Document rejNZXd7zg1gN6Jy4nw873eX7

TABLE OF CONTENTS CHAPTER 12: BIOLOGICAL EFFECTS OF LEAD 12.1 INTRODUCTION ........................... ...................... ............................. 12.2 SUBCELLULAR EFFECTS OF LEAD ......................................................... 12.3 EFFECTS OF LEAD ON HEME BIOSYNTHESIS AND ERYTHROPOIESIS/ ERYTHROCYTE PHYSIOLOGY ................................................ ...................... 12.4 NEUROTOXIC EFFECTS OF LEAD .................................................... .......... 12. 5 EFFECTS OF LEAD ON THE RENAL SYSTEM ........................................... 12.6 EFFECTS OF LEAD ON REPRODUCTION AND DEVELOPMENT ........................ 12.7 GENOTOXIC AND CARCINOGENIC EFFECTS OF LEAD .......... .......... ............ 12.8 EFFECTS OF LEAD ON THE IMMUNE SYSTEM ........ .................................... 12.9 EFFECTS OF LEAD ON OTHER ORGAN SYSTEMS __________ ___________ 12.9.1 THE HEPATIC SYSTEM ........ .......... 12.9.2 THE CARDIOVASCULAR SYSTEM ______ 12.9.3 LEAD EFFECTS ON HORMONAL SYSTEMS 12.9.4 THE GASTROINTESTINAL SYSTEM ____ REFERENCES APPENDIX 12A . with references APPENDIX 12B . with references 12-1 12-4 12-5 12-38 12-149 12-180 12-217 12-238 12-255 12-255 12-258 12-259 12-262 12-263 N33763 TEH 0530863 DUP050031776 PRELIMINARY DRAFT CHAPTER 12. BIOLOGICAL EFFECTS OF LEAD EXPOSURE 12.1 INTRODUCTION As noted in Chapter 2, air quality criteria documents evaluate current scientific knowledge of relationships between pollutant concentrations and their effects on the environment and public health. Early chapters of this document have discussed: sources of emissions; transport, transformation, and fate; ambient concentrations, and other aspects related to exposures of the U.S. population to lead in the ambient environment. Later chapters have thus far discussed aspects related to the uptake, distribution, toxicokinetics and excretion of lead and the relationships of various external and internal lead exposure indices to each other. This chapter assesses important information regarding and biological effects of lead exposure with particular emphasis on both (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 the biological effects of lead, one should note at the outset that, to date, lead has not been demonstrated to have any biological effect in human populations which can be considered beneficial. Some investi* gators have, on the other hand, hypothesized that lead may serve as an essential element in certain other mammalian species (e.g., the rat) and have reported experimental data interpreted as supporting such a hypothesis. However, critical evaluation of these studies as presented in Appendix 12-A of this chapter raises serious questions regarding interpretation of the reported findings. Therefore, given the lack of any clear evidence demonstrating the essentiality of lead in man or other mammalian species, consideration here and later (in Chapter 13) of the effects of lead on man is carried out in the absence of any demonstrated health benefit/health cost balance. It is clear from the wealth of available literature reviewed below 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 erytropoietic pyrimidine metabolism, are detectable. Heme biosynthesis is a generalized process in mammalian species, PB12A/A 12*1 1-5*83 TEH 0530864 DUP050031777 PRELIMINARY DRAFT inculding man. 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 in creasingly 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 nervous systems are among the earliest effects observed as a function of increasing lead exposure. With increasingly intense exposure, more severe disruption of the erythropoietic 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, impairment of immunological functions, and a variety of 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 or , non-fatal lead poisoning, long-lasting sequelae such as permanent mental retardation. The etiologies of many of the various types of functional disruption of different mammalian organ systems can be seen to derive (at least in their earliest stages) from the impact of lead on certain subcellular organelles which results in biochemical derangements (e.g., disruption of heme synthesis processes) common to and affecting many tissues and organ systems. Some of the major effects of lead on subcellular organelles (especially those common to numerous organ systems) in mammalian species are discussed below in Section 12.2, with particular emphasis on the effects of lead on mitochondrial functions. The remaining sections of Chapter 12 cover the biological effects of lead in terms of the various organ systems of the body affected by that element and its compounds, except for Section 12.7, which is devoted to discussion of geootoxic 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 vulnerability of each organ system to lead. Major emphasis is placed first on diseusssion of the three systems classically considered most sensitive to the effects of lead (i.e.; the hematopoietic, the nervous, and the renal systems). Additional discussion then follows in the next sections PB12A/A TEH 0530865 12-2 1-5-83 DUP050031778 PRELIMINARY DRAFT on the effects of lead on reproduction and development in view of the impact of lead on the fetus (and, therefore, on pregnant women), as well as gametoxic effects of lead. Genotoxic effects of lead and data reported in regard to possible carcinogenic effects of lead are then reviewed, followed by discussion of effects of lead on the immune system and, lastly, an overview of lead effects on other organ systems. Subdividing much of this chapter on the biological effects of lead according to organ systems affected was done for the purpose of easier discussion. It must be kept in mind that, in reality, all systems function in delicate con cert to preserve the physiological integrity of the whole organism. Furthermore, all systems are interdependent, so that not only do effects in a critical organ often exert impacts on other organ systems, but low-level effects, which may be construed as less important in a single specific system, also contribute to the cumulative or aggregate impact of what might be seen otherwise as relatively minimal biological responses in a number of different organ systems if each is viewed in isolation. 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 retention, and the partitioning in soft tissues and hard tissues. The greater sensitivity of children to lead toxicity, their inability to recognize symptoms, and their dependence on parents and health care professionals all make them a particularly vulnerable population in need of special consideration in regulatory activities concerning lead (Lin-Fu, 1972; U.S. Environmental Protection Agency, 1977). PB12A/A 12-3 1-5-83 TEH 0530866 DUP050031779 PRELIMINARY DRAFT 12.2 5UBCELLULAR EFFECTS OF LEAD NOTE: There is to be inserted here a general discussion of the subcellular effects of lead, with particular emphasis on mitochondrial effects and other important effects on subcelluar organelles common to many different organ systems. PB12A/A TEH 0530867 12-4 1-5-83 DUP050031780 PRELIMINARY DRAFT 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. In view of this, then, this section is 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 sub-divided into effects of lead on hemoglobin production, cell morpoholgy and survival, and erythropoietic nucleotide metabolism. The inter relationship of effects of lead on heme biosynthesis and neurotoxic effects of lead are discussed in a final sub-section. 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 both because of their prominence 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 cullular energetics. Hence, any effects of lead on heme biosynthesis will, per force, pose the potential for multi-organ toxicity, regardless of the current level of recognition of the magnitude of such effects in any specific organ system. At present, much of the available information concerning the effects of lea'd on heme biosynthesis concern the erythropoietic system, in large part due to the relative ease of assessing such effects via measurements in blood as well as blood being the vehicle for movement of metabolites from other organ systems. On the other hand, a number of reports have concerned 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, information describing erythropoietic effects usually appearing first, followed by studies involving other tissues. The process of heme biosynthesis results in the 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. PB12A/C 12-5 1-5-83 TEH 0530868 DUP050031781 PRELIMINARY DRAFT As may be noted in Figure 12-1, lead interferes with heme biosynthesis by disturbance of the activity of 3 major enzymes: (1) the indirect stimulation, by feedback derepression, of the mitochondrial enzyme delta-aminolevulinic acid synthase (ALA-5) which mediates the condensation of glycine and succinylcoenzyme A to form delta-aminolevulinic acid; (2) direct inhibition of the cytosolic enzyme delta-aminolevulinic acid dehydratase (ALA-D) which catalyzes the cyclocondensation of two units of ALA to porphobilinogen; and (3) the mitochondrial enzyme ferrochelatase, found in liver, bone marrow, and other tissues, which catalyzes the insertion of iron (II) into the protoporphyrin ring to form heme. Disturbance of ferrochelatase occurs by either direct inhibition or alteration of intramitochondrial transport of iron, the enzyme in mammals being situated in the inner mitochondrial membrane (McKay et al., 1969). 12.3.1.1 Effects of Lead on d-Aminolevulinic Acid Synthase--In the heme biosynthetic pathway, the activity of the enzyme ALA-S is the rate-limiting step of heme formation. With decreased heme formation at other steps downstream or because of increased heme oxygenase activity, an increase of ALA-S activity is signalled by feedback derepression in an effort to compensate by enhancing the rate of heme formation. Hence, excess ALA formation is due to both stimulation of ALA-S and direct inhibition of ALA-D (vide infra). In lead workers, increased ALA-S activity has been reported (Takaku et al., 1973; Campbell et al., 1977; Meredith et al., 1978), and in the investigations of Meredith et al. (1978), leukocyte ALA-S in lead workers was seen to be stimulated at a blood lead value of 40 pg/dl, a level at which ALA-D activity is- significantly inhibited. To the extent'that mitochondria in leukocytes show a comparable dose-effect relationship to the bone marrow and hepatic systems, it appears that most of the excess ALA formation below the observed threshold value is due to ALA-D inhibition. From the authors' data, blood ALA had increased ca. 2-fold in these workers in going from the value of 18 pg/dl to 40 pg/dl. PB12A/C TEH 0530869 12-6 1-5-83 DUP050031782 * fa 2 M ITOCHONDRIAL MEMBRANE PB12A/C 9 g fi \ 8fa csl 0) 0> 12-7 1-5-% TEH 0530870 DUP050031783 PRELIMINARY DRAFT. In vitro and in vivo experimental data have provided mixed results in terms of the direction of the effect of lead on ALA-5 activity. Silbergeld et al. (1982) observed that ALA-S activity is 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 in utero, through development, and up to 9 months of age. Meredith and Moore (1979) noted a steady increase in hepatic ALA-5 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 affects 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-synthetase activity. Stimulation of activity was observed at lead levels as low as 5 x 10 M, with maximum stimulatron at ID-5 M. The authors report that the activity increase was associated with biosynthesis of more enzyme, rather than stimulating the pre-existing enzyme. Lead-stimulated ALA-S formation was also not limited to liver cells, rat gliomas and mouse neuroblastomas showing similar results. 12.3.1.2 Effects of Lead on d-Aminolevulinic Acid Dehydratase and ALA Accumulation/Excretion--Delta-aminolevulinic acid dehydratase (5-aminplevuTinate hydrolase; porphobilinogen synthase; E.C, 4.2.1.24; ALA-D) is a sulfhydryl, allosteric enzyme in the heme biosynthetic pathway which catalyzes the conversion of 2 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-0 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). In this connection, Dresner and co-workers (1982) found that ALA-D activity in rat bone marrow suspensions was significantly inhibited in 35 percent of controls in the presence of 5 x ID-7 M (0.5 pM) lead. This potency, on a comparative molar basis, was unmatched by any other metals tested. PB12A/C TEH 0530871 12-8 1-5-83* DUP050031784 PRELIMINARY DRAFT Hernberg and Nikkanen (1970) found that enzyme activity was correlated inversely (logarithmic) with blood lead values in a group of urban, non-exposed subjects, inhibition being 50 percent, compared to lower level subjects, at a blood lead level of 16 pg/dl. Other reports have confirmed these observations across age groups and exposure categories (Alessio et al., 1976; Reels et al., 1975a; Nieberg et al-, 1974; Wada et al., 1973), The use of a ratio of activated to inhibited enzyme activity (versus a single activity measurement, which does not accommodate genetic intersubject variability) measured against blood lead in children with values between 20-90 pg/dl was employed by Granick et al. (1973) to obtain an estimated threshold of 15 pg/dl for an effect of lead, while Hernberg and Nikkanen (1970) observed no threshold in their subjects, all at or below 16 pg/dl. In the former case, the lowest blood lead measured was higher than the actual values obtained by Hernberg and Nikkanen (1970), Kuhnert et al. (1977) reported that ALA-D activity in erythrocytes from pregnant women and cord blood of infants at delivery are both correlated with the corresponding blood lead values, using the activated/ inhibited activity ratio method of Granick et al. (1973). In fetfl erythrocytes, the coefficient of correlation, r ~ -0.58, p <0.01, of activity with lead level was higher 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. While several factors other than lead may affect the activity of erythrocyte ALA-0, much of the available information would suggest that these do not materially compromise the interpretation of enzyme activity and lead relationships or the use of this relationship in a screening context. Border et al. (1976) questioned the reliability of ALA-D activity measurement in subjects concurrently exposed to both lead and zinc, the latter also affecting the activity of the enzyme. The data of Meredith and Moore (1980) refute this objection. In subjects without exposure and having serum zinc values of 80-120 pmol/liter, there was only a minor activating effect with increasing zinc when contrasted to the correlation of activity vs, blood lead in these same subjects. In workers having both lead and zinc exposure, serum zinc values were greater than in subjects with just lead exposure, but the mean level of enzyme activity 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 materially affects ALA-D activity. The obverse of PB12A/C 12-9 1-5-83 TEH 0530872 DUP050031785 PRELIMINARY DRAFT this, lead exposure in the presence of zinc deficiency, is probably the more significant issue, but one which has not been well studied. Since AlA-D is a zinc-requiring enzyme, one woul expect that optimal activity would be governed by in vivo zinc availability; Furthermore, zinc deficiency could potentially have a dual deleterious effect on ALA-D activity, first by reduced activity with reduced zinc availability and secondly, via enhanced lead absorption in the presence of zinc deficiency (see Chapter 10, Section 5), the increased lead burden further inhibiting ALA-D activity. The recent study of Roth and Kirchgessner (1981) indicates that ALA-D activity is significantly decreased in zinc deficiency. In zinc-deficient rats showing reduced serum and urinary zinc levels, the activity of erythrocyte ALA-D activity was pnly 50 percent that of pair-fed controls while urinary ALA was significantly elevated. Although these investigators 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 increased lead absorption from diet. Given the available information documenting zinc deficiency in children (Chapter 10, Section 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 further, careful study. Similarly, Moore and Meredith (1979) noted the effects of carbon monoxide on the activity of this enzyme, comparing moderate or heavy smokers with non-smokers. At the highest level of carboxyhemoglobin measured in their smoker groups, the depression of ALA-D activity was 2.1 percent. In these subjects, a significant inverse correlation of activity and blood lead persisted, while there was no significant correlation of 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 alcohol intoxication. Hence, relevance of this observation to screening is limited. 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 PB12A/C TEH 0530873 12-10 1-5-83 DUP050031786 PRELIMINARY DRAFT activity and blood lead in the range 12-56 pg/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 RoeIs et al. (1977), tissue ALA-D changes were not observed, while the recent report of Silbergeld et al, (1982) described moderate inhibition of ALA-D activity in brain and significant inhibition of activity in kidney, liver, and spleen when adult rats were acutely exposed to lead given intraperitoneally, while chronic exposure was associated with reduced activity in kidney, liver, and spleen. Gerber and coworkers (1978) found that neonate mice exposed to lead from birth through 17 days of age at a level of 1.0 mg/ml in water showed significant decreases in brain ALA-D activity (p < 0.01) at all time points studied. These reults support the data of Millar et al. (1970) for the suckling rat. In this same study, exposure of rats 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 that brain ALA-D activity in the developing animal is more sensitive to lead than in the adult. The study of Dieter and Finley (1979) sheds light on the relative sensitivity of brain ALA-D activity in several brain regions and permits comparison of blood vs. brain ALA-D activity 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 ppnj lead in liver and 0.5 ppm lead in brain. Cerebellum ALA-D activity was reduced 50 percent at a lead level below 0.5 ppm, erythrocyte enzyme activity was lowered 75 percent while liver was comparable to cerebellum or somewhat less, although lead level in'liver was 5-fold higher. Cerebellum ALA-D activity was significantly below that for cerebrum. In the avian, then, at blood lead levels where 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-0 is reflected in increased levels of its substrate, ALA, in urine (Haeger, 1957) as well as in whole blood or plasma (Meredith et al., 1978; McGee et al., 1977; Chisolm, 1968; Haeger-Aronsen, 1960). The detailed study of Meredith et al. (1978), which involved both control subjects and lead workers, indicates that in elevated lead exposure, the PB12A/C 12-11 l-=;-sn TEH 0530874 DUP050031787 PRELIMINARY DRAFT increase in urinary ALA is preceded by a significant rise in circulating levels of ALA, the overall relationship of blood ALA to blood lead being expo nential and showing a perceptible continuation of an ALA-blood lead correlation into the non-exposure group, to include the lowest value, 18 pg/dl. The relationship of blood ALA to urinary levels of the precursor was found to be exponential, indicating that as blood ALA increases, a greater proportion undergoes excretion into urine. Inspection of the plot of urinary vs. blood ALA in these subjects shows that the correlation persists down to the blood ALA concentration corresponding to the lowest blood lead level, 18 pg/dl. Cramer and co-workers (1974) have demonstrated that ALA clearance into urine parallels glomerular filtration rate across a range of lead exposure, suggesting that increased urinary output with increasing circulating ALA is associated with decreased tubular reabsorption (Moore et al., 1980). 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. Specter et al. (1971) and Blanksma et al. (1969) pointing out the failure of urinary ALA analysis in detecting lead exposure when compared to blood lead values. This is due to the fact that an individual subject will show a wide variation in ALA-U with random sampling. Chisolm et al. (1976) showed that reliable levels Could only be obtained with 24 h, collections. In children with blood lead levels above 40 pg/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; Reels et al., 1975a; Wada et al., 1973; Selander and Cramer, 1970) and in children (NAS, 1972). Selander and Cramer (1970) reported that two different correlation curves were obtained, one for individuals below 40 pg/dl blood lead, and a different one for values above this, although the degree of correlation was less than with the group data. Meredith et al. (1978) found that the correlation curve for blood ALA vs. urinary ALA was linear below a blood lead of 40 pg/dl, as was the relationship of blood ALA to blood lead. Hence, there was a linear relation ship between blood lead and urinary ALA below 40 pg/dl, i.e., a continuation of the correlation below the commonly accepted threshold blood lead value of 40 pg/dl (see below). Tsuchiya et al, (1981) have questioned the relevance of PB12A/C TEH 0530875 12-12 1-5-83 DUP050031788 PRELIMINARY DRAFT using single correlation curves to describe the blood lead-urinary ALA relationship across a broad range of exposure, having found that this relationship in workers showing moderate, intermediate, and high lead exposure was describable by 3 correlation curves of differing slope, consistent with the observations of Selander and Cramer (1970) and the results of Meredith et al. (1978). An exponential correlation was described (Chisolm et al., 1976) between bipod lead and urinary ALA in children 5 years old or younger, with blood lead ranging from 25-75 pg/dl. In adolescents with blood lead below 40 pg/dl, no clear correlation was observed. It is apparent from the above reports (Tsuchiya et al., 1981; Meredith et al., 1978; Selander and Cramer, 1970) that circulating and urinary ALA are elevated and correlated at blood lead values below the accepted concentration of 40 pg/dl. This is consistent, as in the Meredith et al. study, with the significant and steady increase in ALA-D inhibition at blood lead values considerably below 40 pg/dl concomitant with rising blood levels of ALA. Increases of ALA in tissues of experimental animals exposed to lead have been documented. In the study of Silbergeld et al. (1982), acute lead admini stration to adult rats was associated with an elevation in spleen and kidney ALA vs. controls, while in chronic exposure, there was a moderate increase in ALA in the brain level 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 dosing group. 12.3.1.3 Effects of Lead on Heme Formation from Protoporphyrin--The accumulation of protoporphyrin in the erythrocytes of individuals with lead intoxication has been recognized since the 1930s (Van den Bergh, 1933), but it has only recently been possible to study this effect via the development of sensitive and specific analytical techniques to permit quantitative measurement. In particular, the development of laboratory micro techniques 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, ca. 95 percent of the protoporphyrin in circulating erythrocytes is zinc protoporphyrin (2PP) 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. PB12A/C 12-13 1-4-82 TEH 0530876 DUP050031789 PRELIMINARY DRAFT in lieu of the native iron, with the resulting zinc protoporphyrin (ZPP) tightly bound in the available heme pockets for the life of the erythrocyte (120 days). In lead poisoning, the accumulation of protoporphyrin differs from that seen in the congenital disorder, erythropoietic protoporphyria. In the latter case, there is a defect in ferrochelatase function, leading to loose attachment of the porphyrin, accumulated without uptake 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 absent 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; Kamholtz et al., 1972; Sassa, 1973; Lamola et al., 1975; Roels et al., 1976) and in adult workers (Valentine et al., 1982; Lilts et al., 1978; Grandjean and Lintrup, 1978; Alessio et al., 1976; Roels et al., 1975b; Lamola et al., 1975), As a sensitive biological indicator of lead burden, measurement of erythro cyte protoporphyrin has been held to be as good as other biological indicators, including blood lead (CDC, 1978). Reigert et al. (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 character 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 erythropoietic 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 erythrocytes long after sig nificant lead exposure has ceased appears to be a better indicator of re sorption of stored lead in bone than other measurements. Alessio et al. (1976b) reported that former lead workers, removed from exposure at the PB12A/C TEH 0530877 12-14 1-4-82 DUP050031790 PRELIMINARY DRAFT workplace for more than 12 months In all cases, still showed the typical logarithmic correlation with blood or urinary lead. However, 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 relationship for adults clearly indicates that significant levels of hemato logical! y toxic lead continue to circulate long after exposure to lead has ceased. Data reported for children by Chisolm et al. (1976) indicate that the relationship of EiP to chelatable lead was not as good as was ALA-U. In a report of relevance to the problem of multi-indicator measurement to assess the degree of lead exposure, Helsey and Wimbish (1981) studied two groups of lead workers, newly exposed or removed from significant exposure, with respect to changes in blood lead and ZPP. 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 workers logically conclude that difficulty reported in demonstrating reliable blood lead-ZPP relationships may reflect the non-concordance of plateau achievement at particular points in time. 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 exposure to the element. 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 pg/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 threshold for effect of 15.5 pg/dl. In the age range of children studied here, iron deficiency is uncommon and these workers 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 PB12A/C 12-15 1-4-82 TEH 0530878 DUP050031791 PRELIMINARY DRAFT study* then, iron deficiency was unlikely to be a confounding factor in the primary relationship. PiomeTli et al. (1977) obtained a comparable threshold value for lead's effect on ZPP elevation, 15.5 pg/dl, in children who were older than 4 years and 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 pg/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 dichotomous, being greater in females for a given blood lead (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, using both 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 Stuick, 1974; Roels et al.; 1975a, Zielhuis et al., 1978; Odone et al., 1978; and Toriumi and Kawai, 1981. The effect of age was observed, although all age groups were combined for the study population of 395 subjects in whom no correlation was apparent below 40 pg/dl. Piomelli et all. (1982) investigated both the threshold for the effect of lead on ZPP accumulation and a dose-response relationship in 2,004 children, 1,852 of whom had blood lead values below 30 pg/dl. In this study, blood lead and EP measurements were done in facilities with a high proficiency for both blood lead and ZPP analyses and employed 2 statistical approaches: segmental line techniques and probit analysis. Both methods revealed an averaged thres hold blood lead level of 16.5 pg/dl in either the full group or the children with blood values below 30 pg/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 dose-response relationships, it was found that blood lead values corresponding to significant EP elevation (>1 or >2 PB12A/G TEH 0530879 12-16 1-4-82 DUP050031792 PRELIMINARY DRAFT S.D.s) above reference mean EP in 50 percent of the subjects were 28.6 and, 35.6 pg/dl blood lead respectively. At a blood lead level of 30 pg/dl, furthermore, it was determined that 27 percent of children will have an EP greater than 53 pg/dl. Comparison of ZPP elevation among adult males and females and children at a given blood lead level generally indicate that children and adult females are more sensitive to this effect of lead. Lamola et a!. (1975) demonstrated that the slope of ZPP vs. blood lead was steeper in children than in adults. Roe Is et al. (1976) found that women and children were equally more sensitive in response than adult males, a finding also observed in the population studied by Odone et al. (1978). 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 (Stuick, 1974; RoeIs et al., 1975b; Roels et al., 1978, 1979; Toriumi and Kawai, 1981). The heightened response of females to lead-associated ZPP elevation was investigated in rats (Roels et al., 1978) and shown to relate to hormonal interactions with lead in the response, confirming the human data of Roels et al. (1975b, 1978, 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. Formation of the heme-containing protein cytochrome P-450, which is an integral part of the hepatic mixed function oxidase system, has been documented as being affected with lead exposure, particularly with 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., 1978). 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 detoxifying 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 decreasing microsomal P-450 content, total heme content of microsomes, and increased ALA-synthase activity. Heme oxygenase activity is elevated with lead intoxication in animals (Maines and Kappas, 1976; Meredith and Moore, 1979) where relatively high PB12A/C 12-17 1-4-82 TEH 0530880 DUP050031793 PRELIMINARY DRAFT 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. 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 biosynthesis capability (Whetsell et al., 1978) and that this cell system, in the presence of lead, elaborates increased porphyrinic material (Sassa et al., 1979). Chronic administration of lead to neonate rats indicates that at low levels of exposure, with modest elevation 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 Hsu, 1976; Bull et al. , 1979). These effects on the developing organism are accentuated by the discussion in Chapter 10 of increased whole body lead retention in both developing experimental animals and children, as well as higher retention of lead in brain of suckling rats compared to adults. The mechanism(s) underlying derangement of heme biosynthesis leading to ZPP accumulator in lead intoxication rests with either ferrochelatase inhibition, impaired mitochondrial transport of iron, or a combination of both. Inferential1y, the resemblance of lead-associated ZPP accumulation to a similar effect of iron deficiency is consistent with the unavailability 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, leadinduced effects on mitochondrial morphology and function are well known (Goyer and Rhyne, 1973; fowler, 1978), and such disturbances may include impaired iron transport (Borova, 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 hematopoietic response, the earlier one resulting in significant formation of free vs, zinc protoporphyrin with considerable hemolysis, and a later phase where ZPP is formed and which otherwise resembles the common features of lead intoxication. Subacute exposure shows more of the typical 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 PB12A/C TEH 0530881 12-18 1-4-82 DUP050031794 PRELIMINARY DRAFT hematological derangement, keeping in mind the rabbit's sensitivity to the hemolytic action of lead, compared to other species. Of interest is the report of Fowler et al. (1980). In rats maintained on a regimen of oral lead, starting with exposure of their dams to tap water lead and continuing through 9 months after birth at levels up to 250 ppm lead, it was observed that kidney mitochondrial ALA synthase and ferrochelatase activity, but not that of the cytosolic enzyme ALA-D, were 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 mito chondrial 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 mitochondrial inability to use the 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 suggests, as do the mitochondrial function data of Holtzman and 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 derangement in terms of inhibited ALA-D activity or accumulation of ZPP, This suggests that there is a difference in relative dose-effect 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 biosynthesis derangement in other organs. 12.3,1.4 Other Heme-Related Effects of Lead--An increased excretion of copropor phyria 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 PB12A/C 12-19 1-5-m TEH 0530882 DUP050031795 PRELIMINARY DRAFT in terms of differentiating among direct enzyme inhibition, accumulation of substrate secondary to inhibition of heme formation, or impaired movement of the coproporphyrin intramitochondrially. 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 (Piotnelli 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 uropor phyrinogen UR0-I- synthetase (Piper and Tephly, 1974), In vitro studies of Piper and Tephly (1974) using rat and human erythrocyte and liver preparations indicate that it is the erythrocyte UR0-I- synthetase in both rats and humans which is sensitive to the inhibitory effect of lead, activity of the hepatic enzyme being relatively insensitive. Significant inhibition of the enzyme's activity occurs at 5 pM lead with virtually total inhibition of activity in human red cell hemolysates at 10*^ M. According to Piper and Van Lier (1977), the lower sensitivity of hepatic URO-I- synthetase activity to lead is due to a protective effect afforded by a pteridine derivative, pteroylpolyglutamate. It appears that the protection does not occur via 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 which account 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 usually normocytic. It is associated with reticulocytosis, owing to shortened cell survival, and the irregular presence of basophilic stippling. Its genesis lies in both decreased erythrocyte 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 (WHO, 1977; NAS, 1972; Lin-Fu, 1973; 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 including reticulocytosis, basophilic stippling and a signifi cantly lower total iron binding capacity (TIBC). These latter features suggest PB12A/C TEH 0530883 12-20 1-5-83 DUP050031796 PRELIMINARY DRAFT that, in young children, iron-deficiency anemia is exacerbated by a further effect of 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 Lippmann, 1977) surveys and is known to be correlated with increased lead absorption in humans (Yip et al., 1981; Chisolm, 1981; Watson, 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 hemo globin level and blood lead levels (Adebenajo, 1974; Rosen et al., 1974; Betts et al,, 1973; Pueschel et al., 1972). These studies generally involved children under 6 years old where iron deficiency may have been a factor. In adults, similarly, a negative correlation at blood lead values usually below 80 pg/dl was observed (Grandjean, 1979; Li1 is et al., 1978; Roels et al., 1975a; Wada, 1976), while several studies did not report any relationship below 80 pg/dl (Valentine et al., 1982; 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) having 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 threshold in blood lead for effects on hemogloblin has not been conclusively established. In children, this value appears to be somewhat lower than in adults (Adebenajo, 1974; Rosen et al., 1974; Betts et al., 1973; Pueschel et al., 1972) being about 40 pg/dl (WHO, 1977), In adults, Tola et al. (1973) observed no effect of lead on new workers until the blood lead had risen to a value of 50 pg/dl after ca. 100 days. The regression 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 pg/dl. Dose-response data for the lead-hemoglobin relationship are limited for human populations. 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 pg/dl; 14 percent at blood lead of 60-79; and 36 percent at values above 80 pg/dl. In 202 lead workers, Grandjean (1978) noted the following percentage of workers having a hemoglobin lead below 14.4 g/dl as a function of blood PB12A/C 12-21 1-5-83 TEH 0530884 DUP050031797 PRELIMINARY DRAFT lead: < 25 pg/dl, 17 percent; 25-60 pg/dl, 26 percent; > 60 pg/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. Effects 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 a!., 1972; White and Harvey, 1972; Kassener et al., 1957). White and Harvey (1972) reported a decrease of globin synthesis in reticulo cytes in vitro in the presence of lead at levels as low as 1.0 pM, corresponding to a blood lead level of 20 pg/dl. These data are in accord with the observation of Dresner et al. (1982), who observed a reduced globin synthesis (75 percent vs. controls) in rat bone marrow suspensions exposed to 1.0 pM lead. White and Harvey (1972) also noted that there was a decreased synthesis of alpha chains vs. the 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 disturbance 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 Erythrocyte Morphology and Survival It is clear that there is a hemolytic component to lead-induced anemia in < humans owing to shortened erythrocyte survival, 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 in vivo and in vitro conditions. Waldron (1966) has discussed the frequent reports of increased mechanical fragility of erythrocytes from lead-poisoned workers, dating to the work of Aub et al. (1925). Decreased osmotic fragility of erythrocytes from subjects with lead intoxication is a parallel finding, both i_n 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, 1958). Using an apparatus called a coil planet centrifuge, Kara! et al. (1981) studied erythrocytes of lead workers and found significant increases in osmotic resistance at the same PB12A/C TEH 0530885 12-22 1-5-83 DUP050031798 PRELIMINARY DRAFT time that mean corpuscular volume and reticulocyte counts were not different from controls. These workers suggest that one mechanism involves impairment of hepatic lecithin-cholesterol acyltransferase, leading to a build-up of cholesterol in the cell membrane, thereby resembling the increased osmotic resistance seen in obstructive jaundice where increased membrane cholesterol has been observed (Cooper et al., 1975). In this connection, Fukumoto et al. (1980) have reported an increased cholesterol-phospholipid ratio in lead worker 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 dif1uorophosphonate. Berk et al. (1970) used detailed isotope studies of a subject with severe lead intoxication to determine shorter erythrocyte life span while Leiken and Eng (1963) observed shortened cell survival in 3 of 7 children. These studies, as do the reports of Landow et at. (1973), White and Harvey (1972), Albaharry (1972), and Oagg et al, (1965), indicated that hemolysis is not the exclusive mechanism of anemia and that diminished erythrocyte production also plays a role. The molecular basis for increased cell destruction with lead exposure includes the inhibition by lead of the activities of the enzymes (Na+, K+)-ATPase and pyrimidine-51"nucleotidase. Erythrocyte membrane (Na+, K+)-ATPase is a sulfhydryl enzyme and inhibition of its activity by lead has been well documented (Raghavan et al., 1981; Secchi et al., 1968; Hasan et al., 1967; Hernberg et al., 1967b). In the report of Raghavan et al. (1981), it was found that 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 enzyme inhibition, there is irreversible loss of potassium ion from the cell with undisturbed input into the cell of sodium, 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). Both with lead exposure and in subjects with a genetic deficiency of the enzyme pyrimidine-5'-nucleotidase, activity is reduced, leading to impaired phosphorolysis of the nucleotides cytidine and uridine phosphate, which are then retained in the cell, causing interference with the conservation of the purine nucleotides necessary for cellular energetics (Angle and Mctntire, PB12A/C 12-23 1-4-82 TEH 0530886 DUP050031799 PRELIMINARY DRAFT 1982; Valentine and Paglia, 1980), A more detailed discussion of lead's interaction with this enzyme is presented in a later section. In a series of studies dealing with the hemolytic relationship of lead and vitamin E deficiency in animals, Levander and coworkers (19$0) observed that lead exposure exacerbates the experimental hemolytic anemia associated with vitamin E deficiency by enhancing mechanical fragility, i.e., retarded cell deformabi1ity. These workers note that vitamin E deficiency is seen with children having elevated blood lead levels, especially subjects having glucose6-phosphate dehydrogenase (G-6-PD) deficiency, indicating that the synergistic relationship seen in animals may exist in humans, 12.3.2.3 Effects of Lead on Pyrimidine-51 Nucleotidase Activity and Erythropoietic Pyrimidine Metabolisnr-The presence in lead intoxication of basophilic stippling and an anemia having hemolytic character is similar to what is seen in subjects having a congenital deficiency of pyrimidine-5`nucleotidase (Py5N), 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 degraded ribosomal fragments accounts for the phenomenon of basophilic stippling. In characterizing the enzyme Py5N, Paglia and Valentine (1975) observed that its activity was particularly sensitive to inhibition by certain metals, particularly lead, prompting further investigation of the interplay of lead intoxication and disturbances of erythropoietic pyrimidine metabolism. Paglia et al. (1975) observed that in subjects occupationally exposed to lead but having no evidence of basophilic stippling or significant frequency of anemia, the activity of Py5N was reduced to ca. 50 percent of control subjects and was most impaired in one worker with anemia, ca. 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 pM, with consistent 50 percent inhibition at ca, 10 pM. Subsequently, these investigators (Valentine et al., 1976) observed that an individual with severe lead intoxication had an 85 percent decrease in Py5N activity, basophilic stippling and accumulation of pyrimidine nucleotides, mainly cytidine triphosphate, approaching values in these parameters to those seen in the congenital deficiency PB12A/C TEH 0530887 12-24 1-4-82 DUP050031800 PRELIMINARY DRAFT of Py5N. These data suggested a common etiology for the hemolytic anemia and stippling in both lead poisoning.and the congenital disorder. Several other reports of investigations of PySN activity and pyrimidine nucleotide levels in lead workers have been published (Paglia et al., 1977; Buc and Kaplan, 1978). In 9 workers haying overt lead intoxication and blood lead values of 80-160 pg/d1; Py5N activity was significantly inhibited while the pyrimidine nucleotides comprised 7-80 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 PySN, 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 MeIntire (1978) observed that in 21 children 2-5 years old, with blood lead levels of 7-80 pg/dl, there was a negative linear correlation between Py5N 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 2 subjects had reticulocytosis. While adults tend to show a threshold for inhibition of Py5N at a blood lead level ca. 44 pg/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 pg/dl, Angle and coworkers (1982) noted that there was (1) an inverse correlation (r - -0.64, p <0.001) between the logarithm of Py5N 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 (HPLC). In these studies, there was no threshold of effects of lead on either enzyme activity or cell nucleotide content to below 10 pg/dl. Finally, there was a significant positive correlation of pyrimidine nucleotide accumulation and the accumulation of ZPP, In subjects undergoing therapeutic chelation with EDTA, PySN activity was observed to be increased while there was no effect on pyrimidine nucleotides (Swanson et al., 1982), indicating that the pyrimidine accumulation is associated with the reticulocyte. PB12A/C * 12-25 1-5-83 TEH 0530888 DUP050031801 PRELIMINARY DRAFT The metabolic significance of Py5N activity inhibition and nucleotide accumulation with lead exposure is derived from its effects on red cell membrane stability and survival by alteration 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, two lead-associated disturbances of erythroid tissue function potentiate the effects of each other. 12.3.3 Effects of Alkyl Lead on Heme Synthesis and Erythropojesis In Section 10.7, the discussion of alkyl lead metabolism, it was noted that transformations of tetraethyl- and tetramethyl lead in vivo result not only in generation of the neurotoxic trialkyl lead metabolites but products of further dealkylation, including inorganic lead. One would therefore expect that alkyl lead exposure would be associated, in addition to other effects, with some of those 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 gasoline, the potential exists for lead intoxication. Boeckx and coworkers (1977) carried out two surveys of children in remote Canadian communities with respect to the-prevalence of gasoline sniffing and associated indicators of chronic lead exposure. In one group of 43 children, all engaging in the practice of gasoline sniffing, the mean ALA-0 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 SO children revealed similar results. These investigators also reported that two children having acute lead intoxication associated with gasoline sniffing showed markedly lowered hemoglobin, elevated urinary ALA and elevated urinary coproporphyrin. These authors estimated that > 50 percent of children from disadvantaged backgrounds and residing in rural or remote areas of Canada may have chronic lead exposure via this habit. This is consistent with the estimate of Kaufman (1973) of 62 percent for children in rural American Indian communities in the Southwest. Robinson (1978) described 2 cases of pediatric lead poisoning arising from habitual sniffing of gasoline, one of whom showed basophilic stippling, while Hansen and Sharp (1978) reported that a young adult chronically sniffing gasoline and showing acute lead poisoning showed a 6-fold increase in urinary PB12A/C TEH 0530889 12-26 1-5-83 DUP050031802 PRELIMINARY DRAFT ALA, elevated urinary coproporphyrin and an EP level ca. 4-fold above normal as well as basophilic stippling. In the reports of Boeckx et al. (1977) and Robinson (1978), increased lead levels were measured in urine in the course of chelation therapy, indicating that significant amounts of inorganic lead was 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 neurological effects of lead have been studied because of: (1) the recognized similarity of many, but not all, of the neurological components of the congenital disorder, acute intermittent porphyria, with the classical signs of lead neurotoxicity; and (2) some of the 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 (Dagge et al., 1965; Moore et al., 1980; Silbergeld and Lamon, 1980). According to Angle and Mclntire (1982), some of the unusual features of lead neurotoxicity are consistent with a possible role for deranged hematopoiesis: (1) a lag in production of neurological symptoms; (2) the incongruity of earAly deficits in affective and cognitive function with the regional distribution of lead in the brain; and (3) a better correlation of neurobehavioral function 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 the excessive systemic accumulation and excretion of delta-aminolevulinic acid, ALA, and this common feature has been the starting point for a number of studies of the connection between hemato- and neurotoxicity. In vitro data (Whetsell et al., 1978) have shown that the central nervous system has heme biosynthesis capability in the white matter or support structures of chick dorsal root ganglion. Sassa et al. (1979) indicated that the presence of lead in these preparations increases the production of porphyrinic material, i.e., there is disturbed heme biosynthesis with accumulation of one or more porphyrins and presumably accumulation of ALA. Millar et al, (1970) have reported inhibited brain ALA-D activity in suckling rats exposed to lead, while Silbergeld et al. (1982) observed similar inhibition in brain of adult PB12A/C 12-27 1-5-83 TEH 0530890 DUP050031803 PRELIMINARY DRAFT rats acutely exposed to lead. In the latter study, chronic lead exposure was associated with a moderate increase in brain ALA without inhibition of ALA-D, suggesting an extra-neural source of formation of the heme precursor. Moore and Meredith (1977), who administered ALA to rats, have observed that exogen ous ALA can penetrate the blood-brain barrier. These reports suggest that ALA can either be generated j_n situ in the nervous system or can enter the nervous system from elsewhere. Neurochemical investigations of ALA action in the nervous system have included interaction 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 intoxication, convulsions (Silbergeld and Lamon, 1980). Of particular interest is the similarity in chemical structure between ALA and GABA, differing only in the presence of a carbonyl group in the former, between the alpha and beta carbons. While chronic: lead exposure appears to alter neural pathways involving GABA function (Piepho et al., 1976; Silbergeld et al., 1979), this behavior cannot be duplicated in vitro using various levels of lead (Silbergeld et al., 1980), suggesting that either lead does not impart this effect by direct interaction or that an intact multi-pathway system is required. In vitro studies (Silbergeld et al., 1980; Nicoll, 1976) do demonstrate that ALA can displace GABA from synaptosomal membranes associated with synaptic function of the neurotransmitter on the GABA receptor, but is less potent than GABA by a factor of 103-104 , 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 observation that GABA release is subject to negative feedback control through presynaptic receptors on GABAergic terminals (Snodgrass, 1978; Mitchell and Martin, 1978). In this connection, Brennan and Cantrell (1979) found that ALA inhibits K+-stimulated release of GABA from pre-loaded synaptosomes, ALA functioning as an agonist at the presynaptic receptors. The effect is evident at 1.0 pM concentration of ALA while the inhibiting effect is abolished by the GABA antagonists bicuculline and picrotoxin. Of interest also is the demonstration (Silbergeld et al., 1980) that synaptosomal release of preloaded H-GABA, P812A/C TEH 0530891 12-28 1-4-82 DUP050031804 PRELIMINARY DRAFT resting and K+-stimulated, Is also inhibited in animals chronically treated with lead, paralleling the in vitro data of Brennan and Cantrell (1979) using ALA. The recent report of Silbergeld et al. (1982) described the comparative behavior of lead and the agent succinyl acetone given acutely and chronically to adult rats, in terms of disturbances in heme synthesis 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 stimulation of ALA synthase (Tschudy et al., 1980; Tschudy et al., 1982). Both agents, in vivo, showed significant inhibition of the high affinity Na+-dependent uptake of 14C-GABA by cortex, caudate, and substantia nigra. However, neither agent affected GABA uptake in vitro. Similarly, both agents reduced the seizure threshold to the GABA antagonist picrotoxin in the case of chronic or acute lead treatment and with chronically administered succinylacetone. While these % agents may involve entirely different mechanisms of toxicity to the GABAergic pathway, the fact remains that 2 distinct agents which are potent inhibitors of the heme biosynthetic pathway and ALA-dehydratase, and which do not impart a common neurochemical effect by direct action on a neuro transmitter function, have a common neurochemical action in vivo. Human data offering a connection between the hemato- and neurotoxicity of lead are limited. Hammond and coworkers (1980) reported that the best correlates of the frequency of neurological symptoms in 28 lead workers were urinary and plasma ALA, showing a better level of correlation than EP. These data support a connection between heme biosynthesis impairment and neurological effects through ALA, Of interest is the clinical report of Lemon 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, there was a significant drop in urinary coproporphyrin and ALA and a diminishing of such symptoms as lower extremity numbness and aching. Blood lead levels were not altered in the course of this treatment. While remission of symptoms in this one subject may have been spontaneous, this outcome parallels that observed in hematin treatment of subjects with acute porphyria in terms of similar reduction of heme indicators and relief of symptoms (Lamon et al., 1979). PB12A/C 12-29 TEH 0530892 DUP050031805 PRELIMINARY DRAFT Taken collectively, all of the available data strongly suggest a role for ALA, formed jbn situ or entering the brain, as a neurotoxic agent in GABAergic function, in particular, inhibition of K+-stimulated GABA release from presynaptic receptors where ALA appears to be particularly potent at very low levels based on 111 vitro results. As was described in the section on heme biosynthesis, lead can affect both cellular respiration and cytochrome C levels in the nervous system of the developing rat, this effect having a potential role in some of the symptoms of lead neurotoxicity. Hence, there is more than the issue of ALA neurotoxicity to be considered in assessing the connection between lead-induced hemato- and neurotoxicity. 12.3.5 Summary and Overview Lead has well-recognized effects on heme biosynthesis, a general tissue process, as well as on erythropoiesis and red cell physiology. 12.3.5.1 Effects of Lead on Heme Biosynthesis--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 human and experimental animals. The process of heme biosynthesis starts with glycine and succinyl-coenzyme A and proceeds through formation of protoporphyrin IX and culminates with the insertion of divalent iron into the porphyrin ring, forming heme. In addition to being a constituent of hemoglobin, heme is the prosthetic group of a number of tissue hemoproteins having variable function such as myoglobin, the P-450 component of the mixed function oxidase system and the cytochromes of cellular energetics. Hence, disturbance of heme biosynthesis by lead poses the potential for multi-organ toxicity. At present, the steps in the heme synthesis pathway which have been best studied with respect to lead's effects involve three enzymes: (1) stimulation of mitochondrial delta-aminolevulinic acid synthase, ALA-S, which mediates the formation of deltaaminolevulinic acid; (2) direct inhibition of the cytosolic enzyme, delta-aminolevulinic acid dehydratase (ALA-d) which catalyzes formation of porphobilinogen from 2 units of ALA; (3) inhibition of the insertion of iron (II) into protoporphyrin IX to form home, a process mediated by the enzyme ferrochelatase, Increased ALA-S activity has been documented in lead workers as well as lead-exposed animals, although an actual decrease in enzyme activity has also been observed in several experimental studies. It would appear then, that PB12A/C TEH 0530893 12-30 1-5-83 DUP050031806 PRELIMINARY DRAFT enzyme activity increase via feedback or derepression or activity inhibition may be dependent on the nature of the exposure. In an in vitro study using rat liver cells in culture, it was noted that ALA-S activity could be stimulated at levels as low as 5.0 pM, or 1.0 pg Pb/g preparation. In the same study, increase of activity was seen to be due to biosynthesis of more enzyme. The apparent threshold for lead stimulation of ALA-S activity In humans, based upon a study using leukocytes from lead workers, appears to be ca. 40 pg 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-0 activity inhibition, to the extent that at 40 pg Pb/dl blood lead, ALA-0 activity is significantly depressed while ALA-S activity only begins to be affected. Erythrocyte AlLA-0 activity is very sensitive to lead inhibition, the inhibition being reversed by reactivation of the sulfhydryl group with agents such as dithiothreitol, zinc or zinc plus glutathione. Although zinc appears to offset the inhibitory effects of lead in vitro and in animal studies, lead workers exposed to both zinc and lead do not show significant changes in the relationship of ALA-0 activity with blood lead compared to just lead exposure, nor does the range of physiological zinc in non-exposed subjects affect the activity. By contrast, zinc deficiency in animals has been shown to significantly inhibit activity with concomitant accumulation of ALA in urine. Since zinc deficiency has also been associated with increased lead absorption 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 the effect on activity due to reduced zinc availability, as well as (2) the effect of increased lead absorption leading to further inhibition of 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 remains to be determined in either adults or children, A further measure of this enzyme's sensitivity to lead is a report noting that rat bone marrow suspensions show inhibition of ALA-D activity by lead at a level of 0.1 pg/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 PB12A/C 12-31 1-5-83 TEH 0530894 DUP050031807 PRELIMINARY DRAFT lead workers, with both the erythrocyte activity as well as with blood lead. Of significance are the experimental 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, 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 substrate, 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 pg/dl, while urinary ALA was seen to rise exponentially with blood ALA. Urinary ALA has been 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 and more satisfactory data can be obtained In cases where 24-hour collections are feasible. A large number of independent studies have documented that there is a direct correlation between blood lead and the logarithm of urinary ALA in adult humans and children that the threshold for this with respect to blood lead is commonly accepted as being 40 pg/dl. Several studies of lead workers also indicate that the correlation of urinary ALA with blood lead continues below this value. Furthermore, a Japanese report has demonstrated that 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, to the extent that the "reserve capacity" of ALA-D activity is assumed by some to be such that only the level of inhibition associated with significant accumulation of the enzyme's substrate, ALA, in accessible indicator media may be significant. One difficulty with this view is that it is not possible to quantify 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 threshold for potential neurotoxicity of ALA in terms of blood lead may be different from the level associated with urinary accumulation. PB12A/C TEH 0530895 12-32 1-5-83 DUP050031808 PRELIMINARY DRAFT 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 sensitive, specific micro methods of analysis. Accumulation of protoporphyrin IX in erythrocytes is the result of impaired placement 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 (zinc protoporphyrin, ZPP) and is tightly bound in available heme pockets for the life of the erythrocytes. This tight sequestration is in contrast to the relatively mobile non-metal, or free, protoporphyrin (FEP) accumulated in the congenital disorder, erythropoietic protoporphyria. Elevation of erythrocyte ZPP has been extensively documented as being exponentially correlated 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 eryth.roid 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 accumulation in erythrocytes of newly-employed lead workers continues to increase when blood lead'increase has already achieved a plateau, which would influence the relative correlation of ZPP and blood lead in workers with a shorter exposure history. In individuals removed from occupational exposure, the ZPP level in blood declines much more slowly, even years after removal from exposure, and when blood lead has declined by a relatively greater amount. Hence, ZPP level would appear to be a more reliable indicator of continuing intoxication from lead resorbed from bone. The measurable threshold for the effect of lead on ZPP accumulation is affected by the relative spread of blood lead and corresponding ZPP values measured. In young children, under 4 years old, the ZPP elevation associated with iron-deficiency anemia should be taken into account. In adults, a number of studies indicate that the threshold for ZPP elevation with respect to blood lead is ca. 25-30 pg Pb/dl, In children 10-15 years old the threshold is ca. 16 pg Pb/dl; in this age group, iron deficiency is not a factor. In one report, it was noted that children over 4 years old showed the same threshold, 15.5 pg/dl, as a second group under 4 years old, indicating that iron deficiency was not a factor in the study. PB12A/C 12-33 1-5-83 TEH 0530896 DUP050031809 PRELIMINARY DRAFT Within the blood lead range considered "normal," i.e., below 30-40 pg/dl, any assessment of the ZPP-blood lead relationship will be strongly influenced by the relative analytical proficiency for measurement of both blood lead and EP. The types of statistical treatments given the date 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 dose-effect threshold and dose-response. An averaged blood lead threshold for the effect using both statistical techniques yielded a value of 16,5 pg/dl for either the full group or those subjects with blood lead below 30 pg/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 > 1 and > 2 S.D.s above the reference mean in 50 percent of the children was 28,6 and 35.7 pg Pb/dl, respectively. Hence, fully half of the children were seen to have significant elevation in EP at blood lead around the accepted cut-off value for undue lead exposure of 30 pg/dl. ' From various reports, children and adult women appear to be more sensitive to the effects of lead on EP accumulation with respect to any given blood lead level, with children being somewhat more sensitive than women. Effects of lead on ZPP accumulation and reduced heme formation is not restricted to the erythropoietic system. The heme-containing protein cytochrome P-450, which is an integral part of the hepatic mixed function oxidase system is known to be affected with lead exposure, particularly with acute intoxication, of humans and animals. 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 biosynthesis capability but that such preparations elaborate porphyrinic material in the presence of lead. In the neonate rat, chronic lead exposure to lead resulting in levels of blood lead which are only moderately elevated, are associated with retarded growth in the hemoprotein cytochrome C and disturbed electron-transport in the developing rat cerebral cortex. These data are a parallel of the earlier discussion concerning the effect of lead on ALA-D activity and ALA accumulation in neural tissue and when both these effects are viewed in the toxicokinetic context of increased retention of lead in both developing animals and children, there is PB12A/C TEH 0530897 12-34 1-5-83 DUP050031810 PRELIMINARY DRAFT then an obvious, serious potential for impaired heme-based metabolic function in the nervous system of lead-exposed children. Ferrochelatase is an intramitochondria] enzyme, so that impairment of its activity, either directly by lead or via impairment of iron transport to the enzyme, is evidence of lead's presence in mitochondria. As set forth in the previous 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. 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 to, say, the central nervous system. This is, obviously, a distinct issue from that concerned with which system is most accessible to measurement of the effect. In this connection, 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, although these have not been as heavily studied on a biochemical or molecular level. Levels of .coproporphyrin are increased in urine in lead intoxication, this effect reflecting active intoxication. Lead also affects the activity of the enzyme uroporphyrinogen-I- synthetase, resulting in an accumulation of its substrate, porphobilinogen. It has been reported that the erythrocyte enzyme is much more sensitive to lead than the hepatic species and presumably accounts for much of the accumulated substrate. 12.3.5.2 Effects of Lead on Erythropoiesis and Erythrocyte Physiology--Anemia is a manifestation of chronic lead intoxication, being characterized as mildly hypochromic and usually normocytic. It is associated with reticulocytosis, owing to shortened cell survival, and the variable presence of basophilic stippling. Its occurrence is due to both decreased erythrocyte production and increased rate of red cell destruction. In young children, under 4 years of age, the anemia of iron deficiency is exacerbated by the further effect of lead, and vice versa. PB12A/C 12-35 1-5-83 TEH 0530898 DUP050031811 PRELIMINARY DRAFT Reduced hemoglobin production is negatively correlated with blood lead in young children, where iron deficiency my be a confounding factor, as well as in lead workers. In one lead study, blood lead values which were usually below 80 pg/dl were inversely correlated with hemoglobin content. In these subjects, iron deficiency was found to be absent. The threshold in blood lead for reduced hemoglobin is ca. 50 pg Pb/dl in adult lead workers and somewhat lower in children, 40 pg Pb/dl. 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 rests with disturbances of both heme and globin biosynthesis. The hemolytic component to lead-induced anemia appears to be due to increased cell fragility and decreased osmotic resistance. In one rat study, it was noted that the hemolysis associated with vitamin E deficiency, via reduced cell deformability, 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 leading to cell "shrinkage" and inhibition of the latter resulting 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- and tetramethyl lead, components of leaded gasoline, undergo transformation jin 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 there exists chronic lead intoxication in children from socially deprived backgrounds and residing in rural or remote areas resulting from the habit of sniffing leaded gasoline. Notable in these subjects is evidence of impaired heme biosynthesis which has mainly been assessed as significantly reduced ALA-0 activity. In a number of 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. 12.3.5.4 The Inter-Relationship of Lead's Effects on Heme Synthesis and the Nervous System--Lead-assoclated disturbances of heme biosynthesis as a possible factor in the neurological effects of Lead have been the object of PB12A/C TEH 0530899 12-36 1-5-83 DUP050031812 PRELIMINARY DRAFT considerable interest because of the recognized similarity of a number of the neurological components of the congenital disorder, acute intermittent porphyria, with the classical signs of lead neurotoxicity as well as some of the unusual aspects of lead neurotoxicity. There are two possible points of connection between lead's effects on both heme biosynthesis and the nervous system. Concerning the similarity of lead neurotoxicity to acute intermittent porphyria, there is the common feature of excessive systemic accumulation and excretion of ALA. Secondly, lead neurotoxicity reflects, to some degree, impaired synthesis of heme and hemoproteins involved in crucial cellular functions. Available information indicates that ALA levels in brain are elevated in the brain of lead-exposed animals, arising via in situ inhibition of brain ALA-D activity, known to occur in various species, or via transport to the brain after formation in other tissues. In this connection, ALA is known to traverse the blood-brain barrier. Hence, ALA is accessible to" or in brain and available upon lead exposure to express any neurotoxic potential. Based on various in vitro and in vivo data obtained in the context of neurochemical mechanisms of lead neurotoxicity, it appears that ALA can readily play a role in GABAergic function, in particular inhibition of release of the neurotransmitter GABA from presynaptic receptors where ALA appears to be very potent, even at low levels. In an In vitro study, agonist behavior by ALA was demonstrated at levels as low as 1.0 pM ALA. This in 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-GA8A, Further evidence for an effect of some agent acting directly other than lead are the observations that in vivo effects of lead on neurotransmitter function cannot be duplicated with in yitrp preparations in which lead is added. The connection of impaired heme and hemoprotein synthesis in the brain of the neonate rat was noted earlier. In these studies there was observed 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, it is also these regions where selective lead accumulation appears to occur. Human data on lead-induced associations between disturbed heme synthesis and neurotoxicity, while limited, also suggest that ALA may function as a neurotoxicant. PB12A/C 12-37 1-5-83 TEH 0530900 DUP050031813 PRELIMINARY DRAFT 12.4 NEUROTOXIC EFFECTS OF LEAD 12,4.1 Preliminary Considerations Neurotoxic effects have long been recognized as some of the more severe consequences of exposure to lead (Blackfan, 1917; Tanqueral Des Planches, 1839; Oliver, 1911; Prendergast, 1910; Stewart, 1895). Lead exposure levels necessary to produce specific neurotoxic effects and the reversibility/irre versibility of such effects, however, have been controversial issues that have stimulated considerable research and debate since the early 1900s. Such research has contributed to progressively increasing concern that lead exposure levels previously accepted as harmless are actually sufficient to cause sig nificant neurological or behavioral impairments. It is now generally accepted that at high levels of lead exposure pro ducing blood-lead concentrations greater than 80 to 100 pg/dl a person is likely to experience the clinical syndrome of fulminant lead encephalopathy (Chisolm, 1971, 1980). This syndrome includes neurological and other symptoms of such severity that immediate medical intervention and, frequently, hos pitalization are necessary. The onset of encephalopathy is heralded by gross ataxia, persistent and forceful vomiting, periods of lethargy or stupor'inter spersed with lucid intervals, and finally coma and intractable convulsions. Based on the literature reviewed below, it now also appears that lower levels of lead exposure, yielding blood levels below 80 pg/dl, produce less welldefined but still medically significant neurotoxic effects in both adults and children. These effects include nonspecific, and often difficult to detect, neural or behavioral changes that occur in some humans in the absence of severe neurological symptoms or other signs that typify acute lead intoxication. The lowest levels of lead exposure sufficient to produce nonspecific (sometimes termed "subclinical") neurobehavioral deficits are difficult to estimate with certainty and remain a matter of considerable controversy (Needleman, 1980a). Some evidence reviewed below suggests that no clear threshold may exist for certain neurological effects, whereas others may only become detectable once blood-lead levels exceed 30 to 40 pg/dl. The speci fication of which neurotoxic effects constitute "adverse health effects11 is also highly controversial, due to the lack of clear boundaries between increasingly severe manifestations of neurotoxicity observed along what appears to be a steadily ascending dose-response continuum of varying slope for different individuals. PB12A/D TEH 0530901 12-38 1/4/82 DUP050031814 PRELIMINARY DRAFT 12.4.1.1 Objectives of the Neurotoxicity Section^--The aims of this section are: (1) to characterize dose-effect relationships between lead exposure indices and various behavioral and electrophysiological measures that have been used to evaluate neurotoxic effects; (2) to examine these effects in terms of important end points of behavioral function, i.e., (a) sensory and perceptual-motor performance (e.g., sensory acuity, speed of reaction, vigil ance), (b) cognitive performance (e.g., learning, memory, language), and (c) social adaptation (e.g., conduct disorders, hyperactivity, attention deficit); and (3) to identify critical interactive factors that covary with the effects of lead, i.e., (a) age at exposure vs. age at expression, (b) lead-nutrition interactions (e.g., iron deficiency), (c) social factors (e.g., socio-economic status, parental IQ, quality of the caregiving environment), and (d) type of exposure (e.g., acute vs. chronic, occupational vs. non-occupational). The following review of the neurobehavioral effects of lead has been derived from studies of both humans and other mammalian species. Such effects have been indexed by means of a variety of approaches, including*. (1) assess ment of neuropathology by classical histological and ultrastructural analyses of morphological damage; (2) analysis by biochemical assays of altered neuro chemical parameters or processes; (3) assessment of altered electrophysiological responses in both the central and peripheral nervous system; (4) assessment of neurobehavioral effects both by neurological examinations and various types of behavioral testing methods; and (5) assessment of alterations in oeuropharmacological responses affecting many of the above variables assessed by the other approaches. The effects of high-level toxic exposure to lead have been well documented by most of these approaches. At lower exposure levels, however, the demonstration of the effects of lead by the above types of assessments has been complicated by numerous methodological considerations that should be noted as a prelude to any critical review of the literature. 12.4.1.2 Methodological Issues in Studies of Neurotoxicity--Defining doseresponse relationships between lead and particular neurotoxic responses involves two basic steps. First, there must be an assessment of the internal lead burden resulting from external doses of lead received via exposure to various environmental sources (such as air, water, food, occupational hazards, pica, etc.). Internal lead burdens may be indexed by: lead concentrations in blood, teeth, hair, or other tissue; EP levels; Or other biological indicators. PB12A/D 12-39 1/4/82 TEH 0530902 DUP050031815 PRELIMINARY DRAFT The second step involves an assessment of the relationship of internal exposure indices to behavioral and 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. Some important considerations related to such methodological issues are briefly discussed next. It should be noted that these are issues relevant to all clinical research and not just to lead research alone. Although epidemiological studies have immediate environmental relevance at the human level, difficult problems are often associated with the inter pretation of the findings, as has been pointed out in several recent reviews (Bornschein et a!., 1980; Cowan and Leviton, 1980; Needleman and Landrigan, 1981; Rutter, 1980; Valciukas and Lilis, 1980; Repko and Corum, 1979). The main problems, as discussed below, 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) inappropriate statis tical analyses; (6) inappropriate generalization and interpretation of results; and (7) the need for "blind" evaluations by experimenters and technicians. Each major exposure route--food, water, air, dust and soil--contributes to a person's total daily intake of lead (Chisolm and Baritrop, 1979). The relative contribution of each, exposure route, however, is a matter of contro versy (see chapter 13). The most commonly used measure of internal dose is the concentration of lead in the blood. Blood-lead values vary complexly as a function of age, sex, race, geographic location, and exposure source. The blood-lead level is an indirect marker of current exposure but may not reflect total body burden (including that from long past exposures) as well as the concentration of lead in dentine or teeth (Needleman, 1980b). Subjects in epidemiological studies may be misclassified as to exposure level unless careful choices of exposure indices are made based upon the hypotheses to be tested, the accuracy and precision of the biological media assays, and the collection and assay procedures involved. Cowan and Leviton (1980) summarize the measures of internal exposure to lead and their respective advantages and disadvantages (see Table 12B-1 of Appendix 12-B). Future research may indentify a more standard exposure index, but for now it appears that a risk classification similar to that of the Center for Disease Control (1978) in terms of blood-lead and FEP levels PB12A/D TEH 0530903 12-40 1/4/82 DUP050031816 PRELIMINARY DRAFT will continue for the near future to be the standard approach most often used for lead exposure screening and evaluation. Much of the ensuing discussion appearing later in this chapter is therefore focussed on definition of dose-effect relation ships for various neurotoxic effects in relation to associated blood lead levels. Frequency and timing of sampling for internal lead burdens is another important factor in evaluating studies of the effects of lead on neurological and behavioral functions. For example, epidemiological studies often rely on blood-lead and/or erythrocyte protoporphyorin (EP) levels determined at a single point in time to retrospectively estimate or characterize internal exposure his tories 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. Very few prospec tive studies exist with reliable estimates of critical lead exposure levels asso ciated with observed effects. Several prospective longitudinal studies on the effects of lead on early development of infants and young children (e.g., Bornschein, 1982) are currently in progress, but the results of these studies are not available as yet. Therefore, the present assessment of the neurotoxic effects of lead in humans must rely heavily on published retrospective epidemiological studies. Obviously, parameters such as exposure levels and durations can be defined with much more precision in experimental studies carried out in the laboratory. Unfortunately, however, adequate experimental designs are frequently lacking, and the environmental relevance of experimental laboratory data is limited by two major considerations. The most serious is the fact that nonhuman models are, of necessity, used to determine dose-response curves. However, since large species differences exist in sensitivity to lead (Pentschew and Garro, 1966), adequate nonhuman exposure models are difficult to devise. A second problem is that animal experiments frequently use doses of lead much higher than would be expected to occur in the ambient environment. In addition, investigators must guard against confounding variables such as possibly reduced nutrition levels because of altered food pal stability (if the lead dose delivery system is via food or water). Furthermore, if central nervous system (CNS) alterations are noted, it is often difficult to separate damage caused by direct versus indirect effects on neural tissue. Despite these difficulties, much useful data on the neurotoxic effects of lead with potential implications for understanding human exposure effects have been obtained through animal studies. PB12A/D 12-41 1/4/82 TEH 0530904 DUP050031817 PRELIMINARY DRAFT Key variables that have emerged in determining effects of lead on the nervous system include (1) the duration and intensity of exposure and (2) age at exposure. Evidence suggests that young organisms with developing nervous systems are more vulnerable than adults with fully matured nervous systems. Particular attention will, therefore, be accorded to discussion of the neuro toxic effects of lead in children and developing animals as a special group at risk. Precision of measurement is a Critical methological issue, especially when research on neurotoxicity leaves the laboratory setting. Neurotoxicity is most often measured indirectly with psychometric or neurometric techniques in epidemiological studies (Valciukas and Li 1is, 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 measurement theory. Most 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. Standard ization 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 thresholds for neurotoxic effects of lead exposure in children. Table 12B-2 (Appendix 12B) lists some of the major tests used, together with their advantages and weaknesses. The subsequent review places more weight on age-normed and standardized test instruments. Other measures, such as reaction time, finger tapping, and various electrophysiological measures (e.g., cortical evoked and slow-wave potentials, brain-stem evoked responses) are potentially more sensitive, but still experimental, indices whose clinical utility and psychometric properties with respect to the neurobehavioral 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 parent popula tion. 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 because a reference group is 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 PB12A/D TEH 0530905 12-42 1/4/82 DUP050031818 PRELIMINARY DRAFT effects of confounding can be reduced by choosing a more homogeneous stratified sample, but the .generalinability of the results of such cohort studies is thereby limited. Perhaps the most vulnerable methodological point in epidemological studies is controlling for confounding covariates, so that residual effects can be more confidently attributed to lead. Among adults, the most important covari ates 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 develop mental covariates are additionally important: parental socioeconomic status (Needleman et a!., 1979); maternal IQ (Perino and Emhart, 1974); pica (Barltrop, 1966); quality of the caregiving 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. The latter is the case because, for the forms of lead existing in the normal diet, the rate of absorption in children is higher than in adults (Hammond, 1982), Children below the age of three years appear to be particularly vulnerable, in ' that the rate of accumulation of even a low body-lead burden is higher for them than for adults (National Academy of Sciences, 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 also to all epidemiological studies are: adequate sample size (Hill, 1971); 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. It is often difficult and expensive to use large sample sizes in complex research such as that 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 which 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 PB12A/D 12-43 1/4/62 TEH 0530906 DUP050031819 PRELIMINARY DRAFT chance alone. This is a serious problem since threshold effects are often small and variable. A final crucial question in this (and all other research) revolves around the care that was taken to assure that investigators were protected from any information that might identify a subject as high or low in lead 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 (and if possible, 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 key studies on neurotoxic effects of lower-level lead exposures in children. Then a review of animal studies on neurotoxic effects of lead is presented, followed by a summary and conclusions section integrating information from both human and animal studies in an interpretive manner. Special emphasis is placed on assessment of effects associated with the lower end of the lead exposure continuum during early development, since children are placed at special risk (National Academy of Sciences, 1980), for reasons stated below and in Chapter 13. 12.4.2 Human Studies 12.4.2.1 Neurotoxic Effects of Lead Exposures in Adults--Severely deleterious effects of exposures to high levels of lead, especially for prolonged periods that produce overt signs of acute lead intoxication* are by now well documented in both adults and children and were extensively reviewed in the 1977 EPA document Air Quality Criteria for Lead. The most profound effects occurring in adults are referred to as the clinical syndrome of lead encephalopathy, described in detail by Aub et al. (1925), 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, restless ness, irritability, poor attention span, headaches, muscular tremor, halluci nations, and loss of memory. These symptoms may progress to delirium, mania, convulsions, paralysis, coma, and death. The onset of such serious 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 central nervous 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, 1961 a,b,c), PB12A/D TEH 0530907 12-44 1/5/83 DUP050031820 PRELIMINARY DRAFT In addition to the above CNS effects, lead also clearly damages the peripheral nervous system (PNS) at toxic, high exposure levels which predominantly affect large myelinated motor fibers (Fullerton, 1976). Pathologic changes in the PNS include segmental demyelination and, in some fibers, axonal degeneration (Fullerton, 1976). These changes appear to reflect the effects of lead on Schwann cells, with concomitant disruption of myelin membranes (Lampert and Schochet, 1968). 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). Reports of ges cavus defor mities due to old peripheral neuropathies (Emmerson, 1968), however, suggest that lead-induced neuropathies of sufficient severity could result in permanent peripheral nerve damage. Morphologically, the neuropathy is usually detect able only after high or prolonged exposure to lead, with distinctly different sensitivities existing among mammalian species. 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 acute lead intoxication exert more nonspecific (and more difficult to detect) neurobehavioral effects in otherwise apparently asymptomatic adults or children. Attention has focused in particular on whether exposures leading to blood lead below 80 to 100 pg/dl may lead to neurobehavioral deficits in the absence of classical signs of lead encephalopathy. In adults, if such neurobehavioral deficits occurred with great frequency, one might expect this to be reflected by performance measures in the workplace, such as higher rates of absences, reports of neurologically related symptoms, and 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. No correlation between elevated blood-lead levels and sickness rates or symptom types were found (Shannon et al., 1976) for groups of workers in a lead storage battery factory from high-medium-, and low-exposure areas vs. control workers in nohexposure areas of the same plant. However, mean blood-lead values for workers in the three exposure groups were 60, 50, and 42 pg/dl, respectively, differing little from the mean of 45 pg/dl for the so-called nonexposure control group. Similarly, Robinson (1976) reported no increased sickness rates for tetraethyl-lead (TEL) workers having mean blood lead values PB12A/D 12-45 1/4/82 TEH 0530908 DUP050031821 PRELIMINARY DRAFT of 43 pg/dl and daily urinary excretion of 0.089 mg/liter urine over an 8- to 10year period (3 to 4 times the control group rate). Sickness rates were deter mined based on a retrospective study of records over a 20-year period. Another study (Tola and Nordman, 1977) failed to find notable symptoms among workers with blood-lead levels in the 60 to 80 pg/dl range. Absence or sickness reports, however, may not be sensitive enough measures to detect nonspecific neurobehavioral symptoms. Several other recent studies have found neurological, gastrointestinal, and other lead-related symptoms in smelter workers with blood-lead levels between 60 and 80 pg/dl (Oahlgren, 1978; Hammond et al., 1980; Irwig et al., 1978; Li 1is et al., 1977; Roels et al., 1978). One study (Baker et al., 1979) found a dose-effect relationship between extensor muscle weakness and blood-lead levels in 11 percent of workers in the 40 to 59 pg/dl range and 16 percent in the 60 to 79 pg/dl range, Hammond et al. (1980) and Spivey et al. (1979) both found significant differences between non-exposed controls and lead workers with blood-lead levels above 60 pg/dl, but not below that level. However, there was a positive correlation between frequency of symptoms and blood-lead levels when all groups of lead workers were combined. Spivey et al. (1979) also found a negative correlation between number of symptoms reported and length of. employ ment. They attributed this result to biological resistance, better hygiene, and more stoicism among long-term employees. Apparently other factors such as general educational level, occupational education, and knowledge of symptom susceptibility affect frequency of reporting symptoms, and need to be controlled in these studies along with the other covariates mentioned in Section 12,4.1.2 (Araki et al., 1982). Nevertheless, the weight of evidence points toward a dose-response relationship between blood-lead levels and frequency and severity of symptoms among adults down to at least 60 pg/dl and perhaps as low as 40 pg/dl. The evidence for the 40 pg/dl level is very uncertain for any clinical symptoma tology, including classical indications of neurotoxicity. Only a few studies have employed more sensitive psychometric and/or nuerological testing procedures in an effort to demonstrate nonspecific 1ead-induced. neurobehavioral effects in adults. For example, Morgan and Repko (1974) reported preliminary results of an extensive study of behavioral functions in 190 leadexposed workers (mean blood-lead level = 60.5 17.0 pg/dl. The majority of the subjects were exposed between 5 and 20 years. The authors examined 36 noninde pendent measures of general performance and obtained 44 measures and a-ALA-D PB12A/D TEH 0530909 12-46 1/4/82 DUP050031822 PRELIMINARY DRAFT changes correlated with effects on hand-eye coordination, suggesting that some behavioral changes did occur in adult workers below a blood-lead level of 80 jjg/dl. In addition, variability of performance increased with increasing blood-lead level; however, only during periods of high-demand performance did a worker's capacity clearly decrease as a result of lead exposure. Aspects of the Morgan and Repko work can be criticized because of methodological problems, including reported apparatus failures during testing of subjects and the use of multiple statistical comparisions without correcting for chance significance. Also, findings analogous to those reported by Morgan and Repko were not obtained in a similar study (Milburn et al., 1976) that 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; Haenninen et al., 1978; Valciukas et al., 1978) have found difficulties in visual motor performance, IQ test performance, mood, ner vousness, and coping in lead workers with blood-lead levels of 50 to 80 pg/dl. Again, a graded dose-effect relationship is indicated by such studies. In addition to the above studies suggesting possible CNS dysfunctions, numerous investigations have provided electrophysiological data indicating that peripheral neuropathy symptoms in adults can be associated with bloodlead values < 80 pg/dl. As reviewed by SeppSlainen and Hernberg, 1972; Sessa et al., 1965) More recently, such peripheral nerve deficits were established by SeppalSinen et al. (1975) for lead workers whose blood-lead levels were as low as 50 pg/dl and had never exceeded 70 pg/dl during their entire exposure period (mean = A.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 highpressure oils. Results of a multielemental analysis of the worker's blood for lead, chromium, copper, nickel, and manganese indicated a clear association between lead exposure and peripheral nerve damage. Half of the workers (10 to 20) had elevated blood-lead levels (60 to 120 pg/dl) and showed definite electromyographic deficits. Mean bloodlead level for the control group was 18.6 pg/dl. Melgaard et al. (1976) reported additional results on associating lead exposures with polyneuropathy of unknown etiology in 10 cases from the ` general population. Another study reported by Araki (1976) provides further confirmation of the Seppalainen et al. (1975) and Melgaard et al. (1976) PB12A/D 12-47 1/5/83.. ... TEH 0530910 DUP050031823 PRELIMINARY DRAFT findings in that evidence for peripheral neuropathy effects were reported for lead-industry workers with blood-lead values of 29 to 70 pg/dl. More recent studies by Ashby (1980), Seppalainen et al. (1979), and Seppalainen and Hernberg (1980) have confirmed a slowing of nerve conduction velocity (NCV) in lead workers with blood-lead levels as low as 60, 45, and 30 pg/dl, respectively, whereas Buchtal and Behse (1979), Lilts et al. (1977), and Paulev and dossing (1979) found no neuropathy below 80 pg/dl. Buchtal and Behse (1979, 1981), using nerve biopsies from workers with encephalopahty, found histological changes indicative of axonal degeneration resulting in conduction velocity reductions that corresponded to loss of large fibers and decreased amplitude of sensory potentials, whereas these changes were not seen in asymptomatic workers. The mechanism of the lesion may be different in the two cases. Araki et al. (1980) examined the changes in NCV of asymptomatic lead workers with blood-lead levels below 70 pg/dl and health nonexposed controls over repeated assessments and found that a change of 4.0 m/s in NCV was associated with a change of only 14 pg/dl in blood-lead levels. Bloodlead was more highly correlated with NCV than ZPP levels were. They concurred with Buchtal and Behse (1979) that recovery in NCV may be due to recovery from a minor defect in the excitable membrane of the nerve fiber. Seppalainen and Hernberg (1980) suggested that this slowing of conduction velocities should be interpreted as an early sign of neuropathy. The low blood-lead levels (below 50 pg/dl) reported in some of the above studies should be viewed with caution until further confirmatory data are reported on samples of larger size using well verified blood assay results, but once again a continuous dose-response relationship between blood-lead concentration and extent and degree of neuropathy was seen. In summary, the above studies, provide convergent evidence for peripheral neuropathies occurring in adults having blood-lead levels in the 50 to 70 pg/dl range. Futhermore, although it might be argued that peak levels of lead may have been important and that substantially higher lead body burdens existing before the time of some of the studies were actually responsible for producing the neuropathies, it appears that in several cases (Araki et al., 1970; Seppalainen, 1975; SeppdlSinen and Hernberg, 1980) blood levels that had never exceeded 70 pg/dl were treated to increased peripheral nerve dysfunctions. The studies by Seppalainen et a'L are generally methodologically sound, having been well controlled for the possible effects of extaneous factors such as history, PB12A/D TEH 0530911 12-48 1/5/83 DUP050031824 PRELIMINARY DRAFT length and type of exposure, multiple assessments of different nerves, temper ature differences at the nerve conduction velocity assessment sites, plus relevant confounding covariates. However, it should be noted that, in the 1975 study, the data reported for control subjects were obtained at an earlier time (1971 to 1973) than data for the lead exposed subjects (early 1973), and no blood-lead levels were reported for the control subjects. Still, when the SeppalSinen et al. (1975) results are viewed collectively with the data from other studies reviewed here, strong evidence exists for peripheral neuropathies occurring in adults at blood-lead levels of 50 to 70 pg/dl or lower levels. PB12A/D 12-49 1/5/83 TEH 0530912 DUP050031825 PRELIMINARY DRAFT 12.4.2,2 Neurotoxic Effects of lead Exposure in Children*"Symptoms of encepha lopathy similar to those that occur in adults have been reported to occur in infants and yound children (Blackfan, 1917; Chisolm, 1968; Cumings, 1959; Giannattsig et al.,{ 1952; McKhann and Vogt, 1926; Oliver, 1911; Prendergast, 1910; Tapper, 1963), with a markedly higher incidence of severe encephalopathic symptoms and deaths occurring in them than in adults. This may reflect the greater difficulty in recognizing early symptoms in young children, which allows intoxication 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 theraphy as standard medical practice (Grenngard et al., 1965; National Academy of Sciences, 1972). The following mortality rates have been reported for children 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, BAL was used. From 1950 to 1960, CaEDTA completely replaced BAL. Beginning in 1960, combined therapy with BAL and CaEDTA (Chisolm, 1968; Coffin et al., 1966) resulted in a very substantial reduction in mortality. Morphological findings in cases of fatal lead encephalopathy vary (Blackman, 1937; Pentschew, 1965; Popoff et al., 1963). On macroscopic examination the brains are often edematous and congested. Microscopically, cerebral edema, altered capillaries (endothelial hypertrophy and hyperplasia), and perivascular glial proliferation often occur. Neuronal damage is variable and may be caused by anoxia. In some cases gross and microscopic changes are minimal (Krigman et al., 1978; Pentschew, 1965). Reported neuropathologic findins are essentially the same for adults and children. 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 PB12B/A TEH 0530913 12-50 1/6/83 DUP050031826 PRELIMINARY DRAFT capillaries with swelling of endothlial cells. Diffuse astrocytic prolifera tion is the earliest morphological response to increased permeability of the blood-brain barrier (dysoria). Concurrent with the dysotic alterations, especially evident in the cerebellum, were changes that Pentschew (1965) attri buted to hemodynamic disorders, i.e., ischemic changes manifested as cell ne crosis, perineuronal incrustations, and loss of neurons, especially in isocortex and basa ganglia. Pentschew concluded that structural changes in infantile lead encephalopathy are a mixture of dysorie and hemodynamic parenchymal alterations. Also of great concern in cases of severe or prolonged nonfatal episodes of lead encephalopathy are neurological sequelae qualitatively similar to those often seen following traumatic or infectious cerebral injury, with permanent sequelae being more common in children than in adults (Chisolm, 1962; Mellins and Jenkins, 1955; Tepper, 1963). The most severe sequelae in children are cortical atrophy, hydrocephalus, convulsive seizures, and severe mental retardation (Chisolm, 1968; Mellins and Jenkins, 1955; Perlstein and Attala, 1966; Tepper, 1963). Children who recover from acute lead encephalopathy but are re-exposed to lead almost invariably show evedence of permanent central nervous system damage (Chisolm and Harrison, 1956), Even if further lead exposure is minimized, 25 to 50 percent show severe permanent sequelae, such as seizure disorders, blindness, and hemiparesis (Chisolm and Baritrop, 1979). Lasting neurotoxic sequelae of lead poisoning in the absence of acute en* cephalopahty were first reported by Byers and Lord (1943). They reported that 19 out of 20 children with previous mild lead poisoning later made unsatisfac tory progress in school, presumably due to sensorimotor deficits, short attention span, and behavioral disorders. 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 Harrisio, 1956; Cohen and Ahrens, 1959; Kline, I960). Perlstein and Attala (1966) also reported neurological sequelae in 140 of 386,children (37 percent) following lead poiso ning without encephalopathy. Such sequelae included mental retardation, seizures, cerebral palsy, optic atrophy, and visual-perceptual problems in some children with minimal intellectual impairment. The severity of sequelae was related to severity of earlier-observed symptoms. For 9 percent of those children who appeared to be asymptomatic at the time of diagnosis of lead poisoning, mental retardation was the only effect observed upon later follow-up. PB12B/A 12-51 1/4/82 TEH 0530914 DUP050031827 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 en cephalopathy, chelation therapy tends to reverse the neurotoxic effects of lead poisoning (especially cognitive, perceptual, and behavioral deficits), whereas one study recently found a residual effect on fine motor performance after chelation (Kirkconnell and Hicks, 1980). However, none of these studies are conclusive because of failure to document adequately prior exposure or developmental histories, inappropriate control groups, non-blind evaluations, and/or selection biases. In summary, pertinent literature definitively demonstrates that lead poisoning with encephalopathy results in a greatly increased incidence of permanent neurological and cognitive impairments. The Perlstein and Attala (1966) study further indicates that children with symptomatic lead poisoning in the absence of encephalopathy also evidence a later increased incidence of neurological and behavioral impairments. Unfortunately, methodological short comings of studies reviewed to this point make it difficult to determine if apparently "asymptomatic" lead exposure results in later nonspecific, neurolo gical, cognitive, or behavioral deficits. Earlier studies (e.g. , Mel 1 ins and Jenkins, 1955) failed to distinguish between children with and without encepha lopathy. Some studies (Byers and Lord, 1943; Mel Tins and Jenkins, 1955; Perlstein and Attala, 1966) also did not include a suitable control group. Lead poisoning often occurs in children from low socioeconomic status (SES) families who reside in old, inner-city housing. Robinson and Robinson (1976) point out that children's IQs tend to vary with the status of their families. Taking into account the SES of children in the Mellins and Jenkins (1955) study, the distribution of the children's IQ scores may be representative of the popula tion from which these children were drawn. Likewise the 9 percent incidence of mental retardation following "asymptomatic" lead poisoning found by Perl stein and Attala (1966) may be no different that the base rate for a population of that SES, Other shortcomings, such as relying on maternal interviews to determine previous development (Mellins and Jenkins, 1955) and failure to take into account base rates of such problems as short attention span, hyperactivity, perceptualmotor deficits, fine motor deficits, and deficits in test-taking behavior in children of lower SES (Byers and Lord, 1943; Mellins and Jenkins, 1955; Perl stein and Attala, 1966), make it difficult to conclude with certainty that "asympto matic" lead poisoning has a detrimental effect on neurological, behavioral, or PB12B/A TEH 0530915 12-52 1/4/82 DUP050031828 PRELIMINARY DRAFT cognitive functioning. An earlier review of the literature by Weiner (1970) came to the same conclusion. Determining precise values for lead exposures necessary to produce acute symptoms such as lethargy, vomiting, irritability, loss of appetite, dizziness, etc. (Weiss, 1980), or later neurotoxie sequelae in humans is difficult in view of the usual sparsity of data on environmental lead levels experienced by the client, period(s) of exposure, or body burdens of lead existing prior to manifestation of symptoms. Nevertheless, enough information is available to permit reasonable estimates to be made regarding the range of blood-lead levels associated with acute encephalopathic symptoms or death. Lead levels found to produce permanent encephalopathy appear to be lower for children than for adults. According to Kehoe (1961a,b,c) blood-lead levels well in excess of 120 pg/dl are usually necessary to produce such irreversible effects for adults. Recurrent bouts of lead intoxication in the absence of acute encephalopathy may also lead to progressive mental deterioration. Other data (Smith et al., 1938), suggest that acute lead intoxication, including severe gastrointestinal symptoms and/or signs of encephalopathy, can occur in adults at blood-lead levels around 100 pg/dl; but ambiguities make these data difficult to interpret. Data on effective lead exposure levels indicate that lower blood-lead levels among children than among adults are associated with the occurence of acute encephalopahty symptoms and death. The most extensive early compilation of infor mation concerning this question 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 encephalopahty and death in children in Baltimore to blood-lead levels determined by the Baltimore City Health Depart ment (using the dithizone method) between 1930 and 1970. Blood-lead levels associated with asymptomatic cases or milder 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 pg/dl (mean - 105 pg/dl), Acute lead poisoning symptoms, other than signs of encephalopathy, were observed from approximately 6o to 450 pg/dl (mean = 178 pg/dl). Signs of encephalopathy (hyperirritability, ataxia, convulsions, stupor, and coma) were associated with blood-lead levels of approximately 90 to 700 or 800 pg/dl (mean = 330 pg/dl). The distribution of blood-lead levels associated with death (mean - 327 pg/dl) was essentially the same as for levels yielding encephalopathy. These data PB12B/A 12-53 1/4/82 TEH 0530916 DUP050031829 PRELIMINARY DRAFT suggest that bipod-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 chidren starting at blood-lead levels of approximately 100 pg/dl. Certain other evidence from scattered medical reports (Bradley et al., 1956; Bradley and Baumgartner, 1958; Cumings, 1959; Gant, 1938; Rough et al., 1979; Smith et al., 1938), however, suggests that acute encephalopathy in the most highly-susceptible children may, in some rare instances, be associated with blood-lead levels somewhat below the 100 pg/dl figure derived from the above-mentioned Baltimore data compilation. Those reports are evaluated in detail in the 1977 EPA docuemt Air Quailty Criteria for Lead. From the preceding, 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 irreversible CNS damage or death at blood-lead levels around 100 pg/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 pg/dl or higher range. This diversity of symptomatic response severity may be due to: (1) individual biological variation in lead uptake or susceptibility to lead effects; (2) changes in blood-lead values from the time of initial damaging intoxication; (3) better tolerance for a gradually accumulating lead burden; (4) other interacting factors, such as nutritional state or inaccurate deter minations of blood lead; or (5) lack of use of blind evaluation procedures on the part of the evaluators. For adults, it appears that the most susceptible individuals do not exhibit encephalopathy symptoms until blood-lead levels of 100 pg/dl are reached or, more typically, substantially exceeded. For children, the majority of cases showing acute encephalopathic symptoms have blood-lead levels of 100 pg/dl or more, although in a very few cases, levels as low as 80 pg/dl have been reported. It should also be noted that a continuous gradation of frequency and severity of neurotoxic symptoms extends into the lower ranges of lead exposure. In addition to neurotoxic effects of relatively low-level lead exposures in adults, there is growing evidence that lead exposures producing blood-lead levels of 40 to 80 pg/dl or less in children induce nonspecific neurological damage, especially in the young, developing CNS. This issue has attracted much attention and generated considerable controversy during the past decade. PB12B/A TEH 0530917 12-54 1/6/83 DUP050031830 PRELIMINARY DRAFT 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 review of methods employed in the various important studies on the subject. There are now several excellent reviews of over forty studies published on this topic. The two most thorough reviews are by Bornschein et al. (1980) and by Rutter (1980). Based on the five criteria listed in section 12.4.2.1 (i.e., adequate markers of exposure to lead, sensitive measures, appropriate subject selection, control of confounding covariates, appropriate statistical analysis), the 17 population studies in Table 12-1 were conducted rigorously enough to permit at least some conclusions to be drawn. Even so, no epidemiological study is completely flawless. Therefore, overall interpretation of such findings must be based on evaluation of both (1) the internal consistency and quality of each study and (2) the consistency of results obtained across independently conducted studies (Hill, 1971). One of the study approaches employed has been retrospective analysis of lead levels existing in populations of apparently asymptomatic children that are then divided into nonexposed control and one or more lead-exposed experi mental groups for comparison of their performance in various neurological and psychometric tests. Only a few studies have employed subsequent follow-up reevaluation of the same children by the same investigators to assess possible persistence of neurotoxic effects. Among studies employing this population sampling approach, those of Albert et al. (1974); de la Burde and Choate (1972, 1975); Landrigan et al. (1975); Needleman et al. (1979); Peri no and Ernhart (1974); Rummo et al., 1979; Thatcher et al. (1982); Winneke et al. (1982a,b); and Yule et al. (1981) suggest significant effects of low-level lead exposure in the absence of classical lead intoxication symptoms. In contrast, the studies of Kotok (1972), Lansdown et al. (1974), McBride et al, (1982), McNeil et al. (1975), Ratcliffe (1977), and Ernhart et al. (1981) report generally negative results. Two other studies (Landrigan et al., 1976; Kotok et al., 1977), although not reporting clearly statistically-significant differences between moderately lead-exposed and control subjects, nevertheless report certain findings highly suggestive of a relationship between cognitive impairment and moderate lead exposures not causing overt lead intoxication symptoms. PB12B/A 12-55 1/6/83 TEH 0530918 DUP050031831 T a b le 1 2 - 1 . SUMMARY OF RESULTS OF HUMAN S TU O IE S ON NEUROBEHAVIORAL EFFECTS AT MODERATE BLO O D -LEAD LEVELS 8 o cm a V> N*--. - >c p at 04 VI OJU) V * OJ vy OIOJP o rM V * ca vt oj p V JVU I *0104 QI la^,i0l 4w> alp v) v) .04 .> Z * ai vi v 3C </> V> V) zzz JSTS 52 V II p 4a k ex. v> o*- o <5 e 1? Po uS *r >> 5*2 -C u tit 5MS Jl /V J w 4^ Vt S2 H -3 CD VJ ra |S <k k ep <9 4S PO ut o CH to >> n j s*j ot oe O- 4- qot3v da. M Ja O*4' 13 35 2 P V .v3t flj * *" o k u .k V p 3-c ZZQ PlA nm <9 U> O +* V> LA ^5 ii n ii a jp o o. II A o <j ipPO P^ CPOfZilO-Pf II II II A A uoy uu to V) q> p oi H- 0*-C " .PMt- P ^ pwe p o wt p -r~w <* v> o S5r Ot ^ U ".vi wii + uu m io p >P f5e.g' '*i uuu t/> v>- Ma.a..u Z*-+ + O </> O <J tn a. v> v) 3 M O* *-* to-* P 3 3 -vt 4^ .>2c*-.> +* at+* u o j<o vm pu M H ,4- O CO Qe a. o A a. -AJ V) & u* + u at ap al at at > o S '**" V) I-- -r- ** W O -H U V> kPp 3 U'H+I PO zo I P* P P o p f-J it*! cn HHH II II II cOu aO. ^a3. com m HHH II II II II U uuu > P o C <9 *> p 3 e wt X v<A- *O- HP Vt > +J O O, Tt 5 q> p k H- HI k O - V) i- O ft. o Vk P < U. O. o ot o a. <9 0 Vt O V P-4 >0 OI -M't oo 4) W 33 0o r1o fi* Svt P II (I <33 cp- i' ll so. 4Cf P .19 P U -J ro ro P 90 II II P I xi x A mm HH ti CO CO O O to p in rt r t n II H II II s <9 p S3 SB e-- r* 3 "O 0.3 O + i?*o c uo kl PCS *Mf" COs HV VI 4- U p CL*1 3 -Q pc: 3s z > <U- &. SC v 5n -/ P r>. 3 H H H M CO H ED s Jl 41 II II rX *X IX IX P CM <Rf P <9 P ro mPmP ii II II x *X `X IX p P 4h pi P vf> in o o 9f fH CM H II II II Vt V>| 9 PI MI. PI Z CL. Za. Z z k o k <*> 4^ k o. P cn .8 eOc o p z p Ho P OS 51 . - <U kP P T3 Cf~ C> *- o I< P St* ^ *G *139 S3 cOl UJ p k* <9 k k *P3 P VI M p -* x: Pu p 3 c g~ CM f>. VP *3 -4 k N- <p.g 19 <0 o S? r*p o> 'Pk.-c 2. 3 r* p S5 5c tn w r ap C f< 10 TEH 0530919 p.*-\ oR 13 3w 12-56 r<*T** u 4-> p DUP050031832 Table 12-1 (C ontinued) SUMMARY OF RESULTS OF HUMAN STUDIES ON NEUROBEHAVIORAL EFFECTS AT MODERATE BLOOD-LEAD LEVELS o Vi noo --S- 0**> .e 4j OS V> <o*- p** O nj vs H l. V) . +< e> 0 V -c: re ' cu Aoj.> u v> z I/) 0 0*~s vs t3 p re --3 r0n p oo > V. V. 4-S IP to o u `0>1 > *2 it s* u vs OVr 0V) v. e - a * 5" % i t0 .O3' <M Ui Cr-5 " v vi e UJ v<iu*o*- O55' wre voi CSJ fO cn c m n o <r cs Cl Ol " H H " S J3 XI P ^ O. 4. O. o vfii m o HO$i.0rN U U II Ij It o oo o o 90 re uvs (A CO P c. A O o : C"V * P f O P w re p *- c s ** re v> 0 .0 883* Pge I > .o SSEmS S- 6) 0 -- 8<a0. oa to vo .3 vs *3 .c>u <a. c v~0 a t33o Ift s A S II .aP. II tj O to VOI-J ro r-4 rs. P-> o <SJ s Cl flO 7" II r* ll ll CM || 5 *5 P CU O CM CO C05IrOH S s r* CM II II tl ti II ll u U o o O 0 'S rue 2 841 e'Uo. 8 Sofae. C^O >4>vo >.5 v> up p < **. _5.* pelo* re 0)0 C P O OV),Pi- O O H-M-4 VS re ps Q<41J. o u41. eS *-a(E rP**<*--"9*,PVO2v 2OP3O'.*O--L--'-f 9 oca -0a 0 Z% O Ol O =L s CM OS 4T-O *Cr* P 0 V) < pOJ 01 P vo o .<*5 II It IX N-/ IsXp VO ioni int vCOo CrOl O C II pmm II Z z c uo p 08 P *53 re cu 0.3 oP Q. V) o u or P 8 vs 0 -C o r> in.ro 5 ift r-4 VO o A trs, CO cmcm 1 II it l XI X CO sN09* to go S3 in in II ini ini to to r-to CM H IS s iCnM CM rC-M* co p CO CM tl II II 11 II If i 22o ZH O0 COl -.J0 ao P0s *5 c wS |3 *-4 0) z t ->* OS uc U 'UJ 8c S S0 <0 o* to VS v4oi >re 3 re Cro*%M P V) <71 C<e >P*I -- vft MC 12-57 TEH 0530920 DUP050031833 Table 12-1 (C ontinued) SUMMARY OF RESULTS OF HUMAN STUDIES OH NEUROBEHAVIORAL EFFECTS AT MODERATE BlOOD-LEAO LEVELS O (Q U vs r-r W .>v*ae-t u Vo *Z o P **- IS" 5 02 St 5 vs vi a. p<u CM O O O fOPO . O .* . > OOWOirt 'a* ' 3C V v V v v v a. ci. a. M3 PO V* CM csj <ri*4<g\*Ct Q <'J'tCri MH CM rH UflJ .<> ii it" ii ! Op tp crs p * tp o to c s un to m cd ** c m pi o o O c m eg rH rH rH rH Or II II II If II II A II puuo o u o o t<O3 Vy a. - a. M> VaI r0H9 .*--ourraHmCM II *"" II It II II O PPU OS rH rH 3 S vi S <-0 ** 3B as vv v y Q.Q. a. o. $ 3 ggggpp a9*.o*.o**.^a*.a.^a*. .8 42 P A A A AAA it rarararamra S'S'<3"<< AAAAA A eo CM CM CM CM CM CM 2 yvva Vv 11 g TO C 1- d i- f* . o 01 44 t2v- 02 US --OrO-*' c Ps u s :> 3-5 8 o. u a vir-- trs *-- JZ 3 02 J5 o ac u a> 02 C *Va>)) H- o0c2 <9 Cp 42 co v* St? ,C 4S2 02 4) >J= 44 $U0t 0 t/) 04 V> VS <9 02 N-4 j_ fro* 00 O 3.3'3v)h vC<>u U is rO4- hIaB> K E S.& isv A * os oo IA 2 vs v>h+jr* V> *r- v> 8Cl54V1 3)V> '> 41 Xi VO _ S*Zfi> ** > + u 5 g.5 ? ZJ4-JC5 S- > tn- 9 e o 4i a^#-uro -ios its MS - 41 s- i4cxJl>cov a c gcq w v> Pr*HHa-#HA 4< vs 44 42 VS H*< <P 44 HU i" er*Pp h02* u 0'S 02 _ U? H t - H O) f t - CD r*r P r" -- U.4J*rO s o* a a.% <o u. *-* vs < as OBr-SfiC* aI o<9 vO cO ci--<P c a s- t- s- C <a M 41 a> V 01 4) 3>&>>X P9 to urs to OOrH^Os V) .&p 02 VS VI to - ra a> P *-p <0 -c u 9 02 g >r *r CM x0u1: *-gpc 00 02 Cmu P *5 - CcHi Sv s01 >or* r- > vws eP 02 44 ut-s O Sr .01 q *0 <9 Co> lv X 03 e Co <J V) 44 QS) Um cru. 44 44 036 ra VUI 02 to g* 4) VS OS 4) "S 3 T5 a0o.w3v ^*5 PO>-022 Co--ms 44 2 Q.< 01 VS Jgs>* =53 02Sp F 42 .a: TEH 0530921 2 11 S3 .3 U J *r^> --O vs O OS rH CM <V CM CM II 11 11 II i- CM C9 ** <0 <3 CO 43 m02 oCs s- 44 <oJ A 9 VS s; 12-58 02 C4 <u 3 DUP050031834 Table 12-1. (C ontinued) SUMMARY OF RESULTS OF HUMAN STUDIES ON NEUROBEHAVIORAL EFFECTS AT MODERATE BLOOO-LEAO LEVELS J9 Pu **- fS O0 U VI *r-- <* '>C P9 CD f>vn <si r-iCMCMCl a a ti a a 43 42 43 43 43 Q. Q. OU A* Om II 43 wCMn<<9-n0 fs T'** HHHHP4 a ii n ii a O u <J U P Jl g * O U .0 'Z o* -W rp a. 6 P JSm >U+LA o-5LA| p ar*> p 0 3U- Mf- s. 2 co o' up +> aPs ; *r* c -a? _ F 2* H gOfl A __ E V tA : ofir-- C - 0 4-> t : co V>Af)i^VCO v> ZZ v S o. 0 U> VO r-4 CM i ? + + c m w 1 1 2 >S W iT> r-4 OOO Oo OoOo 'V VVv Q.O.Q.3 V V5 508500 CO 1-4 OT CO CM CM CM CM W oS CM O s0- o. 4-> u Lrt - "I o0. g i. M . 03 Cj V> P 4- P U.UIZ 00 -4 CP F-- 39 O'2,0 ' -- 8 Sh V> (A p e -Cl.iBaJ5.f-C 0 O T? LA .0 0 O'43 V S c p c g 0 >-4Pt.J-.pCw0 --CV) o & e5v> o Xf c .0 0u C3 C3 P t- 0 P L/> S 0 33 g .<**- up 3 O' C '0 Cl - g W CDJJV O.P p ^'.1 v> t c. < u a_* 0) oa Q9 42 43 O.Q- a* 0.3. 3 0- C l O <-4 Si!LA C A3 g0 0.0. c4o9 trc>>pi.. Zb. H O.S 0* in cr OWe *-pp3l 4J * ra p 3 T3 CL 9 O 4- 0 t- pbp> 0 MS -M (A .a-- * P3 40-> 0 0ac C0S0J p cn c ~i s 0 u- t0 C.> U3 0 42 0 3 a 2? 00 +a>t p Jia sa C s/ s 0 J3 U0 P CM 43 CD IQV 12-59 TEH 0530922 DUP050031835 g 01iHt/i*`4f**' *' >C <v p> in in V V)W)W> V v> o-a: ac a: q.a= + I; IX^ it s* 2 4) s X % S'rH 2TJ 2vO | troeOg cHo * ov .eg 5 a. n I! II II if i u 'X 3" ** Ow u Va. i/> ^0.^ 0. ^ Is 2 *** c p-4 <Q Q <T g O2l v&*Oo no o n . O. N fO ifKL H ii A II |t II A II o uuuuuu ul > aa 21 O) --.* a. io<si *oir3 a oa. o v> AO > 0V>I x41 X oo > *<- sm QO.1I3 *>* isc u o <J <u rs* X 4t-1 Ufo <LI r 4.-U> >v X41 4<10 5 3U 9 *- UP -- 4,-V>> r<~0 >f a. Uh - x41 03 * u _ <u eg V) o w> eg :S^ x y> >o wP o> fQ <-- d< 5 +> SAP^Si; 0 *Ji Or J3 4l$OCLiC^ (OH C 010.(3 IQ +U roe- 5* c 41 O cOl O) 6 01 r 91 4 3 O. u-o. t-- co m oc sin vin v> & ' ** o <Q V 5 d '=9 o> 3. o o> o a. cr t m o s* .> od d t> C OH VPi 2 t- v> O +* H- U 01 22 > IA UVI T3 a 0v1 <0 ns41 IX I 41 " * S +a5i Si wo n IQ *'V u t - "13 x jds w VI IQ I 41 >O iji 32 W 2* 1 .<a0 J- . .o O i.(* & v> iIQ So u 3 O t- e x 2 2 s 23- 0 mU w d II 12 tA 2- IQ <-- c2 21 O O vi *r~ U o. .*- .3 *0 O e >>41 V ."I,i-4"1i 41 O 41 V) o IQ Xoi 01 .OO3e*w*>^4y1O- l3i4oni IQ f-- -41 4S-1- *0 IQ VO c*4If--Oax * 8 f SIXO? IQ" .p ,, -o n . om v 15 9S W (Aa I** H eo e n-0 01 d <n* o o 2,,S_*V^V 9.4r- &v 2* `5 r-- J^IB^ w V* ^ M ^ W fl|r" ' p 91 (A i. Odd UiA^UQQHaiO * <0 * Q Q <Q 0! <0 t* % - * >* i! <*> ,ooi - oi cun 41 >> 1 *o cn U " W 0135 d >U <*- I9<-VU) 41 &> 01 - a. e i*> 49^) u t.<*<*5 oao> *vo v.i ei_-- 9>'Ql Q J3V- IQ 01 fc. 4) +j > C e U It u 9 2BS'lc0=5+.2 3 i- m cxn i- c e o -- t. r- U. fQ 4) 01 V C 41 O V ,W W Vt c -g QIC H 3 I <*- o au O0 0.r- Q.Q91-n- b--Q^*xZ3--*j Uoww3(>-xV-0>---1I-Q-i----0>w"o"1 eovcO*ifQo^^0--1 O O.C 034-UU3 4/0 H3o 5a 9in1 U*No.iHO* i. &. 4*aa<-->.OeU*nnldi j4W0=>CC>/.2^. iVft><V0H*1-HvV0i-! ViolH- *O- u01 4Sfi1V^4C1t4* 4^1*' <Q w / S3 iq o > v- n ii w o *o *c. n ro n 3 c -p a. iq _ 0Q1 X01 .f01l W9W) f--UU|/vV>ni. U9wwi)(w9uI)>Jr"r-0e*MQ O* 4-IfQ-- r4 jEas ao<33Xazao. nr^ n <j is o j V no *- TEH 0530923 12-60 DUP050031836 PRELIMINARY DRAFT Among the several pediatric studies presenting evidence for CMS deficits being associated with blood-lead levels of less than 80 pg/dl, all are either retrospective or cross-sectional studies except the work of de la Burde and Choate (1972, 1975) and Perino and Ernhart (1974). De la Burde and Choate (1972) observed dysfunctions of the CNS, fine motor dysfunction, impaired concept formation, and altered behavioral profiles in 70 preschool children exhibiting pica and elevated blood-lead levels (in all cases above 30 pg/dl; mean ~ 59 pg/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, 1982). The child development study at the Medical College of Virginia in Richmond, which participated in this collaborative study, had a total popula tion of 3,400 mothers. De la Burde's population was drawn from this group in which all mothers were followed throughout pregnancy and delivery and all children were postnatally evaluated by regular pediatric neurologic examina tions, psychological testing, and medical interviews, A careful re-reading of her reports indicates that all children subject to prenatal, perinatal, and early postnatal insults were excluded from her group. 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 her study. It is unfortunate that blood-lead data were not regularly obtained; however, at the time of the study in the 1960s, 10 to 20 ml of venous blood was required for a blood lead determination. 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 confirmation of continuing CNS impairment as assessed by a variety of psychological and neurological tests. This was despite the fact that many of the blood-lead levels of the lead-exposed children had 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 find ings. For example, there were appreciable numbers of children (67 leadexposed and 70 controls) whose blood lead values were obtained in preschool PB12B/A 12-61 1/4/83 TEH 0530924 DUP050031837 PRELIMINARY DRAFT years and who were old enough (7 years) during the follow-up study to cooperate adequately for reliable psychological testing. The specific psychometric tests employed were well standardized and accepted as sensitive indicators of minimal brain damage, and the neurobehavioral evaluations were carried out in a blind fashion (i.e., without the evaluators knowing which were control or lead-exposed subjects). The de la Burde and Choate (1972, 1975) studies might be criticized on several points, but none of them provide sufficient grounds for rejecting the 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 very improbable that appreciable numbers of lead-exposed subjects might have been wrongly assigned to the control group, thereby causing an underestimation of the effect of lead. Subjects in the control group did, in fact, have a history of pica, but not for paint. Also, results indicating no measurable copro porphyrins in the urine of control subjects 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 probable inappropriate use of multiple chi-square statistical analyses. One last problem concerns ambiguities in subject selection which complicate interpretation 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 pg/dl with "positive radiographic findings of lead Tines in the long bones, metallic deposits in the intestines, or both," observed deficits might be attributed to blood-lead levels as low as 30 pg/dl. Other evidence (Betts et al., 1973), however, suggests that such a simple interpretation is not likely accurate. That is, the Betts et al. (1973) study indicates that lead lines are usually seen only if blood levels exceed 60 pg/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 pg/dl. Virtually none have lead lines at levels below 40 pg/dl. In view of this, the de la Burde and Choate results can probably be most reasonably interpreted as showing lasting neurobehavioral deficits at blood-lead levels in excess of 40 to 60 pg/dl. PB12B/A TEH 0530925 12-62 1/6/83 DUP050031838 PRELIMINARY DRAFT Similar conclusions are also suggested by results of the Perino and Ernhart (1974) study, which demonstrated a relationship between neurobehavioral deficits and blood-lead levels ranging from 40 to 70 pg/dl in a group of 80 inner-city preschool black children. A key result is that the normal correla tion of 0.52 between parents1 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. Many methodological strengths were present in the Perino and Ernhart (1974) work, including sound blood-lead determinations and statistical analyses. One of the few possible alternative explanations 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 negatively related to blood-lead levels of the children participating in the Perino and Ernhart (1974) study. Parents of children in the lead-exposed group had significantly poorer educational back grounds than control group parents. The importance of this point lies in the fact that several other studies (Elardo et al., 1975; Ivanans, 1975; McCall et al., 1972) have demonstrated that higher parental education levels are asso ciated with more rapid development and higher intelligence quotients (IQs) for their children. Perino and Ernhart*s finding (1974) that the correlation between parental IQ and child IQ is suppressed in groups of lead-exposed children is interest ing and suggestive. However, this does not preclude the fact that differences in rearing environment are largely responsible for differences in child IQ. For example, heritability studies find that correlations of individual differ ences for measures of intellectual accomplishment between child and biological parent are higher than between child and adoptive rearing parent, a result that presumably favors a genetic interpretation (reference needed). However, the average performance of the children is closer to that of the rearing than their biological parents, a result that emphasizes rearing environment. The rearing environment appears to have an effect on developmental function but not on the stability of individual differences. P812B/A 12-63 1/6/83 TEH 0530926 DUP050031839 PRELIMINARY DRAFT Two other studies with positive findings had serious methodological limitations. Albert et al. (1974) reported that asymptomatic children (5 to 15 years old) whose blood-lead levels at an earlier age were elevated (> 60 pg/dl) later had significantly more mental disorders and poorer school per formance than a control group with lower lead levels in both blood and decidu ous teeth. However, no assay of the lead burden, in either blood or teeth, was done for about one-half of the children in the control group; and no significant effects were reported for children with blood levels < 60 pg/dl. Also, another major criticism is that some children in the control group had relatively high blood-lead levels (> 40 pg/dl). In another study, Landrigan et al. (1975) found that asymptomatic, lead-exposed children living near an El Paso smelter scored significantly lower than matched controls on measures of performance 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 statistically significant after appropriate adjustments were made for age differences, McNeil et al. (1975) found that another sample of children living near the same lead smelter in El Paso was generally comparable medically and psy chologically to matched controls living elsewhere in the same city other than in terms of direct effects of lead (blood-lead level, free erythrocyte proto porphyrin levels, and X-ray findings). Lead-exposed children in the group living near the smelter, however, had significantly different personality test results, which were ascribed by the authors to recent upheaval in the lives of the lead-exposed children who had been recently forced to move from near the smelter. Much community unrest did exist at the time of both the McNeil et al, study (1975) and the work of Landrigan et al. (1975) on the El Paso smelter area population. The uncertainties in selecting control groups and the impact of the extraneous unrest on both studies clouds interpretation of their respec tive results which, in turn, have become quite controversial. An extensive critique of these studies is available in the Muir Report (Appendix E, U.S. Environmental Protection Agency, 1977). Other studies of children living in the vicinity of smelters or factories, have typically not found significant neurobehavioral effects at moderate elevations of blood-lead levels. For example, Lansdown et al. (1974) found a relationship between blood-lead level in children and the distance they lived PB12B/A TEH 0530927 12-54 1/4/83 DUP050031840 PRELIMINARY DRAFT from lead-processing facilities, but no relationship between blood-lead level and mental functioning was found. However, only a minority of the lead-exposed cohort had blood-lead levels over 40 pg/dl. Furthermore, this study failed to Consider adequately social factors such as socio-economic status. Similarly a study by Radcliffe (1977) of children living near a battery factory in Man chester, England, found no relation between their blood levels taken at two years of age (28 pg/dl vs. 44 gg/dl) 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.e., 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; potentially higher blood lead levels occurring after age two among control children may have lessened exposure differences between the low- and high-lead groups. In contrast, Rummp et al, (1979) found significant neurobehavioral deficits (hyperactivity, lower scores on McCarthy scales of cognitive function, etc.) among 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. Children with moderate blood-lead elevation but not manifesting enceph alopathy symptoms, however, were not statistically significantly different from controls on any measure of cognitive functioning, psychomotor perform ance, 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 plotted graphically, a rather interesting relationship between test performance and level/duration of lead exposure becomes apparent. That is, although the scores for short-term moderate-exposure subjects are essentially the same as control values, the scores for long-term moderateexposure subjects consistently fall below those for control subjects and lie between the latter and the encephalopathy group scores. Thus, it appears that long-term moderate lead exposure may, in fact, exert nonspecific neurobehavioral effects. This might be shown to be statistically significant if other types of analyses were used or if larger samples were assessed. However, control for confounding variables in the different exposure groups would have to be PB12B/A 12-65 1/4/83 TEH 0530928 DUP050031841 PRELIMINARY DRAFT considered. Note that (1) the maximum blood-lead levels for the short-term and long-term exposure subjects were all > 40 pg/dl (means = 61 + 7 and 68 13 pg/dl, respectively) whereas control subjects all had blood-lead levels below 40 pg/dl (mean =23+8 pg/dl), and (2) the control and lead-exposed subjects were inner-city (Providence, Rhode Island) 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 bloodlead levels < 40 pg/dl were compared with 31 children having distinctly elevated blood-lead levels (61 to 200 pg/dl) but no classical lead intoxication symptoms. Both groups were well matched on important background factors, notably includ ing their propensity to exhibit pica. Again, no clearly statistically signifi cant differences between the two groups were found on numerous tests of cogni tive and sensory functions. However, the mean scores of the control-group children were consistently higher than those of the lead-exposed group for all six of the ability classes listed. Also, in one case the level of signifi cance achieved borderline significance, a pattern of results hinting at a trend toward lower ability levels for the lead-exposed group. The authors cautiously stated that "the data do not prove that these children have sus tained no neurologic damage by lead" and that "later longitudinal testing may demonstrate cognitive or educational deficiencies." Other inner-city studies have produced mixed or negative results in attempts to determine whether a relationship exists between lead exposure and CNS deficits using various standardized psychometric techniques, neurologic examinations, and ratings by teachers, parents, or experimenters. For example, Kotok (1972) reported earlier that developmental deficiencies (using the Denver Development Screening test, which is a somewhat insensitive measure of development) in a group of asymptomatic children having elevated lead levels (58 to 137 pg/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 pg/dl). The deficiencies could be correlated with inadequacies in the children's environment. Children in the lead-exposed group, however, had blood-lead levels as high as 137 pg/dl, whereas some control children had blood-lead levels as high as 55 pg/dl. PB12B/A TEH 0530929 12-66 1/4/83 DUP050031842 PRELIMINARY DRAFT Thus, the study was essentially a comparison of two groups with different degrees of elevation in lead exposure rather than one of lead-exposed vs. nonexposed control 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 Oraw-A-Child test, and the Conners Teacher's Rating Scale for hyperactivity. The children's blood-lead levels correlated 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 from 46.5 to 32.4 pg/dl. When control variables of sex and parent IQ were extracted by multivariate analyses, the magnitude of observed differences was greatly reduced but remained statistically significant for three of seven tests on the McCarthy scales. This led Ernhart et al. (1981) to discount their 1974 (Perino and Ernhart) results in which they had not controlled for parental education. The Conners Teachers' Questionnaire, one of the most widely accepted measures of attention deficit disorder did not differentiate the lead groups. Nor were they able to replicate the significant differences in correlations between parental IQ and child IQ between moderate and low lead groups which they had found in 1974. On the whole, the Ernhart et al. (1981) study was well-conducted. The use of three correlated exposure variables, i.e., FEP, blood-lead, and toothlead, a multivariate approach, control for covariates, careful subject selec tion, and long-term follow-up lends weight to their results. However, if the markedly poorer scores for a single high-lead group extreme outlier are deleted from the analyses, then the differences between the high- and low-lead groups are no longer statistically significant at p <.05 and the percent of the common variance accounted for by lead drops from 7.7 percent to 4.4 percent (personal communication from Ernhart). Based on the above results, Ernhart et al. (1981) concluded that the neuropsychologic effects observed by them are not likely due to lead, or, if due to lead in the exposure range studied, they are minimal. Alternatively, since the source and extent of exposure both before 1974 and between 1974 and 1979 cannot be accounted for, it may be that the original Perino and Ernhart (1974) results are indicative of an actual lead effect existing earlier in childhood and that subsequent reductions in PB12B/A 12-67 1/4/83 TEH 0530930 DUP050031843 PRELIMINARY DRAFT lead exposure resulted in later decreases In deleterious effects on IQ and related school behaviors, as measured in the Ernhart et al. (1981) follow-up study. If so, then this might provide confirmatory support for a small effect on IQ of relatively low-level lead exposures (originally 40 to 70 pg/dl blood, later dropping to )( = 32.4 pg/dl) early in childhood. The apparent reduction over time in neuropsychologic effects attributable to lead and their small or negligible magnitude by early school years for the Ernhart study children, however, suggest that such effects of low-level lead exposure diminish with time and, possibly, may disappear if lead exposure is discontinued. Probably the most comprehensive of the population studies to date is that of Needleman et al. (1979), who used shed deciduous teeth as an index of lead exposure. Mean blood-lead levels for the high tooth-lead group were reported to be 35 pg/dl when assayed for 15 percent of the high-lead group children four years before neurobehavioral testing. Teeth were donated from 70 percent of a total population of 3329 first and second grade children from two towns near Boston. After excluding various subjects for control reasons, two groups (<10th and >90th percentiles) remained, 58 high lead children (14.03 ppm dentine lead) and 100 low lead (8.5 ppm dentine lead) children. Almost all children who donated teeth (2146) were rated by their teachers on an elevenitem classroom behavior scale devised by the authors to assess attention deficit disorders. No differences between included and excluded children's lead or rating scale results were found. A preliminary analysis on 39 non lead variables showed significant differences between lead groups for maternal IQ and education, maternal age at time of birth, paternal SES, and family size. They were entered as covariates into an analysis of covariance with lead as the main effect. Significant effects were found on the fun-scale WISC-R, on 9 of 11 items of the classroom behavior scale, and on several experimental measures of perceptual-motor behavior. Particularly interesting was an apparent dose-response function shown on the classroom behavior scale, although this was not tested statistically. In a later paper, Needleman and Bellinger (1982) showed that controlling for pica did not affect these class room rating scale results. Parent ratings, though collected, were not reported apparently because they were regarded as less trustworthy than teacher ratings. The latter finding differs from many other studies which failed to find effects PB12B/A TEH 0530931 12-68 1/4/83 DUP050031844 PRELIMINARY DRAFT of lead on parent or teacher behavior ratings (Baloh et al., 1975; Ernhart et al., 1981; Landrigan, 1975; Lansdown et al., 1974; McNeil et al., 1975; Milar at al,, 1981; Rummo et al., 1979; Winneke et al., 1982a,b). Since the classroom behavior questionnaire used by Needleman et al, (1979) was not an age-normed, validated scale. Yule et al. (1982) performed a concurrent validation study of the Needleman scale with the Conners Teachers' Questionnaire and the Teacher Rating Scale by Rutter and Yule. They concluded that the Needleman Scale is more sensitive to lead body burden because it taps behavior related to focusing of attention rather than the more multi factorial scales that have been validated for assessing attention deficit disorders with or without hyperactivity in other childhood disorders. The same subjects were used in both the Yule et al, (1981 and 1982) studies to be examined below. This will be important research for the future in relating lead deficits to currently highly controversial concepts of attention deficit, hyperactivity, and minimal brain dysfunction (Rutter, 1982). While many questions raised by Needleman et al. (1979) remain to be answered (e.g., the relation and timing of exposure, the relation of blood lead and tooth lead to dose-response over a broad range of values, and the impact of social factors in different populations), overall this study pro vides the strongest evidence, to date, indicative of likely associations between neurotoxicity and low-level lead exposures in children. Unfortu nately, the study results do not allow for clear-cut estimates to be made of specific blood-lead levels associated with the reported effects, since bloodlead samples had been taken 4 to 5 years prior to the study. Four other studies conducted since 1979 tend to support, though not fully replicate the Needleman findings. Yule et al. (1981, 1982) 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 of lead in the population. The blood-lead concentrations ranged from 7 to 32 pg/dl, and the children were assigned to four quartiles according to these values: 7 to 10 pg/dl; 11 to 12 pg/dl; 13 to 16 pg/dl; and 17 to 32 pg/dl. The tests of attainment and intelligence were similar to those used in the Landsdown et al. (1974) and Needleman et al, (1979) studies. There were significant associations between blood-lead levels and attainment PB12B/A 12-69 1/4/83 TEH 0530932 DUP050031845 PRELIMINARY DRAFT scores on tests of reading, spelling, and intelligence, but not on mathematics (Yule et al., 1981), These differences in performance largely remained after age, sex, and social class were taken into account. The paper by Yule et al. (1982) 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 the ques tionnaire items showed a significant dose-response function ("Day Dreamer," "Does not Follow Sequence of Direction," "Low Overall Functioning"). Nine of 11 items were highly correlated 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 behavior (Yule et al., 1982). The hyperactivity factors on the Conners and Rutter scales were related to blood-lead levels (7 to 12 vs^ 13 to 32 pg/dl). 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 (possibly higher) lead exposures may not be fully reflected and the reported blood-lead levels cannot be accepted confidently as those with which any of the demonstrated effects are associated. Also, parental IQ and educa tion and home environment were not evaluated. 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) also provide partial replication of the Needleman et al. (1979) findings. 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 pg/m . Two extreme groups were formed, with 2.4 pg/g tooth-lead (n = 26) and 7 pg/g tooth-lead (n = 16), and matched for age, sex, and father's occupational status. The two groups did not differ significantly on confounding covariates, except that the high-lead group showed more peri natal risk factors. Parental IQ and quality of the home environment were not among the 52 covariables examined. They found a nearly significant decrease (p <0.10) of 5 to 7 IQ points, and a significant decrease in perceptual-motor integration (p <0.05), but not hyperactivity as measured by the Conners Teachers* PB12B/A TEH 0530933 12-70 1/4/83 DUP050031846 PRELIMINARY ORAFT 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. The use of the Conners Scale during psychometric testing only renders these negative results suspect. Even though the size of the groups was small, it was a generally well-conducted but only suggestive study. The same can be said for their extended study which involved 115 children aged 9.4 years living in the lead smelter town of Stolberg (Winneke et al., 1982b). Tooth-lead (x = 6.16 ppm, range - 2.0 to 38.5 ppm) and blood-lead levels (x " 14.3 pg/dl; range - 6.8 to 33.8 pug/dl) correlated significantly (r 0.47; p <0.001). Using stepwise multiple regression analysis, they found significant (p <. 05) or near-significant (p <0.10) associations between toothlead levels and measures of perceptual-motor integration, reaction perform ances, and four behavioral rating dimensions, including distractibility. This was true even after taking into account age, sex, duration of labor at birth, and socio-hereditary 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 sodio-hereditary background were eliminated. The Winneke results, then, only partially replicate the Needleman et al. (1979) results in that significant attentional deficits attributable to lead were found, but verbal IQ deficits were not found. It is possible that they failed to replicate the Needleman et al. (1979) findings more closely because of differences in social backgrounds or social stratification of the Boston and Stolberg populations or because of actual differences in tooth-lead and blood-lead levels. Average blood-lead levels for a 15 percent sample of the lead-exposed group in the Boston study 4 to 5 years prior to testing was 35 pg/dl and could have been even higher 4 years later, whereas mean blood-lead levels in the Stolberg study were only 14 pg/dl. Thus, differences between the two studies may reflect a dose effect, due to differences in lead exposure levels present in the two study populations. It is also possible (or even likely), however, that the blood-lead values were higher for the Stolberg i subjects earlier in childhood. Overall, then, the Winneke et al. (1982b) study provides suggestive evidence for small, but significant, neuropsycho logical (especially attentional) deficits being attributable to lead in some what older school-age children who likely experienced relatively low-level PB12B/A 12-71 1/4/83 TEH 0530934 DUP050031847 PRELIMINARY DRAFT lead exposures earlier in childhood. It is impossible, however, to state with any confidence that effective blood-lead levels of < 30 pg/dl were associated with the observed effects. The results of a recent hair-lead study of 149 rural school children aged 5 to 16 years from four eastern shore counties in Maryland have been reported by Thatcher et al. (1982a,b). Subjects were recruited by newspaper advertise ment and with the help of the local county boards of education. Subjects were grouped into four groups according to scores on the short form of the WI5C-R and Wide Range Achievement Test (WRAT) from gifted (IQ > 130) to very low achievers (IQ < 84). They also received a test of balance and dexterity (Motor Impairment Test) and the Purdue Pegboard Test to assess fine motor movements. All tests were age-normed and standardized, A minimum of 0.25 gm hair sample was taken from the nape of the neck as close to the scalp as possible and was analyzed in U.S. F.D.A, Laboratories, using induction-coupled argon plasma (ICAP) spectroscopy. Analysis of variance significantly dis criminated between IQ groups, and hierarchial multiple regression analysis was reported to yield highly significant lead effects after taking into account age, sex, race, and SE$. Lead accounted for 16.54 percent of the variance in the full scale IQ, which held constant over the full range of IQ scores. Unfortunately, this study did not control adequately for birth history, several important potentially confounding social factors (e.g., parental IQ and educa tion), or sampling bias. Questions have also been raised regarding whether degrees of freedom for statistical analyses were correctly determined, render ing suspect the reported statistical significance of the Thatcher et al, (1982a,b) findings. In addition to the above problems, lead exposure histories are inadequately assessed and essentially no data are provided by which to relate lead exposure levels (indexed by hair-lead) to blood-lead levels in the subjects studied. Overall, then, in view of the numerous shortcomings noted above and the lack of any basis for judging blood-lead levels present in the study population, the Thatcher et al. (1982a,b) results cannot be accepted as providing even suggestive evidence indicative of neuropsychologic deficits being associated with low blood-lead levels (< 30 to 40 pg/dl). Nevertheless, the results are consistent with other recent studies. 4 PB12B/A TEH 0530935 12-72 1/6/83 DUP050031848 PRELIMINARY DRAFT The final study of at-risk urban children to be reviewed is a study of 454 preschoolers, aged four to five years, from Sydney, Australia with bloodlead levels ranging from 2 to 29 pg/dl. Children born at the Women's Hospital in Sydney were recruited via personal letter, 57 percent of whom consented to participate, 3 percent of whom declined, 3 percent of whom had moved and 37 percent of whom could not be contacted. No blood-leads measures were avail able on non-participants. A comparison of socio-economic 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 entire Australian work force. There apparently was some self-selection bias due to a high proportion of professionals living near the hospital. Other demographic variables such as mother's IQ, pica, and caregiving environment were not evaluated. Evaluations were apparently performed blind since blood levels were evaluated after neurobehavioral testing. Exposure history appar ently was not assessed. Using a multiple statistical comparison procedure and Bonferroni correc tion to protect against study-wise error, they found no statistically signifi cant differences between two groups with blood-lead levels more than one standard deviation above and below the mean (> 19 pg/`dl vs^ < 9 pg/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 (track ing), the five-year old boys in the higher lead group performed worse than the boys in the lower lead group. In one test of gross motor skill (walking balance), the results for the two age groups were conflicting. This study suffers from many of the methodological weaknesses of the Thatcher et al. (1982a,b) and Yule et al. (1981, 1982) studies, so that the results cannot be regarded as conclusive evidence for or against an effect of low-level lead exposure at blood-lead levels < 30 pg/dl. Conflicting results are likely due to the many procedural differences among these studies. Lead studies on special risk populations have shown such populations to have consistently higher lead levels than the general population. Whether disorders such as mental retardation, hyperactivity, autism, etc. are the causes or effects of lead exposure is difficult to prove or generalize to PB12B/A 12-73 1/4/83 TEH 0530930 DUP050031849 PRELIMINARY DRAFT other risk populations as a result of sampling bias in these groups. Never theless, they are important groups to study in their own right and may offer indirect corroborative evidence as to the hazards of lead by using a different strategy from that used in population studies (David et al., 1979). The approach consists of identifying populations of children with diagnosed neurobehavioral deficits of unknown etiology and assaying blood lead or making other assessments in order to evaluate possible links between past lead expo sures and the children's present neurobehavioral impairments. Thus, for example, efforts have been made to implicate moderate- or low-level lead expo sures as a causative factor in at least some cases of hyperactivity of unknown etiology. The possibility that such low-level lead exposures induce hyper activity gained some credence through the well documented (Byers and Lord, 1943; Cohen and Ahrens, 1959) fact that hyperactivity is one of the frequent neurobehavioral sequelae observed in children who survive episodes of acute encephalopathy resulting from high-level lead exposures. The evidence for and against the hypothesis that lower-level lead exposures produce hyperactivity has been accumulating at a rapid rate and has generated considerable contro versy. Only a few of the more salient findings are reviewed below and under the section on animal studies (section 12.4.3). In a case-control study, David et al. (1972) compared the incidence of elevated blood-lead levels in five groups of children: (1) a pure hyperactive group with no apparent cause for hyperactivity; (2) a group of hyperactive children with a highly probable cause of hyperactivity, e.g., prematurity; (3) a group of hyperactive children with a possible cause; (4) a group of children who had recovered from lead poisoning; and (5) a nonhyperactive control group. Pure hyperactive children had statistically significantly higher blood-lead levels (mean = 26.2 8 pg/dl) than controls (mean ~ 22.2 9.6 pg/dl), where as children with a highly probable cause did not (mean = 22.9 6.6 pg/dl). Similarly, the pure hyperactive children tended to excrete more lead than controls or probable-cause hyperactives when given a single dose of penicil lamine. Although the causal relationship between lead exposure and hyperactivity cannot be said to be proved by this study, the data of David et al. (1972) might be interpreted as supporting the hypothesis that a relationship between moderate lead exposure and altered motor activity exists. On the other hand, PB12B/A TEH 0530937 12-74 1/4/83 DUP050031850 PRELIMINARY DRAFT several other points argue against acceptance of such a thesis at this time. The David et al. (1972) study itself and the authors' conclusions can be questioned on several bases. For example, the closeness of the match of subjects in the five groups on variables other than age and sex is not clearly specified by the authors. Also, the interpretation of differences in bloodlead levels in the 7-to-8-year-o1d children is fraught with numerous problems, not the least of which is the fact that such levels are probably not very accurate indices of long-past lead exposures that presumably occurred during preschool years. Many factors in the interim between presumed lead exposure and assay for lead could affect the results, including possible differen tially higher incidences of pica in the hyperactive children than control subjects. Klein et al. (1974) have noted that pica may be part of certain behavioral syndromes that exist even in the absence of lead exposure, but that would predispose the affected child toward more lead ingestion by virtue of the habit's presence. Indeed, there is evidence that, among mentally sub normal children whose mental deficiency can be definitely attributed to etiol ogies other than lead poisoning, there is both a high incidence of pica and moderately elevated blood lead (Bicknell et al., 1968), Lastly, it should be noted that a number of other investigators (Baloh et al., 1975; Landrigan et al., 1975; Lansdown et al., 1974; McNeil et al., 1975; Milar et al., 1981; Rummo et al,, 1979), who expressly looked for evidence of lead-induced hyper activity as part of their screening for neurobehavioral deficits associated with blood-lead levels as low as 30 pg/dl, failed to find any significant effects that support the thesis that low-level lead exposures induce hyper activity. Thus, even though the hypothesis is intriguing, insufficient evi dence exists to establish hyperactivity as a neurobehavioral deficit clearly associated with low or moderate lead exposures. A recent study by David et al. (1982) attempted to link low-level lead exposure and mental retardation by invoking the concept of biological variabil ity. They reasoned that if an etiological association between lower lead levels and CNS dysfunctions exists, then, in a population of retarded people, CNS dysfunctions less severe than pre-encephalopathy should occur in fewer people relative to the total number having lower lead levels. Further, a direct correlation between the severity of illness and the quantity of lead present should exist. PB12B/A 12-75 1/4/83 TEH 0530938 DUP050031851 PRELIMINARY DRAFT The authors searched the records of two hospital clinic populations, one a Developmental Evaluation Clinic for mentally retarded children (35 of whom had unknown etiology and 48 of whom had probable organic etiology) and one a general pediatric outpatient clinic (40 non-retarded children). Children varied in age from 4 to 12 years and from 55 to 84 in IQ (WISC, Stanford-Binet, Merrill-Palmer). The pediatric outpatient children were not tested, but were alleged to be average academic achievers by their teachers and guidance coun selors. It was found that there was a significantly higher blood level in the unknown-etiology group (25.02 pg/dl) than in the probable-etiology group (18.83 pg/dl) and the control sample (18.88 pg/dl). There was a significant negative correlation between IQ and lead levels (r = -.28, p <.05) in the unknown-etiology group, but not the other groups. In addition to all of the criticims of the David et al. (1972) study, this study also did not consider any of the important social factors related to lead exposure, viz. exposure history, selection bias, and possible bias in the information gleaned from the clinical records. Therefore, this study does not conclusively support or refute a connection between lead and mental retard ation. In addition to the above data on possible links between lower-level lead exposures and the induction of hyperactivity, certain data (Pihi and Parkes, 1977) provide evidence implicating increased heavy metal absorption, including lead uptake, in the etiology of learning disabilities. More specifically, children identified for other classification purposes as having learning disabilities were found to have significantly elevated levels of lead, as well as cadmium and some other metals, in their hair when compared with control children not classed as learning-disabled. In fact, a discriminant function analysis yielded 98 percent accuracy in classifying children as normal or learning-disabled based on a combined factor of cadmium, cobalt, manganese, chromium, and lithium levels. Lead was not included in this five-metal dis criminant function, since its predictive value was well served by cobalt and cadmium because of a significant negative correlation between lead and cobalt (r = -0.57; < 0.0.1) and a significant positive correlation between lead and cadmium (r = +.53; < 0.01). Unfortunately for present purposes, no bloodlead levels or possible past exposure histories were provided for the children in the above study (Pihl and Parkes, 1977). PB12B/A TEH 0530939 4 12-76 1/4/83 DUP050031852 PRELIMINARY DRAFT Other studies analogous in basic approach to that employed by David et al. (1972) and Pi hi and Parkes (1977) have provided intriguing information tending to link prenatal lead exposures to the later development of mental retardation. For example, Beattie et al. (1975) identified 77 retarded children and 77 normal children matched on age, sex, and geography. The residence during the gestation of the subject was identified, and a first-flush morning sample of tap water was obtained from the residence. Of 64 matched pairs, no normal children were found to come from homes served with water containing high lead levels (> 800 pg/liter), whereas 11 of the 64 retarded children came from homes served with water containing high lead levels. The authors con cluded that pregnancy in a home with high lead in the water supply increases by a factor of 1.7 the risk of bearing a retarded child. In a follow-up to the Beattie et al, study, Moore et al. (1977) obtained lead values from blood samples drawn during the second week of life and stored on filter paper. These samples had been obtained as part of a routine phenyl ketonuria screening study and were kept on file. Blood samples were available for 41 of the retarded and 36 of the normal children in the original study by Beattie et al. (1975), Blood-lead concentrations in the retarded children were significantly higher than values measured in normal children. Mean blood-lead for retardates was 1.23 + 0.43 pMol/liter (25,5 + 8,9 ug/dl) and was 1.0 0.38 pMol/liter (20.9 7.9 ug/dl) for normals. The difference in lead concentrations was significant ( = 0.0189) by the Mann-Whitney test. These two studies suggest that lead exposure of the fetus during the critical period of brain development may cause perturbations in brain organi zation that are expressed later in mental retardation syndromes and they raise for careful scrutiny the risks of intrauterine exposure to lead. Insufficient information exists, however, to allow estimation of the levels of lead expo sure of pregnant women that might cause those prenatal effects in the fetus that may result in later neurobehavioral impairments. There are several reports of previous overexposure to heavy metals in amyotrophic lateral sclerosis (ALS) patients. Conradi et al. (1976, 1978, 1980) found elevated cerebrospinal fluid lead levels in ALS patients as com pared to controls. The possible pathogenic significance of lead in ALS needs to be considered. PB12B/A 12-77 1/4/83 TEH 0530940 DUP050031853 PRELIMINARY DRAFT Another adverse effect of lead on neural function in children remains to be considered, and that is the possible induction of peripheral neuropathies by low- to moderate-lead exposures. It is generally accepted that lead-induced peripheral neuropathies, although frequently 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 electro myography, assessment of nerve conduction velocity, and observation of other overt neurological signs, such as tremor, wrist and foot drop. Frank neuro pathic effects have been observed at blood-lead levels of 60 to 80 pg/dl (Erenberg et al., 1974). In other cases peripheral neuropathy was associated with blood-lead values of 30 pg/dl. In the latter cases, lead lines in long bones suggest probable past exposures leading to prior blood lead levels at least as high as 40 to 60 pg/dl and probably in excess of 60 pg/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 were reported after treatment for lead poisoning. A tentative association has also been hypothesized be tween the existence of sickle cell disease and increased risk of peripheral neuropathy as a consequence of childhood lead exposure. Most of the cases reported (7 out of 15) involved inner-city black children, several with sickle cell trait (Imbus et al., 1978). In summary, (1) evidence exists for frank peripheral neuropathy in children; (2) such neuropathy can be associated rather well with blood-lead levels at least as low as 60 pg/dl. Further evidence for lead-induced peripheral neuropathies in children is provided in the data 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 study are presented in Figure 12-2 in the form of a scatter diagram relating peroneal nerve conduction velocities to blood-lead levels. No clearly pathologic conduction velocities were observed, although a statis tically 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 in creased blood-lead levels), but do not allow clear statements to be made regarding a threshold for pathologic slowing of NCV. PB12B/A TEH 0530941 12-78 1/4/83 DUP050031854 CONDUCTION VELO C ITY, m/sec Figure 12-2, Peroneal nerve conduction velocity versus blood lead level, Idaho, 1974. Source: Landrigan et al. (1976). 12-79 TEH 0530942 DUP050031855 PRELIMINARY DRAFT Several other electrophysiological measures of lead neurotoxicity have been examined recently, Seppalainen (1978) compared the sensitivity of NCV and somatosensory evoked potentials (SEP) as dose-response measures of peri pheral neuropathy in lead workers and found that SEP reflected neuropathy at lower blood-lead levels than NCV measures did. Guerit et al, (1981) found no differences in a composite index including frequency analysis of an electro encephalogram (EEG), visual evoked potentials (VEP), and brainstem auditory evoked potentials (BAEP) in 150 children aged 11 years attending five schools near a lead smelter. Blood-lead levels varied from 7 to 44 pg/dl (mean s 18 pg/dl). However, lack of details on the measures derived and statistical analyses employed render this study uninterpretable. Burchfiel et al. (1980), using computer-assisted spectral analysis of a standard EEG examination on 41 of the children from Need!eman et al. (1979) study, found significant EEG spectrum differences in percentages of low frequency delta 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 dealy, etc.) obtained for the same children were then employed as input variables (or "features") in direct and stepwise discriminant analyses. The separation determined by these analyses for combined psychological and EEG variables (p < 0.005) was 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. Unfortunately, no dentine-lead or blood-lead values were reported for the specific children from the Need!eman et al, (1979) study who underwent the EEG evaluations reported by Burchfield et al, (1980). It is therefore impossible to determine with any confidence the specific lead-exposure levels (including blood-lead values) that may have been associated with the observed EEG effects. Nor is it possible to state with much confidence what the health or medical significance of the observed brain wave alterations might be. The relationship of low-level lead exposure and neurobehavioral function in children aged 13-75 months was extensively explored in another study at the University of North Carolina carried out in collaboration with the U. S. Environmental Protection Agency. Psychometric evaluation (Milar et al., 1980, 1981) revealed lower IQ scores for children with elevated blood-lead (PbB) PB12B/A TEH 0530943 12-80 1/4/83 DUP050031856 PRELIMINARY DRAFT levels (> 3D pg/dl) compared to children with PbB levels < 30 pg/dl. IQ scores, however, were confounded by poorer home-care environment scores in children with elevated PbB levels (milar et al,, 1980). Furthermore, no relationship between PbB levels and hyperactive behavior (as indexed by stand ardized playroom measures and parent-teacher rating scales) was observed in these children (Milar et al., 1981). Electrophysiological assessments, including analyses of slow cortical potentials during sensory conditioning (Benignus et al., 1981; Otto et al., 1981) and EEG spectra, were also carried out in the same children. In contrast to psychometric and behavioral findings, a significant linear relationship between PbB (ranging from 6 to 59 pg/dl) and slow wave voltage (SW) was observed (Otto et al., 1981) as depicted in Figure 12-3 below. Analyses of quadratic and cubic trends in 5W voltage, moreover, did not reveal any evidence of a threshold for this effect. The slope of the PbB x SW voltage function, however, varied systematically with age. No effect of PbB on EEG power spectra or coherence measures was observed, but the relative amplitude of synchronized EEG between left and right hemispheres (gain spectra) increased relative to PPbB 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 pg/dl. This finding suggests that EEG gain is altered at PbB levels below 15 pg/dl, although the clinical and functional significance of this measure has not been established. A follow-up study of slow cortical potentials and EEG 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 condition ing again varied as a linear function of PbB, even though the mean PbB level had declined 11 pg/dl (the original PbB mean was 32.5 pg/dl; follow-up mean was 21.1 pg/dl). The similarity of SW results obtained at initial and follow-up assessments suggests that the observed alterations in this parameter of CNS function are persistent, despite the significant decrease in mean PbB level during the two-year interval. EEG gain did not vary as a function of PbB at follow-up. 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 EEG measures'was found in the initial (Benignus et al., 1981; Otto et al., 1981) or follow-up study. SW voltage and EEG gain thus PB12B/A 12-81 1/6/83 TEH 0530944 DUP050031857 Figure 12-3*: predicted SW voltage and 95% confidence bounds In 13- and 75-month-old children as a func tion of PbB. b: scatter plots of SW data from children aged 13-^7 months with predicted regression lines for ages 1$, 30, and 42 months, c: scatter plots for children aged 48--75 months with predicted regres sion lines for ages 54 and 66 months. These graphj depict the linear interaction of FbB and age. Source: Otto et al. (1981), TEH 0530945 12-82 O DUP050031858 , PRELIMINARY DRAFT appear* to provide sensitive CN5 indices of Pb exposure effects that are inde pendent of standardized psychometric measures used in other studies. Electrophysiological measures such as these hold considerable promise as indicators of CNS function that are free of cultural bias and other linguistic and motor constraints attendant to traditional paper-and-pencil or behavioral tests. Observation of a linear relationship between SW voltage and PbB within a range of 6 to 59 jjg/dl without evidence of any threshold effect level is also provo cative, particularly in view of the apparent persistence of the effect over a two-year interval. The inflection point in the EEG gain function at 15 pg/dl provides additional evidence of the effect of Pb exposure on CNS function in young children at levels considerably below what is currently considered to be safe (30 pg/dl). Interpretation of these intriguing electrophysiological data, however, must be carefully tempered by several considerations: (1) SW voltage and EEG gain are both experimental measures, the clinical and func tional significance of which is presently unknown; (2) blood-lead measures of body burden are problematic because observed CNS effects could have resulted from higher PbB levels prior to the reported studies; and (3) the study sample was relatively 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. The authors, therefore, concluded that CNS electrophysiological effects of lead exposure were observed at levels as low as 15 pg/dl. They further noted, however, that I.Q. decrements were not correlated with the electro physiological changes and that the functional significance of such changes remains to be clarified. The studies reviewed here have important bearing on a key issue, i.e., that of dose-effect relationships for neurotoxic effects in children and, especially, whether such effects occur at blood-lead levels below 30 pg/dl. Among the most important and controversial of the studies reviewed here are those that involved the evaluation of neuropsychological or electrophysiological effects associated with low-level lead exposures in children. None of the studies, individually, can be said to prove conclusively that significant adverse health effects occur in children at blood-lead levels < 30 pg/dl. The Needleman et al. (1979) study, for example, can be most reasonably interpreted as clearly demonstrating an association between neuropsychologic deficits and low-level lead-exposures in young children resulting in blood-lead levels of PB12B/A 12-83 1/6/83 TEH 0530946 DUP050031859 PRELIMINARY DRAFT approximately 30 to 50 pg/dl or, possibly (though doubtfully), at somewhat lower blood-lead levels. Furthermore, the generally small (though statistically significant) 4 to 5 point average decrement in overall IQ scores and small decrements in verbal ability test scores for high dentine-lead children in their study, the relatively small (but significant) differences in teacher rating scores indicative of poorer classroom behavior of such children, the somewhat more marked and significant attentional deficits indicated for such children by delayed reaction-time test results and teacher ratings of distractability, and the difficulty of demonstrating the association of such effects with lead in the range studied (independent of other potentially confounding variables, e.g., parental IQ, perinatal risk factors, etc.), all tend to suggest the presence of relatively subtle, difficult-to-detect effects of a magnitude likely to be seen near an effective ''threshold" level for lead-induced neuropsychologic effects in young children. Other studies noted above, at least qualitatively, collectively provide highly suggestive partial replication of certain aspects of the Needleman et al. (1979) findings. Among the more striking results consistent with the Needleman findings that emerge from several of the studies are observations of small, but statistically significant, lead-associated decreases in IQ scores as measured by several different specific IQ tests and/or by versions of the same test (e.g., the WISC-R) in the hands of independent investigators. Similarly, findings of evidence of attentional deficits (as indexed by ratings of "distractability" by teachers, parents, or experimenters) among lead-exposed children by several independent research groups are also striking and consistent across the studies reviewed. However, due to specific methodological problems with each of the studies (as noted earlier), much caution is warranted that precludes conclusive acceptance of the observed effects being due to lead rather than other (at times uncontrolled) potentially confounding variables. Analogously, inadequate indexing of lead-exposure histories in most of the other studies (especially in terms of blood-lead levels) limits or precludes credible interpretation of them as demonstrating associations between any reported neuropsychologic effects and blood-lead levels 30 pg/dl. Only the studies by Perino and Ernhart (1974) and Ernhart et al. (1981) provided suffi cient exposure indices by which to estimate that IQ deficits observed by them, if due to lead, would likely be associated with blood-lead levels in young children in the range of 40 to 70 pg/dl. Again, however, the small size of PB12B/A TEH 0530947 12-84 1/6/83 DU P050031860 PRELIMINARY DRAFT reported IQ deficits observed five years later among some of the same children (and mainly due to a single "outlier") tends to suggest that the neuropsycho logic effects of lead at blood-lead levels around or somehwat greater than 30 pg/dl are relatively minimal, may not persist for long into later childhood school years, and are likely indicative of an effective "threshold11 for neuro psychologic effects of lead being in the blood-lead range of 30 to 50 pg/dl. Also of considerable importance are studies by Burchfield et al. (1980) and Otto et al. (1981, 1982a,b), which provide evidence of changes in EEC brain wave patterns and cortical slow-wave potentials in some of the same high-lead children displaying neuropsychological deficits of the above type or other lead-exposed children experiencing low blood-lead levels. The former study (Burchfiel et al., 1980), however, provided no specific exposure infor mation by which to judge what blood-lead levels might be associated with the reported electrophysiological effects. Sufficient exposure information was provided, in contrast, by Otto et al. (1981, 1982a, b); and appropriate statis tical analyses were carried out which demonstrated clear, statistically sig nificant associations between electrophysiological (SW voltage) changes and blood-lead ievels across the range of 6 to 59 pg/dl (with no evident threshold). The continued presence of such electrophysiplogical changes upon follow-up two years later, suggests persistence of such effects even in the face of later declines in blood-lead levels. However, the reported electrophysiological effects were not significantly associated with IQ decrements. The precise medical or health significance of the neuropsychological and elctrophysiological effects associated with low-level lead-exposures as reported in the above studies is difficult 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 affect the intellectual development, school perform ance, 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 reflec tive of persisting, long-term effects of low-level lead exposure in early childhood. The issue of persistence of such lead effects, however, remains to PB12B/A 12-85 1/4/83 TEH 0530948 DUP050031861 PRELIMINARY DRAFT be more clearly resolved, for some study results reviewed above suggest rela tively short-lived or markedly decreasing effects on neuropsychological func tions over a few years from early to later childhood, whereas other studies suggest that significant low-level lead-induced neurobehavioral and EEG effects may, in fact, persist into later childhood. PB12B/A TEH 0530949 12-86 1/6/83 DUP050031862 PRELIMINARY DRAFT 12.4.3 Animal Studies The following sections focus on recent studies of the behavioral, morpho logical, physiological, and biochemical parameters of nervous system develop ment and function in experimental lead exposure. Initial investigations of nervous system involvement in lead intoxication utilized exposure concentra tions and doses which produced encephalopathic symptoms. The best-known example is the study by Pentschew and Garro (1966), which described an animal model of lead encephalopathy in which morphological changes similar to those reported in children occurred. Neonatal rats were indirectly exposed to lead by feeding their mothers a diet containing 4 percent lead carbonate. The lead was then transmitted to the suckling young via the mothers' milk. Between 23 and 29 days of age, 90 percent of the animals developed paraplegia lasting no longer than 2 weeks; 85 to 90 percent of the paraplegic animals died during this period. Neuropathological examination of these animals revealed that the lead encephalopathy of the suckling rat was caused by a disorder in the perme ability of the capillaries, resulting in dysoric encephalopathy. The suckling rat therefore differs from the human in that the latter shows a mixture of both dysoric and hemodynamic alterations. In summary, the histopathological changes associated with lead encephalo pathy in experimental animals vary among species and are characterized by their relative involvement of neuronal degeneration and vasculopathy. The fact that lead encephalopathy is reported to occur in the absence of cerebral edema in man (Pentschew, 1965), mouse (Rosenblum and Johnson, 1968), and guinea pig (Bouldin et al., 1975) is argument for a direct neuronal involve ment of lead. Recent studies have, in fact, suggested such direct neuronal alterations by lead. Furthermore, the relative involvement of the capillary bed in lead encephalopathy may also depend on the degree of maturation at the time of exposure. Bouldin et al. (1975), for example, were able to produce lead encephalopathy in adult guinea pigs with no cerebral edema or increased capillary permeability, whereas in the suckling rat these latter effects were the most evident. 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 PB12B/A 12-87 1/05/83 TEH 0530950 DUP050031863 PRELIMINARY DRAFT exposure of the developing organism. The interpretation of a large number of studies dealing with early exposure to lead has, however, been confounded by a number of flaws in experimental design. Perhaps the most notable of these experimental shortcomings has been the occurrence of undernutrition in experimental animals. Changes in nutritional status during early brain development are known to produce changes in behavior (Altman, 1971; Bobbing, 1970). Neurochemical processes have also been shown to be affected by early undernutrition. For example, Eckhert et al. (1976) reported changes in cholinergic enzyme activities when rats were placed on protein-deficient diets during various periods of development. Their results indicate that "the relationship between the activity of individual cholinergic enzymes, nutritional status and developmental age is complex and is not the same for different brain regions or even the same brain region exposed to undernutrition during different periods of development." Studies of lead neurotoxicity in experimental animals can also be con- founded by the use of relatively 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). Another important factor that may differ among various studies is the route of exposure to lead. For example, 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, intra- gastric gavage allows one to determine precisely the dose and chemical form of administered lead, but the procedure is quite stressful to the animal and does not necessarily reflect the actual amount absorbed by the gut. Injections of lead salts (usually performed intraperitoneally) do not mimic natural exposure routes and can be complicated by local tissue calcinosis at the site of repeated injections. A recent report by Barrett and Livesey (1982) suggests another possible confounding factor in many studies of the neurotoxic effects of lead. Because of its solubility in water, the acetate salt form of lead is PB12B/A TEH 0530951 12-88 1/05/83 DUP050031864 PRELIMINARY DRAFT frequently used in experiments that provide exposure through the subjects' (or, prior to weaning, their mothers') drinking water. Barrett and Livesey examined the role of the acetate radical by comparing the effects of lead acetate and acetic acid solutions (of equal acetate molarity) with distilled water as the dams' drinking water supply. Pups exposed via their mothers to acetic acid' gained significantly more weight than pups exposed to distilled water; the lead acetate subjects were intermediate in weight. Thus, there is some evidence to suggest that the acetate component in lead acetate might tend to offset the nutritional impairment that would otherwise result from exposure to lead alone. Conceivably, then, effects of lead on nutrition or subsequent endpoints might be masked in studies comparing lead-acetate exposure to a sodium-acetate control condition. More work is needed to elucidate the extent of such potential confounding. Another variable in experimental animal studies that merits attention is the species and strain of experimental subjects used in a study. Reports by Mykkanen et al. (1980) and Overmann et a). (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 effects between different species as well. The force of this discussion of various potential confounding factors is to place increased emphasis on the importance of measurements of bipod and tissue concentrations of lead in experimental studies. Without such measures, attempts to formulate dose-response relationships are futile. The following sections will discuss and evaluate the most recent studies of nervous system involvement at subencephalopathic exposures to lead. Older studies covered in the previous Air Quality Criteria Document for Lead (U.S. EPA, 1977) will be cited as needed to illustrate particular points but, in general, discussion will be restricted to the most recent work. In many cases, developing animals were exposed indirectly to lead via maternal milk using a modification of the Peotsehew and Garro (1966) model, but at subence phalopathic doses. In some studies, efforts were made to control for the possible effects of undernutrition either by altering litter size relative to controls or pair-feeding animals. Those studies that did not report any attempt to control for confounding problems (e.g., the effects of undernutrition), PB12B/A 12-89 1/05/83 TEH 0530952 DUP050031865 PRELIMINARY DRAFT as discussed above, will either not be included here or will be qualified appropriately. Recent studies of the effects of lead exposure on the nervous system have focused on four basic areas: (I) behaviorial toxicity, including the develop ment of motor function and alterations in such aspects as social behavior and learning performance; (2) alterations in morphology, including synaptogenesis, dendritic development, myelination and fiber tract formation; (3) perturbations in various electrophysiological parameters, e.g., ionic mechanisms of neuro transmission or conduction velocities in various tracts; and (4) disruptions of biochemical processes such as energy metabolism and chemical neurotrans mission. 12.4.3.1 Behavioral toxicity: Critical periods for exposure and expression of effects. The 1977 (U.S. EPA, 1977) review 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) have pointed to the perinatal period of ontogeny as a particularly critical time for the induction of behavioral effects from exposure to lead. 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). However, alterations in the behavior of rats exposed after weaning or after maturation have also been reported (Angel1 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; Shapiro et al., 1973). These findings stand in contrast to the negative results from other studies compar ing postweaning with preweaning exposure effects (e.g., 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 been noted in adult subjects of other species, including pigeons (Barthalmus et al., 1977; Dietz et al., 1979) and fish (Weir and Hine, 1970). The more specific question of the long-term consequences of lead's effects on the developing organism has been addressed in a number of studies by admin istering behavioral testing some time after the termination of lead exposure. Such evidence of long-term effects has been reported by Bushnell and Bowman (1979b) using rhesus monkeys. Their subjects were fed lead acetate so as to maintain blood-lead (PbB) levels of either 50 10 (low-lead) or 80 + 10 pg/dl PB12B/A TEH 0530953 12-90 1/05/83 DUP050031866 PRELIMINARY DRAFT (high-lead) throughout the first year of life (actual means and standard errors for the year were 31.71 2,75 and 65.17 6.28 pg/dl). Lead treatment terminated at 12 months of age, after which blood-lead levels declined to around 5 to 6 pg/dl at 56 months. At 49 months of age the subjects were re-introduced to a discrimination reversal training procedure using new dis criminative 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 performance levels. This difference was found only on the first discrimination task and 9 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. Studies using rats have also suggested that behavioral perturbations may be evident some time after subjects have been exposed 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 stopped at weaning, at which time average blood-lead values were 29 ( 5) and 65 ( 25) pg/dl, respectively. At 120 days of age the subjects were placed on an operant conditioning simultaneous visual discrimi nation task. (Although Hastings et al. [1979] did not actually measure bloodlead levels in adult subjects at the time of behavioral testing, they presumed the levels for control and experimental groups were by then probably quite similar, i.e,, on the order of 10 pg/dl, based on prior work [Hastings et al., 1977].) Forty-six percent of the high-lead group and 37 percent of the lowlead 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). Addi tional 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 PB12B/A 12-91 1/05/83 TEH 0530954 DUP050031867 PRELIMINARY DRAFT counter-balanced in presentation, 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 pg/dl. At the time of testing (3 to 4 months postnatally) the lead-exposed subjects' blood-lead levels averaged 4.55 pg/dl, compared to 3.68 pg/dl in control subjects. Although the two groups appeared qualitatively similar in their behavior in an open-field test and in preliminary 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 differentially 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. Results from the above studies indicate that behavioral effects may exist as sequelae to past lead exposure, even though blood-lead levels at the time of behavioral assessment are essentially "normal." 12.4.3.1.1 Development of motor function. Reiter et al. (1975) examined the development of rats indirectly exposed to lead prenatally as well as during lactation via the mothers' milk. They reported a delay in the age of appear ance of the air righting reflex in the 0.005 percent treatment group. This exposure level (no blood-lead concentrations were reported) was shown to produce no depression in growth, which suggested a direct effect of lead on nervous system development. No ^difference in the development of the auditory startle response was observed. Similar results with rat pups were reported by Overmann et al. (1979), who found that air righting reflex development was slowed, but neither the auditory startle refelex nor the ability to hang suspended by the front paws was affected at the levels of lead presented (0,02 or 0.1 percent lead as lead acetate in the dams' drinking water). Grant et al. (1980) indirectly exposed rats to lead jk utero, during lactation through the mother's milk, and after weaning through drinking water at the same lead concentrations their respective dams had been given. In addition to morphological and physical effects [see sections 12.6 and 12.11 PB12B/A TEH 0530955 12-92 1/05/83 DUP050031868 PRELIMINARY DRAFT for discussions of this work as reported by Kimmel et al. (1980), Faith et a]. (1978), 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 Pb conditions; other reflexive patterns showed no effect. The average blood-lead concentration for the 0.005-percent subjects at post' natal day (PND)-11 was 35 pg/dl; brain-lead concentration was 0.07 pg/g. Locomotor development generally showed no significant alteration due to lead exposure. Body weight was significantly 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 dams1 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). How ever, data for individual days were .statistically significant only on PND-60 and 150. An adverse effect of lead exposure on rotorod performance at PND-30 to 70 was also found in an earlier study by Overmann (1977) at a higher expo sure level of 30 mg/kg lead acetate by intubation (average PbB value at PND-21 was 173.5 32.0 pg/dl). At blood-lead concentrations averaging 33,2 1.4 pg/dl, however, performance was not impaired. Moreover, other studies using rotorods at average blood-lead concentrations of approximately 61 pg/dl (Zenick et al., 1979) and 30 to 48 pg/dl (Grant et al., 1980) have not found signifi cant 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 PB12B/A 12-93 1/05/83 TEH 0530956 DUP050031869 PRELIMINARY DRAFT pattern occurs and matures. In the study by Crofton et al. (1980), photocell interruptions by pups as they moved through small passageways into an "explor atory cage" adjacent to the home cage were automatically counted on PND-5 to 21. Pups exposed jn utero through the dams' drinking water (0.01 percent solution of lead as lead chloride) lagged controls by approximately one day in their daily activity counts starting at PND-16. (Blood-lead concentrations at PND-21 averaged 14.5 6.8 pg/dl for representative pups exposed to lead in utero and 4.8 1.5 pg/dl for controls.) Another form of developmental lag in gross activity around PND-15 to 18, as measured in an automated activity chamber, was reported by Jason and Kellogg (1981), Rats were intubated on PND-2 to 14 with lead at 25 mg/kg (PbB = 50.07 5.33 pg/dl) and 75 mg/kg (PbB = 98.64 9.89 pg/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; Mel berg 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 darn to her offsprings' slowed development through, for example, 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). 12.4.3.1.2 Locomotor Activity. The spontaneous activity of laboratory animals has been measured frequently and in various ways as a behavioral assay in pharmacology and toxicology. With reports of hyperactivity 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. As the 1977 review (U.S. EPA, 1977) of this material demonstrated, however, and other reviews (e.g., Jason and Kellogg, 1980; Michaelson, 1980; Mullenix, 1980) have since confirmed, the use of activity PB12B/A TEH 0530957 12-94 1/05/83 DUP050031870 PRELIMINARY DRAFT 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 "act ivity." Activity is, quite obviously, a composite of many different motor actions and can comprise diverse behavior patterns including (in rodents) ambulation, rearing, sniffing, grooming, and, depending on one's operational definition, almost anything an animal does. These various behavior patterns may vary independently, so any gross measure of activity that fails to dif ferentiate these components will be susceptible to confounding. Thus, dif ferent investigators' definitions of activity are critical to interpreting and comparing these findings. When these definitions are sufficiently explicit operationally (e.g., activity wheel measures that specify the direction and amount of force required to turn the wheel) they are frequently incommensur able with other operational definitions of activity (e.g., open field activity as measured by photocell interruptions). Moreover, empirical comparisons show that different measures of activity do not necessarily correlate with one another (e.g., Capobianco and Hamilton, 1976; Tapp, 1979). In addition to these rather basic difficulties, activity is 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 could confound measurements of activity. Also, nutritional status has been a frequent confounding variable in experiments examining the neurotoxic effects of lead on activity (see review by U.S. EPA, 1977; Jason and Kellogg, 1980; Michael son, 1980). In general, it appears that rodents exposed neonatally to sufficient concentrations of lead experience undernutri tion and subsequent retardation in growth. But, as Loch et al. (1978) have shown, retarded growth apart from that caused by lead exposure can induce increased activity measures of the same sort attributed to lead alone in some earlier studies. In view of the various problems associated with the use of activity measures as a behavioral assay of the neurotoxic effects of lead, the dis crepant findings summarized in Table 12-3 should come as no surprise. Until the measurement of "activity" can be standardized, there appears to be no basis for comparing or further discussing the results of studies listed in Table 12-3. PB12B/A 12-95 1/05/83 TEH 0530958 DUP050031871 PRELIMINARY DRAFT Table 12-3. Effects of Lead on Activity in Rats and Mice Increased Decreased Age-dependent, qualitative, or no change Driscoll and Stegner, 1978 Goiter and Michaelson, 1975 Kostas et al,, 1974 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, 19! Brown, 1975 Croftoil et al., 1980 Cutler, 1977 Dolinsky et al., 1981 Dubas and Hrdina, 1978 Geist and Balko, 1980 Grant et al., 1980 Gross-Selbeck and Gross-Selbeck, 1981 Hastings et al., 1977 Jason and Kellogg, 1981 Loch et al., 1978 Mullenix, 1980 Sobotka and Cook, 1974 Sobotka et al., 1974 Rafales et al., 1979 Zimering et al., 1982 PB12B/A TEH 0530959 12-96 1/05/83 DUP050031872 PRELIMINARY DRAFT 12.4,3.1.3 Learning Ability, When animal learning studies related to the neurotoxic effects of lead were reviewed in 1977 (U,$. EPA, 1977), a number of criticisms 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 administered, 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-lead values). 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 determined by several independent U.S, laboratories (Kimmel, 1982). The results of this program should help standardize some behavioral testing procedures and perhaps create some refer ence methods in behavioral toxicology. Also, as well-described studies are replicated within and between laboratories, the reliability of certain experi mental paradigms for demonstrating neurotoxic effects is progressively estab lished. Some progress is also being made in dealing with the issue of the validity of animal behavioral assays. As the neurological and biochemical mechanisms underlying reliable behavioral effects become better undersood, the basis for extrapolating from one species to another becomes stronger and more meaningful. An awareness of different species' phylogenetic, evolutionary, and ecological relationships can also help elucidate the basis for comparing behavioral effects in one species with those observed in another (Davis, 1982). Table 12-4 summarizes exposure conditions, testing conditions, and results of a number of recent studies of animal learning (see U.S. EPA, 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 expo sure 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 PB12B/A 12-97 1/05/83 TEH 0530960 DUP050031873 cs x--4 z fX .< Q s g 9< lu o </ a u. u. LU LU Z H Uo* VJ s x Q >os $ 4J C .01 *3 </> O .(/I e0 o -- e x *3a. __ Xa.05Hv-> 'Eg*!-* _V> a. Oc,,lVVT> t*<t3Ju *o>- X 'F* * * 44 0 C O 1 s- H(- <u o vi os ,2 CLV ^3 J ^. LD 0> O OlMD . C3M.A<>IZ CAk> ,*A 4 Z CU ai ra c Sfl S3 O 10 uu o ** i. o S.> 4U <J O CO 8e &. 00..00 u01 2 vo3 0) *n v> . <*- CL S-o <U H C fc> .05 Itj X <11 __ SI T3 *Q ui OJ *t- V o j <fif O O VI O to u a e . > u **-'8 > o *o_*- *3<u *5*~ U0><4- V) c,<- V V H- S..2fd 40 .J3 *D C 4 45 <9 U O**- ** Q, ,Q.g 151Ue +Jo <9. i-4 i- > a o tf w) O VI e 40 9 01 > V) lyo w a. u 01 Oh - 40 1" <- fc <0 _ O W vi <u e O <9 . VI Q 40.X S2.es*5 <U<fc> a 3 o u u S- 40 rx-- u 9 u U S- VI f - S- 4-> C 4 3 013 t0- '*19- 0a1 Xo> Oo a> e _ V *3 C '*30 00 n ac Fi- VI F- ^ 2 2gg 4*-f--1z| Scry. C Z Q. a 4J S5fi ^ 5^1 4f 3 19 "3 .U > U<ZV\ ** a> o <9 v 6- *3 <U L~ az. os 3c l cs o j f -1 39.10 o??Y <9 Gl c l c l S-- ooo Q3U500 ua no Qm r-4 f - -O C^a S3 vi O o o L\H I CHI O *a SO CM v 49 LO is II 6'g <0 H fO 8 a. CLkO o ui aa.V<oJSo o * ii " II V5 + > *lA^ L 0. - 4-9 .. ua a.** (JO. tl)f w* ;S x CM <V O -O O 8 w ooo aoz mH 5.0. i o O $... oo rtcncv I >s. at CMcxS^-> CM -g 3,=rS5' <M1-4 V f--t #v^!S 4 4--k i o <m cn z jp z a. i a. i i -*S2 0 cn 1 . CW 1.2 2 CLL J2 3 <0 2 94 2 oz g. 88 a. a 3 as aa. s- <9 U O oa. aj TEH 0530961 s oc 1~ uvea f-- VJflO F-- <n 053S W 38 at X JS.4-^ <U/> HOr-- z .r^ yr-H u> C9 P I o> 12-98 4- LU IV I Q -1 n cn U r-4 0SV r O DUP050031874 *1 eo b.b ob 5a 19 3 VI o ev a> 2 A iA|M Ml +> r* S >t CL 0)10 4J *P 5 |3 r* C*- V) VI . (A 3 a. f- I c l VI VI < f- i 2*Jo^- 4)5 s: v I V- *m > a. o o -- vi b b r-- 4J 3 U IT) b VI 4) iM/>l I.WO g* 2.*- b **- v>*o v > UV* 49 a. a> v o b e x 6 XJ 9 O A O -- e VQ9.SXOCbV^C9Q:K_OU. .I3L c m oi aub* 4- VI 0*0 *0 yiro c l e o x > O 41 VI 41 b-* <'FCO 41 TJ *> vC'-v *o u eo i o r-* jig*3 5 - 9 u> u> e 0 6 0 oi 8.SS54" 9 4) *0 41 JZ O b Q| 4> i u .e <a 3 4>r- V> ** .9 Ml i e-cf e b 2o c V) * be a a1C_ o_ b -.O* -- a. ob bO vX3 eOs S'9 Jl e o*- vi c _ <b v>i +3 v t* __v> vt).1-v.>O 4Jr- msL Q V . V) b O) 9 4M *o O U -- W <TJ cacc-b sss .es-ss <o x w* bflJ * *b 4) i 5 ) S' 5 141 C 4J b X VI 3 " b 3 :3 *r- b 3. O Ml 41 4> viler 41 *0 Ml 4-> 6 b X -r- O O tn f- o bS t a Cb 4Mb b gvifp u bb Ml b VI b o o> CM OTk ro CM -- rH 4 *0 I S'&S 09 CLU) VI A ty>| V)| 22 Ml 0) +* g. _ bo '<-! Oi b " 0.0) LO E * b a *% bO <U & a, o> CM * M a 4> i-- > Ml Ml voCTc9): oax. a J3 JS Ml -O W O 4) <J 4b. 64) U3_ b_V <0 4) Cl b ro Q> b 3 > r-- Ml a Oi^LlK* Ml I'--' jQ r- J2 b-O. c_ a-a c m 3 VI 8^:=<0 431 a a- a. IMl Ml inn 4) Ml o Q >- +a4J.>C8- cOCLOCCOLhV-) .u10bb41 bu4) c43>'-4-s1 mi <~o w" C u4>> 4>)> ba> *bo ob o4>'Hc-j O b M Cl O o VI b *o Ml 9 OO -o Ml O QO b 4) % a I <o r*^ 4^ *>7h 41 41 W U 41 I mi rs. vi 12-99 ui 41 VI 22* 41 b o vi .9 O I _ 43 00 VM)l `O6-- r--O- r 9 vi> J TEH 0530962 DUP050031875 oeo us vi i/>l c B'io t0f>)*U") U.) O| * 01 SLefcS M- I -O O) C<0 .5 e S*5 Ja <v . S O. S- OVT3 2? JO C V> *9 CL f <S> | 0.>4c)*o0)*--0 <0 0 <Lo2r -1 c l Jb'ro'vi <0 *ra>i-c6l Z0303 ft CI lA L VI $ O ** ZA *5u a. ftt. <M f* ID p> Lii -- `if* i +1 -H +* deMA<B4H0jjj*.+A-gCHM) CL c t_n* ocvitLAd . en r-- ro -+H Ovd9N >>0- HN(0 4-N rcr i- m r4 .. Ol43 VHifl a .. h (s l . <si M fi.fi* 0..A0.0A. L or it 2 v> o 0> 0 4= L CS V VI g **- 0VI f|A c (Mi a * as3>4> VI .01 S &*H L w s- -43VI-TM >>0o I (i'll or > a. VI r e V>| A 15 _I _n .o 6L V L* L> O o > a0X?.O--J*'*~c *0-- CO Al <0 A & VTO U4J 4S .C3L &to. o&i Uoi O O *j > *- 31 1 0000 wsr* s ** 000 co i-4 irt co Aa. V . * rH CM 3*2 k| C * * oe 43CVJ < & 3 r-4 **-0O.-0 yruH oCMcMv0j. U3U1 "H'f*. IcJo 0* A0r.H0A-CM PRELIMINARY DRAFT > gi-4fi iV-) !f5 cm ft.. ..ft 3 88 CMVI s-ft.-ft3*fi *fl 1 <M g A e <n jg U f^ lilt,? CM 'aiO 2* .Ot0fl 4Q3- I S 3.0* oS1 A*T3 *jfj* CAL nX4T C4> T<0Jf4 V UVI46>j Q&L(0 **$ * 25Q. o 1 L C* 1 .AO 36.CU JQ. I * AAfiV *V*>I?U5m>&00J <AH *J A S ;U < I ' ; V> < va1V0. U o*r Of:L<oD>-0rCfgOeCsi0- j>Vf>>P-4v,La(cC0*i LSn au c<0 S0- * voi pc5 00V I 1*9o3 2 01 (0 Lo> 2or 9 t 'IS c as > 0 c u <u0 o0<0 . 0g. a rd td X oV>* --.*a*si C l?9N1 VI 0 <0 TEH 0530963 ..s 04TH *-- OlS-r* or 01 or N01 UH ^u I--ro 0is>. 01 12-100 " .v ' 111! DUP050031876 e u<010 <V4o*1- -J CL :9-s *a t- .eio 0c(-1<ob3>0. su-^:ooi *g jt*o *-+ x*t: r-t <C11 (A MOL as 3 to- i>n"r 4ua- 3-S*. 8 7 fc ,lO-Ui-3+e> *o.sc 4A 4- e o Jr S L M C .O L O o> > rCL.uo4.1 vc UO'<M4- LQ- I*O3 Q11 Ou e 10 3!L a MOO gQ p- a. nr i 01 2 kVfmsO.l d > 0.4*tA fe^ee 01 3 U1& fc. L OS >a; ouwti*e*0" 3 6 6 I *o <0 e x: s- C > OHO V o> .( P4- 4 LL0A1- ei> 01 S- g rH +4 OMS *Acn~ Ol u oC M cS o {Jr*3 2 >V0{o1.)i f L-A r* J- <0 +veii*oSOt*.lH9C .0 O *p 01""5-feS HS.l 1-63 o .* <A 4-1 p i O >uu "8 S*g v.10 o2-oS Hi0.1 Co 6e v<</9> v-tCo u>U- 10 U (A %n 4c| 4Cg-. &*.* (0 LA r3 til LA . LA 01 > (0 44 e44 l a oVIL 3 eai<o S01 'wS -QI LA CU . (A <0 iM 4O4 bO O V)|b V 9i 404 444a440. oi t(05Q a4 *01Z> ez *o a. 4H4 33S bfl 9 bLA as5** QQ 0144 0 4? 04 CO I*- 04 t *H *i tr) i/i f* 04 a.pLf't.e0* 00 CM to CO O a.cKoooj SoH .0**0** trtflO tO OHV 196 -H ff 5 Ol * CO 6 o co >1<010 *,- - bHLOfOHtIOA 1.O0U1LS US 'Uiu to H<A PQ03.a4<J(.U--X&IC&O 43--434 o <a <r ** fee a. gi TABLE 12-4. {c o n tin u e d ) <aAi *9- L. O 0a1. Scn. enr^-s Ol 01 IA o01 et ob. uX o44 so n>ai>F4Jv ,,c ?6 w 6o --3gC3 M3<e0) -Oi4ef4<40^a3af. Hi4b4H4>* C4(MO4H-j S . >> 4100 lo0Xa.0*_+to1o_4 oi s 44 U>*lr/4l U1 9 0 44 vCfifl b >* g a <0 C LA *r- be 41 b > <0 01 "S ag --oi LU g 4.1J0 ^-M . Hit- M WOOJfl aO wS-vX4c1 M9 BU LoaA.*noo W*0 44 2 O O 644 O.vV- anCrLov . *o <a a og 41 3 if a 0M $ -53 1N oaai.. 4-1 4> ,,$ 4J ,H* C*4 44 LA 41 45>qC/> Otyo 44^44 0u1 c01 12-101 TEH 0530964 DUP050031877 Behavioral task Apparatus Reward TABLE 12-4. lead exposure Period1* Levelc (continued) L itte r per te st group Reference Species Task Subjects per test group Blood~TIi. gg/dl Growth*1 rate Learning performance Peak At test ^ I <> > LA .*> <0*3 e(9 < e_ U c .uU41 xV: 3^' o>TO -- ro 3 V) w at is-e g fir - VJ 40 rV-I *q*l' 2S 81 aa. *n *e o5 o- wS-' ro>Sv s_ ' +.e ,o 0> 0 O 8*4- c5t 'o-V).rt8o-- vi 0 u C v. 4 Uks 335 .U-4 V5 xS r-t *.O5'5W 3u 05 (A V > vt >* *r- S/)|U 0 SC 3 (03004- C U u0 s .0 V __ 5U0 V) ^ v>(v> *- o SS2 M % +j U VI 0 5 2 5 v. C C .*0 J0ov-eT3 0 U V>*l- 9 0p aw- 0p- .S5 u _ y- &> V> 0 bO VI O 0.0 0> I ,0 =0 4J 30 X3 CO SO tC- cVp)nc0.av oU3ikg 33 MOf- ca <r M> i!nnts -o o> r . uri U3 CO 3o* ""I cn f2^ QtC. 0.Or.HOC Q3Vo,>H0m^-0H)* *G45Q. 3O. .kf.i*J.nIc-t3a.vu J? CSi 03Lw3C 03. *3 oto ao 9 vt * >tU*0 t3JS? X3w35O VI eT3 v4ii0i U0*3r-rwCHO* V00k)- S3. '#*0*> S".83S (SIV U>r >-- ' i-- (A _ & 3 . . O V- iCM V* o <s >T! CJfi *3. 8. 0 I <9 ? SS *{ ? * ' O O) <M CM >*r. eo e<a V> V e0 35 VO 0 u 33 V0> eo TS vi ** v03t 0O> *o 7c 2 'm ` 5 3 33 4J 0V f 0^1 UC0 1 -C CM i w I e-S 9 5 1-4 CO * CM CO I 0 c v> 3K 4= O vo .a a <i**n. 33 12-102 TEH 0530965 DUP050031878 PRELIMINARY DRAFT U> V 'S'* .5* 2S>, c e a*o vi *2**3 *St 5iq e aiiSs r oxo0.0a 0U 0 0 .c 0 0 *o&4. -- v O .#* VI U **-i C C o o 4- 4- **TJ . vi Cl 3 O I t> i ! o> jaa. .vc op Q-W> z ^ o> " y> $ ti,S * 2. e 2." ? S 5 5 #r3 2'S IT) 00 5 <M =.VO <q P> cj C4q o i-t cn 4rf *0 O H<nri 0v,., .. 0 Sr--0-*.JCO0M403 .0. Is. <3 U ton ** ss CL 0> ft. II *> VuI V0 r-- +J Q.+J w*r* rt fotvaaS.oti9 9 S. oei 4a4t SS 44. 400)> CUrftLm-CX- OOt-400 > <0 4C4 *t- It It It It Q<5M:3* Po la as ii it f CJ m <Q v fT <J 44 V) (0Q/).-Tr3- S. 4* as Of 2 TJ W^ 0 > 3(A O U Q. IQ 0 - 4UC CBO 0 JS .C V) tf ftfi 44 0 i* V) 0 e o"~s 0 C CD JE 4 <t*o rO*. 0 i-- t H 2 28 6 4T"' P" U*Q U VI VI 2 v> .0 uOs c*<- 44 W 0Ou 5- 'oSji'0 * (A U3 . , AO9 tn cn 4>-0 0c p e p c e o c o > <5<5 ns p r VV-I .p44- **p0- *p3- 4S4ts *I4Q +9~* gn>j 5 60 *i9. .3e a > > *r* **r 0<0vi 3 o o c e oU -(/Spfl(9*p8* *fr-i w3 II > >svPl* Pfc C_ 4_4-_^ _44 r<--4-*Ur- 3I c I -- <A S. O ep* tfl 3) 41 0 *0*0II 5 * V> V> I I Tf** f- 45 ft^UO (J . Jiis1 . .cS aaa i-- g ' 0 -- W . , VI JSo ? u h or S ^ II --.< 41^uJu 23 j I g\\ it i oo O C || <Q IQ s &- < cy&a-sV)V)HMU.V) ft 8 * ^08a 8a0^ 44 -wo w ftOOft U 44 44 t. 33 ft W (. oVI 41 V VI as ? a X V> V* X 0 O O 41 0.0. ftftVft >-- ft ft ft ft +4 C C 44 (p Oj 4j XX 00 t-QQ t 0OO0 CL-p - O- b. {. c 0 0 o> O 0.0. 0 4CC6 0OO0 44 ^.P 0 (A 44 44 2 000 0)44 44 44 0 t- VI 0 <4 0 VOI 0.0 4.0, 11 M 11 It 2' 12-1.03 TEH 0530966 DUP050031879 PRELIMINARY DRAFT 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, 1982) and by Cory-Slechta and Thompson (1979) report alterations in learning task performances by rats with blood-lead levels below 30 pg/dl. Winneke et al. (1977) exposed Wistar rats in 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 dis crimination learning tasks using either "easy" stimuli (vertical vs>. hori zontal stripes) or "difficult" stimuli (white circles of differing diameters). Blood-lead concentrations were measured at about PND-16 (26.6 pg/dl) and PND-190 (28.5 pg/dl). Although there were no significant differences between lead-exposed and control subjects on the easy discrimination task, the leadexposed subjects performed significantly (p <0.01) worse than controls on the size discrimination task. The performance of the lead group continued around chance 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). In a more recent study, Winneke et al. (1982) repeated the size discrimi nation experiment and added another test involving shock avoidance. As in the earlier study, exposure started ijn utero and continued through the time of testing. Different concentrations of lead acetate in the diet were used to yield average blood-lead concentrations of 18.3 and 31.2 pg/dl after 130 days of feeding, compared to a control level of 5 pg/dl. It should be noted that these values were not determined directly from the subjects in this study but were based on separate work by Schlipkoter and Winneke (1980). However, 6-ALAD 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. (1982) found that lead-exposed subjects were significantly slower to reach criterion performance 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. PB12B/A 12-104 1/05/83 TEH 0530907 DUP050031880 PRELIMINARY DRAFT Although apparently inconsistent with the impairment found in the discrimina tion task, this 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 Pb in dams' drinking water) to cause a 20 percent weight reduction in the subjects prior to weaning (no PbB concentrations were measured). Cory-Slechta and Thompson (1979) exposed Sprague-Dawley rats to 0.005, 0.03, or 0.1 percent drinking water solutions containing lead as lead acetate starting at PND-20 to 22. Operant conditioning 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 55 to 60. Blood-lead concentrations measured at approximately PND-150 were reported in graphical form roughly as follows: 0.005-percent solution group, 5 to 10 pg/dl PbB; 0.03-percent group, 25 to 30 gg/dl PbB; 0.1-percent group, 40 to 45 pg/dl PbB. Subjects exposed to 0,005 or 0.03 percent lead solutions showed a "significantly11 (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 0,005percent group and the next 30 sessions for the 0.03-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 0.005* and 0.03-percent groups. However, response rates for the group exposed to the 0.1 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 0,02 and 0.03 percent solutions) and exmained the effects on another aspect of operant 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 variability in creased 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 subjects tended to shorten their response durations (p = 0.04 for the 0.01-percent group; p - 0.03 for the 0.03-percent group). This tendency PB12B/A 12-105 1/05/83 TEH 0530968 DUP050031881 PRELIMINARY DRAFT 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 0.03-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 0.01-percent group and 320 to 1080 ng/g for the 0.03-percent group. Given the same exposure regimens in the two studies, blood-lead values should be comparable. A study by Gross-Selbeck and Gross-Selbeck (1981), described in part under section 12.4.3.1, 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 average PbB = 22.7 pg/dl. Although no differ* ences were apparent in preliminary operant bar-press 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 been recently studied in sufficient detail to warrant discussion here. One of the primate studies, that by Bushnell and Bowman (1979b), is discussed under section 12.4.3.1 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 averaged as low as 31.71 pg/dl PbB for one group during that period. When measured following behavioral testing, average blood-lead concentrations were similar to control levels, i.e., 5 to 6 pg/dl. 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 month of life. Lead acetate was fed to the subjects during the first 12 months so as to maintain nominal blood-lead concentrations of 50 and 80 pg/dl in 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 PB12B/A 12-106 1/05/83 TEH 0530969 DU P050031882 PRELIMINARY DRAFT was terminated at 12 months, blood-lead levels were still somewhat elevated over control levels at the completion of behavioral testing (18.75 i 2.87 pg/dl, low-lead group; 46.25 6.74 pg/dl, high-lead group). The basic find ing 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 pg/dl were, compared to control groups with levels of approximately 5 pg/dl, significantly retarded in their ability to learn a visual discrimination 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, which followed extended training in the original discrimination task. Rice and Willies (1979) attempted to replicate Bushnell and Bowman's (1979a) findings by feeding rhesus monkeys lead acetate from day one of life. Blood-lead concentrations in their four experimental subjects ranged between 35 and 70 pg/dl around PN0-200 and dropped tp 20 to 50 pg/dl by PND-400; the four control subjects' levels were generally 5 pg/dl or lower. At 2 to 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 leadtreated subjects appeared to make more errors in performing the reversal tasks; analysis of variance yielded a main effect with 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' reluctance to manipulate the novel negative stimulus after 100 pretraining tries in which only the positive stimulus was presented. Thus, the unexpected initial success of the lead-exposed subjects PB12B/A 12-107 1/05/83 TEH 0530970 DUP050031883 PRELIMINARY DRAFT may have.been an artifact of the pretraining procedure. By this interpreta tion, the lead-treated monkeys in Rice and Willes1 (1979) study and the highlead group of monkeys in Bushnell and Bowman's (1979a) study were both showing perseverative behavior or a refractoriness to modifying 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 fixedinterval/time-out schedule of reinforcement (Rice et al., 1979). This schedule alternated a 10- to 90-sec time-out period, during which responses were unre warded, 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 stabilized at 20 to 30 pg/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 rewarded. In conclusion, it appears that alterations in behavior in rats and monkeys occur as a consequence of chronic exposure to dietary lead resulting in bloodlead levels on the order of 30 to 50 pg/dl. The question that arises, however, is whether such alterations reflect any impairment in functioning of the lead-exposed subjects. As some studies indicate, lead-treated subjects may actually perform better than non-treated control subjects on certain learned tasks. For example, in Winneke et al.'s (1982) study, the task on which lead-exposed rats excelled required the subjects to move from one compartment to the other in a 2-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 Driscoll and Stegner (1976) for shock-avoidance performance. As previously described, a study by Gross-Selbeck and Gross-Selbeck (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. 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 PB12B/A 12-108 1/05/83 TEH 0530971 DUP050031884 PRELIMINARY DRAFT the same tendency unadaptive, as, for example, 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 en hancement or impairment of performance) may be misleading designations for the behavioral alterations measured under arbitrary experimental conditions. Of greater importance 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 gleaned from the results of other studies of the neurotoxic effects of lead (e.g., Angel 1 and Weiss, 1982; Overmann, 1977; Rice et al., 1979). Taken together, these reports could be interpreted as suggesting a possible "hyper-reactivity" (cf. Winneke et al., 1982) in lead-treated animals. Although careful to distinguish hyper-reactivity from the term hyperactivity (as in locomotor activity), Winneke et al. (1982) have not yet provided suf ficient empirical grounding (e.g., an operational definition) for their notion to make it useful as an explanatory concept. They and others (e.g., Petit and Alfano, 1979) have, however, noted the commonality of such behavioral outcomes with experimental studies of lesions to the hippocampus (see section 12.4.3.2.1). 12.4.3.1.'4 Effects of lead on social behavior--The social behavior and organi zation 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 com pelling grounds for including animal social behavior in the present reivew. As in the case of nonsocial behavior patterns, an animal's ability to interact with conspecifics may reflect 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 state or condition (Davis, 1982). Such behavior could potentially provide a sensitive and con venient indicator of toxicological impairment. PB12B/A 12-109 1/05/83 TEH 0530972 DUP050031885 PRELIMINARY DRAFT Two early reports (Silbergeld and Goldberg, 1973; Sauerhoff and Michaelson, 1974) suggested that lead exposure produced increased aggressiveness in rodents. Neither report, however, attempted to quantify these observations of increased aggression. 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 partur ition to weaning at PND-21, at which time exposure terminated. This lead treatment produced no change in growth of the pups. Individual pairs of male offspring (from the same treatment groups) were tested at PND-60 for shockelicited aggression. Both lead-exposed groups (average blood-lead levels of 5 and 9 pg/dl and brain-lead levels of 8 and 14 pg percent) 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 agresssion 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 leadtreated 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 reduc tion 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 160 pg/dl after 2 weeks exposure and 101 pg/dl after 4 weeks expo sure. 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 concentrations were 2.45 nmol/g for controls and 4.38 nmol/g for experimental subjects). At 8 weeks of age social PB12B/A 12-110 1/05/83 TEH 0530973 DUP050031886 PRELIMINARY DRAFT encounters between subjects from the same treatment group were anlayzed In terms of a number of specified, identifiable behavioral and postural elements. The frequency and duration 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 significantly reduced agnostic 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 her coworkers (Donald et a!., 1981) used a similar paradigm of exposure and behavioral evaluation, except that exposure occurred either only prenatally or postnatally and testing occurred at two times, 3 to 4 and 14 to 16 weeks of age. Significant effects were found only in the postnatal exposure condition. 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 to 4 weeks old), lead-treated males (and to some extent, females) showed decreased social investigation 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 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 aggres sive behavior in Donald et al.'s study (cf., e.g., Adams and Boice, 1981). Later testing at 14 to 16 weeks revealed that lead-exposed female sub jects 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 pmol/kg in males versus 191.6 pmol/kg in females (blood-lead concentrations were not measured). Donald et al.'s study. PB12B/A 12-111 1/05/83 TEH 0530974 DUP050031887 PRELIMINARY DRAFT along with the above-mentioned study of Ogilvie and Martin (1982), points to the importance of not focusing exclusively on perinatal exposure in assessing neurotoxic effects of chronic lead exposure. The social behavior of rhesus monkeys has also been evaluated as a func tion of early lead exposure, A study by Allen et al. (1974) reported persis tent perturbations in various aspects of the social behavior of lead-exposed infant and juvenile monkeys, including increased clinging, 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 pg/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 exposure conditions. In experiments 1 and 2, daily ingestion of lead acetate during the first year of life resulted in blood-lead levels of 30 to 100 pg/dl, with consequent suppression of play activity, increased clinging, and greater disruption of social behavior when the play environment was altered. In experiment 3, a comparison of chronic and acute lead exposure, the latter resulting in a peak blood-lead concentration of 250 to 300 pg/dl during weeks 6 to 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 signifi cantly from controls in various categories of social behavior. Experiment 4 of the study showed that prenatal exposure alone, with blood-lead concentra tions of exposed infants ranging between 33 and 98 pg/dl at birth, produced no detectable behavioral effects under the same procedures of evaluation. Over all, neither aggressiveness nor dominance was clearly affected by lead expo sure. Another aspect of social behavior--interaction 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 concentrations averaged 96.14 16.54 pg/dl in the high-exposure group at day 1 of gestation). Dams and their litters were videotaped on PND-1 to 11, and the occurrence of certain behavior patterns PB12B/A 12-112 1/05/83 TEH 0530975 DUP050031888 PRELIMINARY DRAFT (e.g., lying with majority of pups, lying away from pups, feeding) was tabu lated by the experimenters. In addition, dams were tested for their pro pensity 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 behavior 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. Although there is no evidence to indicate that maternal behavior in rats is disrupted as a result of lead exposure, the converse conclusion cannot be strongly supported by existing evidence, 12.4.3.2 Morphological Effects. 12.4.3.2.1 In Vivo Studies. Examinations of the effects of perinatal lead exposure on dendritic development have been carried out by Alfano and Petit (1982), who indirectly exposed suckling rats to either 0, 0,4 or 4.0 percent lead carbonate in drinking water via their mother*s milk, While these groups were not pair-fed, litter size was culled to 10, 10, and 5 pups, respectively, to control for the effects of undernutrition. By PND-25, 2D percent of highlead pups exhibited the hind-limb paraplegia and urinary incontinence typical of lead encephalopathy, although such signs disappeared by PND-30. Light microscopic examination of the hippocampus at that time revealed that the maximal lengths of the dendritic fields of granule cells in the dentate gyrus were reduced in both the low- and high-lead groups, even when body weight was used as a statistical covariate. At the same time, the dendritic fields showed greater branching at 20 pm from the Cell body, although such increases were reversed at points 160 pm or greater from the cell body. It would appear, then, that neonatal lead exposure causes a retardation in the temporal sequence of hippocampal dendritic field development. PB12B/A 12-113 1/05/83 TEH 0530976 DUP050031889 PRELIMINARY DRAFT Averin and Needleman (1980) Indirectly exposed suckling rats by feeding their dams chow containing up to 1 percent lead carbonate, then weaned the pups to normal chow. One group of control dams was pair-fed an equivalent amount of normal chow (animals on a diet containing lead usually eat less (or drink less, if the lead is in their water] than animals on a normal regimen), while another control group received normal chow ad libitum. Neither the experimental nor the pair-fed pups exhibited behavioral abnormalities, histopathological lesions, or any changes in rates of body or brain weight gain. However, both groups had 25 percent more morphological ly-immature synapses in frontal cortex than ad lib, controls when examined at PND-10, 21, 40, and 60. Blood-lead (PbB) levels on these days were 100, 385, 30, and 20 pg/dl, respec tively. The total number of synapses was also reduced in lead-treated and pair-fed control rats at every age; in addition, at P.ND-60 the lead group had a significantly smaller number (20 percent fewer) of synapses than their pair-fed controls. The authors concluded that neonatal lead exposure may retard cortical synaptogenesis over and above any nutritional effects. An earlier study (Petit and LeBoutillier, 1979) of suckling rats whose dams were exposed to 4 percent lead carbonate in their chow revealed both brain weight and cortical thickness to be 13 percent lower in lead-treated animals. Synaptic density on PND-28 was reduced 23 percent in the molecular layer of the occipital cortex, as was dendritic branching 80 pm from the cell body of the large pyramidal cells in the parietal cortex. The same exposure protocol also led to a PbB level of 258 pg/dl and a 35 percent decline in the network length and segment frequency of the dentrites cerebellar Purkinje cells (McConnell and Berry, 1978, 1979), When rats were indirectly exposed from gestation until PND-30 to either 400 or 750 mg lead acetate/kg body weight/ day in drinking water (Murray et al., 1977), the number of dendritic spines on pyramidal and stellate cells in the parietal cortex decreased by 42 percent for the 400-mg exposure and 62 percent for the 750-mg exposure. Rats exposed only from conception until weaning (PND-21) or only post-weaning (PND-21 to 30) showed decreases of 20 to 38 percent. However, the interpre tation of the significance of these latter three studies is complicated by the fact that no attempt was made to control for the possible effects of undernutrition. PB12B/A 12-114 1/Q5/83 TEH 0530977 DUP050031890 PRELIMINARY DRAFT Exposure of neonatal rats to 400 mg lead acetate/kg body weight/day by intragastric gavage (Toews et al., 1980) from PND-2 to 30 produced PbB levels of 400 pg/dl. Marked reductions in body weight (26 percent), brain weight (21 percent), and myelin concentration in forebrain (42 percent) and optic nerve (30 percent) were observed. Starvation controls and animals administered 100 mg of lead did not exhibit these effects, which would indicate that malnutri tion did not play a significant role here. Furthermore, biochemical analyses of myelin protein composition in these animals demonstrated a comparable maturity between controls and 30-day-old lead-treated rats. It would seem from this study, then, that neonatal lead exposure does not delay myelin maturation, but rather inhibits its synthesis. On the other hand, when Stephens and Gerber (1981) examined the lipid composition of whole-brain myelin in rats exposed from conception until PND-32 to 0.1 percent lead acetate in either their dams' or their own drinking water, they found significant reductions in the initial and final concentrations of gangliosides (30 percent) and glycolipids (70 to 80 percent). These findings would seem to argue that perinatal exposure to low levels of lead inhibited both myelin deposition and maturation. However, it is not possible to rule out the contributing effect of undernutri tion in these treated animals, although the concentration of lead used was sufficiently small that such effects would probably have been relatively minor. Reyners et al. (1979) studied the effects of chronic lead exposure on the glial cell composition of rat cerebral cortex. Suckling rats whose dams were fed diets containing 0.01, 0.1, 0.5, or 1 percent lead were weaned to the same chow. At 90 days of age, morphometric analysis of the cerebral cortex revealed a significant increase in the density of astrocytes and microglia, whereas oligodendrocyte density was markedly decreased. Although it is difficult to evaluate these findings from the sparse data presented in the paper, it would seem that these effects do not begin to appear until exposures reach the 0.1 percent level. Studies on the effect of lead on the development of CNS fiber tracts were carried out by Campbell et al. (1982), who indirectly exposed suckling rats via their mothers' milk to 0.2 percent lead acetate in drinking water. They observed reductions of 15 to 30 percent in the density of certain types of synaptic profiles in the suprapyramidal mossy fiber zone proximal to the PB12B/A 12-115 1/05/83 TEH 0530978 DUP050031891 PRELIMINARY DRAFT dentate gyrus. These morphological findings may represent the anatomical substrate underlying the reduced dendritic field development observed in granule cells of the dentate gyrus of the hippocampus by Alfano and Petit (1982). A similar study by Alfano et al. (1982) indirectly exposed suckling rats from PND-1 to 25 by feeding their dams chow containing 4 percent lead carbonate. In lead-treated animals raised in litters culled to 5 pups to compensate for undernutrition and compared with weight-matched controls, the mossy fiber afferent projection (MFP) to the hippocampus was significantly reduced in length and width, as was the hippocampus itself at PND-25. It should be noted that these reductions occurred in the absence of any brain weight changes and that some degree of recovery was observed by PND-60. The effect of lead on the development of the optic nerve was examined by Tennekoon et al. (1979) in suckling mice whose dams were exposed to 0.5 percent lead acetate in their drinking water. Lead-treated litters were culled to 3 pups to control for undernutrition. Electron microscopy on 21-day-old rats revealed a 10 to 15 percent reduction in the total number of axons in the optic nerve and fiber diameter histograms showed a marked skewing to smaller axon diameters. In addition, biochemical markers for myelin (myelin basic protein, 2',3'-cyclic nucleotide phosphodiesterase (CNP), and cerebroside sulfotransferase) were all reduced to as little as 60 percent of control values. However, since there was a linear correlation between axon diameter and myelin content in both experimental and control animals, it is unclear whether these biochemical reductions were direct effects of lead or reflective of secondary hypomyelination due to a reduction in axon diameter. Further studies on the demyelinating effects of lead were carried out by Windebank et al. (1980) on the peroneal, sural, and sciatic nerves of adult rats chronically exposed to 4 percent lead carbonate in their diet for up to 5 months, at which time they had PbB levels of 300 pg/dl. Animals receiving lead initially lost weight more rapidly than did pair-fed controls, but returned to normal within 5 months. Endoneurial water* content increased by 10 percent on PND-50 and remained elevated above controls through PND-150. Endoneurial lead content increased immediately upon treatment, reaching a peak at PND-35 that was almost 300 percent greater than the perineuria! fraction. These values gradually declined until PNO-100, when they were not different from controls. Demyelination of fibers began between PND-20 and 35, then became PB12B/A 12-116 1/05/83 TEH 0530979 DUP050031892 PRELIMINARY DRAFT progressively worse, although there was no correlation between endoneurial lead content and water content or teased fiber abnormalities. However, there did appear to be more of a correlation between endoneurial water content and demyelination. It is conceivable that lead causes an injury to the Schwann cells which precipitates demyelination, but is then removed by some mechanism before the injury manifests itself. Investigations into the effects of lead on the regeneration of crushed or transected peripheral nerves were carried out by Ohnishi and Dyck,(1981), who fed adult rats 4 percent lead carbonate in their diet for 3 months prior to producing injury in the sural nerve. Initially, the lead-treated animals exhibited a twofold increase in the number of fascicular Schwann cell nuclei when compared to their pair-fed controls. However, after nerve crush or transection, treated animals showed a 2-day lag in the reactive Schwann cell increase, which only reached 60 percent of the values exhibited by control animals. In addition, morphometric analysis of regenerating fibers 4 weeks after injury revealed reductions of 12 percent in the mean diameters of mye linated fibers, 181 percent in the number of myelin lamellae, and 32 percent in the cross sectional area of the axis cylinder. The authors concluded that chronic lead treatment induces a delay in the onset and peak of Schwann cell division and axonal regrowth in regenerating peripheral nerves, Organolead compounds have also been demonstrated to have a deleterious effect on the morphological development of the nervous system, Seawright et al. (1980) administered triethyl lead acetate (TEL) by gavage to weanling (40 to 50 gm) and "young adult" (120 to 150 gm) rats. Single doses of 20 mg TEL/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 exposure, with extensive neuronal necrosis evident in several brain regions by three days posttreatment. Weekly expsoures to 10 mg TEL/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, Bjorklund et al. (1980) placed tissue grafts of developing nervous 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 hippocampal, but not cerebellar, PB12B/A 12-117 1/05/83 TEH 0530980 DUP050031893 PRELIMINARY DRAFT 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 anamolous result might be explained by a hyperplasia of one particular cell type at lower concentrations of lead exposure. Organolead compounds have also been demonstrated to affect neuronal growth. Cultured cells from embryonic chick brain (Grundt et al., 1981) were exposed to 3.16 pM triethyl!ead chloride in the incubation medium for 48 hrs, resulting in a 50 percent reduction in the number of cells exhibiting pro cesses. There was no observed effect on glial morphology. Other investigations have focused on morphological aspects of the bloodbrain barrier and its possible disruption by lead intoxication (Kolber et al., 1980). Capillary endothelial cells isolated from rat cerebral cortex and exposed to 100 pM lead acetate in vitro (Silbergeld et al., 1980) were examined by electron microscopy and X-ray microprobe analysis. Lead deposits were found to be preferentially sequestered in the mitochondria of these cells in much the same manner as calcium. This affinity may be the basis for leadinduced disruption of transepithelial transport of Ca++ and other ions. 12.4.3.2.3 Summary of morphological effects of lead exposure. Recent key findings on the morphological effects of 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 chow. Higher exposure concen trations (0.2 to 0.4 percent lead salts) can reduce synaptogenesis and retard dendritic development in the hippocampus of developing rats (Campbell et al. (1982) and Alfano and Petit (1982), respectively). 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, 1890), or a reduction in cortical thickness at 4.0 percent lead carbonate exposure (Petit and LeBoutillier, 1979). This latter exposure PB12B/A 12-118 1/05/83 TEH 0530981 DUP050031894 PRELIMINARY DRAFT Table 12-5. Summary of Key Studies of Morphological Effects of Lead Exposure Species Young rats Adult rats Exposure protocol 0.1% Pb** in chow from PND-0 to 90 0.2% Pb(Ac)2 in dams1 drinking water from PND-0 to 25 0.4% PbCO, in dams' drinking water from PND-0 to 30 0.5% Pb(Ac)2 in dams' drinking water from PND-0 to 21 1% PbCO, in chow from PND-0 to 60 4% PbCO, in dams' chow from PND-0 to 28 4% PbCO- in dams' chow from PND-0 to 25 4% PbCO- in chow for 3 mos. Observed Effect Reference decreased density of oligodendrocytes In cerebral cortex Reyners et al. (1979) 15-30% reduction in synaptic profiles in hippocampus Campbell et al. (1982) retardation in temporal sequence of hippocampal dendritic development Alfano and Petit (1982) 10-15% reduction in number Tennekoon et al. of axons in optic nerve; (1979) skewing of fiber diameters to smaller sizes retardation of cortical synaptogenesis AveriH and Needleman (1980) 13% reduction in cortical thickness Petit and LeBouti11ier (1979) reduction in hippocampal length and width; similar reduction in afferent projection to hippocampus Alfano et al. (1982) delay in onset and peak of Schwann cell division and axonal regrowth in regenerating nerves Ohnishi and Dyck (1981) concentration also causes a delay in the onset and peak of Schwann cell divi sion and axonal regrowth in regenerating peripheral nerves in chronically-exposed adult rats (Ohnishi and Dyck, 1981). In summary, both neuronal and glial components of the nervous system appear to be affected by neonatal or chronic lead exposure. PB12B/A 12-119 1/05/83 TEH 0530982 DUP050031895 PRELIMINARY DRAFT 12.4.3.3 Electrophysiploglcal effects. 12.4.3.3.1 In vivo Studies. Investigations of electrophysiological para meters of the nervous system can provide sensitive indications of altered function. One such indicator is the maximal electroshock seizure (MES) response of experimental animals. Fox et al. (1978, 1979) measured the ontogeny of the MES response at PND-10, 12, 14, 16, 18, and 20 in suckling rats exposed to 0.02 or 0.2 percent lead acetate in their mothers' drinking water. Although lead treatment had no effect on standard developmental markers such as rate of body growth, time of eye-opening, and appearance of auditory startle response, testing of MES responses at PND-10 or 12 showed the 0.2-percent pups to have more severe seizures than the 0.02-percent and control animals. By PND-18 to 20, all groups showed comparable MES responses, although the high-lead group had markedly more severe seizures. Blood-lead levels were 25 and 90 pg/dl in the 0.02-percent and 0.2-percent groups, respectively (Fox et al., 1979). The authors speculated that neonatal lead treatment acts to increase the ratio of excitatory to inhibitory systems in the developing cerebrospinal axis. Suckling rats exposed to 0.2 percent lead acetate in their mothers' drinking water from PNO-1 to 21 (Fox et al., 1977; Impelman et al., 1982) in litters which were culled to 6 pups to control for the possible effects of undernutrition showed no changes in rate of brain- or body weight growth, time of eye-opening, or other developmental landmarks while they were maturing (average PbB level of 65 pg/dl was recorded on PND-21). Yet even at PND-100, their visual evoked responses (VERs) showed longer latencies of the primary and secondary components and altered waveforms, as compared to controls. Development of the VER had been 2 times slower than in control adults, and these rats (which had only been exposed to lead during suckling) showed an increased latency of response to paired flashes and decreased ability to follow repetitive flashes of light. These results seem to indicate that early exposure to lead can lead to permanently altered VERs, probably due to decreased conduction velocities in one or more of the visual projection pathways (Cooper et al., 1980; Impelman et al., 1982). Similar effects of lead on the VER were observed by Winneke (1980) after rats were exposed to 0.2 percent acetate in their diets from birth until 12 months of age. It should be noted here, however, that Feeney et al. (1979) found somewhat different results in adult (70- to 80-day-old) rats exposed indirectly from conception until weaning to PB12B/A 12-120 1/05/83 TEH 0530983 DUP050031896 PRELIMINARY DRAFT 750 mg lead acetate/kg body weight in their dams' drinking water. This exposure caused a decreased latency only in the P2 component of the VER. The authors suggest that low-level perinatal lead exposure may damage the inhibitory systems which impinge on the visual projection pathways. Fox and Wright (1982) exposed suckling rats via their dams' milk to 0.2 percent lead carbonate in water from parturition until weaning (PND-21), then examined the effect of this exposure on visual acuity. Lead-exposed rats exhibited 25 to 50 percent decreases in scotopic acuity and spatial resolu tion, which is a function of the decreased amplitudes and increased latencies of the VER previously observed with this exposure protocol (Cooper et al,, 1980; Fox et al., 1977; Impelman et al., 1982). This same phenomenon was observed by Bushnell et al. (1977) in young rhesus monkeys chronically exposed from birth until PND-365 to solutions of lead acetate sufficient to maintain blood-lead levels of either 55 pg/dl ("low-lead" group) or approximately 85 pg/dl ("high-lead" group). [Note: The "high-lead" group exhibited peak PbB levels as high as 300 pg/dl during the first 2 months of life.] At 18 months after cessation of lead exposure, when blood-lead levels had declined to essentially normal values, the high-lead group showed a severe impairment of discrimination accuracy at differing light intensities. This finding was interpreted by the authors to be a reflection of a loss of scotopic function. Recent studies of the electrophysiological effects of lead exposure on the peripheral nervous system of experimental animals have been limited to that by Hietanen et al.(1980), who exposed adult rabbits to 0.2 percent lead acetate in their drinking water for 4 weeks. They found that motor conduction velocity in the sciatic nerve was reduced to only 15 percent of control. 12.4.3.3.2 In vitro Studies, Palmer et al. (1981) and Olson et al. (1981) looked at intraocular 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 to 5 months. They found no alter ations 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 control animals all exhibited significant levels of spontaneous activity. Taylor et al. (1978) recorded extracellularly from cerebellar Purkinje cells in adult rats both in situ and in intraocular grafts in an PB12B/A 12-121 1/05/83 TEH 0530984 DUP050031897 PRELIMINARY DRAFT effort to determine what effect lead had on the norepinephrine (NE)-induced inhibition of Purkinje cell spontaneous discharge. Application of exogenous NE to both In situ and j u j oculo cerebellum produced 61 and 49 percent inhibi tions of spontaneous activity, respectively. The presence of 5 to 10 pM lead reduced this inhibition to 28 and 13 percent, respectively. This "disinhibition" was specific for NE, as responses to both cholinergic 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 pM). Evidence that lead does indeed resemble other divalent cations, in that it appears to interfere with chemically-mediated synaptic transmission, has been demonstrated in studies of peripheral neural functions. Kostial and Vouk (1957) reported that in vitro perfusion of the cat superior cervical ganglion with 4.8 pM lead nitrate depressed or blocked nerve transmission. Contraction of the nictitating membrane during acetylcholine (ACh) perfusion was unaltered. Also, perfusion of the ganglion with excess calcium (Ca*4) restored ACh release and thus reversed the lead blockade. From these findings Kostial and Vouk concluded that lead depressed synaptic transmission by impairing ACh release from the presynaptic terminals. Manalis and Cooper (1973) and Cooper and Manalis (1974) confirmed the findings of Kostial and Vouk (1957) that lead depresses the phasic release of transmitter evoked by nerve stimulation. They further observed that lead increases spontaneous release of ACh, as evidenced by increased miniature end-plate potentials (MEPP's). Kolton and Yaari (1982) found that this leadinduced increase in spontaneous MEPP's in the same preparation could be observed at concentrations of lead as low as 5 pM, Kober and Cooper (1976) demonstrated that, in the frog, lead blocks synaptic transmission in the sympathetic ganglion .j. by competitive antagonism of spike-evoked entry of Ca into the presynaptic nerve terminals, with a resultant reduction in ACh release. Experiments by Silbergeld et al. (1974a,b) indicated a similar blockade of ACh release by lead in the rat and in the phrenic-nerve/diaphragm preparation from mice exposed to lead from birth through PND-60. PB12B/A 12-122 1/P5/83 TEH 0530985 DUP050031898 PRELIMINARY DRAFT Lead is also capable of blocking neural transmission at peripheral adre nergic 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 an effect on the muscle* Since the response recovered when Cai++ concentration was increased in the bathing solution, it was concluded that lead does not deplete transmitter stores in*the nerve terminals, but more likely blocks NE release. 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/day for 5 days and looked at Ca** retention in brain slices. Treated animals showed a 75 percent increase in retention time, indicating that lead inhibited the mediated efflux of Ca** from the incubated brain slice. Investigation of the in vitro effects of lead on Ca4*'4' binding was carried out by Silbergeld and Adler (1978) on caudate synaptosomes, They AC ^.4, determined that 50 pM lead caused an 8-fold increase in Ca binding and that in both control and lead-treated preparations addition of ATP increased -j-j. binding, while Rutheniun red and Ca decreased it. Further findings in this series of experiments demonstrated that lead inhibits the Na*-stimulated loss of Ca*+ by mitochondria and that blockade of dopamine (DA) uptake by 5 pM benztropine reversed the lead-stimulated increase in Ca uptake by synaptosomes. The authors concluded that lead affects the normal mechanisms of Ca binding and uptake, perhaps by chelating with DA in order to enter the nerve terminal. By inhibiting the release of Ca bound to mitochondria there, lead essentially causes an increase in the Ca concentration gradient across the nerve terminal membrane. As a result, more Ca would be expected to enter the nerve terminal during depolarization, thus effectively increasing synaptic neurotransmission at dopaminergic terminals without altering firing rates. Fox and Siliman (1979) looked at receptor potentials the isolated, per fused 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 pM produced an average 16 percent depression, while 12.5 pM produced an average 23 percent depression. PB12B/A 12-123 1/05/83 TEH 0530986 DUP050031899 PRELIMINARY DRAFT 12.4.3.3.3 Summary of electrophysiological effects of lead exposure. Recent key findings on the electrophysiological effects of lead exposure are sum marized below in Table 12-6. The visual system appears to be particularly susceptible to perturbation by neonatal lead exposure. Suckling rats whose dams were given drinking water containing 0.2 percent lead acetate had signi ficant alterations in their visual evoked responses (VER) and decreased visual acuity at PND-21, at which time their blood-lead levels were 65 pg/dl (Cooper et al., 1980; Fox et al., 1377, 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 (Fox et al, 1978, 1979) who exhibited blood-lead levels of 90 pg/dl. The authors speculated that neonatal lead exposure acts to increase the ratio of excitatory to inhibitory systems in the developing cerebrospinal axis. 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 velocity in the sciatic nerve. Recent in vitro studies have revealed that lead has electrophysiological effects at extremely low in situ concentrations. For example, concentrations as low as 5 pM have been shown to increase the frequency of MEPP's at the frog neuromuscular junction (Kolton and Yaari, 1982) and to decrease rod receptor potentials in the frog retina (Fox and Siliman, 1979). Only 10 pM lead was required to cause signficiant disinhibition of the norepinephrine (NE>-induced reduction in spontaneous activity of cerebellar Purkinje cells (Taylor et al., 1978). This effect was apparently specific for NE, as responses to both cholinergic and parallel fiber stimulation of these cells remained the same. j. i Lead also appears to affect the normal mechanisms of Ca and uptake binding by nerve terminals. Silbergeld and Adler (1978) showed that 50 pM lead caused an 8-fold increase in Ca binding in caudate synaptosomes, perhaps by chelating with dopamine (DA) to enter the nerve terminal. By inhibiting the release of Ca4+ bound to mitochondria there, lead essentially causes an increase in the Ca++ concentration gradient across the nerve terminal membrane. As a result, more Ca would be expected to enter the nerve terminal during depolarization, thus effectively increasing synaptic neurotransmission at dopaminergic terminals without altering firing rates. PB12B/A 12-124 1/05/83 TEH 0530987 DUP050031900 PRELIMINARY DRAFT Table 12-6. Summary of Key Studies of Electrophysiological Effects of Lead Exposure Species Suckling rat Exposure protocol 0.2%.Pb (Ac), in dams1 drinking water from PND-Q to 20 0.2% Pb(Ac), in dams' drinking water from PND-0 to 21 Adult rabbit 0.2% Pb (Ac), in drinking water for 4 wk. Adult frog 5 gM Pb in vitro Adult frog 5-12.5 yM Pb in vitro Adult rat 10 uM Pb in vitro Adult rat 50 yM Pb in vitro dbserved Effect more severe MES response Reference Fox et al. (1978, 1979) 1) increased latencies and decreased amplitudes of primary and secondary components of VER; 2) decreased visual acuity; 3) decreased conduction velocities in visual pathways 85% reduction in motor conduction velocity of sciatic nerve increased MEPP's at neuromuscular junction depression of rod receptor potentials in retina reduction of NE-induced inhibition of spontaneous activity of cerebellar Purkinje cells infeased binding of Ca in caudate synaptosome mi tochondr.ia Fox et al. (1977,1982) Fox and Wright (1982) Cooper et al, (1980) Winneke (1980) Hietanen et al. (1980) Kolton and Yaari (1982) Fox and $iliman (1979) Taylor et al. (1978) Si1bergeld and Adler (1978) 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 pertubations of various neurotransmitter systems. Early research on the effects of lead exposure on neurotransmitter systems concentrated primarily on cholinergic and monoaminergic functions, probably because of the extensive background literature that existed on the basic neurochemistry of PB12B/A 12-125 1/05/83 TEH 0530988 DUP050031901 PRELIMINARY DRAFT those transmitters and because of the documentation extant on the neurophysio logical 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 or glucose transport. A significant portion of this work has, however, been conducted in vitro. 12.4.3.4.1 In vivo Studies. McCauley and Bull (1978) and McCauley et al. (1979) indirectly exposed suckling rats from conception until weaning by giving their mothers 0.02 percent lead chloride in drinking water. Pups at PND-21 exhibited PbB levels of 36 pg/dl. Synapse formation in PND-15 pups was decreased approximately 15 percent and showed a less mature profile in cere bral cortex, slices of which were also examined for effects on energy metab olism. Oxygen and glucose consumption in response to potassium stimulation were increased 40 and 50 percent, respectively, in lead-treated animals, indicating a possible uncoupling of energy metabolism. Bull et al. (1979) examined the development of cytochrome content in the cerebral cortex of rat pups who were indirectly exposed to 0.005 to 0.02 percent lead chloride in their dam's drinking water from conception through weaning. Although the lead-treated pups exhibited no differences in brain or body weight from their controls, there was a 30 percent reduction in cytochrome content between PND-10 and 15 in the 0.02-percent group. This decrease was transient, how ever, and disappeared by PND-21. Other recent studies have focused on the ability of lead to alter heme synthesis and the possible neurological consequences of this, Silbergeld and Lamon (1980) pointed out that 6-ALA, the primary biochemical product which accumulates as a result of lead inhibition of heme synthesis, is very similar in structure to the neurotransmitter y-arainpbutyric acid (GABA). They suggest that this structural similarity accounts for the ability of ALA to displace GABA (albeit weakly) from post-synaptic binding sites and to block its uptake, which is the primary synaptic inactivation process for this particular neuro transmitter. It is therefore possible that ALA, as a product of lead-altered heme synthesis, has a direct toxic effect on GABAergic neuronal systems. (See Section 12.3.5 for a complete discussion of the interrelationship between the effects of lead on the hematopoietic and nervous systems). PB12B/A 12-126 1/05/83 TEH 0530989 DUP050031902 PRELIMINARY DRAFT Most of the more recent studies on the effects of lead on neurotransmitter systems have dealt with alterations of dopamine (DA) function. A series of studies carried out by Govoni and co"Workers indirectly exposed suckling rats to 0.25 percent lead acetate in their dams' drinking water until weaning, then exposed them to the same water until PND-35 (Govoni et al., 1978a). Lead-treated rats weighed 10 to 15 percent less than controls, but did not exhibit any other obvious manifestations of lead toxicity. They did exhibit a 20 percent decline in striatal concentration of the DA metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC). Inhibition of DA synthesis by a-methylparatyrosine (ofMPT) caused no significant decline in levels of DOPAC or HVA in lead-treated animals, while controls showed a 50percent drop. These data indicate an overall decline in the synthesis and turnover of striatal DA following lead exposure. Later studies by this group (Govoni et al., 1979, 1980; Memo et al., 1980a, 1981) replicated these results in the striatum, even at lead concentra tions as low as D.004 percent, but found DA synthesis in nuc. accumbens and frontal cortex to be increased by 10 to 30 and 35 to 45 percent, respectively. Synthesis of DA in substantia nigra was unaffected, nor was any effect of lead found on spiroperidol binding or adenylate cyclase activity in these areas. Injections of d-amphetamine (3 mg/kg body weight, i.p.) in 42-day-old animals' (Memo et al., 1980a) lowered DOPAC levels in both groups by approximately 35 percent. However, when Lucchi et al. (1981) looked more closely at DA receptor binding properties, using the same protocols reported in Govoni et al. (1978, 1979, 1980) and Memo et al. (1980, 1981), they found that while neither adenyl cyclase activity nor spiroperidol-specific binding (to D-^ + D^ receptors) were altered in striatum and nuc. accumbens of control and lead-treated rats, sulperide-specific binding (to D2 receptors only) in treated animals was elevated by 50 percent and diminished by 33 percent in striatum and nuc. accumbens, respectively. The elevated binding in striatum might not only be a reflection of sensitization phenomena resulting from decreased DA turnover and concentration (Govoni et al ., 1979, 1980), but might also indicate that a specific subset of DA receptors is affected. PB12B/A 12-127 1/05/83 TEH 0530990 DUP050031903 PRELIMINARY DRAFT Wince et al. (I960) examined DA synthesis, uptake and release in the forebrain of young rats who were exposed from birth until weaning by feeding their dams chow containing 4 percent lead carbonate, followed by a 0.004 percent lead carbonate-containing diet until PND-35. (This latter concentra tion of lead was equivalent to what they received in maternal milk during suckling.) Lead-treated rats showed no signs of encephalopathy. Although there were no significant differences in the synthesis of DA in forebrain synaptosomes or its uptake or amphetamine-stimulated release by forebrain slices, concentration-dependent, KC1-stimulated release of DA from these slices was reduced up to 15 percent at KC1 concentrations >15 mM. Further more, DA- or apomorphine*stimulated adenylate cyclase activity in neostriatal homogenates was decreased 10 and 35 percent, respectively. Lead carbonate alone had no such effect in vitro. A similar study was carried out by Jason and Kellogg (1981), who gave rat pups 25 or 75 mg lead acetate/kg body weight/day via gastric intubation from PND-2 to 14, No differences in growth patterns or histopathologic lesions were found in the brains of treated animals, yet PbB levels were 50 and 98 pg/dl at PND-15; by PND-35 these values had declined to 4 and 10 pg/dl for the 25 and 75 pg treatments, respectively. However, at both PND-15 and 35, 'striatal DA concentration was depressed by 20 percent in the 75 mg-treated (Pb75) rats. Inhibition of DA synthesis by aMPT revealed a decreased turnover rate begin ning at PND-18 which culminated in a 35 percent decline by PND-35. Uptake of DA into striatal slices from Pbyg rats was reduced 50 to 60 percent compared with controls or Pb^g rats, but only at PND-15. There were no apparent dif ferences in KCl-stimulated release of DA from striata of any animals. The authors concluded that at PND-15, lead may interfere with mechanisms that sustain DA levels, such as membrane-dependent processes. By PND-35, higher lead concentrations have reduced the turnover rate, but are not affecting maintenance systems. The lowered DA concentration found at PND-35 may there fore be a reflection of decreased dopaminergic nerve terminal density. The effects of neonatal lead exposure on norepinephrine (NE) chemistry in suckling rats indirectly exposed to 0.05, 0.1, or 0.2 percent lead acetate in their dams' drinking water were investigated by Goldman et al. (1980). Litter PB12B/A 12-128 1/05783 TEH 0530991 DUP050031904 PRELIMINARY DRAFT sizes were not culled to control for possible undernutrition, but equivalent volumes of water were consumed by control dams. While no differences in behavior or time of eye-opening were observed in any group, the 0.2-percent rats (whose PbB levels averaged 47 pg/dl) showed a 23 percent increase in NE concentration in hypothalamus and striatum, but no change in brainstem or cerebellum on PND-21. Activity of catechol-0-methyl transferase, the enzyme responsible for degradation of NE, was unaltered in any region studied, although the activities of both dopamine B-hydroxylase (reflective of the rate of HE synthesis) and phenylethanolamine N-methyltransferase (reflective of the rate of NE conversion to adrenaline) were elevated by 36 and 42 percent, respec tively, in the brainstem of 0.2 percent-treated rats. This would seem to indicate an increased turnover of NE in brainstem, and perhaps a decrease in turnover rate in hypothalamus and striatum. Wysocka-Paraszewska and Blel-Baranowska (1979) found decreases in whole brain NE concentration after chronic treatment (12 weeks) of adult rats with 0.2 percent lead acetate in drinking water. When Ewers and Erbe (1980) looked at adenylate cyclase activity in adult rats exposed for 12 weeks to diets con taining 0.15, 0.4, or 1 percent lead acetate, they found 10 to 30 percent reductions in NE levels in cerebellum and brainstem, although these decrements were not statistically significant. These treatments produced PbB levels of 39, 61 and 122 gg/dl, respectively. Dubas et al, (1978) examined the effects of chronic lead exposure on levels of all the biogenic amine neurotransmitters [DA, NE, serotonin (5-HT)] in the CNS of rats. Suckling pups were indirectly exposed to 2 percent lead acetate in their dams' drinking water from birth until weaning, then received 0.002, 0.004 or 0.008 percent in their own drinking water until 8 weeks of age. They exhibited no gross behavioral changes alterations in body weight gain, from controls. Regional analyses of neurotransmitter levels revealed non-dose-dependent elevations of NE by 60 to 90 percent in midbrain, depression of NE in hypothalomus and striatum by 20 to 30 percent, and elevations of DA and 5-HT in midbrain, striatum and hypothalamus by 15 to 30 percent. However, when Dubas and Hrdina (1978) repeated the experiment using higher doses and longer exposure times, the results were quite different. Rat pups received 50 pg lead acetate by gavage from birth until weaning, then 0,008 percent lead in their drinking water for 8 to 12 weeks. Lead-treated rats showed no differences PB12B/A 12-129 1/05/83 TEH 0530992 DUP050031905 PRELIMINARY DRAFT in body weight gains, time of eye-opening, etc. The highest concentrations of lead were found in hypothalamus and striatum* which had much more than cortex or midbrain. Eight-week-old animals showed 26 percent decreases in NE content of striatum and cortex, 20 percent decreases in DA content of hypothalamus, cortex and midbrain, and 11 to 20 percent decreases in 5-HT in hypothalamus and cortex, interestingly, the NE content of midbrain was elevated by 16 percent. By 12 weeks, however, the only changes noted in treated animals were 10 to 30 percent reductions in 5-HT content of striatum and cortex. Neonatal exposure to lead also seems to affect the cholinergic systems of the CNS. Modak et al. (1978) indirectly exposed suckling mice via their dams' drinking water to 0.25, 0.5, or 1 percent lead acetate from birth until weaning, at which time they continued on the same solution these respective mothers had received. Blood-lead levels at PND-21 were 72, 70, and 91 pg/dl, respectively. While only the 1 percent lead treatment retarded body growth, all 3 concentra tions decreased whole brain acetylcholine (ACh) content 40 to 50 percent by PNQ-21 and approximately 36 percent by PND-30, Although the effect was transi tory (animals returned to control values by PND-60), the regional differences observed at PND-30 were striking. For example, treatment with 1 percent lead reduced the ACh content of the midbrain, diencephalon, and hypothalamus 15 to 20 percent, while ACh in the cerebellum and striatum was reduced by 34 and 51 percent, respectively. Whether these decreases were due to decreased avail ability of choline, increased ACh turnover, and/or decreased synthesis is not known, Louis-Ferdinand et al. (1978) chose to measure cholinergic enzyme activity, rather than ACh content, in the CNS of neonatal rats given injections of 7.5 mg lead acetate/kg body weight (i.p.) from birth until PND-10, Animals sacri ficed on PND-10, 15, 20, or 30 were examined for acetylcholinesterase (AChE) and butyrocholinesterase (BuChE) activities. On PND-10, AChE activity was depressed by 40 percent in the hippocampus and 17 percent in the medulla, but not at all in the striatum, cerebellum, midbrain, or cerebral Cortex. At PND-20, the only region exhibiting a reduction was the cerebral cortex (17 percent). By PND-30, AChE activity in all areas appeared normal. In contrast, BuChE activity (which is regarded as a "marker" for glial cells and the bloodbrain barrier) was decreased by approximately 35 percent in cerebrum, hippo campus, and midbrain of 15- and 20-day-old rats, but it, too, returned to PB12B/A 12-130 1/05/83 TEH 0530993 DUP050031906 PRELIMINARY DRAFT normal by PND-30. The concurrent observation of 10 to 15 percent reductions In both hippocampal cell layer thickness and distance from the hippocampus to the cerebral cortex tend to suggest that early lead exposures can cause marked morphological changes in these systems which outlast perturbations of cholin ergic neurotransmission. Studies of the effects of neonatal lead exposure on the development of CNS pathways utilizing GABA as a neurotransmitter have been done by Govoni et al. (1978b, 1980), who indirectly exposed rats from gestation until weaning to 0.25 percent lead acetate in their dams' drinking water, then continued the pups on the same solution until PNO-42. (Blood- and brain-lead concentrations at this time were 87 pg/dl and 85 pg/dl, respectively.) GABA-specific binding 3 sites, as measured with H-GABA, were increased 31 percent in cerebellum and decreased 36 percent in striatum. No alterations were observed in substantia nigra, nuc. accumbens, hypothalamus, or cerebral cortex. Cyclic guanosine 5-phosphoric acid (GMP) activity paralleled these findings, being elevated 53 percent in Cerebellum and decreased 47 percent in striatum, while remaining unchanged elsewhere. Memo et al, (1980b) also looked at the effects of chronic lead treatment on GABA systems in the rat. Indirect exposure to 0.25 percent lead acetate in the dams' drinking water began at birth, followed by weaning to water con taining 0.004 or 0.25 percent lead acetate until PNO-42. They, like Govoni et al., found GABA-specific binding to be elevated, by 12 and 34 percent in the cerebellum of both the 0.004- and 0.25-percent groups, respectively, and de creased in striatum by 20 to 45 percent, depending on lead dose. Cyclic GMP levels paralled these receptor alterations, being elevated 25 and 50 percent in cerebellum and depressed 20 and 50 percent in striatum of 0.004 and 0.25 percent rats, respectively. The authors suggest that decrements in striatal DA function, which may represent disinhibition of GABA systems, might enhance GABA turnover and perhaps lead to a decrease of GABA receptor function. Silbergeld et al. (1979, 1980) treated suckling rats with 0.5 to 1 percent lead acetate in their dams' drinking water, then weaned the pups to the same lead-contaminated water for 45 to 60 days. Treated rats were more sensitive to seizures induced by the GABA blockers picrotoxin, isoniazid, and strychnine, although this sensitivity was not correlated with lead concentration, GABA levels in cortex, caudate nucleus, substantia nigra, and cerebellum were all PB128/A 12-131 1/05/83 TEH 0530994 DUP050031907 PRELIMINARY DRAFT normal in 0.5 percent-treated (Pbg g) animals, but depressed by 20 percent in cerebellum of 1 percent-treated (Pb-^) rats. GABA synthesis, as measured by glutamic acid decarboxylase activity, was elevated by 18 and 27 percent in caudate of PBq g and Pb^ animals, respectively. GABA-transaminase (GABA-T) activity in cortex of PBq g rats was decreased by 23 percent. The overall indication that lead treatment caused an increase in GABA synthesis was re inforced by the finding that accumulation of GABA after pharmacological inhi bition of GABA-T was up to twofold greater in cortex and striatum of PB1 rats. Synaptosomes from cerebellum, substantia nigra, and caudate (but not cortex) of Pbp g and Pb^ animals exhibited 5 to 25 percent and 25 to 50 percent inhi bitions of GABA uptake, respectively; both resting and KC1-stimulated release of GABA by these same synaptosomes was also inhibited, although in a non-dosedependent manner. Specific binding of H-GABA to receptors in cerebellum increased by 70 percent. The authors concluded that lead affects GABA presynaptic function, causing an increased sensitivity of postsynaptic receptors, thereby sensitizing animals to convulsive agents. 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/day by intragastric gavage. On PND-25, they found that even 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 pM produced the same effects. Purdy et al. (1981) examined the effects in rats of varying concentra tions of lead acetate on the whole-brain synthesis of tetrahydrobiopterin (BH^), a cofactor for many important enzymes, including those regulating catecholamine synthesis. Concentrations of lead as low as 0.01 pM produced a 35 percent inhibition of BH^ synthesis, while 100 pM inhibited the BH^ salvage enzyme, dihydropteridine reductase, by 40 percent. This would result in a decreased conversion of phenylalanine to tyrosine and thence to DOPA (the initial steps in dopamine synthesis), as well as decreases in the conversion of trytophan to its 5-hydroxy form (the initial step in serotonin synthesis). These decrements, if occurring ijn vivo, could not be ameliorated by increased dietary intake of BH^, as it does not cross the blood-brain barrier. PB12B/A 12-132 1/05/83 TEH 0530995 DUP050031908 PRELIMINARY ORAFT Lead has also been found to have an inhibitory effect on mitochondrial respiration in the cerebrum and cerebellum of immature or adult rats at con centrations greater than 50 pH (Holtzman et al., 1978). This effect, which was equivalent in both brain regions at both ages studied, is apparently due to an inhibition of NAD-linked dehydrogenases within the mitochondrial matrix. These same authors found that this lead-induced effect, which is an energydependent process, could be blocked in vitro by addition of Ruthenium red to the incubation medium (Holtzman et al., 1980). In view of the fact that Ca uptake and entry into the mitochondrial matrix is also blocked by Ruthenium red, it is possible that both lead and Ca share the same binding site/ carrier in brain mitochondria. These findings are supported by the work of Gmerek et al. (1980) on adult rat cerebral mitochondria, with the exception that they observed respiratory inhibition at 5pM lead acetate, which is a full order of magnitude lower than the Holtzman et al. (1978, 1980) 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 concentrations of triethyl lead (TEL) chloride as low as 5 to 7 pM caused a 40 percent decrease in the incorporation of S0^ or serine into myelin galacto-lipids in cerebellar slices from 2-week-old rats. Similarly, Konat and coworkers (Konat and Clausen, 1977, 1980; Konat et al., 1979) observed that 3 pM TEL chloride preferentially inhibited the incor poration 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 protein synthesis (approxi mately 10 vs, 20 percent, respectively). In addition, acute intoxication of these animals by i.p, injection of 8 mg TEL chloride/kg produced equivalent 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 pM TEL chloride (the lowest concentration examined), a cell-free system prepared from the same tissue Was not affected by TEL chloride concen trations as high as 200 pM. This result, coupled with a similar, although not PB12B/A 12-133 1/05/83 TEH 0530996 DUP050031909 PRELIMINARY DRAFT as severe, inhibitory effect of TEL chloride on oxygen consumption in the cell suspension (20 percent inhibition at 20 pM) would tend to indicate that the inhibition of rat forebrain protein synthesis is related to an inhibition of cellular energy-generating systems. The effects of organslead compounds on various neurotransmitter systems have been investigated in adult mouse brain homogenates. Bondy et al. (1979a,b) demonstrated that micromolar concentrations (5 pM) of tri-n-butyl lead (TBL) acetate were sufficient to cause not only a 50 percent decline in the high affinity uptake of BABA and DA in such homogenates, but that TBL acetate stimulated a 25 percent increase in GABA and OA release. These effects are apparently selective for DA neurons at lower concentrations, as only DA uptake or release was affected at 0.1 pM, albeit mildly so. The effect of TBL acetate on DA uptake appears to be specific, as there is a clear dose response rela tionship down to 1 pM TBL (Bondy and Agarwal, 1980) for inhibition (0 to 60 percent) of spiroperidol binding to rat striatal DA receptors. A concomitant inhibition of aderiyl cyclase in this dose range (50 percent) suggests that TBL may effect the entire postsynaptic binding site for DA. 12.4.3.4.3 Summary of Biochemical effects of lead exposure. Recent key findings on the biochemical effects of lead exposure are summarized below 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 particularly vulnerable to perturbation by lead exposure. McCauley and coworkers have demonstrated that exposure of suckling 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 PNQ-21, it occurs at blood-lead levels as low as 36 pg/dl (McCauley and Bull, 1978; McCauley et al., 1979). There does not appear to be a selective vulnerability of one or another of the more well-studied neurotransmitter systems to the effects of lead exposure. Pathways utilizing dopamine (DA), norepinephrine (ME), serotonin (S-H7) and y-aminobutyric acid (GABA) are all perturbed in neonatal animals at lead-exposure concentrations of 0.2 to 2.0 percent lead salts in dams' drinking water. Although the blood-lead values reported following exposure to the lower lead concentrations (0.2 to 0.25 percent lead acetate or lead chloride) PB12B/A 12-134 1/05/83 TEH 0530997 DUP050031910 PRELIMINARY DRAFT Table 12-7. Summary of Key Studies on Biochemical Effects of Lead Exposure Species Suckling rat Exposure protocol 0.02% PbCl2 in dam's drinking water from PND-0 to 21 0.2% Pb(Ac)2 in dam's drinking water, from PND-0 to 21 0.25% Pb(Ac)? in dam's drinking water from PND-0 to 35 0.25% Pb(Ac)2 in dam's drinking water from PND-0 to 35 0.25% Pb(Ac)2 in dam's drinking water from PND-0 to 35 0.25% Pb(Ac)2 in dam's drinking water from PND-0 to 42 0.25% Pb(Ac)2 in dam's drinking water from PND-0 to 21; 0.004% or 0.25% unit! PND-42 Observed Effect Reference 1) 30% reduction in cyto chrome. content of cerebral cortex; 2) possible uncoupling of energy metabolism McCauley and Bull (1978) McCauley et al. (1979) Bull et al. (1979) 1) 23% decrease in NE levels of hypotholamus and striatum; 2) increased turnover of NE in brainstem Goldman et al. (1980) decline in synthesis and turnover of striatal DA Govoni et al. (1978a) increase in DA synthesis in frontal cortex and nuc. accumbens Govoni et al. (1979, 1980) Memo et al. (1980a, 1981) 1) 50% Increase in DA Lucchi et al. binding to striatal (1981) D, receptors; 2) 33% decrease in DA binding to nuc. accumbens D2 receptors 1) 31% increase in GABA Govoni et al. specific binding in (1978b, 1980) cerebellum; 53% increase in GMP activity; 2) 36% decrease in GABA- specific binding in striatum; 47% decrease in GMP activity 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 PB12B/A 12-135 1/05/83 TEH 0530998 DUP050031911 PRELIMINARY DRAFT Species Young rat Adult rat Suckling rat Adult rat Adult rats Table 12-7. (continued) Exposure protocol Observed Effect Reference 0.5 to 1% Pb(Ac)2 in drinking water from PND-0 to 60 1) increased sensitivity Silbergeld et a to seizures induced (1979, 1980) 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 2% Pb(Ac)? in dam's drink ing water^from PND-0 to 21 then 0.002-0.008% until PND-56 1) non-dose-dependent Dubas et al. elevations of NE in (1978) 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%). 0.1 uM Pb in vitro 35% inhibition of wholebrain BH^ synthesis Purdy et al. (1981) 0.1 uM Pb in vitro blockade of sugar specific transport sites in capil lary endothelial cells Kolber et al. (1980) 5 mM Pb in vitro inhibition of cerebral mitochondrial respira tion Gmerek et al. (1980) 50 uM Pb in vitro inhibition of cerebral and cerebellar mitochondrial respiration Holtzman et al. (1978, 1980) PB12B/A 12-136 1/05/83 TEH 0530999 DUP050031912 PRELIMINARY DRAFT range from 47 pg/dl (Goldman et al., 1980) to 87 pg/dl (Govoni et al., 1980), a few general observations can be made: 1. Synthesis and turnover of DA and N are depressed in the striatum, and elevated in midbrain, frontal cortex, and nuc. accumbens.* This seems to be paralleled by concomittant increases in DA-specific binding in striatum and decreases in DA-specific binding in nuc, accumbens, possibly involving a specific subset (Dg) of DA receptors (Lucchi et al., 1981), These findings are probably reflective of sensitization phenomena resulting from changes in the availability of neurotransmitter at the synapse. 2. The findings for pathways utilizing GABA show similar parallels. Increases in GABA synthesis in striatum are coupled with decreases in GABA-specific binding in that region, while the converse holds true for the cerebellum. In both eases, cyclic GMP activity mirrors the apparent changes in receptor function. This increased sensitivity of cerebellar postsynaptic receptors (probably a response to the lead-induced depression of presynaptic 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), In vitro studies reveal that certain aspects of intermediary metabolism are especially vulnerable to perturbation by lead exposure. Concentrations of lead as low as 0.1 pM can cause marked inhibitions of tetrahydrobiopterin ((BHy) synthesis in the brains of adult rats, or effectively block the sugarspecific transport sites in capillary endothelial cells of neonatal rat brain. Other studies have shown that cerebral mitochondrial respiration can be inhib ited at 5 pM exposure. 12.4.3.5 The relationship between levels of lead in blood and the brain. All top infrequently, experimental studies of the neurotoxic effects of lead exposure do not report the blood-lead (PbB) levels achieved by the expo sure protocols used. Even less frequently reported are the concomittant tissue levels found in brain. From the recent information that is available, however, it is possible to draw some limited conclusions about the relation ship 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. 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O w 00 H c-- Ml <4 D CD Q 30 H> "3 3 O TEH 0531004 DUP050031917 PRELIMINARY DRAFT The most thorough information comes from studies of neonatal animals. For example, Fox et al. (1979) exposed suckling rats from birth through PND-21 to either 0.02 or 0.2 percent lead acetate in their dams1 drinking water. Blood-lead and brain-lead levels at PND-10 were 21.7 pg/dl and 0.063 pg/g for 0.02 percent exposure, and 49,6 pg/dl and 0.19 pg/g for 0,2 percent.- At PND-21, the values were 25 pg/dl and 0,13 pg/g, and 90 pg/dl and 0.82 pg/g, respectively. An earlier study by Fpx et al, (1977), looking only at 0.2 percent lead acetate in the same exposure protocol, yielded blood- and brainlead levels at PND-21 of 65 pg/dl and 0.53 pg/gm, respectively. Virtually identical values are reported by Cooper et al. (1980). Similar results were also obtained by Hastings et al. (1979), who exposed suckling rats from birth until weaning to either 0.02 or 0.2 percent lead acetate in their dams' drink ing water. On PND-21, blood-lead levels in the pups were 29 and 65 g/dl for the 0.02 and 0.2 percent exposures, respectively, while brain-lead levels were 29 and 65 g/lOOg, respectively. A study by Bull et al. (1979) measured blood- and brain-lead levels in suckling rats exposed from birth to PND-21 to .0005, 0.003, and 0.02 percent lead chloride in their dams' water. Blood-lead values at each of these con centrations were 12, 21, and 36 pg/dl, respectively, while brain-lead levels were 0.08, 0.11, and 0.25 pg/g, respectively. Grant et al. (1980) exposed rats from conception to 0,005 and 0.01 percent lead acetate in their dams' drinking water. Blood-lead levels for these two exposure concentrations at PND-11 were 22 and 35 pg/dl, while brain-lead levels were 0.03 and 0.07 pg/g, respectively. At PND-30, these values Were: PbB - 18 and 48 pg/dl, and brainlead ~ 11 and 22 g/g, respectively. Looking at higher concentrations, Goldman et al. (1980) measured blood- and brain-lead levels in suckling rats exposed from birth to PND-21 to 0,05, 0,1, and 0.2 percent lead acetate in their dams' drinking water. Blood-lead values were 12, 20, and 42 pg/dl, respectively, while brain-lead levels were 0,2, 0.5, and 0.8 pg/g, respectively. Using even higher concentrations in the same protocol (0.25, 0.5, or 1 percent lead acetate from birth to PND-21), Modak et al. (1978) observed blood-lead levels of 72, ,70, and 91 pg/dl, repectively, while brain-lead levels were 2.3, 2.8, and 2.7 pg/g. These values are in contrast to those of Wince et al. (1980), who found a brain-lead level of only 1.36 pg/g on PND-27 after rats had been PB12B/A $ 12-142 1/05/83 TEH 0531005 DUP050031918 PRELIMINARY DRAFT exposed from birth to 4 percent Pb carbonate in their dams' chow. These differences may reflect properties of either the exposure protocols or leadassay techniques. When Jason and Kellogg (1981) exposed rats by gastric intubation to either 25 or 75 mg/kg lead acetate during PND-2 to 14, they obtained bloodlead levels of 50 or 98 pg/dl and brain-lead levels of 0.1 and 0.6 pg/g, respectively, on PND-15. When Modak et al. (1978) continued chronic exposure of their suckling rats (see above) to the same levels for 40 days after weaning (until PND-60) they produced the following blood- and brain-lead levels for 0.25, 0.5, and 1 percent lead acetate, respectively: 91, 194, and 223 pg/dl, and 4.1, 3.6, and 8.1 pg/g. A sequential analysis of blood-lead levels during exposure of suckling rats to 0.1 percent lead acetate in their dams' drinking water from birth through PND-21, followed by continued exposure to the same level until PND-70 (Michaelson and Bradbury, 1982), yielded the following concentrations: 15 pg/dl on PND-2, 35 to 40 pg/dl on PND-13 to 22, and 55 pg/dl at PN0-55, with a slow decline thereafter. Other studies of chronic exposure that have measured blood- and brainlead levels have used adult animals. For example, Bull et al. (1979) exposed* nursing dams to 0.0005, 0.003, and 0.02 percent lead chloride in their drink ing water for 21 days. Blood- and brain-lead levels were 9, 11, and 29 pg/dl, and 0.10, 0.12 and 0.28 pg/g, respectively. Ewers and Erbe (1980) exposed adult rats for 3 months to 0.15, 0.4, or 1 percent lead acetate. Blood-lead levels ranged from 31 to 69 to 122 pg/dl, respectively. Depending on the particular brain region assayed, brain-lead levels were 0.12-0.18, 0.16-0.34, or 0.37-0.72 pg/g. 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 falls below unity. This would indicate that, even as blood-lead levels fall due to excretion or some other mechanism, that lead in brain is retained to a greater extent. PB12B/A 12-143 1/05/83 TEH 0531006 DUP050031919 PRELIMINARY DRAFT 12.4.4 Integrative Summary of Animal and Human Studies of Neurotoxicity. An attempt is made here to integrate information derived from the above assessment and to focus on certain key issues concerning the impact of lead on human and animal neurobehavioral functions. Among the key points to be addressed are: (1) the internal exposure levels, as indexed by bloodlead levels, at which various adverse neurobehavioral effects occur; (2) the reversibility of such deleterious effects; and (3) the population(s) that appear to be most susceptible to neural damage. In most cases with humans, the effects of undue lead exposure on the development of the organism are difficult to trace. Once detected in humans, there are strong ethical demands to treat such effects, therefore, develop mental animal models of lead exposure have been useful in the experimental study of neurotoxicity. There generally have been three strategies used to establish such animal models: (I) induction in animals of the same etiological factors suspected to play a role in the human behavior, e.g., discrimination performances; (2) mimicking symptoms, i.e., generating behaviors in animals similar in topography to those observed in humans, e.g., hyperactivity; (3) using laboratory techniques whose functions are known, so that clues as to the neurobiological substrate may be inferred, e.g., onset of regeneration of Schwann cells following transection of peripheral nerves. Usually, it is better to use a combination of all three, since one alone may lead to ambiguous conclusions. For example, low-level lead exposure seems to be related to increased locomotor activity in developing rats but not children. Although the behaviors of both species seem phenotypically similar, it appears that in children, but not necessarily in animals, hyperactivity is a multidimensional situationspecific disorder which is mediated by attention deficit. Thus, caution in modeling across species is necessary. It is not known how comparable different species are in terms of dose-response functions, exposure histories, blood-tissue concentrations, topographies of behavioral endpoints, or environmental covariates. On the other hand, both the animal and the human studies show great internal consistency in that (1) they support a dose-response functional relationship between lead and behavioral, morphological, electrophysiological, and biochemical effects and (2) they nearly unanimously identify all of these effects as deleterious. PB12B/A 12-144 4 1/05/83 TEH 0531007 DUP050031920 PRELIMINARY DRAFT 12.4.4.1 Interna] Exposure levels at Which Adverse Neurobehavioral Effect? Occur. Regarding this issue, 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 or chronic encephalopathic symptoms, or both) in both humans and and animals. For most human adults, such damage typically does not occur until blood-lead levels in excess of 120 pg/dl are reached. Evidence does exist, however, for acute encephalopathy and death occurring in some human adults at blood-lead levels below 120 pg/dl. In children, the effective blood-lead level for producing encephalopathy or death is lower, starting at approximately 100 pg/dl, Again, however, evidence exists for encephalopathy occurring in some children at lower blood-lead levels, i.e., at 80 to 100 pg/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 tend to exacerbate matters and push the outcome toward fatality if the nature and severity of the problem are not fully recognized or are misdiagnosed. It is also crucial to note the rapidity with which acute encephalopathic symptoms or death can develop in apparently asymptomatic individuals or in those only apparently mildly affected by elevated body burdens of lead. It is not unusual for rapid deterioration to occur, with convulsions or coma suddenly appearing and progression to death within 48 hours. This strongly suggests that, even in apparently asymptomatic 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 conclusion is further supported by numerous studies showing that children with high blood-lead levels (over 80 to 100 pg/dl), but not observed to manifest acute encephalopathy symptoms, are permanently cognitively impaired, as are individuals who survive acute episodes of lead encephalopathy. Other evidence tends to confirm that some type of neural dysfunction exists in apparently asymptomatic children, and at much lower levels of blood lead. The body of studies on low or moderate level lead effects on neuro behavioral functions, as summarized in Table 12-1, presents an overall rather impressive array of data pointing to that conclusion. Several well-controlled PB12B/A 12-145 1/05/83 TEH 0531008 DUP050031921 PRELIMINARY DRAFT studies have found effects that are clearly statistically significant, whereas others have found nonsignificant but borderline effects. Even some studies reporting generally nonsignificant findings at times contain data confirming 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 blood-lead levels associated with significant neurobehavioral deficits both in apparently asymptomatic children and developing rats and monkeys generally appear to be in the range of 30 to 50 pg/dl. However, smaller 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 below the age of three years, at significant risk. However, it must be acknowledged that nutrition covariates, as well as demographic social factors, have been poorly controlled in most of the previously reviewed studies. Socioeconomic status also is a crude measure of parenting and family structure that requires further assessment as a factor possibly contributing to observed results of neuro behavioral studies* Timing, type, and duration of exposure are also important factors in both animal and human studies. It is often undertain whether observed bloodlead 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 neuro behavioral assessment, in most human studies, only one or two blood-lead values are provided per subj*ect. 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 complexity 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 different studies. PB12B/A 12-146 1/05/83 TEH 0531009 DUP050031922 PRELIMINARY DRAFT 12.4.4.2 The question of irreversibility. Little research oh humans is available oh persistence of effects. The work of Araki et al. (1980) and Buchtal and Behse (1979) suggest the possibility of reversing mild forms of peripheral neurophathy in lead workers. Little is known regarding reversibility of lead effects on central nervous system function in humans. Otto et. al. (1982) have recently conducted at two-year follow-up of 28 of the original children of battery factory workers and found a persistent relation between blood lead and altered slow wave voltage of cortical slow wave potentials. However, other early childhood studies from different highrisk populations suggest a continuum of caretaker casualty (Rutter, 1980; Sameroff and Chandler, 1976) where many of the effects of early damage disappear through remediation or for unknown reasons in later childhood. The current population studies will have to be supplemented by prospective longitudinal studies of lead on development to answer such questions of reversibility/irreversibility satisfactorily. Animal studies of the long-term consequences of low lead exposures are somewhat inconsistent, probably due to methodological differences. However, several investigations with rats suggest that disruption of discrimination performance may persist for some time after subjects have been exposed to lead. These results are also consistent with morphological, electrophysiological, and biochemical studies on animals which suggest lasting changes in synaptogenesis, dendritic development, myelin and fiber tract formation, ionic mechanisms of neurotransmission, and energy metabolism. The extent to which remediation can compensate for or reverse these effects in animals is largely unknown and remains to be investigated. 12.4.4.3 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 lead, which suggests that the order of susceptibility to lead effects is young > female > male (Davison, 1977; RoeIs et al., 1978). 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; PB12B/A 12-147 1/05/83 TEH 0531010 DUP050031923 PRELIMINARY DRAFT (3) it is a period where the caregiver environment is vital to normal development. Whether there is a critical period of exposure in animals is subject to some disagreement. However, there is general agreement that human infants and toddlers below the age of three years are at special risk because of in utero exposure and because of mouthing and pica of lead-containing objects. PB12B/A 12-148 1/05/83 * TEH 0531011 DUP050031924 PRELIMINARY DRAFT 12.5 EFFECTS OF LEAD ON THE RENAL SYSTEM 12.5.1 Historical Aspects The first description of renal disease due to lead was published by E. Lancereaux in 1862 (1862). In a painter with lead encephalopathy and gout, Lancereaux noted tubulo-interstitial disease of the kidneys at post-mortem. Distinctions between glomerular and tubulo-interstitial forms of Bright's disease were not, however, clearly established in the mid-nineteenth Century, In 1863, Ollivier reported observations in 37 cases of lead poisoning with renal disease in a paper entitled, "De 1'Albuminurie Saturnine," and thus introduced the idea that lead nephropathy was a proteinuric disease, a confu sion with primary glomerular disease which persisted for over a century. Under the leadership of the great French Neurologist, 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; Geppert, 1882; Lorimer, 1886), More than ninety years ago the English toxicologist, Thomas Oliver (1885, 1891), distinguished 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, later, a "true interstitial nephritis" developed, 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), are the primary injury to the kidney In lead poisoning. Many subsequent studies have shown patho logical 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) the development of func tional 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 hyperglycemia in childhood pica by McKhann in 1926. Subsequently, it was shown that the proximal tubule transport defect included aminoaciduria PB12B/8 12-149 1/05/83 TEH 0531012 DUP050031925 PRELIMINARY DRAFT (Wilson et al., 1953). In 1955 Chisolm et al., found that the full Fanconi syndrome was present; glycosuria, aminoaciduria, hyperphosphaturia (with hypophosphatemia) often associated with rickets (1955). Proximal tubular transport defects appeared only when blood lead levels exceeded 80 pg/dl. Aminoaciduria was seen more consistently in Chisolm's studies (1962, 1968) than were other manifestations of renal dysfunction. The aminoaciduria was generalized in that the amino acids excreted in greatest amounts were those normally present in urine. The condition was related to the severity of clinical toxicity and the complete Fanconi syndrome occurred in children with encephalopathy when blood lead concentrations exceeded 150 pg/dl (NAS 1972). These children who were under 3 years of age excreted 4 to 12.8 mg of lead chelate during the first day of therapy using CaEDTA 50 mg/kg/day. The amino aciduria 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 Puesche) et al. (1972) generalized aminoaciduria was found in 8 of 43 children with blood lead levels of 40 to 120 pg/dl. It should be noted that the children reported to have aminoaciduria in this study were selected because of a blood lead.of 50 pg/dl or more or a provocative chelation test of >500 gg of lead chelate per 24 hours. 12.5.3 Lead Nephropathy in Adults There is convincing evidence in the literature that prolonged lead expo sure in humans can result in chronic lead nephropathy in adults. This evidence is conveniently reviewed in five major categories: 1) Lead nephropathy in Australia, 2) "Moonshine" lead nephropathy, 3) Occupational lead nephropathy, 4) Lead and gouty nephropathy, 5) Lead and hypertension. 12.5.3,1 Lead nephropathy in Australia A series of reports from Queensland, Australia (Gibson et al., 1892; Nye, 1933,; Emmerson, 1963) points to a strong association between severe lead poisoning in childhood 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 contamination of their fingers or by drinking PB12B/B 12-150 1/05/83 TEH 0531013 DUP050031926 PRELIMINARY DRAFT lead-sweetened rain water as it flowed over the weathered surfaces. Two fingers brushed against the powdery paint have been shown to pick up about 2 mg of lead (Murray, 1939), Henderson (1954) followed up 401 untreated children who had been diag nosed as having lead poisoning in Brisbane between 1915 and 1935. Of these 401 subjects, death certificates revealed 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 albuminuria. In a more recent study, Emmerson (1963) presented a criterion for impli cating lead as an etiological factor in such patients: the patients should have an excessive urinary excretion of lead following administration of calcium EDTA. Leckie and Tompsett (1958) had shown that increasing the CaEDTA dosage above 2 gm/day intravenously had little effect on the amount of lead-chelate excreted by adults. They observed little difference in chelstable lead excre tion when 1 gm was compared to 2 gm I.V. Similarly, the magnitude of lead chelated when 1 gm is given intravenously or 2 gm intramuscularly (over 12 hrs) 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 filtration rate (GFR) at which the EDTA lead-mobilization test is no longer reliable has not been precisely defined but probably exceeds a reduction of 853 (serum creatinine concentrations in excess of about 6 mg/dl), In Emmerson's study (1963) 32 patients with chronic renal disease attrib utable to lead poisoning had elevated excretion of lead-chelate. The presence of intranuclear inclusion bodies is very helpful in establishing a relation ship 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). The Australian investigators established the validity of the EDTA leadmobilization test for the detection of excessive past lead absorption and further demonstrated that the body lead stores were retained primarily in bone PB12B/B 12-151 1/05/83 TEH 0531014 DUP050031927 PRELIMINARY DRAFT (Emmerson, 1963; Henderson, 1954; Inglls et al., 1978). Bone -lead concentra tion averaged 94 pg/gm wet weight in the young adults dying of lead nephro pathy in Australia (Henderson and Inglis, 1957; Inglis et al., 1978) compared to mean values ranging from 14 to 23 pg/gm wet weight in bones from non-exposed individuals (Barry, 1975; Gross et al., 1975; Emmerson, 1963). 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 (1973) found no evidence of 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. One hundred sixty-five patients were entered into this study (after review of 524 case records) who met the following criteria: 1) A definite history of pica or use of lead nipple shields; 2) X-ray evidence of lead-induced skeletal alterations; 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 including urinalysis, endogenous creatinine clearance, urine culture, concentrating ability, 24 hour protein excretion and PSP excre tion, Only one patient was believed to have died of lead nephropathy and 3 with creatinine clearances under 90 ml/min were said to have had inadequate urine collections. Likewise, Chisolm et al. (1976) found no evidence of renal disease in 62 adolescents known to have been treated for lead intoxication 11 to 16 years earlier. An important distinction between the Australian group and those reported by patients in the United States was that none of the subjects of Chisolm et al, (1976) showed evidende of increased residual body lead burden following the EDTA lead-mobilization test. This difference has suggested to Chisolm that lead toxicity in the Australian children must 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 prevented the develop ment of renal failure later in life. Most children in the United States who suffer from lead toxicity do so early in childhood, between the ages of 1 and 4, the source usually being oral ingestion of flecks of wall paint and plaster containing lead. Although the transition from acute to chronic lead nephro pathy has not been observed prospectively in humans, progressive hypertension and renal disease have been reported to develop in lead-fed young rats follow ing a latent period (Aviv et a., 1980). PB12B/B 12-152 1/05/83 TEH 0531015 DUP050031928 p r e l imin a r y d r a f t 12.5.3.2 "Moonshine11 lead nephropathy In the United States, chronic lead nephropathy in adults first came to attention among illicit whiskey consumers in the Southeastern States. The pre-revolutionary tradition of homemade whiskey had been modernized during the Prohibition era for large scale production. The copper condensors tradi tionally used in the illegal stills were replaced by truck radiators with welded parts. The locally produced "white lightening" contained up to 74 mg of lead per liter (Eskew et al., 1961), The enormous variability in moonshine lead content has recently been reiterated in studies of 12 samples from Georgia in which 5 contained less than 10 pg/L but one contained 5.3 mg/L (Gerhardt et al., 1980). Renal disease frequently accompanied by hypertension and gout was common among the moonshiners (Eskew et al., 1961; Morgan et al., 1966; Ball and Sorenson, 1969a,b). 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 intravenous EDTA lead-mobilization test (Morgan, 1968; Morgan and Brush, 1972). While acute symptomatology including azotemia sometimes improved during chelation therapy, residual chronic renal failure, gout and hyper* tension frequently proved refractory 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; Editor, 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 sequeal to overt lead intoxication in the Industrial setting (Albahary et al., 1961, 1965; Cramer et al,, 1974; Danilovic 1959; Galle and Morel-Maroger, 1965; Lejeune and To!at, 1969; Lilis et al,, 1967, 1968; Radosevic et al., 1961; Radulescu et al., 1957; Richet et al., 1964, 1966; Tara and Francon, 1957; Vigdortchik, 1935), While it would be Inappropriate to review all available reports here, a few of the more important recent studies will be summarized. PB12B/B 12-153 1/05/83 TEH 0531016 DUP050031929 PRELIMINARY DRAFT Radosevic et al. (1961) described 44 patients with occupational lead exposure and 9 farmers whose excessive absorption derived from lead-glazed pottery. All these Yugoslavian patients had acute lead colic and/or neuro logic symptoms. Seven had elevated serum urea nitrogen concentrations (BUN) which disappeared spontaneously within a week of hospitalization. Similarly, hypertension was transient in four patients in association with symptoms of acute toxicity but eventually became permanent in 3. Evidence of chronic lead nephropathy was found in only 2 of the 53 patients. These observations indi cate the importance of distinguishing transient acute renal dysfunction asso ciated with colic and encephalopathy from chronic renal impairment due to lead retention. Richet et al, (1964) reported renal findings in 8 lead workers all of whom had experienced repeated episodes of lead poisoning including colic. Intravenous EDTA lead-mobilization tests ranged from 587 to 5930 pg leadchelate 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 inclu sions and ballooning of mitochondria in proximal tubule cells. Richet et al. (1966) subsequently recorded renal findings in 23 sympto matic lead workers. Blood leads ranged from 30 to 87 pg/dl, six had diastolic pressures over 90 mmHg, 3 had proteinuria exceeding 200 mg/day and five had gout. In five of 21 renal biopsies 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 was, however, seen in all but two biopsies. Intranuclear inclusion bodies were noted in 13 cases. Electron microscopy showed loss of brush borders, iron-staining intracellular vacuoles and ballooning of mitochondria in proximal tubule epithelial cells, Galle and Morel-Moroger (1965) described light and electron microscopy in 10 renal biopsies from lead-intoxicated subjects 9 of whom had EDTA leadmobilization tests exceeding 800 pg Pb/24 hrs. These investigators found PB12B/B 12-154 1/05/83 TEH 0531017 DUP050031930 PRELIMINARY DRAFT major changes in proximal tubules including loss of brush borders and intra nuclear and cytoplasmic inclusions. In two patients proliferative glomerular changes were noted by light microscopy* One of these had intercapillary hyalin deposits while the other showed fibrinoid intercapillary deposits. Endarteritis and interstitial nephritis were frequent in this series but, the authors noted, were distinguished from the effects of age only with diffi culty. Effective renal plasma flow (Cpg^, plasma clearance of p-aminohippuric acid) by the single injection disappearance technique was measured in 14 lead-poisoned Rumanian workers before and after chelation therapy by tills et al. (1967). Cpah increased from a pre-treatment mean of 428 ml/min (sig nificantly 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. Lilts et al. interpreted the change in effective renal plasma flow as indicating reversal of the renal vasoconstriction which 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 series of 102 cases of occupational lead poisoning studied by Lilts et al. (1968), 7 cases of clinically verified chronic nephropathy were found. In this group, endogenous creatinine clearances were less than 80 ml/min two weeks or more after the last episode of lead colic. The mean blood lead level approximated 80 pg/dl, with a range of 42 to 141 pg/dl. All patients excreted over 10 mg lead-chelate over 5 days during therapy consisting of 2 gm CaNagEDTA intravenously daily. Nephropathy was more common among those who had been exposed to lead for more than 10 years than among those who had been exposed for less than 10 years. Most of the Rumanian lead-workers had experi enced lead colic and 13 of 17 had persistent hypertension which followed the appearance of renal failure by several years. Proteinuria was absent except in two cases who excreted 250 and 500 mg/1. Hyperuricemia was not evident in the absence of azotemia. In both Rumanian studies, reduced urea clearance preceded reduced creatinine clearance. PB12B/B 12-155 1/05/83 TEH 0531018 DUP050031931 PRELIMINARY DRAFT Cramer et al. (1974) examined renal biopsies from five lead workers exposed for 0.5 to 20 years in Sweden. Their blood leads ranged from 71 to 138 (jg/dl, GRFs from 65 to 128 ml/min but Cpa^ exceeded 600 ml/min in all. Although plasma amino acid concentrations were reduced, tubular amino acid reabsorption was not significantly different from controls. A proximal tubule reabsorptive defect might, therefore, have been present which was undetected because of low circulating levels of amino acids. Albuminuria and glycosuria were not present. Glomeruli were normal by electron microscopy. Intranuclear inclusions in proximal tubules were found in two patients with normal GFRs and peritubular fibrosis was present in the remaining three patients who had had the longest occupational exposure (4 to 20 years). In the Danilovic (1959) study, 7 of 23 cases due to lead-contaminated flour had blood' lead levels of 100 to 200 pg/dl. Nineteen of these Yugo slavian patients had azotemia and 20 had hypertension. In the studies of Albahary et al. (1965), blood levels were not reported but exposure must have been quite high because mean ALA excretion was about 37 mg/24 hr for 29 workers. These studies suggest that the acute effect of lead is pre-renal (possibly volume depletion or renal vasospasm) since the reduction in urea clearance preceded the reduction in creatinine clearance. On the other hand, sustained reductions in endogenous creatinine clearance indicate intrinsic renal par enchymal damage. Wedeen et al. reported on renal dysfunction in 14D occupationally exposed men (1975, 1979). These investigators relied upon the EDTA lead-mobilization test (1 gm CaEDTA with 1 ml 2% procaine given intramuscularly twice, 8 to 12 hrs apart) to detect workers with excessive body lead stores. In contrast to workers with concurrent lead exposure (Alessio et al., 1979), blood leads have proven unsatisfactory for the detection of lead exposure in the past (Baker et al., 1979*, Havelda et al., 1980; Vitale et al., 1975). One hundred and thirteen of the 140 workers tested excreted 1000 pg or more of lead-chelate in 24 hrs compared to an upper limit of normal of 650 pg/day (Albahary et al., 1961; Emmerson, 1973; Wedeen et al., 1975), Glomerular filtration rates measured by 125 I-iothalamate clearance in 57 men with increased mobilizable lead revealed reduced renal function in 21 (GFR less than 90 ml/min/1.73 m body surface area). When workers with gout, hypertension, other possible cause of renal P812B/B 12-156 1/05/83* TEH 0531019 DUP050031932 PRELIMINARY DRAFT disease or over age 55 were excluded, 15 remained who had previously unsus pected lead nephropathy. Their GFRs ranged between 52 and 88 ml/rain/1.73 m2. Only 3 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 over 80 pg/dl, 3 repeatedly had blood leads under 40 pg/dl and 11 had blood leads between 40 and 80 pg/dl at the time of the study. Thus, blood leads were poorly correlated with the degree of renal dysfunction. Percutaneous renal biopsies from 12 of the lead workers with reduced GFRs revealed focal interstitial nephritis in 6. Non-specific changes were present in proximal tubules including loss of brush borders, deformed mitochondria and increased lysosomal bodies. Intranuclear inclusion bodies were not found in the renal biopsies from these men who had been subject to long-term occupa tional exposure and who had had chelation tests shortly before biopsy. In experimental animals, chelation results in the rapid disapperance of leadinduced intranuclear inclusions (Goyer and Wilson, 1975). The presence of a variety of immunoglobulin microscopy suggests (but does not prove) the possi bility that some stages of lead nephropathy in adults may be mediated by immune mechanisms. Eight patients with pre-azotemic occupational lead nephropathy were treated with 1 gm CaEDTA (with procaine) intramuscularly three times weekly for 6 to 50 months. In 4 the GFR increased by 20% or more by the time the EDTA test had fallen to less than 850 pg Pb/day. The rise in GFR was paral leled by increases in effective renal plasma flow (Gpah) during the course of treatment. These findings indicate that chronic lead nephropathy may be reversible by chelation therapy at least during the pre-azotemic phase of the disease (Wedeen et al., 1979). However, considerably 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 accompanied by disappearance of tubulo-interstitial changes in the kidney. Chronic volume depletion, for example, might be re flected in the depression of the renin-angiotension-aldosterone system induced by lead (McAllister et al., 1971) or by direct inhibition of Na++ K+ATPase PB128/8 12-157 1/05/83 TEH 0531020 DUP050031933 PRELIMINARY DRAFT mediated sodium transport (Nechay and Williams, 1977). The value of chelation therapy in chronic: lead nephropathy after azotemia has become established is unknown. The prevalence of azotemia among lead workers has recently been confirmed in health surveys conducted at industrial sites (Baker et al., 1979; Hammond et al., 1980; Lanclrigan et al., 1981; Lilis et al., 1979, 1980). Interpreta tion 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 information on the workers found to have renal dysfunction. Moreover, the BUN is a relatively poor indicator of renal function because it is sensitive to a variety of physiologic variables other than GFR including protein anabolism, catabolism and hydration. Several other measures of renal function are more reliable than the serum urea. In order of increasing clinical reliability these are: the serum creatinine, the endogenous creatinine clearance and the 125I-iothalamate or inulin clearance. It should be noted that none of these measures of GFR can be considered reliable in the presence of rapidly changing renal function as in the presence of any acute illness including lead colic or encephalopathy. Elevated BUNs in field surveys may, therefore, sometimes represent transient acute functional changes rather than chronic intrinsic renal disease. An epidemiologic survey in Scotland of households with water lead con centrations in excess of WHO recommendations (100 pg/1) revealed a close correlation between water lead content, blood lead and serum urea concen trations (Campbell et al,, 1977), In this study, the frequency of renal dysfunction in individuals with elevated blood lead concentrations (> 41 pg/dl) was significantly greater than that of age- and sex-matched controls. None of the azotemic subjects were hypertensive. Despite reservations con cerning the use of the BUN for assessing renal function (described 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 relation ship, i.e., that renal failure had caused elevated blood leads in their study group. A carefully matched control population of azotemic individuals from low lead households would have been helpful for this purpose. PB12B/B 12-158 1/05/83 TEH 0531021 DUP050031934 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 usually exposed to a variety of metals, some of which, such as cadmium (Buchet et al., 1980), are themselves nephrotoxic. Multiple interactions between environmental toxins may enhance susceptibility to lead nephrotoxicity. Similarly, nephrotoxicity may be modulated by non-renal effects such as lead-induced reductions in 1,25-dihydroxy vitamin D-j, increased 6-beta-hydroxycortisol production (Saenger et al., 1981, 1982) or Immunologic alterations (Buchet et al., 1980). Reduc tions in dietary intake of calcium, copper, or iron similarly appear to increase susceptibility to lead intoxication (Mahaffey and Michael son, 1980). The slowly progressive chronic lead nephropathy resulting from years of relatively low-dpse lead absorption observed in adults is strikingly different from the acute lead nephropathy 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 adult. (Cramer et al., 1974; Wedeen et al., 1975). Although aminoaciduria has been found to be greater in groups of lead workers than in controls (Coyer et al., 1972; Clarkson and Kench, 1956), 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 remembered, however, that children with the Fanconi syndrome have far more severe acute lead intoxication 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 occupationally exposed adults are uncommon and subtle; clearance measure ments 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., 1980) in contrast to the reduced serum uric acid levels usually associated with the Fanconi syndrome. 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 to para-aminohippuric acid (PAH) in 5 of 6 lead-workers they studied. PAH transport has not been con sistently altered beyond that expected in renal failure of any etiology (Hong et al., 1980; Wedeen et al, 1975). Biagini et al. (1977) have, however, PB12B/B * 12-159 1/05/83 TEH 0531022 DUP050031935 PRELIMINARY DRAFT reported a good negative linear correlation between the one day EDTA leadmobilization test and C ^ in 11 patients with histologic evidence of leadinduced 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 nephropathy) 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 proportion to the effec tive 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 hyperuricemia nephropathy with intratubular uric acid depo sition (Bluestone et al., 1977). In the absence of intra- or extra-renal urinary tract obstruction, the frequency, mechanism and even the existence of a renal disease peculiar to gout remains in question. While some investi gators 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).Glomerulo-nephritis, vaguely defined "pyelonephritis" (Heptinstall, 1974), or intra- and extrarenal 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 nonspecific and cannot usually be differentiated from that of "pyelonephritis," nephrosclerosis or lead nephropathy 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 hypertensives have been reported to be identical to those found in gout patients (Cannon et al., 1966). In these hypertensive patients, serum uric acid levels paralleled the BUN. Confusion between glomerular and interstitial nephritis can in part be explained by the tendency of proteinuria to increase as renal failure PB12B/B 12-160 1/05/83 TEH 0531023 DUP050031936 PRELIMINARY DRAFT progresses regardless of the underlying etiology (Batuman et al., 1981a). In the absence of overt lead intoxication it may, therefore, 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, I960), have, more recently, been reported in end-stage kidneys from patients with no history of gout (Inglis et al,, 1978; Ostberg, 1968; Verger et al., 1967). Urate deposits are, therefore, 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 coexis tence of 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; Yu and Berger, 1982a,b,; Yu, 1982). Over the past four decades the reported incidence of renal disease has varied from greater than 25% (Fineberg and Altschul, 1956; Hench and Vanzant, 1941; Talbott, 1949; Talbott and Terplan, 1960; Wyngaarden, 1958) to less than 2% in 707 patients followed by Yu from 1970 to 1980 (Yu, 1982). 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 en countered 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 and North, 1967; Levin and Abrahams, 1966; Ogryzlo, 1966; Rosenfeld, 1974; Wilson et al., 1967) suggests that renal disease in gout may in part be due 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; Briney et al., 1975). While the contribution of uric acid to the renal disease of gout remains controversial, the hypothesized deleteri ous effect of hyperuricemia on the kidney has no bearing on other potential mechanisms of renal damage in these patients. PB12B/8 12-161 * 1/05/83 TEH 0531024 DUP050031937 PRELIMINARY DRAFT Although hyperuricemia is universal in patients with renal failure, gout is rare except in patients with saturnine gout in whom gout is present in approximately one half of the renal failure patients (Emmerson 1963, 1973; Ball and Sorenson, 1969a; Richet et al., 1965). The long association of lead poisoning with gout raises the possibility that lead absorption insufficient to produce overt lead intoxication may, nevertheless, cause gout with slowly progressive renal failure. Garrod (1859), Ball and Sorenson (1969a) and Emmerson (1971) have demonstrated that lead reduces uric acid excretion, thereby creating the internal millieux in which gout can be expected. More over, among non-lead exposed gout patients in Scotland, blood lead levels were found to be higher than in non-gouty controls (Campbell et al., 1978). Having specifically excluded patients with gout or hypertension from their study of occupational lead nephropathy, Wedeen and his collaborators went on to examine the possible role of lead in the etiology of the gouty kidney (Batuman et al., 1981a). To test the hypothesis that surreptitious 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 specific ally excluded from this study. Collection of urine during the EDTA leadmobilization test was extended to 3 days because reduced GFR delays excretion of the lead-chelate (Emmerson, 1963). Half of the gout patients had renal failure as indicated by serum creatinines over 1.5 mg/dl (mean 3.00.4 SEM mg/dl) indicating approximately a 70% reduction in renal function, and half had normal renal function, The groups were comparable with respect to age, duration of gout, incidence of hypertension and history of past lead exposure. Blood leads were uniformly within acceptable limits; the mean blood lead was 26+3 pg/dl in the patients with reduced renal function and 243 in the gout patients with normal kidney function. The gout patients with renal dysfunc tion, however, excreted significantly more lead-chelate than did those without renal dysfunction (mean 8Q690 and 47052 pg Pb/3 days respectively). Ten control patients with comparable renal failure excreted 42472 pg Pb during the 3 day EDTA (2 gm IM) test. The non-gout control patients with renal failure had "normal" lead stores (Emmerson, 1973; Wedeen et al., 1975) indicating that the excessive mobilizable lead in the gout patients with renal PB12B/B 12-162 1/05/83 TEH 0531025 DUP050031938 PRELIMINARY DRAFT failure was not a consequence of reduced renal function ger se. These studies suggest that excessive lead absorption may sometimes be responsible for the gouty kidney in contemporary patients just 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 is the most probable or 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 contribution of airborne lead, industrial sources and illicit whiskey to the excessive body lead stores demonstrated by the EDTA lead-mobilization test Could not, therefore, be determined. 12.5.3.5 lead and Hypertension--Hypertension is another complication of excessive lead absorption that has a long and controversial history. Hyper tension has long been associated with lead poisoning (Beevers et al,, 1980; Dingwell-Fordyce and Lane, 1963; Emmerson, 1963; Janeway, 1912; Legge, 1901; Lorimer, 1886; Oliver, 1891; Morgan, 1976; Richet et al., 1966; Vigdortchik, 1935) although a number of investigators have failed to find an association (Belknap, 1936; Brieger and Rieders, 1959; Cramer et al., 1966; Fouts, 1942; Malcolm, 1971; Mayers, 1947; Ramirez-Cervantes et al., 1978). Because of the absence of uniform definitions of excessive lead exposure and the absence of 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 involving vasoactive hormones or sodium transport. Definitive epidemiologic 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 cor relate with blood lead levels (Beevers et al., 1976), Moreover, the kidneys of patients with chronic lead nephropathy may show uric acid microtophi and the vascular changes of "benign essential hypertension" even in the absence of PB12B/B 12-163 * 1/05/83 TEH 0531026 DUP050031939 PRELIMINARY DRAFT 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, Yu and Berger (1982b) reported that while hyperuricemia alone had no deliterious effect on renal function in their series, decreased renal function was more likely to occur in gout patients with hypertension and/or ischemic heart disease than in those with uncomplicated gout. Like gout, hypertensive patients were 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 (KincaidSmith, 1982). In order to determine if unsuspected excessive body lead stores might contribute to the renal disease of hypertension 3 day EDTA (2 gm IM) lead mobilization tests were performed in hypertensive Armed Service veterans with and without renal failure who were living in New Jersey (Batuman et al., 1981b; Wedeen, 1982), A significant increase in mobilizable lead was found in hypertensives with renal disease compared to those without renal disease. Control renal failure patients again demonstrated normal mobilizable lead supporting the view that renal failure is not responsible for the excess mobilizable lead in the patients with hypertension and renal failure. These findings suggest that patients who would otherwise be deemed to have "essen tial" 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 hyper tension, renal arteriolar histologic 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 indicated the presence of hyporeninemic hypoaldosteronism. A blunted plasma renin response to salt depletion occurs in lead poisoned patients which can be restored to normal by chelation therapy (McAllister et al., 1971; Gonzalez et al., 1978; Sandstead et al., 1970). The diminished renin-aldosterone responsiveness found in moonshine drinkers could not, however, be demonstrated in occupationally PB12B/B 12-164 1/05/83 TEH 0531027 DUP050031940 PRELIMINARY DRAFT 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 hyppaldosteronism due to lead might contribute to the hyperkalemia (Morgan, 1976) and the exaggerated natriuresis (Fleischer et al., 1980) of some patients with "benign essential hyper tension." 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 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). Reports of renal carcinoma among lead workers are distinctly unusual (Baker et at., 1980). In a more limited study of 241 employees of the Broken Hill Associated Smelters in New South Wales, Australia who were diagnosed as lead poisoned between 1928 and 1959 by a government medical board, 140 deaths were identi fied between 1930 and 1977 (McMichael and Johnson, 1982). Standard propor tional mortality rates of the lead exposed workers compared to 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. 12.5.5 Summary and Conclusions One hundred and twenty years ago lead nephropathy was identified because of the association of interstitial nephritis with overt lead intoxication; known exposure followed by unmistakable symptoms of colic and encephalopathy. In the twentieth century diagnostic acumen was increased by assessment of heme synthesis defects induced by lead and the development of accurate blood lead measurements. More recently, objective assessment of cumulative past lead absorption has been attempted by use of the EDTA lead-mobilization test. Because of the invasive nature of the EDTA test and the serious difficulties PB12B/B 12-165 1/05/83 TEH 0531028 DUP050031941 PRELIMINARY DRAFT encountered in the collection of accurate 24 hour urine specimens, this pro cedure has limited application in the field. If this cumbersome procedure could be replaced by a non-invasive method for assessing cumulative body lead stores, e.g. X-ray induced X-ray fluorescence, considerable important epidemi ologic data might be forthcoming. Such a procedure would have to be validated against the EDTA lead-mobilization test and specific organ damage before it could be generally adopted, A number of major questions remain to be answered concerning the effect of lead on the kidney. Can a distinctive lead-induced renal lesion be identi fied either in functional or histologic terms? What biologic measurements are most reliable for the prediction 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? 12,5.6 Experimental Studies of Lead Nephrotoxicity 12.5.6.1 Pathophysiology of Lead Nephropathy 12.5.6.1.1 Lead uptake by the kidney. Lead uptake by the kidney has been studied in'vivo (Vander et a!., 1977; Victery et al., 1979a, b) and in vitro using renal slices (Vander et al., 1979; Vander and Johnson, 1981), Vander et al. (1977) performed renal clearance studies in dogs 2 hours after a single intravenous dose of 0.1 or 0.5 mgPb acetate containing 1-3 mCi of ^Pb or 1 hour after continous intravenous infusion of 0,1-0.15 mgPb/kg/ hour. These investigators reported that 43-44 percent of the plasma Pb was ultrafiltrable with kidney reabsorption values of 89-94 percent for the ultrafiitrable fraction. Subsequent stop-flow analysis investigations from this laboratory (Victery 1979a) using dogs given a single intravenous dose of 0.2 or 10,0 mg/Pb/kg showed both proximal and distal tubular reabsorption sites for Pb. Distal reabsorption was not linked to sodium chloride or calcium transport pathways. Proximal tubule reabsorption was demonstrated in all animals tested during citrate or bicarbonate infusion. Other studies (Victery et al., 1979b) concerning the influence of acid-base status on renal accumula tion and excretion of Pb in dogs given 0.5-50 pg/kg/hr as an infusion or rats given access to drinking water containing ,500 pgPb/ml for 2-3 months showed that alkalosis increased lead entry into tubule cells via both luminal and PB12B/B 12-166 1/05/83 TEH 0531029 DUP050031942 PRELIMINARY DRAFT basolateral membranes with a resultant increase in both renal tissue accumu lation and urinary excretion of Pb. Similarly, acutely-induced alkalosis increased Pb excretion in rats previously given access to drinking water containing 500 ppm Pb for 2-3 months. These authors also concluded that the previously reported acute experiments concerning the renal handling of Pb were at least qualitatively similar with results of the chronic Pb experiments and that rats were an acceptable model for investigating the effects of alkalosis on the excretion of Pb following chronic exposure. In vitro studies (Vander et al., 1979) using slices of rabbit kidney -- 203 incubated with Pb acetate at concentrations of 0.1 or 1.0 p MPb over 180 minute time intervals showed that a steady-state condition (10-42 slice/medium ratio of 203Pb) was reached after 90 minutes and that Pb could enter the slices as a free ion. Tissue slice uptake was reduced by a number of metabolic inhibitors suggesting a possible active transport mechanism. Tin (Sn IV) was found to markedly reduce 203Pb uptake into the slices but not to affect Pb efflux or para-aminohippurate accumulation raising 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 203Pb into the slices in the presence of organic anions such as cysteine, citrate, glutathione, histidine or serum ultrafiltrate was relatively small compared with uptake due to ionic Pb. In summary, it is clear from the above in vivo and in vitro studies using several different animal species that the renal accumulation of Pb is an efficient process which occurs in both proximal and distal portions of the nephron and at both luminal and basolateral membranes. The transmembrane movement of Pb appears to be mediated by an uptake process which is subject to inhibition by a number of metabolic inhibitors and acid-base status of the organism. 12.5,6,1.2 Intracellular Binding of Lead in the Kidney The bioavailability of Pb inside renal tubule cells under low or Pb tracer exposure conditions is mediated in part by binding 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 intranuclear inclusion bodies (Goyer, 1970a), These inclusion bodies have PB12B/8 12-167 1/05/83 TEH 0531030 DUP050031943 PRELIMINARY DRAFT been shown by both cell fractionation (Goyer et al, 1970a) and X-ray micro analysis (Fowler, 1980) to contain the highest intracellular concentrations of Pb. Saturation analysis of the renal 63,000 dalton (63K) cytosolic binding protein has shown that it possesses an approximate Kd of 10 M (Mistry et al., 1982). These data quantify the high affinity nature of this protein for Pb and explain the previously reported (Oskarsson et al-, 1982) finding that this protein constituted a major intracellular Pb binding site in the Kidney cytosol. Biochemical studies on the protein components of isolated rat kidney intra nuclear inclusion bodies (Moore et al, 1973; Shelton and Egle, 1982) have shown that the main component has an approximate molecular weight of 27,000 (Moore et al, 1973), or 32,000 (Shelton and Egle, 1982) and that it is rich in the dicarboxylic amino acids glutamate and aspartate (Moore et al., 1973). The isolectric point of the main nuclear inclusion body protein has been reported to be pi = 6.3 and appeared from 2-D gel analysis to be unique to nuclei of Pb-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 Pb burden that they sequester, to some degree, Pb away from sensitive renal organelles or metabolic pathways (i.e., heme biosynthetic) until their capacity is exceeded. The same argument would apply to the high affinity cytosolic Pb-binding proteins at Pb exposure levels below those which cause formation of inclusion bodies. It is also presently unclear, whether Pb-binding to these proteins is an initial step in the formation of the cytoplasmic or nuclear inclusion bodies (Oskarsson et al,, 1982). 12.5.6.1.3 Pathological Features of Lead Nephropathy The main morphological effects of Pb 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 12-9. 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 cytomegally, 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). PB12B/8 12-168 1/05/83 TEH 0531031 DUP050031944 _cr_ v) c> a Bo ' Table 12-9. Morphological Features of Pb Nephropathy in Various Species i *S9m> ,4xye1s ** .u*> ff 3 e .8 o OS I 41 Q. S <*- **-5 w J3 1S 2^: MV 31 1S? 8 * *H i i* 1 \X> * \Vt 12# m- o* 2 S - 4-o nm' fi <5 a o" * ^ *3 to < . % a. F* <l) OJ C c > acr- oo 3 ^e 3 H 44-> , r*"2- f=O2 4> So Us to *0 F* Ol I 01 <7> C *8o 4e8) *a_4n(*_0** >tO T<30 +u> * IQ Oft 3 I F=- *fC 3f 8 j& f 8 8 *4v0>i 2e-- 3 3 : S ]2-169 TEH 0531032 DUP050031945 PRELIMINARY DRAFT Another feature reported in two of these studies (Hass et al., 1964; White, 1977; Fowler et al., 1980) was the primary localization of morphological changes in the straight (S3) segments of the proximal tubule indicating that not all cell types of the kidney are equally involved in the toxicity of Pb to this organ. Interstitial fibrosis have also been reported in rabbits (Hass et al., 1964) given diets containing 0.5% Pb acetate for up to 55 weeks and rats (Goyer, 1971) given drinking water containing Pb acetate for 9 weeks. 12.5.6.1.4 Functional Studies 12.5.6.1.4.1 Renal Blood Flow (RBF) and Glomerular Filtration Rate (GFR) Studies by Aviv et al. (1980) concerning the impact of Pb on renal func tion as assessed by RBF and GFR have reported significant (p < .01) reduction in both these parameters in rats exposed to 1 percent Pb acetate in drinking wate at 3 and 16 weeks after termination of exposure, relative to controls . Statistically significant (p<.05) reduction of GFR has also been recently described (Victery et al., 1981) in dogs 2.5-4 hours after a single intravenous dose of 3.0 mgPb/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 experimental variables. 12.5.6.1.4.2 Tubular Function Exposure to Pb has also been reported to produce tubular dysfunction (vonStudnitZ and Haeger-Aronsen, 1962; Goyer, 1970; Mouw et al., 1978; Suketa et al., 1979; Victery et al., 1981, 1982). Early studies, (von Studnitz and Haeger-Aronsen, 1962) reported aminoaciduria in rabbits given a single dose of 125 mgPb/kg and urine collected over a 15-hour period. Goyer et al. (1970) described aminoaciduria in rats following exposure to 1 percent Pb acetate In the diet for 10 weeks, Wapnir et al. (1979) confirmed a mild hyperaminoaci duria in rats injected with 20 mgPb/kg five times a week for 6 weeks but found no changes in urinary excretion of phosphate or glucose. Other investigators (Mouw et al,, 1978; Saketa et al., 1979; Victery et al., 1981, 1982) have focused attention on increased urinary excretion of electrolytes, Mouw et al., 1978 reported increased urinary excretion of PB12B/B 12-170 1/05/83 TEH 0531033 DUP050031946 PRELIMINARY DRAFT sodium, potassium, calcium and water in dogs given a single intravenous injec tion of 0.6 mgPb/kg or 3.0 mgPb/kg over a 4-hour period despite a constant glomerular filtration rate (GFR) indicating decreased tubular reabsorption of these substances. Suketa et al. (1979) treated rats with a single oral dose of Pb at 0, 5.0, 50 or 200 mgPb/kg and killed the animals at 0, 6, 12 or 24 hours after treatment. These investigators observed a dose-related increase in urinary sodium, potassium and water over time. Victery et al., 1981, 1982 studied zinc excretion in dogs over a 4-hour period following intravenous injection of 0.3 mgPb/kg or 3.0 mgPb/kg, These investigators reported maximal increases in zinc excretion in the dog of 140 ng/min at the 0.3 mgPb/kg dose and 300 ng/min at the 3.0 mgPb/kg dose at the end of the 4-hour period. In contrast, with studies by Mouw et al., 1978, no changes in urinary excretion of sodium or potassium were noted. Urinary protein or magnesium excretion were also observed to be unchanged. The results of the above studies indicate that acute or chronic Pb treat ment is capable of producing tubular dysfunction in several species of animals as manifested by increased urinary excretion of amino acid nitrogen, and some electrolytes such as Zn , Ca Na , K , and water. 12.5.6.2 Biochemical Aspects of Lead Nephrotoxicity 12.5.6.2.1 Membrane Marker Enzymes and Transport Functions The biochemical effects of Pb in the kidney appear to be preferentially localized in the cell membranes, mitochondrial, and nuclear compartments fol1owing either acute or chronic Pb exposure regimens. Oral exposure of rats to Pb acetate in the diet at concentrations of 0, 1 or 2 percent for 10-40 weeks (Hirsch, 1973) was found to produce no signifi cant changes in renal slice water content or accumulation of paraminohippurate (PAH) or tetraethyl-ammonium (TEA) but to significantly (p<.05) reduce tissue glucose synthesis at 40 weeks and to markedly reduce pyruvate metabolism, Wapnir et al, (1979) examined the biochemical effects of Pb in kidneys of rats injected with Pb acetate (20 mgPb/kg) five days per week for 6 weeks. They observed a significant (p<,05) reduction in renal alkaline phosphatase activity and an increase in Mg+2ATPase but no significant changes in Na+-K+ ATPase, glucose-6-phosphatase, fructose 1-6 diphosphatase, tryptophan hydroxy lase or succinic dehydrogenase, indicating that preferential effects were only being observed in marker enzymes localized in the brush border membrane and PB12B/B 12-171 1/05/83 TEH 0531034 DUP050031947 PRELIMINARY DRAFT mitochondrial inner membrane. Suketa et al. (1979) reported marked (50-90 percent) decreases in a renal Na+-K+ ATPase at 6-24 hours following a single oral administration of Pb acetate at dose of 200 mgPb/kg, Subsequent studies (Suketa et al,, 1981) using this regimen, showed marked decreases in renal K+-ATPase but no significant changes in Mg+^ATPase after 24 hours indicating inhibition of a cell membrane marker enzyme prior to changes in a mitochon drial marker enzymes. 12.5.6.2.2 Mitochondrial Respiration/Energy-Linked Transformation--The effects of Pb on renal mitochondrial structure and function have been studied by a number of investigators (Goyer et al., 1968; Coyer and Krai1, 1969, Fowler et al., 1980, 1981a, b). Examination of proximal tubule cells of rats exposed to drinking water contained 0.5-1.0 percent Pb acetate for 10 weeks (Goyer et al., I960; Goyer and Krall, 1969) or 250 ppm Pb acetate for 9 months (Fowler et al., 1980) have shown swollen proximal tubule cell mitochondria in situ. Common biochemical findings 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 (1969) found that these respiratory effects were associated with a decreased capacity of the mito chondria to undergo energy-linked structural transformation. In vitro studies (Garcia-Canero, 1981) using 10~^M Pb have reported decreased renal mitochondrial membrane transport of pyruvate or glutamate associated with decreased respiration for these two substrates. Other in vitro studies (Fowler et al., 1981a, b) have reported decreased renal mitochondrial membrane energization as measured by the fluorescent phobes ANS or ethidium bromide following exposure to Pb acetate at concentrations of 10 -10 MPb. High amplitude mitochondrial swelling was also observed by light scattering. The results of the above studies indicate that Pb produces mitochondrial swelling both in situ and in vitro which is 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 Pb- PB12B/B 12-172 1/05/83 TEH 0531035 DUP050031948 PRELIMINARY DRAFT alteration of mitochondrial membrane energization. 12.5.6.2.3 Renal Heme Biosynthesis There are several reports concerning the effects of Pb on renal heme bio synthesis following acute or chronic exposure. Silbergeld et al, (1982) injected rats with 10 pmPb/kg/day for 3 days and examined effects on several tissues including kidney. These investigators found an increase in ALAS following acute injection and no change following chronic exposure first indirectly viat their dams' drinking water containing 10 mgPb/ml until 30 days of age and then directly via this drinking water to 40-60 days of age. Renal tissue content of ALA was increased in both acutely and chronically exposed rats. Renal ALAD was found to be inhibited in both acute and chronic treat ment groups. Gibson and Goldberg (1970) injected rabbits with a subcutaneous administration of Pb acetate at doses of 0, 10, 30, 150 or 200 mgPb/week for up to 24 weeks. The mitochondrial enzyme 6-aminolevulinic acid synthetase (ALAS) in kidney was found to show no measurable differences from controls. Renal 6-aminolevulinic acid dehydratase (ALAD) which is found in the cytosol fraction showed no differences from controls when glutathione was present but was significantly reduced (pc,05) to 50 percent of control values for the pooled lead-treated groups when glutathione was absent. Mitochondrial heme synthetase (ferrocnelatase) was not significantly decreased in Pb-treated versus control rabbits but this enzyme in the kidney was inhibited by 72 and 94 percent at Pb-acetate concentrations of 10~4 and 10~3 M Pb respectively. Renal accumulation (12-15 fold) of both 6-aminolevulinic acid (ALA) and por phobilinogen (PBG) were also observed in kidney tissue of Pb-treated rabbits relative to controls. Zawirska and Medras (1972) injected rats with Pb acetate at a dose of 3 mgPb/rat/day for up to 60 days and noted a similar renal tissue accumulation of uroporphyrin, coproporporphyrin and protoporphyrin. Studies by Fowler et al. (1980) in rats exposed through 9 months of age to 50 and 250 ppm Pb acetate in drinking water showed significant inhibition of the mito chondrial enzymes ALAS and ferrochelatase but no change in the activity of the cytosolic enzyme ALAD. Similar findings have been reported for ALAD following acute intraperitoneal injection of Pb acetate at doses of 5-100 mg Pb/kg at 16 hours prior to sacrifice (Woods and Fowler, 1982). In both the above studies, reduced glutathione was present in the assay mixture. P812B/B 12-173 1/05/83 TEH 0531036 DUP050031949 PRELIMINARY DRAFT A summary of the data from the above studies (Table 12-10) indicates that the pattern of Pb alteration of renal heme biosynthesis is somewhat different from that usually observed with this agent in other tissues (see hematopoietic effects). A general lack of Pb inhibition of renal ALAO is one frequently reported observation in this tissue except under conditions of high level exposure. Such a finding could result from the presence of the recently described high affinity cytosolic Pb-binding proteins (Oskarsson et al., 1982*, Mistry et al., 1982) in the kidney and/or the formation of Pb-containing intranuclear inclusion bodies in this tissue (Goyer, 1971; Fowler et al*, 1980) which would sequester most of the intracellular Pb 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 con centrations of glutathione may also be of importance to the effects of Pb on this enzyme. The observed lack of ALAS induction in kidney mitochondria reported by the above studies may be due decreased mitochondrial protein synthetic capacity or as previously suggested (Fowler et al., 1980) by over whelming Pb-inhibition of this enzyme such that any inductive effects were not measureable. Further research is needed to resolve these questions. 12.5.6.2.4 Lead alteration of Renal Nucleic Acid/Protein Synthesis Studies by Choie and Richter (1972a,b; 1973, 1974a,b; Cihak and Seifertorca, 1976; Stevenson et al., 1977) have shown marked increases in renal nucleic acid or protein synthesis following acute or chronic exposure to Pb acetate. Early studies (Choie and Richter, 1972a) conducted on rats given a 3 single intraperitoneal injection of Pb acetate showed an increase in Hthymidine incorporation. Subsequent studies (1972,b) involved rats given intraperitoneal injections of 1 to 7 mgPb/rat-once per week over a 6-month period. Autoradiography of H-thymidine incorporation into tubule cell nuclei showed a 15-fold increase in proliferative activity in the Pb-treated rats relative to controls. The proliferative response involved both cells with and without intranuclear inclusions. Follow-up autoradiographic studies in rats given 3 intraperitoneal injections of Pb acetate (0.05 mgPb/kg) 48 hours apart showed a 40-fold increase in H thymidine incorporation 20 hours after the first Pb dose and 6 hours after the second and third doses. Subsequent studies in mice (Choie and Richter, 1974a) given a single intracardiac injection of Pb (5 pg Pb/g) demonstrated a 45-fold maximal increase PB12B/B 12-174 1/05/83 TEH 0531037 DUP050031950 *FC - Ferro chelatase **NC - Not changed re la tive to controls ***NM - Not measured M (/> VO *! CO h J Co cr 0) (rQe a. re + 0* ># 5re0 c+ n o so: *o re o. V) -j a 3 M Cl U3 03 ro 30C0>+ h-1 -n to 00 o X "5 c* fli Ml* 1* 5re0 C* Hi O-5 M O COO var<e. in O t&o s-c3or. **> * /--S TP H* TC. 3C IQ IQ?<r 00 3 rQe. Q. < k E X-SJ -cor cn .* O -s N7T ta VPi#nO o irnine --rr-eeHu rf* *C 5 'w' Mcn a-sr <jn 1o* MOO 3 03 CO to rt3O3o3r* -aa 3 -cor 3 *c# n *CJt *PcOn O . cn s o -s rcon o r3e JreNI a-5. Qi trt 1tn *reT M re 3 ro Q. cro IgMvl o. in Ore ' o2 *5 IQ* re a3. M V-OO o 5re0 r*ef> -re5 3 nre r3e0 C+ 5rcce0rr CO ro_eau CO o ? o <3o w -O" >-re$s, >care**N. t< 3 XXX (3Q M* "cOr CO \ jp t>nor X Hi cn 5C o f"N cn ro n o * v-* 3O X X -o cr orcen 5Qre0 mmtm 3re 3 >or3n>- l-f 3C>P n * >CO rmiP--O1 m"Tn| mO in o m mx"tcOn 5m0 m cHooH i To-Oo5 ,<3<" **oOO35 V# cO->5 .<# ^4 M> *? 5 S' "O 5re0 o 3re o H .i3n "O-oco5+ T3 X 03 w tn ^5 in 3 tn in 3re o m 3 > -7<3 O 3 TEH 0531038 DUP050031951 PRELIMINARY DRAFT In DNA synthesis in proximal tubule cells as judged by 3H thymidine autoradio* graphy 33 hours later which was preceded by a general increase in both ENA and protein synthesis (Choie and Richter, 1974b). The above findings were essen tially confirmed with respect to lead-induced increases nucleic acid synthesis by more recent studies of Cihak and Seifertova (1976) who found a 13-fold increase in H thymidine incorporation into mouse kidney nuclei of mice 4.0 hours after an intracardiac injection (5ggPb/g) of Pb acetate. This finding was associated with a 34-fold increase in the mitotic index but no change in the activities of thymidine kinase or TMP-kinase. Stevenson et al. (1977) have also reported a 2-fold increase in 3H thymidine or ^C-orotic acid incor poration into kidney DNA or RNA of rats given a single intraperitoneal injec tion of Pb chloride 3 days earlier. Results of the above stuides clearly demonstrate that acute or chronic administration of Pb stimulates renal nucleic acid and protein synthesis in kidneys of rats and mice. The relationship between this proliferature response and formation of intranuclear inclusion bodies is presently unknown. Nor is the basic mechanism underlying this response and the formation of renal adenomas in rats and mice following chronic Pb exposure presently understood. 12.5.6.2.5 Lead Effects on the Renln-Anglotension System Studies by Mouw et al. (1978) using dogs given a single intravenous injection of Pb acetate at doses of 0.6 or 3.0 mgPb/kg and followed over a 4-hour period showed a small but significant decrease in plasma renin activity (PRA) at 1 hour followed by a large significant (p<.05) increases from 2.5-4.0 hours. Follow-up studies in dogs (Goldman et al., 1981) given a single intra venous injection of Pb acetate at 3.0 mgPb/kg showed changes in the reninangiotensin system over 3 hour period. The data demonstrated an increase in PRA but also that increased renin secretion occurred in only 3 of 9 animals. Hepatic extraction of renin was virtually eliminated in all animals thus providing an explanation for the increased blood levels of PRA. Despite the large, observed increases in PRA, blood levels of angiotensin II (All) did not increase after Pb treatment suggesting Pb-inhibition of Ail converting enzyme. Exposure of rats to drinking water containing 0.5 mg/Pb/ml for 3 weeks to 5 months (Fleischer et al., 1980) produced an elevation of PRA after 6 weeks of exposure in those rats on a sodium-free diet. No change in plasma renin substrate (PRS) was observed. At 5 months, PRA was significantly higher in PB12B/B 12-176 1/05/83 TEH 0531039 DUP050031952 PRELIMINARY DRAFT the Pb-treated group on a 1 percent NaCl diet but the previous difference in renin levels between animals on a sodium-free diet and I percent sodium diet had disappeared. The lead-treated animals had a reduced ability to decrease sodium excretion following removal of Na from the diet. Victery et al. (1982a) exposed rats to Pb in utero and to drinking water solution containing 0, 100 or 500 ppm Pb as Pb acetate for 6 months. Male rats on the 100 ppm Pb dose level became significantly hypertensive at 3.5 months and remained in that state until termination of the experiment at 6 months. All female rats remained normotensive a$ did males on the 500 ppm dose-level. PRA was found to be significantly reduced in the 100 ppm Pb male rat treatment group and normal in the 500 ppm Pb treatment group. Dosedependent decreases in AII/PRA ratios and renal renin content were also ob served. Pulmonary All converting enzyme was not significantly altered. It was concluded that since the observed hypertension in the 100 ppm Pb male rat group was actually associated with reduction of PRA and AH in that the reninangiotensin 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 Pb for 7 months. These investigators found that the mild hypertension associated with this lead-treatment regimen was associated with increased vascular responsiveness to oradrenergic agonists. Male rats exposed to Pb in utero by administration of drinking water containing 0, 5 or 25 ppm Pb as Pb acetate followed by continuation of expo sure on these dose-levels for 5 months (Victery et al., 1982b) showed no change in systolic: blood pressure. Rats exposed to the 25 ppm Pb dose-level showed a significant (p<.05) decrease in basal PRA. Stimulation of renin relase by administration of polyethylene glycol (PEG) showed a significant increase in PRA relative to controls, but low All values yielding a signifi cant (p<.001) decrease in the AII/PRA ratio. Basal renal renin concentrations were found to be significantly reduced (p<.05-p<.01) in both the 5 and 25 ppm Pb dose groups relative to controls. Victery et al. (1983) exposed rats in utero to Pb by maternal administra tion of 0, 5, 25, 100 or 500 ppm Pb as Pb acetate. The animals were continued on their respective dose levels through 1 month of age. All exposure groups has PRA values significantly (p<.05) elevated relative to control. Renal PB12B/B 12-177 1/05/83 TEH 0531040 DUP050031953 PRELIMINARY DRAFT renin concentration was found to be similar to controls in the 5 and 25 ppm Pb groups but significantly increased (p<,05) in the 100 and 500 ppm Pb groups. The plasma AII/PRA ratio was similar to controls in the 100 ppm Pb group but significantly reduced (p<,05) in the 500 ppm Pb 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 magnitude of these changes is mediated by a number of factors including dose level, age and sex of the species tested, and dietary sodium content, Pb also appears capable of directly altering vascular respon siveness to a-adrenergic agents. The mild hypertension observed with chronic low level lead exposure appears to stem in part from this effect and not from changes in the renin-angiotensin system, 12.5.6.2.6 Effects of Pb on Uric Acid Metabolism Studies by Mahaffey et al. (1981) in rats exposed concurrently to Pb, Cd, or As alone or in combination have reported significantly (p<.05) increased serum concentrations of uric acid in the Pb only treatment group. While the biochemical mechanism of this effect is presently unclear, these data do support some of the observations made in humans concerning hyperuricemia as a result of Pb exposure. These data confirm an earlier report by Goyer (1971) showing increased serum uric acid concentration in rats exposed to 1 percent Pb acetate in drinking water for 84 weeks, 12.5.6.2.7 Effects of Pb on Vitamin D Metabolism in the Kidney--Smith et al. (1981) fed rats vitamin D-deficient diets containing either low or normal Ca+2 or P04~ for 2 weeks. The animals were subsequently given the same diets supplemented with 0.82% lead as lead acetate. The ingestion of Pb at this dose level significantly reduced plasma levels of 1, 25 dihydrocholecalciferol in cholecalciferol-treated rats. Rats fed either a low phosphorous or low calcium diet while it had no effect in rats fed either a high calcium diet or normal phosphorous diet. These data suggest decreased renal production of 1, 25-dihydrocholecalciferol in the kidney. PB128/B 12-178 1/05/83 TEH 0531041 DUP050031954 PRELIMINARY DRAFT 12.5.7 General Summary and Comparision of Lead Effects in Kidneys of Humana and Animal Models It seems clear from the preceding review that, in general, results of animal Studies have confirmed and extended to the mechanistic level findings reported in kidneys of humans exposed to Pb for prolonged time periods. The nature of the morphological/pathological lesions in kidneys of humans and animals following chronic Pb exposure are in clear agreement with respect to formation of reversible lesions such as nuclear inclusion bodies, cytomegally, 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. Physiological/functional renal changes observed in humans have also been confirmed in animal model systems with respect to increased excretion of amino acids elevated blood urea nitrogen (BUN) and serum uric acid concentrations. The inhibitory effects of acute/chronic Pb exposure on renal blood flow (RBF) and glomerular filtration rate (GFR) are presently less clear in experimental model systems and further research is needed to clarify the effects of Pb on these functional parameters in animals. Similarly, while Pb-induced perturbation of the renin-angiotensin system has been demonstrated in experimental animal models, further research is needed to clarify the exact relationships between Pb exposure (particularly chronic low-level exposure), alteration of the renin-angiotensin system and hypertension in both humans and animals. On the biochemcial level, it appears that Pb exposure produces changes at a number of sites. Inhibition of membrane marker enzymes, decreased mito chondrial respiratory function/cellular energy production, inhibition of renal heme biosynthesis and altered nucleic acid synthesis are the most marked changes which have been reported. The extent to which these biochondrial alterations occur is probably mediated in part by the intracellular bioavail ability of Pb which is determined by Pb binding to high affinity kidney cyto solic binding proteins and deposition within intranuclear inclusion bodies. PBI2B/B 12-179 1/05/8* TEH 0531042 DUP050031955 PRELIMINARY DRAFT 12.6 EFFECTS OF LEAD ON REPRODUCTION AND DEVELOPMENT Extensive evidence for adverse effects of lead on reproduction and development has been accumulating in the literature and has become a matter of increasing concern. Data from human and animal studies indicate that lead exerts gametotoxic, embryotoxic,, and teratogenic effects that could influence the survival and development of the fetus and newborn. It appears that prenatal viability and development may also be markedly affected by lead indirectly, via adverse effects on various health parameters of the expectant mother. The vulnerabil ity of the conceptus to such lead effects has contributed to concern that the unborn may be a group at risk for lead poisoning, and certain information regarding adverse lead effects on male reproductive functions has led to additional 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 reproduc tive functions have existed in the literature since before the turn of the century. For example, Paul (I860) observed that severely lead poisoned 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 in 1897, found that of 212 pregnancies in 77 female lead workers, only 61 living children were produced. Fifteen workers had never become pregnant. There were 21 stillbirths, miscarriages occurred 90 times, and of 101 children born, 40 died in the first year. Legge also noted that when pregnant animals were fed lead, they typically aborted. He con cluded that maternal exposure to lead resulted in a direct action of the element on the fetus. Four years later. Hall (1905) discussed the increasing use of lead in nostrums sold as abortifacients in Britain. Hall cites nine previous reports of the use of diachylon ("lead plaster") in attempts to cause miscarriage. He further describes 30 cases of known or apparent use of lead in attempts to terminate real or suspected pregnancy. Of 22 cases described in detail, 12 resulted in miscarriage. Of these 12, all exhibited marked signs of plumbism, including a blue gum line, and 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 Hales (1905), who described three additional women who miscarried following consumption of lead-containing pills. PB12D/G 12-180 1/05/83 TEH 0531043 DUP050031956 PRELIMINARY DRAFT Lead compounds were also said by Tanssig (1936) to be known for their embryotoxic properties and to have been used to induce criminal abortion. Oliver (1911) then published statistics in Britain on the effect of lead on pregnancy (Table 12-11) which showed that the miscarriage rate was elevated among women employed in industries in which they were exposed to lead. TABLE 12-11. STATISTICS ON THE EFFECT OF LEAD ON PREGNANCY (OLIVER, 1911) Sample Number of Number of abortions and neonatal deaths stillbirths per (first year) per 1000 females 1000 females Housewives Female workers (mill work) Females exposed to lead premaritally Females exposed to lead after marriage 43.2 47.6 86.0 133.5 150 214 157 271 Such studies as those of Legge, Hall, and Oliver may suffer from methodo logical inadequacies; however, they are mentioned because they provide evidence that effects of lead on reproduction occurred at times when women were exposed to high levels of lead. Nevertheless, evidence for adverse reproductive outcomes in women with obvious lead poisoning is of little help in defining lead effects at significantly lower exposure levels. 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 excluded from occupational exposure to lead, and lead is no longer used to induce abortion. In a more 3 recent study by Lane (1949), women exposed to lead levels of 750 pg/m were examined 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 pg/liter. Little new information is available on reproductive effects of chronic exposure of women to lead. Various 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 invariably result in abortion or in major problems readily detectable in the first few years of PB12D/G 12-181 1/05/83 ... TEH 0531044 DUP050031957 PRELIMINARY DRAFT life. These findings are based on only a few case histories, however, and are obviously not an adequate 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. (1978) found an increased frequency of spontaneous abortions among women living closest to the smelter. In addition to the multiple exposure to 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., 1979) 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 deficien cies as the preceeding study, The above studies clearly demonstrate an adverse effect of lead at apparently high levels on human reproductive functions, and include evidence of Increased incidences of miscarriages and stillbirths when women are exposed to lead during pregnancy. The mechanisms underlying these effects 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 fertility or offspring viability during gestation. Pregnancy is a stress that may place a woman at higher risk for lead toxicity exposure. Both iron deficiency and calcium deficiency increase the susceptibility to lead, and women have an increased risk of both deficiencies during pregnancy and postpartum (Rom, 1976). Efforts have been made to define more precisely the points at which lead may affect reproductive functions both in the human female and male, as well as in animals, as reviewed below. 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 1 year (1 to 12 months) where ambient air levels were <7 pg lead/m . Using a classification of various age groups (20 to 25, 26 to 35, and 36 to 40) and type of ovarian cycle (normal, anovular, and disturbed lutein phase), Panova claimed that statistically significant differences existed between the PB12D/G 12-182 1/05/83 TEH 0531045 DUP050031958 PRELIMINARY DRAFT lead-exposed and control groups in the age range 20 to 25 years. The report, however, does not show the age distribution, the level of significance, or data on the specificity of his method for classification. Zielhuis and Wibowo (1977), in a critical review of the above study, concluded that study design and presentation of data are such that it is difficult to evaluate the author's conclusion that chronic exposure to low lead in air leads to a disturbed function of ovaries. 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 report appears to exist in the literature in regard to assessing lead effects on human ovarian function or other factors 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. 12.6.1.2.2 Effects associated with exposure of men to lead. Lead induced effects on male reproductive functions have been reported in several instances. Among the earliest of these was the review of Stofen (1974), where data from the work of Neskov in the USSR were reported 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. Lancranjan et al. (1975) reported lead-related interference with male reproductive functions. Moderately increased lead absorption (blood lead mean = 52.8 pg/dlj 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. 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 (74.5 ug/dl) and those showing moderate (52.8 pg/dl), slight (41 pg/dl), or physiologic (23 pg/dl) absorption of lead. 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 incidences of these difficulties were invariably lower than those of the "lead exposed" groups, however, so the lack PB12D/G 12-183 1/05/83 TEH 0531046 DUP050031959 PRELIMINARY DRAFT of a clear cut dose-response may have been due to inappropriate assignment of individuals to the high, moderate, and low exposure groups, rather than to Tack of a true effect. Data on the reproductive success of the workmen's wives were not given, as it was based only on interviews with the husbands and was thus believed to be of questionable accuracy. The Lancranjan et al. (1975) study has been criticized by Zielhuis and Wibowo (1977), who stated that the distributions of blood levels appeared to be skewed, so that the means for each putative exposure group may not be representative of their relative distributions. They also suggest that the lead exposure groups overlapped considerably in terms of lead intake. It is difficult to discern from such data, however, if the men in the study were improperly assigned to exposure level groups, as blood lead levels may have varied considerably on a short term basis. Zielhuis and Wibowo also state that the measured urinary ALA levels were unrealistically high for individuals with the stated blood lead levels. Such data suggest that if the ALA values are correct, blood lead levels may have been underestimated. Zielhuis and Wibowo also criticize the Lancranjan et al. study for deficiencies such as failing to use matched controls and for excluding different proportions of individuals per exposure group when semen analysis was done. 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 between lead content of the semen and sperm counts or blood lead levels. The range of lead values was not great, the group size was small, and no mention was made of sperm morphology or vitality. Thus, these findings are not definitive in terms of possible relationships between lead content and semen parameters. Hypothalamic-pituitary-testicular relationships were investigated by Braunstein et al. (1978) in a group of men occupationally exposed at a lead smelter. Six subjects had 2-11 year lead exposure histories, exhibited marked symptoms of lead toxicity, and had decreased libido and frequency of intercourse. All had been away from the smelter for three or more months prior to testing and had received one or more courses of EDTA chelation therapy. This group was referred to as "lead-poisoned" (LP). Four additional men from the same smelter had no obvious signs of lead toxicity, but had been exposed for from 1-23 years, and were designated "lead-exposed" (LE). Three of these men had noted some decrease in libido and sexual activity. The control (C) group PB12D/G 12-184 1/05/83 TEH 0531047 DUP050031960 PRELIMINARY DRAFT consisted of nine male volunteers from "similar socioeconomic backgrounds" to the smelter workers. One control complained of decreased libido, but no biochemical or physiological basis could be found for his complaint. The blood lead levels (pg/dl) for the LP, IE, and C groups were 38.7 3, 29.0 5, and 16.1 1.7, respectively, at the time of the study. Previously, however, the LP arid LE groups had exhibited values as high as 88.2 4 and 80 0, respectively. All three groups were chelated and 24-hour lead excretion values were 999 141, 332 17, and 225 31 pg for the LP, LE, and C groups, respectively. Frequency of intercourse was significantly less in both lead exposed groups than in controls (1 0.3 and 1.6 0.4 vs. 4.7 0.5 times/week for LP, LE, and C, respectively). Sperm concentrations in semen of the LP and LE men ranged from normal to severely oligospermic and one from the LP group was unable to produce an ejaculate. Testicular biopsies were performed on "the two most severely lead-poisoned men," one with aspermia and one with testicular pain. The findings were similar in both men, with increased peritubular fibrosis, decreased spermatogenesis, and Sertoli cell vacuolization. Both lead groups exhibited reduced basal serum testosterone levels, but had normal SeX steriod binding globulin, estradiol, LH, FSH, and prolactin levels. They displayed a normal increase in serum testosterone following stimulation with hCG. A similar rise in serum FSH was seen following treatment with clomiphene citrate or gonadotrophin releasing hormone, although the LP men exhibited a lower than expected increase in LH. The LE men also appeared to have a decreased LH response, but the difference from the control values was not statistically 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 therapy. Thus, further studies on lead intoxicated men prior to chelation would be helpful. Additionally, the numbers of men involved in the study were quite small, so that additional numbers of test subjects are needed to increase the reliability of the results. In a more recent study, Wildt et al. (1982) compared two groups of men exposed to lead in a Swedish battery factory. The study subpopulations were matched with regard to age and to "ethnic and* social factors." The 29 men PB12D/G 12-185 1/05/83 TEH 0531048 DUP050031961 PRELIMINARY DRAFT comprising the high lead group had had blood leads of 50 pg/dl or higher at least once during the six months prior to the study, while the 30 "controls11 seldom exceeded .30 pg/dl. There were two test periods eight months apart. Fifteen and 24 men were in the high lead and control groups, respectively, in the first test and 17 in each group in the second test. Fourteen and 15 of these men took part in both tests from the high lead and control groups, respectively. Blood lead values (pg/dl) were obtained periodically over a six-month period. For the two high lead groups, blood leads were 46,1 and 44.6, respectively, (range 25-75); like values for the controls were 31.1 and 21.5 (range 8-39). The high lead men appeared 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 statistically significant difference was seen only in the case of zinc content. When semen volume was examined, more men in the high lead group than in the controls had low values, but the numbers of individuals involved were too low to allow a reliable statistical analysis. There were no differences among groups in sperm morphology, number, or motility. The heads of sperm of high lead individuals were more likely to swell when exposed to a detergent (SDS) solution, a test of functional maturity, but values were still said to be within the normal range. Conversely, the leakage of lactate dehydrogenase isoenzyme X (LDH-X) was greater in the control semen samples. The values for live and for motile sperm were lower in the control group when averaged across both sampling times. 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 confusing factor is 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, as opposed to none in the high lead group. This large number of men among the controls with urogenital problems may explain the incidence of control samples with lowered sperm motility and viability. Also, according to Wildt et al. (1982), the observed decrease in SDS resistance in sperm of men from the high lead group may have been related to their apparent abnormal prostatic function, or to an effect of lead on sperm maturation. The authors also stated that the fertility of the high lead men was unaffected, but they offered no data to support that contention. In evaluating the above results, it must be kept in mind that even the "control" PB12D/G 12-186 1/05/83 TEH 0531049 DUP050031962 PRELIMINARY DRAFT individuals had elevated blood lead levels. Thus, any effects of lead exposure were likely to have been more difficult to detect. 12.6.1.3 Placental Transfer of Lead--The transfer of lead across the human placenta and its potential threat to the conceptus have been recognized for more than a century (Paul, 1860). Nevertheless, documentation of placental transfer of lead to the fetus and data on relevant parameters, e.g., fetal blood lead levels resulting from such transfer, help to build the case for a potential, but as yet not clearly defined, threat for subtle teratogenic and 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. The relevant data on prenatal lead absorption have been reviewed 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. That the prenatal exposure of the fetus to lead, even in the absence of teratogenic effects, may be of consequence in regard to other adverse health effects is suggested by studies relating fetal and cord-blood levels to changes in fetal heme synthesis and claimed incidences of premature births. For example, Haas et al. (1972) examined 294 mother-infant pairs for blood lead and for urinary ALA levels. The maternal blood mean was 16.89 pg/dl, and the fetal blood mean was 14.98, with a correlation of 0.54 ( <0.001), although the data were based on spot urines, which tends to limit their value. In the infants, the levels of blood lead and urinary ALA were positively correlated (r = 0.19, <0,01). Whether a biological significance exists here, however, is not clear. According to the authors, the positive correlation between lead in blood and urinary ALA for the group as a whole indicated there was already an effect at lower blood lead levels, i.e., increased susceptibility of heme synthesis. Subsequently, Kuhnert et al. (1977) measured ALAD activity and levels of erythrocyte lead in pregnant urban women and their newborn offspring. Cord erythrocyte lead levels ranged from 16 to 67 pg/100 ml of cells, with a mean of 32.9, Lead levels were correlated with inhibition of ALAD activity (r 0,58, <0.01), suggesting that typical urban lead exposures could affect fetal enzyme activity. In a later study, Lauwerys et al. (1978) found no lead-related increase in erythrocyte porphyrin levels in 500 mothers or their PB12D/G 12-187 1/05/83 TEH 0531050 DUP050031963 PRELIMINARY DRAFT offspring. They did, however, report a negative correlation between orami no levulinic acid dehydratase activities and blood lead levels in both mothers and their newborns. Such results indicate that ALAD activity may act as a more sensitive indicator of fetal lead toxicity than would the use of urinary ALA levels. Maternal blood lead levels averaged 10.2 pg/dl, with a range of 3.1-31. Similar values for the newborns were 8.4 and 2.7-27.3. 12.6.1.4.2 Other toxic effects of in utero lead exposure. In a study by Fahim et al. (1976) on maternal and cord-blood lead levels, blood lead values were determined 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 areas delivering at full term. A confusing aspect of this study, however, is the similarity of blood lead levels in women from nonlead and lead belt areas. In fact, no evidence was presented that women included in the lead belt group had actually received greater lead exposures during pregnancy than did control individuals, and problems may be seen with the analytical aspects of this study. For example, other workers (e.g., see summary table in Clark, 1977) have typically found the levels of lead in mothers and their newborn offspring to be much more similar than those given by Fahim et al. (1976). Fahim et al. (1976) noted that among the 249 pregnant women in the control group outside the lead belt area, the percentages of women having preterm deliveries and premature _ rupture were 3 and 0.4, respectively, whereas the corresponding values for the lead area (n = 253) were 13.04 and 16.99, respectively. That the differences presented were in fact due to differential lead exposures, however, does not obtain from the evidence presented. With reference to more subtle prenatal effects, Palmisano et al. (1969) noted failure to thrive and neurological deficits in a 10-week-old infant whose mother had lead poisoning, although any possible lead effect was confounded by maternal alcoholism during pregnancy. When this infant was challenged with a chelating agent, an abnormal urinary excretion of lead was observed, indicating intrauterine exposure. Clark (1977) failed to detect any adverse 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 were 41.2 14.4 and 37.9 15.3 pg/dl, respectively. Like values for control mothers and offspring were 14.7 7.5 and 11.8 5.6 pg/dl. PB12D/G 12-188 1/05/83 TEH 0531051 DUP050031964 PRELIMINARY DRAFT 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. Nordstrom et al. (1979) examined the birth weight records for offspring of female employees of the RSnnskar smelter. They found decreased birth weights related to employment at the smelter and to the distance that the mothers lived from the smelter, in comparison with a control population. Low birth weights were also related to the proximity of the mother's job to the actual smelting process. The smoking habits of experimental and control mothers appeared to be similar, but it is not clear from the information presented whether appropriately matched controls were used in terms of factors such as socio-economic status. Similar results with regard to birth weights were also seen for children born to mothers merely living near the smelter (Nordstrom et al, 1978a). Nordstrom et al. (1979b) also investigated the frequencies of birth defects in offspring of the female smelter workers and in populations living at various distances from the RonnskHr 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 smelter workers with malformed offspring was relatively small (39), making any conculsions drawn less reliable. The incidence of children with birth defects whose mothers worked while pregnant was 58.4%, however, and five of the six offspring with multiple malformations were in this group, suggesting that the effect found was a real one. Nevertheless, the crucial factor in evaluating all of the Ronnskar studies is the exposure of workers and the nearby population to a number of toxic substances in addition to lead. These include arsenic, mercury, cadmium, and sulfur dioxide. Thus, any adverse effects seen could have been due to any of these agents or to a combination thereof. 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 pg/dl, respectively ( <0.001). There was no demonstrable effect of the higher maternal blood-lead levels on any aspect of perinatal health, since there were no significant differences in the rates for stillbirth, PB12D/G 12-189 1/05/83 TEH 0531052 DUP050031965 PRELIMINARY DRAFT neonatal death, and perinatal mortality for the two areas. The rate for congenital abnormality was higher in the rural area, suggesting that whatever the cause, it was unlikely to be related to the reported maternal levels of lead. Also, the differences in lead levels associated with the two populations were so slight that it is not surprising that no differential effect was seen. Additional studies of placental lead and stillbirths have not clarified this situation. Khera et al. (1980) measured placental and stillbirth tissue lead in occupationally exposed women in the United Kingdom. Regardless of the incidence of stillbirths, placental lead concentrations were found to increase with duration of occupational exposure, from 0.29 pg/g at < 1 yr exposure to 0.48 pg/g at > 6 yr exposure for a group of 26 women aged 20-29 years. Placental lead concentrations also increased with age of the mother, independently of time of occupational exposure, and ranged from 0.30 0.16 pg/g at < 20 yrs to 0.51 + 0.44 pg/g at > 30 yrs. Average placental lead concentrations for 20 occupationally exposed women whose babies were stillborn were higher (0.45 0.32 pg/g) than the average level of 0.29 0.09 pg/g for 8 mothers who had hot been occupationally exposed for at least two years. The authors concluded that it was not possible to say whether occupational exposure caused any of the stillbirths that were examined 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., 1978). These differences were attributed to methodological changes and to changes in concentration during storage of placentas at -20C (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 for 7 stillbirths and 1.27 0.48 for 7 matched healthy controls. None of the women were occupationally exposed. The wide range of concentrations reported for the healthy controls (0.34 to 5.56 pg/g) and the differences in concentration 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. In addition, placental lead was said to be associated with decreased activity of a placental enzyme, steriod sulfatase (Kaye and Robertson, 1977). A similar association was found for mercury, suggesting that either metal or PB12D/G 12-190 1/05/83 TEH 0531053 DUP050031966 PRELIMINARY DRAFT both together could have been affecting the enzyme activity or that the authors had merely uncovered a spurious correlation. 12.6.1.4.3 Paternal ly-medlated effects of 1ead. There is increasing evidence in the literature that exposure of male laboratory animals to toxic agents can result in adverse effects on their offspring, including decreased litter size, birth weight, survival, and learning ability (Soyka and Joffe, 1980). Possible mechanisms involved may include mutagenic or other effects. In the following cases, exposure of human males to lead is implicated as the cause of adverse effects on the conceptus. According to Koinuma (1926) in a brief industrial study, 24.7 percent of workmen exposed to lead in a storage battery plant had sterile marriages, while the value for men not so exposed was 14.8 percent. Rates for miscarriage or stillbirths in wives of lead exposed men and controls were 8,2 percent and 2.8 percent, respectively, while Tike figures for neonatal deaths were 24.2 percent and 19.2 percent, Koinuma compared 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 cannot be determined, due to the brevity of the report. In a study of the pregnancies of 104 Japanese women married to lead-exposed workers before and after their husbands began lead work, miscarriages were said to increase to 84.2/1000 preganacies from a pre-exposure rate of 45.6/100 (Nogaki, 1957), The miscarriage rate for 75 women whose husbands were not exposed to lead was 59.1/1000. In addition, exposure to lead was related to a significant increase in the ratio of male to female offspring at birth. Lead content of the paternal blood was found to range from 11 to 31,7 pg/dl, but was not correlated with reproductive outcome, except in the case of the male to female ratio. Also, a report (Van Assen, 1958) on fatal birth defects in children conceived during a period of time when their father was lead poisoned, but neither before nor after, hints at possible effects of lead on the fetus being mediated via human males as well as females. In the previously discussed study by Nordstrom et al. (1979), women employed at the Rb'nnskSr 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 PB12D/G 12-191 1/05/83 TEH 0531054 DUP050031967 PRELIMINARY DRAFT long terra exposure of the male gametogenic stem cells. Whether this was a lead effect or resulted from exposure to other toxins from the smelter was impossible to determine. The literature reviewed here on lead effects on human reproduction and development leaves little doubt that lead can, in fact, exert significant adverse health effects on reproductive functions. Most studies, however, have 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 are observed. Also the human data were derived from studies involving relatively small numbers of individuals and therefore do not allow for discrimi nating statistical analysis. These reports are often additionally confounded by failure to obtain appropriate controls and in some cases by the presence of additional toxic agents or disease states. These and other factors obviously make interpretation of the data difficult, yet it appears possible that effects on sperm or on the testis may occur due to chronic exposures resulting in blood lead values of 40-50 pg/dl. Exposure 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 infants are also quite meager, although the data of Haas et al. (1972) and Lauwerys et al. (1978) suggest possible effects on heme metabolism at maternal blood levels of considerably less than 30 pg/dl. The human data on actual absorbed doses is even more lacking than that on blood lead values, adding to the imprecision of conclusions relating lead exposure to reproductive outcome. 12.6,2 Animal Studies Animal experiments have demonstrated that levels of lead that are com patible with life have interfered with normal reproduction and development. The most relevant such articles are summarized in Table 12-12, in order to allow the reader to gain a perspective on the numerous studies in the litera ture. 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 males due to lead exposure were Puhac et al. (1963), who exposed rats via their diet. Ability to sire offspring returned, however, 45 days after cessation of treatment. PB12D/G 12-192 1/05/83 TEH 0531055 DUP050031968 r^-4. 9f *14 so o-4i V* s9- &g 8* 0 a a c IA a m vu o* ^u U9 1 ra*i a + * 5 - oS) Se v IS ^ * a r *3 r<"0 rH H i r9r*114l H^ot>1 12 sa s oT 91 s i 0<i00 10 1r-104 s 10 10100 a t GC ' **foa* a> *u ^s s* -. 5S' !> 2 -a -Sj. ! z -It D> ^ ^ 8. 1 if > ?UJ o 11 ft i as u m* w CM 9c m ? 0 < f <71 ui ?w M tf> iii ui I 2 N. I 3 S' a> +j +> Q) *o +> 4*> 88 U01l IS 13 _ 22 22 4) V 88 1V3 IS 12-193 TEH 0531056 DUP050031969 . ______ Treatment________ w Effect on the Offspring* Hartali-ty Fetotoxicity Halforoation Species Test Agent Dose*1 and Mode Timingc Reference sa\ frCv-T.k oo C 8s .j* 3 00 s *9 o .X 1 e mr- e<a9 1 4* 1 s con 2U 12 c* + * + c** <v. I ++ 3 ot- rO-*Ui-0*l f--" r-- IH M <H*> HfO rH 2. XV +01 u S at mj X19 e e f c 5*m a > i s. 111 * ft: * to p in in f-4 r4 iH Cl 9 U<9 sa s 19 IW 12-194 TEH 0531057 DUP050031970 . ________ Treatment. Effect en the Offspring3______ M ortality Fetotoxicity Malformation Species Test Agent Doseb and Node Timing0 Reference u& I* e 8L O. art Timft $j m cm . a 41 t| e>* i <91 M i * f*Hi IihrHO > f > a (N <m o & H<n> N 3 A .2 (fl 3 *u_n !leAg aa w . oo r* r* <H0 ri6t H O) &\ p( rt H ***o e 44 * 41 44 i * . 4. c c i "<* S a & ^ 7<*i n m 5 M 4* .3 5 44 *- % A O f3 3 41 44 nc 1v3 <o T4S> s i<w <oe *41 444) 2 9 44 <P 9u 44 3* m9a <aQ 41 12-195 W3 1 TEH 0531058 DUP050031971 Reference ninmr> on poH> ft 9> mph PPhH s 5a Cl s0 > ,e (9 +* 0 + sr 0r- ltCO>l 40J1 H 4O3<R >40^1 <TCT O <J 0* O3W O* U r> p<rh> 1 19o* U *-- 0 * 9Qcr 9 -0 s 0 *0>) O s 3 4 3-3 -3 *00 't9o i M ortality Fetotoxicity Maitarnation e ffe ct on the Offspring" lining : Treatment Dose end Node a ID 00 ID sM3 is i 0 0 a 0Q. Oc l . 0 a a !| I I |1 S* ^a 3\ an j^< o |9 mr mm! 3tao 9f ID NmO s 55 -^ a *3 A A U o u o a m ` 5 0eo)* . = aa o. a. 0p 0 * 5 Ic s to m Oa s $ S' S' 00 00 A4 ONf c i I 10 Ofc. a A 9 c aa. mmm 3+0* 00U 33 23 0 00 O$0 3 30u 0 3 0 0 s s 3 *9 0 0 a 0 0 "?09 3 12-196 TEH 0531059 DUP050031972 Test Agent Species Reference ra*> tpOos*. S e & I 1 tXJT PS cn I . :Uar 00 TJ 8H 41 O'* *- i .9 g9 |L ,-T <*a0 m vJS>. M ortality Fetotoxicity Malformation _______ Effect on the Offspring*1 Timing0 sTreatment_________________________ Dose and Mode o* CM b. 9 t oO 00 h p*. i s* |222 N CtM 8 ^> tmo <n S' i <ji 81 ^& OJ r-4 # 9 (0 8 | n to m H H lii rt 9 atope HI a9 "?:5 24bm* 0 9 *0 9 +> 2 22 C fef- Um0C 1 9 T0ol t9s-u5e 5 a9 T93 9 2! 12-197 TEH 0531060 DUP050031973 Test Agent Species a + = effect present, - = effect not seen, = e ffe ct ambiguous, ? = effect not examined o r in s u ffic ie n t data. b As elemental lead. * ALAO a c tiv ity was decreased. p L itte r size values fo r the high dose group ar c Specific gestation days. - Hematocrit was decreased. 4 No fetuses survived to be examined fo r malfor *to >t. *aa a 2 41 oe </> ~0e S (A s X Xi9m c > S eg tbo oS dn 1st o? S sflj <-0 tflj O SStois-a 12-198 TEH 0531061 DUP050031974 PRELIMINARY DRAFT More recently, Varma et al. (1974) fed a two percent aqueous solution of lead subacetate in drinking water to male Swiss mice for 4 weeks. The total mean intake of lead amounted to 1,65 g. They placed the males with females for one week. The fertility of the treated males was reduced by 50 percent. Varma et al, calculated the mutagenicity index (number of early fetal deaths2/ total implants) to be 10.4 for lead-treated mice versus 2.98 for controls (X =10.4, <0.05). The major differences in fecundity appeared to have been due to differing pregnancy rates, however, rather than prenatal mortality. Lead effects on male fertility rather than lead-induced mutagenicity were thus likely to have been the primary toxic effect observed. It has been suggested by Leonard et al, (1972), however, that effects seen following administration of lead acetate in the water may be due to the resulting acidity, rather than to lead. Also, Eyden et al. (1978) found no decrease in fertility of male mice given 0.1 percent lead acetate in the diet for 64 weeks. A number of animal studies have found lead associated damage to the testes or to the prostate, generally at relatively high doses. Among these were the report of Hildebrand et al. (1973), who noted testicular damage in male rats given oral lead acetate (100 pg/day for 30 days). Also, Egorova (cited in Stofen, 1974), injected lead at a dose of 2 pg/kg six times over a ten-day period and claimed to have observed testicular damage. More recently, Ivanova-Chemishanska et al. (1980) investigated the effect of lead on male rats. According to the authors, the rats were given lead acetate "per os for 4 months," and dose levels were "0.2 mg/kg (0.0001 percent aqueous solution)" or "20 mg/kg (0,01 percent aqueous solution) in the water." It appears from this description that lead was offered in the drinking water. It seems unlikely, however, that these dose levels could have been achieved at the lead acetate concentrations given. Nevertheless, Ivanova-Chemishanska et al, (1980) state that changes in enzymatic activity were observed in testicular homogenates from the high-dose rats. Decreases in activity were seen for acid phosphatase and cytochrome oxidase. Testicular ATP levels were also diminished, and for both lead doses, disulfide groups were also decreased. No histopathological changes in testicular tissue were found, but the fertility index for high dose males was decreased. Also, offspring of those males exhibited "failure to thrive" and stunting at one and three weeks postpartum. Treatmentrelated behavioral changes were claimed for offspring of lead exposed males in both dosage groups. Such data indicate important biological effects due to PB12D/G 12-199 1/05/83 TEH 0531062 DUP050031975 PRELIMINARY DRAFT chronic lead exposure of the male, but are difficult to interpret without more specific knowledge of the dose levels actually received. In the most current study of lead effects on the male reproductive tract, no histopatbological changes were seen during an examination of the testes of rabbits (Willems et al., 1982). Five males per group Were dosed subcutane ously with up to 0.5 mg/kg lead acetate three times weekly for 14 weeks. 81ood lead levels at termination of treatment were 6.6 and 61.5 pg/dl for control and high dose rabbits, respectively. Lead related effects on spermatozoa have also been published. For example, in 1973, Stowe et al. 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 (cytoplasmic swelling). ^ In the study of Maisin et al. (1975), male mice received 0.1 and 1 percent lead, as the acetate, in the diet. With increasing lead exposure, the percentage of abnormal spermatozoa increased, including some with ultrastructural changes. Then in 1978, lead effects in the male were investigated by Eyden et al., who fed lead acetate at a level of 1 percent in the diet to male mice. By the eighth week, an increase in abnormal sperm had occurred. The affected mice showed weight loss and other signs of general toxicity, however, so the effect on spermatogenesis was not an indication of differential sensitivity of the gonad to lead. The effect of lead acetate on sperm morphology was also tested in male mice that were given approximately one sixteenth to one half an LD^ dose by ip injection on five consecutive days (Bruce and Heddle, 1979; Wyrobek and Bruce, 1978; Hedle and Bruce, 1977). The doses used were not precisely specified, but the two lowest doses (apparently 100 and 250 mg/kg) resulted in a modest increase in morphologically abnormal sperm 35 days after treatment. Doses of 500 or 900 mg/kg resulted in considerably greater effects, with up to 21% abnormal sperm. Effects on sperm were seen in lead-intoxicated rats when Krasovskii et al. (1979) observed decreased motility, duration of motility, and osmotic stability of sperm from males given 0.05 mg/kg lead orally for 20-30 days. Damage to gonadal blood vessels and to Leydig cells was also seen. Rats treated for 6-12 months exhibited abnormal 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. PB12D/6 * 12-200 1/05/83 TEH 0531063 DUP050031976 PRELIMINARY DRAFT In addition to its effects in mammals, lead has been said to affect the reproductive system of male doves (Kendall et al., 1981), Birds that had ingested lead shot were rendered oligospermic or aspermic. Morgan et al. (1975) had previously noted reduced testis weights in coturnix quail subjected to 1,000 ppm dietary lead. That lead could directly affect developing sperm or their cellular precursers `is made more plausible by the data of Timm and Schulz (1966), who found lead in the seminiferous tubules of rats and even in the tails of their sperm. The mechanisms for lead effects on the male gonad or gamete are unknown, however, although Gloubovieh et al. (1968) found decreased RNA levels in the testes of lead exposed rats. They suggested that the testicular damage they observed 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 the observed lead effects. A more likely mechanism for lead effects on the testis may be found in the work of Donovan et al. (1980), who found inhibition by lead of androgen binding by the cytosolic receptors of mouse prostate. This could provide a mechanism for the observation of Khare et al. (1978) 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). They 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% lead (C57B1/6J) or 0.5% 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 of lead effects on sperm involves its affinity for sulfhydryl groups. Mammalian sperm possess high concentrations of sulfhydryls that are 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. A number of studies have focused on lead exposure effects in females. For example, effects of lead on the reproduction of female rats were reported by Hildebrand et al. PB12D/G 12-201 1/05/83 TEH 0531064 DUP050031977 PRELIMINARY DRAFT (1973). The ani nulls were orally fed lead acetate at doses of 5 and 100 pg for 30 days. Control rats of both sexes possessed the same blood lead levels. However, for the treated animals, the blood lead levels of the females were higher than those of similarly treated males: 30 pg/dl versus 19 pg/dl at the low dose, and 53 pl/dl versus 30 pg/dl at the high dose. The females exhibited irregular estrus cycles at both doses. When blood lead levels reached 50 pg/dl, they developed ovarian follicular cysts, with a reduction in the number of corpora lutea. In a study subsequent to that of Hildebrand et al. above, but from the same laboratory (Der et al., 1974), lead acetate was injected subcutaneously for 40 days in weanling female rats at a dose of 100 pg of lead per day. Lead treated rats received a diet low (4 percent) or adequate in protein (20 percent); 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 said to be 23 pg/dl in the two control groups, while values for the adequate and low protein diet lead treated groups were 61 and 1086 pg/dl, respectively. The results of Der et al. (1974) thus suggest that lead chronically administered in relatively high doses can interfere with sexual development in rats and that the body burden of lead could be greatly increased by protein deprivation. Maker et al. (1957) noted a delay in age at first conception in female mice of two strains exposed to 0.08% (C57B1/6J) or 0.5% lead (Swiss-Webster) via the maternal diet while in utero, while nursing, and up to 60 days of age. These females were retarded in growth and tended to conceive only when reaching weights approximating those at which untreated mice normally first conceive. Litters from such females developmentally exposed to at least 0.5% lead themselves were said to have lower survival rates and retarded development. More recently. Grant et al. (1980) reported delayed vaginal opening in rats whose mothers were given 25 or more ppm lead (as lead acetate) during gestation and lactation. The pups also received a like dose after weaning. Although most animal studies employed rodents, Vermande-Van Eck and Meigs (1960) administered lead chloride intravenously to female rhesus monkeys. The PB12D/G 12-202 1/05/83 TEH 0531065 DUP050031978 PRELIMINARY DRAFT monkeys were given 10 mg/week for four weeks and 20 mg/week for the next seven months. The monkeys' right ovaries were then removed and examined histologically. Lead treatment was discontinued, and eight months later, the remaining ovary and the uterus were removed and examined. Lead treatment resulted in cessation of menstruation, loss of color of the "sex skin" (presumably due to decreased estrogen production), and pathological changes in the ovaries. One to 5 months after lead treatment was withdrawn, menstrual periods resumed, the sex skin returned to a normal color, and the ovaries had 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 previous studies indicate that pre- and/or postnatal exposure of female animals to lead can affect pubertal progression and hypothalamic-pituitaryovarian- uterine functions. The observations of delayed vaginal opening may reflect delayed ovarian estrogen secretion, suggesting toxicity to the ovary, hypothalamus, or pituitary. An additional investigation demonstrated decreased levels of circulating FSH (Petrusz et al., 1979), and others discussed previously have shown lead induced ovarian atrophy (Stowe and Coyer, 1971; Vermande-Van Eck and Meizs, i960), again suggesting toxicity involving the hypothalamicpituitary-ovarian-endometrial axis. 12.6.2.2 Effects of Lead on the Offspring 12.6.2.2.1 Male mediated effects. Other studies have focused on malemediated lead effects on the offspring. For example, the data from various studies indicate 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 confirm experimentally the paternal effects of lead intoxication. In their study, the litters of dams sired by lead-toxic male rabbits were smaller than those sired by control males. Weller (1915) similarly demonstrated reduced birth weights and survival among offspring of lead-toxic male guinea pigs. Offspring of lead-treated males from the Ivanova-Chemishanska et al. (1980) study described above were said to have been affected in a variety of ways. For example, they exhibited "failure to thrive" and lower weights than did control progeny at one and three weeks postpartum. Treatment-related behavioral changes were also claimed for offspring of lead exposed males. Such data indicate important biological effects due to chronic lead exposure of the male. These results are difficult to interpret, however, without more specific knowledge of the dose levels actually received by the rats. PB12D/G * 12-203 1/05/83 Te h 0531066 DUP050031979 PRELIMINARY DRAFT 12,6.2.2.2 Results of lead exposure of both parents. Studies have assessed the effects of lead exposure of both parents on reproduction, Schroeder and Mitchener (1971), for example, showed a reduction in the number of offspring of rats and mice that were given drinking water containing lead at a concen tration of 25 ppm. In another report (Schroeder et al., 1970), however, it was noted that animals in the earlier study were chromium deficient. No effects were found in animals with normal diets. The combined effects of maternal and paternal oral lead intoxication upon reproductive performance had also been studied fay Morris et al. (1938), who reported no significant reduction in weaning percentage among offspring of rats fed 512 ppm of lead. In an Important study, Stowe and Goyer (1971) assessed the relative paternal and maternal effects of lead as measured by effects on the progeny of F- lead-toxic rats. Female rats fed laboratory chow with or without 1 percent lead (as lead acetate) were bred to normal, mature males, the pregnant rats were continued on their respective rations with or without lead throughout gestation and lactation. Offspring of these matings, the F^ 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 lead-toxic male (CF-PbM), lead-toxic female to control male (PbF-CM), and lead-toxic female to lead-toxic male (PbF-PbM). The results identifying specific effects of lead toxicity upon rat reproduction are shown in Table 12-13. The paternal effects of lead included reductions of 15 percent in number of pups per Titter, 12 percent in mean pup birth weight, and 18 percent in pup survival rate. The maternal 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 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 effects of lead seemed to be irreversible and had additive male and female components. Intrauterine effects were presumed to be due to lead uptake by the conceptus, plus gametotoxic effects. 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. (1972), however, found no effect on the reproductive performance of groups of 20 pairs of mice given lead in their drinking water PB12D/G 12-204 1/05/83 TEH 0531067 DUP050031980 Table 12-13. REPRODUCTIVE PERFORMANCE OF F, LEAD-TOXIC RATS (STOWE ANO COVER, 1971) itte r w *- * * T? T3 i,f*N*0 0* O oo uo in ps CM S s AO 9 <D d d P** o 9 +l -H +i H H H H H 19 Is* 3 8 3 3 9 91 P> <si dr* r~ f-- P<*si d o *o %% en f*-- 99o -H H +9 PCSO. * 5r CO ui n s fs + s si (PAO d 1g d H CO cy s (A coyf dd +1 H Ps U) - d o ui % ps o 9 d C) o P> H H +f H O 91 3 O Ps d if! Ps R is d H 3 Si s d +1 fs. Cl dr* S |s* od H H 8 p) 9 5d * o s** tfl S s 9CO 900 s8 ddd d d e d w -H H H +4 H H 8 s: 3 S 9 i n PO 01 d d d co o6 CM d .* p PO 5 b& iLiiSS (JUU&O. UUt-UL. Wo *o- Wo vo ^o gs efgl e1g0 sfsfeg 5e c ee|c *3 v + +J 2 IWdAl .VUV>) (WVA1WU1UOU)I X* ooooo II1- S 8 SI %9U oi V s *s a . o. X -r : t 2* 2S rz +j +J O 9 is S5 * -.J|MQ .-lid PS* 9s *< o* Sfc Q. <t" C S' *5. 9& <8r v yv v v >|> >*>!>* . 4J PV 4J UM.i U4c0 Ue19U4eUc10U0e) M V" ** " ,e s e e e e iO 0 9)0)010) X < ) yi u> v> v> m A U"a Q> >- 12-205 TEH 0531068 DUP050031981 PRELIMINARY DRAFT 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, equivalent to ingestion of 2.2 kg lead by a 70 kg man over the same time period. 12.6.2.2.3 Effects of lead exposure on implantation and early development. A series of studies have been performed to elucidate the 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 later will be examined primarily in Section 12.6.2.3. Maisin et al. (1975) exposed female mice to lead in the diet (0.1 or 0.5 percent) for 18 days after mating. The number of pregnancies decreased, and the number of embryos succumbing after implantation increased. Similarly, female mice were exposed to lead via their diet (0.125, 0,25, 0,50, and 1.00 percent) from vaginal plug to 16 to 18 days afterward (Jacquet, 1976; Oacquet et al., 1975), At the 0.25 percent dosage, pregnancy incidence decreased, as did the number of corpora lutea. At the two highest dosages, the number of embryos dying after implantation increased. Decreases in -body weight of surviving fetuses .were seen even at the lowest dose by day 18. Doses of 0.5 percent and above often resulted in prenatal death, and the high dose was fatal to a number of the treated dams. Jacquet and his co-workers also described the effect of maternal dietary lead exposure on pre-implantation mouse embryos (Jacquet, 1976; Jacquet et al., 1976). They found that 0,125 to 1.0 percent lead in the diet beginning on the first day of pregnancy was associated with retardation of cleavage in embryos examined 48 hours later. The trophoblastic giant cells failed to differentiate and there was failure of the uterine decidual reaction, Maisin et al, (1978) fed mice a diet containing 0.1 percent lead acetate from 7 or 30 days prior to mating and for up to 7 days after, and they too found delayed cleavage. Examination of cleaving embryos revealed no ultrascructural changes, however, except that seven day old embryos had lead deposits in their mitochondria. Wide and Nilsson (1977) had reported that inorganic lead could interfere with implantation in mice. In an attempt to ascertain the mechanism of action involved, the same authors (Wide and Nilsson, 1979) later observed implanting mouse embryos and their surroundings by both TEM and SEM. The pregnant mothers had been given an intravenous lead dose of 5$ mg/kg on gestation day four. Lead treatment caused failure of attachment of the trophoblast cells to the endometrial surface and the normal closure of the uterine lumen was blocked. PBI2D/G 12-206 1/05/83 TEH 0531069 DUP050031982 PRELIMINARY DRAFT More recently, Jacquet (1978) was able to force implantation by use of high doses of injected progesterone, while Wide (1980) determined that administration of estradiol-170 and progesterone could reverse the effects 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 (1977b, 1976) 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 supported 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 the effects of lead early in gestation. Wide and Nilsson (1977) examined mouse embryos from untreated mice and from mothers that were given 1 mg lead chloride per mouse on days 3, 4, or 6 of pregnancy. The pregnant mothers were sacrificed two days later. In the first two treatment groups, embryonic mortality was greater in the lead-treated litters, but morphology appeared normal by light microscopy. In the day 6 group, mortality was also higher in the treated litters, and some abnormal embryos were observed. In a later experiment. Wide (1978) removed blastocysts from mice given one mg lead chloride on the third day of gestation. She found that they attached and grew normally during three days of in vitro culture. Other blastocysts from untreated mothers were cultured in media containing lead concentrations of 0, 5, 10, or 20 pmo1/1 iter, and a dose-dependent decrease in the number of normally developing embryos was seen. Giavini et al. (1980) further confirmed the ability of lead to affect the preimplantation embryo when they reported a reduced number of blastomeres in rat blastocysts transplacentally exposed to lead nitrate (50 mg/kg). Treatment was given intraperitoneally on gestation day three, and the blastocysts were examined on day five. 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 0.5-16 mg/kg/day in an effort to keep blood lead levels near 40 pg/dl (actual levels ranged from 30-70 pg/dl). Such treatment resulted in a greatly decreased lambing percentage but no gross malformations. The numbers examined were low, however. PB12Q/G 12-207 1/05/83 TEH 0531070 DUP050031983 PRELIMINARY DRAFT 12.6.2.2.4 Teratogenicity and prenatal toxicity of lead in animals. 12.6.2.2.4.1 High dose effects on the conceptus. In the following discussion, as well as throughout this chapter, teratogenic effects have been considered to be those that result in physical defects (malformations) in the developing offspring. Prenatal toxicity (embryotoxicity, fetotoxicity) includes premature birth, prenatal death, stunting, histopathological effects, and transient biochemical or physiological changes. Behavioral teratogenicity, consisting of behavioral alterations or functional (e.g., motor, sensory) deficits resulting from jin utero effects has been dealt with in Section 12.4 of this document. Teratogenicity of lead, at least at high exposure levels, has been demonstrated in rodents and in birds. The results obtained to date suggest a species-related specificity of some of the gross teratogenic effects. For example, Perm end Carpenter (1967) as well as Perm and Ferm (1971) reported increased embryonic resorption and malformation rates when various lead salts were administered intravenously to hamsters on the 8th day of gestation at doses of 25 or 50 mg/kg. The teratogenic effect of lead was largely restricted to the tail region, and included malformations of the sacral and caudal vertebrae, resulting in absent or stunted tails. Gale (1978) reported the same effects in similarly treated hamsters but also saw hydrocephalus. He tested six strains of hamsters and found differences in susceptibility among strains, indicating a genetic component capable of affecting lead-induced teratogenicity. In another of the earlier animal studies, James et al. (1966) gavaged two pregnant ewes daily with 9 mg/kg lead acetate from the day of mating. Both ewes lost weight, aborted their fetuses, and died. Two additional ewes given doses of 5 mg/kg/day gave birth to apparently normal offspring. McLellan et al. (1974) in a brief report described a study in which pregnant mice were given 74.6 mg/kg lead on gestation day nine (no treatment route was specified). Treatment resulted in delayed ossification and in decreased birth weight. Perinatal mortality was also increased, and growth of the surviving pups was retarded. Zegarska et al. (1974) performed a study with rats given lead acetate at a dose of 25 mg/kg on gestation day 9, They reported 75 percent embryonic mortality, with 20 percent of the survivors malformed. Defects seen included meningocele and hypoplasia of the eyes and incisors. McClain and Becker (1975) subsequently administered doses of 25 to 70 mg/kg of lead nitrate PB12D/G 12-208 1/05/83 TEH 0531071 DUP050031984 PRELIMINARY DRAFT intravenously to rats on one of days 8-17 of gestation. A urorectocaudal syndrome of malformations as well as missing cervical vertebrae were seen when lead was administered on day nine. Lead was increasingly embryo- and fetotoxic when administered on later days of gestation but not teratogenic except for the production of hydrocephalus in day 16 treated litters. Kennedy et al. (1975) dosed pregnant rats and mice during early organogenesis with up to 10 mg/kg lead acetate. Material intoxication, developmental retardation, and decreased offspring survival were associated with the high doses, although due to maternal toxicity, the highest dose was administered for only three days. Hackett et al. (1982; 1978) gave 5 or 25 mg/kg lead nitrate intravenously to rats, and found high incidences of prenatal mortality following treatment at the high dose on the only gestation days tested, days 9 and 15. After day 9 treatment, fetal stunting, gastroschisis, and skeletal defects were seen, while day 15 treatment caused major brain hemorrhage. Granahan and Huber (1978) reported teratogenicity in developing rats following maternal treatment with 1,000 ppm lead in the diet throughout gestation, while lead-induced malformations were also seen by Coro and Amoedo (1980), who administered intra venous lead nitrate to pregnant rats. Among the most recent studies employing the rat as an animal model was that of Minsker et al. (1982), who gave 5 or 25 mg/kg lead intravenously to females on gestation day 17. Both doses were associated with decreased birth weights, while both weight and survival by postpartum day 7 were reduced at the high dose. Three pups from 2 high dose litters were found to have hydro cephalus, a defect rare in historical controls. In another recent study with Long-Evans rats, Miller et al, (1982) used oral doses of 50, 75, or 100 mg/kg lead acetate given daily for three weeks before breeding and throughout pregnancy. They reported fetal stunting at the high dose, but no effects on fetal survival, gross or skeletal morphology (viscera were not examined), brain weight, or DNA content. Maternal blood lead values ranged from 80 to 92 pg/dl prior to mating and 53 to 92 pg/dl during pregnancy. Pretreatment and control blood leads averaged 6 to 10 pg/dl. Such results indicate that the Long-Evans rat is relatively resistant to prenatal lead exposure. Ferm (1969) reported that in the presence of cadmium, the teratogenic effect of lead in hamsters is potentiated, leading to severe caudal dysplasia. This finding was duplicated by Hilbelink (1980), using the same .conditions, including treatment by intravenous injection. PB12D/G 12-209 1/05/83 TEH 0531072 DUP050031985 PRELIMINARY DRAFT In addition to caudal malformations, lead appears to influence the morphology of the developing brain. For example, Murray et al. (1978) described a significant decrease in numbers of dendritic spines and a variety of morphological abnor malities of such spines in the parietal cortex of 30 day old rat pups exposed to lead during gestation and nursing, postweaning only, or during both periods. Morphometric analysis of rats transplacentally exposed to lead indicated that cellular organelles were altered in a dose- and exposure-stage-related manner (Klein et al. 1978). These results indicate that morphologically apparent lead effects on the brain could be produced by exposure during pregnancy alone, a question not addressed by Murray et al, (1978). Prenatal effects of lead exposure on the immune system were investigated by Thind (1978). He fed pregnant rats lead chloride before mating and during pregnancy and lactation. The offspring, on the same diet, were inoculated intracerebrally with West Nile Egypt 101 strain virus. Lead-exposed offspring were said to have displayed decreased natural resistance to the infection. A$ part of an investigation of a teratogenicity test system, Beaudoin and Fisher (1981) gave 50 mg/kg lead nitrate to pregnant rats at midgestation, recovered the embryos 4 or 24 hours later, and cultured them for 24 or 48 hours. Lead exposure resulted in delayed in vitro development, as did a variety of other test agents. A variety of studies relating behavioral deficits to prenatal lead exposure have also been published. These studies are covered in Section 12.4.3.2 of this document and will not be repeated here. 12.6.2.2.4.2 Low dose effects on the conceptus. There is a paucity of information regarding the teratogenicity and developmental toxicity of chronic lead exposure. Kimmel et al. (1980) exposed female rats chronically to lead acetate via drinking water (0.5, 5, 50, and 250 pg/g) from weaning through mating, gestation, and lactation. No teratogenic effects were observed, although exposure to 250 pg/g lead acetate caused a decrease in fetal body length of female offspring. The lead-treated animals produced litters of normal numbers, but the female offspring from the 50- and 250-pg/g groups weighed less at weaning and showed delays in physical development. Maternal toxicity was evident in the rats given 25 pg/g or higher doses, corresponding to blood lead levels of 20 pg/dl or higher. Reiter et al. (1975) have also observed delays in the development of the nervous system in offspring exposed to 50 pg/g lead throughout gestation and lactation. Whether these delays in PB12D/G 12-210 1/05/83 TEH 0531073 DUP050031986 PRELIMINARY DRAFT development result from a direct effect of lead on the nervous system of the pups or reflect secondary changes (resulting from malnutrition, hormonal imbalance, etc.) is not clear. Whatever the mechanisms involved, these studies suggest that low-level,, chronic exposure to lead may induce postnatal developmental delays in rats, 12.6.2.2.4.3 Prenatal effects of organolead compounds. The initial study of the effects of organolead compounds in animals appears to be that of McClain and Seeker in 1972. They treated rats orally with 7.5 to 30 mg/kg tetraethyl lead (TEL), 40 to 160 mg/kg tetramethyl lead, or 15 to 38 mg/kg trimethyl lead chloride, given in three divided doses on gestation days 9-11 or 12-14. The latter compound was also given intravenously 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 ossification was observed. In addition, direct intra-amniotic injection of trimethyl lead chloride at levels up to 100 pg/fetus was associated with increasing fetal mortality. Kennedy et al. (1975) administered tetraethyl lead by gavage to mice and rats during the period of organogenesis. Dose levels of up to 10 mg/kg were used. Maternal toxicity, prenatal mortality, and developmental retardation were noted at the highest doses in both species, although due to excessive toxicity, maternal treatment was discontinued after only three days. In a subsequent study involving alkyl lead, Odenbro and Kihistrom (1977) treated female mice orally with triethyl lead at doses of 0.5, 1.5, 2.2, or 3,0 mg/kg/day on days 3 to 5 following mating. Treatment at the two highest dose levels resulted in decreased pregnancy rates, while at 1.5 mg/kg a lower implantation rate was seen. In an attempt to elucidate the mechanism of implantation failure in organolead-intoxicated mice, Odenbro et al. (1982) quantitated 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 doses of 6 mg/kg on days three and four of gestation. Progesterone levels were also lower in females given 3 mg/kg doses. Such results suggest a hormonal mechanism for blockage of implantation, a finding also suggested for inorganic lead (Wide, 1980; Jaquet et al., 1977a). PB12D/G 12-211 1/05/83 TEH 0531074 DUP050031987 PRELIMINARY DRAFT 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 recent investigations, as possible indicators of subtle prenatal effects. Hubermont et al. (1976) exposed female rats to lead in drinking water for three weeks before mating, during pregnancy, and three weeks after delivery. In the highest exposure group (10 ppm), maternal and offspring blood lead values were elevated and approached 6$ and 42 pg/dl, respectively. Inhibition of ALA-D and elevation of free tissue porphyrins were also noted in the newborns. Lead treatment was also associated with increased fetal porphyrins and decreased ALA-0 activity by Jacquet et al. (1977). These authors fed pregnant mice diets containing up to 0.5 percent lead and assayed their fetuses for protein, DNA, ALA-D, porphyrins, and heme. Fetuses in the high dose group had decreased weight, but no data were presented on maternal food consumption (which could have Influenced fetal weight) or weight gain. In the only tests involving inhalation exposure, rats were subjected throughout gestation to an aerosol containing 1, 3, or 10 mg lead/m or to a combination of 3 mg lead/m and 500 ppm carbon monoxide (Prigge and Greve, 1977). 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, however, fetal (but not maternal) ALA-D activity was higher than in the group given lead alone. This effect may have been due to the increase in total ALA-D seen in the CO plus Pb treated fetuses. Fetal body weight and hematocrit were also decreased in the high dose lead group, while maternal values were unchanged. This discrepancy suggests that the fetuses were more sensitive to lead effects than were the mothers. Granahan and Huber (1978) also found decreased, fetal hematocrit, as well as reduced hemoglobin levels, in fetal rats from lead intoxicated dams (1,000 ppm in the diet throughout gestation). Gerber and Maes (1978) fed pregnant mice diets containing up to one percent lead from day 7-18 of pregnancy and determined heme synthesis. Incorporation of Fe into fetal heme was inhibited, but glycine incorporation into heme and protein was unaffected, Gerber et al. (1978) also found that dietary lead (0.25 or 0.5 percent) given late in gestation resulted in diminished placental blood flow but did not decrease uptake of a non-metabolizable amino acid, alpha-amino isobutyrate. The authors concluded that they 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, PB12D/G 12-212 1/05/83 TEH 0531075 DUP050031988 PRELIMINARY DRAFT 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. 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 interference with specific enzymatic events during early development. Lead alters mitochondrial 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 malformations, Carpenter and Ferm (1977) examined hamster embryos treated at mid-gestation. The initial effects were edema of the tail region of embryos 30 hours after maternal exposure, followed by blisters and hematomas. These events disrupted normal caudal development, presumably by mechanical displacement. 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 fetoxicity. Nutritional 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 intraperitoneal injections of lead during early organogenesis. Lead treatment had a greater effect on the offspring of mothers consuming a calcium deficient diet during gestation. Also, in several treatment groups, especially at the high dose (35 mg/kg), lead-treated Ca-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 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* and litter numbers were small, A further study on the interaction of lead with other elements was per formed by Dilts and Ahokas (1979), who exposed rats to levels of 10 to 500 mg of lead per liter in their drinking water throughout gestation. Controls were pair fed or fed ad libitum. According to the authors, lead treatment was associated with decreased fetal weight, and dietary zinc supplementation was associated with an apparent protective effect against fetal stunting. Fetal PB12D/G 12-213 1/05/83 TEH 0531076 DUP050031989 PRELIMINARY DRAFT deaths also appeared to Increase with increasing lead exposure, although the differences were not statistically significant. 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, making statistical comparisons less meaningful, and some of the data were confusing (e.g., a lead-treated and a pair-fed group with very similar litter sizes and total Titter weights, but rather dissimilar average fetal weights; live litter weight divided by live litter size does not give the authors1 values for average fetal weight). Also, no data were given on maternal or fetal lead or zinc levels. In another report on apparently the same animals as in the previous study, pilts and Ahokas (1980) stated that lead inhibited cell division and decreased protein contents of the fetal placentas, eviscerated carcasses, and livers. Such lead-related effects were not influenced by maternal zinc supplementation. 12.6.2.2.4.7 Lead effects on avian embryos. Several researchers have employed avian embryos as test systems for investigating the developmental toxicity of lead. Although the relevance of bird embryos in clarifying the effects of lead or other potential teratogens on developing humans is subject to question, such studies will be cited here in the interest of completeness. In 1941, Catizone and Gray reported production of CNS abnormalities in chick embryos exposed to lead chloride. According to those authors, similar results had been reported by Gray in 1939. Ridgway and Karnofsky (1952) confirmed the finding of CNS lesions, while Hammett and Wallace in 1928 had noted growth retardation after injection of lead nitrate into eggs, Butt et al. (1952) reported that lead nitrate injected into albumin of chicken eggs prior to incubation decreased survival of the embryos and induced the formation of meningoceles. Studies by Gilani (1973a,b) also show that lead is teratogenic to chick embryos. When 2-day-old embryos were given varying doses of lead acetate and were examined on the 8th day of incubation, congenital cardiac anomalies and other defects as well as stunting were demonstrated (Gilani, 1973a,b). The incidence of cardiac anomalies rose with increasing doses of lead. Hirano and Kochen (1973) confirmed the production of lead-induced CNS lesions in chick embryos. When 4-day-old embryos were exposed to 47.8 pg lead (as lead acetate) per egg injected into the yolk sac, a massive cyst was produced due td distension of the subarachnoid space and opening into the PB12D/G 12-214 1/05/83 TEH 0531077 DUP050031990 PRELIMINARY DRAFT fourth ventricle. Damage to blood vessels supplying the brain resulting in severe hemorrhage appeared to be the cause of the brain malformation. Later, De Gennaro (1978) injected lead nitrate into hens' eggs and found growth retardation, lesions of the CNS, and curled toes. None of the treated embryos survived to hatching. It should be noted that in these studies, high, acute doses of lead were administered. Members of another avian species, the ringed turtle dove (Streptope1 la risoria), were given lead in their drinking water (0.1 mg/liter) for two weeks before pairing and throughout a breeding cycle (Kendall and Scanlon, 1981). Lead treatment had no effect on fertility, although testis weights and sperm production were reduced in the treated males. Bone lead levels in the treated females were over ten times as high as those of the males. This difference was presumed to be due to the higher metabolic activity of bone in the females during calcium mobilization for egg shell production. Previous studies by Edens et al. (1976) had indicated that Japanese quail produced fewer eggs when given diets containing lead at levels of 0.001/mg/g or greater. 12.6.3 Summary The most clear cut data described in this section on reproduction and development are derived from studies employing high lead doses in laboratory animals. There is still need for more critical research to evaluate the possible subtle toxic effects of lead to the fetus, perhaps using biochemical, ultrastructural, or behavioral endpoints. This overall evaluation in the offspring may require correlation with the possible additive effects of paternal lead burden, as well as possible exacerbation of lead effects on both parents by other environmental factors (e.g., dietary influences, maternal hypothermia, hypoxia, cometal exposure). Neonatal lead intake via consumption of milk from lead-exposed mothers may also be a factor at times. The paucity of human exposure data force an examination of the animal studies for indications of threshold levels for effects of lead on the conceptus, It must be noted that the animal data is almost entirely derived from rodents. Based on this rodent data, it seems likely that fetpxic effects have occured in animals at chronic exposures to 600-1,000 ppm lead in the diet. Subtle effects may be observed at 10 ppm in the drinking water, while effects of inhaled lead were seen at levels of 10 mg/m . When acute exposure is by PB12D/G 12-215 1/05/83 TEH 0531078 DUP050031991 PRELIMINARY DRAFT gavage or by injection, the values become 10-16 mg/kg and 16-30 mg/kg, respec tively. Since humans are most likely to be exposed to lead in their diet, air, or water, the parenteral data are of less value in estimating harmful exposures. Indeed, it seems likely that teratogenic effects, whether in animals or humans, occur only when the maternal dose is given by injection. The animal evidence indicates a variety of possible effects of lead exposure on reproduction and development, although it is recognized that human and animal responses may be dissimilar. It also indicates 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 to clarify the issues of exposure levels and durations and blood lead volumes associated with significant effects. PB12D/G 12-216 1/05/83 TEH 0531079 DUP050031992 12.7 Genotoxic and Carcinogenic Effects of Pb 12.7.1 Introduction--The carcinogenic and genotoxic*1 or mutagenic role of Pb can be addressed from several avenues. Epidemiological studies examining causes of mortality in populations where excessive exposure to Pb is evident by occupational or environmental considerations address the issue of its Causality in the induction of human neoplasia. These studies are not conclusive in supporting or eliminating the possible etiological role of Pb in human neoplasia. For example the largest and most comprehensive of the epidemiological studies (Cooper and Gaffey, 1975) borders on statistical significance in implicating Pb in the induction of all deaths from malignancies observed in the study, but the nature of the excess in these human malignancies is not of renal origin, the most prominent target site for induction of tumors by Pb in rats. This key study is discussed extensively to evaluate the role of Pb in human carcinogenesis. The carcinogenic role of Pb is further substantiated by the ability of PbCC2H302)2 to induce renal cancer in numerous feeding studies conducted with rats and several studies in mice. The most comprehensive of these studies is that of Azar et al. (1973) which is discussed in considerable detail. Pb(C2H302)2 also induces transformation in cultured Syrian hamster embryo cells. It will be shown that these studies form the basis for concluding that human exposure to Pb compounds at relatively high doses which would produce clinical signs of Pb intoxication are of considerable concern in the induction of malignant disease. The potential mutagenic role of Pb is also addressed in studies of chromosomal aberrations of human populations with extensive exposure to unspecified chemical forms of this metal. Chromosomal aberrations in lymphocytes isolated from experimental animals exposed to specific Pb compounds are also factors in the mutagenic role of Pb. Assays of a purely mutagenic nature particularly in bacterial systems but even using mammalian cells as targets are generally negative with this metal, but are also negative with other metals of more established carcinogenic activity such as Ni compounds. Biochemical studies which examine lesions produced upon ONA by Pb have* received limited attention but are rapidly emerging as useful, sensitive, and reproducible systems for 1 Genotoxicity refers to the alteration of the structure and metabolism of DNA induced by toxic agents. PB12C/B 12-217 1/05/83 i I,-- - TEH 0531080 DUP050031993 PRELIMINARY DRAFT determining mechanisms and genotoxic activity of carcinogenic metals. The genotoxic and mutagenic properties of metal compounds warrant particular concern with respect to choices of suitable systems that produce reliable responses consistent with carcinogenic potency demonstrated In human epidemi ological and experimental animal studies. 12.7.2 Carcinogenesis Studies With Pb 12.7.2.1 Human Epidemiological Studies--Various studies have attempted to correlate an increased incidence of cancer with geographically associated elevations in Pb concentrations. Alien-Price (1975) found that the incidence of cancer in Devon was greater in people residing in areas of Devonian geological formation than people living in adjacent carboniferous formations. However, a number of other potentially carcinogenic minerals in addition to Pb were present at higher concentrations in these mineralized formations, and a relation ship between Pb exposure and the reported excess of cancer incidence was difficult to establish. A number of other studies on human populations with both an increased cancer incidence and a greater environmental Pb exposure have been documented (Berg and Burbank, 1972; Granata et al., 1970; Keen, 1974), but it was difficult to establish Pb as the cause*of these increased tumor incidences and many of the types of tumors documented did not correlate with those induced by soluble Pb salts in experimental animals. Additionally, exposure to a number of other Carcinogenic metals was prevalent in these populations. Blumer and Reich (1976) have demonstrated an increase in cancer mortality in individuals living adjacent to an urban motorway compared with a traffic-free neighborhood; however, in addition to higher Pb exposure the excess of cancer may have also been due to elevated exposure to hydrocarbons. Finally, Schrauzer et al. (1977) found no association between dietary Pb intake and an increased incidence of cancer at any site in the body. Epidemiological studies of industrial workers exposed to Pb have failed to conclusively implicate or rule out Pb as an etiological factor in the induction of human neoplasia (Dingwall-Fordyce et al., 1963; Lane, 1964; Cooper, 1976; Cooper and Gaffy, 1975; Chrusciel, 1975; Neal et al., 1941; Nelson et al., 1973; and Cooper, 1978). The largest and most comprehensive of these epidemiological surveys are the studies of Cooper (1976) and Cooper and PB.12C/3 12-218 1/05/83 TEH 0531081 DUP050031994 PRELIMINARY DRAFT Gaffey (1975). The earlier studies of Cooper (1976) and Cooper and Gaffey (1975) reported an excess of mortality from all malignant neoplasms for smelter workers but not battery plant workers. An excessive but not statistically significant mortality from cancers of the respiratory system and digestive organs was Seen in both smelter and battery plant workers. It was noted that Cooper and Gaffey (1975) made errors in their statistical analysis of the data they had collected which upon reanalysis by a corrected form of their own statistical equations and by what was claimed to be more appropriate statistical tests yielded significant excess of mortality from all neoplasms, from cancer of the digestive organs, and from cancer of the respiratory system in Pb smelter workers (Kang, Infante, and Carra Letter to Science Vol. 207, p. 935, 1980). For battery plant workers there were no excessive deaths from all types of malignant neoplansms but for respiratory cancer, and cancer of the digestive organs there was a statistically significant excess of mortality. The statistical significance was maintained in these categories using a number of different types of data analyses. Gaffey responded to this criticism in a letter to Science indicating that errors in the statistical equations used were typographical and the correct formula was used in the data evaluations. In this same letter by.Kang it was also noted that the excess of cancer related deaths could have been further underestimated because the latency period for 59 percent of the smelter workers and 36 percent of the battery plant workers was less than 20 years. The International Agency for Research on Cancer (IARC) recognizes a 20- or more year latency period as a more sensitive means to determine carcinogenic risk by epidemiological studies. It should also be noted, however, that a recent IARC monograph on carcinogenic risk of Pb (Vol. 23, 1980) also re-evaluated the data of Cooper and Gaffey (1975) by the Poisson statistical method and found no statistically significant excess of mortality from cancer in any group whether the mortality was from total neoplasia or from specific types of cancer. It is apparent from these studies that the evidence for induction of malignant cancers by Pb borders on statistical significance. It should also be noted that this mortality pattern was not maintained in a 5-year follow-up study over 5,000 workers from the previous cohort (Cooper, 1978). In this study, a small deficit in malignant neoplasms in smelterers and a small but significant excess in battery plant workers largely accounted for by malignancies of unknown primary site was reported. PB12C/B 12-219 1/05/83 TEH 0531082 DUP050031995 PRELIMINARY DRAFT The earlier excess mortality from cancer of the digestive organs was not observed. Only one renal tumor was reported in this study. The overall conclusion of this follow-up in contrast to the earlier report indicated no statistically significant carcinogenic role of Pb in humans (Cooper, 1978). A population of 7,032 battery and smelter workers was examined in the original study of Cooper and Gaffey (1975) and 2,275 recorded average mean urinary Pb levels of 129 pg/L for battery workers and 173 pg/L for smelter workers (uncorrected for specific gravity). These were analyzed from data obtained from men providing 10 or more urine samples. Blood Pb levels for 1,326 battery workers averaged 62.7 pg/dL while 537 smelter workers averaged 79.7 pg/dl. (It should be noted that the authors indicated thse blood Pb concentrations in pg/L which is not consistent with their discussion of these similar levels as pg/lOOg and with other reports of normal blood Pb levels.) Again the data represents only men from whom 3 or more samples were taken, Although the Pb level information is severely limited, the Pb exposure determined by biological monitoring was substantial in that at least a portion of the population that was examined for mortality due to neoplasia. The study did not, however, correlate the blood or urine Pb level with mortality because data were not available for many workers and because the distribution between active, terminated, and deceased employees was uneven. Despite these problems and considering the likely high exposure to Pb received by these workers it is evident that this study evaluated the potential carcinogenic role of Pb adequately in exposed humans. The study did not examine the influence of smoking or other parameters which might affect the incidence of cancers and considering the low incidence of kidney tumors it is difficult to attribute the excess neoplasia which borders on statistical significance to Pb. However, respiratory and digestive tumors may have arisen as a result of Pb exposure to these sites. Pb, like most metal carcinogens may produce cancers at the site of exposure since metabolic activation is probably not an essential feature of its carcinogenic mechanism. Additionally, kidney tumors were induced in experimental animals by exposure to water soluble Pb salts such as Pb (C2H302)2* Smelter and battery plant workers were probably exposed to a number of different Pb compounds, which may distribute differently from Pb(C2H302)2 and thus by virtue of their pharmacokinetics or by other factors may induce other types of neoplasia. For example, oral administration of Pb powder resulted in leukemias and lymphomas in rats (Furst et a!., 1976). PB12C/B 12-220 1/05/83 TEH 0531083 DUP050031996 PRELIMINARY DRAFT 12.7.2.2 Induction of Tumors in Experimental Animals---Table 12-14 summarizes carcinogenesis studies conducted with Pb compounds in experimental animals. These studies were used by the IARC (International Agency for Research on Cancer) to evaluate the carcinogenic activity of Pb. It is clear from this table that feeding studies in rats with Pb (C^H302)2 at relatively high doses in both male and female animals result in kidney tumors. In most instances administration of 1 % Pb(C2H3Q2)2 (10,000) ppm resulted in substantial deaths and gross kidney damage was evident. This dose must therefore be considered rather toxic. However, kidney tumors were evident at lower dosages (0.1% diet) which resulted in less death but in some instances evident kidney damage. The induction of renal tumors by Pb acetate appears to be directly related to the metal accumulating in that organ. As discussed earlier in this chapter renal damage, neurotoxicity, and its effects on maturation of rbc are the three primary toxic effects of Pb. At 0.1% Pb (1000 ppm) in the diet the kidney levels were 30 pg/g weight while 1% Pb resulted in 300 pg/g weight of Pb in the kidney. In most of the studies with rats fed 0.10% or 1.0% Pb in the diet incidence of kidney tumors increased with the higher dosage, suggesting a relationship between deposition of Pb in the kidney and the carcinogenic response. Renal tumors were also induced in mice (Table 12-14) at the 0.1% oral dosage of Pb subacetate but not in similarly exposed hamsters. It is difficult to evaluate the carcinogenic role of Pb based upon these studies because Pb acetate [Pb(C2H302)2] or Pb subacetate [Pb20H(C2H302)3] were admin istered over a relatively narrow dosage range and only a limited number of other Pb compounds have been examined for carcinogenic activity in experimental animals. Lead nitrate produced no tumors when tested at very low concentrations (Table 12-14) but lead phosphate administered subcutaneously at relatively high doses induced a high incidence of renal tumors in two rat studies (Table 12-14). Lead powder administered orally resulted in lymphomas and leukemia but given intramuscularly only one fibroma was produced in 50 animals (Table 12-14). Lead naphthenate applied as a 20 percent solution in benzene two times each week for 12 months resulted in the development of 4 adenomas and 1 renal carcinoma in a group of 59 mice (Baldwin et al., 1964), However, in this study control mice were not painted with benzene. Tetraethyl lead at 0.6 mg given in 4 divided doses between birth and 21 days to female mice resulted in 5/36 surviving animals developing lymphomas while 1/39 controls and 1/26 males PB12C/B 12-221 1/05/83 TEH 0531084 DUP050031997 TABLE 12-14. EXAMPLES OF THE INCIDENCE OF CANCERS IN EXPERIMENTAL ANIMALS EXPOSED TO Pb COMPOUNDS uU0>> t V) tO-sp-- 0a>.. ao0. l|_ c wC00 --V1Me0I -->(otA oC oO0L*3>- HC 01 JS r-4 W lilt UJ C ID re 0 eo re u > sc re S_ s 3 4-> r-* 10 Sc T > <u c x 2 x O in 44 vn x> re *s. 3 0 i* n -o il ii 08 t O 3$ re o pH r-- -- re u jp J i r*re f~ Il X CM WO 0> rl T3 *> C (0 -- 1-- 10 0s ffi 0 s on re ii in ut in 1- c re * re u C -o 32 2 o> Jrde. rl in `*'2 2 <-- c re 3 re t. C "o r5 CM VO 9 rl <4 * in . UJ i-- re e re 44 >0 00 13 9 re re 1! i re re 13 -o r- rre re i: e 00 ac ac oo in in Su re # 5e 44 -f _y ^ c. 0 re cu 13 r-- F- ac re 'v. s lO 0 r-t iN in rer-1 p H re r- 4-i in 3 .-- (- 01-- O -p - 0 i 44 u 3 in 44 0 9 44 f - P- 'M.'O re w >, e 00 0 (-- _i ac re w si VO __ V) c m re- t- rNi ^on.12 CM fO 5 IM CM 44 m re C o 44 c l e r 3 3 in -44 44 re *o SLj-g ^ *->9**4>=> 8 a 44 re c e 0i- -- oc re (-- s l re o <ca c- 9 0 0 44 SLLre 9 0-0+4 co u re m in in t. fc- is 44 44 frnm fm 10 10 ee 01 01 uu 9 CM NN 1-4 9 r-t rH VO 9 rH . 44 . 0 ---- in re in re 44 X0 hWO rH cu <0 <0 c O f-- ss in VI U s- o i s 35 44 4^ r-- pmm re cc 9* (- a> U \ < o CM or "V re- H |H fO o L. in 0 II sVO- re 0 J4C^ w3 9o --I 50- 3 001 S VI 8- S w 4- CM O > in u re 0 > CM cr* in re- m VN vi in i. u i re 00 > > CM CM "O 0 4>f* > U 3 in VO f- N *re 0 >> H re w 08? . -- Ortt r-s _ 44 e0 n" f WO 04 00 do 44 e0 f" 4wo . 0 VO a in *. re 0 >. in pH o CL in >> _ >4T re 4= re 44 jo 4g4 f-o ie IX> m CM ^ rl 9 X. in u re 0 >i CM rH o" a. >i re 13 S' ro CM 9 "VCM in 9 L-- re in 0 t>> re 0 CM CM reCM V in <re 0 > re- H o sf \ v w 40 0> > CM 44 C0 -T-- -rM4 13 08 * O rH 91 00 r* 1 T3 mc r Ow <P 01 r* XJ c w 0 ieno ~o 3O CL " u 0 44 re +4 0 .3 u a. re .a 030 PB12C/B 0 f- 0 re -- ac re we 0.. u0_ w O3 VC X0 0 0 44 44 re re g c re St W(0 (0 U -O A o. a. 0 44 re 44 y0 re X3 o. 0) 0 r* f -- <e WU. 4-> T3 (0 C 00 (0 0 #-- p or 0 4> 44 2re 44 00 <4 u re re 43 A CL a. 0 0 r-- -- re WU. 4-> *0 <0 C10 0 r<0-0 SI ^U. 4^ TO C<C0 <C0 12-222 0) <(M0 4^ 01 u C. (0 XI 9 VI F-N 01 01 r#-- <0 SI WLU 4-> *0 <0 e QO <0 2(0 *> O) X) u a. <0 9 V> a> <0 0) X* o & (0 xa 9 v> r-s 4T>, 0 01 r* s sv P 0 oc e4x0 1/05/83 TEH 0531085 DUP050031998 Furst et al. 1976 eStchraol .e,der1970 1/50 fibrosarcoma No significant tumor incidence over control r 00 01 WD Ol ro (3 0> m* H to iH i-- i--i rH s- jr os a* o o C JC U) .e * 05 P P 1--'> \ft 10 U ia suu ^ 01 C "O to fr * i *"* *o ip p <8 TJ O >|C4> 10 18 V) -O E *r+ . rOS xs OS PC P w id c c frm--m UI to O 01 pp 3P 10 C 01 10 P P <8 a. id LU 3* ip rH CO OS sc sc Ui s fsl CO <A 0 <0 .O JC Ol O.J* p3 r-- r--' h*. d* in ia IS IA ip r-- (0. o <0 IS 01 Jo ,3S IA E A iP r-f CLP O os ? e p P IP U C P^ OS C 0) ho c V CP 01 . N mo C iD VX 1 o> oc N3 C r<0 -Q o x: v. +J CM -- M 2 in v-* 0) t- C <D OVU ro fc. c O Q.U oi CO IA --* H O Sa p MO c c CM <P O IA>s U JO Hi <A ' OS C O * -- *f-- JC IA Id a. os e ia . r- -IP >> IP IP p s i- 5 O J= J= h v aa . s \ c >> >, i/)*r r-i- tA to 6 3 P pv (0 c i- 01 CM >% 01 tH (A fc. 5 p c s. o 00 01 CM Weaning u n til death 25 g/L in drinking water 10 mg/monthly 1 year fo r 9 months; then 3 monthly injections of 5 mg (A *- SL> to IP 0) >% >> t H CsJ > .5-- .e (O P hc O <A O Ol 6 05 E 6 r-- JZ PO O ^ <0 rH CM hs p c os M" *r VP CO U> . rH O h IP OS >> P c OS N CM C1 OS tH CO P e >> .r- E r OS OP P CMS-* > H IP OS >1 fH i""'* id <A 1 Cl ip 1-- o c U i- id P 0s P S. CUO) f- U os ip *H H C rH H +J *r V jC P id os *0 P c {. Ipys IP O (A OS -- Q. >><H 3 1- rH C S1 p) css UI *r <A C O W*r {A >> IP XS > ` fH CM (A *0 wC IA C IP cn as o j IA OS JS Sp to *o P s- OS *r* O ^ 0 p <A xz p c o O' i. . >1 rH /-s U <A N. O) 5 iA O OX 00 to r- 1 OP CM O rH P S-J <A s >> CO pH H r--> u p* O IA P w . 05 05 CO <A rH *0 5- o o a. .0 cl . PB12C/B /"N -- r-- 111 (0 wU. P "O e Q (0 0) P ip P 0) u CL IP JQ 3 CO OS p IP c OS JO -Q P CL. j C fiu IP S T3 (A e i- ip 4^ os P r-- IA r- Is IP 6 IP S OS :c v i l OS OS r-- r-- IP <P se w os LL. OS U *0 r- c S IP c 1-- #> rOS JS > *o PCSp fiSL X i0 Ip o LPU P Id *r x> u a. h- r-- p T3 V) C J- P 00 (J Sir iflr* O E <0 E 0 W zvu. 12-223 0 *<0 2 as LU U T3 r* C z P JS CL IA O JZ XX X* a. / s *o r* H .r*> a Q. tn (A Pe 3 av Rats Pb powder P xz a. (A o x: CL J3 eu to P 00 1/05/83 TEH 0531086 DUP050031999 Pb N itrate Rat (Male) PRELIMINARY DRAFT treated with this compound developed lymphomas (Epstein and Mantel, 1968). Lead subacetate has also been tested in the mouse adenoma lung bioassay (Stoner et al., 1976). This assay measures the incidence of nodules forming in the lung of strain A/strong mice following parental 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 measure of carcinogenic potency independent of lung tissue. Lead subacetate was administered at 150, 75, and 30 mg/mouse (total dose) which represented the maximum tolerated dose (Mtd), 1/2 Mtd and 1/5 Mtd, respectively, over a 30 week period using 15 separate ip injections (Stoner et al., 1976). Survivals at the 3 doses were 15/20 (Mtd), 12/20, and 17/20 mice, respectively, with 11/15 (Mtd), 5/12 (1/2 Mtd), and 6/17 (1/5 Mtd) survivors having lung nodules. Only at the highest doses was the incidence of lung nodules statistically significant (P < 0.05). However, these authors concluded that on a Molar-dose basis lead subacetate was the most active metallic compound examined. The compounds examined included nickelous acetate, Cupric (III) acetate, and cobalt acetate to name a few. Injection of 0.13 mmole/kg of lead subacetate was required to produce 1 lung tumor per mouse, indicating that this compound was 3 times more potent than urethane (0.5 mmol/kg) and 10-11 times more potent than nickelous acetate (1.15 mmole/kg), a compound having carcinogenic activity. 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 human carcinogenic hazard. There is little doubt that lead subacetate is active in this system. 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 (Table 12-14,. Kobayashi and Okamoto, 1974). Other types of tumors have been reported in rats following administration of lead acetate, e.g. testicular, adrenal, thyroid, pituitary, prostate, lung (Zirwiska and Madras, 1968) and cerebral gliomas (Oyasu et al., 1970). However, in other animal species such as dogs (Azar et al., 1973; Fouts and Page, 1942), hamsters (Van Esch and Kroes, 1969), and mice (Kamisawa and Schroeder, 1969), lead acetate induced either no tumors or kidney tumors only (Table 12-14). PB12C/B 12-224 1/05/83 TEH 0531087 DUP050032000 PRELIMINARY'DRAFT Pb(H2C2H302) is a highly water soluble Pb compound but is also poorly ionized and therefore considerably lipid soluble. Most of the animal studies were conducted with this Pb compound while other Pb compounds such as lead nitrate, carbonate, or arsenate did not produce renal tumors in rats (Table 12-14). Lead nitrate did, however, result in renal tumors while Pb powder administered orally caused lymphomas and leukemias but not kidney tumors. Chemical considerations have not been adequately addressed in animal carcinogenic studies with Pb and possible differences in pharmacokinetic distribution of the Pb given in distinctly different chemical forms may help explain the differences in tumor incidence and sites of tumor induction (see Chapter 3). The study by Azar et al. (1973) represents one of the most thorough examinations of the carcinogenic role of Pb in experimental animals. Doses of lead acetate examined in rats included D, 10, 50, 100, 1,000 and 2,000 ppm during a two year feeding study. Fifty male and fifty female rats were utilized at doses of 10 to 500 ppm while 100 animals of each sex were used as controls. Four male and four female beagle dogs were also utilized at dosages of Pb ranging from 0 to 500 ppm in this two year feeding study. During this study, the clinical appearance and behavior of the animals was observed. Food consumption, growth, and mortality were recorded. Periodic blood, urine, fecal, and tissue lead analyses were done using atomic absorption spectrophotometry. A Complete blood count, hemoglobin, hematocrit, stippled cell count, prothrombin time, alkaline phosphatase, urea nitrogen, glumatie-pyruvic transaminase, cholesterol, and albumin to globulin ratio were done periodically on the blood specimens. Routine and microscopic urinalyses were performed. The activity of the enzyme alpha-aminoevulinic acid dehydrase (ALAD) in the blood and the excretion of its substrate, delta-aminolevulinic acid (DALA) in the urine were also determined. A thorough necropsy, including both gross and histologic examination was done on all animals that died, survived, or were sacrificed during the two-year period. A three-generation, six-litter, reproduction study was done using the rats fed 0, 10, 50, 100, 1000 and 2000 ppm lead. Table 12-15 demonstrates the mortality and incidence of kidney tumors obtained in rats at the conclusion of the study. At 500 ppm and above male rats developed a significant number of renal tumors. Female rats did not develop tumors unless fed 2000 ppm Pb (C2H302)2. The precise Pb concentration fed to the animals is also shown in this study. PB12C/B 12-225 1/05/83 TEH 0531088 DUP050032001 PRELIMINARY DRAFT Table 12-15. MORTALITY AND KIDNEY TUMORS IN RATS FED LEAD ACETATE FOR TWO YEARS Dietary Pb (ppm) No. of Rats of Each Sex % Mortality Male Female % Kidney Tumors Male Female 5 100 37 34 0 0 18 50 36 30 0 0 62 50 36 28 0 0 141 50 36 28 0 0 548 50 52 36 10 0 3 1130 2102 20 50 35 0 0 20 50 50 50 0 20 80 35 80 35 a) Measured concentration of Pb in diet. b) Includes rats that died or were sacrificed in extremis. (Table from Azar et al., 1973) i Female dogs fed Pb(C2H302)2 up to 500 ppm had no pathological change in any organ system while four male dogs fed 500 ppm developed a slight degree of cytomegaly in the proximal convoluted tubule without the development of any kidney tumors. Since this study was relatively complete and the data obtained was consistent with other studies reported in Table 12-14 a second table (12-16) is shown to illustrate other toxicological parameters measured in rats that developed renal tumors*, this facilitates an understanding of the level of Pb intoxication required for the development of neoplasia. The number of stippled rbc cells increased at 10 ppm Pb in the rat but not in the dogs until 500 ppm Pb. ALAD was decreased at 50 ppm in the rats but not until 100 ppm in the dogs. However, hemoglobin and hematocrit were not depressed in the rats until 1000 ppm Pb., These results illustrate that the induction of kidney tumors coincides with moderate to severe toxicological doses of Pb(C2H302)2- Additionally, it should be noted that at 500-1000 ppm Pb in the diet, a significant increase in mortality of the animals was obtained (Table 12-15), further indicating that the dose range which caused neoplasia was toxic. Pb(C2H302)2 up to 2000 ppm, however, had no reproductive effects in this same PB120/B 12-226 1/05/83 TEH 0531089 DUP050032002 Dietary Pb (ppm) TABLE 12-16, EFFECT OF DIETARY Pb ON HEMOPOIETIC SYSTEM (Mean 95% C, L.a)) Hemoglobin Hematocrit Stippled Cells ALAD (gm %) (%) (cells/50 mg) (units/ml rbc) OALA (mg %) 5 Rat (2) (Dog) 18 (16) 62 (57) 141 (155) 548 (576) 15.39 (16.23) 15.27 (16.04) 15.42 (16.12) 15.32 (16.27) 15.03 (15.64) 42.9 (44.1) 42.2 (43.1) 42.3 (43.6) 42.1 (44.1) 42.1 (42.4) 0,05 (0.00) 0.18* (0.01) 0.33 (0,00) 0.50 (0,04) 2,11 (0.23)* 22.0 (23.4) 22.2 (21.8) 14.4* (17.8) 9.3 (10.6)* 3.6 (3.9) 0.42 (0.26) 0,43 (0.27) 0.42 (0.29) 0.44 (0,27) 0.97* (0.57) * 95% C. L. 0.24 (0.57) 0.7 (1.8) 0.14 (0.11) 2.2 (4.1) 0.53 (0.08) 3 1130 2102 15.72 14.73* 14.37 45.2 43.1* 41.1 0,04 4.27 7.57 17.3 2.4* 1.6 0,49 2,09* 2.38 = 95% C. L. 0.29 0.8 0.63 0.7 0.21 a) C, L. = Confidence Limits * Lowest level at which significant difference from control occurred (p < .05) (Table from Azar et al., 1973) PB12C/8 12-227 1/05/83 TEH 0531090 DUP050032003 PRELIMINARY DRAFT study while at 1,000 and 2,000 ppm Pb 21 day old weaning rats (F^B) showed histological changes in the kidney comparable to those seen in adult receiving > 500 ppm Pb. An additional interesting finding of this study was the direct correlation obtained in dogs between blood Pb level and kidney Pb concentrations. Dietary Pb levels of 500 ppm produced blood Pb levels of 80 pg/100 ml which corresponds to levels of Pb in humans that begin to show clinical signs of Pb poisoning (see earlier parts of this chapter). The kidney Pb concentration corresponding to this blood Pb level was 2.5 pg/g (wet weight), while at 50 pg/dl in blood the kidney levels were 1.5 pg/g. Presumably blood and kidney Pb were determined at approximately the same time interval (Azar et al,, 1973). At this level of Pb, kidney tumors were induced in the rat but not the dogs. However, it is apparent that dogs tolerate higher levels of Pb than rats (vide supra). It is concluded that in rats chronic exposure to Pb at levels where clinical signs of toxicity would be evident in humans resulted in the induction of kidney tumors. Several lines of evidence suggest that Pb directly induces neoplasia as opposed to it being caused secondarily to other effects (i.e., inhibition of heme synthesis and build-up of respective metabolites): 1) Pb is able to directly transform cells in culture and 2) the induction of kidney tumors correlates well with the build-up of Pb in this tissue. 12.7.2.3 Cell Transformation--Lead acetate has been shown to induce morphological transformation in Syrian hamster embryo cells following a continuous exposure to 1 pg/ml or 2.5 pg/ml for 9 days (DiPaolo et al., 1978), The incidence of transformation increased from 0 in untreated to 2.0 and 6.0 percent, respectively, in lead acetate treated cultures. Morphologically transformed cells were capable of forming fibrosarcomas when cloned and administered to "nude" mice and Syrian hamsters while no tumor growth resulted from similar inoculation of 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 caused significant enhancement (1.9-3.10 fold) at 100 and 200 pg/ml following 3 hours of exposure (DiPaolo et al., 1978). In another study PbQ also enhanced SA-7 transformation of Syrian hamster embryo cells 3.8 fold at 50 pM following 3 hours of expsoure (Castro et al ., 1979), The induction of neoplastic transformation by lead acetate suggests that this agent is potentially carcinogenic at the cellular level. The cell PB12C/B 12-228 1/05/83 TEH 0531091 DUPO 50032004 PRELIMINARY DRAFT transformation assay is a highly reliable and efficient system to examine potential carcinogenic activity of metals and their compounds at the cellular level, since an excellent correlation exists between the activity displayed by metals in this assay compared with their carcinogenic response in experimental animals and in human epidemiological investigations. Morphological transformation induced by lead acetate was correlated with the ability of the transformed cells to form tumors in appropriate hosts (vide supra). Since the carcinogenic activity of Pb compounds based upon epidemiological in humans has not been as well studied as some other metal compounds such as nickel or chromate it is useful to compare the activity of Pb compounds in inducing cell transformation with other metal compounds. PbO and Pb acetate were slightly more potent than the soluble nickel salts in inducing cell transformation but less potent than chromate (Heck and Costa, 1980), 12.7.3 Genotoxlcity of Lead 12.7.3.1 Chromosomal Aberrations In Lymphocytes--The use of cultured human lymphocytes allows a specific probe to evaluate potential exposure to genotoxie agents. Cultured lymphocytes from a Pb exposed worker or lymphocytes cultured from a normal individual and exposed to Pb in vitro have been utilized. The latter method allows for better control of exposure condition but is disadvan tageous in that it does not mimic the in vivo exposure conditions. Contradictory reports exist on the effect of Pb in inducing chromosomal aberrations (Table 12-17 and 12-18). The results of human studies from Pb exposed workers are summarized in Table 12-1? where Pb was found to induce aberrations in 9 separate studies and in Table 12-18 where negative studies are indexed. In a number of the positive studies, lymphocytes were cultured for 72 hr which exceeded the desired 48 hr culture time and is considered to result in the appearance of chromosomal aberrations in control lymphocytes. Unfortunately a number of unknown variables makes it difficult to fully evaluate each study in terms of the ability of Pb to induce chromosomal aberrations (i.e., absence of sufficient evidence of Pb intoxication, no dose response, 72 hour culture time, and absence of information regarding lymphocyte culture time) (Schwanitz et al., 1970). However, it is evident that in the negative studies the blood Pb concentation was considerably lower < 400 pg/L than in the studies reporting a positive effect of Pb on chromosomal aberrations where in most instances blood Pb levels indicate severe exposure. In some of these positive studies there PB12C/B 12-229 1/05/83 TEH 0531092 DUP050032005 +M*k&f> sc-s^ u pf> is e<0 ** tf kit* f w.41 m XZ tbm io g Ssa= XKI V) - 10 *i j a *6 .o nil: 5 ss O ss5 |i 3 5*2 * s: a tir ^ * C ^ e0 *>451 '> .4-* . fitt* 'is!lo*;-:5 t.f V Sa<Qi 4gpA- S3 S ip53-lp IS I-S 3 u2oo_ P_oe 2yso*3v**--- 5**8 li _2 2> S| J= V 6. * 3ia TABLE 12-17. CYTOGENIC INVESTIGATIONS OF CELLS FROM INDIVIDUALS EXPOSED TO LEAD: 9 POSITIVE STUDIES PB12C/B w fc.A0)-#<s- P* Ow 9A *-- i? -cS\i t'~ SSOfciXort O.C0O9 TAO1 9$ "l ?1 W s X?u3e5I'.s<xf0fnLi*s/3v*^> U ws* S*t" S__ -4 4 2*f3c 2 If * 2 " 9 U% 52 &3ufc*.Zb10 VPe *U rv~ s|3 ^s *32 <2 s s i* * + *<--IA0 fl *M. O JS T3 fl '3- 0^ 8* ?> lis 1 5.2.*A0*.3 C jj r n V* 3s-Kg 1* S3, IrH f A S~ aNfJIf>i) sV9f/ S!"l iH Ie0TOi-- ,W-*"S ?1 . A2 tf>?0 r*. o iZ eISg. 5F m S S u> Ss O u Lri "O 119 Hi- CmOa 12-230 1/05/83 TEH 0531093 DUP050032006 RReeffeerreenncceess PB12C/B Neuxapboesreodf Ncuomnbterrolosf culCteulrle bUlrionoed loervel sEuxpbjoescetds dToyepoeooef Remarks subjects time (h rs .) (Mg /l) IS sfc. scn 10 r4 12 -<n <n Co O * X 2p eo o> CV 495i-- ir r--" v !i C: Jso u SSS S oi jjr ># ss St , fe is IS So>i u.ja S oe- 7* 5 b> o *ur 0| I to 2 C frl 2* *2 me % I s se *0i>*S-- c5 Uo> SJwQ SO) i +> 58 OQ 1 roItwrjae tA Ox-*x ftf>)J lO43f NV 9 9 U 9 e 3 8 + > Oe *o > SIS ^ 0 01b _w 6 -3 * * ofi 12-231 1/05/83 TEH 0531094 DUP050032007 TABLE 12-18. CYTOGENETIC INVESTIGATIONS OF CELLS FROM INDIVIDUALS EXPOSED TO LEAD: 6 NEGATIVE STUDIES M I I 4* V CAI 631005F^<; p 3 9pp 9fys 9v 9te MS? 2 is 55 ?**p &< C4 TgSI 2 t9. v OW I II sis ii r(0 >1*5 p S* 55 *5* 4* <lf. ff to 5*52 2 -! uJ: 5* U4HJZ W (1J to S3 4* WJ *1 VI <9 g1 X0>t M iLcS *c : .> * a ew5 H3.S- Su-S g*4* ge .e-se u^< x <e gVg HI I?g ig SS-i5* 32 V30P^o- IowS 43f Mb 4(0.-*PU* Jt S o 3p p <r St c in *sc 4* O (A ML i I *> .59 M9 P.d) p sum VI u e9*pe IS *Eo +ft* 6 II tr *S! C 3~ '4S# e 5, -8 *8e e-s ll3t 9 01 O) 4>*0,& S3 2 eg >.(* o, fci>$>f S: coo In is If C tf SS. 8 or * i ^f0s*" JS w,, mw $ 8 S* *J2 is.o V is .9 I4* 3 LA lA A PH *g PB12C/B 41U*0 ? Si * a II as o * 3 3 12-232 1/05/83 TEH 0531095 DUP050032008 PRELIMINARY DRAFT was a positive correlation In the Incidence of gaps, fragments, chromatid and chromosomal aberrations with blood Pb levels (Sarto et al., 1978 and Nordenson et al., 1978). However, as indicated in Table 12-18, in other studies there were no direct correlations between indices of Pb exposure (i.e, deltaaminoevulinic acid excretion) and incidence of chromosomal aberrations. Nutritional factors such as CA2+ levels in vivo or in yitro are also important since it is possible that the effects of Pb on cells may be antagonized by Ca2+. As with all studies of a human population exposed to Pb, exposure to other metals that may produce chromosomal aberrations was prevalent (Zn, Cd, and Cu). In a recent study by Forni et al. (1980), 18 healthy females with occupational exposure to Pb were evaluated for chromosomal aberrations in their lymphocytes cultured for 2 or 3 days. The results demonstrated a greater incidence of abnormal metaphases at the two culture times in the Control group, but these differences were not statistically significant. Statistically significant differences from the 72-hour Controls were noted in the 72-hour culture obtained from the Pb exposed group. These results demonstrate that the extended 72-hour culture time results in increased chromosomal aberrations in the control lymphocytes. The longer culture time was required to demonstrate the effects of Pb on chromosomal structure; however, the blood Pb levels in the exposed females ranged from 24 to 59 pg/100 ml while control females had blood Pb levels of 22 to 37 pg/100 ml. Again in this study there was a marginal effect of Pb on chromosomal aberration but the individuals were probably not sufficiently intoxicated with Pb to induce measurable chromosomal aberrations. Collectively, these studies show that Pb can in fact induce lymphocyte-chromosomal aberrations in vivo but only at very high concentrations where clinical symptoms of intoxication are evident. Several studies have also been conducted on the direct effect of soluble Pb salts on cultured human lymphocytes (Seek and Obe, 1974; Deknudt and Deminatti, 1978). In the former study longer culture time was used (72 hours) and Pb acetate was found to induce chromosomal aberrations at 100 pM. Pb acetate had no effect on chromatid aberrations induced with X-rays or alkylating agents (Beek and Obe, 1975). In another study Pb acetate at 1 mM and 100 pM caused minimal chromosomal aberrations (Deknudt and Deminatti, 1978). Both CdCl-j and ZnCl2 were more potent than Pb acetate in causing these changes; however, CdCl2 was more toxic than ZnCla, which displayed greater toxicity than Pb PB12C/B 12-233 1/05/83 TEH 0531096 DUP050032009 PRELIMINARY DRAFT acetate, ZnCl2 was shown to cause significant chromosomal aberrations (Deknudt and Deminatti, 1978), Lead acetate has been shown to induce chromosomal aberrations in cultured cells other than lymphocytes (Chinese hamster ovary cells; Bauchinger and Schmid, 1972). Chromosomal aberrations have been demonstrated in lymphocytes from cynomolyus 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., 1977), These aberrations accompanying a low Ca2+ diet were characterized by the authors as severe (chromatid exchanges, dispiralization, translocations, rings and polycentric chromosomes). Similar results were observed in mice (Deknudt and Gerber, 1969), The effect of low calcium on chromosomal aberrations induced by Pb is most likely due to interaction of Ca2+ and Pb2+ at the level of the chromosome. Lead acetate injected intraperitoneally did not produce micronuclei in mouse bone marrow cells (Oacquet et al., 1977). Some investigators found no effect of lead on the incidence of chromosomal aberrations in cattle (Leonard et al., 1974) and in mice given 1 gram of Pb per liter of drinking water for 9 months (Leonard et al., 1973), However, Muro and Goyer (1969) found gaps and chromatid aberrations in bone marrow cells cultured for 4 days isolated from mice maintained on 1 percent dietary lead acetate for 2 weeks. Lead acetate did not increase the frequency of sister chromatid exchange in lymphocytes at 0.01 mM (Beek and Obe, 1975). Chromosomal loss has been reported in Drosophila exposed to triethyl-Pb (4 mg/1) but inorganic Pb had no effect (Ahlberg et al., 1972; Ramel, 1973). 12.7.3,2 Effect of Pb on Bacterial and Mammalian Mutagenesis Systems--This section is included purely for the sake of completeness and to indicate that these test systems are not suited at the present time to screen for the potential mutagenicity of metal compounds (Heck and Costa, 1982). Use of bacterial systems for assaying of metal genotpxicity must await further development of bacterial strains that are appropriately responsive to known mutagenic metals (Rosenkranz and Poirier, 1979; Simmon, 1979a; Simmon et al., 1979; Nishioka, 1975; Nestmann et al., 1979). In plants, chromosomal aberrations (root tips; Mukherji and Maitra, 1976) and other mutagenic activity (chlorophyll mutations; Reddy and Vaidyanath, 1978) have been demonstrated with Pb, Mammalian cell mutagenic systems which screen for specific alterations in a defined gene mutation have not been useful in detecting activity with known carcinogenic metals (Costa and Heck, 1982). PB12C/B 12-234 1/05/83 TEH 0531097 DUP050032010 PRELIMINARY DRAFT 12.7.3.3 Effect of Pb on Parameters of DNA Structure and Function--A number of very sensitive techniques are in existence for examining the effect of metals on DNA structure and function in Intact cells. However these techniques have not been extensively utilized with respect to metal compounds, but future research will be directed in this area since these are obvious gaps in knowledge which investigators are becoming aware of. Considerable work with metals has been done in understanding their effects on enzymes involved with ONA transcription. Si rover and Loeb (1976) have examined the effect of Pb and other metal compounds upon the fidelity of transcription of DNA by a viral DNA polymerase, Relativey high concentrations of metal ions (in some cases mM range) were required to decrease the fidelity of transcription but there was a good correlation between metal ions which 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 result in 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. Pb at 4mM was among the metals listed as mutagenic or carcinogenic which caused a decrease in the fidelity of transcription (Sirover and Loeb, 1976), Other metals active in decreasing fidelity in addition to Pb included Ag , Be2 , Cd2 > Co2+, Cr2+, Cr3+, Cu2+, Mn2+, and Ni2+, No change in fidelity was produced by A1S+, Ba2+, Ca2+, Fe3+, K+, Rb\ Mg2+, Mg+, $e2+, Sr2+, and Zn2+. Thus this assay system appears sensitive in detecting an activity which may alter the genetic basis of a cell that may be involved in the carcinogeneic process. In a similar study, Hoffman and Niyogi (1977) demonstrated that PbCl2 was the most potent of 10 metals tested in inhibiting RNA synthesis (i.e., Pb2+ > Cd2+ > Co2+ > Mn2+ > Li+ > Na+ > K+) for both types of templates tested (calf thymus DNA and T4 Phage ONA). These results were explained by the authors in terms of the binding of these metal ions to the bases of the DNA more than phosphate groups (i.e., Pb2+ > Cd2+ > Zn2+ > Mn2+ > Mg2+ > Li+ = Na = K+). Additionally, metal compounds such as PbCl2 with carcinogenic or mutagenic activity were found to stimulate mRNA chain initiation at 0,1 mM concentrations. PB12C/B 12-235 1/05/83 TEH 0531098 DUP050032011 PRELIMINARY DRAFT Again it is evident that Pb has activity in an in vitro system which can lead to alterations in gene expression, an integral part of the neoplastic process. 12.7.4 Mechanisms of Pb Carcinogenesis It is evident from the relevant studies discussed that Pb must be implicated as a carcinogen but only at relatively high concentrations. An agent may act as a carcinogen at two distinct levels, 1) initiator or 2) promotor. By definition an initiator must be able to interact in some way with the DNA to produce a genetic alteration while a promotor acts in a way that allows the expression of the altered genetic change responsible for cancer. Since Pb is capable of transforming cells directly in culture and affecting DNA to DNA and DNA to RNA transcription it has some initiating activity. Its ability to induce chromosomal aberrations is also indicative of initiating activity. There are no studies which implicate or support a promotional activity of Pb although its similarity to Ca + may suggest that it can alter regulation of this cation which may be involved in many processes related to promotion (i.e.> cell growth). Intranuclear Pb inclusion bodies are an interesting observation which may be involved with its carcinogenic effects, since both the formation of these bodies and induction of tumors occur at relatively high doses of Pb. The interaction of Pb with key non-histone chromosomal proteins in the nucleus to form these inclusion bodies or the presence of these inclusion bodies in the nucleus may alter genetic function leading to cell transformation. Obviously elucidating the mechanism of Pb carcinogenesis requires further research efforts and only theories can be formulated regarding its oncogenic action at the present time. 12.7.5 Conclusion A recent IARC monograph (Vol. 32, p. 380, 1982) offered the following conclusion Concerning the carcinogenic role of Pb: "There is sufficient evidence that lead subacetate is carcinogenic to mice and rats and that lead acetate and lead phosphate are carcinogenic to rats. In the absence of adequate human data, it is reasonable for practical purposes to regard these compounds as if they presented a carcinogenic risk to humans." It is noteworthy to add that epidemiological studies of mortality of humans with excessive exposure to unspecified Pb compounds from all malignancies, and those of digestive and respiratory origins border on statistical significance. Pb compounds (Pb acetate) generally induce tumors in experimental animals at concentrations PB12C/B 12-236 1/05/83 TEH 0531099 DUP050032012 PRELIMINARY DRAFT where clinical signs of Pb toxicity are evident. At similarly high concentrations Pb compounds induce chromosomal aberrations in human lymphocytes, induce transformation of cells in culture and affect the DNA-+DNA and DNA-RNA transcription process in cells. The latter effects are produced at 4.0 mM and 0.1 mM PbCl2 respectively which was added to an isolated enzyme system. It is not unreasonable that cells may accumulate similarly high concentrations of Pb2+ in the nucleus. The ability of lead acetate to induce morphological transformation of cells in culture along with its activity in inducing chromosomal aberrations support the view that Pb and/or its compounds may initiate the carcinogenic process. PB12C/B 12-237 1/05/83 TEH 0531100 DUP050032013 PRELIMINARY DRAFT 12,8 EFFECTS OF LEAD ON THE IMMUNE SYSTEM 12.8.1 Introduction The immune system is comprised of cells (lymphocytes, macrophages, etc.), organs (spleen, thymus, lymph nodes, etc.), and secretory products (antibody, lymphokines, etc.). When a chemical is introduced into the body, it is absorbed into the blood stream, deposited into organs, and eventually eliminated. Immune cells and their products can essentially remain in constant contact with the chemical while in circulation as well as after deposition in organs. Thus, the immune system, which is a major line of defense for the body against invasion by infectious agents and perhaps neoplasia is possibly one of the first body systems provoked to respond to chemical exposure. The immune system of animals and man is complex and cooperation between the three major components, humoral, cell-mediated, and macrophage systems, is often necessary for optimal expression. Thus, when a compound is suspected of altering the immune response of a host, the humoral (B lymphocyte) as well as cellular (T lymphocyte) systems must be examined both collectively and individually. The third cell type is the macrophage which functions as an accessory cell in cooperation with B and T lymphocytes. The essential role of the macrophage is to sustain the immune response by immunoregulatory mechanisms. Finally, other cells such as polymorphonuclear neutrophils, natural killer, null cells, etc., also have important immune properties. 12.8.2 Host Susceptibility One method of initially ascertaining if a chemical may affect the immune response of an animal is to challenge an exposed animal with an LD^ dose of an infectious agent. These procedures are generally simple to perform and establish if the compound actually interferes with the course of a pathogenic disease. Agents most commonly used are endotoxins, bacteria, viruses, and parasites. 12.8.2.1 Bacteria and Virus--Lead impairs the ability of an animal to combat infectious agents (Table 12-19). Mice (Swiss Webster) injected intraperitoneally (i.p.) for 30 days with 100 or 250 pg of lead nitrate and inoculated with an LDj j q of Salmonella typhimurium had greater mortality than non-lead-exposed animals (Hemphill et al., 1971), These concentrations of lead were subclinical (without clinical manifestations) since none of the lead-treated mice showed toxic manifestations during the 30-day exposure period. Similar results were PB12D/D 12-238 1-05-83 TEH 0531101 DUP050032014 PRELIMINARY DRAFT observed in rats exposed to lead and challenged with Escherichia coli (Cook et al., 1975). In another study (Lawrence, 1981a), mice (CBA/J) exposed to 400 and 2,000 ppm lead for four weeks had reduced resistance to Listeria monocytogenes These experiments present data that lead impairs the ability of animals to resist bacterial infections. Lead has also resulted in increased susceptibility of mice challenged with viral agents. In one experiment (Gainer, 1977), CD-I mice administered 2,000 or 10,000 ppm lead in drinking water for 14 days and subsequently inoculated with eneephalomyocarditis virus (EMCV) had a significant increase in mortality compared with control EMCV-injected mice. In another study (Exon et al., 1979), Swiss-Webster mice were exposed to 13, 130, 1,300, or 2,600 ppm of lead for 10 weeks in the drinking water. After 10 weeks of exposure, the mice were inoculated with an LD^q dose of EMCV. As many as 80 percent (P > 0.05) of EMCV-inoculated mice exposed to the lowest dose (13 ppm) of lead died compared with 50 percent of non-lead-exposed EMCV-infected mice. These findings were confirmed in yet another investigation (Thind and Singh, 1977) in which leadinoculated mice (ICR) suffered 40 to 50 percent increased mortality upon challenge with Langat virus. Inhalation of lead chloride impaired elimination of bacteria (Serratia marcesens) from the lungs of mice (Schl'ipkoter and Frieler, 1977). The mice (NMRI) were exposed to an aerosol of 13.14 pg/m3 lead chloride for one to three hours for five days. The animals then inhaled fluorescent labelled non-pathogenic bacteria. Lead significantly reduced the normal clearance of bacteria from the lungs. Phagocytosis of the bacteria by alveolar macrophages was unaffected by lead. 12.8.2.2 Endotoxins--The ability of lead salts to induce a profound sensitiza tion to endotoxicosis in rats, mice, and chickens is well documented. A single intravenous (i.v.) injection of 5 mg of lead acetate rendered rats (Sprague-Dawley) up to 100,000 times more susceptible to concurrent i.v. administration of Escherichia colj endotoxin (Selye et al., 1966). These data were confirmed in rats (Holtzman) by others (Filkins, 1970; Cook et al., 1974) while a similar sensitivity to bacterial endotoxin occurred in adrenalectomized rats (Sprague-Dawley) treated with lead acetate (Schumer and Erve, 1973). Further, mice injected i.v. with 2 mg of lead and 5 pg of endotoxin from Salmonella typhimuirium had greater mortality than control mice (ICR) injected PB12D/D 12-239 1-05-83 TEH 0531102 DUP050032015 PRELIMINARY -ORAFT Table 12-19. Effect of Lead on Host Susceptibility to Endotoxins and Infectious Agents Species Parameter Effect Rat Rat Rat Rat Mouse Chick Mouse Rat Mouse Mouse Mouse Mouse E. coli endotoxin E. coli endotoxin S. enteriditis endotoxin . coli endotoxin 5. typhimurium endotoxin E. coli endotoxin ' $. typhimurium 1 E. coli L. monocytogenes EMC virus EMC virus Langat virus I I I I I I I I I I I I Reference Selye et al., 1966 Filkins, 1970 Cook et al., 1974 Schumer and Erve, 1973 Rippe and 8erry, 1973 Truscott, 1970 Hemphill et al., 1971 Cook et al., 1975 Lawrence, 1981a Gainer, 1977 Exon et al., 1979 Thind and Singh, 1977 PB12D/D 12-240 1-05-83 TEH 0531103 DUP050032016 PRELIMINARY DRAFT with endotoxin alone (Rippe and Berry, 1973). Lead potentiated the toxicity of bacterial endotoxin by 1000-fold in chicks dosed with 2.8 mg of lead acetate i.v. per 100 g body weight (Truscott, 1970). It must be kept in mind that the animals in these studies were exposed to lead parenterally (i.v.) which is an unnatural route of exposure to environmental lead, A pattern was established from those early host susceptibility studies which incriminated lead as a chemical which could perhaps be detrimental to health by methods other than direct toxicity. As noted in Table 1, lead conclusively results in increased susceptibility of animals to infectious agents and endotoxins. Lead is a pollutant which is ubiquitous in the environment. Moreover, since lead rendered animals, regardless of species or strain, more susceptible to infectious agents, the next step was to ascertain if lead had an effect on humoral immunity. 12.8.3 Humoral Immunity 12.8,3.1 Antibody Titers--A low antibody titer in animals exposed to lead could explain the Increased sensitivity of animals to endotoxins and bacterial infections (Table 12-20). In fact, lead acetate resulted in as much as a nine fold decrease in antibody titer in New Zealand white rabbits challenged with pseudorabies virus (Koller, 1973). In another study, Wistar dams were exposed to 5,000, 10,000, or 20,000 ppm lead for 20 days following parturition (Stankovic and Jugo, 1976). The progeny were weaned at 21 days of age and given standard laboratory chow for an additional month. At that time, they were injected with Salmonella typhimuriurn and serum antibody titers were assessed. Each dosage of lead resulted in significantly reduced antibody titers. More recently, rats (Sprague-Dawley) administered 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 8$A non-lead-exposed rats (Koller et al., 1983). Development of a highly sensitive, quantitative assay, enzyme-linked immunosorbent assay (ELISA) contributed to detecting the immunosuppressive activity of lead at this dosage. It should be mentioned at this point that normal rodent chow contains 1 to 2 ppm lead which must be considered as a baseline for control animals. Tetraethyl lead has also 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 three weeks. A significant reduction in PB12D/0 12-241 1-05-83 TEH 0531104 DUP050032017 PRELIMINARY DRAFT Table 12-20. Effect of Lead on Humoral Immunity Species Parameter Effect Reference Rabbit Rat Rat Mouse Rat Mouse Mouse Mouse Mouse Antibody titer - virus Antibody titer - S. typhimurium Antibody titer - BSA Antibody titer - SRBC Antibody synthesis - SRBC Antibody synthesis - SRBC DNP-Ficpll Antibody synthesis - LPS Memory C3 receptor, B cell D Roller, 1973 D Stankovic and Jugo, 1976 D Roller et al., 1973 D Blakley et al.,1980; Roller et al., 1976; Blakley et al., 1980 D Blakley and Archer, 1981 N N Blakley and Archer, 1981 D Roller and Roan, 1980 D Roller and Brauner, 1977 PB12D/D 12-242 1-05-83 TEH 0531105 DUP050032018 PRELIMINARY DRAFT hemagglutination titers to sheep red blood cells (SRBC) occurred at all levels of exposure. It was obvious from these investigations that lead, even at very low dosages, consistently suppressed antibody-mediated responses in animals. 12.8.3.2 Memory Response and B Cell Surface Receptors--The primary immune response represents the individual's first Contact with that particular antigen. The secondary immune response (memory, anamnestic response) represents 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 of a substantial number of antigen-sensitive memory cells after initial contact with the antigen. Impairment of the memory response, therefore, results in serious impairment of humoral immunity in the host. It has been documented that lead affects the memory response of lymphocytes. Mice exposed to 1,300 ppm lead for 10 weeks had marked suppression of the memory response (Roller and Roan, 1980). Since it is well established (LaFrenz and Feldbush, 1977; Braley-Mullen, 1978; Hosokawa et a!., 1979) that the B cell memory response is highly dependent on T lymphocytes, these data suggested that perhaps the helper T lymphocyte was also affected by lead. Subpopulations of lymphoid cells can be differentiated on the basis of characteristic cell surface antigens and receptors (Gone!ig-Meyling et al., 1976; Gormus and Shands, 1975). Since lead inhibits B cell function, lead could perhaps alter membrane receptor activity. In fact, when mice were exposed to 13, 130, or 1,300 ppm lead acetate for 10 weeks, the complement receptor sites on the surface of the 8 cell were altered (Roller and Brauner, 1977). 12.8.3.3 Enumeration of Antibody Producing Cells (Synthesis)--Lead appears to reduce antibody titers by impairing antibody synthesis. Mice (Swiss Webster) exposed orally to 13, 137, and 1,375 ppm of lead for eight weeks had reduced numbers of spleen cells producing IgM antibody in each dose.of lead-exposed mice (Roller and ICovacic, 1974). Even those that received the lowest lead dose (13 ppm) had a significant decrease in the number of IgG (secondary response) plaque-forming cells. This study disclosed that chronic exposure to lead produced a significant decrease in antibody synthesis, particularly IgG. Similar results occurred when Swiss Webster mice were exposed to a single 4-mg dose of lead either orally or i.p. (Roller et al., 1976). The secondary (IgG) response was significantly reduced while the IgM response was enhanced. PB12D/D 12-243 1-05-83 TEH 0531106 DUP050032019 PRELIMINARY DRAFT Female Sprague Cawley rats were exposed to 25 or 50 ppm lead acetate for seven weeks prior to breeding, three weeks during gestation, and three weeks while nursing their young (Luster et a!., 1978). The progeny were then weaned and continued on the same lead exposure regimen as their mothers. Both dosages of lead resulted in a significant reduction of antibody synthesis. In a recent report (Blakley and Archer, 1981), BDFj mice exposed to 50, 200, or 1.000 ppm lead for three weeks had diminished antibody responses at all dosages. One study (Lawrence, 1981a) using CBA/J mice failed to demonstrate an effect of lead (16-2,000 ppm) On antibody synthesis. These experiments indicate that lead modulates antibody synthesis in both laboratory mice and rats and that the humoral immune system in these animals fails to respond properly when exposed to lead. Lead, therefore, inhibits antibody synthesis in several species of animals and at dosages as low as 13 ppm lead. In summary, the humoral immune system is adversely affected by lead. Lead deters antibody production, impairs memory, and reduces activity of surface receptors on B lymphocytes (Table 2). Whether these effects result from direct action on B lymphocytes or other cells and substances which regulates activation and differentiation of B cells remains to be determined. 12.8.4 Cell-Mediated Immunity 12.8.4.1 Delayed-Type Hypersensitivitv--T lymphocytes mediate cell-mediated immunity. The T cell does not secrete antibody and must come in contact with the antigen to be effective. However, T cells do secrete a variety of soluble factors which regulate other immune cells. Groups of mice injected i.p. daily for 30 days with 0.5, 1.0, 1.5, or 2.0 ppm were subsequently sensitized iv with SRBC. The delayed-type hyper sensitivity (DTH) reaction was suppressed in these animals in a dose related fashion (Muller et al., 1977) (Table 12-21). 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 through gestation and lactation. The progeny were weaned at three weeks of age and continued on the respective lead exposure regimen as their mothers for an additional 14 to 24 days. Thymic weights and DTH responses were significantly decreased at both lead dosage levels responses. These results indicated that chronic low levels of lead suppress cell-mediated immune function. PB12D/D 12-244 1-05-83 TEH 0531107 DUP050032020 PRELIMINARY DRAFT Table 12-21. Effect of Lead on Cell'Mediated Immunity and Interferon Sped es Parameter Effect Reference Mouse Rat Mouse Mouse Delayed-type hypersensitivity De1ayed-type hypersens i11vity Mixed lymphocyte culture Interferon D Muller et al., 1977 D Faith et al., 1979 D Lawrence, 1981a N Gainer, 1974; Gainer, 1977 Blakley et al., 1982; AlPerson et al., personal communication PB12D/D 12-245 1-05-83 TEH 0531108 DUP050032021 PRELIMINARY DRAFT 12.8.4.2 Mixed Lymphocyte Reaction--When two populations of allogeneic lymphoid cells are cultured together, cellular interactions provoke blast transformation and proliferation of a portion of the cultured cells (Cerottini and Brunner, 1974; Bach et el., 1976). The mixed lymphocyte reaction is an in vitro assay of cell"mediated immunity analogous to in vivo host versus graft reactions. Mice (0BA/2J) fed 13, 130, or 1,300 ppm lead for 10 weeks' were evaluated for responsiveness in mixed lymphocyte cultures. The 130 ppm lead dose tended to stimulate the lymphocyte reaction (Koller and Roan, 1980). In another study (Lawrence, 1981a), mice (CBA/J) were fed 16, 80, 400, or 2,000 ppm lead for four weeks. The 16" and 80-ppm dose slightly stimulated while the 2,000-ppm dose suppressed the mixed lymphocyte reaction. Little data are available to evaluate the effects of lead on CMI. However, those data which are available indicate that low dosages of lead result in diminished CMI responses in both the rat and mouse. Thus, exposure to lead consistently suppresses both the humoral and CMI responses of laboratory animals. 12.8.4.3 Interferon--A1though susceptibility of lead-treated mice to infection could have been due to an alteration of interferon, a substance released by cells exposed to a virus that subsequently protects other cells against the viral infection, lead has not affected its production. Lead does not appear to inhibit the action of interferon in vivo (Gainer, 1974) or in vitro (Gainer, 1977). In a more recent study (Blakley et al., 1982) where mice (BDFX) were exposed to 1,000 ppm lead for three weeks, the production of immune interferon induced by T lymphocyte mitogens was unaffected. Similar results recently obtained indicated that injection of an interferon inducer in mice (Swiss Webster) exposed to lead prior to challenge with an LDgo dosage of EMC virus resulted in complete protection from lethality of the virus (Alderson and Woodard). Therefore, it was hypothesized that increased host susceptibility to viral agents associated with chronic ingestion of lead is by mechanisms other than interference with production of interferon. 12.8.5 Lymphocyte Activation by Mitogen 12.8.5.1 B Specific Mitogens---Polyclonal B cell-activators are substances which directly activate immunoglobulin cells to proliferate and secrete antibody by interacting with receptors that are not immunoglobulin receptors (Primi et al., 1977). The activation is non-specific since the variable regions of the PB12D/D 12-246 1-05-83 TEH 0531109 DUP050032022 PRELIMINARY DRAFT immunoglobulin receptors are not involved. This indicates that some B lymphocytes can be activated in the absence of antigen stimulation to secrete non-specific antibody. Escherichia coli 1ipopolysaccharide (LPS) stimulates B lymphocytes while pokeweed mitogen (PWM) activates both T and B lymphocytes. Interference of B blastogenesis may indicate impaired humoral immunity. However, it is important to realize that macrophages influence mitogen responsiveness of lymphocytes and that any change in function or concentration of macrophages may be reflected in lymphocyte transformation. Lymphocytes from Swiss Webster mice given 2,000 ppm lead orally for 30 . days had significantly reduced responses to PWM when compared with lymphocytes from non-lead-exposed mice (Gaworski and Sharma, 1979) (Table 12-22). Lead failed to alter LPS stimulation of B lymphocytes in two other studies. In one, mice (CBA/0) were exposed to 13, 130, or 1,300 ppm lead acetate for 10 weeks (Koller et al., 1979), while in the other, mice (BDFjJ were exposed to 50, 200 or 1,000 ppm lead acetate for three weeks (Blakley and Archer, 1982). Lymphocytes obtained from spleens of normal mice (Balb/c) have been cultured in the presence of lead in vitro and subsequently subjected to trans formation by LPS. Lead was found to have mitogenic properties (Shenker et a!,, 1979) as well as reduced viability of lymphocytes (Gallagher et al., 1979). Further, lead stimulated blastogenesis of the cultured lymphocytes. This reaction was confirmed in three other studies (Gaworski and Sharma, 1979; Lawrence, 1981a, b,c) in which lymphocyte responsiveness was enhanced when cells were cultured in 104 , 10 , or 10-fi M concentrations of lead acetate and stimulated with LPS or PWM. These contrasting results from in vitro and in vivo exposure to lead tend to question the validity of the Jjn vitro responses. Apparently, many immune cells and their soluble products which interact and regulate each other were missing from the culture systems and thus, the response was not representative of in vivo exposures. 12.8.5.2 T lymphocyte Mitogens--Mitogens induce blast formation in normal lymphocytes. Interference of mitogenic proliferation induced by Coneanavalin A (Con A) and phytchemagglutinin (PHA) could suggest an alteration of cell-mediated immune reponses. Lymphocytes collected from mice (Swiss Webster) given 250 ppm lead orally for 30 days had significantly reduced responses to PHA when compared with lymphocytes from non-lead-exposed mice (Gaworski and Sharma, 1979). In another study (Koller et al., 1979), lymphocyte responses to Con A were similar PB12D/D 12-247 1-05-83 TEH 0531110 DUP050032023 PRELIMINARY DRAFT Route Exposure Table 12-22. Effect of Lead on Mitogen Activation Mitogen Effect Reference In vivo In vivo PWM LPS In vitro LPS D N I In vitro In vivo In vivo In vivo In vivo In vivo In vivo In vitro In vitro In vitro PWM PHA Con A Con A, PHA Con A, PHA Con A, PHA Con A, PHA, SEA Con A, PHA Con A, PHA PHA I D N I D D I I N I Gaworski and Sharma, 1978 Roller et al., 1979; Blakley and Archer, 1982 Shenker et al., 1977; Gallagher et al., 1979; Lawrenee, 1981a,b,c Gaworski and Sharma, 1978 Gaworski and Sharma, 1978 Roller et al., 1979 Lawrence, 1981a Faith et al., 1979 Neil an et al., 1980 Blakley and Archer, 1982 Lawrence, 1981a Lawrence, 1981b Gaworski and Sharma, 1978 PB12D/D 12-248 1-05-83 TEH 0531111 DUP050032024 PRELIMINARY DRAFT for control and Iead-exposed mice (CBA/J). Lymphocytes collected from mice (CBA/J) exposed to 16, 80, 400, or 2,000 ppm lead for four weeks had responses to PHA that did not differ from control cells, except for the lowest dosage (16 ppm) which produced a significant enhancement (Lawrence, 1981a). The Con A-induced response was enhanced at the 80-ppm lead dosage while the other levels failed to alter the blastogenic response of T lymphocytes. Rats (Sprague-Dawley) were exposed to 25 or 50 ppm lead both In utero and post parturient for 46 and 56 days (Faith et al., 1979). Splenic lymphocyte responses to Con A and PHA were diminished at both lead exposure levels. A similar response occurred in lymphocytes collected from mice (C57B1/6) exposed to 1,300 ppm lead for eight weeks and subsequently stimulated with Con A and PHA (Neilan et al., 1980). Lead impaired transformation of lymphocytes by both mitogens. In a more recent study (Blakley and Archer, 1982) in which mice (BOFi) were exposed to 50, 200, or 1000 ppm lead for three weeks, blasto genesis of lymphocytes was increased by Con A, PHA, and staphylococcal enterotoxin (SEA). When lymphocytes were cultured (in vitro exposure) in the presence of -4 -5 -c ~ '.......... lead (10 , 10 , and 10 M), a significant increase in lymphocyte transformation occurred at the highest dosage when stimulated with Con A or PHA compared with non-lead-exposed cultures (Lawrence, 1981a). In another study by the same .4 -5 .6 author (Lawrence, 1981b), lead (10 , 10 , and 10 M) lead failed to signifi cantly alter the Con A and PHA proliferative responses of lymphocytes. On the other hand, lymphocytes cultured in the presence of 0.1, 0.5, or 1.0 M lead had a significantly enhanced response to PHA (Gaworski and Sharma, 1978). It must be kept in mind that lead by itself has been demonstrated to be directly mitogenic and stimulate blastogenesis of lymphocytes (Shenker et al., 1977). Further, direct exposure of lymphocytes in culture to lead has resulted in reduced viability of the lymphocytes (Gallagher et al., 1979). These investigations using polyclonal activators have generated data which is rather confusing. In two studies (Faith et al., 1979; Neilan et al., 1980), lead inhibited the PHA- and Con A- induced proliferation of lymphocytes while in another (Gaworski and Sharma, 1978), PHA- and PWM-induced blastogenesis was significantly decreased following lead exposure. However, lead has also produced a dose-dependent enhancement of T cell mitogenesis to Con A, PHA, and SEA (Blakley and Archer, 1982). In another study (Koller et al, 1979), lead did not alter transformation of lymphocytes. Similar results have been obtained PB12D/D 12-249 1-05-83 TEH 0531112 DUP050032025 PRELIMINARY DRAFT from mitogen activation of lymphocytes following i_n vitro exposure to lead. It has been stated that "Comparisons and extrapolations between the in vivo and in vitro mitogen responses with similar lead exposures are not meaningful" (Blakley and Archer, 1982). Thus, mitogen responses to lead not only are inconsistent within a mitogen group but at the same time do not parallel the humoral or CMI immune responses when cells are exposed in vivo in a fully competent immune system. 12.8.6 Macrophage Activity Macrophages have an important role as an accessory cell by interacting with both 8 and T lymphocytes. Macrophages are not only immunoregulators but also destroy and eliminate foreign antigens from the body. These cells also function in destruction of neoplastic cells. A single iv injection of lead impaired the phagocytic ability of Kupffer cells in the liver (Trejo et al., 1972). Similarly, lead injected iv depressed the intravascular clearance of lipids (Cook et al., 1974) and colloidal carbon (Trejo et al., 1972; Filkins and Buchanan, 1973). It was shown that the decreased vascular clearance resulted from impaired hepatic phagocytosis of the foreign particles (Trejo et al., 1972), However, lead given orally to mice (CBA/J) for 10 weeks stimulated phagocytosis and increased acid phosphatase levels in peritoneal macrophages (Roller and Roan, 1977). A single intravenous injection of lead would be similar to an acute oral exposure. When macrophages are tested in vivo, they are exposed to a variety of factors such as opsonins, alpha-2-globulins, lymphokines, etc., which regulate their activity. Thus, to assess the holistic effects of lead, the complexity of the immune system negates in vitro exposure and, in some cases, in vitro assays after _[n vivo exposures. Cells obtained from guinea pigs and cultured in the presence of lead inhibited the motility of guinea pig macrophages and decreased or abolished the effect of MIF on their migration (Klremidjian-Schumacher et al., 1981a.,b). Rat alveolar macrophages cultured in media which contained lead had depressed oxidative metabolic processes (Castranova et al., 1980). Lead has been demonstrated to suppress macrophage-dependent immune responses (Blakely and Archer, 1981). Lead (50 ppm) administered to mice (BDFi) for three weeks resulted in suppressed immune responses to a macrophage-dependent antigen, SRBC, but failed to affect the response to a practically macrophageindependent antigen, E, coli LPS. The immunosuppressive effect of lead was PB12D/D 12-250 1-05-83 TEH 0531113 DUP050032026 PRELIMINARY DRAFT restored by use of 2-mercaptoethanol, an In vitro substitute for macrophanges. The authors postulated from these investigations that lead inhibits the immune response by compromising macrophage function. Therefore, lead impairs three major components of immunity, i.e., humoral, cell-mediated, and macrophage. (See Table 12-23 for a summary of studies on the effects of lead on macrophages.) 12.8.7 Neoplasia Lead causes neoplasia in laboratory animals. The neoplastic activity of lead was best illustrated in a recent report (Koller et al., 1982) where 81 percent of rats exposed to lead for 18 months developed tumors. Lead also promotes other carcinogens. Lead acetate potentiated the oncogenicity of Rauscher leukemia virus in mice (Gainer, 1972). Intratracheal administration of a combination of lead oxide and benzo[a]pyrene resulted in adenomas and adenocarcinomas in the lungs of hamsters when neither alone produced tumors (Kobayashi and Qkamoto, 1974). Lead acetate also enhanced the formation of N(4'-fluoro-4-biphenyl) acetamide-induced renal carcinomas by reducing the latent period, increasing the number of tumors per animal, and increasing the percentage of tumor-bearing rats from 70 to 100 percent (Hinton et al., 1980), Growth of Moloney sarcoma virus-induced tumors was enhanced in animals exposed to lead levels of 130 to 1,300 ppm (Kerkvliet and Baecher-Steppan, 1982). Since lead compromises most all major components of the immune response in laboratory animals, perhaps lead-induced inhibition of the immune system contributes to development of certain types of neoplasia in a primary target organ such as the kidneys. Macrophage function was impaired in mice exposed to lead and inoculated with Moloney sarcoma virus (Kerkvliet and Baecher-Steppan, 1982). The action of lead on natural killer cells warrants investigation. 12.8.8 Mechanisms of Lead Immunomodulation The mechanism of toxic action of lead to cells is complex and presently not understood. The effect is most likely at the biochemical level of cellular metabolism. Lead has a high affinity for sulfhydryl groups which are important in structure of antibody molecules. A blockage of essential sulfhydry1-dependent enzymes or non-enzymatic sulfhydryl groups could result in disruption of antibody function. Since lead impairs antibody production, the site of action would most likely occur during synthesis of antibody rather than by catabolism of normally formed antibody molecules. Lead ions tend to accumulate on cell surfaces (Goyer and Rhyne, 1973), thereby possibly affecting receptor and membrane activity. Interference of PB12D/D 12-251 1-05-83 TEH 0531114 DUP050032027 PRELIMINARY DRAFT Table 12-23. Effect of Lead on Macrophages Sped es Parameter Effect Reference Rat Vascular clearance lipids Rat Vascular clearance colloidal carbon Mouse Mouse Clearance Serratia marcesens Phagocytosis * Mouse Phagocytosis Guinea pig Macrophage migration Rat Mouse Macrophage oxygen metabolism Macrophage-dependent antigens D Cook et al., 1974 D Trejo et al., 1972; Filkins and Buchanan, 1973 D Trejo et al., 1972; Kervliet and Steppan, 1982 I Koller and Roan, 1977 D Kiremidjian-Schumacher et al., 1981 D Castranova et al., 1980 D Blakley and Archer, 1981 PB12D/D 12-252 1-05-83 TEH 0531115 DUP050032028 PRELIMINARY DRAFT immune receptors or membrane function could deter the cascade of immune events. For instance, lead did impair the activity of complement receptors on the surface of B lymphocytes (Koller and Brauner, 1977), Further studies are necessary to elucidate the role of these structures in lead induced immuno suppression. The essential role of the macrophage in sustaining immune responses has been thoroughly established. Recent research has indicated that macrophages play a central role in immunoregulation via activities that not only enhance other cells of the immune system, but have inhibitory effects on various mediators. It has been proposed that lead may actually produce its immuno suppressive effect by compromising on early macrophage function (Blakley and Archer, 1981). The ability of macrophages to produce reactive oxygen metabolites correlates closely with macrophage metabolic activity. Suppression of cellular oxidation-reduction reactions have been reported to occur in lead intoxication (Gmerek et al., 1981). This presents another pathway by which lead could modulate immune responses. 12.8.9 Lead Blood and Tissue Residues The renal concentrations of lead from mice exposed 10 weeks to either 13, 130, or 1300 ppm were comparable in two separate experiments. The kidneys in mice exposed to 13 ppm lead contained wet weight residues which ranged from 0.36 to 0.65 ppm, the lead residues in the 130-group were from 3,00 to 3.77 while those in the 1300 group ranged from 12.20 to 13.00, Lead levels were not measured in the blood. Mice exposed to 50, 200, or 1000 ppm lead for three weeks had blood lead concentrations of 0.254, 0.386, and 0.826 pg/ml, respectively. Antibody synthesis was affected at the 50-ppm dosage in mice (BDFi) with blood lead concentrations of 0.254, 0,386, and 0.826 pg/dl. These investigations illustrate that blood lead levels considered permissible for man resulted in significant suppression of the immune system in mice, 12.8.10 Human Studies Few studies have evaluated the effects of lead on the immunologic response of man. Reigart and Garber (1976) studied 12 preschool children having elevated free erythrocyte protoporphyrin and blood levels 5 40 pg/dl and seven non-leadburdened children for evidence of impairment of their immunological responses. They found no differences between the control group and the lead-exposed group PB12D/D 12-253 1-05-83 TEH 0531116 DUP050032029 PRELIMINARY DRAFT with reference to complement levels, immunoglobulins, or anamnestic response to tetanus toxoid antigen. It has been noted that children with persistent blood lead levels and infected with Shigella enteritis had prolonged diarrhea (Sachs, 1978). 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 man. The paucity of information cannot currently support the formulation of any dose-response relationship for man. Epidemiologic investigations may help to determine if lead alters the immune system of man and consequently increases susceptibility to infectious agents and neoplasia. 12.8.11 Summary Lead suppresses the three major components of the immun'e system (i.e., humoral, cellular, and macrophage). Lead also renders animals highly susceptible to endotoxins and infectious agents. Host susceptibility and the humoral immune system appear to be particularly sensitive but as postulated in recent studies, the macrophage may be the primary immune target cell of lead. Leadinduced immunosuppression occurs at very low subclinical dosages and, therefore, may be detrimental to the health of animals and perhaps of man by mechanisms other than the well-documented neurotoxicity which occurs at larger dosages. The data accumulated to date provide good evidence of that lead affects immunity, but additional studies are necessary to elucidate the actual mechanism by which lead exerts its immunosuppressive action. The effect of lead on the immune system of man is virtually lacking and must be properly ascertained in order to determine permissible levels for human exposure. However, since this chemical modulates immunity in laboratory animals and is immunosuppressive at very low dosages, an awareness of its potential serious effects in man should be considered for regulatory purposes. PB12D/D 12-254 1-05-83 TEH 0531117 DUP050032030 PRELIMINARY DRAFT 12,9 EFFECTS OF LEAD ON OTHER ORGAN SYSTEMS 12.9.1 THE HEPATIC SYSTEM The effect of lead poisoning on liver function has not been extensively studied. In a laboratory study of 301 workers in a lead-smelting and refining facility, Cooper et al. (1973) found serum glutamic oxalacetic transaminase (SGOT) activity at an increased value of 11.5 percent in subjects with blood lead levels below 70 pg/dl, 20 percent in those with a blood lead level of about 70 pg/dl, and 50 percent in workers with a blood lead level of about 100 pg/dl. The correlation between blood lead levels and SGOT was not statistically significant. In the absence of information on the possible influence of diet, infection, or personal habits, however, the authors were unable to draw any definite conclusions concerning the etiology of these changes. In lead workers with moderate effects on the hematopoietic system and no obvious renal signs, GOT was not increased compared to controls on repeated examinations (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 acute lead poisoning, the mixed-function oxidase system of liver endoplasmic reticulum is impaired (Alvares et al., 1972). The activity of this enzyme system, involved in the hepatic biotransformation of medicaments, hormones, and many environmental chemicals, is closely related to the availability of the microsomal heinoprotein, cytochrome P-450 (Remmer et al., 1966), It has been shown that in rats lead induces inhibition of heme synthesis and, therefore, causes a reduction in cytochrome P-450 levels, with consequent impairment of the mixed-function oxidase system (Scoppa et al., 1973), Drug-metabolizing activities were significantly decreased in the lead-poisoned animals. Intensity and duration of these changes were dose-dependent. In vivo experiments, based on the duration of pentobarbital sleeping time, provided further evidence for the inhibition of drug metabolism in lead-poisoned rats. These data would suggest that an enhanced sensitivity to xenobiotics (drugs, pesticides, food additives, etc.) should be expected to occur in lead-poisoned animals. Alvares et al. (1975) studied the effect of lead exposure on drug metabolism in children and adults. There were no differences between two normal children and eight lead-poisoned children in their capacities to metabolize two test drugs, antipyrine and phenylbutazone. This might suggest that low plasma concentrations of lead do not have an PB12D/8 12,-255 1-05-83 TEH 0531118 DUP050032031 PRELIMINARY DRAFT effect on the hepatic cytochrome P-450-dependent enzymatic activities in children. In 2 acutely poisoned children in whom plasma levels of lead exceeded 60 pg/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. Meredith et al. (1977) also demonstrated enhanced hepatic metabolism of antipyrine in lead-exposed workers following chelation therapy. The significance of this evidence of restored hepatic mixed oxidase function is, however, unclear because the pretreatment antipyrine biologic half-life and clearance were not significantly different in lead-exposed compared with control subjects. Moreover, the effect of chelation on antipyrine metabolism in non-lead-exposed control subjects was not determined. In 11 children with blood lead levels between 43 and 52 pg/dl, Saenger et al. (1981) found a decrease in 24-hour urinary 6-beta-hydroxycortisol excretion which correlated closely (r = 0.85, < 0.001) with a standardized EDTA lead-mobilization test (1000 mg E0TA/m^ body surface area). This glucocorticoid metabolite is produced by the same hepatic microsomal mixed function oxidase system that hydroxylates antipyrine. The EDTA provocative test proved to be more sensitive to the presence of excessive body lead stores than did blood lead or erythrocyte protoporphyrin levels (Saenger et al., 1982). These 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). 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 hepatic 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 two authors concluded that chronic lead exposure results in significant inhibition of the heme biosynthetic pathway without causing significant changes in hepatic Cytochrome P-450-associated enzymatic activities. In a few animal studies special attention has been paid to effects of lead on the liver. White (1977) gave 8 beagle dogs oral doses of lead carbonate, 50- PB12D/B 12.-256 1-05-83 TEH 0531119 DUP050032032 PRELIMINARY DRAFT 100 mg PB/kg bw, for 3-7 weeks. Lead concentrations were not measured in blood or tissues. In two dogs exposed from 5 weeks to 50 mg/kg, morphological changes were noted. Changes in enzyme activities were noted in most exposed animals, e.g., some dehydrogenases showed increased activity after short exposure and decreased activity after longer exposures, mainly seen 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 bw. It can be concluded that effects on the liver may be expected to occur only at high exposures. If effects on more sensitive systems, i.e., the nervous and hematpoietic systems, are prevented, no effects should be noted in the liver. PB12D/B 12.-257 1-05-83 TEH 0531120 DUP050032033 PRELIMINARY DRAFT 12.9.2 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 hypertension is reviewed in Section 12.5.3.5 above. Under conditions of long-term lead exposure at high levels, arterio sclerotic changes have been demonstrated in the kidney. Dingwall-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 recently. There are conflicting reports regarding whether lead can cause athero sclerosis in experimental animals. Scroczynski et al. (1967) observed increased serum lipoprotein and cholesterol, and cholesterol deposits in the aortas of rats and rabbits receiving large doses of lead. On the other hand, Prerovska (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 of the myocardium have been noted in 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 coincidental. In many cases in which encephalopathy is present, the electrocardiographic abnor malities 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 children (70 percent) having symptoms of lead toxicity. After chelation therapy, the electrocardiograms remained abnormal in only four (13 percent) of the patients. Electron micro scopy of the myocardium of lead-intoxicated rats (Asokan, 1974) and mice (Khan et al., 1977) have shown diffuse degenerative changes . Kopp et al. have demonstrated depression of contractility, isoproterenol responsiveness, cardiac protein phosphorylation (1980a) and high energy phosphate levels (1980b) in hearts of lead-fed rats. Similarly, persistent increased susceptibility to PB12D/C 12-258 1-05-83 TEH 0531121 DUP050032034 PRELIMINARY DRAFT norepinephrine-incluced arrhythmias has been observed in rats fed lead during the first 3 weeks of life (Hejtmancik and Williams, 1977, 1978, 1979; Williams et al., 1977). 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. Kosmider and Petelnz (1962) examined 38 adults over 46 years of age with chronic lead poisoning. They found that 66 percent had electrocardiographic changes, which was four times the expected rate for that age group. Makasev and Krovdina (1972) observed a two-phase change in the permeability of blood vessels (first, increased permeability; second, 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 cardiovascular pathology of lead poisoning. The susceptibility of the myocardium to toxic effects of lead was supported by Jj2 vitro studies in rat mitochondria by Parr and Harris (1976). These investigators found that the rate of Ca++ removal by rat heart mitochondria is decreased by 1 nMol Pb/mg protein. 12.9.3 THE ENDOCTINE SYSTEM The endocrine or hormonal system effects of lead are not well defined at the present time, but some evidence exists for such effects, at least at high Pb-exposure levels. Lead is thought, for example, to decrease thyroid function in man and experimental animals. Porritt (1931) suggested that Pb 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) ob served impaired concentration of I by thyroid glands in 10 out of 41 patients with industrial plumbism. Subsequently, Zel'tser (1962) showed that in vivo 131 " ' ~ I uptake and thyroxine synthesis by rat thyroid were decreased by lead when doses of 2 and 5 percent lead acetate solution were administered. Uptake of 131 I, sometimes decreased in men with lead poisoning, can be offset by treatment with thyroid-stimulating hormone (TSH) (Sandstead et al., 1969; Sanstead and PB12D/C 12-259 1-05-83 TEH 0531122 DUP050032035 PRELIMINARY DRAFT Galloway, 1967). Lead may act to depress thyroid function by inhibiting SH groups or by displacing iodine in a protein sulfonyl iodine carrier (Sanstead 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 lower ranges of blood-Pb levels (i.e., below 30-40 pg/dl). Sandstead et al., (1970a) 'studied the effects of lead intoxication on pitu itary and adrenal function in man and may produce clinically significant hypo pituitarism in some. P'b effects 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, suggesting a possi ble Pb impact on pituitary-adrenal hormonal functions. That excessive oral in gestion of lead may in fact result in pathological changes in the pituiraryadrenal axis is also indicated by reports of Pb-induced decreased metapyrone responsiveness, a depressed pituitary reserve, the decreased immunoreactive ACTH (Murashov, 1966; Pines, 1965), These same events may also affect adrenal gland function in as much as decreased urinary excretion of 17-hydrorhy-corticosteroids was observed in these patients. Also, suppresion of responsiveness to exogenous ACTH in the zona fasciculate of the adrenal cortex has been reported in leadpoisoned subjects (Makotchenko, 1965), and impairment of the zona glomerulosa of the adrenal Cortex has also been suggested (Sanstead et al., 1970). Once again, however, none of these effects (on adrenal hormone function) have been shown to occur at blood-Pb levels as low as 30-40 pg/dl. Other studies provide evidence suggestive of Pb-exposure effects on endo crine systems controlling reproductive functions. For example, evidence of ab normal leutinizing hormone (LH) secretory dynamics was found in secondary lead smelter workmen (Braunstein et al., 1978). Reduced basal serum testosterone levels with normal basal LH levels but a diminished rise in LH following stimu lation Indicated suppression of hypothlamic-pituitary function. Testicular biop sies in two lead poisoned workmen showed pertitubular fibrosis suggesting direct toxic effects of lead in the testes as well as effects at the hypothalamicpitui tary level. No evidence was provided, however, suggestive of effective blood-Pb levels being as low 30-40 pg/dl. Lancranjan et al. (1975) also reported leadrelated interference with male reproductive functions. Moderately increased lead absorption (blood lead mean - 52.8 pg/dl), among a group of 150 workmen who had PBI2D/C 12-260 1-05-83 TEH 0531123 DUP050032036 PRELIMINARY DRAFT 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 test for hypothalamopituitary influence were negative. 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 1 to 2 months, where ambient air Pb levels were <7 pg/m . Using a classification of various age groups (20 to 25, 26 to 35, and 36 to 40) 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. It should be noted that the report does not show the age distribution, the level of significance, or the data on specificity of the method used for classification. Also, Zjelhuis and Wibowo (1977), in a critical review of the above study, concluded that study design and presentation of data are such that it is difficult to evaluate the author's conclusion that chronic exposure to low air Pb-levels leads to disturbed ovarian function. It should 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 report exists in the literature in regard to assessing lead effects on human ovarian function or other factors 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 indicated that at least high-level Pbexposure induces stillbirths and abortions). Also, only the results of one animal study (Petrusz et al., 1978) are available that indicate that orally administered Pb can exert effects on pitui tary and serum gonadotropins, which may represent one mechanism by which Pb affects reproductive functions. However, the blood-Pb levels at which alter ations in serum and pituitary follicle stimulating hormone (FSH) were observed in neonatal rats far exceeded 30 pg/dl, as reported by Petrusz et al. (1978). PB12D/C 12-261 1-05-83 TEH 0531124 DUP050032037 PRELIMINARY DRAFT 12.9,4 THE GASTROINTESTINAL SYSTEM Colic is usually a consistent early symptom of lead poisoning, warning of much more serious 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 present in infants and young children. Beritic (1971) reported on cases of 13 of 64 men exposed on their jobs to occupational levels of lead. The 13 had colic, probably lead-related, and constipation. They had blood lead levels ranging from a little less than 40 to $0 pg/dl as determined by polarography, a technique that tends to yield values lower than the actual blood lead levels. The diagnosis of lead-caused colic was supported by findings of high urinary coproporphyrin, excessive baso philic stippling, reticulocytosis, and some degree of anemia, all of which are other clinical signs of lead poisoning. Although these symptoms are well documented in the literature, there are insufficient data by which to establish dose-effect or dose-response relationships for lead effects on the gastrointestinal system, particularly at exposure levels expected to be encountered by the general population in the ambient environment. PB120/E 12-262 1-05-83 TEH 0531125 DUP050032038 PRELIMINARY DRAFT REFERENCES Adams; Boice (1981). Adebenajo, F. 0. (1974) Hematologic status of urban black children in Phila delphia. 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Yip, R.; Norris, T. N,; Anderson, A, S. (1981) Iron status of children with elevated blood lead concentrations. J. Pediatr. (St. Louis) 98: 922-925. Yu, T. A. (1982). Yu, T. A.; Berger, L. (1982) Impaired renal function in gout: its association with hypertensive vascular disease and intrinsic renal disease. Am. J. Med. 72: 95-100, Yu, T. F,; Berger, L. (1982) Clinical aspects of nephropathy in gout and hyperuricemic states. In: The kidney in gout and hyperuricemia. Mt. Kisco, New York: Futura Pub. Com. Yule, W.; Lansdown, R. (1981) Blood lead concentrations and school perform ance. Br. Med, J. 283: 1336. PB12R/F 12-320 l-OS-ft* TEH 0531183 DUP050032096 PRELIMINARY DRAFT APPENDIX 12A: ASSESSMENT OF STUDIES REPORTING DATA REGARDING 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 essentiality studies involving effects of lead deficiency be done using synthetic or semi-synthetic diets prepared from components extrememly low in lead or by the use of chemical agents to reduce the level of background lead in the components. Such procedures, particularly the use of chelating agents to remove lead, can entail risk in terms of the potential effect of such procedures 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 measur able enhancement in growth rate compared to low-lead animals. In a critique of the Schwarz results, Nielsen (1980) points out that all of the animals in the Schwarz study, both low-lead and supplementation groups, showed sub-optimal growth which can be ascribed to riboflavin deficiency (Moran 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 questioned the statistical methods used in the Schwarz studies and also pointed out that lead addition to the diet was of no apparent benefit to deficient controls in subsequent studies. The problems associated with such studies can be seen in the fact that Schwarz was not able to duplicate any of the growth rate date for an extended period of time, attributed by the author to 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 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, PBAPP/F 12A-1 1/05/83 TEH 0531184 DUP050032097 PRELIMINARY DRAFT 1981b; 1981c; 1981d; Kirchgessner and Reichlraayr-Lais, 1981a; 1981b) and changes in certain enzyme activities and metabolite levels (Reichimayr-Lais and Kirchgessner, 1981e; Kirchgessner and Reichimayr-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 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. 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 experiement, only the starch and cellulose components were treated, with APDC (Schnegg, 1975). Although the report of Reichimayr-Lais and Kirchgessner (1981b) indicated that the cellulose and starch extraction treatment was done on all of the material, a communication in this regard (M. Kirchgessner to ECAO, 10/26/82) noted that only a portion of the starch and cellulose 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 assumed to also remove the chelants. Caution must be exercised in interpreting these studies as they currently stand, owing to the use of the chelating agents EDTA and/or APDC. Retention of free chelating agent(s) in the diets employed could potentially affect the bioavailability of certain metals and concerns regarding this are noted below. In the report of Davis and coworkers (1962), it was noted that diets containing soy bean protein which had been extracted with EDTA to lower iron content, followed by supplementation with iron and copper, were associated with iron deficleny in chicks maintained on these diets when compared to chicks fed the same level of iron and copper in untreated diets. Clearly, EDTA treatment of the soy bean protein affected iron bioavail ability in this study. Subsequently, the authors (Davis and coworkers, 1964) attempted to determine the presence of EDTA in the diets, simultating those used earlier. The crude methodology employed made an accurate quantitation difficult, but the amounts PBAPP/F 12A-2 1/05/83 TEH 0531185 DUP050032098 PRELIMINARY DRAFT of EDTA measured ranged up to 67 pg/g diet. Other investigations through the years have documented that EDTA will affect iron absorption/retention and utilization in various species (e.g., Larsen et al., I960;. Brise and Hallberg, 1962; Saltman and Helbock, 1965; Guenther et al., 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 (Shimamura and Shimamura, 1982) and human fibrinogen (Nieuwenhuizer et al., 1981). Furthermore, complete removal of EDTA from these rather diverse proteins are reported to involve forcing conditions and the washing procedure used by the authors of the studies in question gives no assurance of being adequate for chelant removal. Available information also suggests that diets retaining free ADTA and/or APDC, even at quite low levels, may pose problems in the subject studies by affecting the bioavailability of the essential metal, nickel. The studies of Schnegg and Kirchgessner (see review of Kirchgessner and Schhegg, 1980) have shown that nickel deficiency in rats followed over several generations is associated with reduced growth rate, disturbed hematological indices, lowered tissue iron, reduced iron absorption, and disturbances in enzyme activities and metabolite levels. According to Nielsen (1980), nickel has 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 Reichimayr-Lais, 1981b), The interaction of nickel with the chelants EDTA and/or APDC in the context of bioavailability has been documented. Dithipcarbamates such as APDC are effective chelation therapy agents in protecting against nickel toxicity (see review of Sunderman, 1981) while the report of Solomons and coworkers (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 PBAPP/F 12A-3 , 1/05/83 TEH 0531186 DUP050032099 PRELIMINARY DRAFT nickel monitored. When nickel was co-ingested with EDTA (40 mg of Na2EDTA. H20, a 1,3:1 EDTA/Ni ratio), 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 fj-generation 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 used can only be inferred, given the absence of any further experimental 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 immediately affect nickel bound up as the EDTA (as the common 1:1 complex) or APDC (as the common 1:2 complex) 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., 1959), 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 nutritional requirement is noted to be ca. 50 ppb (Kirchgessner and Schnegg, 1980; Nielson, 1980). The corresponding amounts of APDC and EDTA required to bind up 1.0 ppm nickel calculate to be 5,4 pg/g (1:2 complex, Ni-APDC) and slightly under 5 pg/g (1:1 complex, Ni-EDTA respectively. Hence only traces of free chelants would be required to 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 PBAPP/F 12A-4 1/05/83 TEH 0531187 DUP050032100 PRELIMINARY DRAFT 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 in which Pb-EDTA if formed by exchange of ligand from Fe-EDTA, is dissociated in -vivo, and the displacement process repeated, would have to be invoked. Nickel supplementation at 20 ppm in the Schnegg and Kirchgessner studies, where a similar APDC procedure was used to purify starch and cellulose com ponents, 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 disparity in the level of supplementation. Given the above concerns, it would appear that: 1) further experiments are necessary to conclusively determine that diet preparation in the ReichlmayrLais and Kirchgessner studies did not involve retention of free chelating agents, using methodology such as scintillography and labeled chelants. 2) determination of levels of nickel and lead in tissues, blood and excreta would greatly help to elucidate the true role of lead 3) replication of the results in the authors' or another laboratory, preferably with minimal chelant treat ment of components, should be done. It appears that the various reports describe basically single experiments over several generations, one at a diet level of 18 ppb lead, and one at 45 ppb lead. PBAPP/F 12A-5 1/05/83 TEH 0531188 DUP050032101 PRELIMINARY DRAFT APPENDIX 12A - REFERENCES Brise, H., Hallberg, L. (1962). Iron absorption studies. A method for method for comparative studies on iron absorption in man using 2 radio-rion 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 in absorption of dietary non-heme iron. Amer. J. Clin. Nutr. 29: 614-620. Csopak, H., Szajn, H. (1973). Factors affecting the zinc content of E. coll alkaline phosphatase. 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. Guenther, R. (1969). Distribution and excretion of radio iron in the rat as influenced by chelating agents. Arch. Pharmacol. Exp. Pathol. 262: 405-418. Hegenauer, J., Saltman, P., Nance, G. (1979). Iron (III) - photophoprotein chelates: Stoichiometric equilibrium constants for interaction of iron and phosphoryl serine residues of phosvitin and casein. Amer. 0. Clin. Nutr. 32: 809-816. Kirchgessner, M., Schnegg, A. (1980). Biochemical and physiological effects of nickel. In: Nickel in the Environment (J. 0. Nriagu, Ed.), Wiley, New York, pp. 635-652. Kirchgessner, M., Reichlmayr-Lais, A. M, (1982). Concentrations of various metabolites in experimental lead deficiency. Ann. Nutr, Metab. 26: 50-55, Kirchgessner, M., Reichlmayr-Lais, A. M. (1981a). Changes of iron concentra tion and iron-binding capacity in serum resulting from aliementary lead deficiency. Biol, Trace Elem, Res. 3: 279-285. Kirchgessner, M., Reichlmayr-Lais, A. M. (1981b). Retention, absorbierbarkeit und intermeditare neifugbarkeit von eisen bei alimentarem bleimangel. Inti. J. Vitamin. Nutr. Res. 51: 421-424, Larsen, B, A., Bidwell, R. E. S., Hawkins, W. W. (1960). The effect of ingestion of disodium ethylene diamine tetra-acetate on the absorption and metabolism of radioactive iron by the rat. Can. 0. Biochem. Physiol. 38: 51-55. PBAPP/F 12A-6 1/05/83 TEH 0531189 DUP050032102 PRELIMINARY DRAFT Morgan, J. K., Schwarz, K. (1978), Light sensitivity of riboflavin in amino acid diets. Fed. Proc. 37:. 671. Nielsen, F. H. (1980). Possible functions and medical significance of the abstruse trace metals. In: Inorganic Chemistry in Biology and Medicine, ACS Symposium Series, No. 140, American Chemical Society, Wash., O.C., pp, 23-42. Nielsen, F. H., Zimmerman, T, J., Colling, M. ., Myron, D. R. (1979). Nickel deprivation in rats: nickel-iron interactions. J. Nutr. 109: 1623-1632. Nieuwenhuizen, W., Vermond, A. , Hermans, T. (1981), Human firninogen binds EDTA and citrate. Thromb. Res. 22: 659-663, Pribl, R, (1972). Analytical Applications of EDTA and Related Compounds. International Series of Monographs in Analytical Chemistry, Vol. 52. Pergamon, New York. p. 27. Price, E. M., Gibson, J. F. (1972). A re-interpretation of bocarbanatofree ferric transferrin E.P.R. epertico. Biochem. Biophys. Res. Commun, 46: 646-651, Reichlmayr-Lais, A. M,, Kirchgessner, M. (1981a), Zur essentialitat von blei fur das tierisehe Wachstum. Z. Tierphysiol. Tierernnahrg. u. Futtermi ttel kde. 46: 1-8. Reichlmayr-Lais, A. M., Kirchgessner, M. (1981b). Depletions studien zur essentialitat von blei an wachsenden ratten. Arch. Tierernnahrung 31: 731-737. Reichlmayr-Lais, A. M., Kirchgessner, M. (1981c), Hamatologische veranderungen bei alimentarem blei mangel. Ann. Nutr. Metab. 25: 281-288. Reichlmayr-Lais, A, M., Kirchgessner, M. (1981d). Eisen-, kupfer- und zinkgehalte in neugeborenen sowie in leber und milz wachsender ratten bei alimentarem blei-mangel, 1. Tierphysiol, Tierernahrg, u. Futtermi ttel kde. 46: 8-14, Reichlmayr-Lais, A. M., Kirchgessner, M. (1981e). Activities-Veranderungen verschiedener Enzyme im alimentaren Blei-Mangel, Z Tierphysiol. Tiernahr. Futtermittelk. 46: 145-150. Saltman, P., Helbock, H. (1965). The regulation and control of intestinal iron transport. Proc. Symp. Radio-isotyope Anim. Nutr. Physiol., Prague, Czeckoslovakia. pp, 301-317, Sastri, V. S., Aspila, K. I., Chakrabarti, C. L, (1969). Studies on the solvent extraction of metal dithiocarbamates. Can. J. Chem, 47: 2320-2323. Schnegg, A. (1975). Ph.D. Dissertation, Technical University, Munich-- Weihenstephan. PBAPP/F 12A-7 1/05/83 TEH 0531190 DUP050032103 PRELIMINARY DRAFT Schwarz, K. (1975). Potential essentiality of lead. Ark. Rada Toksikol. 26 (Suppl.): 13-28. Shimomura, 0., Shimomura, A. (1982). EDTA-binding and acylation of the Ca(2+)-sensitive photoprotein aequorin. F.E.B.S. Lett, 138: 201-204. Solomons, 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 nicke. J. Nutr. 112: 39-50. Sunderman, F. W., Chelation therapy in nickel poisoning (1981). Ann, Clin. Lab. Sci. 11: 1-8, P8APP/F 12A-8 1/05/83 TEH 0531191 DUP050032104 PRELIMINARY DRAFT APPENDIX 12-B; EVALUATION OF CLINICAL MEASURES OF LEAD. EXPOSURE TABLE 12B-1, ADVANTAGES AND DISADVANTAGES OF DIRECT MEASURES OF LEAD EXPOSURE Method Advantages Disadvantages Blood lead EPa Examination of red cells basophilic stippling Useful for screening programs Measures only recent exposures; grams variable laboratory measurements; affected by physiologic state (e.g., infections, acidosis); collection procedures may be difficult in young children Useful for screening; earlier Levels increased in iron defi- indication of exposure than ciency as well as with lead blood lead; unaffected by exposure; measures only recent contamination with environ* exposure mental lead; can also be used as indicator of pre-anemia state -- Not always found in chronic clinical lead poisoning; rela tively insenstive to lesser degrees of lead exposure Inhibition of 6-ALAD^ activity assayed in in yitro in circu1ating' erythrocytes Coproporphyrin excretion in urine 6-ALA excretion in urine Calcium disodium EDTAC mobi1ization test X-ray of long bone Measurable effects at fairly low levels of lead exposure (20 pg/dl) Measureable effects at fairly low levels of lead exposure (40 pg/dl) Measurable effects at fairly low levels of lead exposure (40 pg/dl) Provides measure of mobile or potentially toxic fraction of total body lead burden Measure of past exposure Limited availability of test Measures only at recent expos sure Measures only recent exposure Potentially dangerous if bloodlead level >70 pg/dl; difficult to collect speciments in young children Not useful for measurement of recent exposure; "lead lines" seldom seen in chil dren <24 months of age; high false negative rate; radiation exposure PB11A/E 12B-1 12/10/82 EH 0531192 DUP050032105 PRELIMINARY DRAFT TA8LE 12B-1 (continued). Method Advantages Disadvantages Flat plate of abdomen ZPPd Dentine lead Hair -- Shows ingested foreign material only within preceding 24-36 hr; high false negative rate; radiation exposure. Measures average exposure over approximately the prior 4 months; less variable under Conditions of variable expo sure than blood lead Measures only relatively re cent exposure Quantitates cumulative expo Unable to determine precise sure; no loss of lead over age at exposure time; dose-related measure of exposure; not affected by chelation; easily sampled in young children; can accurately measure low level exposures Easily sampled Uncertain whether hair re flects a body metal burden; easily contaminated by envi ronmental lead. aErythrocyte protoporpyrin. bS-Arainolevo1inic acid dehydratase, cEthylene diaminetetraacetate. dZinc prbtoporphyrin. Reprinted from H, L. Needleman (ed.), tow Level Lead Exposure, by permission of Raven Press. PB11A/E 128-2 12/10/82 TEH 0531193 DUP050032106 TABLE 12B-2. TESTS COMMONLY USEQ IN A PSYCHO-EDUCATIONAL BATTERY FOR CHILDREN 2 mJt c o * 5*2 !- w S. io a2 C Im ssw *>4* - e ?42 +> ggs^*s5 to .5 Av ?SS opes. <A ** 0 ,P to o >- S >.11 JC C V Sv V* (to 4* r(tAo VV M*5(A S- w oOV r t*o-r fV. 3p 9m 4*X9 W *10- Ctt c.H*- * X % S51 o s. U?P> C4M- XI T) *" *P c <3 VO <0 c fc 2 nr- nC Co Cb H *4 HN a OCl Vu >p OO.P X ! *> rl at 4* ,.a ..S9,,.. OJ *H*(A* 11>to %bom v> e su 3 -sfesa *52 VI O Is-5: ,-S -->2 IJt <to0 g PJL 4tHC4 ** a Si" S VI POl to* *-4 -to* >Wp- ecHtoc* O'ii at *)* sr&fia 5g_5 au 4su*> > IcoL too> 3t_o r/i>. s&g u _8 > 3 fit? V CT O 2"Z `5 safe# A1A3* 8 8 SIX v < to* C -- p - g * -o p-r- a e VEM*ttoAo***> to<A* >P O 40 P P Z VI to CO s" t.. 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SAs *o* * 'S A uo ^& * *sti s* . *- AHV3d>*-*40M0) yLt S 0d b-.c0p V* G :>.i..p-- U*" B O 0 r- 0 X I*SX,U 3 a 2*C -*> 43 V> OC 5iz5h3a8v)S*>* 1-4 caen 1<4* 4>0 *- e eB24FXa*- Www2 SS'g >|*K ill Ul 0 r5 w S% us ea b- uir 11 #^<o ^*A gg TJ S G U TABLE 12B-Z. (continued) i Ot- 0 0u fi ss.- 1M ,x 9&At30S3O fCO*M* sit AA a8 M3 <A e> 2> I s ifc >% 5e 5 2 Cl . & S. <0 U S3 2s S7A3 a<0A 40- A0 is sa g fl *gov sl 128-4 TEH 0531195 DUP050032108 e o5 S Si *5 -- o s> sc 9 2C' uo I itj10 2 2 u'S e9 ..* Sr-b (0 9 cr<-- is 4J C *r c W1I CO v <o J fl ss st; O_) V +C* eo 3 a. *s u '.c u> c c " -C 3 i. Ov 4 CL vi . I ftv 2 5333>02.?SSu g'o *5 <3 ,oo V* 9 A0v--> ^C s3 * i- -s >% e voi Oo>W** Oo4rMu8 aO 3 q )-9*i tu --3 a .3 WCO v> So<- J!v *" > C O.l-- >? V AC 3 C 3 5 t- 10 V) 9 4c* VOo 4v U. > I- > J r- X vi ** : <s| piw-C* O g>^ $ s lA Ov* .c0) cfl) c*- 4 t O 4* 9 9 e g c o a< Xj? U0 (*0!O f IfV) 0)4* e *. 3 A- 1 dia sW<-f 4* *-- c - e O' r* 0*>3- e 251 E 0 4- 9 t.2-5 trow ~ <or 5 oV*N- Q U. WO. g O9 6VvOi 4^* LKW2 V <9 rt PRELIMINARY DRAFT XUI4J 3 ^1 i; 3 2* v * 4- 2 2 s* <0 4* fc *g| *" o * V B V) o 5 tUJO o 4* <u0 2 O a o,,3. 3 S3 lor S2& 9 s o rAsOt U X 93 i? e CT <;>0 .M 4r* 5 S' 8 b ! 4) sasS!-=Ssf X a t *9 9 -(uO0 5 3 3 u Vor!t 52 12B-5 *- 4o 55 ii 9 A0 *- p V) O* *U) o tts III u t oo 3 VI .4* ** 0.3 4* O.T3 u -o >wV '<i0-9r 9 9 (0 *53 a9. vU> 49> (si co c 8 9 vi <CO --4* 9V) 9h 3oj" I* .:s m2 03 d A *4 a a, 99 >1 & Ol 19 3i*vp* g5^ TEH 053119$ DUP050032109 PRELIMINARY ORAFT A>t lA P- R) * C ** -O 555 s 'S'* t. 2*8* +j eo oe 4?J 14-)4o? +1J.19H9-3 SM e - lisx w <9 3*8 3 .**5 i:g&8. V "-- !lg(j D6 S ft I 5 ;* o w o *5 vJ s:f * si* . 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No V>(/'o| c 3X5 2 S <o22 I*. 9U<P AO o s PI 03 S> f P pH p 3* I3 * f ** 33 *3 5g 5t ifeM C Sv po-- V* O U 91 43 9 v*- a2. 0to +* e 0) <b >a> C3 Ib>A1 2 W to v! f 2 3> 0> t b >i ?* I 41 I S9 ?8 b LO TEH 0531197 DUP050032110 A 0> *! i y<-STuJ M*0 ..e$*..5*i*'tO iP5sCl *9D*t S, L 22 33 & 3+2AJft 23 WO 22 WO 2 !&* (0 : Easily Long ti aafces tool f 4.w I <9 5 * 5*1 IS 12 a0* *?tr4*>*49t-*iO*--M,,c<&Od j*-wea 3 35 35 M >13 IA % 5SO ves t3t.<sw > <9 iin a o o + _o 0 22|5*> 5* Hios oIk kovg 9 2o of. _-sw "ef* 4* If >.2 if Umi <o3 W --9>4+0*> *499 * - r- (A 2*. st S* S3g S'" 35 2 c9qe5 O9gen9Sv*0t&C~Qt>B4VJ"(9A-I ^k2 PRELIMINARY DRAFT 1 t * rH I 1*. -T PH*tKO!K2bU5C 8 I 4 3 3 L l >s> 53 I N oet 9g sis 21* 3# 5 a= 9 Q> 12B-7 534! M8V&*5c w o cS3rL SS * 5v S32o e 3 si *roJ>rj>2u 1 a2>> ac< TEH 0531198 DUP050032111