increased 6-ALA
excretion
No correlation with
blood lead levels
Thought to be caused
by lead, not cadmium
or zinc
No correlation with
6-ALA excretion or
blood lead levels
No correlation
with ALA-D activity
in red cells
Positive correlation
with length of expo-
sure
Factors other than
lead exposure may be
required for severe
aberrations
Positive correlation
witn blood lead levels
Positive correlation
with blood lead levels
References
Schwanitz et al.
(1970)
Gath & Thiess
(1972)
Deknudt et
al. (1973)
Schwanitz et
al. (1975)
Forni et al .
(1976)
Garza-Chapa
et al. (1977)
Deknudt et al.
(1977b)
Sarto et al.
(1978)
Nordenson et al .
(1978)
Forni et al . (1980)
-o
JO
m
»— i
3:
•z.
•30
3O
-n
— i
Source: International Agency for Research on Cancer (1980), with modifications.
-------
TABLE 12-21. CYTOGENETIC INVESTIGATIONS OF CELLS FROM INDIVIDUALS EXPOSED TO LEAD: NEGATIVE STUDIES
Number of Number of
exposed subjects controls
29 20
32 20
35 35
24 15
9 9
30 20
Cell culture Blood lead
time (hrs.) level (ug/dl)
46-48 Not given, stated
to be 20-30%
higher than controls
46-48 Range not given;
highest level was
590 mg/1 [sic]
45-48 Control, <4. ; ex-
posed, 4. - >12.
48 19.3 (lead)
0.4 (cadmium)
72 40.0 ±5.0, 7
weeks
48 Control, 11.8-13.2;
exposed, 29-33
Exposed subjects
Policemen "permanently in
contact with high levels of
automotive exhaust"
Workers in lead manufacturing
industry; 3 had acute lead
intoxication
Shipyard workers employed as
"burners" cutting metal struc-
tures on ships
Nixed exposure to zinc, lead,
and cadmium in a zinc-smelting
plant; significant increase in
chromatid breaks and exchanges.
Authors suggest that cadmium
was the major cause of this
damage
Volunteers ingested capsules
containing lead acetate
Children living near a lead
smelter
References
Bauchinger et al.
(1972)
Schmid et al. (1972)
O'Riordan and Evans
(1974)
Bauchinger et al.
(1976)
Bulsma & De France
(1976)
Bauchinger et al.
(1977)
m
t— 4
2
»— 1
•z.
o
•30
>
-n
— (
Source: International Agency for Research on Cancer (1980).
-------
PRELIMINARY DRAFT
In some of these positive studies there was a correlation in the incidence of gaps, fragments,
chromatid exchanges, and other chromosomal aberrations with blood lead levels (Sarto et al.,
1978; Nordenson et al., 1978). However, as indicated in Table 12-20, in other studies there
were no direct correlations between indices of lead exposure (i.e., 6-ALA excretion) and
numbers of chromosomal aberrations. Nutritional factors such as Ca levels iji vivo or rn
vitro are also important since it is possible that the effects of lead on cells may be antag-
2-t-
onized by Ca (Mahaffey, 1983). As is usually the case in studies of human populations ex-
posed to lead, exposure to other metals (zinc, cadmium, and copper) that may produce chromoso-
mal aberrations was prevalent. None of the studies attempted to determine the specific lead
compound that the individuals were exposed to.
In a more recent study by Form" et al. (1980), 18 healthy females occupationally exposed
to lead were evaluated for chromosomal aberrations in their lymphocytes cultured for 48 or 72
hours. There were more aberrations at the 72-hour culture time compared with the 48-hour cul-
ture period in both control and lead-exposed groups, but this difference was not statistically
significant. However, statistically significant differences from the 72-hour controls were
noted in the 72-hour culture obtained from the lead exposed group. These results demonstrate
that the extended 72-hour culture time results in increased chromosomal aberrations in the
control lymphocytes and that the longer culture time was apparently necessary to detect the
effects of lead on chromosomal structure. However, the blood lead levels in the exposed fe-
males ranged from 24 to 59 pg/dl, while control females had blood lead levels ranging from 22
to 37 ug/dl. Thus, there was a marginal effect of lead on chromosomal aberration, but the two
groups may not have been sufficiently different in their lead exposure to show clear differ-
ences in frequency of chromosomal aberrations.
Some studies have also been conducted on the direct effect of soluble lead salts on cul-
tured human lymphocytes. In a study by Beek and Obe (1974), longer (72-hr) culture time was
used and lead acetate was found to induce chromosomal aberrations at 100 uM. Lead acetate had
no effect on chromatid aberrations induced with X-rays or alkylating agents (Beek and Obe,
1975). In another study (Deknudt and Deminatti, 1978), lead acetate at 1 and 0.1 mM caused
minimal chromosomal aberrations. Both cadmium chloride (CdCl2) and zinc chloride (ZnCl2) were
more potent than lead acetate in causing these changes; however, both CdCl2 and ZnCl2 also
displayed greater toxicity than lead acetate.
Chromosomal aberrations have been demonstrated in lymphocytes from cynomolgus monkeys
treated chronically with lead acetate (6 mg/day, 6 days/week for 16 months), particularly when
they were kept on a low calcium diet (Deknudt et al., 1977a). These aberrations accompanying a
2+
low Ca diet were characterized by the authors as severe (chromatid exchanges, dispiraliza-
tion, translocations, rings, and polycentric chromosomes). Similar results were observed in
mice (Deknudt and Gerber, 1979). The effect of low calcium on chromosomal aberrations induced
CPB12/A 12-191 9/20/83
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PRELIMINARY DRAFT
2+ 2+
by lead is most likely due to interaction of Ca and Pb at the level of the chromosome
(Mahaffey, 1983). Leonard and his coworkers found no effect of lead on the incidence of
chromosomal aberrations in accidentally intoxicated cattle (Leonard et al., 1974) or in mice
given 1 gram of lead per liter of drinking water for 9 months (Leonard et al., 1973). How-
ever, Muro and Goyer (1969) found gaps and chromatid aberrations in bone marrow cells cultured
for four days after isolation from mice that had been maintained on 1 percent dietary lead
acetate for two weeks. Chromosomal loss has been reported (Ahlbert et al. , 1972) in Droso-
phila exposed to triethyl lead (4 mg/1), but inorganic lead had no effect (Ramel, 1973). Lead
acetate has also been shown to induce chromosomal aberrations in cultured cells other than
lymphocytes, viz. Chinese hamster ovary cells (Bauchinger and Schmid, 1972).
These studies demonstrate that under certain conditions lead compounds are capable of in-
ducing chromosomal aberrations _in vivo and in tissue cultures. The ability of lead to induce
these chromosomal changes appears to be concentration-dependent and highly influenced by cal-
cium levels. In lymphocytes isolated from patients or experimental animals, relatively long
(72-hr) culture conditions are required for the abnormalities to be expressed.
Sister chromatid exchange represents the normal movement of DNA in the genome. The sister
chromatid exchange assay offers a very sensitive probe for the effects of genotoxic compounds
on DNA rearrangement, as a number of chemicals with carcinogenic activity are capable of in-
creasing these exchanges (Sandberg, 1982). The effect of lead on such movement has been exam-
ined in cultured lymphocytes (Beek and Obe, 1975), with no increase in exchanges observed at
lead acetate concentrations of 0.01 mM. However, one study with lead at one dose in one sys-
tem is not sufficient to rule out whether lead increases the incidence of these exchanges.
The ability of agents such as lead to cause abnormal rearrangements in the structure of
DNA, as revealed by the appearance of chromosomal aberrations, and sister chromatid exchanges
has become an important focus in carcinogenesis research. Current theories suggest that can-
cer may result from an abnormal expression of oncogenes (genes that code for protein products
associated with virally induced cancers). Numerous oncogenes are found in normal human DNA,
but the genes are regulated such that they are not expressed in an carcinogenic fashion.
Rearrangement of these DNA sequences within the genome can lead to oncogenic expression. Evi-
dence has been presented suggesting that chromosomal aberrations such as translocations are
associated with certain forms of cancer and with the movement of oncogenes in regions of the
DNA favoring their expression in cancer cells (Shen-Ong et al. , 1982). By inducing aberra-
tions in chromosomal structure, lead may enhance the probability of an oncogenic event.
12.7.3.2 Lead Effects on Bacterial and Mammalian Mutagenesis Systems. Bacterial and mamma-
lian mutagenesis test systems examine the ability of chemical agents to induce changes in DNA
sequences of a specific gene product that is monitored by selection procedures. They measure
the potential of a chemical agent to produce a change in DNA, but this change is not likely to
CPB12/A 12-192 9/20/83
-------
PRELIMINARY DRAFT
be the same alteration in gene expression that occurs during oncogenesis. However, if an
agent affects the expression of a particular gene product that is being monitored, then it
could possibly affect other sequences which may result in cancer. Since many carcinogens are
also mutagens, it is useful to employ such systems to evaluate genotoxic effects of lead.
Use of bacterial systems for assaying metal genotoxicity must await further development
of bacterial strains that are appropriately responsive to known mutagenic metals (Rosenkranz
and Poirier, 1979; Simmon, 1979; Simmon et al., 1979; Nishioka, 1975; Nestmann et al., 1979).
Mammalian cell mutagenic systems that screen for specific alterations in a defined gene muta-
tion have not been useful in detecting mutagenic activity with known carcinogenic metals (Heck
and Costa, 1982b). In plants, however, chromosomal aberrations in root tips (Mukherji and
Maitra, 1976) and other mutagenic activity, such as chlorophyll mutations (Reddy and Vaidya-
nath, 1978), have been demonstrated with lead.
12.7.3.3 Lead Effects on Parameters of DNA Structure and Function. There are a number of
very sensitive techniques for examining the effect of metals on DNA structure and function in
intact cells. Although these techniques have not been extensively utilized with respect to
metal compounds, future research will probably be devoted to this area. Considerable work has
been done to understand the effects of metals on enzymes involved in DNA transcription.
Si rover and Loeb (1976) examined effects of lead and other metal compounds upon the
fidelity of transcription of DNA by a viral ONA polymerase. High concentrations of metal ions
(in some cases in the millimolar range) were required to decrease the fidelity of transcrip-
tion, but there was a good correlation between metal ions that are carcinogenic or mutagenic
and their activity in decreasing the fidelity of transcription. This assay system measures
the ability of a metal ion to incorporate incorrect (non-homologous) bases using a defined
polynucleotide template. In an intact cell, this would cause the induction of a mutation if
the insertion of an incorrect base is phenotypically expressed. Since the interaction of
metal ions with cellular macromolecules is relatively unstable, misincorporation of a base
during semi-conservative DNA replication or during DNA repair synthesis following breakage of
DNA with a metal could alter the base sequence of DNA in an intact cell. Lead at 4 mM was
among the metals listed as mutagenic or carcinogenic that caused a decrease in the fidelity of
transcription (Sirover and Loeb, 1976). Other metals active in decreasing fidelity included:
+ 2+ 2+ 2+ 2+ 3+ 2+ 2+ 2+
Ag , Be , Cd , Co , Cr , Cr , Cu , Mn , and Ni . No change in fidelity was produced
by Al *, Ba +, Ca +, Fe *, K*. Rb+, Mg2+, Hg+, Se2"*", Sr2+, and Zn +. Metals that decreased
fidelity are metals also implicated as carcinogenic or mutagenic (Sirover and Loeb, 1976).
In a similar study, Hoffman and Niyogi (1977) demonstrated that lead chloride was the
most potent of 10 metals tested in inhibiting RNA synthesis (i.e., Pb * > Cd + > Co * > Mn + >
Li* > Na > K ) for both types of templates tested, i.e., calf thymus DNA and T4 phage DNA.
CPB12/A 12-193 9/20/83
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PRELIMINARY DRAFT
These results were explained in terms of the binding of these metal ions more to the bases
2+ 2+ 2+ 2+ 2+ + .
than to the phosphate groups of the DMA (i.e., Pb > Cd > Zn > Mn > Mg > Li = Na =
K ). Additionally, metal compounds such as lead chloride with carcinogenic or mutagenic
activity were found to stimulate mRNA chain initiation at 0.1 mM concentrations.
These well-conducted mechanistic studies provide evidence that lead can affect a molecu-
lar process associated with normal regulation of gene expression. Although far removed from
the intact cell situation, these effects suggest that lead may be genotoxic.
12.7.4 Summary and Conclusions
It is evident from studies reviewed above that, at relatively high concentrations, lead
displays some carcinogenic activity in experimental animals (e.g. the rat). An agent may act
as a carcinogen in two distinct ways: (1) as an initiator or (2) as a promoter (Weisburger
and Williams, 1980). By definition, an initiator must be able to interact with DMA to produce
a genetic alteration, whereas a promoter acts in a way that allows the expression of an
altered genetic change responsible for cancer. Since lead is capable of transforming cells
directly in culture and affecting DNA-to-DNA and DNA-to-RNA transcription, it may have some
initiating activity. Its ability to induce chromosomal aberrations is also indicative of
initiating activity. There are no studies that implicate or support a promotional activity of
2+
lead; however, its similarity to Ca suggests that it may alter regulation of this cation in
processes (e.g., cell growth) related to promotion. Intranuclear lead inclusion bodies in the
kidney may pertain to lead's carcinogenic effects, since both the formation of these bodies
and the induction of tumors occur at relatively high doses of lead. The interaction of lead
with key non-histone chromosomal proteins in the nucleus to form the inclusion bodies or the
presence of inclusion bodies in the nucleus may alter genetic function, thus leading to cell
transformation. Obviously, elucidating the mechanism of lead carcinogenesis requires further
research efforts and only theories can be formulated regarding its oncogenic action at
present.
It is hard to draw clear conlusions concerning what role lead may play in the induction
of human neoplasia. Epidemiological studies of lead-exposed workers provide no definitive
findings. However, statistically significant elevations in respiratory tract and digestive
system cancer in workers exposed to lead and other agents warrant concern. Also, since lead
acetate can produce renal tumors in some experimental animals, it may be prudent to assume
that at least that lead compound may be carcinogenic in humans. However, this statement is
qualified by noting that lead has been observed to increase tumorogenesis rates in animals
only at relatively high concentrations, and therefore does not appear to be an extremely
potent carcinogen. J.n vitro studies further support the genotoxic and carcinogenic role of
lead, but also indicate that lead is not extremely potent in these systems either.
CPB12/A 12-194 9/20/83
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PRELIMINARY DRAFT
12.8 EFFECTS OF LEAD ON THE IMMUNE SYSTEM
12.8.1 Development and Organization of the Immune System
Component cells of the immune system arise from a pool of pluripotent stem cells in the
yolk sack and liver of the developing fetus and in the bone marrow and spleen of the adult.
Stem cell differentiation and maturation follows one of several lines to produce lymphocytes,
macrophages, and polymorphonuclear leukocytes. These cells have important roles in immunolog-
ical function and host defense.
The predominant lymphocyte class develops in the thymus, which is derived from the third
and fourth pharyngeal pouches at 9 weeks of gestation in man (day 9 in mice). In the thymus
microenvironment they acquire characteristics of thymus-derived lymphocytes (T-cells), then
migrate to peripheral thyinic-dependent areas of the spleen and lymph nodes. T-cells are
easily distinguished from other lymphocytes by genetically defined cell surface markers that
allow them to be further subdivided into immunoregulatory amplifier cells (helper T-cells) and
suppressor T-cells that regulate immune responses. T-cells also participate directly as cyto-
lytic effector cells against virally infected host cells, malignant cells, and foreign tissues
as well as in delayed-type hypersensitivity (DTH) reactions where they elaborate lymphokines
that modulate the inflammatory response. T-cells are long-lived lymphocytes and are not read-
ily replaced. Thus, any loss or injury to T-cells may be detrimental to the host and result
in increased susceptibility to viral, fungal, bacterial, or parasitic diseases. Individuals
with acquired immune deficiency syndrome (AIDS) are examples of individuals with T-cell dys-
function. There is ample evidence that depletion by environmental agents of thymocytes or
stem cell progenitors during lymphoid organogenesis can produce permanent immunosuppression.
The second major lymphocyte class differentiates from a lymphoid stem-cell in a yet un-
defined site in man, which would correspond functionally to the Bursa of Fabricius in avian
species. In man, B-lymphocyte maturation and differentiation probably occur embryologically
in gut-associated lymphoid tissue (GALT) and fetal liver, as well as adult spleen and bone
marrow. This is followed by the peripheral population of thymic-independent areas of spleen
and lymph nodes. Bone marrow-derived lymphocytes (B-cells), which mature independently of the
thymus, possess specific immunoglobulin receptors on their surfaces. The presence of cell
surface immunoglobulin (slg) at high density is the major characteristic separating B-cells
from T-cells. Following interaction with antigens and subsequent activation, B-lymphocytes
proliferate and differentiate into antibody-producing plasma cells. In contrast to the long-
lived T-cell, B-cells are rapidly replaced by newly differentiating stem cells. Therefore,
lesions in the B-cell compartment may be less serious than those in the T-cell compartment
since they are more easily reversed. Insult to B-cells at the stem cell or terminal matura-
tion stage can result in suppression of specific immunoglobulin and enhanced susceptibility to
infectious agents whose pathogenesis is limited by antibodies.
CPB12/B 12-195 9/20/83
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PRELIMINARY DRAFT
Pluripotent stem cells also give rise to lymphocytes whose lineages are still unclear.
Some possess natural cytolytic activity for tumor cells (natural killer cell activity), while
others, devoid of T- and B-cell surface markers (null cells), participate in antibody-depen-
dent cell-mediated cytotoxicity (ADCC). The pluripotent stem cell pool also contains precur-
sors of monocyte-macrophages and polymorphonuclear leukocytes (PMN). The macrophage has a
major role in presentation and processing of certain antigens, in cytolysis of tumor target
cells, and in phagocytosis and lysis of persistent intracellular infectious agents. Also, it
actively phagocytizes and kills invading organisms. Defects in differentiation or function of
PMNs or macrophages predispose the host to infections by bacteria and other agents.
This introduction should make it evident that the effects of an element such as lead on
the immune system may be expressed in complex or subtle ways. In some cases, lead might pro-
duce a lesion of the immune system not resulting in markedly adverse health effects, espe-
cially if the lesion did not occur at an early stem cell stage or during a critical point in
lymphoid organogenesis. On the other hand, some lead-induced immune system effects might
adversely affect health through increasing susceptibility to infectious agents or neoplasti-
cally transformed cells if, for example, they were to impair cytocidal or bactericidal
function.
12.8.2 Host Resistance
One way of ascertaining if a chemical affects the immune response of an animal is to
challenge an exposed animal with a pathogen such as an infectious agent or oncogen. This pro-
vides a general approach to determine if the chemical interferes with host immune defense
mechanisms. Host defense is a composite of innate immunity, part of which is phagocyte activ-
ities, and acquired immunity, which includes B- and T-lymphocyte and enhanced phagocyte reac-
tivities. Analysis of host resistance constitutes a holistic approach. However, dependent on
the choice of the pathogen, host resistance can be evaluated somewhat more selectively.
Assessment of host resistance to extracellular microbes such as Staphylococci, Salmonella
typhimurium, Escherichia coli, or Streptococcus pneumom'ae and to intracellular organisms such
as Listeria monocytogenes or Candida albicans primarily measures intact humoral immunity and
cell-mediated immunity, respectively. Immune defense to extracellular organisms requires
T-lymphocyte, B-lymphocyte, and macrophage interactions for the production of specific anti-
bodies to activate the complement cascade and to aid phagocytosis. Antibodies can also
directly neutralize some bacteria and viruses. Resistance to intracellular organisms requires
T-lymphocyte and macrophage interactions for T-lymphocyte production of lymphokines, which
further enhance immune mechanisms including macrophage bactericidal activities. An additional
T-lymphocyte subset, the cytolytic T-cell, is involved in resistance to tumors; immune
defenses against tumors are also aided by NK- and K-lymphocytes and macrophages.
CPB12/B 12-196 9/20/83
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PRELIMINARY DRAFT
12.8.2.1 Infactivity Models. Numerous studies designed to assess the influence of lead on
host resistance to infectious agents consistently have shown that lead impairs host resis-
tance, regardless of whether the defense mechanisms are predominantly dependent on humoral- or
cell-mediated immunity (Table 12-22).
TABLE 12-22. EFFECT OF LEAD ON HOST RESISTANCE TO INFECTIOUS AGENTS
Species
Mouse
Rat
Rat
Mouse
Mouse
Mouse
Mouse
Infectious agent
S. typhimurium
E. coli
S. epidermidis
L. monocytogenes
EMC virus
EMC virus
Langat virus
Lead dose
200 ppm
2 mg/100 g
2 mg/100 g
80 ppm
2000 ppm
13 ppm
50 mg/kg
Lead exposure
i.p. ; 30 days
i. v. ; 1 day
i . v . ; 1 day
oral ly; 4 wk
orally; 2 wk
orally; 10 wk
orally; 2 wk
Mortality
54% (13%)
96% (0%)
80% (0%)
100% (0%)
100% (19%)
80% (50%)
68% (0%)
Reference
Hemphill et al. (1971)
Cook et al. (1975)
Cook et al. (1975)
Lawrence (1981a)
Gainer (1977b)
Exon et al. (1979)
Thind and Kahn (1978)
aThe percent mortality is
altered host resistance.
infected control group.
reported for the lowest dose of lead in the study that significantly
The percent mortality in parentheses is that of the non-lead-treated,
Mice (Swiss Webster) injected i.p. for 30 days with 100 or 250 ug (per 0.5 ml) of lead
nitrate and inoculated with Salmonella typhimurium had higher mortality (54 and 100 percent,
respectively) than non-lead-injected mice (13 percent) (Hemphill et al., 1971). These concen-
trations of lead, by themselves, did not produce any apparent toxicity. Similar results were
observed in rats acutely exposed to lead (one i.v. dose of 2 mg/100 g) and challenged with
Escherichia col i (Cook et al., 1975). In these two studies, lead could have interfered with
the clearance of endotoxin from the §^ typhimurium or E. coli, and the animals may have died
from endotoxin shock and not septicemia due to the lack of bacteriostatic or bactericidal
activities. However, the study by Cook et al. (1975) also included a non-endotoxin-producing
gram-positive bacterium, Staphylococcus epidermidis. and lead still impaired host resistance.
In another study, lead effects on host resistance to the intracellular parasite Listeria
monocytogenes were monitored (Lawrence, 1981a). CBA/J mice orally exposed to 16, 80, 400, and
2000 ppm lead for four weeks were assayed for viable Listeria after 48 and 72 hours, and for
mortality after 10 days. Only 2000 ppm lead caused significant inhibition of early bacteri-
cidal activity (48-72 hr), but 80-2000 ppm lead produced 100 percent mortality, compared with
0 percent mortality in the 0-16 ppm lead groups. Other reports have suggested that host
CPB12/B 12-197 9/20/83
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PRELIMINARY DRAFT
resistance is impaired by lead exposure of rodents. Salaki et al. (1975) indicated that lead
lowered resistance of mice to Staphylococcus aureus, Listeria, and Candida; and observed
higher incidence of inflammation of the salivary glands in lead-exposed rats (Grant et al.,
1980) may be due, in part, to lead-induced increased susceptibility to infections.
Inhalation of lead has also been reported to lower host resistance to bacteria.
Schlipkoter and Frieler (1979) exposed NMRI mice to an aerosol of 13-14 ug/m3 lead chloride
and clearance of Serratia marcesens in the lungs was reduced significantly. Microparticles of
lead in lungs of mice were also shown to lower resistance to Pasteurella multocida, in that
6 pg of lead increased the percentage of mortality by 27 percent (Bouley et al., 1977).
Lead has also been shown to increase host susceptibility to viral infections. CD-I mice,
administered 2,000 and 10,000 ppm lead in drinking water for two weeks and subsequently inocu-
lated with encephalomyocarditis (EMC) virus, had a significant increase in mortality (100 per-
cent at 2,000 ppm; 65 percent at 10,000 ppm) compared with control EMC virus-infected mice (13
percent) (Gainer, 1977b). In another study (Exon et al., 1979), Swiss Webster mice were ex-
posed to 13, 130, 1300, or 2600 ppm lead for 10 weeks in their drinking water and were infec-
ted with EMC virus. Although as low as 13 ppm lead caused a significant increase in mortality
(80 percent) in comparison with the non-lead-treated EMC virus-infected mice (50 percent),
there were no dose-response effects, in that 2600 ppm lead resulted in only 64 percent mortal-
ity. The lack of a dose-response relationship in the two studies with EMC virus (Gainer,
1977b; Exon et al., 1979) suggests that the higher doses of lead may directly inhibit EMC
infectivity as well as host defense mechanisms. Additional studies have confirmed that lead
inhibits host resistance to viruses. Mice treated orally with lead nitrate (10-50 mg/kg/ day)
for two weeks had suppressed antibody titers to Langat virus (Type B arbovirus) and increased
titers of the virus itself (Thind and Singh, 1977), and the lead-inoculated, infected mice had
higher mortalities (25 percent at 10 mg/kg; 68 percent at 50 mg/kg) than the non-lead-infected
mice (0 percent) (Thind and Khan, 1978).
The effects of lead on bacterial and viral infections in humans have never been studied
adequately; there is only suggestive evidence that human host resistance may be lowered by
lead. Children with persistently high blood lead levels who were infected with Shigella
enteritis had prolonged diarrhea (Sachs, 1978). In addition, lead workers with blood lead
levels of 22-89 ug/dl have been reported to have more colds and influenza infections per year
(Ewers et al., 1982). This study also indicated that secretory IgA levels were suppressed
significantly in lead workers with a median blood lead level of 55 ug/dl. Secretory IgA is a
major factor in immune defense against respiratory as well as gastrointestinal infections.
Hicks (1972) points out that there is need for systematic epidemiological studies on the
effects of elevated lead levels on the incidence of infectious diseases in humans. The cur-
rent paucity of information precludes formulation of any clear dose-response relationship for
CPB12/B 12-198 9/20/83
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PRELIMINARY DRAFT
humans. Epidemiological investigations may help to determine if lead alters the immune system
of man and consequently increases susceptibility to infectious agents and neoplasia.
12.8.2.2 Tumor Models and Neoplasia. The carcinogenicity of lead has been studied both as a
direct toxic effect of lead (see Section 12.7) and as a means of better understanding the
effects of lead on the body's defense mechanisms. Studies by Gainer (1973, 1974) demonstrated
that exposure of CD-I mice to lead acetate potentiated the oncogenicity of a challenge with
Rauscher leukemia virus (RLV), resulting in enhanced splenomegaly and higher virus titers in
the spleen presumably through an immunosuppressive mechanism. Recent studies by Kerkvliet and
Baecher-Steppan (1982) revealed that chronic exposure of C57BL/6 mice to lead acetate in
drinking water at 130-1300 ppm enhanced the growth of primary tumors induced by Moloney sar-
coma virus (MSV). Regression of MSV-induced tumors was not prevented by lead exposure, and
lead-treated animals resisted late sarcoma development following primary tumor resistance.
Depressed resistance to transplantable MSV tumors was associated with a reduced number of
macrophages, which also exhibited reduced phagocytic activity.
In addition to enhancing the transplantability of tumors or the oncogenicity of leukemia
viruses, lead has also been shown to facilitate the development of chemically induced tumors.
Kobayashi and Okamoto (1974) found that intratracheal dosing of benzo(a)pyrene (BaP) combined
with lead oxide resulted in an increased frequency of lung adenomas and adenocarcinomas over
mice exposed to BaP alone. Similarly, exposure to lead acetate enhanced the formation of
N(4'-fluoro-4-biphenyl) acetamide-induced renal carcinomas from 70 to 100 percent and reduced
the latency to tumor appearance (Hinton et al., 1980). Recently, Keller et al. (1983) found
that exposure to lead for 18 months increased the frequency of spontaneous tumors, predomi-
nantly renal carcinomas, in rats. Similarly, Schrauzer et al. (1981) found that adding lead
at 5 ppm to drinking water of C3H/St mice infected with Bittner milk factor diminished the up-
take of selenium and reduced its anticarcinogenic effects, causing mammary tumors to appear at
the same high incidence as in selenium-unsupplemented controls. Lead likewise significantly
accelerated tumor growth and shortened survival in this model.
The above studies on host susceptibility to various pathogens, including infectious
agents and tumors, indicate that lead could be detrimental to health by methods other than di-
rect toxicity. In order to understand the mechanisms by which lead suppresses host resistance
maintained by phagocytes, humoral immunity, and/or cell-mediated immunity, the immune system
must be dissected into its functional components and the effects of lead on each, separately
and combined, must be examined in order that the mechanism(s) of the immunomodulatory poten-
tial of lead can be understood.
12.8.3 Humoral Immunity
12.8.3.1 Antibody Titers. A low antibody titer in animals exposed to lead could explain the
increased susceptibility of animals to extracellular bacteria and some viruses (see Table
CPB12/B 12-199 9/20/83
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PRELIMINARY DRAFT
12-23), as well as to endotoxins (Selye et al., 1966; Filkins, 1970; Cook et al. , 1974;
Schumer and Erve, 1973; Rippe and Berry, 1973; Truscott, 1970). Specific antibodies can
directly neutralize pathogens, activate complement components to induce lysis, or directly or
indirectly enhance phagocytosis via Fc receptors or C3 receptors, respectively. Studies in
animals and humans have assayed the effects of lead on serum immunoglobulin levels, specific
antibody levels, and complement levels. Analysis of serum immunoglobulin levels is not a good
measure of specific immune reactivity, but it would provide evidence for an effect on B-
lymphocyte development.
TABLE 12-23. EFFECT ON LEAD ON ANTIBODY TITERS
Species Antigen
Lead dose and
exposure
Effect
Reference
Rabbit Pseudorabies virus 2500 ppm; 10 wk Decrease
Rat S. typhimurium 5000-20000 ppm; 3 wk Decrease
Rat Bovine serum albumin 10-1000 ppm; 10 wk Decrease
Mouse Sheep red blood cells 0.5-10 ppm ; 3 wk Decrease
Koller (1973)
Stankovid and Jugo
(1976)
Koller et al.
(1983)
Biakley et al.
(1980)
Lead was administered as tetraethyl lead; other studies used inorganic forms.
Lead had little effect on the serum immunoglobulin levels in rabbits (Fonzi et al.,
1967a), children with blood lead levels of 40 ug/dl (Reigart and Garber, 1976), or lead
workers with 22-89 ug/dl (Ewers et al., 1982). On the other hand, most studies have shown
that lead significantly impairs antibody production. Acute oral lead exposure (50,000 ppm/kg)
produced a decreased titer of anti-typhus antibodies in rabbits immunized with Typhus vaccine
(Fonzi et al., 1967b). In New Zealand white rabbits challenged with pseudorabies virus, lead
(oral exposure to 2500 ppm for 70 days) caused a 9-fold decrease in antibody titer to the
virus (Koller, 1973). However, lead has not always been shown to reduce titers to virus.
Vengris and Mare (1974) did not observe depressed antibody titers to Newcastle disease virus
in lead-treated chickens, but their lead treatment was only for 35 days prior to infection.
Lead-poisoned children also had normal anti-toxoid titers after booster immunizations with
tetanus toxoid (Reigart and Garber, 1976). In another study, Wistar rat dams were exposed to
5,000, 10,000, or 20,000 ppm lead for 20 days following parturition (Stankovid and Jugo,
1976). The progeny were weaned at 21 days of age and given standard laboratory chow for an
CPB12/B
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PRELIMINARY DRAFT
additional month. At that time, they were injected wi Lh Salmonella typhimurium, and serum
antibody titers were assessed. Each dosage of lead resulted in significantly reduced antibody
liters. More recently, rats (Sprague-Dawley) given 10 ppm lead acetate orally for 10 weeks
had a significant suppression in antibody titers when challenged with bovine serum albumin
(BSA) and compared with BSA-immunized non-lead-exposed rats (Keller et al. , 1983). Develop-
ment of a highly sensitive, quantitative, enzyme-linked immunosorbent assay (ELISA) contrib-
uted to detecting the immunosuppressive activity of lead at this dosage.
Tetraethyl lead also has been responsible for reduced antibody titers in Swiss-cross mice
(Blakley et al., 1980). The mice were exposed orally to 0.5, 1.0, and 2.0 ppm tetraethyl lead
for 3 weeks. A significant reduction in hemagQlutination titers to sheep red blood cells
(SRBC) occurred at all levels of exposure.
12.8.3.2 Enumeration of Antibody Producing Cells (PIague-Forming Cells). From the above re-
sults, it appears that lead inhibits antibody production. To evaluate this possible effect at
the cellular level, the influence of lead on the number of antibody producing cells after pri-
mary or secondary immunization can be assessed. In primary humoral immune responses (mostly
direct), IgM plaque-forming cells (RFC) are measured, whereas in secondary or anamnestic
responses (mostly indirect), IgG PFC are counted. The primary immune response represents an
individual's first contact with a particular antigen. The secondary immune response repre-
sents re-exposure to the same antigen weeks, months, or even years after the primary antibody
response has subsided. The secondary immune response is attributed to persistence, after
initial contact with the antigen, of a substantial number of antigen-sensitive memory cells.
Impairment of the memory response, therefore, results in serious impairment of humoral immun-
ity in the host.
Table 12-24 summarizes the effects of lead on IgM or IgG PFC development. Mice exposed
orally to tetraethyl lead (0.5, 1, or 2 ppm) for three weeks produced a significant reduction
in the development of IgM and IgG PFC (Blakley et al., 1980). Mice (Swiss Webster) exposed
orally to 13, 137, or 1375 ppm inorganic lead for eight weeks had reduced numbers of IgM PFC
in each lead-exposed group (Keller and Kovacic, 1974). Even the lowest lead group (13 ppm)
had a decrease. The secondary response (IgG PFC, induced by a second exposure to antigen SRBC
seven days after the primary immunization) was inhibited to a greater extent than the primary
response. This study indicated that chronic exposure to lead produced a significant decrease
in the development of IgM PFC and IgG PFC. When Swiss Webster mice were exposed to 13, 130,
and 1300 ppm lead for 10 weeks and hyper immunized by SRBC injections at week 1, 2, and 9, the
memory response as assessed by the enumeration of IgG PFC was significantly inhibited at 1300
ppm (Koller and Roan, 1980a). This suggests that the temporal relationships between lead
exposure and antigenic challenge may be critical. Other studies support this interpretation.
CPB12/B 12-201 9/20/83
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PRELIMINARY DRAFT
TABLE 12-24. EFFECT OF LEAD ON THE DEVELOPMENT OF ANTIBODY-PRODUCING CELLS (RFC)
Species
Mouse
Mouse
Mouse
Mouse
Mouse
Rat
Mouse
Mouse
Antigen3
SRBC (in vivo)
SRBC (in vivo)
SRBC (in vivo)
SRBC (in vivo)
SRBC (in vivo)
SRBC (in vitro + 2-ME)
SRBC (in vivo)
SRBC (in vitro)
SRBC (in vitro + 2-ME)
SRBC (in vitro + 2-ME)
Lead dose and exposure
13-1370 ppm; 8 wk
0.5-2 ppm tetraethyl lead;
3 wk
13-1370 ppm; 10 wk
4 mg (i.p. or orally)
16-2000 ppm; 1-10 wk
16-80 ppm; 4 wk
2000 ppm; 4 wk
25-50 ppm; pre/postnatal
50-1000 ppm; 3 wk
50-1000 ppm; 3 wk
2-20 ppm (in vitro)
Effect6
IgM PFC (D)
IgG PFC (D)
IgM PFC (D)
IgG PFC (D)
IgG PFC (D)
IgM PFC (I)
IgG PFC (D)
IgM PFC (N)
IgM PFC (I)
IgM PFC (D)
IgM PFC (D)
IgM PFC (D)
IgM PFC
(N or I)
IgM PFC (I)
Reference
Koller and
Kovacic (1974)
Blakley et al .
(1980)
Koller and
Roan (1980a)
Koller et al.
(1976)
Lawrence
(1981a)
Luster et al.
(1978)
Blakley and
Archer (1981)
Lawrence
(1981b,c)
The antigenic challenge with sheep red blood cells (SRBC) was ui vivo or jji vitro after HI
vivo exposure to lead unless otherwise stated. The ir\ vitro assays were performed in the
presence or absence of 2-mercaptoethanol (2-ME).
The letters in parentheses are defined as follows: D = decreased response; N = unaltered
response; I = increased response.
Female Sprague-Dawley rats with pre- and post-natal exposure to lead (25 or 50 ppm) had a
significant reduction in IgM PFC (Luster et al., 1978). In contrast, CBA/J mice exposed
orally to 16-2000 ppm lead for 1-10 weeks did not have altered IgM PFC responses to SRBC
(Lawrence, 1981a). Furthermore, when Swiss Webster mice were exposed to an acute lead dose (4
mg lead orally or i.p.), the number of IgG PFC was suppressed, but the number of IgM PFC was
enhanced (Koller et al., 1976).
The influence of lead on the development of PFC in mice was assessed further by ui vivo
exposure to lead, removal of spleen cells, and jn vitro analysis of PFC development. Initi-
ally it appeared that low doses of lead (16 and 80 ppm) enhanced development, and only a high
CPB12/B
12-202
9/20/83
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PRELIMINARY DRAFT
dose (2000 ppm) inhibited the development of IgM RFC (Lawrence, 1981a). However, a later
study by Blakley and Archer (1981) indicated that 50-1000 ppm lead consistently inhibited IgM
RFC. Through the analysis of mixed cultures of lead-exposed lymphocytes (nonadherent cells)
and unexposed macrophages (adherent cells), and vice versa, as well as of in vitro responses
to antigens that do not require macrophage help (i.e., 1 ipopolysaccharide, LPS), their data
indicated that the effects of lead may be at the level of the macrophage. This was substan-
tiated by the fact that 2-mercaptoethanol (2-ME, a compound that can substitute for at least
one macrophage activity) was able to reverse the inhibition by lead. This may explain why in
vivo lead exposure (16 and 80 ppm) appeared to enhance the ui vitro IgM RFC responses in the
study by Lawrence (1981a), because 2-ME was present in the uj vitro assay system. Further-
more, i_n vitro exposure to lead (2 or 20 ppm) in spleen cell cultures with 2-ME enhanced the
development of IgM RFC (Lawrence, 1981b,c).
These experiments indicate that lead modulates the development of antibody-producing
cells as well as serum antibody titers, which supports the notion that lead can suppress hu-
moral immunity. However, it should be noted that the dose and route of exposure of both lead
and antigen may influence the modulatory effects of lead. The adverse effects of lead on hu-
moral immunity may be due more to lead's interference with macrophage antigen processing
and/or antigen presentation to lymphocytes than to direct effects on B-lymphocytes. These
mechanisms require further investigation.
12.8.4 Cell-Mediated Immunity
12.8.4.1 Delayed-Type Hypersensitivity. T-lymphocytes (T-helper and T-suppressor cells) are
regulators of humoral and cell-mediated immunity as well as effectors of two aspects of cell-
mediated immunity. T-cells responsive to delayed-type hypersensitivity (DTH) produce lym-
phokines that induce mononuclear infiltrates and activate macrophages, which are aspects of
chronic inflammatory responses. In addition, another subset of T-cells, cytolytic T-cells,
cause direct lysis of target cells (tumors or antigenically modified autologous cells) when in
contact with the target. To date, the effects of lead on cytolytic T-cell reactivity have not
been measured, but the influence of lead on inducer T-cells has been studied (Table 12-25).
Groups of mice injected i.p. daily for 30 days with 13.7 to 137 ppm lead were subsequently
sensitized i.v. with SRBC. The DTH reaction was suppressed in these animals in a dose-related
fashion (Muller et al., 1977). The secondary DTH response was inhibited in a similar fashion.
In another study (Faith et al., 1979), the effects of chronic low level pre- and post-natal
lead exposure on cellular immune functions in Sprague-Dawley rats was assessed. Female rats
were exposed to 25 or 50 ppm lead acetate continuously for seven weeks before breeding and
through gestation and lactation. The progeny were weaned at three weeks of age and continued
on the respective lead exposure regimen of their mothers for an additional 14 to 24 days.
CPB12/B 12-203 9/20/83
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PRELIMINARY DRAFT
TABLE 12-25. EFFECT OF LEAD ON CELL-MEDIATED IMMUNITY
Species
Mouse
Rat
Mouse
Mouse
Lead dose and exposure
13.7-137 ppm; 4 wk
25-50 ppm; 8 wk
13-1300 ppm; 10 wk
16-2000 ppm; 4 wk
Parameter*
DTH
DTH
MLC
MLC
Effect
Decrease
Decrease
None
Decrease
Reference
Muller et al . (1977)
Faith et al. (1979)
Koller and Roan (1980b)
Lawrence (1981a)
*DTH =delayed-type hypersensitivity; MLC = mixed lymphocyte culture.
Thymic weights and DTH responses were significantly decreased by both lead dosages. These
results indicate that chronic low levels of lead suppress cell-mediated immune function.
The iji vitro correlate of the analysis of DTH responsive T-cells in vivo is the analysis
of mixed lymphocyte culture (MLC) responsive T-cells. When two populations of allogeneic lym-
phoid cells are cultured together, cellular interactions provoke blast cell transformation and
proliferation of a portion of the cultured cells (Cerottini and Brunner, 1974; Bach et al.,
1976). The response can be made one-way by irradiating one of the two allogeneic prepara-
tions, in which case the irradiated cells are the stimulators (allogeneic B-cells and macro-
phages) and the responders (T-cells) are assayed for their proliferation. The mixed lympho-
cyte reaction is an in vitro assay of cell-mediated immunity analogous to |n vivo host versus
graft reactions.
Mice (DBA/2J) fed 13, 130, or 1300 ppm lead for 10 weeks were evaluated for responsive-
ness in mixed lymphocyte cultures. The 130-ppm lead dose tended to stimulate the lymphocyte
reaction, although no change was observed at the other dose levels (Koller and Roan, 1980b).
In another study (Lawrence, 1981a), mice (CBA/J) were fed 16, 80, 400, or 2000 ppm lead for
four weeks. The 16 and 80 ppm doses slightly stimulated, while the 2000 ppm dose suppressed,
the mixed lymphocyte reaction. It is important to note that in these jn vitro MLC assays,
2-ME was present in the culture medium, and the 2-ME may have reversed the in vivo effects of
lead, as was observed for the jri vitro PFC responses (Blakley and Archer, 1981).
The data on the effects of lead on humoral and cell-mediated immunity indicate that in
vivo lead usually is immunosuppressive, but additional studies are necessary to fully under-
stand the temporal and dose relationship of lead's immunomodulatory effects. The in vitro
analysis of immune cells exposed to lead j_n vivo suggest that the major cell type modified is
the macrophage; the suppressive effects of lead may be readily reversed by thiol reagents
possibly acting as chelators.
CPB12/B 12-204 9/20/83
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PRELIMINARY DRAFT
12.8.4.3 Interferon. Interferons (IF) are a family of low molecular weight proteins which
exhibit antiviral activity in sensitive cells through processes requiring new cellular RNA and
protein synthesis (Stewart, 1979). It has been speculated that the enhanced susceptibility of
lead-treated mice to infectious virus challenge might be due to a decreased capacity of these
animals to produce viral or immune interferons or to respond to them. Studies by Gainer
(1974, 1977a) appeared to resolve this question and indicated that exposure of CD-I mice to
lead does not inhibit the antiviral action of viral IF ui vivo or in vitro. In the later of
the two studies, lead exposure inhibited the protective effects of the IF inducers Newcastle
disease virus and poly I:poly C against encephalomyocarditis virus (EMC)-induced mortality.
These data suggest that, although lead did not directly interfere with the antiviral activity
of interferon, it might suppress viral IF production in vivo. Recently, Blakley et al. (1982)
re-examined this issue and found that female BDft mice exposed to lead acetate in drinking
water at concentrations ranging from 50 to 1000 ug/ml for three weeks produced amounts of IF
similar to controls given a viral IF inducer, Tilorone. Similarly, the |n vitro induction of
immune IF by the T-cell mitogens phytohemagglutinin, concanavalin A, and staphylococcal
enterotoxin in lymphocytes from lead-exposed mice were unaltered compared with controls
(Blakley et al., 1982). Thus, lead exposure does not appear to significantly alter the lym-
phocyte's ability to produce immune interferon. Therefore, it must be assumed that increased
viral susceptibility associated with chronic lead exposure in rodents is by mechanisms other
than interference with production of or response to interferon.
12.8.5 Lymphocyte Activation by Mitogens
Mitogens are lectins that induce activation, blast-cell transformation, and proliferation
in resting lymphocytes. Certain lectins bind specifically to (1) T-cells (i.e., phytohemag-
glutinin [PHA] and concanavalin A [Con A]), (2) B-cells (i.e., lipopolysaccharide [IPS] of
gram-negative bacteria) or (3) both (i.e., pokeweed mitogen [PWM]). The blastogenic response
produced can be used to assess changes in cell division of T- and B-lymphocytes. The biologi-
cal significance of the following studies is difficult to interpret since exposure to lead was
either iji vivo or iji vitro at different doses and for different exposure periods.
12.8.5.1 In Vivo Exposure. Splenic lymphocytes from Swiss Webster mice exposed orally to
2000 ppm lead for 30 days had significantly depressed proliferative responses to PHA (Table
12-26) which were not observed after 15 days of exposure (Gaworski and Sharma, 1978). Sup-
pression was likewise observed with PWM, a T- and B-cell mitogen. These observations with
T-cell mitogens were confirmed in Sprague-Dawley rats exposed orally to 25 or 50 ppm lead pre-
and postnatally for seven weeks (Faith et al., 1979). Splenic T-cell responses to Con A and
PHA were significantly diminished. A similar depression of Con A and PHA responses occurred
CPB12/B 12-205 9/20/83
-------
TABLE 12-26. EFFECT OF LEAD EXPOSURE ON MITOGEN ACTIVATION OF LYMPHOCYTES
Species
Lead dose
Mitogen
Effect
Reference
Mice
Mice
Rats
- Mice
PO
r\3
§. Mice
Mice
Mice
Mice
Mice
In vivo. 250 and 2000
ppffl, 30 days
In vivo. 13, 130 and 1300
ppm, 10 weeks
I_n vivo, pre/postnatal
25 and 50 ppm, 7 weeks
In vivo. .08 - 10 mM, 4 weeks
In vivo. 1300 ppm, 8 weeks
In vivo. 50, 200 and 1000 ppm
3 weeks
In vitro, lo"4 - 10"6 for
full culture period
In vitro. 0.1, 0.5, 1.0 mM
for full culture period
In vitro. 10"3 - 10"7 M
PHA (T-Cell)
PWM (T and B-Cell)
Con A (T-Cell)
LPS (B-Cell)
Con A
PHA
Con A, PHA
LPS
Con A, PHA
LPS
Con A, PHA, SEA
LPS
Con A, PHA
LPS
PHA
PWM
LPS
Significantly depressed at
2000 ppm on day 30 only1
Significantly depressed at
2000 ppm on both days 15
and 301
No effect
No effect
Significantly depressed at
25 and 50 ppm
Significantly depressed at
50 ppm only
No effect
Depressed at 2 and 10 mM
Significantly depressed
No effect
Increased to all2
No effect
Slightly increased at
highest dose at day 2, no
effect at day 3.5
Increased up to 245%1
Increased at all doses by
up to 453%3
Increased by approximately
250% at 0.1 and 0.5 mM only
Increased by up to 312%
Gaworski and
Sharma (1978)
Koller et al. (1979)
Faith et al. (1979)
TJ
•yo
Lawrence (1981c) ~
K-*
Neilan et al. (1980) |
O
Blakley and Archer (1982)5
~n
—l
Lawrence (1981a,b)
Gaworski and
Sharma (1978)
Shenker et al. (1977)
Gallagher et al. (1979)
1. Difficult to interpret since data were reported only as % of control response rather than
2. Untreated control values unusually low for T-cell response. Lead treated had much higher
showing cytotoxicity.
3. Noted white precipitate thought to be lead carbonate in cultures.
CPM of 3H-TdR incorporation.
response with highest dose
-------
PRELIMINARY DRAFT
in lymphocytes from C57B1/6 mice exposed to 1300 ppm lead for 8 weeks (Neilan et al., 1980).
Lead impaired blastogenic transformation of lymphocytes by both T-cell mitogens, although the
B-cell proliferative response to LPS was not impaired.
In contrast to reports that lead exposure suppressed the blastogenic response of T-cells
to mitogens, several laboratories have reported that lead exposure does not suppress T-cell
proliferative responses (Keller et al. , 1979; Lawrence, 1981c; Blakley and Archer, 1982).
These differences are not easily reconciled since analysis of the lead dose employed and ex-
posure period (Table 12-25) provides little insight into the observed differences in T-cell
responses. In one case, a dose of 2000 ppm for 30 days produced a clear depression while a
lesser dose of 1300 ppm produced no effect at 10 weeks in another laboratory. These data are
confusing and may reflect technical differences in performing the T-cell blastogenesis assay
in different laboratories, a lack of careful attention to lectin response kinetics, or the
influence of suppressor macrophages. Thus, no firm conclusion can be drawn regarding the
ability of jn vivo exposure to lead to impair the proliferative capacity of T-cells.
The blastogenic response of B-cells to LPS was unaffected in four different jji vivo stud-
ies at lead exposure levels from 25 to 1300 ppm (Koller et al. , 1979; Faith et al., 1979;
Neilan et al., 1980; Blakley and Archer, 1982). Lawrence (1981c), however, reported that the
LPS response was suppressed after 4 weeks exposure at 2 and 10 mM lead. The weight of the
data suggests that the proliferative response of B-cells to LPS is probably not severely im-
paired by lead exposure.
12.8.5.2 In Vitro Exposure. The biological relevance of immunological studies in which lead
was added jm vitro to normal rodent splenocytes in the presence of a mitogen (Table 12-26) is
questionable since differences probably reflect either a direct toxic or stimulatory effect by
the metal. These models may, however, provide useful information regarding metabolic and
functional responses in lymphocytes using lead as a probe.
_4 -5 -6
In one study, lymphocytes were cultured in the presence of lead (10 , 10 , and 10 M).
A slight but significant increase in lymphocyte transformation occurred on day 2 at the high-
est lead dosage when stimulated with Con A or PHA (Lawrence, 1981b). In a follow-up study
-4 —5
where the kinetics of the lectin response were examined (Lawrence, 1981a), lead (10 , 10 ,
_6
and 10 M) significantly suppressed the Con A- and PHA-induced proliferative responses of
lymphocytes on day 2, but not on days 3 to 5. In yet another ui vitro exposure study, lympho-
cytes cultured in the presence of 0.1, 0.5, or 1.0 mM lead had a significantly enhanced
response to PHA (Gaworski and Sharma, 1978). It should be kept in mind when considering these
ID vltro exposure observations that lead has been demonstrated to be directly mitogenic to
lymphocytes (Shenker et al., 1977). The data discussed here suggest that lead may also be
slightly co-mitogenic with T-cell mitogens. Direct exposure of lymphocytes in culture to lead
can also result in decreased lymphocyte viability (Gallagher et al., 1979). In vitro studies
CPB12/B 12-207 9/20/83
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PRELIMINARY DRAFT
on the effect of lead on the B-cell blastogenic response to LPS indicated that lead is
potently co-mitogenic with LPS and enhanced the proliferative response of B-cells by 245 per-
cent (Lawrence 1981b,c) to 312 percent (Shenker et al., 1977; Gallagher et al., 1979).
12.8.6 Macrophage Function
The monocyte/macrophage is involved with phagocytosis, bactericidal activity, processing
of complex antigens for initiation of antibody production, interferon production, endotoxin
detoxification, and immunoregulation. Since some of these functions are altered in lead-
treated rodents (Table 12-27), the monocyte/macrophage or comparable phagocytic cell in the
liver has been suggested as a possible cellular target for lead (Trejo et al., 1972; Cook et
al., 1974; MUller et al., 1977; Luster et al. , 1978; Blakley and Archer, 1981).
Several laboratories have shown that a single i.v. injection of lead impaired the phago-
cytic ability of the reticuloendothelial system (RES) (Trejo et al., 1972; Cook et al., 1974;
Filkins and Buchanan, 1973). Trejo et al. (1972) found that an i.v. injection of 5 mg lead
impaired vascular clearance of colloidal carbon that resulted from an impaired phagocytic
ability of liver Kupffer cells. Similarly, others have confirmed that lead injected i.v. de-
pressed intravascular clearance of colloidal carbon (Filkins and Buchanan, 1973) as well as a
radiolabeled lipid emulsion (Cook et al., 1974). Opposite effects on RES function have been
seen when lead was given orally (Koller and Roan, 1977). Similarly, Schlick and Friedberg
(1981) noted that a 10-day exposure to 10-1000 ug lead enhanced RES clearance and endotoxin
hypersensitivity.
Lead has likewise been demonstrated to suppress macrophage-dependent immune responses
(Blakley and Archer, 1981). Exposure of BDF1 mice to lead (50 ppm) for three weeks in drink-
ing water suppressed in vitro antibody RFC responses to the macrophage-dependent antigens,
sheep red blood cells or dinitrophenyl-Ficoll, but not to the macrophage-independent antigen
E. coli lipopolysaccharide. The macrophage substitute 2-mercaptoethanol and macrophages from
non-exposed mice restored lead-suppressed response. Castranova et al. (1980) found that
cultured rat alveolar macrophages exposed to lead had depressed oxidative metabolism.
The effects of heavy metals on endotoxin hypersensitivity were first observed by Selye et
al. (1966), who described a 100,000-fold increase in bacterial endotoxin sensitivity in rats
given lead acetate. The increased sensitivity to endotoxin was postulated to be due to a
blockade of the RES. Filkins (1970) subsequently demonstrated that endotoxin detoxification
is primarily a hepatic macrophage-mediated event that is profoundly impaired by lead exposure
(Trejo and Di Luzio, 1971; Filkins and Buchanan, 1973). The several types of data described
above suggest that macrophage dysfunction may be contributing to impairment of immune func-
tion, endotoxin detoxification, and host resistance following lead exposure.
CPB12/B 12-208 9/20/83
-------
TABLE 12-27. EFFECT OF LEAD ON MACROPHAGE AND RETICULOENDOTHELIAL SYSTEM FUNCTION
Species
Rat
Rat
House
r\5
§ Guinea Pig
Rat
Mouse
Mouse
Lead dose
and exposure
2.25 umol i.v. ,
single injection
5 rag i.v. ,
single injection
13, 130, 1300 ppm
oral , 10-12 weeks
.3 .6
10 -10 M
_3 _6
10 -10 M
50-1000 ppm oral ,
3 weeks
10-1000 ug,
10 days
Parameter
Vascular clearance;
lipid emulsion
endo toxin sensitivity
Vascular clearance;
colloidal carbon
endotoxin sensitivity
Phagocytosis
Macrophage migration
Macrophage oxygen
metabolism
Macrophage dependent
antigens RFC response
Vascular clearance
Effect
Depressed
Increased
Depressed
Increased
Depressed
Depressed
Depressed
Depressed
Enhanced at
10 days;
no effect
at >30 days.
Reference
Cook et al. (1974);
Trejo et al. (1972)
Trejo et al. (1972);
Filkins and
Buchanan (1973)
Kervliet and Braecher-
Steppan (1982)
Kiremidjian-
Schumacher et al. (1981)
Castranova et al. (1980)
Blakley and Archer
(1981)
Schlick and
Friedberg (1981)
-o
TO
m
I—
i — i
2
t— <
J>
TO
O
TO
3>
-n
—t
Endotoxin sensitivity
Increased
-------
PRELIMINARY DRAFT
12.8.7 Mechanisms of Lead Immunomodulation
The mechanism of toxic action of lead on cells is complex (see Section 12.2). Since lead
has a high affinity for sulfhydryl groups, a likely subcellular alteration accounting for the
immunomodulatory effects of lead on immune cells is its association with cellular thiols.
Numerous studies have indicated that surface and intracellular thiols are involved in lympho-
cyte activation, growth, and differentiation. Furthermore, the study by Blakley and Archer
(1981) suggests that lead may inhibit the macrophage's presentation of stimulatory products to
the lymphocytes. This process may rely on cellular thiols since the inhibitory effects of
lead can be overcome by an exogenous thiol reagent. Goyer and Rhyne (1973) have indicated
that lead ions tend to accumulate on cell surfaces, thereby possibly affecting surface recep-
tors and cell-to-cell communication. A study by Koller and Brauner (1977) indicated that lead
does alter C3b binding to its cell surface receptor.
12.8.8 Summary
Lead renders animals highly susceptible to endotoxins and infectious agents. Host sus-
ceptibility and the humoral immune system appear to be particularly sensitive. As postulated
in recent studies, the macrophage may be the primary immune target cell of lead. Lead-induced
immunosuppression occurs at low dosages that induce no evident toxicity and, therefore, may be
detrimental to the health of animals and perhaps of humans. The data accumulated to date pro-
vide good evidence that lead affects immunity, but additional studies are necessary to eluci-
date the actual mechanism by which lead exerts its immunosuppressive action. Knowledge of
lead effects of lead on the immune system of man is lacking and must be properly ascertained
in order to determine permissible levels for human exposure. However, since this chemical
affects immunity in laboratory animals and is immunosuppressive at very low dosages, its
potential serious effects in man should be carefully considered.
CPB12/B 12-210 9/20/83
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PRELIMINARY DRAFT
12.9 EFFECTS OF LEAD ON OTHER ORGAN SYSTEMS
12.9.1 The Cardiovascular System
Since the best understood pathophysiologic mechanisms of hypertension in humans are those
resulting from renal disease, the clinical evidence for a relationship between lead and hyper-
tension is reviewed in Section 12.5.3.5 above. Under conditions of long-term lead exposure at
high levels, arteriosclerotic changes have been demonstrated in the kidney. Oingwall-Fordyce
and Lane (1963) reported a marked increase in the cerebrovascular mortality rate among heavily
exposed lead workers as compared with the expected rate. These workers were exposed to lead
during the first quarter of this century when working conditions were quite bad. There has
been no similar increase in the mortality rate for men employed in recent times.
There are conflicting reports regarding whether lead can cause atherosclerosis in experi-
mental animals. Scroczynski et al. (1967) observed increased serum lipoprotein and choles-
terol levels and cholesterol deposits in the aortas of rats and rabbits receiving large doses
of lead. On the other hand, PrerovskS (1973), using similar doses of lead given over an even
longer period of time, did not produce atherosclerotic lesions in rabbits.
Structural and functional changes have been noted in the myocardium of children with
acute lead poisoning, but to date the extent of such studies has been limited. Cases have
been described in adults and in children, always with clinical signs of poisoning. There is,
of course, the possibility that the coexistence of lead poisoning and myocarditis is coinci-
dental. In many cases in which encephalopathy is present, the electrocardiographic abnorma-
lities disappeared with chelation therapy, suggesting that lead may have been the original
etiological factor (Freeman, 1965; Myerson and Eisenhauer, 1963; Silver and Rodriguez-Torres,
1968). Silver and Rodriguez-Torres (1968) noted abnormal electrocardiograms in 21 of 30 chil-
dren (70 percent) having symptoms of lead toxicity. After chelation therapy, the electro-
cardiograms remained abnormal in only four (13 percent) of the patients. Electron microscopy
of the myocardium of lead-intoxicated rats (Asokan, 1974) and mice (Khan et al., 1977) have
shown diffuse degenerative changes. Kopp and coworkers have demonstrated depression of con-
tractility, isoproterenol responsiveness, and cardiac protein phosphorylation (Kopp et al.,
1980a), as well as high energy phosphate levels (Kopp et al., 1980b) in hearts of lead-fed
rats. Similarly, persistent increased susceptibility to norepinephrine-induced arrhythmias
has been observed in rats fed lead during the first three weeks of life (Hejtmancik and
Williams, 1977, 1978, 1979a,b; Williams et al., 1977a,b).
In a review of five fatal cases of lead poisoning in young children, degenerative changes
in heart muscle were reported to be the proximate cause of death (Kline, 1960). It is not
clear that such morphological changes are a specific response to lead intoxication. Kdsmider
and Petelnz (1962) examined 38 adults over 46 years of age with chronic lead poisoning. They
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found that 66 percent had electrocardiographic changes, a rate that was four times the expec-
ted rate for that age group.
The cardiovascular effects of lead in conjunction with cadmium have been studied in rats
following chronic low level exposure by Perry and coworkers (Perry and Erlanger, 1978; Kopp
etal., 1980a,b). Perry and Erlanger (1978) exposed female weanling Long-Evans rats to
cadmium, lead, or cadmium plus lead (as acetate salts) at concentrations of 0.1, 1.0, or 5.0
ppm in deionized drinking water for up to 18 months. These authors reported statistically
significant increases in systolic blood pressure for both cadmium and lead in the range of
15-20 mm Hg. Concomitant exposure to both cadmium and lead usually doubled the pressor ef-
fects of either metal alone. A subsequent study (Kopp et al., 1980a) using weanling female
Long-Evans rats exposed to 5.0 ppm cadmium, lead, or lead plus cadmium in deionized drinking
water for 15 or 20 months showed similar pressor effects of these two metals alone or in com-
bination on systolic blood pressure. Electrocardiograms performed on these rats demonstrated
statistically significant prolongation of the mean PR interval. Bundle electrograms also
showed statistically significant prolongations. Other parameters of cardiac function were not
markedly affected. Phosphorus-31 nuclear magnetic resonance (NMR) studies conducted on
perchloric acid extracts of liquid nitrogen-frozen cardiac tissue from these animals disclosed
statistically significant reductions in adenosine triphosphate (ATP) levels and concomitant
increases in adenosine diphosphate (ADP) levels. Cardiac glycerol 3-phosphoryl-choline (GPC)
were also found to be significantly reduced using this technique, indicating a general
reduction of tissue high-energy phosphates by lead or cadmium. Pulse-label ing studies using
32P demonstrated decreased incorporation of this isotope into myosin light-chain (LC-2) in all
lead or cadmium treatment groups relative to controls. The results of these studies indicate
that prolonged low-dose exposure to lead (and/or cadmium) reduces tissue concentrations of
high-energy phosphates in rat hearts and suggest that this effect may be responsible for
decreased myosin LC-2 phosphorylation and subsequent reduced cardiac contractility. Other
studies by these authors (Kopp et al., 1980b) were also conducted on isolated perfused hearts
of weanling female Long-Evans rats exposed to cadmium, lead, or lead plus cadmium in deionized
drinking water at concentrations of 50 ppm for 3-15 months. Incorporation of 32P into cardiac
proteins was studied following perfusion on inotropic perfusate containing isoproterenol at a
concentration of 7 x 10" M. Data from these studies showed a statistically significant reduc-
tion in cardiac active tension in hearts from cadmium- or lead-treated rats. Phosphorus-32
incorporation was also found to be signficantly reduced in myosin LC-2 proteins. The authors
suggested that the observed decrease in LC-2 phosphorylation could be involved in the observed
decrease in cardiac active tension in lead- or cadmium-treated rats.
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Makasev and Krivdina (1972) observed a two-phase change in the permeability of blood ves-
sels (first increased, then decreased permeability) in rats, rabbits, and dogs that received a
solution of lead acetate. A phase change in the content of catecholamines in the myocardium
and in the blood vessels was observed in subacute lead poisoning in dogs (Mambeeva and
Kobkova, 1969). This effect appears to be a link in the complex mechanism of the cardiovas-
cular pathology of lead poisoning.
The susceptibility of the myocardium to toxic effects of lead was supported by iji vitro
studies in rat mitochondria by Parr and Harris (1976). These investigators found that the
2+
rate of Ca removal by rat heart mitochondria is decreased by 1 nmol Pb/mg protein.
12.9.2 The Hepatic System
The effect of lead poisoning on liver function has not been extensively studied. In a
study of 301 workers in a lead-smelting and refining facility, Cooper et al. (1973) found an
increase in serum glutamic oxaloacetic transaminase (SCOT) activity in 11.5 percent of
subjects with blood lead levels below 70 ug/dl, in 20 percent of those with blood lead levels
of about 70 ug/dl, and in 50 percent of the workers with blood lead levels of about 100 ug/dl.
The correlation (r = 0.18) between blood lead levels and SGOT was statistically significant.
However, there must also have been exposure to other metals, e.g., cadmium, since there was a
zinc plant in the smelter. In lead workers with moderate effects on the hematopoietic system
and no obvious renal signs, SGOT was not increased compared with controls on repeated examina-
tions (Hammond et al. , 1980). In most studies on lead workers, tests for liver function are
not included.
The liver is the major organ for the detoxification of drugs. In Section 12.3.1.3 it is
mentioned that exposure to lead may cause altered drug detoxification rates as a result of in-
terference with the formation of heme-containing cytochrome P-450, which is part of the
hepatic mixed function oxidase system. This enzyme system is involved in the hepatic bio-
transformation of medicaments, hormones, and many environmental chemicals (Remmer et al.,
1966). Whereas a decrease in drug-metabolizing activity clearly has been demonstrated in
experimental animals given large doses of lead resulting in acute toxicity, the evidence for
effects of that type in humans is less consistent. Alvares et al. (1975) studied the effect
of lead exposure on drug metabolism in children. There were no differences between two normal
children and eight children with biochemical signs of lead toxicity in their capacities to
metabolize two test drugs, antipyrine and phenylbutazone. In two acutely poisoned children
in whom blood levels of lead exceeded 60 ug/dl, antipyrine half-lives were significantly
longer than normal, and therapy with EDTA led to biochemical remission of the disease and
restoration of deranged drug metabolism toward normal. One of the "normal" children in this
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study had a blood lead level of 40 (J9/d1» but normal ALA-D and EP values. No data were given
on the analytical methods used for indices of lead exposure. Furthermore, the age of the
children varied from 1 to 7.5 years, which is significant because, as pointed out by the
authors, drug detoxification is age-dependent.
Meredith et al. (1977) demonstrated enhanced hepatic metabolism of antipyrine in lead-
exposed workers (PbB: 77-195 M9/dl) following chelation therapy. The significance of this
evidence of restored hepatic mixed oxidase function is, however, unclear because the pretreat-
ment antipyrine biologic half-life and clearance were not significantly different in lead-
exposed and control subjects. Moreover, there were more heavy smokers among the lead-exposed
workers than controls. Smoking increases the drug-metabolizing capacity and may thus counter-
act the effects of lead. Also, the effect of chelation on antipyrine metabolism in non-
exposed control subjects was not determined.
Hepatic drug metabolism in eight adult patients showing marked effects of chronic lead
intoxication on the erythropoietic system was studied by Alvares et al. (1976). The plasma
elimination rate of antipyrine, which, as noted above, is a drug primarily metabolized by he-
patic microsomal enzymes, was determined in eight subjects prior to and following chelation
therapy. In seven of eight subjects, chelation therapy shortened the antipyrine half-lives,
but the effect was minimal. The authors concluded that chronic lead exposure results in sig-
nificant inhibition of the heme biosynthetic pathway without causing significant changes in
enzymatic activities associated with hepatic cytochrome P-450.
A confounding factor in the above three studies may be that treatment with EDTA causes an
increase in the glomerular filtration rate (GFR) if it has been reduced by lead (Section
12.5.3.3). This may cause a decrease in the half-times of drugs. There are, however, no data
on the effect of chelating agents on GFR in children or adults with moderate signs of lead
toxicity.
In 11 children with blood lead levels between 43 and 52 ug/dl, Saenger et al. (1981)
found a decrease in 24-hour urinary 6-beta-hydroxycortisol excretion that correlated closely
(r = 0.85, p <0.001) with a standardized EDTA lead-mobilization test (1000 mg EDTA/m2 body
surface area). This glucocorticoid metabolite is produced by the same hepatic microsomal
mixed function oxidase system that hydroxylates antipyrine. The authors suggest that the
depression of 6-beta-hydroxylation of cortisol in the liver may provide a non-invasive method
for assessing body lead stores in children (Saenger et al., 1981).
In a few animal studies special attention has been paid to morphological effects of lead
on the liver. White (1977) gave eight beagle dogs oral doses of lead carbonate, 50-100 mg
Pb/kg b.w., for 3-7 weeks. Lead concentrations were not measured in blood or tissues. In two
dogs exposed from 5 weeks of age to 50 mg/kg, morphological changes were noted. Changes in
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enzyme activities were noted in most exposed animals; for example, some dehydrogenases showed
increased activity after short exposure and decreased activity after longer exposures, mainly
in animals with weight losses. The small number of animals and the absence of data on lead
concentrations makes it impossible to use these results for risk evaluations.
Hoffmann et al. (1974) noted moderate to marked morphological changes in baboon livers
after a single intravenous injection of large doses of lead acetate (25 mg/kg b.w.). It can
be concluded that effects on the liver may be expected to occur only at high exposure levels.
If effects on more sensitive systems, viz., the nervous and hematopoietic systems, are pre-
vented, no adverse effects should be noted in the liver.
12.9.3 The Endocrine System
The effects of lead on the endocrine or hormonal system are not well defined at the pre-
sent time, but some evidence exists for such effects, at least at high levels of lead expo-
sure. Lead is thought, for example, to decrease thyroid function in man and experimental ani-
mals. Porritt (1931) suggested that lead dissolved from lead pipes by soft water was the
cause of hypothyroidism in individuals living in southwest England. Later, Kremer and Frank
(1955) reported the simultaneous occurrence of myxedema and plumbism in a house painter.
Monaenkova (1957) observed impaired concentration of 131I by thyroid glands in 10 of 41
patients with industrial plumbism. Subsequently, Zel'tser (1962) showed that iji vivo 131I up-
take and thyroxine synthesis by rat thyroid were decreased by lead when doses of 2 and 5 per-
cent lead acetate solution were administered. Uptake of 131I, sometimes decreased in men with
lead poisoning, can be offset by treatment with thyroid-stimulating hormone (TSH) (Sandstead
et al., 1969; Sandstead and Galloway, 1967). Lead may act to depress thyroid function by in-
hibiting thiol groups or by displacing iodine in a protein sulfonyl iodine carrier (Sandstead
and Galloway, 1967), and the results suggest that excessive lead may act at both the pituitary
and the thyroid gland itself to impair thyroid function. None of these effects on the thyroid
system, however, have been demonstrated to occur in humans at blood lead levels below 30-40
ug/dl.
Sandstead et al. (1970a) studied the effects of lead intoxication on pituitary and adre-
nal function in man and found that it may produce clinically significant hypopituitarism in
some. The effects of lead on adrenal function were less consistent, but some of the patients
showed a decreased responsiveness to an inhibitor (metapyrone) of 11-beta-hydroxylation in the
synthesis of cortisol. This suggests a possible impact of lead on pituitary-adrenal hormonal
functions. That excessive oral ingestion of lead may in fact result in pathological changes
in the pituitary-adrenal axis is also supported by other reports of lead-induced decreased
metapyrone responsiveness, a depressed pituitary reserve, and decreased immunoreactive ACTH
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(Murashov, 1966; Pines, 1965). These same events may also affect adrenal gland function as
much as decreased urinary excretion of 17-hydroxy-corticosteroids was observed in these
patients. Also, suppression of responsiveness to exogenous ACTH in the zona fasciculata of
the adrenal cortex has been reported in lead-poisoned subjects (Makotchenko, 1965), and
impairment of the zona glomerulosa of the adrenal cortex has also been suggested (Sandstead et
al., 1970b). Once again, however, none of these effects on adrenal hormone function have been
shown to occur at blood lead levels as low as 30-40 pg/dl.
Other studies provide evidence suggestive of lead exposure effects on endocrine systems
controlling reproductive functions (see also Section 12.6). For example, evidence of abnormal
luteinizing hormone (LH) secretory dynamics was found in secondary lead smelter workers
(Braunstein et al., 1978). Reduced basal serum testosterone levels with normal basal LH
levels but a diminished rise in LH following stimulation indicated suppression of hypo-
thai amic-pituitary function. Testicular biopsies in two lead-poisoned workmen showed peritu-
bular fibrosis suggesting direct toxic effects of lead in the testes as well as effects at the
hypothalamic-pituitary level. Lancranjan et al. (1975) also reported lead-related interfer-
ence with male reproductive functions. Moderately increased lead absorption (blood lead mean
= 52.8 ug/dl) among a group of 150 workmen who had long-term exposure to lead in varying
degrees was said to result in gonadal impairment. The effects on the testes were believed to
be direct, however, in that tests for hypothalamic-pituitary influence were negative.
In regard to effects of lead on ovarian function in human females, Panova (1972) reported
a study of 140 women working in a printing plant for 1 to 2 months, where ambient air lead
levels were <7 ug/m3. Using a classification of various age groups (20-25, 26-35, and 36-40
yr) and type of ovarian cycle (normal, anovular, and disturbed lutein phase), Panova claimed
that statistically significant differences existed between the lead-exposed and control groups
in the age range 20-25 years. It should be noted that the report does not show the age dis-
tribution, the level of significance, or the data on specificity of the method used for class-
ification. Also, Zielhuis and Wibowo (1976), in a critical review of the above study, con-
cluded that the design of the study and presentation of data are such that it is difficult to
evaluate the author's conclusion that chronic exposure to low air lead levels leads to dis-
turbed ovarian function. Moreover, no consideration was given to the dust levels of lead, an
important factor in print shops. Unfortunately, little else besides the above report exists
in the literature in regard to assessing lead effects on human ovarian function or other fac-
tors affecting human female fertility. Studies offering firm data on maternal variables,
e.g., hormonal state, that are Known to affect the ability of the pregnant woman to carry the
fetus full-term are also lacking, although certain studies do indicate that at least high-
level lead exposure induces stillbirths and abortions (see Section 12.6).
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One animal study (Petrusz et al., 1979) indicates that orally administered lead can exert
effects on pituitary and serum gonadotropins, which may represent one mechanism by which lead
affects reproductive functions. The blood lead levels at which alterations in serum and pitu-
itary follicle stimulating hormone were observed in neonatal rats, however, were well in ex-
cess of 100 pg/dl.
12.9.4 The Gastrointestinal System
Colic is usually a consistent early symptom of lead poisoning, warning of much more seri-
ous effects that are likely to occur with continued or more intense lead exposure. Although
most commonly seen in industrial exposure cases, colic is also a lead-poisoning symptom pres-
ent in infants and young children.
Beritic (1971) examined 64 men suffering from abdominal colic due to lead intoxication
through occupational exposure. The diagnosis of lead colic was based on the occurrence of
severe attacks of spasmodic abdominal pain accompanied by constipation, abnormally high copro-
porphyrinuria, excessive basophilic stippling, reticulocytosis, and some degree of anemia (all
clinical signs of lead poisoning). Thirteen of the 64 patients had blood lead levels of 40-
80 pg/dl upon admission. However, the report did not indicate how recently the patients'
exposures had been terminated or provide other details of their exposure histories.
A more recent report by Dahlgren (1978) focused on the gastrointestinal symptoms of lead
smelter workers whose blood lead levels were determined within two weeks of the termination of
their work exposure. Of 34 workers with known lead exposure, 27 (79 percent) complained of
abdominal pain, abnormal bowel movements, and nausea. Fifteen of the 27 had abdominal pain
for more than 3 months after removal from the exposure to lead. The mean (and SD) blood lead
concentration for this group of 15 was 70 (± 4) jjg/dl. There was, however, no correlation
between severity of symptoms and blood lead levels, as those experiencing stomach pain for
less than 3 months averaged 68 (± 9) ug/dl and the remaining 7 workers, reporting no pain at
all, averaged 76 (± 9) ug/dl.
Hanninen et al. (1979) assessed the incidence of gastrointestinal symptoms in 45 workers
whose blood lead levels had been regularly monitored throughout their exposure and had never
exceded 69 ug/dl. A significant association between gastrointestinal symptoms (particularly
epigastric pain) and blood lead level was reported. This association was more pronounced in
subjects whose maximal blood lead levels had reached 50-69 ug/dl, but was also noted in those
whose blood lead levels were balow 50 ug/dl.
Other occupational studies have also suggested a relationship between lead exposure and
gastrointestinal symptoms (Lilis et al., 1977; Irwig et al., 1978; Fischbein et al., 1979,
1980). For demonstrating such a relationship, however, the most useful measure of internal
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exposure has not necessarily been blood lead concentrations. Fischbein et al. (1980) surveyed
a cross-section of New York City telephone cable splicers exposed to lead in the process of
soldering cables. Of the 90 workers evaluated, 19 (21 percent) reported gastrointestinal
symptoms related to lead colic. The difference between mean blood lead levels in those re-
porting GI symptoms and those not reporting such symptoms (30 vs. 27 |jg/dl) was not statis-
tically significant. However, mean zinc protoporphyrin concentrations (67 vs. 52 ng/dl) were
significantly different (p <0.02)
Although gastrointestinal symptoms of lead exposure are clinically evident in frank lead
intoxication and may even be present when blood lead levels approach the 30-80 ug/dl range,
there is currently insufficient information to establish a clear dose-effect relationship for
the general population at ambient exposure levels.
12.10 CHAPTER SUMMARY
12.10.1 Introduction
Lead has diverse biological effects in humans and animals. Its effects are seen at the
subcellular level of organellar structures and processes as well as at the overall level of
general functioning that encompasses all systems of the body operating in a coordinated,
interdependent fashion. The present chapter not only categorizes and describes the various
biological effects of lead but also attempts to identify the exposure levels at which such
effects occur and the mechanisms underlying them. The dose-response curve for the entire
range of biological effects exerted by lead is rather broad, with certain biochemical changes
occurring at relatively low levels of exposure and perturbations in other systems, such as the
endocrine, becoming detectable only at relatively high exposure levels.
In terms of relative vulnerability to deleterious effects of lead, the developing organism
generally appears to be more sensitive than the mature individual. A more detailed and quan-
titative examination of overall exposure-effect relationships for lead is presented in
Chapter 13.
12.10.2 Subcellular Effects of Lead
The biological basis of lead toxicity is its ability to bind to ligating groups in bio-
molecular substances crucial to various physiological functions, thereby interfering with
these functions by, for example, competing with native essential metals for binding sites,
inhibiting enzyme activity, and inhibiting or otherwise altering essential ion transport.
These effects are modulated by: 1) the inherent stability of such binding sites for lead; 2)
the compartmentalization kinetics governing lead distribution among body compartments, among
tissues, and within cells; and 3) the differences in biochemical organization across cells and
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tissues due to their specific functions. Given the complexities introduced by items 2 and 3,
it is not surprising that no single unifying mechanism of lead toxicity across all tissues in
humans and experimental animals has yet been demonstrated.
In so far as effects of lead on activity of various enzymes are concerned, many of the
available studies concern rn vitro behavior of relatively pure enzymes with marginal relevance
to various effects jjn vivo. On the other hand, certain enzymes are basic to the effects of
lead at the organ or organ system level, and discussion is best reserved for such effects in
the summary sections below dealing with lead effects on particular organ systems. This sec-
tion is mainly concerned with organellar effects of lead, expecially those which provide some
rationale for lead toxicity at higher levels of biological organization. Particular emphasis
is placed on the mitochondrion, because this organelle is not only affected by lead in numer-
ous ways but has also provided the most data bearing on the subcellular effects of lead.
The critical target organdie for lead toxicity in a variety of cell and tissue types
clearly is the mitochondrion, followed probably by cellular and intracellular membranes. The
mitochondria! effects take the form of structural changes and marked disturbances in mitochon-
drial function within the cell, particularly in energy metabolism and ion transport. These
effects in turn are associated with demonstrable accumulation of lead in mitochondria, both
j_n vivo and HI vitro. Structural changes include mitochondrial swelling in a variety of cell
types as well as distortion and loss of cristae, which occur at relatively moderate lead
levels. Similar changes have also been documented in lead workers across a range of exposures.
Uncoupled energy metabolism, inhibited cellular respiration using both succinate and
nicotinamide adenine dinucleotide (NAD)-linked substrates, and altered kinetics of intracellu-
lar calcium have been demonstrated jjn vivo using mitochondria of brain and non-neural tissue.
In some cases, the lead exposure level associated with such changes has been relatively low.
Several studies document the relatively greater sensitivity of this organelle in young vs.
adult animals in terms of mitochondrial respiration. The cerebellum appears to be particular-
ly sensitive, providing a connection between mitochondrial impairment and lead encephalopathy.
Impairment by lead of mitochondrial function in the developing brain has also been consistent-
ly associated with delayed brain development, as indexed by content of various cytochromes.
In the rat pup, ongoing lead exposure from birth is required for this effect to be expressed,
indicating that such exposure must occur before, and is inhibitory to, the burst of oxidative
metabolism activity that occurs in the young rat at 10 to 21 days postnatally.
J_n vivo lead exposure of adult rats also markedly inhibits cerebral cortex intracellular
calcium turnover in a cellular compartment that appears to be the mitochondrion. The effect
was seen at a brain lead level of 0.4 ppm. These results are consistent with a separate study
showing increased retention of calcium in the brain of lead-dosed guinea pigs. Numerous
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reports have described the j_n vivo accumulation of lead in mitochondria of kidney, liver,
spleen, and brain tissue, with one study showing that such uptake was slightly more than
occurred in the cell nucleus. These data are not only consistent with deleterious effects of
lead on mitochondria but are also supported by other investigations J_n vitro.
Significant decreases in mitochondrial respiration jn vitro using both NAD-1inked and
succinate substrates have been observed for brain and non-neural tissue mitochondria in the
presence of lead at micromolar levels. There appears to be substrate specificity in the inhi-
bition of respiration across different tissues, which may be a factor in differential organ
toxicity. Also, a number of enzymes involved in intermediary metabolism in isolated mitochon-
dria have been observed to undergo significant inhibition of activity with lead.
Of particular interest regarding lead effects on isolated mitochondria are ion transport
effects, especially in regard to calcium. Lead movement into brain and other tissue mitochon-
dria involves active transport, as does calcium. Recent sophisticated kinetic analyses of
desaturation curves for radiolabeled lead or calcium indicate that there is striking overlap
in the cellular metabolism of calcium and lead. These studies not only establish the basis of
lead's easy entry into cells and cell compartments, but also provide a basis for lead's im-
pairment of intracellular ion transport, particularly in neural cell mitochondria, where the
capacity for calcium transport is 20-fold higher than even in heart mitochondria.
Lead is also selectively taken up in isolated mitochondria j_n vitro, including the mito-
chondria of synaptosomes and brain capillaries. Given the diverse and extensive evidence of
lead's impairment of mitochondrial structure and function as viewed from a subcellular level,
it is not surprising that these derangements are logically held to be the basis of dysfunction
of heme biosynthesis, erythropoiesis, and the central nervous system. Several key enzymes in
the heme biosynthetic pathway are intramitochondrial, particularly ferrochelatase. Hence, it
is to be expected that entry of lead into mitochondria will impair overall heme biosynthesis,
and in fact this appears to be the case in the developing cerebellum. Furthermore, lead
levels associated with entry of lead into mitochondria and expression of mitochondrial injury
can be relatively moderate.
Lead exposure provokes a typical cellular reaction in human and other species that has
been morphologically characterized as a lead-containing nuclear inclusion body. While it has
been postulated that such inclusions constitute a cellular protection mechanism, such a
mechanism is an imperfect one. Other organdies, e.g., the mitochondrion, also take up lead
and sustain injury in the presence of nuclear inclusion formations.
In theory, the cell membrane is the first organelle to encounter lead and it is not
surprising that cellular effects of lead can be ascribed to interactions at cellular and
intracellular membranes in the form of distrubed ion transport. The inhibition of membrane
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(Na+,K )-ATPase of erythrocytes as a factor in lead-impaired erythropoiesis is noted else-
where. Lead also appears to interfere with the normal processes of calcium transport across
membranes of different tissues. In peripheral cholinergic synaptosomes, lead is associated
with retarded release of acetylcholine owing to a blockade of calcium binding to the membrane,
while calcium accumulation within nerve endings can be ascribed to inhibition of membrane
(Na*,K+)-ATPase.
Lysosomes accumulate in renal proximal convoluted tubule cells of rats and rabbits given
lead over a range of dosing. This also appears to occur in the kidneys of lead workers and
seems to represent a disturbance in normal lysosomal function, with the accumulation of
lysosomes being due to enhanced degradation of proteins because of the effects of lead else-
where within the cell.
12.10.3. Effects of Lead on Heme Biosynthesis, Erythropoiesis. and Erythrocyte Physiology in
Humans and Animals
The effects of lead on heme biosynthesis are well known both because of their prominence
and the large number of studies of these effects in humans and experimental animals. The
process of heme biosynthesis starts with glycine and succinyl-coenzyme A, proceeds through
formation of protoporphyrin IX, and culminates with the insertion of divalent iron into the
porphyrin ring, thus forming heme. In addition to being a constituent of hemoglobin, heme is
the prosthetic group of numerous tissue hemoproteins having variable functions, such as myo-
globin, the P-450 component of the mixed function oxygenase system, and the cytochromes of
cellular energetics. Hence, disturbance of heme biosynthesis by lead poses the potential for
multiple-organ toxicity.
The steps in the heme synthesis pathway that have been best studied in regard to lead
effects involve three enzymes: (1) stimulation of mitochondrial delta-aminolevulinic acid
synthetase (ALA-S), which mediates formation of delta-aminolevulinic acid (ALA); (2) direct
inhibition of the cytosolic enzyme, delta-aminolevulinic acid dehydrase (ALA-D), which cata-
lyzes formation of porphobilinogen from two units of ALA; and (3) inhibition of insertion of
iron (II) into protoporphyrin IX to form heme, a process mediated by the enzyme ferrochelatase.
Increased ALA-S activity has been documented in lead workers as well as lead-exposed ani-
mals, although the converse, an actual decrease in enzyme activity, has also been observed in
several experimental studies using different exposure methods. It would appear, then, that
enzyme activity increase via feedback derepression or that activity inhibition may depend on
the nature of the exposure. In an iji vitro study using rat liver cells in culture, ALA-S
activity could be stimulated at levels as low as 5.0 uM or 1.0 ug Pb/g preparation. In the
same study, increased activity was seen to be due to biosynthesis of more enzyme. The thres-
hold for lead stimulation of ALA-S activity in humans, based upon a study using leukocytes
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from lead workers, appears to be about 40 |jg Pb/dl. The generality of this threshold level to
other tissues is dependent upon how well the sensitivity of leukocyte mitochondria mirrors
that in other systems. It would appear that the relative impact of ALA-S activity stimulation
on ALA accumulation at lower levels of lead exposure is considerably less than the effect of
ALA-D activity inhibition: at 40 ug/dl blood lead, ALA-D activity is significantly depressed,
while ALA-S activity only begins to be affected.
Erythrocyte ALA-D activity is very sensitive to lead inhibition, which is reversed by re-
activation of the sulfhydryl group with agents such as dithiothreitol, zinc, or zinc plus glu-
tathione. The zinc levels employed to achieve reactivation, however, are well above normal
physiological levels. Although zinc appears to offset the inhibitory effects of lead observed
in human erythrocytes jm vitro and in animal studies, lead workers exposed to both zinc and
lead do not show significant changes in the relationship of ALA-D activity to blood lead con-
centration when compared to workers exposed only to lead. By contrast, zinc deficiency in
animals has been shown to significantly inhibit ALA-D activity, with concomitant accumulation
of ALA in urine. Since zinc deficiency has also been associated with increased lead absorp-
tion in experimental studies, the possibility exists for a dual effect of such deficiency on
ALA-D activity: (1) a direct effect on activity due to reduced zinc availability, as well as
(2) the effect of increased lead absorption leading to further inhibition of such activity.
The activity of erythrocyte ALA-D appears to be inhibited at virtually all blood lead
levels measured so far, and any threshold for this effect in either adults or children remains
to be determined. A further measure of this enzyme's sensitivity to lead comes from a report
noting that rat bone marrow suspensions show inhibition of ALA-D activity by lead at a level
of 0.1 ug/g suspension. Inhibition of ALA-D activity in erythrocytes apparently reflects a
similar effect in other tissues. Hepatic ALA-D activity was inversely correlated in lead
workers with both the erythrocyte activity as well as blood lead. Of significance are the ex-
perimental animal data showing that (1) brain ALA-D activity is inhibited with lead exposure
and (2) inhibition appears to occur to a greater extent in the brain of developing vs. adult
animals. This presumably reflects greater retention of lead in developing animals. In the
avian brain, cerebellar ALA-D activity is affected to a greater extent than that of the
cerebrum and, relative to lead concentration, shows inhibition approaching that occurring in
erythrocytes.
The inhibition of ALA-D activity by lead is reflected in increased levels of its sub-
strate, ALA, in blood, urine, and tissues. In one investigation, the increase in urinary ALA
was seen to be preceded by a rise in circulating levels of the metabolite. Blood ALA levels
were elevated at all corresponding blood lead values down to the lowest value determined (18
ug/dl), while urinary ALA was seen to rise exponentially with blood ALA. Numerous independent
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studies have documented that there is a direct correlation between blood lead and the loga-
rithm of urinary ALA in adult humans and children, and that the threshold is commonly accepted
as being 40 ug/dl. Several studies of lead workers also indicate that the correlation of
urinary ALA with blood lead continues below this value. Furthermore, one report has demon-
strated that the slope of the dose-effect curve in lead workers is dependent upon the level of
exposure.
The health significance of lead-inhibited ALA-D activity and accumulation of ALA at low
levels of exposure has been an issue of some controversy. One view is that the "reserve
capacity" of ALA-D activity is such that only high accumulations of the enzyme's substrate,
ALA, in accessible indicator media would result in significant inhibition of activity. One
difficulty with this view is that it is not possible to quantify at lower levels of lead ex-
posure the relationship of urinary ALA to levels in target tissues nor to relate the potential
neurotoxicity of ALA at any level of build-up to levels in indicator media; i.e., the thres-
hold for potential neurotoxicity of ALA in terms of blood lead may be different from the level
associated with urinary accumulation.
Accumulation of protoporphyrin in the erythrocytes of individuals with lead intoxication
has been recognized since the 1930s, but it has only recently been possible to quantitatively
assess the nature of this effect via the development of specific, sensitive microanalysis
methods. Accumulation of protoporphyrin IX in erythrocytes is the result of impaired place-
ment of iron (II) in the porphyrin moiety to form heme, an intramitochondrial process mediated
by the enzyme ferrochelatase. In lead exposure, the porphyrin acquires a zinc ion in lieu of
native iron, thus forming zinc protoporphyrin (ZPP), and is tightly bound in available heme
pockets for the life of the erythrocytes. This tight sequestration contrasts with the rela-
tively mobile non-metal, or free, erythrocyte protoporphyrin (FEP) accumulated in the congen-
ital disorder erythropoietic protoporphyria.
Elevation of erythrocyte ZPP has been extensively documented as being exponentially cor-
related with blood lead in children and adult lead workers and is presently considered one of
the best indicators of undue lead exposure. Accumulation of ZPP only occurs in erythrocytes
formed during lead's presence in erythroid tissue, resulting in a lag of at least several
weeks before such build-up can be measured. It has been shown that the level of such accumu-
lation in erythrocytes of newly-employed lead workers continues to increase when blood lead
has already reached a plateau. This would influence the relative correlation of ZPP and blood
lead in workers with a short exposure history. In individuals removed from occupational expo-
sure, the ZPP level in blood declines much more slowly than blood lead, even years after re-
moval from exposure or after a drop in blood lead. Hence, ZPP level would appear to be a more
reliable indicator of continuing intoxication from lead resorbed from bone.
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The measurable threshold for lead-induced ZPP accumulation is affected by the relative
spread of blood lead and corresponding ZPP values measured. In young children (under four
years old) the ZPP elevation typically associated with iron-deficiency anemia should be taken
into account. In adults, several studies indicate that the threshold for ZPP elevation with
respect to blood lead is approximately 25-30 HO/dl- In children 10-15 years old the threshold
is about 16 (jg/dl; in this age group, iron deficiency is not a factor. In one report, it was
noted that children over four years old showed the same threshold, 15.5 |jg/dl, as a second
group under four years old, indicating that iron deficiency was not a factor in the study.
Fifty percent of the children were found to have significantly elevated EP levels (2 standard
deviations above reference mean EP) or a dose-response threshold level of 25 (jg/dl.
Within the blood lead range considered "normal," i.e., below 30-40 ug/dl, any assessment
of the ZPP-blood lead relationship is strongly influenced by the relative analytical profi-
ciency for measurement of both blood lead and EP. The types of statistical treatments
employed in analyzing the data are also important. In a recent detailed statistical study
involving 2004 children, 1852 of whom had blood lead values below 30 pg/dl, segmental line and
probit analysis techniques were employed to assess the dose-effect threshold and dose-response
relationship. An average blood lead threshold for the effect using both statistical tech-
niques yielded a value of 16.5 (jg/dl for either the full group or those subjects with blood
lead levels below 30 ug/dl. The effect of iron deficiency was tested for and removed. Of
particular interest was the finding that the blood lead values corresponding to EP elevations
more than 1 or 2 standard deviations above the reference mean in 50 percent of the children
were 28.6 or 35.7 ug Pb/dl, respectively. Hence, fully half of the children were seen to have
significant elevations of EP at blood lead levels around the currently accepted cut-off value
for undue lead exposure, 30 ug/dl. From various reports, children and adult females appear to
be more sensitive to the effects of lead on EP accumulation at any given blood lead level,
with children being somewhat more sensitive than adult females.
Effects of lead on ZPP accumulation and reduced heme formation are not restricted to the
erythropoietic system. Recent studies show that reduction of serum 1,25-dihydroxy vitamin D
seen with even low level lead exposure is apparently the result of lead's inhibition of the
activity of renal 1-hydroxylase, a cytochrome P-450 mediated enzyme. Cytochrome P-450, a
heme-containing protein, is an integral part of the hepatic mixed function oxygenase system
and is known to be affected in humans and animals by lead exposure, particularly acute intoxi-
cation. Reduced P-450 content has been found to be correlated with impaired activity of such
detoxifying enzyme systems as aniline hydroxylase and aminopyrine demethylase.
Studies of organotypic chick dorsal root ganglion in culture show that the nervous system
not only has heme biosynthetic capability but also such preparations elaborate porphyrinic ma-
terial in the presence of lead. In the neonatal rat, chronic lead exposure resulting in
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moderately elevated blood lead levels is associated with retarded growth in the hemoprotein
cytochrome C and with disturbed electron transport in the developing rat cerebral cortex.
These data parallel the effect of lead on ALA-D activity and ALA accumulation in neural
tissue. When these effects are viewed in the toxicokinetic context of increased retention of
lead in both developing animals and children, there is an obvious, serious potential for
impaired heme-based metabolic function in the nervous system of lead-exposed children.
As can be seen from the above discussion, the health significance of ZPP accumulation
rests with the fact that such build-up is evidence of impaired heme and hemoprotein formation
in tissues, particularly the nervous system, arising from entry of lead into mitochondria.
Such evidence for reduced heme synthesis is consistent with a diverse body of data documenting
lead-associated effects on mitochondria, including impairment of ferrochelatase activity. As
a mitochondrial enzyme, ferrochelatase activity may be inhibited either directly by lead or
indirectly by impairment of iron transport to the enzyme.
The relative value of the lead-ZPP relationship in erythropoietic tissue as an index of
this effect in other tissues hinges on the relative sensitivity of the erythropoietic system
compared with other systems. For example, one study of rats exposed to low levels of lead
over their lifetime demonstrated that protoporphyrin accumulation in renal tissue was already
significant at levels of lead exposure where little change was seen in erythrocyte porphyrin
levels. The issue of sensitivity is obviously distinct from the question of which system is
most accessible to measurement of the effect.
Other steps in the heme biosynthesis pathway are also known to be affected by lead, al-
though these have not been studied as much on a biochemical or molecular level. Levels of co-
proporphyrin are increased in urine, reflecting active lead intoxication. Lead also affects
the activity of the enzyme uroporphyrinogen-I-synthetase, resulting in an accumulation of its
substrate, porphobilinogen. The erythrocyte enzyme is much more sensitive to lead than the
hepatic species and presumably accounts for much of the accumulated substrate.
Anemia is a manifestation of chronic lead intoxication, being characterized as mildly
hypochromic and usually normppytic. It is associated with reticulocytosis, owing to shortened
cell survival, and the variable presence of basophilic stippling. Its occurrence is due to
both decreased production and increased rate of destruction of erythrocytes. In children
under four years old, the anemia of iron deficiency is exacerbated by lead, and vice versa.
Hemoglobin production is negatively correlated with blood lead levels in young children, where
iron deficiency may be a confounding factor, as well as in lead workers. In one study, blood
lead values that were usually below 80 jag/dl were inversely correlated with hemoglobin content.
In these subjects, iron deficiency was found to be absent. The blood lead threshold for
reduced hemoglobin content is about 50 ng/dl in adult lead workers and somewhat lower in child-
ren, around 40 ug/dl.
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The mechanism of lead-associated anemia appears to be a combination of reduced hemoglobin
production and shortened erythrocyte survival because of direct cell injury. Effects of lead
on hemoglobin production involve disturbances of both heme and globin biosynthesis. The hemo-
lytic component to lead-induced anemia appears to be due to increased cell fragility and in-
creased osmotic resistance. In one study using rats, it was noted that the reduced cell
deformability and consequent hemolysis associated with vitamin E deficiency is exacerbated by
lead exposure. The molecular basis for increased cell destruction rests with inhibition of
(Na , K )-ATPase and pyrimidine-5'-nucleotidase. Inhibition of the former enzyme leads to
cell "shrinkage," and inhibition of the latter results in impaired pyrimidine nucleotide phos-
phorolysis and disturbance of the activity of the purine nucleotides necessary for cellular
energetics.
Tetraethyl lead and tetramethyl lead, components of leaded gasoline, undergo transforma-
tion i_n vivo to the neurotoxic trialkyl metabolites as well as further conversion to inorganic
lead. Hence, one might anticipate that exposure to such agents may show effects commonly
associated with inorganic lead in terms of heme synthesis and erythropoiesis. Various surveys
and case reports make it clear that leaded-gasoline sniffing is associated with chronic lead
intoxication in children from socially deprived backgrounds in rural or remote areas. Notable
in these subjects is evidence of impaired heme biosynthesis as indexed by significantly
reduced ALA-D activity. In several case reports of frank lead toxicity from habitual sniffing
of leaded gasoline, such effects as basophilic stippling in erythrocytes and significantly
reduced hemoglobin have also been noted.
Lead-associated disturbances of heme biosynthesis as a possible factor underlying neuro-
logical effects of lead are of considerable interest because of (1) the recognized similarity
between the classical signs of lead neurotoxicity and numerous neurological components of the
congenital disorder known as acute intermittent porphyria, as well as (2) some unusual aspects
of lead neurotoxicity. There are two possible points of connection between lead effects on
both heme biosynthesis and the nervous system. Concerning the similarity of lead neurotoxi-
city to acute intermittent porphyria, there is the common feature of excessive systemic accum-
ulation and excretion of ALA. Second, lead neurotoxicity reflects, to some degree, impaired
synthesis of heme and hemoproteins involved in crucial cellular functions. Available informa-
tion indicates that ALA levels are elevated in the brain of lead-exposed animals, arising via
jj\ situ inhibition of brain ALA-D activity or via transport to the brain after formation in
other tissues. ALA is known to traverse the blood-brain barrier. Hence, ALA is accessible
to, or formed within, the brain during lead exposure and may express its neurotoxic potential.
Based on various ir\ vitro and ui vivo data obtained in the context of neurochemical
studies of lead neurotoxicity, it appears that ALA can readily affect GABAergic function,
particularly inhibiting release of the neurotransmitter GABA from presynaptic receptors, where
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ALA appears to be very potent even at low levels. In an j_n vitro study, agonist behavior by
ALA was demonstrated at levels as low as 1.0 uM ALA. This j_n vitro observation supports
results of a study using lead-exposed rats in which there was reported inhibition of both
resting and K+-stimulated preloaded 3H-GABA. Further evidence for an effect of some agent
other than lead acting directly is the observation that i_n vivo effects of lead on neurotrans-
mitter function cannot be duplicated with HI vitro preparations to which lead is added. Human
data on lead-induced associations between disturbed heme synthesis and neurotoxicity, while
limited, also suggest that ALA may function as a neurotoxicant.
The connection between impaired heme and hemoprotein synthesis in the brain of the neo-
natal rat was noted earlier. In these studies there was reduced cytochrome C production and
impaired operation of the cytochrome C respiratory chain. Hence, one might expect that such
impairment would be most prominent in areas of relatively greater cellularization, such as the
hippocampus. As noted in Chapter 10, these are also regions where selective lead accumulation
appears to occur.
12.10.4 Neurotoxic Effects of Lead
An assessment of the impact of lead on human and animal neurobehavioral function raises a
number of issues. Among the key points addressed here are: (1) the internal exposure levels,
as indexed by blood lead levels, at which various neurotoxic effects occur; (2) the persis-
tence or reversibility of such effects; and (3) populations that appear to be most susceptible
to neural damage. In addition, the question arises as to the utility of using animal studies
to draw parallels to the human condition.
12.10.4.1 Internal Lead Levels at which Neurotoxic Effects Occur. Markedly elevated blood
lead levels are associated with the most serious neurotoxic effects of lead exposure
(including severe, irreversible brain damage as indexed by the occurrence of acute or chronic
encephalopathic symptoms, or both) in both humans and animals. For most adult humans, such
damage typically does not occur until blood lead levels exceed 120 ug/dl. Evidence does
exist, however, for acute encephalopathy and death occurring in some human adults at blood
lead levels below 120 ug/dl. In children, the effective blood lead level for producing
encephalopathy or death is lower, starting, at approximately 80-100 ug/dl. It should be
emphasized that, once encephalopathy occurs, death is not an improbable outcome, regardless of
the quality of medical treatment available at the time of acute crisis. In fact, certain
diagnostic or treatment procedures themselves may exacerbate matters and push the outcome
toward fatality if the nature and severity of the problem are not diagnosed or fully recog-
nized. It is also crucial to note the rapidity with which acute encephalopathic symptoms can
develop or death can occur in apparently asymptomatic individuals or in those apparently only
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mildly affected by elevated lead body burdens. Rapid deterioration often occurs, with
convulsions or coma suddenly appearing with progression to death within 48 hours. This
strongly suggests that even in apparently asymptomatic individuals, rather severe neural
damage probably exists at high blood lead levels even though it is not yet overtly manifested
in obvious encephalopathic symptoms. This conclusion is further supported by numerous studies
showing that overtly lead intoxicated children with high blood lead levels, but not observed
to manifest acute encephalopathic symptoms, are permanently cognitively impaired, as are most
children who survive acute episodes of frank lead encephalopathy.
Recent studies show that overt signs and symptoms of neurotoxicity (indicative of both
CNS and peripheral nerve dysfunction) are detectable in some human adults at blood lead levels
as low as 40-60 |jg/dl, levels well below the 60 or 80 ug/dl criteria previously discussed as
being "safe" for adult lead exposures. In addition, certain electrophysiological studies of
peripheral nerve function in lead workers, indicate that slowing of nerve conduction veloc-
ities in some peripheral nerves are associated with blood lead levels as low as 30-50 ug/dl
(with no clear threshold for the effect being evident). These results are indicative of
neurological dysfunctions occurring at relatively low lead levels in non-overtly lead intoxi-
cated adults.
Other evidence tends to confirm that neural dysfunctions exist in apparently asymptomatic
children, at similar or even lower levels of blood lead. The body of studies on low-or
moderate-level lead effects on neurobehavioral functions in non-overtly lead intoxicated child-
ren, as summarized in Table 12-1, presents an array of data pointing to that conclusion.
Several well-controlled studies have found effects that are clearly statistically significant,
whereas other have found nonsignificant but borderline effects. Even some studies reporting
generally nonsignificant findings at times contain data confirming some statistically signif-
icant effects, which the authors attribute to various extraneous factors. It should also be
noted that, given the apparent nonspecific nature of some of the behavioral or neural effects
probable at low levels of lead exposure, one would not expect to find striking differences in
every instance. The lowest observed blood lead levels associated with significant neurobehav-
ioral deficits indicative of CNS dysfunction, both in apparently asymptomatic children and in
developing rats and monkeys generally appear to be in the range of 30-50 ug/dl. However,
other types of neurotoxic effects, e.g., altered EEG patterns, have been reported at lower
levels, supporting a continuous dose-response relationship between lead and neurotoxicity.
Such effects, when combined with adverse social factors (such as low parental IQ, low socio-
economic status, poor nutrition, and poor quality of the caregiving environment) can place
children, especially those below the age of three years, at significant risk. However, it
must be acknowledged that nutritional covariates, as well as demographic social factors, have
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been poorly controlled in many of the human studies reviewed. Socioeconomic status also is a
crude measure of parenting and family structure that requires further assessment as a possible
contributor to observed results of neurobehavioral studies.
Timing, type, and duration of exposure are important factors in both animal and human
studies. It is often uncertain whether observed blood lead levels represent the levels that
were responsible for observed behavioral deficits or electrophysiological changes. Monitoring
of lead exposures in human subjects in all cases has been highly intermittent or nonexistent
during the period of life preceding neurobehavioral assessment. In most human studies, only
one or two blood lead values are provided per subject. Tooth lead may be an important cumula-
tive exposure index, but its modest, highly variable correlation to blood lead or FEP and to
external exposure levels makes findings from various studies difficult to compare quantita-
tively. The complexity of the many important covariates and their interaction with dependent
variable measures of modest validity, e.g., IQ tests, may also account for many of the discrep-
ancies among the different studies.
12.10.4.2 Early Development and the Susceptibility to Neural Damage. On the question of
early childhood vulnerability, the neurobehavioral data are consistent with morphological and
biochemical studies of the susceptibility of the heme biosynthetic pathway to perturbation by
lead. Various lines of evidence suggest that the order of susceptibility to lead's effects
is: (1) young > adults and (2) female > male. Animal studies also have pointed to the peri-
natal period of ontogeny as a particularly critical time for a variety of reasons: (1) it is
a period of rapid development of the nervous system; (2) it is a period where good nutrition
is particularly critical; and (3) it is a period where the caregiver environment is vital to
normal development. However, the precise boundaries of a critical period are not yet clear
and may vary depending on the species and function or endpoint that is being assessed. Never-
theless, there is general agreement that human infants and toddlers below the age of three
years are at special risk because of jj\ utero exposure, increased opportunity for exposure
because of normal mouthing behavior, and increased rates of lead absorption due to various
factors, e.g., nutritional deficiences.
12.10.4.3 The Question of Irreversibility. Little research on humans is available on persis-
tence of effects. Some work suggests that mild forms of peripheral neuropathy in lead workers
may be reversible after termination of lead exposure, but little is known regarding the rever-
sibility of lead effects on central nervous system function in humans. A recent two-year
follow-up study of 28 children of battery factory workers found a continuing relationship
between blood lead levels and altered slow wave voltage of cortical slow wave potentials indic-
ative of persisting CNS effects of lead. Current population studies, however, will have to be
supplemented by prospective longitudinal studies of the effects of lead on development in
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order to address the issue of reversibility or persistence of lead neurotoxic effects in
humans more satisfactorily.
Various animal studies provide evidence that alterations in neurobehavioral function may
be long-lived, with such alterations being evident long after blood lead levels have returned
to control levels. These persistent effects have been demonstrated in monkeys as well as rats
under a variety of learning performance test paradigms. Such results are also consistent with
morphological, electrophysiological, and biochemical studies on animals that suggest lasting
changes in synaptogenesis, dendritic development, myelin and fiber tract formation, ionic
mechanisms of neurotransmission, and energy metabolism.
12.10.4.4 Utility of Animal Studies in Drawing Parallels to the Human Condition. Animal
models are used to shed light on questions where it is impractical or ethically unacceptable
to use human subjects. This is particularly true in the case of exposure to environmental
toxins such as lead. In the case of lead, it has been effective and convenient to expose
developing animals via their mothers' milk or by gastric gavage, at least until weaning. In
many studies, exposure was continued in the water or food for some time beyond weaning. This
approach simulates at least two features commonly found in human exposure: oral intake and
exposure during early development. The preweaning period in rats and mice is of particular
relevance to in terms of parallels with the first two years or so of human brain development.
However, important questions exist concerning the comparability of animal models to
humans. Given differences between humans, rats, and monkeys in heme chemistry, metabolism,
and other aspects of physiology and anatomy, it is difficult to state what constitutes an
equivalent internal exposure level (much less an equivalent external exposure level). For
example, is a blood lead level of 30 ug/dl in a suckling rat equivalent to 30 ug/dl in a
three-year-old child? Until an answer is available to this question, i.e., until the function
describing the relationship of exposure indices in different species is available, the utility
of animal models for deriving dose-response functions relevant to humans will be limited.
Questions also exist regarding the comparability of neurobehavioral effects in animals
with human behavior and cognitive function. One difficulty in comparing behavioral endpoints
such as locomotor activity is the lack of a consistent operational definition. In addition to
the lack of standardized methodologies, behavior is notoriously difficult to "equate" or com-
pare meaningfully across species because behavioral analogies do not demonstrate behavioral
homologies. Thus, it is improper to assume, without knowing more about the responsible under-
lying neurological structures and processes, that a rat's performance on an operant condition-
ing schedule or a monkey's performance on a stimulus discrimination task corresponds to a
child's performance on a cognitive function test. Still deficits in performance on such tasks
are indicative of altered CNS function which is likely to parallel some type of altered human
CNS function as well.
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In terms of morphological findings, there are reports of hippocampal lesions in both
lead-exposed rats and humans that are consistent with a number of behavioral findings suggest-
ing an impaired ability to respond appropriately to altered contingencies for rewards. That
is, subjects tend to persist in certain patterns of behavior even when changed conditions make
the behavior inappropriate. Other morphological findings in animals, such as demyelination
and glial cell decline, are comparable to human neuropathologic observations mainly at rela-
tively high exposure levels.
Another neurobehavioral endpoint of interest in comparing human and animal neurotoxicity
of lead is electrophysiological function. Alterations of electroencephalographic patterns and
cortical slow wave voltage have been reported for lead-exposed children, and various electro-
physiological alterations both i_n vivo (e.g., in rat visual evoked response) and i_n vitro
(e.g., in frog miniature endplate potentials) have also been noted in laboratory animals. At
this time, however, these lines of work have not converged sufficiently to allow for strong
conclusions regarding the electrophysiological aspects of lead neurotoxicity.
Biochemical approaches to the experimental study of leads effects on the nervous system
have generally been limited to laboratory animal subjects. Although their linkage to human
neurobehavioral function is at this point somewhat speculative, such studies do provide in-
sight to possible neurochemical intermediaries of lead neurotoxicity. No single neurotrans-
mitter system has been shown to be particularly sensitive to the effects of lead exposure;
lead-induced alterations have been demonstrated in various neurotransmitters, including dopa-
mine, norepinephrine, serotonin, and gamma-aminobutyric acid. In addition, lead has been
shown to have subcellular effects in the central nervous system at the level of mitochondria!
function and protein synthesis.
Given the above-noted difficulties in formulating a comparative basis for internal expo-
sure levels among different species, the primary value of many animal studies, particularly iji
vitro studies, may be in the information they can provide on basic mechanisms involved in lead
neurotoxicity. A number of in vitro studies show that significant, potentially deleterious
effects on nervous system function occur at In situ lead concentrations of 5 uM and possibly
lower, suggesting that no threshold may exist for certain neurochemical effects of lead on a
subcellular or molecular level. The relationship between blood lead levels and lead concen-
trations at such extra- or intracellular sites of action, however, remains to be determined.
Despite the problems in generalizing from animals to humans, both the animal and the human
studies show great internal consistency in that they support a continuous dose-response
functional relationship between lead and neurotoxic biochemical, morphological, electrophysio-
logical, and behavioral effects.
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12.10.5 Effects of Lead on the Kidney
It has been known for more than a century that kidney disease can result from lead
poisoning. Identifying the contributing causes and mechanisms of lead-induced nephropathy has
been difficult, however, in part because of the complexities of human exposure to lead and
other nephrotoxic agents.
Nevertheless, it is possible to estimate at least roughly lead exposure ranges associated
with detectable renal dysfunction in both human adults and children. More specifically,
numerous studies of occupationally exposed workers have provided evidence for lead-induced
chronic nephropathy being associated with blood lead levels ranging from 40 to more than
100 ug/dl, and some are suggestive of renal effects possibly occurring even at levels as low
as 30 ug/dl. Similarly, in children, the relatively sparse evidence available points to the
manifestation of renal dysfunction, as indexed for example by generalized aminoaciduria, at
blood lead levels across the range of 40 to more than 100 ug/dl. The current lack of evidence
for renal dysfunction at lower blood lead levels in children may simply reflect the greater
clinical concern with neurotoxic effects of lead intoxication in children. The persistence of
lead-induced renal dysfunction in children also remains to be more fully investigated, al-
though a few studies indicate that children diagnosed as being acutely lead poisoned experi-
ence lead nephropathy effects lasting throughout adulthood.
Parallel results from experimental animal studies reinforce the findings in humans and
help illuminate the mechanisms underlying such effects. For example, a number of transient
effects in human and animal renal function are consistent with experimental findings of revers-
ible lesions such as nuclear inclusion bodies, cytomegaly, swollen mitochondria, and increased
numbers of iron-containing lysosomes in proximal tubule cells. Irreversible lesions such as
interstitial fibrosis are also well documented in both humans and animals following chronic
exposure to high doses of lead. Functional renal changes observed in humans have also been
confirmed in animal model systems with respect to increased excretion of amino acids and
elevated serum urea nitrogen and uric acid concentrations. The inhibitory effects of lead
exposure on renal blood flow and glomerular filtration rate are currently less clear in exper-
imental model systems; further research is needed to clarify the effects of lead on these
functional parameters in animals. Similarly, while lead-induced perturbation of the renin-
angiotensin system has been demonstrated in experimental animal models, further research is
needed to clarify the exact relationships among lead exposure (particularly chronic low-level
exposure), alteration of the renin-angiotensin system, and hypertension in both humans and
animals.
On the biochemical level, it appears that lead exposure produces changes at a number of
sites. Inhibition of membrane marker enzymes, decreased mitochondria! respiratory function/
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cellular energy production, inhibition of renal heme biosynthesis, and altered nucleic acid
synthesis are the most marked changes to have been reported. The extent to which these mito-
chondrial alterations occur is probably mediated in part by the intracellular bioavailability
of lead, which is determined by its binding to high affinity kidney cytosolic binding proteins
and deposition within intranuclear inclusion bodies.
Recent studies in humans have indicated that the EDTA lead-mobilization test is the most
reliable technique for detecting persons at risk for chronic nephropathy. Blood lead measure-
ments are a less satisfactory indicator because they may not accurately reflect cumulative
absorption some time after exposure to lead has terminated.
A number of major questions remain to be more definitively answered concerning the effect
of lead on the kidney. Can a distinctive lead-induced renal lesion be identified either in
functional or histologic terms? What biologic measurements are most reliable for the predic-
tion of lead-induced nephropathy? What is the incidence of lead nephropathy in the general
population as well as among specifically defined subgroups with varying exposure? What is the
natural history of treated and untreated lead nephropathy? What is the mechanism of lead-
induced hypertension and renal injury? What are the contributions of environmental and
genetic factors to the appearance of renal injury due to lead? At what level of lead in blood
can the kidneys be affected? Is there a threshold for renal effects of lead? The most dif-
ficult question to answer may well be to determine the contribution of low levels of lead
exposure to renal disease of non-lead etiologies.
12.10.6 Effects of Lead on Reproduction and Development
Data from human and animal studies indicate that lead may exert gametotoxic, embryotoxic,
and (according to some animal studies) teratogenic effects that may influence the survival and
development of the fetus and newborn. Prenatal viability and development, it appears, may
also be affected indirectly, contributing to concern for unborn children and, therefore, preg-
nant women or women of childbearing age being group at special risk for lead effects. Early
studies of quite high dose lead exposure in pregnant women indicate toxic--but not tera-
togenic—effects on the conceptus. Effects on reproductive performance in women at lower
exposure levels are not well documented. Unfortunately, currently available human data
regarding lead effects on the fetus during development generally do not lend themselves to
accurate estimation of lowest observed or no-effect levels. However, some studies have shown
that fetal heme synthesis is affected at maternal and fetal blood lead levels as low as
approximately 15 ug/dl, as indicated by urinary ALA levels and ALA-D activity. This observed
effect level is consistant with lowest observed effect levels for indications of altered heme
synthesis seen at later ages for preschool and older children.
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There are currently no reliable data pointing to adverse effects in human offspring fol-
lowing paternal exposure to lead, but industrial exposure of men to lead at levels resulting
in blood lead values of 40-50 pg/dl appear to have resulted in altered testicular function.
Also, another study provided evidence of effects of prostatic and seminal vesicle functions at
40-50 ug/dl blood lead levels in lead workers.
The paucity of human exposure data force an examination of the animal studies for indica-
tions of threshold levels for effects of lead on the conceptus. It must be noted that the
animal data are almost entirely derived from rodents. Based on these rodent data, it seems
likely that fetotoxic effects have occurred in animals at chronic exposures to 600-1000 ppm
lead in the diet. Subtle effects on fetal physiology and metabolism appear to have been ob-
served in rats after chronic maternal exposure to 10 ppm lead in drinking water, while similar
effects of inhaled lead have been seen at chronic levels of 10 mg/m3. With acute exposure by
gavage or by injection, the values are 10-16 mg/kg and 16-30 mg/kg, respectively. Since
humans are most likely to be exposed to lead in their diet, air, or water, the data from other
routes of exposure are of less value in estimating harmful exposures. Indeed, it seems likely
that teratogenic effects occur only when the maternal dose is given by injection.
Although human and animal responses may be dissimilar, the animal evidence does document
a variety of effects of lead exposure on reproduction and development. Measured or apparent
changes in production of or response to reproductive hormones, toxic effects on the gonads,
and toxic or teratogenic effects on the conceptus have all been reported. The animal data
also suggest subtle effects on such parameters as metabolism and cell structure that should be
monitored in human populations. Well designed human epidemiological studies involving large
numbers of subjects are still needed. Such data could clarify the relationship of exposure
levels and durations to blood lead values associated with significant effects, and are needed
for estimation of no-effect levels.
Given that the most clear-cut data concerning the effects of lead on reproduction and
development are derived from studies employing high lead doses in laboratory animals, there is
still a need for more critical research to evaluate the possible subtle toxic effects of lead
on the fetus, using biochemical, ultrastructural, or neurobehavioral endpoints. An exhaustive
evaluation of lead-associated changes in offspring will require consideration of possible
additional effects due to paternal lead burden. Neonatal lead intake via consumption of milk
from lead-exposed mothers may also be a factor at times. Also, it must be recognized that
lead effects on reproduction may be exacerbated by other environmental factors (e.g., dietary
influences, maternal hyperthermia, hypoxia, and co-exposure to other toxins).
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12.10.7. Genotoxic and Carcinogenic Effects of Lead
It is difficult to conclude what role lead may play in the induction of human neoplasia.
Epidemic logical studies of lead-exposed workers provide no definitive findings. However, sta-
tistically significant elevations in cancer of the respiratory tract and digestive system in
workers exposed to lead and other agents warrant some concern. Since it is clear that lead
acetate can produce renal tumors in some experimental animals, it seems reasonable to conclude
that at least that particular lead compound should be regarded as a carcinogen and prudent to
treat it as if it were also human carcinogen (as per IARC conclusions and recommendations).
However, this statement is qualified by noting that lead has been seen to increase tumorogen-
esis rates in animals only at relatively high concentrations, and therefore does not seem to
be an extremely potent carcinogen, In vitro studies further support the genotoxic and carcin-
ogenic role of lead, but also indicate that lead is not extremely potent in these systems.
12.10.8. Effects of Lead on the Immune System
Lead renders animals highly susceptible to endotoxins and infectious agents. Host sus-
ceptibility and the humoral immune system appear to be particularly sensitive. As postulated
in recent studies, the macrophage may be the primary immune target cell of lead. Lead-induced
immunosuppression occurs at low lead exposures (blood lead levels in the 20-40 yg/dl range)
that, although they induce no overt toxicity, may nevertheless be detrimental to health.
Available data provide good evidence that lead affects immunity, but additional studies are
necessary to elucidate the actual mechanisms by which lead exerts its immunosuppressive action.
Knowledge of lead effects on the human immune system is lacking and must be ascertained in
order to determine permissible levels for human exposure. However, in view of the fact that
lead affects immunity in laboratory animals and is immunosuppressive at very low dosages, its
potential for serious effects in humans should be carefully considered.
12.10.9 Effects of Lead on Other Organ Systems
The cardiovascular, hepatic, endocrine, and gastrointestional systems generally show
signs of dysfunction mainly at relatively high lead exposure levels. Consequently, in most
clinical and experimental studies attention has been primarily focused on more sensitive and
vulnerable target organs, such as the hematopoietic and nervous systems. However, it should
be noted that overt gastrointestinal symptoms associated with lead intoxication have been
observed in some recent studies to occur in lead workers at blood lead levels as low as 40-
60 ug/dl, suggesting that effects on the gastrointestinal and the other above organ systems
may occur at relatively low exposure levels but remain to be demonstrated by future scientific
investigations.
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T-F.;
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A12REF/C 12-301 9/20/83
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PRELIMINARY DRAFT
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A12REF/C 12-302 9/20/83
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PRELIMINARY DRAFT
APPENDIX 12-A
ASSESSMENT OF STUDIES REPORTING THE POTENTIAL ESSENTIALITY OF LEAD
Available information concerning the potential essentiality of lead is quite limited, due
in part to the inherent difficulties surrounding such investigations. The presence of lead as
a ubiquitous contaminant requires that studies of the effects of lead deficiency use synthetic
or semi-synthetic diets prepared from components extremely low in lead or use chemical agents
to reduce the level of background lead in the diet components. Such procedures, particularly
the use of chelating agents to remove lead, can entail risk in terms of their potential effect
on the nutritional integrity of the particular diet used.
Schwarz (1975) used synthetic diets prepared from low-lead constituents with or without
lead supplementation to determine the effect of low lead on the growth rate of adult rats. It
was reported that lead supplementation, usually over the range of 0.5 to 2.5 ppm lead, was
associated with measurable enhancement in growth rate compared to low-lead animals. In a
critique of the Schwarz results, Nielsen (1980) pointed out that all of the animals in the
Schwarz study, both low-lead and supplementation groups, showed sub-optimal growth, which
could be ascribed to riboflavin deficiency (Morgan and Schwarz, 1978); hence, the question
remains as to what the effect of lead supplementation would be in animals not riboflavin-
deficient and growing optimally. Nielsen (1980) has also questioned the statistical methods
used in the Schwarz studies and pointed out that addition of lead to the diet was of no ap-
parent benefit to deficient controls in subsequent studies. Problems associated with lead
deprivation studies are exemplified by the inability of Schwarz to duplicate his growth rate
data over time. He attributed this to the inadvertent use of a dietary component with an
elevated lead content for diets of the low-lead animals.
In a series of recent reports, Reichlmayr-Lais and Kirchgessner have described results
showing that rats maintained on a semi-synthetic diet low in lead (to levels of either 18 or
45 ppb) over several generations showed reduced growth rate (Reichlmayr-Lais and Kirchgessner,
1981a), disturbances in hematological indices, tissue iron and iron absorption (Reichlmayr-
Lais and Kirchgessner, 1981b,c,d; Kirchgessner and Reichlmayr-Lais, 1981a,b), and changes in
certain enzyme activities and metabolite levels (Reichlmayr-Lais and Kirchgessner, 1981e;
Kirchgessner and Reichlmayr-Lais, 1982). Diets containing 18 ppb were associated with the
most pronounced effects on iron metabolism and growth as well as on enzyme activities and
metabolite levels. Animals maintained on a 45 ppb lead diet showed moderate changes in some
hematological indices in the Fj-group. In these studies, controls were maintained on the same
dietary matrix to which 1.0 ppm lead was added.
LEAD12/A 12A-1 9/20/83
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PRELIMINARY DRAFT
In the above reports, EDTA was used to remove lead (and other elements) from casein, and
the chelating agent ammonium pyrrolidinodithiocarbamate (APDC) was employed to remove lead
from the starch and cellulose components to achieve the final diet level of 18 ppb. For the
45 ppb diet experiment, only the starch and cellulose components were treated with APDC
(Schnegg, 1975). Although the report of Reichlmayr-Lais and Kirchgessner (1981b) indicated
that the cellulose and starch extraction treatment was done on all of the material, a communi-
cation in this regard (Kirchgessner, 1982) noted that only a portion of the starch and cellu-
lose for the 45 ppb study was extracted with APDC. After chelant treatment, the components
were washed with solvents to remove the complexed metals originally present. Washing was also
assumed to remove the chelants.
Caution must be exercised in interpreting these studies as they currently stand owi
to the use of the chelating agents EDTA and/or APDC. Retention of free chelating agent(s) in
the diets could 'potentially affect the bioavailability of certain metals. In the report of
Davis et al. (1962), it was noted that diets containing soybean protein that had been ex-
tracted with EDTA to lower iron content, followed by supplementation with iron and CODD
were associated with iron deficiency in chicks maintained on these diets when compared to
chicks fed the same level of iron and copper in untreated diets. Clearly, EDTA treatment f
the soybean protein affected iron bioavailability in this study. Subsequently, the autho
(Davis et al., 1964) attempted to determine the presence of EDTA in the diets by simulatin
those used earlier. The crude methodology employed made accurate quantification difficult
but the amounts of EDTA measured ranged up to 67 ug/g diet. Other investigations through th'
years have documented that EDTA will affect iron absorption/retention and utilization i
various species (e.g., Larsen et al., 1960; Brise and Hallberg, 1962; Saltman and Helbock
1965; GUnther, 1969; Cook and Monson, 1976).
In this connection, retention of EDTA by proteins appears to be a general problem based
on information available for casein (Hegenauer et al., 1979), transferrin (Price and Gibson
1972), the enzyme alkaline phosphatase (Csopak and Szajn, 1973), photoprotein aequonin
(Shimomura and Shimomura, 1982), and human fibrinogen (Nieuwenhuizen et al., 1981). Further-
more, complete removal of EDTA from these rather diverse proteins is reported to involv
forcing conditions, and the washing procedure used by the authors of the studies in questio
gives no assurance of being adequate for chelant removal.
Available information also suggests that diets retaining free EDTA and/or APDC, even
quite low levels, may pose problems by affecting the bioavailability of the essential
nickel. The studies of Schnegg and Kirchgessner (see review of Kirchgessner and Schn
1980) have shown that nickel deficiency in rats followed over several generations is ass '-
ated with reduced growth rate, disturbed hematological indices, lowered tissue iron
LEAD12/A 12A-2 9/2Q/83
-------
PRELIMINARY DRAFT
iron absorption, and disturbances in enzyme activities and metabolite levels. According to
Nielsen (1980), nickel plays a role in the intestinal absorption of trivalent iron. In the
nickel deficiency studies of Schnegg and Kirchgessner, low-nickel diets contained 15 ppb
nickel, while control groups were maintained on the same basal diet supplemented with 20 ppm
nickel. In the lead deficiency studies under discussion, nickel was added back to the treated
diets at a level of 1.0 ppm (Reichlmayr-Lais and Kirchgessner, 1981b; Kirchgessner and
Reichlmayr-Lais, 1981b).
The interaction of nickel with the chelants EDTA and/or APDC in the context of bioavail-
ability has been documented. Dithiocarbamates such as APDC are effective chelation therapy
agents in protecting against nickel toxicity (see review of Sunderman, 1981), while the report
of Solomons et al. (1982) described the significant effect of EDTA on nickel bioavailability
in human subjects. In the latter study, human volunteers ingested a single dose of 5 mg of
nickel, and the resulting effect on plasma nickel was monitored. When nickel was co-ingested
with EDTA (40 mg of Na2EDTA-H20, a 1.3:1 ratio of EDTA to Ni), not only was the rise in plasma
seen with just nickel abolished, but the plasma nickel level was reduced below the fasting
background level.
It is not possible to draw a close comparison of the data of Schnegg and Kirchgessner for
nickel deficiency with the potential effects of impaired nickel bioavailability in the lead
deficiency studies since 1) the actual level of bioavailable nickel in the studies cannot be
defined and 2) the age points for most of the effects seen in nickel deficiency are different
from those in the lead studies. Interestingly, one can calculate that the decrements in body
weight of animals of the Regeneration in both groups of studies at various common time
points, e.g., 20, 22, 30, 38 days, are virtually identical.
Any mechanism by which lead supplementation at 1.0 ppm in the lead deficiency studies
would operate in a situtation of altered bioavailability of nickel or iron in the diets can
only be inferred, given the absence of any further experiemental data which would more fully
elucidate an essential vs. an artifactive role for lead.
In terms of any simple competitive binding mechanism involving lead, chelating agents,
and nickel or iron, the presence of lead at a level of 1.0 ppm would be seen to most immedi-
ately affect nickel bound up with EDTA (as the common 1:1 complex) or APDC (as the common 1:2
complex). Nickel was added back to the diets at a level of 1.0 ppm. Since the binding
constants for lead and nickel with EDTA are roughly comparable (Shapiro and Papa, 1959; Pribl,
1972), while complexes of lead with dithiocarbamates are vastly greater in stability than the
corresponding nickel complexes (Sastri et al., 1969), lead at 1.0 ppm can displace up to its
molar equivalent of nickel from complexation, which calculates to be 0.3 ppm nickel. This
amount of liberated nickel, 0.3 ppm, appears to be nutritionally adequate, since the minimal
LEAD12/A 12A-3 9/20/83
-------
PRELIMINARY DRAFT
nutritional requirement is noted to be around 50 ppb (Kirchgessner and Schnegg, 1980; Nielson
1980). The corresponding amounts of APDC and EDTA required to bind up 1.0 ppm nickel cal-
culate to be 5.4 ug/g (1:2 complex, NiAPDC) and slightly under 5 pg/g (1:1 complex, Ni-EDTA).
Hence, mere traces of free chelants could be a potential problem.
Similar direct competitive binding involving lead and iron cannot be invoked as likely
given the relative amounts of iron and lead in lead-supplemented diets, although lead forms
more stable complexes than divalent iron with EDTA or APDC (Pribl, 1972; Sastri et al., 1969)
A cyclic mechanism would have to be invoked whereby Pb-EDTA is formed by exchange of ligand
from Fe-EDTA, is then dissociated jn vivo, and the displacement process repeated.
Nickel supplementation at 20 ppm in the Schnegg and Kirchgessner studies, where a similar
APDC procedure was used to purify starch and cellulose components, as well as in the study of
Nielsen et al. (1979), where APDC at 10 ppm was employed to assess the role of nickel in iron
metabolism, do not permit comparison with the studies in question because of the 20-fold dis-
parity in the level of supplementation.
Given the above concerns, it would appear that: 1) further experiments, using methodol-
ogy such as scintillography and labeled chelants, are necessary to conclusively determine that
diet preparation in the Reichlmayr-Lais and Kirchgessner studies did not involve retention
free chelating agents, 2) determination of levels of nickel and lead in tissues, blood and
excreta would greatly help to elucidate the true role of lead, and 3) replication of the
results in the authors' or another laboratory, preferably with minimal chelant treatment of
components,, should be done. It appears that the various reports describe basically si 1
experiments over several generations, one at a diet level of 18 ppb lead, and one at 45 n h
lead.
LM12/A - 9/20/83
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PRELIMINARY DRAFT
12A REFERENCES
Brise, H. ; Hallberg, L. (1962) Iron absorption studies: a method for comparative studies on
iron absorption in man using 2 radio-iron isotopes. Acta Med. Scand. 171 (Suppl.): 23-27.
Cook, J. D.; Monsen, E. R. (1976) Food iron absorption by man. II: The effect of EDTA on
absorption of dietary non-heme iron. Am. J. Clin. Nutr. 29: 614-620.
Csopak, H. ; Szajn, H. (1973) Factors affecting the zinc content of E. coli alkaline phos-
phatase. Arch. Biochem. Biophys. 157: 374-379.
Davis, P. N.; Norris, L. C. ; Kratzer, F. H. (1962) Iron deficiency studies in chicks using
treated isolated soybean protein diets. J. Nutr. 78: 445-453.
Davis, P. N. ; Norris, L. C.; Kratzer, F. H. (1964) Iron deficiency studies in chicks. J. Nutr.
84: 93-94.
Gunther, R. (1969) Der Einfluss von chelatbildnern auf die Verteilung und Ausscheidung von
Radioeisen bei der Ratte. [Distribution and excretion of radioiron in the rat as
influenced by chelating agents.] Naunyn Schmiedebergs Arch. Pharmakol. Exp. Pathol. 262:
405-418.
Hegenauer, J. ; Saltman, P.; Nace, G. (1979) Iron (Ill)-phosphoprotein chelates: stoichiometric
equilibrium constants for interaction of iron and phosphoryl serine residues of phosvitin
and casein. Am. J. Clin. Nutr. 32: 809-816.
Kirchgessner, M. (1982) [Letter to D. Weil]. October 6. Available for inspection at: U.S.
Environmental Protection Agency, Environmental Criteria and Assessment Office, Research
Triangle Park, NC.
Kirchgessner, M.; Reichlmayr-Lais, A. M. (1982) Konzentrationen verschiedener Stoffwechsel-
metaboliten im experimentellen Bleimangel. [Concentrations of various metabolites with
experimental lead deficiency.] Ann. Nutr. Metab. 26: 50-55.
Kirchgessner, M. ; Schnegg, A. (1980) Biochemical and physiological effects of nickel defi-
ciency. In: Nriagu, J. 0., ed. Nickel in the environment. New York, NY: John Wiley &
Sons; pp. 635-652.
Kirchgessner, M.; Reichlmayr-Lais, A. M. (1981a) Changes of iron concentration and iron-
binding capacity in serum resulting from alimentary lead deficiency. Biol. Trace Elem.
Res. 3: 279-285.
Kirchgessner, M.; Reichlmayr-Lais, A. M. (1981b) Retention, Absorbierbarkeit und intermedita're
Verfligbarkeit von Eisen bei alimentarem Bleimangel. [Retention, absorbability and
intermediate availability of iron with alimentary lead deficiency.] Int. J. Vitam. Nutr.
Res. 51: 421-424.
Larsen, B. A.; Bidwell, R. G. S.; Hawkins, W. W. (1960) The effect of ingestion of disodium
ethylenediaminetetraacetate on the absorption and metabolism of radioactive iron by the
rat. Can. J. Biochem. Physiol. 38: 51-55.
B12REF/D 12A-5 9/20/83
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PRELIMINARY DRAFT
Morgan, J. K.; Schwarz, K. (1978) Light sensitivity of riboflavin in amino acid diets Fed
Proc. Fed. Am. Soc. Exp. Biol. 37:671.
Nielsen, F. H. (1980) Effect of form of iron on the interaction between nickel and iron in
rats: growth and blood parameters. J. Nutr. 110: 965-973.
Nielsen, F. H. ; Zimmerman, T. J. ; Ceilings, M. E.; Myron, D. R. (1979) Nickel deprivation in
rats: nickel-iron interactions. J. Nutr. 109: 1623-1632.
Nieuwenhuizen, W. ; Vermond, A.; Hermans, T. (1981) Human fibrinogen binds EDTA and citrate
Thromb. Res. 22: 659-663. citrate.
Pribl, R. (1972) Analytical applications of EDTA and Related Compounds. New York NY-
Pergamon Press; p. 27. (International series of monographs in analytical chemistry v
Price, E. M.; Gibson, J. F. (1972) A re-Interpretation of bicarbonate-free ferric
E.P.R. spectra. Biochem. Biophys. Res. Commun. 46: 646-651.
Reichlmayr-Lais, A. M.; Kirchgessner, M. (1981a) Zur Essentialitat von Blei f(jr das
Wachstum. [Why lead is essential for animal growth.] Z. Tierphysiol
Futtermittelkd. 46: 1-8. '
Reichlmayr-Lais, A. M.; Kirchgessner, M. (1981b) Depletionsstudien zur Essentialitat von Ri •
an wachsenden Ratten. [Depletion studies on the essentiality of lead in arnwinn Z I
Arch. Tierenaehr. 31: 731-737. growing rats.]
Reichlmayr-Lais, A. M.; Kirchgessner, M. (1981c) Hamatologische Veranderungen bei alimo +s
Bleimangel. [Hematological changes with alimentary lead deficiency.] Ann Nutr u +?"
25: 281-288. ' lr' Metab-
Reichlmayr-Lais, A. M.; Kirchgessner, M. (1981d) Eisen-.Kupfer- und Zinkgehalte in Noi
borenen sowie in Leber und Milz wachsender ratten bei alimentarem Blei-Manqel rI
copper and zinc contents in newborns as well as in the liver and spleen of qrowina t'
in the case of alimentary lead deficiency.] Z. Tierphysiol. Tierernaehr FuttermitLif^
46: 8-14. ' '•'•"'""'•teilcd.
Reichlmayr-Lais, A. M.; Kirchgessner, M. (1981e) Aktivitats-veranderungen verschiedener fm
im alimentSren Blei-Mangel. [Activity changes of different enzymes in alimentary i^
deficiency.] Z. Tierphysiol. Tierernaehr. Futtermittelkd. 46: 145-150. y ad
Saltman, P.; Helbock, H. (1965) The regulation and control of intestinal iron transoort T
Proc. Symp. Radio-isotope Anim. Nutr. Physiol., Prague, Czeckoslovakia; pp. 301-317'
Sastri, V. S.; Aspila, K. I.; Chakrabarti, C. L. (1969) Studies on the solvent extrarti
metal dithiocarbamates. Can. J. Chem. 47: 2320-2323. extraction of
Schnegg, A. (1975) [Dissertation.] Technische UniversitHt MUnchen-Weihenstephan CTprh • ,
University, Munich-Weihenstephan, West Germany.] Available for inspection at- lie
Environmental Protection Agency, Environmental Criteria and Assessment Offic*. oL
Triangle Park, NC. ' Kesearch
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PRELIMINARY DRAFT
Schwarz, K. (1975) Potential essentiality of lead. Arh. Rada Toksikol. 26 (Suppl): 13-28.
Shapiro, S.; Papa, D. (1959) Heavy metal chelates and cesium salts for contrast radiography.
Ann. N.Y. Acad. Sci. 78: 756-763.
Shimomura, 0.; Shimomura, A. (1982) EDTA-binding and acylation of the Ca(2+)-sensitive photo-
protein aequorin. FEBS Lett. 138: 201-204.
Solomns, N. W.; Viteri, F.; Shuler, T. R.; Nielsen, F. H. (1982) Bio-availability of nickel in
men: effects of foods and chemically-defined dietary constituents on the absorption of
inorganic nickel. J. Nutr. 112: 39-50.
Sunderman, F. W. (1981) Chelation therapy in nickel poisoning. Ann. Clin. Lab. Sci. 11: 1-8.
B12REF/D 12A-7 9/20/83
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PRELIMINARY DRAFT
APPENDIX 12-B
SUMMARY OF PSYCHOMETRIC TESTS USED TO ASSESS COGNITIVE
AND BEHAVIORAL DEVELOPMENT IN PEDIATRIC POPULATIONS
LEAD12/B 12B-1 9/20/83
-------
CD
TABLE 128. TESTS COMMONLY USED IN A PSYCHCKEDUCATIONAL BATTERY FOR CHILDREN
Age range
Noras
Scores
Advantages
Disadvantages
General Intelligence Tests
Stanford-Binet (Font L-N) 2 yn - Adult 1972
Wechsler Preschool & Primary 4 - 6% yrs 1967
Scales of Intelligence (WPPSI) Best for 5-yr-olds
Hechsler Intelligence Scale 6-16 yrs
for Children-Revised (WISC-ft)
1974
ro
09
ro
McCarthy Scales of Children's 2»» - 8S yrs 1972
Abilities (MSCA) Best for ages
4-6
Bayley Scales of Mental
Development
2-30 mos.
1969
1. Deviation IQ:
Mean = 100 SO = 16
2. Menu I Age Equivalent
1. Deviation IQ:
Mean = 100 SD = 15
2. Scaled Scores for
10 sub tests:
Mean = 10 SO = 3
1. Deviation IQ:
Mean = 100 SD = 15
2. Scaled Scores for
10 subtests: Mean = 10
SO = 3
1. General Cognitive Index:
Mean = 100 SO = 16
2. Scaled scores for 5
subtests: Mean = 50
SO = 10 Age equivalents
can be derived.
1. Standard scores
(M = 100 SD = 16)
2. Mental Development
Psychonotor Index
1. Good reliability & validity
2. Predicts school performance
3. Covers a wide age range
1. Good reliability & validity
2. Predicts school performance
3. Gives a profile of verbal &
non-verbal skills.
4. Useful in early identifica-
tion of learning disability
1. Good reliability & validity
2. Predicts school performance
3. Gives a profile of verbal
and non-verbal skills
4. Useful in identification of
learning disability
1. Good reliability & validity
2. Good predictor of school
performance
3. Useful in identification of
learning disabilities when
given with a WISC-R or
Stanford-Binet
4. Gives good information for
educational programming
1. Norms are excellent
2. Satisfactory reliability
and validity
3. Best measure of infant
development
1. Tests mostly verbal skills
especially after 6 yrs
2. Does not give a profile
of skills
1.
Narrow age range
Mentally retarded children
find this a disproportionate
difficult test
-o
yo
Gives a lower IQ than •—
Stanford-Binet for normal 2
and bright children z
Children score much lower :
than on WISC-R or
Stanford-Binet
Narrow age range
Not a good predictor of
later functioning in
average as in below average
children
<£>
ro
O
CD
CO
-------
t«0
00
TABLE 12B (continued)
Age range
Noras
Scores
Advantages
Disadvantages
Slosson Intelligence Test
Infancy - 27 yrs 1963
Peabody Picture Vocabulary
Test
- 18 yrs
1959,rev.1981
White,
Middle class
sample
no
CD
GO
Visual-Motor Tests
Frostig Developmental Test of
Visual Perception
3 - 8 yrs & older
learning disabled
(L.D.) children
1963
White, niddle
class sample
1. Ratio IQ. is not
related to general
population
Verbal IQ
Age equivalent
Good reliability & validity
Quick to administer. A
good screening test
Easily administered
Does not require language
or motor skills
1. Perceptual Quotient:
Median = 100 Quartile
Deviation = 10
2. Perceptual Age Equivalent
3. Scaled Scores for 5 sub-
tests
1. Good reliability for L.D.
children
1. Many items taken from
Stanford Binet
2. Responses require good
language ski 1 Is
3. Measures a narrow range
of skills
4. A screening test: not to
be used for classification
or placement
1. Fair reliability and
validity
2. Tests only receptive
vocabulary
3. Lower class children score
lower
4. Mentally Retarded children
score higher than on other
tests
S. Not to be used for classi-
fication or placement.
1. Fair reliability for normal
children
2. Poor Validity
3. No known relationship to
reading or learning
4. Remedial program based on
test of questionable value
5. Not useful in identifying
children at risk for 1.0.
-o
73
Bender-Gestalt
4 yrs - Adult
1964
Normal and
Brain-injured
Children
1. Age equivalent
ro
O
oo
CO
Beery-Buktenica
Developmental Test of
Visual Motor Integration (VMI)
2-15 yrs
1967
1. Age equivalent
1. Easily administered
2. Long history of research
makes it a good research
tool
1. Easily administered
2. Good normative sample
1. Fair reliability
2. Poor predictive and
validity
3. Responses influenced by
fatigue & variations in
administration
4. No known relationship to
reading or subtle neuro-
logical dysfunction
1. Moderate reliability and
validity
2. Correlates better with
mental age than chrono-
logical age
-------
ro
•^
03
TABLE 12B (continued)
Age range
Noras
Scores
Advantages
Disadvantages
Educational Tests
Wide Range Achievement Test
(WRAT)
Peabody Individual
Achievement Test (PIAT)
5 yrs - Adult
5 - 18 yrs
1976 1. Standard Score:
Revised mean = 100 SD = 15
2. Grade equivalent
1969 1. Standard Scores:
Mean = 100 SD = 15
2. Grade equivalent
3. Age equivalent
Woodcock Reading
Mastery Tests
Spache Diagnostic
Reading Scales
Kgn - 12 grade
1st - 8th grade
1971-72 1. Grade equivalent
adjusted for 2. Standard Score
social class 3. Percent!le Rank
1972
1. Instructional level of
reading (grade equiva-
lent).
2. Independent level of
reading.
3. Potential level of
reading
1. Good reliability & validity 1.
Reading scores predict
grade level 2.
2. Tasks similar to actual
work
1. Tests word recognition and 1.
2. Breaks down skills into 5
areas 2.
1. Good reliability
2. Breakdown of reading skills
useful diagnostically and in
planning remediation
3. Easy to administer and score
1. Independent level score
predicts gains following
remediation
2. Good breakdown of reading
skills
Reading portion tests
word recognition only
Responses require good
organizational skills
(could be an advantage)
Moderate reliability. Low
stability for Kindergarten
No data on predictive
validity
A multiple choice test
requiring child to recog-
nize correct answer (could
be an advantage).
Heavily loaded on verbal
reasoning.
Factor structure changes
with age.
1. No data on validity
1. Fairly complex scoring
2. Moderate reliability
3. No good data on validity
•J*
TO
Key Math Diagnostic
Arithmetic Test
Pre-school - 6th 1971
grade
1. Grade equivalent
1. Excellent breakdown of Math
skills
2. Easy to administer and
score
1. Moderate reliability
2. No data on validity
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TABLE 12B (continued)
Age range
Norms
Scores
Advantages
Disadvantages
Tests of Adaptive Functioning
Vineland Social Maturity Scale Birth • 25 yrs
AAMO Adaptive Behavior Scale 3 yrs - Adult
Progress Assessment Chart of
Social Development (PAC)
Developmental Profile
Conner* Rating Seal*
Birth - Adult
Birth - 12 yrs
3 yrs - 17 yrs
1983 1. Social Quotient (Ratio)
Revised 2. Social Age Equivalent
1974
Institu-
tionalized
Retardates;
Public School
Children (1982)
1. Percent!le Ranks
2. Scaled scores
1976
1972
1978
No Scores
1. Age equivalents in 5
5 areas
2. IQ equivalency (IQE)
1. Age equivalents
1. Easily administered
2. Good reliability for normal
and MR chidren
1. Discriminates between EMR
and regular classes
2. Useful for class placement
and monitoring progress
1. Poor norms
2. No data on validity
3. Items are limited past
preschool years
4. Scores decrease with age
for MR children
1. Moderate reliability for
independent living skills
scale. Poor reliability
for maladaptive behaviour
scale.
2. Lengthy administration
3. Items & scoring are not
behaviorally objective
1. Useful for training and
assessing progress
2. Gives profile of skills
1. Good reliability and valid-
ity. Excellent study of
construct validity reported
in manual.
2. Gives a profile of skills
1. Most widely used measure of
attention deficit disorder
2. Four factors: conduct prob-
lems; hyperactivity;
inattentive-passive; hyper-
activity index
No data on reliability or £
validity 3
IQE underestimates IQ of
above average children,
overestimates IQ of below
average children.
Parents' ratings don't pre-
dict as well as teachers'
ratings
Works best Middle class
children
Werry-Weiss-Peters Hyperactivity 1 yr - 9 yrs
Scale
1974, 1977 1. Age equivalents
1. Good measure of hyperac- 1.
tivity 2.
2. Seven Factors
Limited age range
Standardized on middle
class children
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APPENDIX 12-C
WILL BE FORTHCOMING UNDER A SEPARATE COVER.
12C-1
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APPENDIX 12-D
ABSTRACT OF A REVIEW OF THREE STUDIES
ON THE EFFECTS OF LEAD SMELTER
EMISSIONS IN EL PASO, TEXAS
Presented by Warren R. Muir
Council on Environmental Quality
Washington, D.C.
At the International Conference on Heavy
Metals in the Environment
Toronto, Ontario, Canada
October 1975
The committee reviewed two independent studies conducted in 1973 by Dr. Landrigan (CDC) and Dr.
McNeil (1LZRO) to determine the effects of community lead exposures near the ASARCO smelter in El
Paso, Texas. The CDC study used a random sample approach to group participating children, and in the
ILZRO study match paired groups were selected on the basis of residence. In both studies the criteria for
subclassification with regard to lead exposure were blood lead levels. Neuropsychological dysfunction was
evaluated by several tests including W1SC, WPPSI, and McCarthy scales. Statistical differences in test results
could not be directly correlated to blood lead levels.
The opinion of the committee was that no firm conclusions could be drawn from the studies as to whether or
not there are subclinical effects of lead on children in El Paso and that the reports and data made available
have not clearly demonstrated any psychologic or neurologic effects in the children under study. It noted the
absence of major chronic clinical effects, and concluded that these studies therefore do not bear upon the con-
clusions of other investigations under different conditions and those in which clinical effects have been con-
firmed. However, because of inherent problems of study design and the limitations in the tests used, this find-
ing should not lead to a conclusion that low levels of lead have no effects on neuropsychological performance.
Ellen Silbergeld, Ph.D., N1H, Eileen Higham, Ph.D., and Mr. Russell Jobaris, Johns Hopkins University,
Department of Medical Psychology, served as special consultants.
The committee decided to limit its focus to a review of the three studies, and to attempt to account for and
interpret the differences between the studies. Thus, aspects not related to differences were not emphasized.
The committee limited its consideration to the following materials: (1) reports of the three studies under
consideration; (2) other materials provided by the authors of the studies; (3) background information and
documents collected by Dr. Muir in El Paso. This presentation today consists of excerpts from a draft com-
mittee report.
D.I HISTORY
El Paso is situated on the Mexican border in the western part of Texas. A lead smelter owned by American
Smelting and Refining Company (ASARCO) has been located on the southwestern border of the city, on the
Rio Grande River, since 1887. The area most conspicuously involved in the studies, Smeltertown, was a 2 x 6
block area located between the plant and the river. Smettertown is no longer in existence, having been
destroyed in December 1972. About 2 km south of Smeltertown is Old Fort Bliss, a considerably smaller
community, whose inhabitants were considered in some, hut not all, of the studies.
The ASARCO smelter produces lead, zinc, copper, and cadmium. Paniculate matter is removed from air-
borne wastes in a series of baghnuses; remaining emissions contain approximately 40 Ib of lead per day.
The El Paso City County Health Department began an investigation of the ASARCO smelter in early 1970,
in preparation for an air pollution suit filed by the city in April 1970. As part of this investigation. Dr.
12D-1
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Bertram Carnow was hired by the city as a consultant. At his suggestion, the city began to sample the blood
lead levels of El Paso children to determine whether any had been over-exposed to lead. This included a large
number of Smeltertown children. Based upon early results in 1971, Dr. Carnow visited El Paso, and saw a
selected group of children with high blood lead levels. He interviewed the children, and reviewed their medi-
cal records. The information contained in the medical histories, and Dr. Carnow's interviews, constitute the
observations reported by Dr. Carnow in the paper presented to the American Pollution Control Association
(APCA). The clinical observations were in a paragraph of a paper otherwise devoted to a consideration of the
effects of the smelter on the environment as a whole, and the extent of its emissions. This report contains no
details on the age, exposures, individual signs and symptoms, or diagnostic criteria used in the ten cases re-
ported. Our committee focused its attention, therefore, upon the two full-scale follow-up epidemiological
studies conducted by Dr. Landrigan (CDC) and Dr. McNeil (ILZRO).
In 1973 ASARCO began a separate investigation of the population of Smeltertown, and asked Dr. James
McNeil of the International Lead Zinc Research Organization (ILZRO) for his assistance in the examination
and possible treatment of children with elevated blood levels greater than 60 mg/100 ml.
As a result of public concern over widespread lead poisoning throughout the city of El Paso, the mayor re-
quested aid from the Federal Government. A separate protocol for a Center for Disease Control (CDC) study
was submitted to and approved by the Public Health Board in 1973 with the understanding that the two
studies would proceed independently, with those children in the ILZRO sponsored study being excluded
from the CDC study.
In the summer of 1973, CDC and ILZRO proceeded independently to collect data for their respective
studies. CDC's examinations were done in two weeks in June 1973, while McNeil's were carried out over the
course of the summer with the aid of the El Paso public school system.
The CDC group supplied to the Committee data in detail, which were sufficient to allow the committee to
conduct statistical tests and analyze characteristics of groups. For the ILZRO study, this committee requested
data sufficient to carry out similar in-depth analyses. All of the requested data were supplied; however, they
were not in such a form as to allow recalculation of most of the statistical findings of the study or to allow
comparison with the CDC findings.
D 2 STUDY DESIGN
The environmental sampling that was performed was common for both of these studies. In the selection of
study and control populations, the Landrigan CDC study used a classical approach of a random sample
survey to determine the prevalence of abnormal blood lead values. The 13 census tracts most adjacent to the
smelter were divided into three areas. The sampling frame was designed to obtain about 100 study subjects
from each area for various age groups. Of 833 occupied residences, interviews were obtained from 758 study
subjects in the 1-19 age group. The participating children were divided into a lead-absorption group (40-80
Mg/100 ml) of 46 and a control group (< 40 /xg/100 ml) of 78. There is no detailed description as to how the
children were chosen.
CDC used these same children as the basis for the later study of neuropsychological dysfunction. All but 3
children chosen for study came from the 1972 prevalence survey; 5 children with known preexisting defects
such as with a history of symptoms compatible with acute lead poisoning or acute lead encephalopathy and
those who had received chelation therapy were excluded.
While it is understood that a number of Smeltcrtown children with blood lead levels over 40 fig/100 ml
were eventually involved in litigation, most of them took part in the studies. However, on the recommenda-
tion of the lawyers representing the children, at least one group of 18 did not participate in the ILZRO study.
In the absence of identification by names of the individuals in the three studies, it has been impossible to
evaluate the effects of non-participation.
The ILZRO study was very different; 138 children from Smeltertown agreed to participate in a study: Resi-
dence, not blood lead, was the selection criterion. Two control groups were chosen, and were reported to have
been matched on age, sex, ethnic background, and income, with one set chosen from El Paso and another set
for those 8 years of age or under from a rural area about 12 miles from the smelter. This classification had the
effect of grouping together children who, under the CDC criteria, would have been in "lead" and "control"
groups.
12D-2
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The criteria used for subclassification of children with regard to lead exposure were based in both studies
on the blood lead level. Whereas the CDC study utilized blood lead values obtained at only two points in
time. ILZRO, which was faced with the problem that many children had repeated blood lead measurements
with marked variations over a period of 18 months (the levels being generally lower after exposure was dis-
continued), classified children on the basis of the average of the "two highest" recorded values.
This criterion results in a substantial increase in the number of children in the apparently higher blood lead
category and a corresponding decrease in the number of those in the apparently lower blood lead level
category.
Although it is understandable that this type of selection was used to avoid underestimating the problem of
lead intoxication in the population examined, it ultimately resulted in muddling of the separation between
groups (and possibly obscuring eventual differences). For example, the selection for analysis of children from
the same geographical area, subclassified according to blood lead level, in the ILZRO study, may give the im-
pression that the effects of lead itself are being studied in a homogeneous population. However, since ex-
posure was geographically the same, other factors inherent to each individual child may be responsible for the
difference in blood lead level observed.
An additional method of classification could have been the use of free erythrocytic protoporphyrin
measurements (FEP) which have been shown to provide an indication of metabolic effects of lead absorption
on metabolism, particularly useful in blood lead level ranges (40-60 *ig/100 ml) where analytical and
biological fluctuation may result in uncertain classification. (The ILZRO study included this test but did not
include it as a basis for data analysis ) Absence of elevation of free erythrocytic protoporphyrin may indicate
those instances where high blood lead levels were spurious.
The following psychometric tests were employed by the two studies:
1. Wechsler Intelligence Scale for Children. WISC (CDC, ILZRO)
2. McCarthy Scales of Children's Abilities (ILZRO)
3. Wechsler Preschool and Primary Scale of Intelligence. WPPSI (CDC)
4. Lincoln-Oseretsky Motor Development Scale (ILZRO)
5. California Test of Personality Adjustment (ILZRO)
6. Frosting Perceptual Quotient (ILZRO)
7. Bender Visual-Motor Gestalf Test (CDC. ILZRO)
8. Peabody
9i WRAT
10. Wepman
11. Draw-a-person
All of the tests selected by both studies were appropriate for the ages of the children to whom they were ad-
ministered. Since the common ground for these studies is the WISC test, with the WPPSI used by CDC and the
McCarthy Scales by ILZRO for the younger children in their studies, the Committee concentrated on these
three tests and the results obtained for them.
C.3 RESULTS
The study by CDC reports results for 27 children given the WPPSI (12 with blood lead levels 40-80 jig'100
ml and !5 with blood lead levels less than 40//g/100 nil) and for 97 children tested with the WISC (34 in the
"lead group" and 63 in the "control group"). Statistical analyses were performed on grouped data with one-
tailed tests. Significant differences between lead and control groups are reported in this study for the perfor-
mance IQ's of the WICS and WPPSI. In subtest scores, significant differences were found in Coding on the
WISC and Geometric Design on the WPPSI. When data from both tests are combined, a significant difference
between lead and control groups on performance IO is found. No differences were found between groups in
verbal IQ's or full-scale IW's of the WISC or WPPSI.
The ILZRO study based on match pairing solely by residences reports no significant differences in scores
on the WISC or McCarthy scales between groups with increased lead absorption and pair-matched controls.
Statistical analysis was by means of two-way analysis of variance by age and blood lead levels.
The two studies base much of their conclusions upon psychometric and neurological testing of children
from El Paso and Smeltertown. The reported significant differences and psychometric and neuromolor func-
tions in the CDC study were clouded by potentially important methodological difficulties. These included
12D-3
-------
age differences between case and control groups, limited statistical treatment of the psychometric data col-
lected, and. in the ILZRO study, the use of an average of the two highest blood lead levels to categorize lead
exposure.
In addition, both the studies shared the following inherent problems:
I. Non-random exclusion of large groups of children
2. Uncertainties as to the selection of control groups
3. Reliance upon blood lead as the indicator of lead exposure and intoxication in analyses of data
4. Measurement of a limited aspect of psychological behavior
5. Lack of consideration of the potentially disruptive influences on test taking of the razing of Smelter-
town, closing of its school, resettlement, litigation, and public controversy
6. Inability to rule out possible preexisting conditions
The Committee stressed the last issue, noting the likelihood that any behavioral or genetic factors that pre-
dispose an individual child to ingest or absorb more lead than another child equally exposed may itself be
correlated to he result of psychometric testing. In other words an increased blood lead level may reflect,
rather than cause, a preexisting difference in intelligence or behavior, an issue inherent in virtually all
retrospective studies of the effects of low level blood lead.
The opinion of the committee was that no firm conclusions could be drawn from the studies as to whether or
not there are subclinical effects of lead on children in El Paso and that the reports and data made available
have not clearly demonstrated any psychologic or neurologic effects in the children under study. It noted the
absence of major chronic clinical effects, and concluded that these studies therefore do not bear upon the con-
clusions of other investigations under different conditions and those in which clinical effects have been con-
firmed. However, because of inherent problems of study design and the limitations in the tests used, this find-
ing should not lead to a conclusion that low levels of lead have no effects on neuropsychological performance.
12D-4
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PRELIMINARY DRAFT
13. EVALUATION OF HUMAN HEALTH RISKS ASSOCIATED WITH
EXPOSURE TO LEAD AND ITS COMPOUNDS
13.1 INTRODUCTION
This chapter attempts to integrate, concisely, key information and conclusions discussed
in preceding chapters into a coherent framework by which interpretation and judgments can be
made concerning the risk to human health posed by present levels of lead contamination in the
United States.
Towards this end, the chapter is organized into seven sections, each of which discusses
one or more of the following major components of the overall health risk evaluation: (1)
external and internal exposure aspects of lead; (2) lead metabolism, which determines the
relationship of external lead exposure to associated health effects of lead; (3) qualitative
and quantitative characterization of key health effects of lead; and (4) identification of
population groups at special risk for health effects associated with lead exposure.
The various aspects of lead exposure discussed include: (1) an historical perspective on
the input of lead into the environment as well as the nature and magnitude of current lead
input; (2) the cycling of lead through the various environmental compartments; and (3) levels
of lead in those media most relevant to lead exposure of various segments of the U.S. popula-
tion. These various aspects of lead exposure are summarized in Section 13.2.
With respect to lead metabolism, some of the relevant issues addressed include: (1) the
major quantitative characteristics of lead absorption, distribution, retention, and excretion
in humans and how these differ between adults and children; (2) the toxicokinetic bases for
external/internal lead exposure relationships with respect to both internal indicators and
target tissue lead burdens; and (3) the relationships between internal and external indices of
lead exposure, i.e., blood-lead levels in relation to lead concentrations in air, food, water,
dust/soil. Section 13.3 summarizes the most salient features of lead metabolism, whereas
Section 13.4 addresses experimental and epidemiological data concerning various blood lead-
environmental media lead relationships.
In regard to various health effects of lead, the main emphasis here is on the identifica-
tion of those effects most relevant to various segments of the general U.S. population and the
Placement of such effects in a dose-effect/dose-response framework. In regard to the latter,
a crucial issue has to do with relative response of various segments of the population in
terms of effect thresholds as indexed by some exposure indicator. Furthermore, it is of
interest to assess the extent to which available information supports the notion of a conti-
nuum of effects as one proceeds across the spectrum of exposure levels. Finally, it is of
interest to ascertain the availability of data on the relative number or percentage of members
23PB13/A I3~l 9/20/83
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PRELIMINARY DRAFT
(i.e., "responders") of specific population groups that can be expected to experience a par-
ticular effect at various lead exposure levels in order to permit delineation of dose-response
curves for the relevant effects in different segments of the population. These matters are
discussed in Sections 13.5 and 13.6.
Melding of information from the sections on lead exposure, metabolism, and biological
effects permits the identification of population segments at special risk in terms of physio-
logical and other host characteristics, as well as heightened vulnerability to a given effect;
and these risk groups are discussed in Section 13.7. With demographic identification of indi-
viduals at risk, one may then draw upon population data from other sources to obtain a
numerical picture of the magnitude of population groups at potential risk. This is also dis-
cussed in Section 13.7.
13.2 EXPOSURE ASPECTS
13.2.1 Sources of Lead Emission in the United States
The important issues to be raised concerning the sources of lead in the human environment
are: What additional pathways to human consumption have been added in the course of civiliza-
tion? What are the relative contributions of natural and anthropogenic lead? From the avail-
able data, what trends can be expected in the potential consumption of lead by humans? What
is the impact of normal lead cycling processes on total human exposure? And finally, arc
there population segments particularly at risk due to a higher potential exposure?
Figure 13-1 is a composite of similar figures appearing in Chapters 7 and 11. This
figure shows that four of the five sources of lead in the human environment are of anthropo-
genic origin. The only significant natural source is from the geochemical weathering of
parent rock material as an input to soils. Of the four anthropogenic pathways, two are close-
ly associated with atmospheric emissions and two (pigments and solder) are more directly
related to the use of metallurgical compounds in products consumed by humans.
It is clear that natural sources contribute only a very small fraction to total lead in
the biosphere. Levels of lead in the atmosphere, the main conduit for lead movement from
sources into various environmental compartments are 10,000 to 20,000-fold higher in some urban
areas than in the most remote regions of the earth. Chronological records assembled using
reliable lead analysis techniques which show that atmospheric lead levels were at least
2,000-fold lower than at present before abrupt anthropological inputs accelerated with the
industrial revolution and more recently, with the introduction of leaded gasoline. For actual
comparison, estimates indicate a general background air lead level of 0.0005 ug Pb/m3 versus
23PB13/A 13-2 9/20/83
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INDUSTRIAL
EMISSIONS
CRUSTAL
WEATHERING
SURFACE AND
GROUND WATER
FECE8 URINE
Rgure 13-1. Pathways of lead from the environment to man.
13-3
-------
PRELIMINARY DRAFT
urban air lead concentrations frequently approaching 1.0 ug Pb/m^. A recent measurement of
0.000076 |jg Pb/m3 at the South Pole, using highly reliable lead analysis, suggests an anthro-
pogenic enrichment factor of 13,000-fold compared to the same urban air level of 1.0 ug Pb/m^.
Lead occupies an important niche in the U.S. economy, with consumption averaging 1.36 x
106 metric tons/year over the period 1971-1980. Of the various categories of lead consump-
tion, those of pigments, gasoline additives, ammunition, foil, solder and steel products are
widely dispersed and therefore unrecoverable. In the United States, about 41,000 tons are
emitted to the atmosphere each year, including 35,000 tons as gasoline additives. Lead and
its compounds enter the atmosphere at various points during mining, smelting, processing, use,
recycling, or disposal. Leaded gasoline combustion in vehicles accounted for 86 percent of
the total anthropogenic input into the atmosphere in the U.S. in 1981. Of the remaining 14
percent of total emissions from stationary sources, 7 percent was from the metallurgical
industry, 2 percent was from waste oil combustion, and 2 percent from coal combustion.
Atmospheric emissions have declined in recent years with the phase-down of lead in gasoline.
The fate of emitted particulate lead depends on particle size. It has been estimated
that, of the 75 percent of combusted gasoline lead which immediately departs the vehicle in
exhaust, 46 percent is in the form of particles <0.25 [im mass median equivalent diameter
(MMED) and 54 percent has an average particle size of >10 urn. The sub-micron fraction is in-
volved in long-range transport, whereas the larger particles settle mainly near the roadway.
13.2.2 Environmental Cycling of Lead
The atmosphere is the main conduit for movement of lead from emission sources to other
environmental compartments. Removal of lead from the atmosphere occurs by both wet and dry
deposition processes, each mechanism accounting for about one-half of the atmospheric lead
removed. The fraction of lead emitted as alkyl lead vapor (1 to 6 percent) undergoes subse-
quent transformation to other, more stable compounds such as triethyl- or trimethyl lead, as a
complex function of sunlight, temperature and ozone level.
Studies of the movement of lead emitted into the atmosphere indicate that air lead levels
decrease logarithmically with distance away from the source: (1) away from emission sites,
e.g., roadways and smelters; (2) within urban regions away from central business districts;
(3) from urban to rural areas; and (4) from developed to remote areas.
By means of wet and dry deposition, atmospheric lead is transferred to terrestrial sur-
faces and bodies of water. Transfer to water occurs either directly from the atmosphere or
through runoff from soil to surface waters. A sizeable fraction of water-borne lead becomes
lodged in aquatic sediments. Percolation of water through soil does not transport much lead
to ground water because most of the lead is retained at the soil surface.
23PB13/A 13-4 9/20/83
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PRELIMINARY DRAFT
The fate of lead particles on terrestrial surfaces depends upon such factors as the
mechanism of deposition, the chemical form of the particulate lead, the chemical nature of the
receiving soil, and the amount of vegetation cover. Lead deposited on soils is apparently
immobilized by conversion to the carbonate, by binding to humic or fulvic acids, or by ion
exchange on clays and hydrous oxides. In industrial, playground, and household environments,
atmospheric particles accumulate as dusts with lead concentrations often greater than 1000
ug/g. It is important to distinguish these dusts from windblown soil dust, which typically
has a lead concentration of 10 to 30 ug/g.
It has been estimated that soils adjacent to roadways have been enriched in lead content
by as much as 10,000 ug Pb/g soil since 1930, while in urban areas and sites adjacent to
smelters as much as 130,000 ug Pb/g has been measured in the upper 2.5 cm layer of soil.
Soil appears to be the major sink for emitted lead, with a residency half-time of
decades; but soil as a reservoir for lead cannot be considered as an infinite sink, because
lead will continue to pass into the grazing and detrital food chains and sustain elevated lead
levels in them until equilibrium is reached. It was estimated in Chapters 7 and 8 that lead
in soils not adjacent to major sources such as highways and smelters contain 3 to 5 ug/g of
anthropogenic lead and that this lead has caused an increase of lead in soil moisture by a
factor of 2 to 4. Thus, movement of lead from soils to other environmental compartments is at
least twice the prehistoric rate and will continue to increase for the foreseeable future.
Lead enters the aquatic compartment by direct transfer from the atmosphere via wet and
dry precipitation as well as indirectly from the terrestrial compartment via surface runoff.
Water-borne lead, in turn, may be retained in some soluble fraction or may undergo sedimenta-
tion, depending on such factors as pH, temperature, suspended matter which may entrap lead,
etc. Present levels of lead in natural waters represent a 50-fold enrichment over background
content, from 0.02 to 1.0 ug Pb/1, due to anthropogenic activity. Surface waters receiving
urban effluent represent a 2500-fold and higher enrichment (50 |jg Pb/1 and higher).
13.2.3 Levels of Lead in Various Media of Relevance to Human Exposure
Human populations in the United States are exposed to lead in air, food, water, and dust.
In rural areas, Americans not occupationally exposed to lead consume 50 to 75 ug Pb/day. This
level of exposure is referred to as the baseline exposure because it is unavoidable except by
drastic change in lifestyle or by regulation of lead in foods or ambient air. There are
several environmental circumstances that can increase human exposures above baseline levels.
Most of these circumstances involve the accumulation of atmospheric dusts in the work and play
environments. A few, such as pica and family home gardening, may involve consumption of lead
from chips of exterior or interior house paint.
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PRELIMINARY DRAFT
13.2.3.1 Ambient Air Lead Levels. Monitored ambient air lead concentration values in the
U.S. are contained in two principal data bases: (1) EPA's National Air Sampling Network
(NASN), recently renamed National Filter Analysis Network (NFAN); and (2) EPA's National Aero-
metric Data Bank, consistting of measurements by state and local agencies in conjunction with
compliance monitoring for the current ambient air lead standard.
NASN data for 1982, the most current year in the annual surveys, indicate that most of
the urban sites show reported annual averages below 0.7 ug Pb/m3, while the majority of the
non-urban locations have annual figures below 0.2 |jg Pb/m3. Over the interval 1976-1981,
there has been a downward trend in these averages, mainly attributable to decreasing lead
content of leaded gasoline and the increasing usage of lead-free gasoline. Furthermore,
examination of quarterly averages over this interval shows a typical seasonal variation,
characterized by maximum air lead values in winter and minimum values in summer.
With respect to the particle size distribution of ambient air lead, EPA studies using
cascade impactors in six U.S. cities have indicated that 60 to 75 percent of such air lead was
associated with sub-micron particles. This size distribution is significant in considering
the distance particles may be transported and the deposition of particles in the pulmonary
compartment of the respiratory tract. The relationship between airborne lead at the monitor-
ing station and the lead inhaled by humans is complicated by such variables as vertical
gradients, relative positions of the source, monitor, and the person, and the ratio of indoor
to outdoor lead concentrations. To obtain an accurate picture of the amount of lead inhaled
during the normal activities of an individual, personal monitors would probably be the most
effective. But the information gained would be insignificant, considering that inhaled lead
is only a small fraction of the total lead exposure, compared to the lead in food, beverages,
and dust. The critical question with respect to airborne lead is how much lead becomes
entrained in dust. In this respect, the existing monitoring network may provide an adequate
estimate of the air concentration from which the rate of deposition can be determined. The
percentage of ambient air lead which represents alkyl forms was noted in one study to range
from 0.3 to 2.7 percent, rising up to about 10 percent at service stations.
13.2.3.2 Levels of Lead In Dust. The lead content of dusts can figure prominently in the
total lead exposure picture for young children. Lead in aerosol particles deposited on rigid
surfaces in urban areas (such as sidewalks, porches, steps, parking lots, etc.) does not
undergo dilution compared to lead transferred by deposition onto soils. Dust can approach
extremely high concentrations. Dust lead can accumulate in the interiors of dwellings as well
as in the outside surroundings, particularly in urban areas.
Measurements of soil lead to a depth of 5 cm in areas of the U.S., using sites near road-
ways, were shown in one study to range from 150 to 500 ug Pb/g dry weight close to roadways
23PB13/A 13-6 9/20/83
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PRELIMINARY DRAFT
(i.e., within 8 meters). By contrast, lead in dusts deposited on or near heavily traveled
traffic arteries show levels in major U.S. cities ranging up to 8000 ug Pb/g and higher. In
residential areas, exterior dust lead levels are 1000 ug/g or less. Levels of lead in house
dust can be significantly elevated. A study of house dust samples in Boston and New York City
revealed levels of 1000 to 2000 ug Pb/g. Some soils adjacent to houses with exterior lead-
based paints may have lead concentrations greater than 10,000 ug/g.
Thirty-four percent of the baseline consumption of lead by children comes from the con-
sumption of 0.1 g of dust per day (Tables 13-1 and 13-2). Ninety percent of this dust lead is
of atmospheric origin. Dust also accounts for more than ninety percent of the additive lead
attributable to residences in an urban environment or near a smelter (Table 13-3).
13.2.3.3 Levels of Lead in Food. The route by which adults and older children in the base-
line population of the U.S. receive the largest proportion of lead intake is through foods,
with reported estimates of the dietary lead intake for Americans ranging from 60 to 75 ug/day.
The added exposure from living in an urban environment is about 30 ug/day for adults and 100
ug/day for children, all of which can be attributed to atmospheric lead.
Atmospheric lead may be added to food crops in the field or pasture, during transporta-
tion to the market, during processing, and during kitchen preparation. Metallic lead, mainly
solder, may be added during processing and packaging. Other sources of lead, as yet undeter-
mined, increase the lead content of food between the field and dinner table. American
children, adult females, and adult males consume 29, 33 and 46 ug Pb/day, respectively, in
milk and nonbeverage foods. Of these amounts, 38 percent is of direct atmospheric origin, 36
percent is of metallic origin and 20 percent is of undetermined origin.
Processing of foods, particularly canning, can significantly add to their background lead
content, although it appears that the impact of this is being lessened with the trend away
from use of lead-soldered cans. The canning process can increase lead levels 8-to 10-fold
higher than for the corresponding uncanned food items. Home food preparation can also be a
source of additional lead in cases where food preparation surfaces are exposed to moderate
amounts of high-lead household dust.
13.2.3.4 Lead Levels in Drinking Water. Lead in drinking water may result from contamination
of the water source or from the use of lead materials in the water distribution system. Lead
entry into drinking water from the latter is increased in water supplies which are plumbo-
solvent, i.e., with a pH below 6.5. Exposure of individuals occurs through direct ingestion
of the water or via food preparation in such water.
The interim EPA drinking water standard for lead is 0.05 ug/g (50 ug/1) and several
extensive surveys of public water supplies indicate that only a limited number of samples ex-
ceeded this standard on a nationwide basis. For example, a survey of interstate carrier water
supplies conducted by EPA showed that only 0.3 percent exceeded the standard.
23PB13/A 13-7 9/20/83
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TABLE 13-1. SUHHARY OF BASELINE HUMAN EXPOSURES TO LEADT
GO
i
oo
Soil
Source
Child 2-yr old
Inhaled Air
Food
Water & beverages
Dust
Total
Percent
Adult female
Inhaled Air
Food
Water & beverages
Oust
Total
Percent
Adult Bale
Inahaled air
Food
Water & beverages
Oust
Total
Percent
Total
Lead
Consumed
0.5
28.7
11.2
21.0
61.4
100%
1.0
33.2
17.9
4^5
56.6
100%
1.0
45.7
25.3
4
76.3
100%
Percent
of
Total
Consumption
0.8%
46.7
18.3
34.2
1.8%
58.7
31.6
7.9
1.3%
59.9
32.9
5.9
Natural
Lead
Consumed
0.001
0.9
0.01
0.6
1.5
2.4%
0.002
1.0
0.01
0.2
1.2
2.1%
0.002
1.4
0.1
0.2
1.7
2.2%
Indirect
Atmospheric
Lead*
-
0.9
2.1
'-
3.0
4.9%
-
1.0
3.4
_I_
4.4
7.8%
-
1.4
4.7
-
6.1
8.0%
Direct
Atmospheric
Lead*
0.5
10.9
1.2
19.0
31.6
51.5%
1.0
12.6
2.0
2.9
18.5
32.7%
1.0
17.4
2.8
2.9
24.1
31.6%
Lead from
Solder or
Other Metals
-
10.3
7.8
_1-
18.1
29.5%
-
11.9
12.5
— Z—
24.4
43.1%
-
16.4
17.5
-
33.9
44.4%
Lead of
Undetermined
Origin
-
17.6
-
1.4
19.0
22.6%
-
21.6
-
1.4
23.0
26.8%
-
31.5
-
1.4
32.9
27. IX
"Indirect atmospheric lead has beet previously incorporated into soil, and will probably remain in the soil for decades or
longer. Direct atmospheric lead has been deposited on the surfaces of vegetation and living areas or incorporated during
food processing shortly before human consumption. It may be assumed that 85 percent of direct atmospheric lead derives
from gasoline additives.
tunits are in ug/day.
;o
-<
o
-------
PRELIMINARY DRAFT
TABLE 13-2. RELATIVE BASELINE HUMAN LEAD EXPOSURES EXPRESSED PER KILOGRAM BODY WEIGHT*
Total
Lead
Consumed
Total Lead Consumed
Per Kg Body Wt
ug/Kg-Day
Atmospheric Lead
Per Kg Body Wt
ug/Kg-Day
Child (2 yr old)
Inhaled air
Food
Water and beverages
Dust
Total
Adult female
Inhaled air
Food
Water and beverages
Dust
Total
(ug/day)
0.5
28.7
11.2
21.0
61.4
1.0
33.2
17.9
4.5
56.6
0.05
2.9
1.1
2.1
6.15
0.02
0.66
0.34
0.09
1.13
0.05
1.1
0.12
1.9
3.17
0.02
0.25
0.04
0.06
0.37
Adult male
Inhaled air
Food
Water and
Dust
beverages
Total
1.0
45.7
25.1
4.5
76.3
0.014
0.65
0.36
0.064
1.088
0.014
0.25
0.04
0.04
0.344
*Body weights: 2 year old child = 10/kg; adult female = 50 kg; adult male = 70 kg.
The major source of lead contamination of drinking water is the distribution system it-
self, particularly in older urban areas. Highest levels are encountered in "first-draw" sam-
ples, i.e., water sitting in the piping system for an extended period of time. In a large
community water supply survey of 969 systems carried out in 1969-1970, it was found that the
prevalence of samples exceeding 0.05 pg/g was greater where water was plumbo-solvent.
Most drinking water, and the beverages produced from drinking water, contain 0.008 to
0.02 ug Pb/g. The exceptions are canned juices and soda pop, which range from 0.033 to 0.052
ug/g. About 11 percent of the lead consumed in drinking water and beverages is of direct
atmospheric origin, 70 percent comes from solder and other metals.
23PB13/A
13-9
9/20/83
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PRELIMINARY DRAFT
TABLE 13-3. SUMMARY OF POTENTIAL ADDITIVE EXPOSURES TO LEAD
Baseline exposure:
Child (2 yr old)
Inhaled air
Food, water & beverages
Dust
Total baseline
Additional exposure due to:
urban atmospheres: 1
air inhalation
dust
family gardens2
interior lead paint3
residence near smelter:4
air inhalation
dust
secondary occupational5
Baseline exposure:
Adult Male
Inhaled air
Food, water & beverages
Dust
Total baseline
Additional exposure due to:
urban atmospheres:1
air inhalation
dust
family gardens2
interior lead paint3
residence near smelter:4
air inhalation
dust
occupational6
secondary occupational5
smoking
wine consumption
Total
Lead
Consumed
(ug/day)
0.5
39.9
21.0
61.4
7
72
800
85
60
2250
150
1.0
70.8
4.5
76.3
14
7
2000
17
120
250
1100
21
30
100
Atmospheric
Lead
Consumed
(ug/day)
0.5
12.1
19.0
31.6
7
71
200
-
60
2250
~
1.0
20.2
2.9
24.1
14
7
500
-
120
250
1100
-
27
7
Other
Lead
Sources
(ug/day)
"
27.8
2.J
29.8
0
1
600
85
-
-
"
-
50.6
1.6
52.2
-
-
1500
17
-
.
-
-
3
?
1 includes lead from household and street dust (1000 ug/g) and Inhaled air (.75 ug/m3)
2assumes soil lead concentration of 2000 ug/g; all fresh leafy and root vegetables, sweet
corn of Table 7-15 replaced by produce from garden. Also assumes 25* of soil lead is of
atmospheric origin.
3assumes household dust rises from 300 to 2000 ug/g. Dust consumption remains the same as
baseline. Does not include consumption of paint chips.
4assumes household and street dust increases to 25,000 ug/g, inhaled air increases to 6
ug/m3.
5assumes household dust increases to 2400 ug/g.
^assumes 8 hr shift at 10 ug Pb/m3 or 90X efficiency of respirators at 100 ug/ Pb/m3. and
occupational dusts at 100,000 ug/m3.
13-10
-------
PRELIMINARY DRAFT
13.2.3.5 Lead in Other Media. Flaking lead paint in deteriorated housing stock in urban
areas of the Northeast and Midwest has long been recognized as a major source of lead exposure
for young children residing in this housing stock, particularly for children with pica.
Individuals who are cigarette smokers may inhale significant amounts of lead in tobacco smoke.
One study has indicated that the smoking of 30 cigarettes daily results in lead intake equiva-
lent to that of inhaling lead in ambient air at a level of 1.0 |jg Pb/m3.
13.2.3.6 Cumulative Human Lead Intake From Various Sources.
Table 13-1 shows the baseline of human lead exposures as described in detail in Chapter
7. These data show that atmospheric lead accounts for at least 30 percent of the baseline
adult consumption and 50 percent of the daily consumption by a 2 yr old child. These percent-
ages are conservative estimates because a part of the lead of undetermined origin may
originate from atmospheric lead not yet accounted for.
From Table 13-2, it can be seen that young children have a dietary lead intake rate that
is 5-fold greater than for adults, on a body weight basis. To these observations must be
added that absorption rates for lead are higher in children than in adults by at least 3-fold.
Overall, then, the rate of lead entry into the blood stream of children, on a body weight
basis, is estimated to be twice that of adults from the respiratory tract and 6 and 9 times
greater from the GI tract. Since children consume more dust than adults, the atmospheric
fraction of the baseline exposure is ten-fold higher for children than for adults, on a body
weight basis. These differences generally tend to place young children at greater risk, in
terms of relative amounts of proportions of atmospheric lead absorbed per kg body weight, than
adults under any given lead exposure situation.
13.3 LEAD METABOLISM: KEY ISSUES FOR HUMAN HEALTH RISK EVALUATION
From the detailed discussion of those various quantifiable characteristics of lead toxi-
cokinetics in humans and animals presented in Chapter 10, several clear issues emerge as being
important for full evaluation of the human health risk posed by lead:
1) Differences in systemic or internal lead exposure of groups within the general popula-
tion in terms of such factors as age/development and nutritional status; and
2) The relationship of indices of internal lead exposures to both environmental levels of
lead and tissues levels/effects.
Item 1 provides the basis for identifying segments within human populations at increased
risk in terms of exposure criteria and is used along with additional information on relative
sensitivity to lead health effects for identification of risk populations. The chief concern
23PB13/A 13-11 9/20/83
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PRELIMINARY DRAFT
with item 2 is the adequacy of current means for assessing internal lead exposure in terms of
providing adequate margins of protection from lead exposures producing health effects of con-
cern.
13.3.1 Differential Internal Lead Exposure Within Population Groups
Compared to adults, young children take in more lead through the gastrointestinal and
respiratory tracts on a unit body weight basis, absorb a greater fraction of this lead intake,
and also retain a greater proportion of the absorbed amount.
Unfortunately, such amplification of these basic toxicokinetic parameters in children vs.
adults also occurs at the time when: (1) humans are developmentally more vulnerable to the
effects of toxicants such as lead in terms of metabolic activity, and (2) the interactive re-
lationships of lead with such factors as nutritive elements are such as to induce a negative
course toward further exposure risk.
Typical of physiological differences in children vs. adults in terms of lead exposure im-
plications is a more metabolically active skeletal system in children. In children, turnover
rates of bone elements such as calcium and phosphorus are greater than in adults, with corre-
spondingly greater mobility of bone-sequestered lead. This activity is a factor in the obser-
vation that the skeletal system of children is relatively less effective as a depository for
lead than in adults.
Metabolic demand for nutrients, particularly calcium, iron, phosphorus, and the trace
nutrients, is such that widespread deficiencies of these nutrients exist, particularly among
poor children. The interactive relationships of these elements with lead are such that defi-
ciency states both enhance lead absorption/retention and, as in the case of lead-induced
reductions in 1,25-dihydroxyvitamin D, establish increasingly adverse interactive cycles.
Quite apart from the physiological differences which enhance internal lead exposure in
children is the unique relationship of 2- to 3-year-olds to their exposure setting by way of
normal mouthing behavior and the extreme manifestation of this behavior, pica. This behavior
occurs in the same age group which studies have consistently identified as having a peak in
blood lead. A number of investigations have addressed the quantification of this particular
route of lead exposure, and it is by now clear that such exposure will dominate other routes
when the child's surroundings, e.g., dust and soil, are significantly contaminated by lead.
Information provided in Chapter 10 also makes it clear that lead traverses the human pla-
cental barrier, with lead uptake by the fetus occurring throughout gestation. Such uptake of
lead poses a potential threat to the fetus via an impact on the embryological developement of
the central nervous and other systems. Hence, the only logical means of protecting the fetus
from lead exposure is exposure control during pregnancy.
23PB13/A 13-12 9/20/83
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PRELIMINARY DRAFT
Within the general population, then, young children and pregnant women qualify as defin-
ale risk groups for lead exposure. Occupational exposure to lead, particularly among lead
workers, logically defines these individuals as being in a high-risk category; work place con-
tact is augmented by those same routes and levels of lead exposure affecting the rest of the
adult population. From a biological point of view, lead workers do not differ from the gene-
ral adult population with respect to the various toxicokinetic parameters and any differences
in exposure control—occupational vs. non-occupational populations—as they exist are based on
factors other than toxicokinetics.
13.3.2 Indices of Internal Lead Exposure and Their Relationship To External Lead Levels and
Tissue Burdens/Effects
Several points are of importance in this area of lead toxicokinetics. They are: 1) the
temporal characteristics of indices of lead exposure; 2) the relationship of the indicators to
external lead levels; 3) the validity of indicators of exposure in reflecting target tissue
burdens; 4) the interplay between these indicators and lead in body compartments; and 5) those
various aspects of the issue with particular reference to children.
At this time, blood lead is widely held to be the most convenient, if imperfect, index of
both lead exposure and relative risk for various adverse health effects. In terms of ex-
posure, however, it is generally accepted that blood lead is a temporally variable measure
which yields an index of relatively recent exposure because of the rather rapid clearance of
absorbed lead from the blood. Such a measure, then, is of limited usefulness in cases where
exposure is variable or intermittent over time, as is often the case with pediatric lead ex-
posure.
Mineralizing tissue, specifically deciduous teeth, accumulate lead over time in propor-
tion to the degree of lead exposure, and analysis of this material provides an assessment
integrated over a greater time period and of more value in detecting early childhood exposure.
These two methods of assessing internal lead exposure have obvious shortcomings. A blood
lead value will say little about any excessive lead intake at early periods, even though such
remote exposure may have resulted in significant injury. On the other hand, whole tooth or
dentine analysis is retrospective in nature and can only be done after the particularly vulne-
rable age in children under 4 to 5 years-- has passed. Such a measure, then provides IHtle
utility upon which to implement regulatory policy or clinical intervention.
The dilemmas posed by these existing methods may be able to be resolved by iji situ analy-
sis of teeth and bone lead, such that the intrinsic advantage of mineral tissue as a cumula-
tive index is combined with measurement which is temporally concordant with on-going exposure.
Work in several laboratories offers promise for such ij} situ analysis (See Chapters 9 and 10).
23PB13/A 13-13 9/20/83
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PRELIMINARY DRAFT
A second issue concerning internal indices of exposure and environmental lead is the
relationship of changes in lead content of some medium with changes in blood content. Much of
Chapter 11 was given over to description of the mathematical relationships of blood lead with
lead in some external medium-- air, food, water, etc., without consideration of the biological
underpinnings for these relationships.
Over a relatively broad range of lead exposure through some medium, the relationship of
lead in the external medium to blood lead is curvilinear, such that relative change in blood
lead per unit change in medium level generally becomes increasingly less as exposure increases.
This behavior may reflect changes in tissue lead kinetics, reduced lead absorption, or in-
creased excretion. Limited animal data would suggest that changes in excretion or absorption
are not factors in this phenomenon. In any event, modest changes in blood levels with expo-
sure at the higher end of this range are in no way to be taken as reflecting concomitantly
modest changes in body or tissue lead uptake. Evidence continues to accumulate which suggests
that an indicator such as blood lead is an imperfect measure of tissue lead burdens and of
changes in such tissue levels in relation to changes in external exposure.
In Chapter 10, it was pointed out that blood lead is logarithmically related to chelata-
ble lead (the latter being a more useful measure of the potentially toxic fraction of body
lead), such that a unit change in blood lead is associated with an increasingly larger amount
of chelatable lead. One consequence of this relationship is that moderately elevated blood
lead values will tend to mask the "margin of safety" in terms of mobile body lead burdens.
Such masking is apparent in one study of children where chelatable lead levels in children
showing moderate elevations in blood lead overlapped those obtained in subjects showing frank
plumbism, i.e. overt lead intoxication.
Related to the above is the question of the source of chelatable lead. It was noted in
Chapter 10 that some sizable fraction of chelatable lead is derived from bone and this compels
reappraisal of the notion that bone is an "inert sink" for otherwise toxic body lead.
The notion of bone lead as toxicologically inert never did accord with what was known
from studies of bone physiology, i.e., that bone is a "living" organ, and the thrust of recent
studies of chelatable lead as well as interrelationships of lead and bone metabolism is more
to a view of bone lead as actually an insidious source of long-term systemic lead exposure
rather than evidence of a protective mechanism permitting significant lead contact in indus-
trialized populations.
The complex interrelationships of lead exposure, blood lead, and lead in body compart-
ments is of particular interest in considering the disposition of lead in young children.
Since children take in more lead on a weight basis, and absorb and retain more of this lead
than the adult, one might expect that either tissue and blood levels would be significantly
elevated or that the child's skeletal system would be more efficient in lead sequestration.
23PB13/A 13-14 9/20/83
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PRELIMINARY DRAFT
Blood lead levels in young children are either similar to adults (males) or somewhat
higher (adult females). Limited autopsy data, furthermore, indicate that soft tissue levels
in children are not markedly different from adults, whereas the skeletal system shows an
approximate 2-fold increase in lead concentration from infancy to adolescence. Neglected in
this observation is the fact that the skeletal system in children grows at an exponential
rate, so that skeletal mass increases 40-fold during the interval in childhood when bone lead
levels increase 2-fold, resulting in an actual increase of approximately 80-fold in total ske-
letal lead. If the skeletal growth factor is taken into account, along with growth in soft
tissue and the expansion of vascular fluid volumes, the question of lead disposition in
children is better understood.
Finally, limited animal data indicate that blood lead alterations with changes in lead
exposure are poor indicators of such changes in target tissue. Specifically, it appears that
abrupt reduction of lead exposure will be more rapidly reflected in blood lead than in such
target tissues as the central nervous system, especially in the developing organism. This
discordance may underlie the observation that severe lead neurotoxicity in children is assoc-
iated with a rather broad range of blood lead values (see Section 12.4).
The above discussion of some of the problems with the use of blood lead in assessing tar-
get tissue burdens or the toxicologically active fraction of total body lead is really a sum-
nary of the inherent toxicokinetic problems with use of blood lead levels in defining margins
of safety for avoiding internal exposure or undue risk of adverse effects.
If, for example, blood lead levels of 40-50 ug/dl in "asymptomatic" children are associ-
ated with chelatable lead burdens which overlap those encountered in frank pediatric plumbism,
as documented in one series of lead-exposed children, then there is no margin of safety at
these blood levels for severe effects which are not at all a matter of controversy. Were it
both logistically feasible to do so on a large scale and were the use of chelants free of
health risk to the subjects, serial provocative chelation testing would appear to be the
better indicator of exposure and risk. Failing this, the only prudent alternative is the use
of a large safety factor applied to blood lead which would translate to an "acceptable" chela-
table burden. It is likely that this blood lead value wquld lie well below the currently
accepted upper limit of 30 ug/dl, since the safety factor would have to be large enough to
protect against frank plumbism as well as more subtle health effects seen with non-overt lead
intoxication. This rationale from the standpoint of lead toxicokinetics is in accord also
with the growing data base for dose-effect relationships of lead's effects on heme biosynthe-
sis, erythropoiesis, and the nervous system in humans as detailed in Sections 12.3 and 12.4.
The future developement and routine use of i_n situ mineral tissue testing at time points
concordant with on-going exposure and the comparison of such results with simultaneous blood
23PB13/A 13-15 9/20/83
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PRELIMINARY DRAFT
lead and chelatable lead measurement would be of significant value in further defining what
level of blood lead is indeed an acceptable upper limit.
13.4 DEMOGRAPHIC CORRELATES OF HUMAN LEAD EXPOSURE AND RELATIONSHIPS BETWEEN EXTERNAL AND
INTERNAL LEAD EXPOSURE INDICES
13.4.1 Demographic Correlates of Lead Exposure
Studies of ancient populations using bone and teeth show that levels of internal exposure
of lead today are substantially elevated over past levels. Studies of current populations
living in remote areas far from urbanized cultures show blood lead levels in the range of 1 to
5 ug/dl. In contrast to the blood lead levels found in remote populations, data from current
U.S. populations have geometric means ranging from 10 to 20 ug/dl depending on age, race, sex
and degree of urbanization. These increases of current exposure appear to be associated with
industrialization and widespread commercial use of lead, for example gasoline combustion.
Age appears to be one of the single most important demographic covariate of blood lead
levels. Blood lead levels in children up to six years are generally higher than those in non-
occupational ly exposed adults. Children aged two to three years tend to have the highest
levels as shown in Figure 13-2. Blood lead levels in non-occupationally exposed adults may
increase slightly with age due to skeletal lead accumulation.
Sex has a differential impact on blood lead levels depending on age. No significant dif-
ferences exist between males and females less than seven years of age. Males above the age of
seven generally have higher blood lead levels than females.
Race also plays a role, in that blacks have higher blood lead levels than either whites
or Hispanics. Race has yet to be fully disentangled from exposure.
Blood lead levels also seem to increase with degree of urbanization. Data from NHANES II
show that blood lead levels in the United States, averaged from 1976 to 1980, increase from a
geometric mean of 11.9 ug/dl in rural populations to 12.8 ug/dl in urban populations less than
one million, and increase again to 14.0 ug/dl in urban populations of one million or more.
Recent U.S. blood lead levels show a downward trend occurring consistently across race,
age and geographic location. The downward pattern commenced in the early part of the 1970's
and has continued into 1980. The downward trend has occurred from a shift in the entire dis-
tribution and not through a truncation in the high blood lead levels. This consistency sug-
gests a general causative factor, and attempts have been made to identify the causative ele-
ment. Reduction in lead emitted from the combustion of leaded gasoline is a prime candidate,
but at present no causal relationship has beon definitively established.
23PB13/A 13-16 9/20/83
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CO
40
36
30
TJ 25
1
0
20
16
o
o
o
10 —
IDAHO STUDY
NEW YORK SCREENING - BLACKS
NEW YORK SCREENING - WHITES
NEW YORK SCREENING HISPANICS
NHANES II STUDY - BLACKS
NHANES II STUDY - WHITES
I I I I I I
6
10
AGE IN YEARS
Figure 13-2. Geometric mean blood lead levels by race and age for younger children in the
NHANES II study, and the Kellogg/Silver Valley and New York Childhood Screening Studies.
-------
PRELIMINARY DRAFT
Blood lead levels, examined on a population basis, have similarly skewed distributions.
Blood lead levels, from a population thought to be homogenous in terms of demographic and lead
exposure characteristics, approximately follow a lognormal distribution. Geometric standard
deviations, an estimation of dispersion, from four different studies discussed in Chapter 11,
including analytic error, are about 1.4 for children and possibly somewhat smaller for adults.
This allows an estimation of the upper tail of the blood lead distribution, which is the popu-
lation segment in the United States at higher risk.
13.4.2 Relationships Between External and Internal Lead Exposure Indices
Because one main purpose of this chapter is to examine relationships of lead in air and
lead in blood under ambient conditions, the results of studies most appropriate to this area
have been emphasized. A summary of the most appropriate studies appears in Table 13-4. At
air lead exposures of 3.2 MS/m? or less, there is no statistically significant difference be-
tween curvilinear and linear blood lead inhalation relationships. At air lead exposures at 10
ug/nr* or more, either nonlinear or linear relationships can be fitted. Thus, a reasonably
consistent picture emerges in which the blood lead air lead relationship by direct inhalation
was approximately linear in the range of normal ambient exposures (0.1 - 2.0 ug/m?) as dis-
cussed in Chapter 7. Differences among individuals in a given study, and among several
studies are large, so that pooled estimates of the blood lead inhalation slope depend upon the
the weight given to various studies. Several studies were selected for analysis, based upon
factors described earlier. EPA analyses of experimental and clinical studies (Griffin et al.,
1975; Rabinowitz et al., 1974, 1976, 1977; Kehoe 1961a,b,c; Gross 1981; Hammond et al., 1981)
suggest that blood lead in adults increases by 1.64 ± 0.22 ug/dl from direct inhalation of
each additional ug/m3 of air lead. EPA analyses of population studies (Yankel et al. , 1977;
Roels et al., 1980; Angle and Mclntire, 1979) suggest that, for children, the blood lead
increase is 1.97 ± 0.39 ug/dl per ug/ma, for air lead. EPA anaylsis of Azar's population study
(Azar et al., 1975) yields a slope of 1.32 ± 0.38 for adult males.
These slope estimates are based on the assumption that an equilibrium level of blood lead
is achieved within a few months after exposure begins. This is only approximately true, since
lead stored in the skeleton may return to blood after some years. Chamberlain et al. (1978)
suggest that long term inhalation slopes should be about 30 percent larger than these esti-
mates. Inhalation slopes quoted here are associated with a half-life of blood lead in adults
of about 30 days. O'Flaherty et al. (1982) suggest that the blood-lead half-life may increase
slightly with duration of exposure, but this has not been confirmed (Kang et al., 1983).
One possible approach would be to regard all inhalation slope studies as equally infor-
mative and to calculate an average slope using reciprocal squared standard error estimates as
23PB13/A 13-18 9/20/83
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PRELIMINARY DRAFT
TABLE 13-4. SUMMARY OF BLOOD INHALATION SLOPES, (p)
(jg/dl per
POPULATION STUDY
Children Angle and
Mclntire, 1979
Omaha, NE
Roels et al .
(1980)
Belgium
Yankel et al.
(1977); Walter
et al. (1980)
Idaho
Adult Males Azar et al.
(1975). Five
groups
Griffin et al.
(1975), NY
prisoners
Gross
(1979)
Rabinovn'tz et
al. (1973,1976,
1977)
STUDY (p) MODEL SENSITIVITY
TYPE N SLOPE OF SLOPE*
ug/dl per ug/m3
Population 1074 1.92 (1.40 - 4.40)1'2'3
Population 148 2.46 (1.55 - 2.46)1'2
Population 879 1.52 (1.07 - 1.52)1'2'3
Population 149 1.32 (1.08 - 2.39)2'3
Experiment 43 1.75 (1.52 - 3.38)4
Experiment 6 1.25 (1.25 - 1.55)2
Experiment 5 2.14 (2.14 - 3.51)5
*Selected from among the most plausible statistically equivalent models. For nonlinear models,
slope at 1.0 ug/m3.
Sensitive to choice of other correlated predictors such as dust and soil lead.
2
Sensitive to linear vs. nonlinear at low air lead.
Sensitive to age as a covariate.
4
Sensitive to baseline changes in controls.
Sensitive to assumed air lead exposure.
23PB13/A
13-19
9/20/83
-------
PRELIMINARY DRAFT
weights. This approach has been rejected for two reasons. First, the standard error estima-
tes characterize only the internal precision of an estimated slope, not its representativeness
(i.e., bias) or predictive validity. Secondly, experimental and clinical studies obtain more
information from a single individual than do population studies. Thus, it may not be appro-
priate to combine the two types of studies.
Estimates of the inhalation slope for children are only available from population
studies. The importance of dust ingestion as a non-inhalation pathway for children is estab-
lished by many studies. A slope estimate has been derived for air lead inhalation based on
those studies (Angle and Mclntire 1979; Roels et al., 1980; Yankel et al., 1977) from which
the air inhalation and dust ingestion contributions can both be estimated.
While direct inhalation of air lead is stressed, this is not the only air lead contribu-
tion that needs to be considered. Smelter studies allow partial assessment of the air lead
contributions to soil, dust and finger lead. Conceptual models allow preliminary estimation
of the propagation of lead through the total food chain as shown in Chapter 7. Useful mathe-
matical models to quantify the propagation of lead through the food chain need to be devel-
oped. The direct inhalation relationship does provide useful information on changes in blood
lead as responses to changes in air lead on a time scale of several months. The indirect
pathways through dust and soil and through the food chain may thus delay the total blood lead
response to changes in air lead, perhaps by one or more years. The Italian ILE study
facilitates partial assessment of this delayed response from leaded gasoline as a source.
Dietary absorption of lead varies greatly from one person to another and depends on the
physical and chemical form of the carrier, on nutritional status, and on whether lead is in-
gested with food or between meals. These distinctions are particularly important for con-
sumption by children of leaded paint, dust and soil. Typical values of 10 percent absorption
of ingested lead into blood have been assumed for adults and 25 to 50 percent for children.
It is difficult to determine accurate relationships between blood lead levels and lead
levels in food or water. Dietary intake must be estimated by duplicate diets or fecal lead
determinations. Water lead levels can be determined with some accuracy, but the varying
amounts of water consumed by different individuals adds to the uncertainty of the estimated
relationships.
Quantitative analyses relating blood lead levels and dietary lead exposures have been re-
ported. Studies on infants provide estimates that are in close agreement. Only one indi-
vidual study is available for adults (Sherlock et al. 1982); another estimate from a number of
pooled studies is also available. These two estimates are in good agreement. Most of the
subjects in the Sherlock et al. (1982) and United Kingdom Central Directorate on Environmental
Pollution (1982) studies received quite high dietary lead levels (>300 ug/day). The fitted
PB13B/C 13-20 9/20/83
-------
PRELIMINARY DRAFT
cube root equations give high slopes at lower dietary lead levels. On the other hand, the
linear slope of the United Kingdom Central Directorate on Environmental Pollution (1982) study
is probably an underestimate of the slope at lower dietary lead levels. For these reasons,
the Ryu et al. (1983) study is the most believable, although it only applies to infants.
Estimates for adults should be taken from the experimental studies or calculated from assumed
absorbtion and half-life values. Most of the dietary intake supplements were so high that
many of the subjects had blood lead concentrations much in excess of 30 ng/ms for a considera-
ble part of the experiment. Blood lead levels thus may not completely reflect lead exposure,
due to the previously noted nonlinearity of blood lead response at high exposures. The slope
estimates for adult dietary intake are about 0.02 ug/dl increase in blood lead per ug/day in-
take, but consideration of blood lead kinetics may increase this value to about 0.04. Such
values are a bit lower than those estimated from the population studies extrapolated to typi-
cal dietary intakes about 0.05 M9/dl per ug/day. The value for infants is much larger.
The relation between blood lead and water lead is not clearly defined and is often de-
scribed as nonlinear. Water lead intake varies greatly from one person to another. It has
been assumed that children can absorb 25 to 50 percent of lead in water. Many authors chose
to fit cube root models to their data, although polynomial and logarithmic models were also
used. Unfortunately, the form of the model greatly influences the estimated contributions to
blood lead levels from relatively low water lead concentration.
Although there is close agreement in the quantitative analyses of the relationship bet-
ween blood lead level and dietary lead, there is a larger degree of variability in results of
the various water lead studies. The relationship is curvilinear, but its exact form is yet to
be determined. At typical levels for U.S. populations, the relationship appears linear. The
only study that determines the relationship based on lower water lead values (<100 pg/l) is
the Pocock et al. (1983) study. The data from this study, as well as the authors themselves,
suggest that in this lower range of water lead levels, the relationship is linear. Further-
more, the estimated contributions to blood lead levels from this study are quite consistent
with the polynomial models from other studies. For these reasons, the Pocock et al. (1983)
slope of 0.06 is considered to represent the best estimate. The possibility still exists,
however, that the higher estimates of the other studies may be correct in certain situations,
especially at higher water lead levels (>100 ug/1).
Studies relating soil lead to blood lead levels are difficult to compare. The relation-
ship obviously depends on depth of soil lead, age of the children, sampling method, cleanli-
ness of the home, mouthing activities of the children, and possibly many other factors. Var-
ious soil sampling methods and sampling depths have been used over time, and as such they may
not be directly comparable and may produce a dilution effect of the major lead concentration
PB13B/C 13-21 9/20/83
-------
PRELIMINARY DRAFT
contribution from dust which is located primarily in the top 2 cm of the soil. Increases in
soil dust lead significantly increase blood lead in children. From several studies (Yankel et
a!., 1977; Angle and Mclntire, 1979) EPA estimates an increase of 0.6 to 6.8 ug/dl in blood
lead for each increase of 1000 jjg/g in soil lead concentration. The values from the Stark et
al. (1982) study of about 2, may rapresent a reasonable median estimate. The relationship of
housedust lead to blood lead is difficult to obtain. Household dust also increases blood
lead, children from the cleanest homes in the Silver Valley/Kellogg Study having 6 |jg/dl less
lead in blood, on average, than those from the households with the most dust.
A number of specific environmental sources of airborne lead have been evaluated for po-
tential direct influence on blood lead levels. Combustion of leaded gasoline appears to be the
largest contributor to airborne lead. Two studies used isotope ratios of lead to estimate the
relative proportion of lead in the blood coming from airborne lead.
From the Manton study it can be estimated that between 7 to 41 percent of the blood lead
in study subjects in Dallas resulted from airborne lead. Additionally, these data provide a
means of estimating the indirect contribution of air lead to blood lead. By one estimate,
only 10 to 20 percent of the total airborne contribution in Dallas is from direct inhalation.
From the ILE data of Facchetti and Geiss (1982), as shown in Table 13-5, the direct in-
halation of air lead may account for 54 percent of the total adult blood lead uptake from
leaded gasoline in a large urban center, but inhalation is a much less important pathway in
TABLE 13-5. ESTIMATED CONTRIBUTION OF LEADED GASOLINE TO BLOOD LEAD
BY INHALATION AND NON-INHALATION PATHWAYS
Location
Turin
<25 km
>25 km
Air Lead
Fraction
From
Gasoline3
0.873
0.587
0.587
Blood
Lead
Fraction
From .
Gasoline
0.237
0.125
0.110
Blood
Lead
From
Gasoline
In Airc
(Mg/dl)
2.79
0.53
0.28
Blood Lead
Net
Inhaled
From .
Gasoline
(ug/dl)
2.37
2.60
3.22
Estimated
Fraction
Gas- Lead
Inhalation
0.54
0.17
0.08
Fraction of air lead in Phase 2 attributable to lead in gasoline.
Mean fraction of blood lead in Phase 2 attributable to lead in gasoline.
Estimated blood lead from gas inhalation = B x (a) x (b), B = 1.6.
Estimated blood lead from gas, non-inhalation = (f)-(e)
eFraction of blood lead uptake from gasoline attributable to direct inhalation = (f)/(e)
Source: Facchetti and Geiss (1982), pp. 52-56.
23PB13/A 13-22 9/20/83
-------
PRELIMINARY DRAFT
suburban parts of the region (17 percent of the total gasoline lead contribution) and in the
rural parts of the region (8 percent of the total gasoline lead contribution). EPA analyses
of the preliminary results from the ILE study separated the inhalation and non-inhalation con-
tributions of leaded gasoline to blood lead into the following three parts: (1) An increase
of about 1.7 ug/dl in blood lead per ug/rn^ of air lead, attributable to direct inhalation of
the combustion products of leaded gasoline; (2) a sex difference of about 2 ug/dl attributable
to lower exposure of women to indirect (non-inhalation) pathways for gasoline lead; and (3) a
non-inhalation background attributable to indirect gasoline lead pathways, such as ingestion
of dust and food, increasing from about 2 ug/dl in Turin to 3 ug/dl in remote rural areas.
The non-inhalation background represents only two to three years of environmental accumulation
at the new experimental lead isotope ratio. It is not clear how to numerically extrapolate
these estimates to U.S. subpopulations; but it is evident that even in rural and suburban
parts of a metropolitan area, the indirect (non-inhalation) pathways for exposure to leaded
gasoline make a significant contribution to blood lead. This can be seen in Table 13-5. It
should also be noted that the blood lead isotope ratio responded fairly rapidly when the lead
isotope ratio returned to its pre-experimental value, but it is not yet possible to estimate
the long term change in blood lead attributable to persistent exposures to accumulated envi-
ronmental lead.
Studies of data from blood lead screening programs suggest that the downward trend in
blood lead levels noted earlier is due to the reduction in air lead levels, which has been at-
tributed to the reduction of lead in gasoline.
Primary lead smelters, secondary lead smelters and battery plants emit lead directly into
the air and ultimately increase soil and dust lead concentrations in their vicinity. Adults,
and especially children, have been shown to exhibit elevated blood lead levels when living
close to these sources. Blood lead levels in these residents have been shown to be related to
air, as well as to soil or dust exposures.
13.4.3 Proportional Contributions of Lead in Various Media to Blood Lead
in Human Populations
The various mathematical descriptions of the relationship of blood lead to lead in indi-
vidual media—air, food, water, dust, soil—were discussed in some detail in Chapter 11 and
concisely in the preceding section (13.4.2) of this chapter. Using values for lead intake/
content of these media which appear to represent the current exposure picture for human popu-
lations in the U.S., these relationships are further employed in this section to estimate
proportional inputs to total blood lead levels in U.S. populations. Such an exercise is of
help in providing an overall perspective on which routes of exposure are of most significance
in terms of contributions to blood lead levels seen in U.S. populations.
PB13B/C 13-23 9/20/83
-------
PRELIMINARY DRAFT
Table 13-6 tabulates the relative direct contributions (in percentages) of air lead to
blood lead at different air-lead levels for calculated typical background levels of lead from
food and water in adults. The blood lead contributions from diet are estimated using the
slope 0.02 ug/dl increase in blood lead ug/day intake as discussed in Section 11.4.2.4.
TABLE 13-6. DIRECT CONTRIBUTIONS OF AIR LEAD TO BLOOD LEAD (PbB)
IN ADULTS AT FIXED INPUTS OF WATER AND FOOD LEAD
Air Lead
(ug/m3)
0.1
1.0
1.5
3 A PbB
PbB (Air)a
0.2
2.0
3.0
fl fn^ 3 ? nni
PbB (Food)b
2.0
2.0
2.0
'm3 nr locc
PbB (Water)c
0.6
0.6
0.6
% PbB
From Air
7.1
43.4
53.5
A Pb Air
Assuming 100 ug/day lead from diet and slope 0.02 as discussed in Section 11.4.2.4.
CAssuming 10 ug/£ water, Pocock et al. (1983).
In Table 13-7 are listed the direct contributions of air lead to blood lead at varying
air lead levels for children given calculated typical background levels of blood lead for food
and water. Diet contribution is based on the work of Ryu et al. (1983). Table 13-8 shows
the relative contributions of dust/soil to blood lead at varying dust/soil levels for children
given calculated background levels of blood lead from air, food, and water. Assuming that
virtually all soil/dust lead is due to atmospheric fallout of lead particles, the percentage
contribution of air directly and indirectly to blood lead becomes significantly greater than
when considering just the direct impact of inhaling lead in the ambient air.
23PB13/A
13-24
9/20/83
-------
TABLE 13-7. DIRECT CONTRIBUTIONS OF AIR LEAD TO 8LOOD LEAD IN CHILDREN AT
FIXED INPUTS OF FOOD AND WATER LEAD
00
1
ro
en
Air Lead
(ug/m*)
0.1
0.5
1.0
1.5
2.5
3 A PbB
PbB (Air)3
0.2
1.0
2.0
3.0
5.0
n fnr ^ ? nn/i
PbB (Food)5
16.0
16.0
16.0
16.0
16.0
m3 or l*»«;s_
PbB (Water)c
0.6
0.6
0.6
0.6
0.6
% PbB
From Air
1.2
5.7
10.8
15.3
23.1
A Pb Air
b
Assuming 100 ug Pb/day based upon Ryu et al. (1983).
c Assuming 10 ug Pb/1 water, using Pocock et al. (1983).
XI
-<
O
-------
OJ
I
ro
(ft
TABLE 13-8. CONTRIBUTIONS OF DUST/SOIL LEAD TO BLOOD LEAD IN CHILDREN AT
FIXED INPUTS OF AIR, FOOD, AND WATER LEAD
Dust-Soil
(MQ/fl)
500
1000
2000
a A PbB
Air Lead
ug/m3
0.5
0.5
0.5
n fnr> t 9 tin
PbB (Air)a
1.0
1.0
1.0
/m nr* IACC
PbB (Food)b
16.0
16.0
16.0
PbB (Water)0
0.6
0.6
0.6
PbB .
(Dust-Soil)
0.3/3.4
0.6/6.8
1.2/13.6
% PbB
From Dust/Soil
1.7/16.2
3.3/27.8
6.4/43.6
o
•33
Assuming 100 pg Pb/day based on Ryu et al. (1983).
^Assuming 10 ug Pb/1 water, based on Pocock et al. (1983).
Based on range 0.6 to 6.8 ug/dl for 1000 ug/g (Angle and Mclntire, 1979).
-------
PRELIMINARY DRAFT
13.5 BIOLOGICAL EFFECTS OF LEAD RELEVANT TO THE GENERAL HUMAN POPULATION
13.5.1 Introduction
It is clear from the wealth of available literature reviewed in Chapter 12, that there
exists a continuum of biological effects associated with lead across a broad range of expo-
sure. At rather low levels of lead exposure, biochemical changes, e.g., disruption of certain
enzymatic activities involved in heme biosynthesis and erythropoietic pyrimidine metabolism,
are detectable. Heme biosynthesis is a generalized process in mammalian species, including
man, with importance for normal physiological functioning of virtually all organ systems.
With increasing lead exposure, there are sequentially more intense effects on heme synthesis
and a broadening of lead effects to additional biochemical and physiological mechanisms in
various tissues, such that increasingly more severe disruption of the normal functioning of
many different organ systems becomes apparent. In addition to heme biosynthesis impairment at
relatively low levels of lead exposure, disruption of normal functioning of the erythropoietic
and the nervous systems are among the earliest effects observed as a function of increasing
lead exposure. With increasingly intense exposure, more severe disruption of the erythropoie-
tic and nervous systems occur and additional organ systems are affected so as to result, for
example, in the manifestation of renal effects, disruption of reproductive functions, and im-
pairment of immunological functions. At sufficiently high levels of exposure, the damage to
the nervous system and other effects can be severe enough to result in death or, in some cases
of non-fatal lead poisoning, long-lasting sequelae such as permanent mental retardation.
As discussed in Chapter 12 of this document, numerous new studies, reviews, and critiques
concerning Pb-related health effects have been published since the issuance of the earlier EPA
lead criteria document in 1977. Of particular importance for present criteria development
purposes are those new findings, taken together with information earlier available at the
writing of the 1977 Criteria Document, which have bearing on the establishment of quantitative
dose-effect or dose-response relationships for biological effects of lead potentially viewed
as adverse health effects likely to occur among the general population at or near existing
ambient air concentrations of lead in the United States. Key information regarding observed
health effects and their implications are discussed below for. adults and children.
For the latter group, children, emphasis is placed on the discussion of (1) heme biosyn-
thesis effects, (2) certain other biochemical and hematological effects, and (3) the disrup-
tion of nervous system functions. All of these appear to be among those effects of most con-
cern for potential occurrence in association with exposure to existing U.S. ambient air lead
levels of the population group (i.e., children ^6 years old) at greatest risk for lead-induced
health effects. Emphasis is also placed on the delineation of internal lead exposure levels,
as defined mainly by blood-lead (PbB) levels, likely associated with the occurrence of such
23PB13/A 13-27 9/20/83
-------
PRELIMINARY DRAFT
effects. Also discussed are characteristics of the subject effects that are of crucial impor-
tance in regard to the determination of which might reasonably be viewed as constituting
"adverse health effects" in affected human populations.
Over the years, there has been superimposed on the continuum of lead-induced biological
effects various judgments as to which specific effects observed in man constitute "adverse
health effects". Such judgments involve not only medical concensus regarding the health sig-
nificance of particular effects and their clinical management, but also incorporate societal
value judgments. Such societal value judgments often vary depending upon the specific overall
contexts to which they are applied, e.g., in judging permissible exposure levels for occupa-
tional versus general population exposures to lead. For some lead exposure effects, e.g.,
severe nervous system damage resulting in death or serious medical sequelae consequent to
intense lead exposure, there exists little or no disagreement as to these being significant
"adverse health effects." For many other effects detectable at sequentially lower levels of
lead exposure, however, the demarcation lines as to which effects represent adverse health
effects and the lead exposure levels at which they are accepted as occurring are neither sharp
nor fixed, having changed markedly during the past several decades. That is, from a histori-
cal perspective, levels of lead exposure deemed to be acceptable for either occupationally ex-
posed persons or the general population have been steadily revised downward as more sophisti-
cated biomedical techniques have revealed formerly unrecognized biological effects and concern
has increased in regard to the medical and social significance of such effects.
It is difficult to provide a definitive statement of all criteria by which specific bio-
logical effects associated with any given agent can be judged to be "adverse health effects".
Nevertheless, several criteria are currently well-accepted as helping to define which effects
should be viewed as "adverse". These include: (1) impaired normal functioning of a specific
tissue or organ system itself; (2) reduced reserve capacity of that tissue or organ system in
dealing with stress due to other causative agents; (3) the reversibility/irreversibility of
the particular effect(s); and (4) the cumulative or aggregate impact of various effects on
individual organ systems on the overall functioning and well-being of the individual.
Examples of possible uses of such criteria in evaluating lead effects can be cited for
illustrative purposes. For example, impairment of heme synthesis intensifies with increasing
lead exposure until hemeprotein synthesis is inhibited in many organ systems, leading to re-
ductions in such functions as oxygen transport, cellular energetics, and detoxification of
xenobiotic agents. The latter effect can also be cited as an example of reduced reserve capa-
city pertinent to consideration of effects of lead, the reduced capacity of the liver to deto-
xify certain drugs or other xenobiotic agents resulting from lead effects on hepatic detoxifi-
cation enzyme systems.
23PB13/A 13-28 9/20/83
-------
PRELIMINARY DRAFT
In regard to the issue of reversibility/irreversibility of lead effects, there are really
two dimensions to the issue that need to be considered, i.e.-: (1) biological reversibility or
irreversibility characteristic of the particular effect in a given organism; and (2) the gene-
rally less-recognized concept of exposure reversibility or irreversibility. Severe central
nervous system damage resulting from intense, high level lead exposure is generally accepted
as an irreversible effect of lead exposure; the reversibility/irreversibility of certain more
difficult-to-detect neurological effects occurring at lower lead exposure levels, however,
remains a matter of some controversy. The concept of exposure reversibility/irreversibility
can be illustrated by the case of urban children of low socioecomomic status showing dis-
turbances in heme biosynthesis and erythropoiesis. Biologically, these various effects may be
considered reversible; the extent to which actual reversibility occurs, however, is determined
by the feasibility of removing these subjects from their particular lead exposure setting. If
such removal from exposure is unlikely or does not occur, then such effects will logically
persist and, defacto, constitute essentially irreversible effects.
13.5.2 Dose-Effect Relationships for Lead-Induced Health Effects
13.5.2.1 Human Adults
Table 13-9 concisely summarizes the lowest observed effect levels (in terms of blood lead
concentrations) thus far credibly associated with particular health effects of concern for
human adults in relation to specific organ systems or generalized physiological processes,
e.g. heme synthesis.
The most serious effects associated with markedly elevated blood lead levels are severe
neurotoxic effects that include irreversible brain damage as indexed by the occurrence of
acute or chronic encephalopathic symptoms observed in both humans and experimental animals.
For most human adults, such damage typically does not occur until blood lead levels exceed
100-120 (jg/dl. Often associated with encephalopathic symptoms at such blood lead levels or
higher are severe gastrointestinal symptoms and objective signs of effects on several other
organ systems as well. The precise threshold for occurrence of overt neurological and gastro-
intestinal signs and symptoms of lead intoxication remains to be established but such effects
have been observed in adult lead workers at blood lead levels as low as 40-60 ug/dl, notably
lower than the 60 or 80 ug/dl levels previously established or discussed as being "safe" for
occupational lead exposure.
Other types of health effects occur coincident with the above overt neurological and gas-
trointestinal symptoms indicative of marked lead intoxication. These range from frank peri-
pheral neuropathies to chronic renal nephropathy and anemia. Toward the lower range of blood
lead levels associated with overt lead intoxication or somewhat below, less severe but impor-
tant signs of impairment in normal physiological functioning in several organ systems are
evident, including: (1) slowed nerve conduction velocities indicative of peripheral nerve
23PB13/A 13-29 9/20/83
-------
TABLE 13-9. SUMMARY OF LOWEST OBSERVED EFFECT LEVELS FOR KEY LEAD-INDUCED HEALTH EFFECTS IN ADULTS
Lowest Observed
Effect Level (PbB)
100-120
80
60
0 50
o
5
40
30
25-30
15-20
<10
ug/dl
Mg/dl
M«/dl
ug/dl
Mg/dl
M9/dl
Mg/dl
Mg/dl
Mg/dl
Heme Synthesis and
Hematological Effects
Frank anemia
Reduced hemoglobin
production
Increased urinary ALA and
elevated coproporphyrins
Erythrocyte protoporphyrin
(EP) elevation In males
Erythrocyte protoporphyrin
(EP) elevation in females
ALA-D Inhibition
Neurological Renal System Reproductive Gastrointestinal
Effects Effects Function Effects Effects
Encephalopathic signs Chronic renal
and symptoms nephropathy
T?
Overt subencephalopathic Altered t
neurological .symptoms funct
i? I 1
Peripheral nerve dysfunction
(slowed nerve conduction)
1
Overt gastrointestinal
symptoms (colic, etc.)
-Q
73
rn
r—
HH
esticular >-<
ion ^
70
o
TO
-n
Abbreviations: PbB = blood lead concentrations.
-------
PRELIMINARY DRAFT
dysfunction (at 30-40 ug/dl» or possibly lower levels); (2) altered testicular function (at
40-50 ug/dl); and (3) reduced hemoglobin production (at approximately 50 ug/dl) and other
signs of impaired heme synthesis evident at still lower blood lead levels. All of these ef-
fects point toward a generalized impairment of normal physiological functioning across several
different organ systems, which becomes abundantly evident as adult blood lead levels approach
or exceed 30-40 ug/dl. Evidence for impaired heme synthesis effects in blood cells exists at
still lower blood lead levels in human adults and the significance of this and evidence of
impairment of other biochemical processes important in cellular energetics are the subject of
discussion below in relation to health effects observed in children.
13.5.2.2 Children
Table 13-10 summarizes lowest observed effect levels for a variety of imporatnt health
effects observed in children. Again, as for adults, it can be seen that lead impacts many
different organ systems and biochemical/physiological processes across a wide range of expo-
sure levels. Also, again, the most serious of these effects is the severe, irreversible cen-
tral nervous system damage manifested in terms of encephalopathic signs and symptoms. In
children, effective blood lead levels for producing encephalopathy or death are lower than for
adults, starting at approximately 80-100 ug/dl. Other overt neurological symptoms are evident
at somewhat lower blood lead levels associated with lasting neurological sequalae. Colic and
other overt gastrointestinal symptoms clearly occur at similar or still lower blood lead
levels in children, at least down to 60 ug/dl and, perhaps, below. Renal dysfunction is also
manifested along with the above overt signs of lead intoxication in children and has been
reported at blood lead levels as low as 40 ug/dl in some pediatric populations. Frank anemia
is also evident at 70 ug/dl, representing an extreme manifestation of reduced hemoglobin syn-
thesis observed at blood lead levels as low as 40 ug/dl along with other signs of marked heme
synthesis inhibition at that exposure level. Again, all of these effects are reflective of
widespread impact of lead on the normal physiological functioning of many different organ
systems in children at blood lead levels at least as low as 40 ug/dl.
Among the most important ahd controversial of the issues discussed in Chapter 12 are the
evaluation of neurbpsychological or electrophysiological effects associated with low-level
lead exposures in non-overtly lead intoxicated children. None of the available studies on the
subject, individually, can be said to prove conclusively that significant neurological effects
occur in children at blood-Pb levels <30 ug/dl. The collective neurobehavioral studies of CNS
(cognitive; IQ) effects, for example, can probably now be most reasonably interpreted as most
clearly being indicative of a likely association between neuropsychologic deficits and low-
level Pb-exposures in young children resulting in blood-Pb levels of approximately 30 to 50
ug/dl.
23PB13/A 13-31 9/20/83
-------
TABLE 13-10. SUMMARY OF LOWEST OBSERVED EFFECT LEVELS FOR KEY LEAD-INDUCED HEALTH EFFECTS IN CHILDREN
Lowest Observed
Effect Level (PbB)
80-100 MQ/dl
70 M9/dl
60 pg/dl
50 pg/dl
V *0 Mfl/dl
ro
30 pg/dl
15-20 pg/dl
10
Heme Synthesis and Neurological Renal Systen Gastrointestinal Other Biochemical
Hewtological Effects Effects Effects Effects Effects
Frank
Encephalopathic Rer
signs and symptoms fur
(at
anemia
T
?
Reduced hemoglobin Cognitive (CNS) deficts
Elevated coproporphyrin Peripheral nerve dysfunction
(slowed NCV's)
Increased urinary ALA
Erythrocyte protoporphyin CNS electrophysiological
elevation deficits
ALA-0 inhibition ?
ial dys- Colic, other overt
iction gastrointestinal symptoms
n'noaciduria)
~o
TO
I—
I-H
1 3
TO
d
Vitamin D metabolism ^
interference
Py-5-N activity
inhibition
Abbreviations: PbB = blood lead concentrations; Py-5-N = pyri«idine-5'-nucleotidase.
-------
PRELIMINARY DRAFT
However, due to specific methodological problems with each of the various studies (as
noted in Chapter 12), much caution is warranted that precludes conclusive acceptance of the
observed effects being due to Pb rather than other (at times uncontrolled for) potentially
confounding variables.
Also of considerable importance are studies by by Benignus et al. (1981) and Otto et al.
(1981, 1982a,b), which provide evidence of changes in EEC brain wave patterns and CNS evoked
potential responses in non-overtly lead intoxicated children experiencing relatively low
blood-Pb levels. Sufficient exposure information was provided by Otto et al. (1981, 1982a,b);
and appropriate statistical analyses were carried out which demonstrated clear, statistically
significant associations between electrophysiological (SW voltage) changes and blood-Pb levels
in the range of 30 to 55 ug/dl and probable analogous associations at blood-Pb levels below 30
ug/dl (with no evident threshold down to 15 ug/dl). In this case, the continued presence of
such electrophysiological changes upon follow-up two years later, suggests persistence of such
effects even in the face of later declines in blood-Pb levels and, therefore, possible non-
reversibility of the observed electrophysiological CNS changes. However, the reported elec-
trophysiological effects were not found to be significantly associated with IQ decrements.
The precise medical or health significance of the neuropsychological and electrophysiolo-
gical effects found by the above studies to be associated with low-level Pb-exposures is dif-
ficult to state with confidence at this time. The IQ deficits and other behavioral changes,
although statistically significant, are generally relatively small in magnitude as detected by
the reviewed studies, but nevertheless may still impact the intellectual development, school
performance, and social development of the affected children sufficiently so as to'be regarded
as adverse. This would be especially true if such impaired intellectual development or school
performance and disrupted social development were reflective of persisting, long-term effects
of low-level lead exposure in early childhood. The issue of persistence of such lead effects,
however, remains to be more clearly resolved, with some study results reviewed in Chapter 12
and mentioned above suggesting relatively short-lived or markedly decreasing Pb-effects on
neuropsychological functions over a few years from early to later childhood and other studies
suggesting that significant low-level Pb-induced neurobehavioral and EEC effects may, in fact,
persist into later childhood.
In regard to additional studies reviewed in Chapter 12 concerning the neurotoxicity of
lead, certain evidence exists which suggests that neurotoxic effects may be associated with
Pb-induced altered heme synthesis, which results in an accumulation of ALA in brain affecting
CNS GABA synthesis, binding, and/or inactivation by neuronal reuptake after synaptic release.
Also, available experimental data suggest that these effects may have functional significance
in the terms of this constituting one mechanism by which lead may -increase the sensitivity of
23PB13/A 13-33 9/20/83
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PRELIMINARY DRAFT
rats to drug-induced seizures and, possibly, by which GABA-related behavioral or physiological
control functions are disrupted. Unfortunately, the available research data do not allow cre-
dible direct estimates of blood-Pb levels at which such effects might occur in rats, other
non-human mammalian species, or man. Inferentially, however, one can state that threshold
levels for any marked Pb-induced ALA impact on CNS GABA mechanisms are most probably at least
as high as blood-Pb levels at which significant accumulations of ALA have been detected in
erythrocytes or non-blood soft tissues (see below). Regardless of any dose-effect levels in-
ferred, though, the functional and/or medical significance of Pb-induced ALA effects on CNS
mechanisms at low-levels of Pb-exposure remains to be more fully determined and cannot, at
this time, be unequivocably seen as an adverse health effect.
Research concerning Pb-induced effects on heme synthesis, also provides information of
importance in evaluating whether significant health effects in children are associated with
blood-Pb levels below 30 (jg/dl. As discussed earlier, in Chapter 12, Pb affects heme synthe-
sis at several points in its metabolic pathway, with consequent impact on the normal func-
tioning of many body tissues. The activity of the enzyme, ALA-S, catalyzing the rate-limiting
step of heme synthesis does not appear to be significantly affected until blood-Pb levels
reach or exceed approximately 40 (jg/dl. The enzyme ALA-D, which catalizes the conversion of
ALA to porphobilinogen as a further step in the heme biosynthetic pathway, appears to be
affected at much lower blood-Pb levels as indexed directly by observations of ALA-D inhibition
or indirectly in terms of consequent accumulations of ALA in blood and non-blood tissues.
More specifically, inhibition of erythrocyte ALA-D activity has been observed in humans and
other mammalian species at blood-Pb levels even below 10 to 15 ug/dl, with no clear threshold
evident. Correlations between erythrocyte and hepatic ALA-D activity inhibition in lead
workers at blood-Pb levels in the range of 12 to 56 ug/dl suggest that ALA-D activity in soft
tissues (eg. brain, liver, kidney, etc.) may be inhibited at similar blood-Pb levels at which
erythrocyte ALA-D activity inhibition occurs, resulting in accumulations of ALA in both blood
and soft tissues.
It is now clear that significant increases in both blood and urinary ALA occur below the
currently commonly-accepted blood-Pb level of 40 (jg/dl and, in fact, such increases in blood
and urinary ALA are detectable in humans at blood-Pb levels below 30 ug/dl, with no clear
threshold evident down to 15 to 20 ug/dl. Other studies have demonstrated significant eleva-
tions in rat brain, spleen and kidney ALA levels consequent to acute or chronic Pb-exposure,
but no clear blood-Pb levels can yet be specified at which such non-blood tissue ALA increases
occur in humans. It is reasonable to assume, however, that ALA increases in non-blood tissues
likely begin to occur at roughly the same blood-Pb levels associated with increases in eryth-
rocyte ALA levels.
23PB13/A 13-34 9/20/83
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PRELIMINARY DRAFT
Lead also affects heme synthesis beyond metabolic steps involving ALA, leading to the
accumulation of protoporphyrin in erythrocytes as the result of impaired iron insertion into
the porphyrin moiety to form heme. The porphyrin acquires a zinc ion in lieu of the native
iron, and the resulting accumulation of blood zinc protoporphyrin (ZPP) tightly bound to ery-
throcytes for their entire life (120 days) represents a commonly employed index of Pb-exposure
for medical screening purposes. The threshold for elevation of erythrocyte protoporphyrin
(EP) levels is well-established as being 25 to 30 |jg/dl in adults and approximately 15 ug/dl
for young children, with significant EP elevations (>1 to 2 standard deviations above refer-
ence normal EP mean levels) occurring in 50 percent of all children studied as blood-Pb
approaches or moderately exceeds 30 ug/dl.
Medically, small increases in EP levels have generally not been viewed as being of great
concern at initial detection levels around 15 to 20 ug/dl in children, but EP increases become
more worrisome as markedly greater, significant EP elevations occur as blood-Pb levels
approach and exceed 30 ug/dl and additional signs of significantly deranged heme synthesis
begin to appear along with indications of functional disruption of various organ systems.
Previously, such other signs of significant organ system functional disruptions had only been
credibly detected at blood-Pb levels somewhat in excess of 30 ug/dl, e.g., hemoglobin synthe-
sis inhibition starting at 40 ug/dl and significant nervous system effects at 50-60 ug/dl.
This served as a basis for CDC establishment of 30 ug/dl blood-Pb as a criteria level for
undue Pb exposure for young children and adoption by EPA of it as the "maximum safe" blood-Pb
level (allowing some margin(s) of safety before reaching levels associated with inhibition of
hemoglobin synthesis or nervous system deficits) in setting the 1978 NAAQS for lead.
To the extent that new evidence is now available, indicative of probable Pb effects on
nervous system functioning or other important physiological processes at blood-Pb levels below
30 to 40 ug/dl, then the rationale for continuing to view 30 ug/dl as a "maximum safe" blood-
Pb level is called into question and substantial impetus is provided for revising the criteria
level downward, i.e., to some blood-Pb level below 30 ug/dl. At this time, such impetus
toward revising the blood-Pb criteria level downward is' gaining momentum not only from new
neuropsychologic and electrophysiological findings of the type summarized above, but also from
growing evidence for Pb effects on other functional systems. These include, for example, the:
(1) disruption of formation of the heme-containing protein, cytochrome c, of considerable
importance in cellular energetics involved in mediation of the normal functioning of many dif-
ferent mammalian (including human) organ systems and tissues; (2) inhibition by Pb of the bio-
synthesis of globin, the protein moiety of hemoglobin, in the presense of Pb at concentrations
corresponding to a blood-Pb level of 20 ug/dl; (3) observations of significant inhibition of
pyrimidine-5'-nucleotidase (Py-5-N) activity in adults at blood-Pb levels £44 ug/dl and in
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PRELIMINARY DRAFT
children down to blood-Pb levels of 10 ug/dl; and (4) observations of Pb interference with
vitamin D metabolism in children across a blood-Pb level range of 33 to 120 jjg/di, with conse-
quent increasingly enhanced Pb uptake due to decreased vitamin D metabolism and likely asso-
ciated increasingly cascading effects on nervous system and other functions at sequentially
higher blood-Pb levels. Certain additional evidence for Pb effects on hormonal systems and
immune system components, thus far detected only at relatively high blood-Pb levels or at
least not credibly associated with blood-Pb levels as low as 30 to 40 ug/dl, also contributes
to concern as blood-Pb levels exceed 30 ug/dl.
Also adding to the concern about relatively low lead exposure levels are the results of
an expanding array of animal toxicology studies which demonstrate: (1) persistence of lead-
induced neurobehavioral alterations well into adulthood long after termination of perinatal
lead exposure early in development of several mammalian species; (2) evidence for uptake and
retention of lead in neural and non-neuronal elements of the CNS, including long-term persis-
tence in brain tissues after termination of external lead exposure and blood lead levels
return to "normal"; and (3) evidence from various in-vivo and in-vitro studies indicating
that, at least on a subcellular-molecular level, no threshold may exist for certain neuro-
chemical effects of lead.
13.6 DOSE-RESPONSE RELATIONSHIPS FOR LEAD EFFECTS IN HUMAN POPULATIONS
Information summarized in the preceding section dealt with the various biological effects
of lead germane to the general population and included comments about the various levels of
blood lead observed to be associated with the measurable onset of these effects in various
populations groups.
As indicated above, inhibition of ALA-D activity by lead occurs at virtually all blood
lead levels measured in subjects residing in industrialized countries. If any threshold for
ALA-D inhibition exists, it lies somewhere below 10 ug Pb/dl in blood lead.
Elevation in erythrocyte porphyrin for a given blood lead level is greater in children
and women than in adult males, children being somewhat more sensitive than women. The thres-
hold for currently detectable EP elevation in terms of blood lead levels for children was
estimated at ca. 16 to 17 ug/dl in the recent studies of Piomelli et al. (1982). In adult
males, the corresponding blood lead value is 25 to 30 ug/dl.
Statistically significant reduction in hemoglobin production occurs at a lower blood lead
level in children, 40 ug/dl, than in adults, 50 ug/dl.
It appears that urinary ALA shows a correlation with blood lead levels to below 40 ug/dl,
but since there is no clear agreement as to the meaning of elevated ALA-U below 40 ug/dl, this
23PB13/A 13-36 9/20/83
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PRELIMINARY DRAFT
value is taken as the threshold for pronounced excretion of ALA into urine. This value
appears to apply to both children and adults. Whether this blood lead level represents a
threshold for the potential neurotoxicity of circulating ALA cannot now be stated and requires
further study.
Coproporphyrin elevation in urine first occurs at a blood lead level of 40 ug/dl and this
threshold appears to apply for both children and adults.
A number of investigators have attempted to quantify more precisely dose-population
response relationships for some of the above lead effects in human populations. That is they
have attempted to define the proportion of a population exhibiting a particular effect at a
given blood lead level. To date, such efforts at defining dose-response relationships for
lead effects have been mainly limited to the following effects of lead on heme biosynthesis:
inhibition of ALA-D activity; elevation of EP; and urinary excretion of ALA.
Dose-population response relationships for EP in children has been analyzed in detail by
Piomelli and et al. (1982) and the corresponding plot at 2 levels of elevation (>1 S.D., >2
S.D.) is shown in Figure 13-3 using probit analysis. It can be seen that blood lead levels in
half of the children showing EP elevations at >1 and 2 S.D.'s closely bracket the blood lead
level taken as the high end of "normal" (i.e., 30 ^g/dl). Dose-response curves for adult men
and women as well as children prepared by Roels et al. (1976) are set forth in Figure 13-4.
In Figure 13-4, it may be seen that the dose-response for children remains greater across the
blood-lead range studied, followed by women, then adult males.
Figure 13-5 presents dose-population response data for urinary ALA exceeding two levels
(at mean + 1 S.D. and mean + 2 S.O.), as calculated by EPA from the data of Azar et at.
(1975). The percentages of the study populations exceeding the corresponding cut-off levels
as calculated by EPA for the Azar data are set forth in Table 13-11. It should be noted that
the measurement of ALA in the Azar et al. study did not account for ami no acetone, which may
influence the results observed at the lowest blood lead levels.
23PB13/A 13-37 9/20/83
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99
95
1°°
§ »
5
o
O 25
2
oc
10
O
I
A
I
O
2
2
u.
O
UJ
I
8
C = NATURAL FREQUENCY _^
• r
I
I
I -r
10
20
50
60
30 40
BLOOD LEAD. Mg/dl
Figure 13-3. Dose-response for elevation of EP as a
function of blood lead level using probit analysis •
Geometric mean plus 1 S.D. = 33 ^g/dl; geometric mean
plus 2 S.D. = 53 fjg/dl.
Source: Piomelli et al. (1982).
40 —
20 —
40
50
20 30
BLOOD LEAD LEVEL ^ Pb/dl
Figure 13-4. Dose-response curve for FEP as a function
of blood lead level: in subpopulations.
Source: Roels et al. (1976).
13-38
-------
2
A
3
I
100 —
90
s •
70
I -
50
I "
30
20
10
1 I I
I I I T~T
O MEAN + 1 S.D.
A MEAN + 2 S.D.
MEAN ALAU = 0.32 FOR
BLOOD LEAD < 13 ug/dl
10 20 30 40 SO 60
BLOOD LEAD LEVEL Mfl
70
80 90
Figure 13-5. EPA calculated dose-response curve for
ALA-U.
Source: Azar et al. (1975).
TABLE 13-11. ERA-ESTIMATED PERCENTAGE OF SUBJECTS
WITH ALA-U EXCEEDING LIMITS FOR VARIOUS BLOOD LEAD LEVELS
Blood lead levels
10
20
30
40
50
60
70
Azar et al. (1975)
(Percent Population)
2
6
16
31
50
69
84
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13.7 POPULATIONS AT RISK
Population at risk is a segment of a defined population exhibiting characteristics asso-
ciated with significantly higher probability of developing a condition, illness, or other ab-
normal status. This high risk may result from either (1) greater inherent susceptibility or
(2) from exposure situations peculiar to that group. What is meant by inherent susceptibility
is a host characteristic or status that predisposes the host to a greater risk of heightened
response to an external stimulus or agent.
In regard to lead, two such populations are definable. They are preschool age children,
especially those living in urban settings, and pregnant women, the latter group owing mainly
to the risk to the conceptus. Children are such a population for both of the reasons stated
above, whereas pregnant women are at risk primarily due to the inherent susceptibility of the
conceptus.
13.7.1 Children as a Population at Risk
Children are developing and growing organisms exhibiting certain differences from adults
in terms of basic physiologic mechanisms, capability of coping with physiologic stress, and
their relative metabolism of lead. Also, the behavior of children frequently places them in
different relationship to sources of lead in the environment, thereby enhancing the opportu-
nity for them to absorb lead. Furthermore, the occurrence of excessive exposure often is not
realized until serious harm is done. Young children do not readily communicate a medical his-
tory of lead exposure, the early signs of such being common to so many other disease states
that lead is frequently not recognized early on as a possible etiological factor contributing
to the manifestation of other symptoms.
13.7.1.1 Inherent Susceptibility of the Young. Discussion of the physiological vulnerability
of the young must address two discrete areas. Not only should the basic physiological differ-
ences be considered that one would expect to predispose children to a heightened vulnerability
to lead, but also the actual clinical evidence must be considered that shows such vulner-
ability does indeed exist.
In Chapter 10 and Section 13.2 above, differences in relative exposure to lead and body
handling of lead for children versus adults were pinpointed throughout the text. The signifi-
cant elements of difference include: (1) greater intake of lead by infants and young children
into the respiratory and gastro-intestinal tracts on a body weight basis compared to adults;
(2) greater absorption and retention rates of lead in children; (3) much greater prevalence of
nutrient deficiency in the case of nutrients which affect lead absorption rates from the GI
tract; (4) differences in certain habits, i.e., normal hand to mouth activity as well as pica
resulting in the transfer of lead-contaminated dust and dirt to the GI tract; (5) differences
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in the efficiency of lead sequestration in the bones of children, such that not only is less
of the body burden of lead in bone at any given time but the amount present may be relatively
more labile. Additional information discussed in Chapter 12 suggests that the blood-brain
barrier in children is less developed, posing the risk for greater entry of lead into the
nervous system.
Hematological and neurological effects in children have been demonstrated to have lower
thresholds in terms of blood lead levels than in adults. The extent of reduced hemoglobin
production and EP accumulation occur at relatively lower exposure levels in children than in
adults, as indexed by blood lead thresholds. With reference to neurologic effects, the onset
of encephalopathy and other injury to the nervous system appears to vary both regarding likely
lower thresholds in children for some effects and in the typical pattern of neurologic effects
presented, e.g., in encephalopathy or other CNS deficits being more common in children versus
peripheral neuropathy being more often seen in adults. Not only are the effects more acute in
children than in adults, but also the neurologic sequelae are usually much more severe in
children. x
13.7.1.2 Exposure Consideration. The dietary habits of children as well as the diets them-
selves differ markedly from adults and, as a result, place children in a different relation-
ship to several sources of lead. The dominance of canned milk and processed baby food in the
diet of many young children is an important factor in assessing their exposure to lead since
both those foodstuffs have been shown to contain higher amounts of lead than components of the
adult diet. The importance of these lead sources is not their relationship to airborne lead
directly but, rather, their role in providing a higher baseline lead burden to which the air-
borne contribution is added.
Children ordinarily undergo a stage of development in which they exhibit normal mouthing
behavior, as manifested, for example, in the form of thumbsucking. At this time they are at
risk for picking up lead-contaminated soil and dust on their hands and hence into their mouths
where it can be absorbed. Scientific evidence documenting at least the first part of the
chain is available.
There is, however, an abnormal extension of mouthing behavior, called pica, which occurs
in some children. Although diagnosis of this is difficult, children who exhibit this trait
have been shown to purposefully eat nonfood items. Much of the lead-based paint problem is
known to occur because children actively ingest chips of leaded paint.
13.7.2 Pregnant Women and the Conceptus as a Population at Risk
There are some rather inconculsive data indicating that women may in general be somewhat
higher risk to lead than men. However, pregnant women and their concepti as a subgroup are
23PB13/A 13-41 9/20/83
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PRELIMINARY DRAFT
demonstrably at higher risk. It should be pointed out that, in fact, it really is not the
pregnant woman per se who is at greatest risk but, rather, the unborn child she is carrying.
Because of obstetric complications, however, the mother herself can also be at somewhat
greater risk at the time of delivery of her child.
Studies have demonstrated that women in general, like children, tend to show a heightened
response of erythorcyte protoporphyrin levels upon exposure to lead. The exact reason for
this heightened response is not known but may relate to endocrine differences between men and
women.
As stated above, the primary reason pregnant women are a high-risk group is because of
the fetus each is carrying. In addition, there is some suggestive evidence that lead expo-
sures may also affect maternal complications at delivery. With reference to maternal compli-
cation at delivery, information in the literature suggests that the incidence of preterm deli-
very and premature membrane rupture relates to maternal blood lead level. Further study of
this relationship as well as studies relating to discrete health effects in the newborn are
needed.
Vulnerability of the developing fetus to lead exposure arising from transplacental trans-
fer of maternal lead was discussed in Chapter 10. This process starts at the end of the first
trimester. Umbilical cord blood studies involving mother-infant pairs have repeatedly shown a
correlation between maternal and fetal blood lead levels.
Further suggestive evidence, cited in Chapter 12, has been advanced for prenatal lead
exposures of fetuses possibly leading to later higher instances of postnatal mental retarda-
tion among the affected offspring. The available data are insufficient to state with any cer-
tainty that such effects occur or to determine with any precision what levels of lead exposure
might be required prior to or during pregnancy in order to produce such effects.
13.7.3 Description of the United States Population in Relation to Potential
Lead Exposure Risk
In this section, estimates are provided of the number of individuals in those segments of
the population which have been defined as being potentially at greatest risk for lead ex-
posures. These segments include pre-school children (up to 6 years of age), especially those
living in urban settings, and women of child-hearing age (defined here as ages 15-44). These
data, which are presented below in Table 13-12, were obtained from a provisional report by the
U.S. Census Bureau (1982), which indicates that approximately 61 percent of the populace lives
in urban areas (defined as central cities and urban fringe). Assuming that the 61 percent
estimate for urban residents also applies to children of preschool age, then approximately
14,206,000 children of the total listed in Table 13-12 would be expected to be at greater risk
23PB13/A 13-42 9/20/83
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PRELIMINARY DRAFT
by virtue of higher lead exposures generally associated with their living in urban versus non-
urban settings. (NOTE. The age distribution of the percentage of urban residents may vary
between SMSA's.)
TABLE 13-12. PROVISIONAL ESTIMATE OF THE NUMBER OF INDIVIDUALS IN URBAN AND
RURAL POPULATION SEGMENTS AT GREATEST POTENTIAL RISK TO LEAD EXPOSURE
Population Segment
Pre-school chi
Women of
child-bearing
Idren
Total
age
Total
Actual Age
(year)
0-4
5
6
15-19
20-24
25-29
30-34
35-39
40-44
Total Number in U. S.
Population
(1981)
16,939,000
3,201,000
3,147,000
23,287,000
10,015,000
10,818,000
10,072,000
9,463,000
7,320,000
6,147^000
53,835,000
Urban ,
Population
10,333,000
1,953,000
1,920,000
14,206,000
6,109,000
6,599,000
6,144,000
5,772,000
4,465,000
3^749,000
32,838,000
Source: U.S. Census Bureau (1982), Tables 18 and 31.
An urban/total ratio of 0.61 was used for all age groups. "Urban" includes central city
and urban fringe populations.
The risk encountered with exposure to lead may be compounded by nutritional deficits (see
Chapter 10). The most commonly seen of these is iron deficiency, especially in young children
less than 5 years of age (Mahaffey and Michaelson, 1980). Data available from the National
Center for Health Statistics for 1976-1980 (Fulwood et al., 1982) indicate that from 8 to 22
percent of children aged 3-5 may exhibit iron deficiency, depending upon whether this condi-
tion is defined as serum iron concentration (<40 (jg/dl) or as transferrin saturation (<16 per-
cent), respectively. Hence, of the 20,140,000 children S5 years of age (Table 13-12), as many
as 4,431,000 would be expected to be at increased risk depending on their exposure to lead,
due to iron deficiency.
As pointed out in Section 13.7.2, the risk to pregnant women is mainly due to risk to the
conceptus. By dividing the total number of women of child-bearing age in 1981 (53,835,000)
into the total number of live births in 1981 (3,646,000; National Center for Health Statis-
tics, 1982), it may be seen that approximately 7 percent of this segment of the populetion
may be at increased risk at any given time.
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13.8 SUMMARY AND CONCLUSIONS
Among the most significant pieces of information and conclusions that emerge from the
present human health risk evaluation are the following:
(1) Anthropogenic activity has clearly led to vast increases of lead input into
those environmental compartments which serve as media (e.g., air, water, food,
etc.) by which significant human exposure to lead occurs.
(2) Emission of lead into the atmosphere, especially through leaded gasoline com-
bustion, is of major significance in terms of both the movement of lead to
other environmental compartments and the relative impact of such emissions on
the internal lead burdens in industrialized human populations. By means of
both mathematical modeling of available clinical/epidemiological data by EPA
and the isotopic tracing of lead from gasoline to the atmosphere to human blood
of exposed populations, the size of atmospheric lead contribution can be con-
fidently said to be 25-50 percent or probably somewhat higher.
(3) Given this magnitude of relative contribution to human external and internal
exposure, reduction in levels of atmospheric lead would then result in signifi-
cant widespread reductions in levels of lead in human blood (an outcome which
is supported by careful analysis of the NHANES II study data). Reduction of
lead in food (added in the course of harvesting, transport, and processing)
would also be expected to produce significant widespread reductions in human
blood lead levels in the United States.
(4) A number of adverse effects in humans and other species are clearly associated
with lead exposure and, from a historical perspective, the observed "thres-
holds" for these various effects (particularly neurological and heme biosynthe-
sis effects) continue to decline as more sophisticated experimental and clini-
cal measures are employed to detect more subtle, but still significant effects.
These include significant alterations in normal physiological functions at
blood lead levels markedly below the currently accepted 30 ug/dl "maxim safe
level" for pediatric exposures.
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(5) Preceding chapters of this document demonstrate that young children are at
greatest risk for experiencing lead-induced health effects, particularly in the
urbanized, low income segments of this pediatric population. A second group at
increased risk are pregnant women, because of exposure of the fetus to lead in
the absence of any effective biological (e.g. placental) barrier during gestation.
(6) Dose-population response information for heme synthesis effects, coupled with
information from various blood lead surveys, e.g. the NHANES II study, indicate
that large numbers of American children (especially low income, urban dwellers)
have blood lead levels sufficiently high (in excess of 15-20 ug/dl) that they
are clearly at risk for deranged herae synthesis and, possibly, other health ef-
fects of growing concern as lead's role as a general systemic toxicant becomes
more fully understood.
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13.9 REFERENCES
Angle, C. R.; Mclntire, M. S. (1979) Environmental lead and children: the Omaha study. J.
Toxicol. Environ. Health 5: 855-870.
Azar, A.; Snee, R. D. ; Habibi, K. (1975) An epidemiologic approach to community air lead ex-
posure using personal air samplers. In: Lead. Environ. Qua!. Saf. Suppl. 2: 254-288.
Benignus, V. A.; Otto, D. A.; Muller, K. E. ; Seiple, K. J. (1981) Effects of age and body lead
burden on CNS function in young children. II: EEC spectra. Electroencephalogr. Clin.
Neurophysiol. 52: 240-248.
Burchfiel, J. L.; Duffy, F. H.; Bartels, P. H.; Needleman, H. L. (1980) The combined discrimi-
nating power of quantitative electroencephalography and neuropsychologic measures in
evaluating central nervous system effects of lead at low levels. In: Needleman, H. L. ,
ed. Low level lead exposure: the clinical implications of current research. New York, NY:
Raven Press; pp. 75-90.
Chamberlain, A. C.; Heard, M. J.; Little, P.; Newton, D.; Wells, A. C. ; Wiffen, R. D. (1978)
Investigations into lead from motor vehicles. Harwell, United Kingdom: United Kingdom
Atomic Energy Authority; report no. AERE-R9198.
Ernhart, C. B. ; Landa, B. ; Schell, N. B. (1981) Subclinical levels of lead and developmental
deficit - a multivariate follow-up reassessment. Pediatrics 67: 911-919.
Facchetti, S. ; Geiss, F. (1982) Isotopic lead experiment: status report. Luxembourg: Commis-
sion of the European Communities; Publication no. EUR 8352 EN.
Greathouse, D. G.; Craun, G. F.; Worth, D. (1976) Epidemiologic study of the relationship
between lead in drinking water and blood lead levels. In: Hemphill, D. D. , ed. Trace sub-
stances in environmental health-X: [proceedings of University of Missouri's 10th annual
conference on trace substances in environmental health]; June; Columbia, MO. Columbia,
MO: University of Missouri-Columbia; pp. 9-24.
Griffin, T. B. ; Coulston, F. ; Wills, H.; Russell, J. C. ; Knelson, J. H. (1975) Clinical
studies of men continuously exposed to airborne particulate lead. In: Griffin, T. B.;
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