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 (Do Not
 Cite or Quote)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.

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                                         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Do Not Quote or Cite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

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                                   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

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                                       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
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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
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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
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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
                                               xi
                                                                 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
                                               xiii
                                                                 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
                                                                                         9/20/83

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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

-------
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

-------
 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

-------
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

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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-

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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-

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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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                                        PRELIMINARY  DRAFT
 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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                                       PRELIMINARY  DRAFT
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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                                      PRELIMINARY DRAFT
                                    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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                                       PRELIMINARY DRAFT
     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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                                       PRELIMINARY DRAFT
     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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                                        PRELIMINARY  DRAFT
 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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                                       PRELIMINARY DRAFT
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
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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
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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
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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-
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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
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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.,
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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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                                       PRELIMINARY DRAFT
     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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                                       PRELIMINARY DRAFT
 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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                                       PRELIMINARY DRAFT


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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                                       PRELIMINARY DRAFT
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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                                       PRELIMINARY  DRAFT


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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                                        PRELIMINARY DRAFT
 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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                                       PRELIMINARY DRAFT
     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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                                       PRELIMINARY DRAFT
     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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                                       PRELIMINARY DRAFT
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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                                        PRELIMINARY  DRAFT
 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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                                      PRELIMINARY DRAFT
      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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                                      PRELIMINARY DRAFT
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).
2BPB12/B                                   12-51                                       9/20/83

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                                      PRELIMINARY DRAFT
     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

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                                      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

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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

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                                                                    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.

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                                      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

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                                      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
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                                      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
 2BPB12/B                                   12-61                                        9/20/83

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                                       PRELIMINARY DRAFT
 (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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                                      PRELIMINARY DRAFT
     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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                                       PRELIMINARY DRAFT
 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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                                       PRELIMINARY  DRAFT
 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

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                        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

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                                       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

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YICONDUCTION VELOCITY) = 54.8 - .045 x (BLOOD LEAD)
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                             BLOOD LEAD, M9/dl

Figure 12-4. Peroneal nerve conduction velocity versus blood lead level, Idaho,
1974.

Source: Landrigan et al. (1976).
                               12-75

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                                       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

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                                       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,

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                                        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).
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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

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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

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                                       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

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                                       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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                                       PRELIMINARY DRAFT


     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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                                       PRELIMINARY DRAFT
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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                                     PRELIMINARY  DRAFT
                  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
                                                              12-101

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                                       PRELIMINARY DRAFT
      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

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                                   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

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                                       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

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                                       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:

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                                       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)
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                                       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

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                                       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

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                                       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

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                                       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

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                          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

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                                             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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                                        PRELIMINARY DRAFT
 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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                                       PRELIMINARY DRAFT
 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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                                       PRELIMINARY DRAFT
     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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                                        PRELIMINARY DRAFT
 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.

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                                                 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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                                        PRELIMINARY  DRAFT
 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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                                       PRELIMINARY DRAFT
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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                                       PRELIMINARY DRAFT
 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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                                       PRELIMINARY  DRAFT
     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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                                       PRELIMINARY DRAFT
 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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                                       PRELIMINARY  DRAFT


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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                                        PRELIMINARY DRAFT
      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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                                       PRELIMINARY DRAFT


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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                                        PRELIMINARY  DRAFT
 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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                                       PRELIMINARY DRAFT
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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                                       PRELIMINARY DRAFT
 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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                                       PRELIMINARY DRAFT


      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
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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
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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.

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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

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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

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                                       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.

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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-
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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
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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
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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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                                        PRELIMINARY DRAFT
 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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                                       PRELIMINARY DRAFT
                  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
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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

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 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

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                                       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

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                                       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

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                              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

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                                       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
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                             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.

 DPB12/G                                    12-167                                      9/20/83

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                                       PRELIMINARY DRAFT
     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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                                       PRELIMINARY DRAFT
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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                                       PRELIMINARY DRAFT
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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                                        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
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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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                                                                                    9/20/83

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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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                                       PRELIMINARY DRAFT
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.
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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.

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                         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).

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                                       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
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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
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                                       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.

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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.
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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.
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     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.
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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
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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
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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
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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
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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.

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       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
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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.

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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

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                              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

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                                       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.

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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

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                                       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.
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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
 CPB12/C                                   12-211                                    9/20/83

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                               PRELIMINARY DRAFT
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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                               PRELIMINARY DRAFT
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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                                       PRELIMINARY DRAFT
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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                                       PRELIMINARY  DRAFT
(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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                                       PRELIMINARY  DRAFT
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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                                       PRELIMINARY  DRAFT
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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                                        PRELIMINARY DRAFT
 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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                                       PRELIMINARY  DRAFT


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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                                       PRELIMINARY DRAFT
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                                       PRELIMINARY DRAFT
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                                         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.
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     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

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                                       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

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                                        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


-------
                                       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

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  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
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        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
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                                                                  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
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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

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 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.
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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.
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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
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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.
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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


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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

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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

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                                        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.

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     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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 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

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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

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                                       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

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                                       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.

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                                       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

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                                       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

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                                       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

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                                       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

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                                       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

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                                       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

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         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).

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                                       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

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                                       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

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                                       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

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                           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.

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                                       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

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                            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

 23PB13/A                                     13-35                                       9/20/83

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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

-------
     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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                                       PRELIMINARY DRAFT
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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                                       PRELIMINARY  DRAFT
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

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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

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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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                                       PRELIMINARY DRAFT
 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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                                      PRELIMINARY DRAFT
    (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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                                       PRELIMINARY DRAFT
13.9  REFERENCES


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Kehoe, R.  A.  (1961a) The metabolism of  lead in man in health and  disease:  the normal  metab-
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                                       PRELIMINARY DRAFT
Kehoe, R. A.  (1961b)  The  metabolism of lead  in  man in health and  disease:  the metabolism of
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Lucas, J. M.  (1981)  Effect  of analytical  variability on measurements of population blood lead
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Mahaffey, K.  R.; Michaelson,  I.  A. (1980)  The interaction  between lead and  nutrition.  In:
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National  Academy  of  Sciences,  Committee on  Lead in the Human Environment. (1980) Lead in the
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0'Flaherty,   E.  J.; Hammond,  P. B.; Lerner, S.  I.  (1982) Dependence  of  apparent blood lead
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Otto, D.  A.; Benignus, V. A.;  Muller, K. E.; Barton,  C.  N. (1981) Effects  of age and  body lead
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Otto, 0.; Benignus,  V.; Muller,  K. ; Barton, C.  (1983) Evidence of  changes  in  CNS  function at
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      hold limits.  In: Rutter,  M.;  Jones,  R.  R.  , eds.  Lead versus  health:  the effects of  low
      level  lead  exposure. New  York,  NY: John Wiley  &  Sons; PAGES. (IN PRESS)

Otto,  D. ;  Benignus,  V.;  Muller,   K.; Barton,  C. ;  Seiple, K. ;  Prah, J. ; Schroeder,  S. (1982)
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Rabinowitz, M. B. ; Wetherill, G. W.; Kopple, J. D.  (1973)  Lead metabolism  in  the  normal  human:
     stable isotope studies. Science (London)  182:  725-727.

Rabinowitz, M,;  Wetherill,  G.  W. ; Kopple,  J. D.  (1974)  Studies  of human lead  metabolism  by
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Rabinowitz, M.  B. ;  Wetherill,  G. W. ; Kopple,  J.  D.  (1976)  Kinetic  analysis of  lead metabolism
     in healthy humans. J. Clin. Invest. 58: 260-270.

Rabinowitz, M.  B. ;  Wetherill,  G. W. ; Kopple,  J.  D. (1977) Magnitude  of lead intake  from  res-
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     Verduyn,  G.  (1980)  Exposure  to  lead  by  the  oral  and  the  pulmonary routes of children
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Roels, H.;   Buchet, J-P.;   Lauwerys,   R. ;   Hubermont,  G.;   Bruaux, P.;  Claeys-Thoreau, F.;
     Lafontaine,  A.;  Van Overschelde,   J.  (1976)  Impact  of air pollution  by  lead on the  heme
     biosynthetic pathway in school-age children. Arch. Environ. Health 31: 310-316.

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Sherlock, J. ;   Smart,  G. ;   Forbes, G.   I.;  Moore,  M. R. ;   Patterson, W. J. ;  Richards,  W. N. ;
     Wilson, T. S. (1982) Assessment of lead intakes and dose-response for a  population  in Ayr
     exposed to a plumbsolvent water supply. Hum. Toxicol.  1: 115-122.

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     mental lead to blood-lead levels in children.  Environ. Res. 27: 372-383.

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     provisional  estimates  of  social, economic,   and  housing  characteristics:  states  and
     selected  standard metropolitan  statistical  areas.  Washington,  DC:  U.S.  Department  of
     Commerce; Bureau of the Census report no.  PHC  80-S1-1. Available  from: U.S.  Department  of
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     Springfield, VA;  PB 280411.

U.S. Public Health  Service.  (1982) Hematological and  nutritional  biochemistry reference  data
     for persons  6  months  - 74 years  of  age: United States,  1976-80.  Hyattsville, MD:  U.S.
     Department  of  Health  and  Human  Services,   National  Center  for  Health  Statistics;  DHHS
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