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The effect of ascorbic acid on the embryotoxic and teratogenic effects of lead acetate in the chick embryo Berg, Janice Marie 1984

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c. -THE EFFECT OF ASCORBIC ACID ON THE EMBRYOTOXIC AND TERATOGENIC EFFECTS OF LEAD ACETATE IN THE CHICK EMBRYO by JANICE MARIE BERG B.A. University of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Division of Human Nutritio n School of Family and N u t r i t i o n a l Sciences We accept t h i s thesis as conforming to the required stan<da)rd_ THE UNIVERSITY OF BRITISH COLUMBIA June 1984 © J a n i c e Marie Berg, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of FAMILY AND NUTRITTONAT, SCTW^.q The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date JUNE 15, 1984 i i ABSTRACT King and Lui (1975) reported that ascorbic acid (1 mg) was ef f e c t i v e in protecting against embryotoxic and teratogenic effects of lead acetate (75 ug/egg) administered to chick embryos on the fourth day of incubation. The purpose of the present study was to confirm the work of King and Lui (1975) and to elucidate the mechanism, by which ascorbic acid may afford such protection. It was hypothesized that i f ascorbic acid afforded protection by meeting an increased requirement for i t , d-isoascorbic acid with only five percent of the antiscorbutigenic a c t i v i t y of ascorbic acid would be less e f f e c t i v e in affording protection than ascorbic acid. Secondly, i t was hypothesized that i f ascorbic acid was acting as a chelator, that other agents known to chelate lead would afford protection against the same effects as ascorbic acid. Since no s t a b i l i t y constants had been published for the lead ascorbate complex, ascorbic acid was compared to ethylenediaminetetraacetate, which has a high s t a b i l i t y constant for lead and i s not metabolized in vivo and c i t r i c acid, which has a low s t a b i l i t y constant for lead and l i k e ascorbic acid is metabolized in vivo. The purpose of these comparisons was to determine i f ascorbic acid does indeed act as a chelator i f i t is a weak chelator such as c i t r i c acid with very l i t t l e p otential for prophylactic and therapeutic use or a more stable chelator l i k e ethylenediaminetetraacetate with good potential for such applications. Administration of lead acetate (75 ug/egg) to chick embryos at 96 hours of incubation resulted in 50 % mortality, 74 % hydrocephalocele and s i g n i f i c a n t decrements in body weight, crown rump length and wet and dry brain weights in comparison to controls on the 19th day of incubation. No r e l a t i o n s h i p was observed between the presence of hydrocephalocele and decrements in body weight and crown rump length in lead acetate treated chick embryos. However, wet and dry brain weights were s i g n i f i c a n t l y less for lead acetate treated embryos exhibiting hydrocephalocele than those not exhibiting t h i s l e s i o n . The administration of lead acetate (75 ug/egg) simultaneously with ascorbic acid (15 to 80 times the molar concentration of lead acetate) or isoascorbic acid (15 to 60 times the molar concentration of lead acetate) resulted in a s i g n i f i c a n t decrease in mortality, hydrocephalocele, growth retardation and decrements in brain weight induced by lead acetate. Ethylenediaminetetraacetate (15 times the molar concentration of lead acetate) administered simultaneously with lead acetate (75 ug/egg) provided a similar degree of protection to that of ascorbic acid and isoascorbic acid (60 times the molar concentration of lead acetate) with respect to lead induced mortality, hydrocephalocele, and decrements in brain weight. C i t r i c acid (15 to 60 times the molar concentration of lead acetate) administered simultaneously with lead acetate (75 ug/egg) provided a similar degree of protection against lead acetate induced mortality and decrements in crown rump length, but was only half as e f f e c t i v e as ascorbic acid (15 to 40 times i v the molar c o n c e n t r a t i o n of l e a d a c e t a t e ) in p r e v e n t i n g hydrocephalocele. These f i n d i n g s suggest that a s c o r b i c a c i d prevents the embryotoxic and t e r a t o g e n i c e f f e c t s of l e a d a c e t a t e in the c h i c k embryo by a c t i n g as a c h e l a t o r , r a t h e r than by meeting an i n c r e a s e d requirement f o r the v i t a m i n . As to c h e l a t o r e f f e c t i v e n e s s the r e s u l t s suggest that a s c o r b i c a c i d i s more e f f e c t i v e than c i t r i c a c i d and l e s s e f f e c t i v e than e t h y l e n e d i a m i n e t e t r a a c e t a t e i n p r e v e n t i n g the embryotoxic^and t e r a t o g e n i c e f f e c t s of l e a d a c e t a t e on c h i c k embryos. V TABLE OF CONTENTS Abstract i i Table of Contents v L i s t of Tables v i i i L i s t of Figures x CHAPTER I 1 INTRODUCTION 1 CHAPTER II 15 REVIEW OF THE LITERATURE 15 Basic Chemical Properties of Lead 15 Lead Metabolism 16 Toxic E f f e c t s of Lead 20 Embryotoxicity, Fetotoxicity and Teratogenicity 25 Embryotoxic, Fetotoxic and Teratogenic E f f e c t s of Lead in Man 25 Embryotoxic, Fetotoxic and Teratogenic E f f e c t s of Lead in the Golden Hamster 28 Embryotoxic, Fetotoxic and Teratogenic Ef f e c t s of Lead in the Mouse 30 Embryotoxic, Fetotoxic and Teratogenic E f f e c t s of Lead in the Rat 32 Hypotheses Regarding Growth Retardation and Skeletal Malformations in the Golden Hamster, Mouse and Rat 34 Embryotoxic and Teratogenic Ef f e c t s of Lead in the Chick Embryo 35 Acute Lead Encephalopathy 46 Chelation 53 Ethylenediaminetetraacetate 59 C i t r i c Acid 67 Ascorbic Acid 71 v i Ascorbic Acid's Mechanism of Action 79 D-Isoascorbic Acid 85 CHAPTER III 90 MATERIALS AND GENERAL METHODOLOGY 90 Materials 90 Method of Lead Injection 90 Experiment 1 (Selection of lead acetate l e v e l for subsequent experiments) ..92 Experiment 2 (Comparison of the effectiveness of increasing levels of ascorbic acid) 92 Experiment 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA) 93 Experiments 4 (Comparison of lower levels of ascorbic acid with isoascorbic acid) and 5 (Comparison of lower levels of ascorbic acid with c i t r i c acid) 94 S t a t i s t i c a l Analyses 95 CHAPTER IV . . 98 RESULTS 98 Mortality 98 Hydrocephalocele 101 External Abnormalities 110 Body Weights 110 Crown Rump Lengths 112 Wet Brain Weights 116 Dry Brain Weights 119 The Relationship of Hydrocephalocele to Growth Retardation and Brain Weight 123 CHAPTER V 125 DISCUSSION 125 The Embryotoxic and Teratogenic E f f e c t s of Lead Acetate in the Chick Embryo 125 v i i The Protective E f f e c t s of Ascorbic Acid in the Lead Treated Chick Embryo 1 3 3 Mechanism of Ascorbic Acid's Protective Effects in the Lead Treated Chick Embryo 138 Bibliography 1 4 3 v i i i L i s t of Tables Table I. Summary of studies of the embryotoxic and teratogenic e f f e c t s of lead on the chick embryo 36 Table I I . The effect on chick embryo mortality of lead acetate administered alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid, or EDTA 99 The ef f e c t on mortality, hydrocephalocele, body weight and crown rump length of administering 75 ug of lead acetate to chick embryos on the fourth day of incubation 102 Table IV. Mortality as determined from experiments 2 to 5, in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or EDTA 103 Table I I I . Table V. The e f f e c t on chick embryo hydrocephalocele of lead acetate administered alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid and EDTA 1 06 Table VI. The frequency of hydrocephalocele as determined from experiments 2 to 5, in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid, or EDTA 109 Table VII. The ef f e c t on chick embryo body weight of lead acetate administered alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or EDTA , 1 1 1 Table VIII Mean body determined weight and standard from experiments 2 to error as in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or EDTA, 1 13 Table IX. The ef f e c t on chick embryo crown rump length of lead acetate administered alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or. EDTA 114 Table X. Mean crown rump length and standard error as determined in experiments 2 to 5, in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid, ix Table XI. Table XII. Table XIII isoascorbic acid, c i t r i c acid or EDTA 115 The effect on chick embryo wet brain weight of lead acetate administered alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or EDTA 118 The effect on chick embryo dry brain weight of lead acetate administered alone or simultaneously with ascorbic acid, isoacorbic acid, c i t r i c acid or EDTA 120 The relationship between hydrocephalocele and chick embryo body weight, crown rump length and wet and dry brain weight 124 X Table of Figures Figure 1. A 5-member ring chelate 54 Figure 2. A multidentate and a bidentate chelate 56 Figure 3. Ethylenediaminetetraacetate 60 Figure 4. Lewin's (1974) proposed structure for ascorbic acid metal chelates 83 Figure 5. L-ascorbic acid and i t s isomer d-isoascorbic acid. .86 Figure 6. External abnormalities observed in chick embryos treated with lead acetate alone or in combination with ascorbic acid or c i t r i c acid 105 Figure 7. The s i g n i f i c a n t positive linear r e l a t i o n s h i p observed between chick embryo crown rump length and increasing levels of ascorbic acid 117 Figure 8. The s i g n i f i c a n t positive linear r elationship observed between chick embryo wet brain weight and increasing le v e l s of ascorbic acid 121 Figure 9. The s i g n i f i c a n t positive linear r elationship observed between chick embryo dry brain weight and increasing le v e l s of ascorbic acid 122 1 CHAPTER I  INTRODUCTION Lead p o l l u t i o n of the biosphere by man began some 5,000 years ago, when lead was f i r s t mined, not for i t s own i n t r i n s i c value, but for the s i l v e r that was a component of i t s ore. The e a r l i e s t reported estimate of annual worldwide production of lead was 160 tons, 4,000 years ago (Settle and Patterson, .1980). 1,300 years l a t e r , the annual worldwide production of lead was estimated to be 10,000 tons (Settle and Patterson, 1980), r e f l e c t i n g man's development of a number of uses for i t including the making of ornaments, cosmetics, jewelry, fi s h i n g net sinkers and the construction of bridges, boats, and such mega projects of the ancient world as the Hanging Gardens of Babylon (Waldron and Stofen, 1974). During the Roman Empire, annual worldwide production of lead increased to 80,000 tons (Settle and Patterson, 1980). Lead was used by the Romans in the construction of their famous aquaducts, as a coating for bronze and copper cooking kettles to improve the taste of cooked foods and as a sweetening agent in the preparation of wine and grape syrup (Waldron, 1973). This intimate association of lead with the food and water supply of the Romans resulted in widespread lead intoxication, which according to G i l f i l l a n (1965) led to the F a l l of the Roman Empire. Worldwide lead production in the Middle Ages decreased 2 considerably from the le v e l of production during the Roman Empire (Settle and Patterson, 1980), due to the lack of slave labour to smelt the ore (Patterson, 1980). However, lead poisoning was s t i l l a f a i r l y common occurrence as attested to by the numerous descriptions of i t in the writings from that time (Grandjeans, 1975). Unfortunately, these writers attributed the cause of the condition to alcoholic beverages (Patterson, 1980), rather than to lead based pottery glazes, pewterware (Grandjean, 1975) and a type of lead sweetened wine (Patterson, 1980) in use at that time. The onset of the i n d u s t r i a l revolution resulted in a further increase in worldwide lead production up to 100,000 tons per year (Settle and Patterson, 1980). During t h i s period, lead was used in medicines, glassware, ceramics, cosmetics, water pipes, paints, bonbons, sugar, paprika, and tobacco (Grandjean, 1975); res u l t i n g in widespread exposure to i t . This widespread exposure was ref l e c t e d in the medical l i t e r a t u r e of the time, by an abundance of reports describing outbreaks of lead, c o l i c (Grandjean, 1975). From the onset of the i n d u s t r i a l revolution u n t i l 50 years ago, the worldwide production of lead continued to r i s e u n t i l i t reached one m i l l i o n tons per year (Settle and Patterson, 1980). This increase in lead production reflected the development of new uses for lead, such that a wide array of lead based products appeared on the market; batteries, paint, c r y s t a l glass, lead shot, water pipes, building cables, pottery glazes, i n s e c t i c i d e s , hair dyes, commercial pigments, soldering agents 3 and gasoline (NRC, 1973; Grandjean, 1978). As these uses for lead expanded and the demand for lead based products increased, lead production underwent a further dramatic increase, such that in the span of a mere 50 years, i t t r i p l e d to today's output, which i s an astounding three m i l l i o n tons per year (Settle and Patterson, 1980). In order to determine i f the dramatic increase in lead production, has had a s i g n i f i c a n t effect on the lead exposure of the current American population, Settle and Patterson (1980) have compared lead in food, water, a i r and the skele t a l burden for Americans l i v i n g today with that for prehi s t o r i c man, whose only environmental source of lead was geologic in o r i g i n . They found that the average dietary intake of lead in Americans today is 200 ng/g of food compared to an estimated prehistoric dietary intake of two ng per g of food. Although 50 ng of lead per g of drinking water i s currently considered acceptable in America, the p r e h i s t o r i c l e v e l of lead in drinking water was estimated to be 0.02 ng/g. The lead content of a i r breathed in urban areas of America was found to be 5 x 10 2 to 1 x 10" ng/m3 in comparison to a pre h i s t o r i c estimate of 0.04 ng/m3. A comparison of the skelet a l lead burden of Peruvians l i v i n g 1,800 years ago, in an environment unpolluted by man-made sources of lead, with the skele t a l lead burden at present, indicated a 100 to 500 fold increase in skel e t a l lead content since that time. These figures suggest that levels of lead exposure for present day Americans are from one to three orders of magnitude greater than natural levels (NRC, 1980). 4 These comparisons in conjunction with the observation that the margin between t y p i c a l body burdens of lead in Americans and those associated with toxic e f f e c t s i s extremely narrow (NRC, 1980), has led to two opposing schools of thought as to whether or not the current l e v e l of lead exposure in the general population i s harmful. One school of thought, headed by Settle and Patterson (1980), contends that exposure to lead is excessive in the American population, resulting in widespread deleterious effects on biochemical processes at the c e l l u l a r l e v e l , which impair general health and i n t e l l e c t u a l functioning (Patterson, 1980). The opposing school of thought, to which the majority subscribes (NRC, 1980), contends that current exposure levels pose no threat for the vast majority of the population, even i f s l i g h t biochemical and physiological changes are occurring, as these changes are readily compensated for by homeostatic mechanisms in the body. At present, both hypotheses are fundamentally unprovable (NRC, 1980). Even the most conservative school of thought, however recognizes that there are certain subgroups within the general population, which may be experiencing s i g n i f i c a n t biochemical and physiological changes, such that their physical and mental well being may be impaired (NRC, 1980). These vulnerable subgroups include fetuses, children, workers in lead related industries and the e l d e r l y (Repko et a l . , 1978; Charney et a l . , 1980; NRC, 1980; Needleman, 1980). The concern regarding fetuses i s directed toward the p o s s i b i l i t y that lead i s an e t i o l o g i c a l factor in current cases 5 of hydrocephalocele (Hirano and Kochen, 1977), miscarriage, s t i l l b i r t h and postpartum behavioral and learning def i c i e n c i e s (NRC, 1980). Lead has not been demonstrated to be an e t i o l o g i c a l factor in current cases of hydrocephalocele. However, reports at the turn of the century (Bell and Thomas, 1980) have c l e a r l y linked heavy exposure to lead with t h i s malformation in humans. Considerable evidence has accumulated associating upper levels of the general population's exposure to lead with increased hazards of miscarrage and s t i l l b i r t h s (Bryce-Smith, 1977; Wibberley et a l . , 1977; Jaworski, 1979; Khera et a l . (1980). As yet, s ensitive designs, free from the e f f e c t s of confounding variables, have not been developed to provide convincing evidence for learning and behavioral d e f i c i t s in children who were exposed in utero to general population l e v e l s of lead (NRC, 1980). However, animal.studies (Bull et a l . , 1979; NRC, 1980) have provided convincing evidence for delays in brain development, and d e f i c i t s in learning and behavior at maternal blood lead l e v e l s (30 ug/100 ml) that have been found to occur in 3.5% of the adult American population (NRC, 1980). The Commmittee on Lead in the Human Environment (NRC, 1980), despite the lack of clear cut evidence, considers fetuses to be p a r t i c u l a r l y vulnerable to the central nervous system effects of lead, due to the finding that a larger percentage of the t o t a l body burden of lead i s concentrated in the central nervous system of fetuses than in children or adults. This has led them to propose further research to assess the interaction between n u t r i t i o n a l factors and lead accumulation in fetuses 6 during pregnancy. Children are considered to be a vulnerable subgroup of the population for detrimental health e f f e c t s from current population exposure lev e l s of lead for three reasons. (1) Their environments frequently contain concentrated sources of lead such as lead based paints; d i r t , dust and a i r contaminated by gasoline emissions or proximally located lead related industries; and weathered lead containing items such as discarded lead storage batteries (Smith, 1974). (2) The common childhood habits of pica and r e p e t i t i v e hand to mouth a c t i v i t i e s increases the l i k e l i h o o d of lead ingestion (Charney et a l . , 1980). (3) Physiological stresses associated with rapid growth and the v u l n e r a b i l i t y of the developing central nervous system to toxic agents during c r i t i c a l periods of i n t e l l e c t u a l , psychological and behavioral development, leaves children p a r t i c u l a r l y vulnerable to detrimental b i o l o g i c a l effects from lead (NRC, 1980). Deaths in children from lead t o x i c i t y are r e l a t i v e l y rare in Canada (Chant et a l . , 1974) and the U.S.A. (Clark et al.,1974). The number of cases of lead encephalopathy have been steadily declining due to hygiene measures that have been i n s t i t u t e d (NRC, 1980). However, increased body lead burdens in children are not so rare. Screening programs car r i e d out on 2,580 children in 31 communities in the U.S.A. (Clark et a l . , 1974), indicated that six percent of children less than three years of age and 4.8% of children over three years of age had blood lead l e v e l s greater than 40 ug/100 ml. In Ontario, a 7 s i m i l i a r study (Chant et a l . , 1974) found 3.6 to 30% of children examined, depending on the proximity of their residences to lead smelters, to have blood l e v e l s above 40 ug/100 ml. The Committee on Lead in the Human Environment (NRC, 1980), associates blood lead le v e l s of this magnitude with uncompensatable changes in the haematopoetic system and s i g n i f i c a n t changes in psychological, sensory and behavioral parameters. The c r i t i c a l concern with regard to children i s that even lower blood lead levels (25 ug/100 ml) (David et a l . , 1976), which would encompass a far greater percentage of the childhood population may have s i g n i f i c a n t e f f e c t s on i n t e l l e c t u a l performance and adaptive behaviors. At present, there i s a growing body of epidemiological l i t e r a t u r e (David et a l . , 1976; Jaworski, 1979; Needleman, 1980) to support this contention. However, such research i s not conclusive, due to the i n a b i l i t y of current researchers to exclude the influence of confounding var iables. In response to the above mentioned body of l i t e r a t u r e , the Committee on Lead in the Human Environment (NRC, 1980), proposes that e f f o r t s be made to est a b l i s h dose-effect relationships for central nervous system changes due to lead. They also recommend that studies be undertaken to determine the potential interactions between lead and toxic substances, essential nutrients, genetic differences and other stresses that affect urban children. There are 110 occupations in the U.S.A. that expose workers to lead (Schoenbord, 1980). Those that are most l i k e l y to result 8 in high lead exposure and lead poisonings are ones in which fine lead p a r t i c l e s are created (Grandjean, 1978). Examples of such occupations are lead smelting, car radiator repair, iron structure demolition, metal foundry and polyvinyl chloride p l a s t i c manufacturing. Although individuals in these industries are at higher risk than the general population for the detrimental health effects from lead," overt lead poisoning as a disease of these occupations has become rather uncommon in recent years, due to a number of hygiene measures i n s t i t u t e d to decrease lead exposure in the workplace and stringent medical monitoring of individuals employed in these industries (Chant et a l . , 1974). There are however, some recent reports in the l i t e r a t u r e of lead poisonings and unacceptably high levels of lead absorption in s i g n i f i c a n t numbers of employees in s p e c i f i c work environments (Fischbein et a l . , 1978; Crawford et a l . , 1980). Of p a r t i c u l a r interest i s the report by Crawford et a l . (1980) of three cases of lead poisoning and 20 cases of excessive lead absorption in an Australian factory producing inorganic lead compounds. The unique aspect of the report was that lead poisoning and excessive lead absorption occurred in 23 out of 35 employees, in spite of the sincere e f f o r t s of management and government regulatory agencies to reduce lead exposure. Of greater concern than lead poisoning in occupationally exposed workers i s the host of subjective and psychological complaints of such workers. L i l i s et a l . (1977) found that 55% of a group of workers whose mean blood lead levels were 60 9 ug/100 ml reported the following subjective and psychological complaints; fatigue, insomnia, headache, loss of appetite, i r r i t a b i l i t y , anxiety, depression, h o s t i l i t y and moodiness. Repko et a l . (1978), in a study on the behavioral effects of lead in occupationally exposed workers reported s i m i l i a r personality changes as well as hearing loss and decreased eye hand coordination at blood lead levels between 40 and 80 ug/100 ml. Although, these studies suggest a possible lead etiology for psychological and subjective complaints of occupationally exposed workers, the non-specific nature of such complaints makes i t d i f f i c u l t to rule out other p o s s i b i l i t e s such as generalized stress syndrome (NRC, 1980). Due to a possible lead etiology for behavioral, sensory and psychological d i f f i c u l t i e s in lead exposed workers, e f f o r t s have been made in the l a s t decade (Chant et a l . , 1974; Schoenbord, 1980) to reduce standards for occupationally safe l e v e l s of lead exposure to levels closer to those recommended for the general population. Considering that some fa c t o r i e s , even though sincere in their e f f o r t s , can not meet current standards (Crawford et a l . , 1980) and other work establishments do not have the economic or technological c a p a b i l t i e s to further reduce lead exposure (Schoenbord, 1980), i t seems necessary that other measures of a medical and n u t r i t i o n a l nature be investigated, in order to determine i f they may be useful adjuncts to hygiene measures in reducing the body burden of lead in occupationally exposed workers. The current medical approach to reducing body burdens of 10 lead in i n d u s t r i a l l y exposed workers is chelation therapy. Unfortunately, the currently used chelating agents produce toxic side e f f e c t s (Bridbord et a l . , 1977), which make them unsuitable for prophylactic use and prevent the administration, in serious cases of lead intoxication, of the most favorable ratio of chelator to body lead (Chisolm, 1968). This suggests that i t may be necessary to investigate the use of nutrients and food factors that have chelating p o t e n t i a l , yet are non-toxic, to determine their s u i t a b i l i t y for prophylaxsis and treatment of lead t o x i c i t y , either alone or in combination with chelating agents already in use (Goyer and Cherian, 1979). There has been l i t t l e work done on the detrimental health e f f e c t s of lead in the e l d e r l y . However, concern has been r i s i n g that they as a group may be, p o t e n t i a l l y , at as great a risk as young children for s u b c l i n i c a l effects from lead (Needleman, 1980). Research (Needleman, 1980) done in this area suggests that s i g n i f i c a n t exposure to lead in the past may result in premature deaths in older individuals from nephropathy, hypertension and vascular disease or be an e t i o l o g i c a l factor in such degenerative diseases in the elderly as amylotrophic l a t e r a l s c l e r o s i s and Alzheimer disease. Needleman (1980). has put forth the interesting hypothesis, that cognitive changes present in the elderly population are the result of mobilization of lead from the bone as part of the aging process in combination with poor dietary intakes of various nutrients that may protect biochemical processes within the c e l l from the adverse e f f e c t s of lead. If t h i s hypothesis has any substance, 11 innovative n u t r i t i o n a l approaches with respect to prevention and treatment of lead induced health related d i f f i c u l t i e s in the elderly w i l l be required. Since hypotheses, such as Needleman's (1980) indicate populations where a narrow margin between t y p i c a l body burdens of lead and those associated with toxic e f f e c t s exists (NRC, 1980), i t was decided in the present study to examine the interaction of 1-ascorbic acid and lead. Ascorbic acid was chosen for investigation over other nutrients that may interact with lead for two reasons. Since the major concern regarding each of the vulnerable population subgroups was central nervous system e f f e c t s from lead, i t seemed important to investigate a n u t r i t i o n a l agent with the potential for preventing or ameliorating such e f f e c t s . Reports in the l i t e r a t u r e (Holmes et a l . , 1939; Pillemer et a l . , 1940; Marchmont-Robinson, 1941; King and Lui, 1975; Goyer and Cherian, 1979) were suggestive of ascorbic acid being such an agent. Secondly, the l i t e r a t u r e (Chisolm, 1968; Bridbord et a l . , 1977) indicated the need to investigate non-toxic chelating agents for use in the prevention and treatment of excessive body burdens of lead. The recent work of McNiff et a l . (1978) and Goyer and Cherian (1979) in rats, suggesting that ascorbic acid may be e f f e c t i v e in reducing body lead burdens by acting as a chelator, made ascorbic acid a l o g i c a l choice for further investigation over other nutrients with chelating potentials, which have not even been tentatively investigated to date. The chick embryo was chosen as the vehicle for 1 2 investigating the potential interaction between ascorbic acid and lead for a number of reasons. Researchers who had worked extensively with chick embryos (McLaughlin et a l . , 1963; Romanoff and Romanoff, 1972) had found them to be economical, with a minimum of space and maintenance requirements. This made them very suitable experimental material for t h i s study, considering the space and economic constraints of the laboratory where the work was done. Secondly, since one of the major purposes of the study was to investigate ascorbic acid's a b i l i t y to protect against central nervous system e f f e c t s of lead, a species was required that consistently exhibited such damage in a readily i d e n t i f i a b l e manner. The chick embryo was the only species to meet these c r i t e r i a . Thirdly, since fetuses had been i d e n t i f i e d as the most vulnerable group for detrimental effects from lead (NRC, 1980), i t seemed desirable to select embryos for study rather than mature animals. Chick embryos were selected over embryos of other species, due to the fact that the hydrocephalocelic malformation they exhibit in response to lead has been observed in human fetuses exposed to excessive amount of lead during gestation (Bell and Thomas, 1980). Fourthly chick embryos were chosen as an e a r l i e r study indicated that ascorbic acid was e f f e c t i v e in protecting them against the teratogenic and embryotoxic e f f e c t s of lead (King and L u i , 1975). This made i t possible to use the present study to confirm the e a r l i e r study and to expand upon i t , by 1 3 investigating two of the proposed mechanisms, by which ascorbic acid may afford such protection. F i f t h l y , the chick embryo was chosen as the model permits controlled doses of ascorbic acid and lead to be in contact with the embryo throughout the period of development, without the agents being subjected to maternal influences. This i s p a r t i c u l a r l y useful in preliminary investigations, such as the present study, as i t s i m p l i f i e s the number of possible results and their i nterpretation. The f i r s t part of the present study was devoted to determining whether or not, ascorbic acid could afford protection against the embryotoxic and teratogenic e f f e c t s of lead in the chick embryo. The second part of the study was d'evoted to an attempt to elucidate the mechanism, by which ascorbic acid afforded such protection. It was hypothesized that i f ascorbic acid afforded protection by meeting an increased requirement for i t , d-isoascorbic with only fiv e percent the antiscorbutigenic a c t i v i t y of ascorbic acid would be less e f f e c t i v e in affording protection than ascorbic acid. Secondly, i t was hypothesized that i f ascorbic acid was acting as a chelator, then other agents known to chelate lead should afford protection against the same parameters as ascorbic acid. Since, no s t a b i l i t y constant has been published for the lead ascorbate complex, ascorbic acid was compared with ethylenediaminetetraacetate, which has a high s t a b i l i t y constant for lead (Reynolds, 1963) and i s not metabolized in vivo, and c i t r i c acid, which has a low s t a b i l i t y constant for lead and 14 l i k e ascorbic acid i s metabolized in vivo. The purpose of these comparisons was to determine i f ascorbic acid does indeed act as a chelator, whether i t i s a weak chelator, such as c i t r i c acid with very l i t t l e potential for prophylactic and therapeutic use or a more stable chelator l i k e ethylenediaminetetraacetate, with good potential for such applications. 1 5 CHAPTER II  REVIEW OF THE LITERATURE Basic Chemical Properties of Lead Lead i s a soft grey d u l l metal belonging to group IVA of the periodic table. It resembles group IVA elements, in that i t forms insoluble halides, hydroxides and phosphates. However, in many other properties i t resembles the a l k a l i n e earth metals, p a r t i c u l a r l y calcium, thereby, explaining why lead metabolism so often p a r a l l e l s calcium metabolism in the body (Gerber et a l . , 1980) . Lead can exist in the form of inorganic s a l t s with varying s t a b i l i t y constants or as synthetic organic compounds such as tetraethyllead or tetramethyllead, which are used as anti-knock additives in f u e l . The more soluble in body f l u i d the lead compound i s , the greater i t s t o x i c i t y , because high s o l u b i l i t y enhances passage of . the compound into the blood (Waldron and Stofen, 1974). It appears from LD50 studies in rats (Fried et a l . , 1956), that organic lead compounds are more toxic and produce somewhat d i f f e r e n t b i o l o g i c a l e f f e c t s than inorganic lead compounds. The difference in t o x i c i t y between organic and inorganic lead compounds may be due to a greater a f f i n i t y of organic compounds for soft tissue, l i p i d . r i c h organs such as brain and l i v e r (Gerber et a l . , 1980), which are more susceptible to the toxic e f f e c t s of lead than bone tissue, which has the greatest a f f i n i t y for inorganic lead (Waldron and 1 6 Stofen, 1974). Additionally, i t may be due to longer lead exposure times in soft tissue after absorption, as a result of slower breakdown of these compounds to inorganic lead, which i s readily excreted or redistributed from soft tissue to bone (Waldron and Stofen, 1974). Lead has not been shown to be essential for any b i o l o g i c a l function (Gerber et a l . , 1980), although, i t has properties that can produce considerable havoc in b i o l o g i c a l systems. Lead has been shown to act as an antagonist to calcium, iron, zinc and cadmium (Chisolm, 1980). It has also been shown to readily bind to s u l f h y d r y l , amino, imidazole, carboxyl, phosphate and hydroxyl groups, with greater a f f i n i t y for groups in which sulphur or nitrogen, rather than oxygen serves as the electron donor (Waldron and Stofen, 1974). These properties in b i o l o g i c a l systems may lead to competition between lead and various metals for absorptive and enzymatic s i t e s (Chisolm, 1980). Such competition may disrupt normal metabolic processes by causing changes in enzymatic a c t i v i t y (DeBruin, 1971), c e l l and organelle membrane structure (Goyer and Rhyne, 1973) and metal d i s t r i b u t i o n within body compartments (Chisolm, 1968). Lead Metabolism Lead can enter the body by way of the ga s t r o i n t e s t i n a l t r a c t , the lungs or the skin. Dermal absorption of lead i s i n s i g n i f i c a n t , except under unusual circumstances, such as when tetraethyllead i s rubbed on the skin (Waldron and Stofen, 1974). Absorption of lead via the lungs is more rapid and complete than 1 7 via the gut (Hammond, 1980). Thus, in some circumstances intake via the lungs may be a more important contributor to body lead burden than uptake via the gut. I n t e s t i n a l absorption of lead in man i s approximately ten percent of the amount ingested (Oehme, 1980). Lead absorption in man and the rat appears to occur mainly from the small intestine, to a lesser extent from the colon and not at a l l from the stomach (Hawkins, 1979; Oehme, 1980). Vitamin D enhances lead absorption, probably in the same manner i t enhances calcium absorption by stimulating synthesis of the acceptor protein (Waldron and Stofen, 1974). Ascorbic acid and other n u t r i t i o n a l factors appear to enhance lead absorption by increasing i t s s o l u b i l i t y , possibly v i a chelation (Conrad and Barton, 1978). Periods of rapid growth in young animals have been found to enhance lead absorption (Waldron and Stofen, 1974). Unfortunately, the mechanism of t h i s enhancement is unknown. Lead uptake via the lungs appears to depend on p a r t i c l e size as well as other factors (Waldron and Stofen, 1974). Of lead p a r t i c l e s inhaled into the lungs, 70% to 75% are discharged into expired a i r (Chisolm, 1971). Of the retained p a r t i c l e s , those 0.9 urn or greater in diameter are deposited in the airways, from which they are swallowed, to follow the same route as ingested lead (Green et a l . , 1980). The smaller p a r t i c l e s , 0.1 urn or less in diameter enter the a v e o l i , where they are cleared by macrophages, lymph drainage or d i r e c t absorption into the blood stream (Waldron and Stofen, 1974). Lead in the c i r c u l a t o r y system exists in dynamic 18 equilibrium bound to plasma microligands and red blood c e l l membranes. Over 90% of the blood lead is bound to the red blood c e l l membranes and i s non-diffusible across c e l l membranes (Waldron and Stofen, 1974). The remaining plasma lead bound to microligands i s d i f f u s i b l e , making th i s f r a c t i o n most important with respect to lead transport and tissue lead content (Goyer and Rhyne, 1973). Following absorption, lead i s i n i t i a l l y deposited in soft tissue, with the highest concentrations being found in the l i v e r and kidney, where i t is strongly bound to mitochondria (Waldron and Stofen, 1974). Over time, a r e d i s t r i b u t i o n occurs from soft tissue to bone, such that 90% or more of t o t a l body lead is found in the bone, l i v e r , aorta and kidney, with only trace amounts present in the brain and muscle (Goyer and Rhyne, 1973). Organic lead compounds have a somewhat di f f e r e n t d i s t r i b u t i o n in the body than inorganic lead compounds. Organic lead compounds, such as tetraethyllead are rapidly taken up by the brain, kidney, l i v e r and blood after absorption (Waldron and Stofen, 1974). They are then broken down in these organs to r e l a t i v e l y stable intermediate compounds such as t r i e t h y l l e a d (Hammond, 1980), which permits them to remain in the tissues for longer periods of time. However, they are eventually broken down into inorganic lead and either excreted or redistributed to bone. Lead present in the bone is r e l a t i v e l y non-toxic. However, lead present in soft tissue can be exceedingly toxic. The severity of the toxic e f f e c t s in soft tissue depends on the 19 concentration of lead, which i s related to the immediate exposure dose, the speed of absorption, and the rate of bone resorption (Green et a l . , 1980). Factors which tend to increase the rate of bone resorption are infe c t i o n , acidosis, fevers, fractures and excessive alcohol intake (Waldron and Stofen, 1974). In general, any factor that leads to the mobilization of calcium from the bone w i l l lead to the mobilization of lead. Concentrations of lead in soft tissue and bone tend to increase with age (Waldron and Stofen, 1974). However, bone concentrations of lead tend to f a l l in the seventh and eighth decades of l i f e , while tissue concentrations do not decrease (Waldron and Stofen, 1974). This is probably due to the loss of mineral content from the bone, which is common in the el d e r l y . This loss of lead from the bone may result in a toxic increase in soft tissue lead, p a r t i c u l a r l y i f large amounts of lead have been stored in the bone in the past during periods of high exposure (Needleman, 1980). Lead i s excreted from the body via the ga s t r o i n t e s t i n a l t r a c t , the kidneys, the skin, the hair and the mammary glands (Oehme, 1980). Of these f i v e modes of excretion, the gas t r o i n t e s t i n a l t r a c t and the kidneys are the most important. Excretion of lead via the ga s t r o i n t e s t i n a l tract i s due to secretion of lead- containing b i l e by the l i v e r and the sloughing off of i n t e s t i n a l c e l l s containing lead (Oheme, 1980). Excretion in the b i l e i s a major route of elimination in cases of excessive lead exposure, but not at normal l e v e l s of environmental exposure (Green et a l . , 1980). 20 Most of the lead absorbed into the body is eliminated by the kidneys. During periods of excessive exposure, lead concentrations in the urine are high. When exposure declines, a rapid decrease in urinary concentrations occur, followed by a more gradual decrease as tissue concentrations continue to diminish (Oehme, 1980). Since most of the body burden of lead i s stored in the bones, excretion generally takes twice as long as accumulation (Green et a l . , 1980). Lead excretion i s enhanced under conditions that favor lead mobilization from bone to soft tissue. Conditions that favor mobilization are dietary imbalances, parathyroid hormone stimulation, acidosis, iodine, bicarbonate and ethylenediaminetetraacetate administration (Oehme, 1980). Toxic E f f e c t s of Lead Lead exerts i t s toxic e f f e c t s on the haematopoietic, nervous, renal, g a s t r o i n t e s t i n a l , immune, reproductive and hormonal systems (Gerber et a l . , 1980). The e a r l i e s t c l i n i c a l manifestations of lead t o x i c i t y in the haematopoietic system are r e t i c u l o c y t o s i s and basophilic s t i p p l i n g (Goyer and Rhyne, 1973). The late c l i n i c a l manifestation of lead t o x i c i t y in the haematopoietic system i s hypochromic microcytic anemia. This anemia i s considered to be due to two defects; an impairment in heme synthesis and shortened erythrocyte lifespans (Goyer and Rhyne, 1973). The impairment in heme synthesis i s primarily the result of the severely i n h i b i t o r y effect of lead on two enzymes; delta-aminolevulinic acid dehydratase, which acts to combine two 21 molecules of delta-aminolevulinic acid into porphobilogen and ferrochetalase, which catalyzes the incorporation of iron into the protoporphyrin molecule to form heme (Moore et a l . , 1980). The decreased erythrocyte lifespan appears to be due to increased f r a g i l i t y of the red blood c e l l membrane, the causes of which may be; the binding of lead to phosphates of the membrane, the impairment by lead of red blood c e l l g l y c o l y t i c a c t i v i t y or excessive red blood c e l l leakage of potassium, due to i n h i b i t i o n by lead of the sodium and potassium dependent adenosine triphosphate (Goyer and Rhyne, 1973). Lead a f f e c t s both the central and peripheral nervous systems. Children exposed to toxic amounts of lead more frequently exhibit central nervous system e f f e c t s , while adults more frequently exhibit peripheral nervous system effects (Goyer and Rhyne, 1973). The c l i n i c a l manifestations of toxic amounts of lead in the central nervous system are drowsiness, ataxia, apathy, coma, convulsions and death (Goyer and Rhyne, 1973). The c l i n i c a l manifestations of toxic amounts of lead in the peripheral nervous system are muscle weakness, palsy, foot and wrist drop (Green et a l . , 1980). Lower motor neuron disease, which i s characterized by symmetrical muscle wasting and weakness may in some cases be a manifestation of the effect of lead on the peripheral nervous system (Campbell et a l . , 1970). The common lesions produced by toxic amounts of lead in the central nervous system are focal areas of necrosis, demylination and neuronal degeneration accompanied by edema, hemorrhage and p r o l i f e r a t i o n of endothelial and g l i a l c e l l s (Goyer and Rhyne, 22 1973). Functionally, changes in the blood brain barrier have been noted, while biochemically changes in calcium metabolism, neurotransmitters and biogenic amine concentrations have been detected (Gerber et a l . , 1980). The common lesions produced by toxic amounts of lead in the peripheral nervous system are segmental demyliniation and/or axonal degeneration depending on the species involved (Waldron and Stofen, 1974). Functionally, there i s decreased ganglionic transmissions accompanied biochemically by a decreased output of acetylcholine, which can be increased to normal by the addition of calcium (Goyer and Rhyne, 1973). This suggests that lead binds to motor end plates i n t e r f e r i n g with calcium release, which in turn interferes with the release of acetylcholine (Gerber et a l . , 1980). Toxic amounts of lead can affe c t the renal system by producing acute damage, which may or may not progress to chronic nephropathy, depending on the length of lead exposure (Waldron and Stofen, 1974). In lead induced acute renal damage, the c e l l s of the proximal tubules swell, mitochondria undergo degenerative changes, intranuclear inclusion bodies consisting of lead, iron, calcium, glycogen and a protein high in sulphydryl groups form and aminoaciduria developes (Goyer and Rhyne, 1973). These changes, none of which are necessarily permanent in nature, are seen in both animal and human studies of acute lead poisoning (Waldron and Stofen, 1974). In chronic renal damage induced by lead, i n t e r s t i t i a l f i b r o s i s occurs accompanied by atrophy, hypertension and 23 hyperuricemia (Goyer and Rhyne, 1973). The incidence of chronic lead nephropathy has declined to exceedingly low levels as a result of i n d u s t r i a l hygiene measures undertaken since the 1950's (Waldron and Stofen, 1974). The g a s t r o i n t e s t i n a l manifestations of lead t o x i c i t y in humans are c o l i c , constipation, and diarrhea (Goyer and Rhyne, 1973). The cause of these manifestations are unknown, but may be related to lead induced changes in neurotransmitters or in smooth muscle contraction (Gerber et a l . , 1980). Further studies in t h i s area are awaiting the development of a suitable animal model for lead induced enteropathy. Lead appears to substantially impair immunological responses in animals. Hemphill et a l . (1971) demonstrated that exposure to low level s of lead for 30 days resulted in greater s u s c e p t i b i l i t y to b a c t e r i a l challenge in mice. In a series of studies discussed by DeBruin (1971), lead treated animals which underwent procedures of active immunization produced smaller quantities of gamma globulins, complement, antibodies and ascorbic acid, when compared to controls. To what extent human immunological responses are impaired by lead i s a question that remains to be answered. Increased incidences of s t e r i l i t y , abortion, s t i l l b i r t h s and neonatal deaths have been reported in man and animals as reproductive responses to lead (Gerber et al.,1980). In animals, paternal ingestion of lead has been associated with reduced b i r t h weights in offspring and reduced survival rates prior to weaning (Goyer and Rhyne, 1973). These paternal effects of lead 24 may be the result of a lead induced defect in the spermatazoa or d i r e c t transfer of lead via the semen to the environment of the conceptus (Bell and Thomas, 1980). In animals, maternal ingestion of lead has been associated with reduced l i t t e r sizes, retarded f e t a l development, impaired postnatal survival and teratogenic effects (Bell and Thomas, 1980). Lead in the maternal reproductive system may exert i t s toxic e f f e c t s d i r e c t l y on the ovum, cross the placenta to exert intrauterine e f f e c t s or be secreted in the milk to produce extrauterine e f f e c t s (Goyer and Rhyne, 1973). When both parents are exposed to lead, the lead burden of each parent contributes i t s own unique, as well as additive e f f e c t s to the offspring (Bell and Thomas, 1980). Lead appears to af f e c t the metabolism of thyroid, adrenal, p i t u i t a r y and sex hormones. Sandstead et a l . (1969) has shown that lead decreases the conversion of iodine to protein bound iodine, r e s u l t i n g in decreased thyroid function in man and animals. The work of Jacquet et a l . (1977a) in mice, suggests that a maternal hormonal imbalance in response to lead, may be responsible for early regression of the corpora lutea and implantation f a i l u r e . Der et a l . (1977) found increased plasma corticosterone le v e l s and adrenal c o r t i c a l histology i n d i c a t i v e of increased adrenal function in rats treated with 50 ug/day of lead for 70 days. 25 Embryotoxicity, Fetotoxcity and Teratogenicity The developing organism i s far more vulnerable to the detrimental effects of lead than the mature organism. This difference in v u l n e r a b i l i t y i s related to differences in the degree to which protective systems have developed and matured (Fahim et a l . , 1976). In the embryonic stage of development, which spans the period of organogenesis, detoxifying and excretory systems, the blood brain barrier, lead binding proteins and tissue such as bone that serve to protect the mature organism from the toxic e f f e c t s of lead are just beginning to develop. Exposing the organism to lead, while these systems are s t i l l immature can result in developmental delays, organ atrophy, growth retardation and malformations (Fahim et a l . , 1976). Embryotoxic, Fetotoxic and Teratogenic Ef f e c t s of Lead in Man The richest source of information, regarding the embryotoxic and teratogenic effects of lead in man i s derived from l i t e r a t u r e dated from the mid 1800's to the 1940's (Bell and Thomas, 1980). During this period, large numbers of women of childbearing age were employed in industries where lead was present in hazardous amounts and lead was widely used as an ab o r t i f a c i e n t . A review of studies from t h i s period (Angle and Mclntire, 1964; B e l l and Thomas, 1980), indicated a d e f i n i t e association between maternal exposure to lead in the workplace and s t i l l b i r t h s , miscarriages, neonatal deaths, 26 macroencephalopathy, growth retardation and neurological damage. In the past 20 years, due to l e g i s l a t i o n in the 1920's, which prohibited female employment in industries where lead exposure was considered to present a hazard to the fetus, and to continuing improvement in lead hygiene measures in the workplace (Angle and Mclntire, 1964), the number of reported cases regarding the embryotoxic and teratogenic e f f e c t s of lead in man has dwindled to a mere f i v e . Wilson (1966) reported three of these cases; a s t i l l b i r t h , an infant with congenital nystagmus, p a r t i a l albinism and haemangioma and a premature b i r t h to a mother with a history of miscarriage in seven out of eight previous pregnancies. In a l l three cases, the mothers had blood lead l e v e l s ranging from 50 to 75 ug/100 ml, as a result of being exposed to drinking water containing lead, that frequently exceeded the 0.05 ug/1 safe l i m i t set by the World Health Organization. These cases do not necessarily indicate a lead etiology, as 73 other pregnancies in which the mothers were exposed to the same levels of lead in the drinking water had normal outcomes and in the one case, where albinism, nystagmus and haemangioma was reported, there has been no other reports in the past or present l i n k i n g these abnormalities with lead. Palmissano (1969) reported a single case of a ten week infant, who exhibited neurological defects, intrauterine growth retardation and postnatal f a i l u r e to thrive, possibly due to the mother regularly ingesting i l l e g a l l y produced alcohol containing lead. In t h i s case, i t was impossible to determine whether the effects observed were due to alcohol, lead or a synergism 27 between the two agents. Angle and Mclntire (1964) reported a single case in which no f e t a l damage to the offspring occurred, in spite of the fact that the mother experienced lead c o l i c and general malaise, as a result of being exposed during the la s t trimester of pregnancy to the dai l y burning of lead battery casings to heat the home. They attributed the normal outcome in this pregnancy, despite the heavy lead exposure, to the fact that exposure occurred in the la s t trimester of pregnancy, rather than in the c r i t i c a l period of organogenesis. In recent years, there has been a considerable body of epidemiological evidence l i n k i n g lead with s t i l l b i r t h s , neonatal deaths, premature bi r t h s , early membrane rupture and f e t a l d i s t r e s s . wibberley et a l . (1977) found that 61% of s t i l l b i r t h s and neonatal deaths had placental lead levels greater than 1.5 ug/g, whereas only seven percent of placentas from infants surviving beyond one week had level s greater than t h i s . Bryce-Smith et a l . (1977) found s t i l l b i r t h infants to have bone lead l e v e l s f i v e to ten times greater than l i v e born infants who subsequently died from c r i b death, heart disease, infections, or accidents between six weeks and ten weeks of age. Khera et a l . (1980) found a s i g n i f i c a n t elevation in the placental lead concentrations in f e t a l d i s t r e s s cases as compared to normal b i r t h s . Fahim et a l . (1976) found the incidence of term pregnancies with early membrane rupture and premature d e l i v e r i e s to be 17 and 13%, respectively, in a population with a high environmental exposure to lead and 0.41 and three percent in a population with low environmental exposure to lead. Thus, in 28 spite of the successful strides made in reducing environmental lead exposure, epidemiological data indicates that lead may be an e t i o l o g i c a l factor in current cases of s t i l l b i r t h s , neonatal deaths and premature d e l i v e r i e s in man. Embryotoxic, Fetotoxic and Teratogenic Effects of Lead in the  Golden Hamster Lead in the form of n i t r a t e , acetate or chloride s a l t s (Ferm and Carpenter, 1967), administered to golden hamsters at a l e v e l of 50 mg/kg body weight, during the 24 hour period between the eighth and ninth days of gestation, when organogenesis is occurring, has been shown to rapidly cross the placenta in substantial amounts (Carpenter et a l . , 1973), resulting in decreased crown rump lengths from days nine to 13 of gestation, intrauterine deaths beginning on day 11 of gestation and increasing to 70% by day 15 and an almost 100% incidence of stunting and deformation of the t a i l in embryos surviving u n t i l day 15 (Carpenter and Ferm, 1977). Other abnormalities reported to occur were ectopia cordis on days ten and 11 of gestation (Carpenter and Ferm, 1977); and infrequently, exencephaly, hydrocephalus, fused ribs and delayed o s s i f i c a t i o n in embryos surviving u n t i l day 15 of gestation (Gale, 1978). Carpenter and Ferm (1977) painstakingly studied the sequence of events leading to lead induced t a i l malformations in the golden hamster. They found the f i r s t v i s i b l e sign to be edematous b l i s t e r s along the dorsum of the t a i l 30 hours post i n j e c t i o n . Subsequently, weakened or defective caudal blood 29 vessels were observed to hemorrhage into the edematous b l i s t e r s to form large hematomas. These b l i s t e r s and hematomas distorted and displaced the surrounding tissue, disrupting normal morphogenetic interactions with the result that shortened, deformed or absent t a i l s occurred. This sequence of events may have been i n i t i a t e d by lead i n t e r f e r i n g with the function of an enzyme system essential to t a i l development, by a direct toxic effect of lead on developing blood vessels in the t a i l that rendered them abnormally permeable and susceptible to hemorrhage or by a toxic e f f e c t of lead at a s i t e remote from the t a i l that provoked a generalized physiological disturbance, which elevated blood pressure and damaged the developing caudal blood vessels. (Carpenter and Ferm, 1977). These studies of the embryotoxic and teratogenic effects of lead in the golden hamster i l l u s t r a t e four important points regarding teratogens. (1) The most susceptible period for teratogenic e f f e c t s i s during the period of organogenesis. Ferm and Carpenter (1967) found golden hamsters to be most susceptible to the teratogenic effects of lead during the 24 hour period between the eighth and ninth days of gestation, when organogenesis was occurring. (2) The effects of teratogens may be s i t e s p e c i f i c or generalized. In the golden hamster, lead appears to be a s i t e s p e c i f i c teratogen as the only consistently occurring malformation i t produces is shortened or absent t a i l s (Ferm and Ferm, 1971). (3) Genetic factors are important in determing s u s c e p t i b i l t i y to a teratogen. Gale (1978) demonstrated that the LSH, LCH, LVG and PD4 strains of golden 30 hamsters were more susceptible to t a i l malformation and intrauterine death than the MHA and CB s t r a i n s , when exposed to lead on the eighth day of gestation. (4) Teratogenic responses may be overlooked as a result of the practice of only examining embryos surviving u n t i l the end of the gestational period for defects. Carpenter and Ferm (1977) observed ectopia cordis as a teratogenic response to lead, when they examined golden hamster embryos on days ten and 11 of gestation, but never observed this abnormality in embryos surviving u n t i l day 15 of gestation. Embryotoxic, Fetotoxic, and Teratogenic E f f e c t s of Lead in the  Mouse Lead administered to pregnant mice appears to consistently produce the following embryotoxic e f f e c t s ; f e t a l resorption (Jacquet et a l . , 1975; Kennedy et a l . , 1975), growth retardation (McClellan et a l . , 1974; Jacquet et a l . , 1975; Jacquet and Gerber, 1975; Kennedy et a l . , 1975) delayed skeletal development (McClellan et a l . , 1974; Kennedy et a l . , 1975) and neonatal deaths (McClellan et a l . , 1974). However, controversy exists as to the occurrence of lead induced teratogenic e f f e c t s in the mouse. Kennedy et al.(1975) observed no teratogenic effects when up to 714 mg/kg body weight of lead acetate was administered by oral intubation to mice on the f i f t h and seventh day of gestation. McClellan et a l . (1974) found no evidence of teratogenesis when lead chloride was administered at the l e v e l of 360 nM/g body weight by gavage on the ninth gestational day. Jacquet et a l . (1975) detected no b i r t h defects in fetuses of 31 mice fed a diet containing 0.125 to 0.50% lead throughout gestation. On the other hand, Jacquet and Gerber (1975) observed a 62% incidence of fusion of two or more vertebrae in the anterior a x i a l skeleton, when lead acetate was administered intr a p e r i t o n e a l l y at the l e v e l of 35 mg/kg body weight on the eighth and ninth gestational days. Likewise, McClain and Becker (1970) observed a 33% incidence of c l e f t palate, following an intravenous inj e c t i o n of lead n i t r a t e at the le v e l of 50 mg/kg body weight on the 12th gestational day. However, they observed no teratogenic e f f e c t s when the same amount of lead n i t r a t e was administered o r a l l y throughout gestation. These findings suggest that the controversy regarding whether or not lead i s a teratogenic agent in the mouse, has arisen as a result of the di f f e r e n t routes of lead administration used in the above studies. From the work of McClain and Becker (1970) and Jacquet and Gerber (1975), i t appears that teratogenic effects of lead are only observed when i t i s administered via the intraperitoneal or intravenous routes. Besides the route of administration, another factor which appears to be important in determining whether embryotoxic or teratogenic e f f e c t s due to lead are observed in the mouse i s the gestational day on which the lead i s administered. McClain and Becker (1970) demonstrated that the in j e c t i o n of pregnant mice with 50 mg/kg body weight of lead n i t r a t e on the tenth day of gestation resulted in a 27% incidence of non-ossification of the ce r v i c a l c e n t r i , while administration on the 12th gestational day resulted in c l e f t palate being the prominent response to 32 lead. These findings suggest that the timing of administration i s c r i t i c a l in the mouse in determining the embryotoxic and teratogenic effects produced by lead. Dietary factors also appear to influence the embryotoxic and possibly, teratogenic e f f e c t s of lead in the mouse. Jacquet et a l . (1975) reported a s i g n i f i c a n t enhancement in the incidence of mortality, growth retardation and delayed o s s i f i c a t i o n in fetuses from pregnant mice who were injected in t r a p e r i t o n e a l l y with 35 mg/kg body weight of lead acetate on days eight or nine gestation, after being fed a calcium d e f i c i e n t d i e t . Thus, these series of studies, regarding the embryotoxic and teratogenic effects of lead in the mouse i l l u s t r a t e at least three important points regarding such agents. They i l l u s t r a t e the importance of the route by which the agent i s administered, of c r i t i c a l periods in development, and dietary factors in determining embryotoxic and teratogenic outcomes. Embryotoxic, Fetotoxic and Teratogenic Effects of Lead in the  Rat The embryotoxic and teratogenic effects of lead have been studied in the rat, with the same type of pattern emerging with respect to routes of administration. Lead administered o r a l l y or intravenously appears to result in s i m i l i a r embryotoxic effects, but only intravenously administered lead appears to result in teratogenic e f f e c t s . A study by McClain and Becker (1970), in which up to 50 mg/kg body weight of lead nit r a t e was 33 administered intravenously in a single inj e c t i o n on day ten of gestation or o r a l l y throughout gestation, found hydronephrosis and non-ossified c e r v i c a l c e n t r i only when the intravenous route of administration was used, but found decreased body weight and size and increased f e t a l resorption, no matter which route of administration was used. Studies by Kimmel et a l . (1976) and Kennedy et a l . (1976) found s i m i l i a r reductions in body weight and increased incidences of f e t a l resorption, but no teratogenic e f f e c t s , when lead was administered o r a l l y at levels of up to 250 ppm throughout gestation and up to 714 mg/kg of body weight for three c r i t i c a l gestational days. McClain and Becker (1975) provide one of the most detailed studies of the teratogenic and embryotoxic effects of lead in the r at. These researchers administered single intravenous injections of lead n i t r a t e , ranging from 25 to 75 mg/kg body weight on days eight through 17 of gestation; k i l l e d the rats on day 22 and recorded the number of l i v e , dead, reabsorbed and malformed fetuses. When lead was administered on days eight or nine, f e t a l resorptions were approximately 20%. However, when lead was administered on days ten through 15, f e t a l resorptions increased to 100% and then, f e l l to eight percent when lead was administered on day 16. When lead was administered on days eight or nine, a wide spectrum of malformations confined to the posterior- portion of the body were observed. These included shortened or absent t a i l s , sirenoform monsters, an absence of the posterior portion of the body, urorectal malformations, absence of external g e n i t a l i a and a x i a l skeletal defects. Few 34 malformations were observed when lead was administered on days ten through 15. However, hydrocephalus was observed when lead was administered on day 16. McClain and Becker (1975) suggest that the increased f e t o t o x i c i t y observed in response to lead, during days ten to 15 of gestation in the rat i s the result of increased amounts of lead reaching the fetus, due to development of the a l l a n t o i c placenta and embryonic c i r c u l a t o r y systems. They suggest that the decreased f e t o t o x i c i t y of lead on day 16 i s the result of lead being deposited in bone developing at that time. They explained the pattern of malformations as being related to the sequence of morphogenic development in the embryo. Hypotheses Regarding Growth Retardation and Skeletal  Malformations in the Golden Hamster, Mouse and Rat In reviewing the above studies in the golden hamster, mouse and rat, i t appears that lead produces a number of s i m i l i a r embryotoxic and teratogenic responses in these species, including embryo mortality, growth retardation, delayed o s s i f i c a t i o n and skeletal defects. At present, two hypotheses have been offered to explain the manner in which lead produces growth retardation, while a single hypothesis has been offered to explain how lead produces skeletal defects. Jacquet et a l . (1977) suggest that the growth retardation may result from lead induced i n h i b i t i o n of f e t a l hemoglobin synthesis, leading to hypoxia, which i s known to decrease mitosis. Gerber et a l . (1978) suggest that growth retardation i s the result of lead 35 accumulating in the placenta, where i t interferes with blood flow and the uptake of substrates required by the growing fetus. Carpenter and Ferm (1977) hypothesize that lead acts to produce the observed skeletal malformations by causing c e l l death or reduced mitotic a c t i v i t y , which results in an i n s u f f i c i e n c y of the mesenchyme, leading to the formation of abnormal blood vessels or inadequate support for existing blood vessels. These changes enhance blood vessel permeability, r e s u l t i n g in the observed sequence of edema and hemorrhage, which always precedes such malformations. The major weakness of t h i s hypothesis is i t s f a i l u r e to explain why the defects are l o c a l i z e d in the posterior region in the hamster and the rat and in the anterior region in the mouse. Embryotoxic and Teratogenic E f f e c t s of Lead in the Chick Embryo Lead has been administered throughout the incubation period in the chick embryo, resulting in comprehensive knowledge of the embryotoxic and teratogenic e f f e c t s of lead in t h i s species (Table I ) . Butt et a l . (1952) injected 0.5 to one mg of lead, as the n i t r a t e s a l t into the albumen of white leghorn eggs prior to incubation. Embryos receiving t h i s treatment, exhibited decreased body weights, occasional meningoceles and high mortality rates up to the 13th day of incubation. Catizone and Gray (1941) injected lead chloride at l e v e l s of 1 x 10"4 to 1 x 10"3 M into the subgerminal cavity of brown leghorn eggs prior to incubation, and at 18 and 24 hours. Lead injected p r i o r to or at 18 hours of incubation resulted in mortality rates that did Table I. Summary of studies of the embryotoxic and teratogenic effects of lead on the chick embryo Day of i n j ect i on Study Lead salt Amount Route Embryotox i c effects Teratogenic effects Butt et al ( 1952) n i trate 0.65-1.30 mg a 1bumen decreased body weight mortali ty (61-79%) 18 hr Catizone & Gray ( 1941) chlor i de 1X10"* 1X10"3M subgerm i na1 cav i ty morta1i ty same as control anter i or end of the CNS destroyed open CNS si nous CNS Karnofsky & Ridgeway (1952) ni trate 0.05-0.25 mg yol k sac LD50=0.13 mg morta1i ty 64% anter i or end of the CNS destroyed hydrocepha1 us (i nfrequent) Gi1 an i (1973b) acetate Gilani (1973a) 0.005-0.08 mg LD50=0.03 mg decreased body weight abnorma1i t i es i nvolv i ng: (0.05 mg or >) neck 100% 1imbs 15% viscera 10% beak 5% eyes 5% brain 5% card i ac anomali es Karnofsky & Ridgeway ( 1952) n i trate 0.15 mg morta1i ty 83% bra i n hemorrhage 70% T a b l e I (cont i nued) Day of i n j e c t i on Study K a r n o f s k y & Ridgeway ( 1952) Ridgeway & K a r n o f s k y (1952) H i r a n o & Kochen (1973) K i n g S ( 1974) Lui Lead sa1t Amount ni t r a t e 0.1-2 mg 0.15 mg a c e t a t e 0.075 mg 0.25 mg 0.20 mg 0.15 mg 0.10 mg 0.05 mg Route Embryotox i c T e r a t o g e n i c e f f e c t s e f f e c t s s t u n t e d growth m o r t a l i ty 75% (0.15 mg) b r a 1 n hemorrhage i n 90% of deaths h y d r o c e p h a l u s 84% m i crome1i a s h o r t e n e d lower beaks bra i n hemorrhage pr i o r to day 8 .hydrocepha1 us a f t e r day 8 m o r t a l i t y h y d r o c e p h a l u s 67% m o r t a l i t y 96% a b n o r m a l i t i e s 90% (0.075 mg) : 85% head 81% 67% d i g i t s 40% 26% beaks 27% eyes 16% v i s c e r a 10% T a b l e I (cont i nued) Day of  i n j e c t i o n Study Lead s a l t Amount Ka r n o f s k y S Ridgeway ( 1952) ni t r a t e 0.05-5.00 mg DeFranc i s i s & B o c c a l a t t e ( 1962) a c e t a t e 10, 20 or 30 mg 10 Ka r n o f s k y & Ridgeway (1952) n i t r a t e 0.15-10.00 mg DeGennaro (1978) 0.5-1.50 mg 12 K a r n o f s k y & Ridgeway (1952) 0.15-5.00 mg 15 K a r n o f s k y & Ridgeway (1952) 2 .00-5.00 mg Route Embryotoxic e f f e c t s T e r a t o g e n i c e f f e c t s LD50=1.5 mg s t u n t e d growth (0.10mg or >) bra i n hemorrhage 96% s h o r t e n e d 1ower beak m i c r o m e l i a c1ubbed f e a t h e r s morta1i ty 70 to 100% mean body we i ght reduced by 35% non-spsc i f i ed abnormali t i es s t u n t e d growth m o r t a l i ty 21% b r a i n i n j u r y s h o r t e n e d lower beaks c l u b b e d f e a t h e r s a i r s p a c e body weight 50% of c o n t r o l s c u r l e d t oes open abdominal and t h o r a c i c cav i t i es y o l k sac growth r e t a r d a t i on m o r t a l i t y 19% (0.25mg or >) b r a i n i n j u r y 32% f e a t h e r and beak abnorma1i t i es morta1i t y 9% bra i n hemorrhage 39 not d i f f e r s i g n i f i c a n t l y from controls. However, lead injected at 24 hours of incubation resulted in a s i g n i f i c a n t increase in mortality. Lead injected prior to or at 24 hours of incubation, most frequently resulted in the anterior end of the central nervous system being destroyed. Lead injected at 18 hours tended to result in an open central nervous system, due to f a i l u r e of the two sides of the central nervous system to grow together or a sinuous central nervous system due to a f a i l u r e of the ectoderm to grow as rapidly or to the same extent as the mesoderm. Gil a n i (1973b) and Karnofsky and Ridgeway (1952) studied the embryotoxic and teratogenic e f f e c t s of in j e c t i n g lead on the second day of incubation. G i l a n i (1973b) administered lead acetate at levels of 0.005 to 0.08 mg/egg, while Karnofsky and Ridgeway (1952) administered lead nit r a t e at levels of 0.05 to 0.25 mg/egg into the yolk sac. G i l a n i (1973b) reported the LD50 for lead acetate in chick embryos injected on day two of incubation to be 0.03 mg/egg, while Karnofsky and Ridgeway (1952) found the LD50 for lead n i t r a t e under s i m i l i a r conditions to be 0.13 mg/egg. Gil a n i (1973b) observed a wide array of embryotoxic and teratogenic e f f e c t s , including reduced body size, micromelia, twisted limbs, shortened and twisted necks, microopthalmia, ruptured brains and everted viscera, which he attributed to lead generally damaging c e l l s that were dividing at that time. Karnofsky and Ridgeway (1952) reported a single embryotoxic e f f e c t , high mortality and a single teratogenic e f f e c t , a low incidence of hydrocephalus in embryos surviving 40 u n t i l day 18. Gila n i (1973a; 1975) in two other papers reported that 0.015 mg of lead acetate injected on day two of incubation, resulted in congenital cardiac abnormalities, including aortic stenosis, vulvular defects and thin ventricular walls. In an electron microscope study ( G i l a n i , 1975) of lead treated embryonic chick heart v e n t r i c l e s , he found malformed mitochondria, disorganized short and scanty myofibrils and an abundance of swollen vacuoles, indicating that microscopic lesions occur as well as gross abnormalities Karnofsky and Ridgeway (1952), Hirano and Kochen (1973), and King and Lui (1974) reported on the embryotoxic and teratogenic e f f e c t s of i n j e c t i n g lead acetate or n i t r a t e into the yolk sacs of chick embryos on the t h i r d and fourth days of incubation. Karnofsky and Ridgeway (1952) observed a 70% incidence of brain hemorrhage, associated with an exceedingly high mortality rate on incubation days four through eight, when day three chick embryos were injected with 0.15 mg of lead n i t r a t e . Using the same amount of lead n i t r a t e on day four of incubation, Ridgeway and Karnofsky (1952) found embryos dying or s a c r i f i c e d on days four through eight to exhibit a 90 and 75% incidence of hemorrhage, respectively. Those surviving to day 18, were found to exhibit an 84% incidence of hydrocephalus. The severity of the hydrocephalic lesion ranged from small herniations of the meninges to massive enlargement of the brain. Infrequent occurrences of generalized stunting, micromelia and shortened lower beaks in lead treated embryos were also 41 reported. When Hirano and Kochen (1973) injected 0.075 mg of lead acetate into the.yolk sac of four day incubated embryos, they observed a 67% mortality rate due to hemorrhage into the central nervous system, which in some cases was so severe that the body of the embryo was depleted of blood. In embryos surviving the treatment, the only abnormality they noted was hydrocephalocele. This abnormality most commonly affected the o c c i p i t a l , but occasionally affected the p a r i e t a l region of the brain. King and Lui (1974) reported the following mortality rates: 96, 90, 85, 67, amd 26%, when the l e v e l of lead acetate injected into four day chick embryos was 0.25, 0.20, 0.15, 0.10 and 0.05 mg, respectively. From t h i s data, they estimated that the LD50 for lead acetate in four day chick embryos was 0.075 mg. At 0.075 mg of lead acetate per egg, they found the incidence of abnormalities involving the head to be 81%, d i g i t s 40%, beak 27%, eyes 16%, viscera ten percent and neck nine percent. Two papers have reported on the effects of injecting lead into the yolk sacs of chick embryos on the eighth day of incubation. DeFrancisis and Boccalatte (1962) observed 70 to 100% mortality rates, a 35% reduction in mean body weight and a 67% incidence of malformations when 10, 20 or 30 mg of lead acetate was administered. Karnofsky and Ridgeway (1952) reported the LD50 for lead n i t r a t e in eight day chick embryos to be 1.5 mg in comparison to an LD50 in four day chick embryos of 0.15 mg. They found the le v e l of lead n i t r a t e required to produce hydrocephalus in 96% of embryos surviving u n t i l day 18 to be 42 0.10 mg/egg; the same le v e l of lead n i t r a t e required to produce a s i m i l i a r incidence of brain injury in four day embryos. However, the hydrocephalocelic lesions were less conspicuous in embryos injected with lead on day eight, due to the more advanced development of the c r a n i a l bones. Other defects ocassionally noted were stunted growth, shortened lower beaks, micromelia and clubbed feathers. DeGennaro (1978) and Karnofsky and Ridgeway (1952) reported on the e f f e c t of i n j e c t i n g lead n i t r a t e at the l e v e l of 0.5 to 1.5 mg or 0.15 to ten mg into the airspace or yolk sac, repectively of f e r t i l e eggs incubated for ten days. DeGennaro (1978) noted that the lead treated embryos weighed half as much as the controls, were smaller in size at the later stages of development, exhibited a high incidence of curled toes (a sign of central nervous system damage) and a low incidence of shortened beaks and open abdominal and thoracic c a v i t i e s with exposed viscera. Karnofsky and Ridgeway (1952) noted severe brain injury, stunted growth, retarded development of the lower beak and clubbed feathers in lead treated embryos surviving u n t i l day 18 of incubation. Karnofsky and Ridgeway (1952) as a result of studying the effects of lead injected into the yolk sac of developing chick embryos on days two through 15 of incubation, concluded that the most vulnerable period for the teratogenic effects of lead in the central nervous system was days three through 12. They hypothesized that lead injected on day two became securely fixed in embryonic tissue or other compartments of the egg, thereby, 43 preventing i t from exerting detrimental e f f e c t s on later developmental stages. However, lead injected late in day three or early in day four readily affected the central nervous system, due to an increased s u s c e p t i b i l i t y or a f f i n i t y for i t , as a result of blood vessels in the brain beginning to form at that time. After day 12, the s u s c e p t i b i l i t y of the central nervous system to lead was hypothesized to decrease as a result of inherent changes in brain tissue or blood vessels or to the development of systems, such as bone, which s e l e c t i v e l y concentrated lead. From these studies, i t is evident that a number of factors influence the embryotoxic and teratogenic e f f e c t s of lead in the chick embryo. The high incidence of central nervous system eff e c t s in the Catizone and Gray (1941) study and the low incidence of central nervous system e f f e c t s in the Karnofsky and Ridgeway (1952) study, when lead was injected during the f i r s t two days of incubation, were l i k e l y the result of d i f f e r e n t routes of lead administration. The less severe brain injury reported in the Karnofsky and Ridgeway (1952) study in embryos injected on day eight with the same amount of lead as administered to day four embryos r e f l e c t s the importance of developmental age in determining the severity of an abnormality. Developmental age i s also a factor in determining the type of defect observed. Clubbed feathers were only observed when lead was injected on day eight of incubation or l a t e r (Karnofsky and Ridgeway, 1952). The size of the eggs and their chemical composition, which r e f l e c t s differences in genetic st r a i n and/or 44 diets of the laying hens, may be a factor serving to explain the wide array of abnormalities reported in the G i l a n i (1973a) study as opposed to the single abnormality reported in the Karnofsky and Ridgeway (1952) study, when two day incubated embryos were injected with lead. Other factors that may a f f e c t the outcome of experiments such as these are the type of lead s a l t administered and i t s s o l u b i l i t y . Butt et a l . (1952) found that chloride, sulfate and n i t r a t e anions f a i l e d to produce meningocele or other teratogenic e f f e c t s , when administered in the same amounts as contained in the lead s a l t s , but did produce s l i g h t increases in mortality in comparison to saline administered controls. However, the increased mortality rates were substantially less than when the same amount of anion was administered as the lead s a l t . McLaughlin et a l . (1963) reported that provided the s a l t was soluble in water and was not administered as a suspension, the emulsifying properties of the egg yolk tended to make i t available for u t i l i z a t i o n by the embryo. In the chick embryo, any embryotoxic or teratogenic e f f e c t s attributed to lead, are considered to be the result of i t s direct action on embryonic development (Romanoff and Romanoff, 1972). In mammalian species, any e f f e c t s attributed to lead are generally considered to have been modified by maternal influences. However, i t would be incorrect to assume that differences in the embryotoxic and teratogenic e f f e c t s of lead between avian and mammalian species can solely be attributed to maternal influences, as other factors may play a r o l e . At high concentrations, lead may upset the physical equilibrium of the 45 yolk, producing osmotic e f f e c t s having s i g n i f i c a n t e f f e c ts on growth and development (McLaughlin et a l . , 1963). A l t e r n a t i v e l y , lead may destroy, a l t e r or combine with vitamins, minerals, fats, proteins, carbohydrates or enzymes in the yolk sac, thereby a l t e r i n g the uptake and u t i l i z a t i o n of these substances by the embryo. Thus, species differences may also be due to differences in the e f f e c t s of lead on the yolk sac. In conclusion, lead appears to have the same ef f e c t s in increasing mortality and retarding growth in the chick embryo as in other species. However, unlike other species, lead administered to the chick embryo during the period of organogenesis consistently produces damage to the central nervous system, res u l t i n g in the development of a f l u i d f i l l e d cyst in the o c c i p i t a l and/or p a r i e t a l regions of the brain in day nine or older embryos. Other abnormalities reported, such as twisted necks, curled toes, microopthalmia and shortened beaks appear to be r e f l e c t i o n s of the central nervous system damage, in t e r f e r i n g with nerve impulses to the body or with normal morphogenic development in the head region. These observations, in conjunction with the work of Ridgeway and Karnofsky (1952), indicating that the central nervous system e f f e c t s produced by lead in the chick embryo were unique to lead alone, suggest that lead in the chick embryo i s a s i t e s p e c i f i c teratogen of the central nervous system. 46 Acute Lead Encephalopathy Cases of acute lead encephalopathy in young children related to excessive lead ingestion, have frequently been reported in the l i t e r a t u r e (Popoff et a l . , 1963; Pentschew, 1965; Clasen et a l . , 1974b). Upon autopsy, gross examinations of the brain of such children reveals them to be f r i a b l e , wet, pale and swollen (Popoff, 1963). Microscopic examinations reveal petechial hemorrhages, perivascular edema, p a r t i a l demylinization, astrocytosis, thickened and disorganized c a p i l l a r y endothelial c e l l s , neuronal degeneration and focal necrosis, p a r t i c u l a r l y of the cerebellum (Popoff et a l . , 1963; Pentschew, 1965; Clasen et a l . , 1974b). In the most severe cases, extensive tissue destruction with cavity formation has been observed (Popoff et a l . , 1963). In order to understand the mechanism, by which lead produces the above changes in the central nervous system of young children, a number of animal models have been developed (Pentschew and Garro, 1966; Hirano and Kochen, 1973; Clasen et a l . , 1974a; O'Tuama et a l . , 1976; Lorenzo et a l . , 1978; Toews et a l . , 1978). The most extensively used model developed by Pentschew and Garro (1966), involves transmitting lead via maternal milk to suckling rats by feeding their mothers a diet of four percent lead carbonate from p a r t u r i t i o n . By the fourth week of l i f e , offspring exhibit weakness, hind limb paralysis and encephalopathy involving the cerebellum. The major weaknesses of t h i s model are the i n a b i l i t y to control the amount of lead received by the offspring and the confounding effects of 47 n u t r i t i o n a l alterations in the maternal milk due to reduced food intake in lead treated mothers (Lorenzo et a l . , 1978). Another important c r i t i c i s m of the model is that the most salient c l i n i c a l feature, hind limb paralysis, is rarely seen in human cases of lead encephalopathy (O'Tuama et a l . , 1976). Lack of control over lead intake has been overcome by Lorenzo et a l . (1978) in the rabbit, O'Tuama et a l . (1976) in the guinea pig and Toews et a l . (1978) in the rat by administering lead via gastric gavage. Clasen et a l . (1974a), O'Tuama et a l . (1976), and Lorenzo et a l . (1978) in the rhesus monkey, guinea pig and rabbit, respectively, have been able to simulate many of the c l i n i c a l features of acute lead encephalopathy observed in humans, making these species, p a r t i c u l a r l y useful in developing an understanding of the pathogenesis of human lead encephalopathy. Hirano and Kochen (1973; 1977) have developed a model to study lead induced central nervous system injury during embryonic development using the chick embryo. They consider t h i s model to simulate the development of hydrocephalocele in human fetuses. The advantages of t h i s model are avoidance of confounding e f f e c t s due to n u t r i t i o n a l d e f i c i e n c i e s as a result of reduced food intakes and due to maternal and placental influences on embryonic development (Hirano and Kochen, 1973). Several researchers (Thomas et a l . , 1971; Clasen et a l . , 1974b; Goldstein et a l . , 1974; Thomas and Thomas, 1974; Ahrens and V i s t i c a , 1977; Toews et a l . , 1978), using the model developed by Pentschew and Garro (1966) or a modification there 48 of, have studied the development of acute lead encephalopathy in the rat during the preparalytic, p a r a l y t i c and postreparative phases. The f i r s t change noted in the preparalytic phase was increased concentrations of lead in c a p i l l a r i e s , p a r t i c u l a r l y of the cerebellum (Toews et a l . , 1978). This lead enrichment of the microvessels was associated with degenerative changes in the c a p i l l a r y endothelial c e l l s consisting of swelling, mitochondrial vacuolation and pinocytosis. These changes were accompanied by increased water, sodium and serum albumin in cerebellar tissue (Clasen et a l . , 1974b; Goldstein et a l . , 1974), indicating increased c a p i l l a r y permeability. The edema f l u i d was i n i t i a l l y l o c a l i z e d around c a p i l l a r i e s in the foot processes of the a s t r o g l i a , resulting in a prominent appearance for affected c a p i l l a r i e s (Thomas et a l . , 1971). As poisoning progressed, the edema f l u i d spread throughout the white matter forming large lakes, coinciding with the development of p a r a l y s i s . The edema lakes increased in size during the early reparative phase through coalescing. However, by the end of the reparative phase evidence of edema had disappeared and the e a r l i e r c a p i l l a r y prominence was no longer evident (Thomas et a l . , 1971; Ahrens and V i s t i c a , 1977). Shortly after the edema appeared in the preparalytic phase, extravasation of red blood c e l l s was observed and lead was detected in the white and grey matter (Goldstein et a l . , 1974). From radioisotope studies (Goldstein et a l . , 1974), the source of the lead was d i f f u s i n g plasma and extravasated red blood c e l l s . Hemorraghes in t h i s phase were described as petechial, 49 but as the day of paralysis approached they became more extensive. In areas of extensive hemorrhaging, c a p i l l a r i e s were observed to be collapsed and evidence of arrested c a p i l l a r y development was observed (Clasen et a l . , 1974b). Towards the end of the preparalytic and in the para l y t i c phase, intravascular thrombi were found in c a p i l l a r i e s and venules, but not in a r t e r i o l e s (Thomas et a l . , 1971; Thomas and Thomas, 1974). In the post reparative phase, evidence of hemorrhage was s l i g h t or non-existent. During th i s phase, a change to normal morphology and function of c a p i l l a r i e s were observed (Toews et a l . , 1978). In the preparalytic phase subsequent to the appearance of edema and hemorraghe, patchy degenerative changes were observed in Purkinje c e l l s (Thomas et a l . , 1971; Thomas and Thomas, 1974). These degenerative changes included loss of nuclear cytoplasmic d i f f e r e n t i a t i o n and the development of mitochondrial vacuolations. The number of c e l l s affected by these degenerative changes were observed to increase u n t i l the onset of paralysis (Thomas et a l . , 1971). During the reparative phase, the damaged Purkinje c e l l s underwent necrosis. However, by the end of the reparative phase, only normal Purkinje c e l l s were observed. The sequence of edema, hemorrhage and neuronal damage, which has been observed in the guinea pig (O'Tuama et a l . , 1976), the rhesus monkey (Clasen et a l . , 1974a) and the rabbit (Lorenzo et a l . , 1978) as well as the rat (Thomas et a l . , 1971), has led to the conclusion that lead increases brain c a p i l l a r y permeability by somehow injurying c a p i l l a r y endothelial c e l l s , p a r t i c u l a r l y of the cerebellum. V i s t i c a et a l . (1977) propose 50 and provide evidence for lead a l t e r i n g the collagen component of c a p i l l a r y basement membranes and i n t r a c e l l u l a r junctions being synthesized in the developing cerebellum, by i n t e r f e r i n g with collagen metabolism. Holtzman et a l . (1980) propose and provide evidence for lead uncoupling mitochondrial respiration in endothelial c e l l s , r esulting by some as yet unidentified sequence of events in increased c a p i l l a r y permeabilty. Goldstein et a l . (1977) suggest that lead decreases calcium uptake by brain mitochondria, resulting in increased i n t r a c e l l u l a r calcium concentrations, which have been shown to interfere with enzymes that regulate c e l l permeabilty. Further research i s required to delineate the precise mechanism by which lead induces microvessel injury in the central nervous system. The degeneration of Purkinje c e l l s in acute lead encephalopathy has been hypothesized by Clasen et a l . (1974b) to be a secondary effect due to anoxia produced as a result of the edema and hemorrhaging. A l t e r n a t i v e l y , the edema and hemorrhaging could permit lead to gain access to the nerve c e l l s , where i t could d i r e c t l y i n t e r f e r e with their metabolism, producing the degenerative changes observed. The normal morphology and function of c a p i l l a r i e s observed in the reparative phase, according to several investigators (Goldstein et a l . , 1974; Ahrens and V i s t i c a , 1977; Toews et a l . , 1978), r e f l e c t s maturation of the brain vascular system, p a r t i c u l a r l y the choroid plexus. According to O'Tuama et a l . (1976) and Holtzman et a l . (1980), the blood brain barrier acts as a lead sink, concentrating lead from the blood and the 51 cerebral spinal f l u i d in a non-mitochondriai compartment in the endothelial c e l l s , where i t s toxic effects are not as marked and from which i t can not gain access as readily to neural t i s s u e . This s h i f t (Holtzman et a l . , 1980) of lead to non-mitochondrial compartments as the endothelial c e l l s mature, may explain why acute lead encephalopathy is nearly impossible to produce in mature animals and man. The model (Hirano and Kochen, 1973; 1977) used to study the development of hydrocephalocele, involves injecting 75 to 100 ug of lead acetate d i r e c t l y into the yolk sac of four day old chick embryos. Within 24 to 48 hours, hemorrhage into the central nervous system occurs, which increases in incidence and intensity, resulting in many deaths and then subsides by the tenth day of incubation. This i s followed, beginning on day ten, by the development of a f l u i d f i l l e d cyst covered with skin in the o c c i p i t a l region of the s k u l l . This cyst tends to increase in s i z e , u n t i l incubation is terminated on days 18 to 21. The cyst i s f i l l e d with a clear f l u i d which occasionally contains hemorrhagic material. It i s an expansion of the subarachnoid space that extends from the floor of the fourth v e n t r i c l e to the subcutaneous tissue over the head. The result of thi s expansion is passive separation of the developing o c c i p i t a l bones. According to microscopic studies (Hirano and Kochen, 1973), the floor of the cyst consists of ependymal c e l l s and the choroid plexus, while the roof of the cyst consists of distended meningeal membrane fused to a dermal covering. The l a t e r a l walls of the cyst consist of interrupted layers of neural tissue that 52 has been progressively depleted from the base to the roof of the cyst. As with other species treated with lead, the sequence of events leading to central nervous system changes in the chick embryo, appear to be related to the development of the central nervous system microvasculature. On days four through ten of incubation, primitive c a p i l l a r i e s consisting of c l o s e l y opposed endothelial c e l l s with l i t t l e or no specialized junctions develop in the chick embryo's central nervous system (Hirano and Kochen, 1977). During this period i t is hypothesized, although no evidence has been obtained due to the d i f f i c u l t i e s in preserving the brains of lead treated embryos for microscopic examination, that lead disrupts the c a p i l l a r i e s , r e s u l t i n g in hemorrhage into the central nervous system (Hirano and Kochen, 1973). By the end of the tenth day of incubation, blood vessels with tight endothelial junctions have developed, leading to the establishment of the blood brain barrier (Hirano and Kochen, 1977). At t h i s point, i t i s hypothesized that lead i s no longer capable of disrupting c a p i l l a r i e s , thereby explaining the observations that lead administered after day ten, occurs in lesser amounts in the brain than when administered before day ten and i s not associated with hydrocephalocele (Hirano and Kochen, 1973). Thus, as in other species, the primary event in the development of lead induced changes in the central nervous system of the chick embryo appears to be related to lead induced c a p i l l a r y injury during the c r i t i c a l period of c a p i l l a r y development. 53 In the chick embryo, the development of the f l u i d f i l l e d cyst i s suspected to be a secondary effect of the extensive central nervous system hemorrhaging. Hirano and Kochen (1977) hypothesize that the hemorrhage, via subsequent hemolysis of red blood c e l l s and the destruction of neuronal tissue, leads to an increase in the osmotic pressure of the cerebral spinal f l u i d , favoring the accumulation of f l u i d in the central nervous system. A l t e r n a t i v e l y , they hypothesize that the hemorrhage results in destruction of the arachnoid granulation, the s i t e of cerebral spinal f l u i d reabsorption or in neural tissue destruction, leading to obstruction of the cerebral spinal f l u i d pathways. Both arachnoid granulation destruction and obstruction of cerebral spinal f l u i d pathways have been observed in cases of hydrocephalocele in human fetuses (Warkany, 1971), suggesting that hydrocephalocele in chick embryos and human fetuses may in some instances, have the common etiology of lead exposure during embryonic development. Chelat ion The word chelate, derived from the Greek word meaning lobster claw, was f i r s t used to describe the c a l i p e r l i k e mode of attachment of two donor atoms of an organic molecule to a metal atom (Mellor, 1964). Currently, the term i s used to describe organic molecules in which two or more electron donor groups coordinate with a polyvalent metal to form one or more ring structures (Anonymous, 1971) (Figure 1). In order for chelation to occur at least two conditions 54 Ha C & HC S CH2OH Pb lead - mono - b r i t i s h a n t i - l e w i s i t e mercaptide Figure 1 . A 5-member rin g chelate 55 must be f u l f i l l e d . (1) The organic molecule acting as a ligand must possess two functional groups with donor atoms capable of combining with the metal atom by donating a pair of electrons (Mellor, 1964). The electrons can be contributed by basic groups such as amino, and hydroxyl or aci d i c groups such as carboxyl, hydroxyl, and sulphydryl, that have lost a proton. (2) The functional groups must be situated in the molecule in such a manner, that they permit the formation of a ring with a metal atom as a closing member (Mellor, 1964). Other factors that influence chelate formation are pH, the oxidation state and the unique properties of the metal atom and the nature of the bonds li n k i n g the metal atom to the chelating molecule (Mellor, 1964). At least five factors influence the s t a b i l i t y of metal chelate complexes. The f i r s t factor is ring s i z e . Experimental work has indicated that f i v e member rings are the most stable (Mellor, 1964) (Figure 1). The second factor is the number of rings binding the metal atom to the ligand. In t h i s regard, multidentate complexes such as lead ethylenediaminetetraacetate are far more stable than bidentate complexes such as lead penicillamine (Mellor, 1964) (Figure 2). The t h i r d factor i s the basic strength (pKa) of the chelating molecule. The general rule i s the more basic the pKa of the chelating molecule, the greater the s t a b i l i t y of the metal chelate (Mellor, 1964). The fourth factor i s the nature of the donor atoms. There are two classes of donor atoms, class A, which includes donor atoms such as nitrogen, oxygen and fluoride and class B, which includes such donor atoms as phosphorus, sulphur and chloride. (Chisolm, l e a d - e t h y l e n e d i a m i n e t e t r a a c e t a t e l e a d - p e n i c i l l a m i n e F i g u r e 2. A m u l t i d e n t a t e and b i d e n t a t e c h e l a t e 57 1968). Most metal atoms form stable complexes with only one of these classes. However, lead is unusual in that i t forms stable complexes with donor atoms from both classes (Chisolm, 1968). The f i f t h factor influencing s t a b i l i t y i s the unique nature of the metal. Lead's unique electron configuration results in i t forming more stable complexes with ethylenediaminetetraacetate than does calcium (Chisolm, 1968). Ideally, the following c r i t e r i a should be met when selecting a chelator for therapeutic use. (1) The chelator should be non-toxic. This i s essential in order to permit the administration of a s u f f i c i e n t molar excess of chelator to metal, in order to bring about a s i g n i f i c a n t enhancement of the metal's excretion (Chisolm, 1968). Unfortunately, the common chelators used in the treatment of lead intoxication frequently can not be administered in s u f f i c i e n t amounts due to their t o x i c i t y (Chisolm, 1968). (2) The chelator should be f a i r l y s p e c i f i c for the metal i t i s to chelate. S t a b i l i t y constant measurements indicate that the a b i l i t y of metal atoms to form chelates i s general and only gradually varies from one metal to another, when a given chelating agent i s used (Mellor, 1964). Thus, absolute s p e c i f i c i t y i s unattainable. However, r e l a t i v e s p e c i f i c i t y may be attainable by adding or removing functional groups on the chelating molecule. For example, the addition of a methyl group in the two position of 8-hydroxyquinoline, a chelating agent used in a n a l y t i c a l chemistry, has been shown to prevent the agent from forming a complex with aluminum (I I I ) , chromium (III) or iron (III) (Mellor, 1964). (3) The metal 58 chelate should be less toxic than the free metal ion. The cadmium-dimercaprol complex, when dimercaprol is in excess, produces more serious damage to the renal tubules than cadmium alone (Schulman and Dwyer, 1964). (4) The chelating agent should be able to penetrate metal storage s i t e s without being readily metabolized. Unfortunately, chelators that are not readily metabolized, such as ethylenediaminetetraacetate, frequently have d i f f i c u l t y penetrating c e l l membranes (Schulman and Dwyer, 1964), while chelators that e a s i l y penetrate c e l l membranes, such as c i t r i c acid, are generally readily metabolized. (5) The chelating agent should be capable of competing with endogenous ligands for the toxic metal. Multidentate chelators such as ethylenediaminetetraacetate are generally more e f f e c t i v e in such competitions than bidentate chelators such as penicillamine, because they occupy a greater number of the potential coordinating positions about the metal, thereby decreasing the opportunity for mixed complex formation with donor groups from endogenous ligands (Schulman and Dwyer, 1964). (6) The chelating agent should form a soluble complex with the metal so that i t can be excreted in the urine or the b i l e . The common chelators used to treat lead t o x i c i t y a l l form soluble complexes with lead (Chisolm, 1968). (7) The chelating agent, when administered o r a l l y should not enhance the uptake of the metal from the ga s t r o i n t e s t i n a l t r a c t . Since ethylenediaminetetraacetate administered o r a l l y , has been found to exacerbate lead symptomology when lead was present in the ga s t r o i n t e s t i n a l t r a c t , i t has been recommended that ethylenediaminetetraacetate 59 only be administered intravenously (Anonymous, 1971). (8) The functional donor groups of the chelating agent should not readily dissociate themselves from the metal atom in vivo. It appears that a substantial portion of the renal damage observed in patients undergoing chelation i s the result of the chelating agent concentrating the metal in the kidney, where i t p a r t i a l l y or completely dissociates from i t , to form complexes with endogenous ligands. These complexes seriously interfere with normal b i o l o g i c a l processes (Chisolm, 1968). In the search to find a suitable chelating agent for the prophylaxis and treatment of lead burdened individuals, many chelators have been investigated and developed with at least some of the above c r t i t e r i a in mind. Of these agents, ethylenediaminetetraacetate, c i t r i c acid, ascorbic acid and isoascorbic acid are reviewed in d e t a i l , as they have a dir e c t bearing on the research undertaken for this t h e s i s . Ethylenediaminetetraacetate Ethylenediaminetetraacetate i s a polyaminocarboxylic acid (Figure 3) that readily forms stable chelates with many metals (Chisolm, 1968). Lead ethylenediaminetetraacetate complexes are ten times more stable than calcium ethylenediaminetetraacetate complexes, making them one of the most stable ethylenediaminetetraacetate complexes known (Garvon, 1964). The s t a b i l i t y of lead ethylenediaminetetraacetate complexes appear to be due to the binding of a single lead atom by a l l six donor atoms (four oxygen and two nitrogen) of 60 OOCH2C OOCH2C F i g u r e 3 . E t h y l e n e d i a m i n e t e t r a a c e t a t e 61 ethylenediaminetetraacetate, such that f i v e - f i v e member chelate rings are formed (Figure 2). The greater s t a b i l i t y of lead ethylenediaminetetraacetate complexes, in comparison to other lead chelates and calcium ethylenediaminetetraacetate complexes, has resulted in calcium ethylenediaminetetraacetate becoming the agent of choice for the treatment of lead t o x i c i t y in the past 20 years (Chisolm, 1968). A considerable amount of information exists regarding the absorption, d i s t r i b u t i o n , metabolism and excretion of ethylenediaminetetraacetate in vivo. Foreman (1960) reports, from studies in rats, that 80 to 95% of an o r a l l y administered dose of ethylenediaminetetraacetate appears in the feces, while two to 18% is absorbed within 24 hours of administration. In man, the maximal absorption of ethylenediaminetetraacetate from the g a s t r o i n t e s t i n a l tract does not appear to exceed five percent, indicating that ethylenediaminetetraacetate i s rather poorly absorbed (Foreman, 1960). Catsch and Harmuth-Hoene (1979) postulate that the poor absorption of ethylenediaminetetraacetate may be due to p r e c i p i t a t i o n in gastric juices or to impermeability of the c e l l membrane to the chelate anion. Ethylenediaminetetraacetate parenterally administered to rats appears to be f a i r l y evenly d i s t r i b u t e d throughout the body, without being concentrated in any p a r t i c u l a r organ or the red blood c e l l (Foreman, 1960). Work with radioactively l a b e l l e d ethylenediaminetetraacetate indicates that i t does not readily permeate c e l l membranes and i s located primarily in the 62 e x t r a c e l l u l a r f l u i d and the plasma (Catsch and Harmuth-Hoene, 1979). Although ethylenediaminetetraacetate does not readily cross c e l l membranes, i t has been shown to slowly tranverse the blood-spinal f l u i d membrane, res u l t i n g in a concentration in the cerebral spinal f l u i d of 1/40th that found in plasma, one hour after intravenous administration in humans (Foreman, 1960). Ethylenediaminetetraacetate i s not metabolized to any extent in the body (Foreman, 1960). The limited metabolism that does occur appears to involve degradation of the acetate group (Catsch and Harmuth-Hoene, 1979). Since this degradation i s i n s i g n i f i c a n t , i t has no effect on the therapeutic e f f i c a c y or t o x i c i t y of ethylenediaminetetraacetate in vivo. Studies with radioactively l a b e l l e d ethylenediaminetetraacetate in rats have shown that 95 to 98% of a parenterally administered dose i s eliminated from the body within six hours of administration (Foreman, 1960). The major route of excretion is via the kidney, although a small percentage of the compound is eliminated via the b i l i a r y route (Catsch and Harmuth-Hoene, 1979). Excretion via the kidney i s id e n t i c a l to that of i n u l i n , indicating that ethylenediaminetetraacetate i s excreted via glomerular f i l t r a t i o n without tubular secretion playing a role (Catsch and Harmuth-Hoene, 1979). Ethylenediaminetetraacetate administered in large doses over a prolonged period of time to animals and man has been shown to produce lesions in the kidney and the small intestine, impair reproduction, decrease i n t e s t i n a l glucose absorption, 63 increase prothrombin time and the urinary excretion of copper, iron, manganese, and zinc, i n h i b i t DNA synthesis, reduce enzymatic a c t i v i t y , result in immunological changes, stress induced changes in the adrenal glands, e l e c t r o l y t e imbalances, disturbances in carbohydrate metabolism, hypocalcemia, hypotension, anemia, glucosuria, dermatitis, electrocardiogram abnormalities, fever, headache, parasthesia and death (Foreman, 1960; Seven, 1960; Swenerton and Hurley, 1971; Bridbord and Blejar, 1977; Catsch and Harmuth-Hoene, 1979). The underlying, as yet unproven, mechanisms for many of the adverse effects of ethylenediaminetetraacetate appear to involve chelation of ess e n t i a l minerals or formation of mixed ethylenediaminetetraacetate and endogenous ligand complexes (Catsch and Harmuth-Hoene, 1979). A study by Swenerton and Hurley (1971) i l l u s t r a t e s the f i r s t mechanism. In t h i s study, three grams of ethylenediaminetetraacetate per 100 g of a n u t r i t i o n a l l y balanced diet was administered to rats from day six to 21 of gestation. These researchers found reduced l i t t e r size, growth retardation, a 44% incidence of hydrocephalus, anencephalus or exencephalus, a 57% incidence of c l e f t palate and fused or missing d i g i t s , a 92% incidence of clubbed legs and a 98% incidence of curly, short or missing t a i l s . When 1000 ppm of zinc was administered in conjunction with ethylenediaminetetraacetate, no embryotoxic or teratogenic changes were observed, suggesting that zinc deficiency as a result of ethylenediaminetetraacetate chelation was l i k e l y 64 responsible for the observed embryotoxic and teratogenic effects of ethylenediaminetetraacetate. The toxic and adverse e f f e c t s of ethylenediaminetetraacetate can be minimized by administering calcium ethylenediaminetetraacetate at a l e v e l in man that does not exceed 75 mg/kg and by using a treatment schedule of fiv e days on, followed by two days off (Chisolm, 1968). Unfortunately, according to Chisolm (1968), this maximum safe dosage may not in a l l cases of severe lead intoxication provide an adequate r a t i o of chelate to metal ion. When an inadequate r a t i o i s used, lead excretion i s not s i g n i f i c a n t l y increased. Ethylenediaminetetraacetate has also been studied from the perspective of i t s effects on the absorption, d i s t r i b u t i o n , metabolism and excretion of lead. Jugo et a l . (1975) found that lead ethylenediaminetetraacetate complexes o r a l l y administered to rats enhance lead absorption in comparison to controls administered the same amount of unchelated lead. However, they found no increase in whole body lead retention after oral lead ethylenediaminetetraacetate administration. McClain and Siekierka (1975a; 1975b) found a s i m i l i a r phenomenon, while studying the teratogenicity and placental transfer of lead ethylenediaminetetraacetate complexes in the rat. They found that ethylenediaminetetraacetate enhanced placental permeabilty to lead, while at the same time affording complete protection against the teratogenic effects of lead. They concluded that rapid maternal excretion of lead ethylenediaminetetraacetate complexes decreased the duration of f e t a l lead exposure, thereby 65 preventing the development of teratogenicity. In man, there is c l i n i c a l evidence of enhanced lead t o x i c i t y , resulting occasionally in death, when ethylenediaminetetraacetate i s o r a l l y administered to patients whose ga s t r o i n t e s t i n a l tracts contain s i g n i f i c a n t amounts of lead (Bridbord and Blejar, 1977). For t h i s reason, ethylenediaminetetraacetate i s not administered o r a l l y . However, the general population is exposed to oral ethylenediaminetetraacetate as a s t a b i l i z e r in food. This has led to Graef's (1980) concern that t h i s practice may be increasing body lead burdens in the general population. However, i f Jugo et a l . ' s (1975) findings in rats applies to man, t h i s may be an unfounded concern. According to Chisolm (1968), i t i s well documented that ethylenediaminetetraacetate enhances urinary lead excretion by 20 to 50 f o l d . In the past 20 years there has been considerable controversy as to the source of the excreted lead. A d e f i n i t i v e study by Hammond (1971) indicates that the major source of lead mobilized by ethylenediaminetetraacetate i s the skeleton, regardless of the magnitude of the body burden of lead or the recency of i t s a c q u i s i t i o n . Ethylenediaminetetraacetate has also been shown to gradually remove lead from soft tissue. Sorensen et a l . (1980) found a s i g n i f i c a n t reduction in brain and kidney, but not l i v e r , lung, spleen and red blood c e l l lead after a single i n j e c t i o n of ethylenediaminetetraacetate. Kochen and Greener (1974) found s i g n i f i c a n t reductions in brain lead when ethylenediaminetetraacetate was administered to chick embryos on 66 day four of incubation, two minutes after lead administration. Goyer and Cherian (1979) found s i g n i f i c a n t reductions in l i v e r and kidney, but not brain and red blood c e l l lead when ethylenediaminetetraacetate was administered at the lev e l of 40 mg/100 g body weight to rats for five consecutive days after consuming drinking water containing 0.1% lead acetate for ten weeks. Hammond and Aronson (1960) and Hoffman and "Segewitz (1975) have questioned the use of ethylenediaminetetraacetate and other chelators in the treatment of lead t o x i c i t y . Hammond (1971) suggests that ethylenediaminetetraacetate may prolong the retention of lead in the body and enhance i t s t o x i c i t y in soft tissues by forming ternary complexes with lead and tissue ligands. Hoffman and Segewitz (1975) suggest that by mobilizing lead from the bone ethylenediaminetetraacetate may cause lead to be red i s t r i b u t e d to soft tissues, which are far more vulnerable than bone to the toxic effects of lead. Since ethylenediaminetetraacetate can produce renal damage and anemia, Catsch and Harmuth-Hoene (1979) suggest that ethylenediaminetetraacetate may act s y n e r g i s t i c a l l y with lead to enhance renal and haematopoietic damage. Bridbord and Blejar (1977) suggest that ethylenediaminetetraacetate may enhance damage to the kidneys by removing lead from inclusion bodies, whose role may be to protect the kidney against the toxic effects of lead. Hammond and Aronson (i960) question ethylenediaminetetraacetate's effectiveness as a chelator, due to i t s i n a b i l i t y to penetrate c e l l membranes. Thus, in spite of 67 ethylenediaminetetraacetate being the agent of choice for the treatment of lead t o x i c i t y , i t may not be as e f f i c a c i o u s as urinary excretion data indicate. C i t r i c Acid C i t r i c acid is a naturally occurring substance in the body. It readily crosses c e l l membranes and i s rapidly metabolized in the body via the Kreb's cycle. In recent years i t has been shown to act as an endogenous chelator. C i t r i c acid complexes of copper and zinc have been shown to be present in human, bovine and goat milk, possibly enhancing the absorption of these minerals in the neonate (Martin et a l . , 1981). In the g a s t r o i n t e s t i n a l t r a c t , c i t r i c acid at physiological concentrations has been shown to complex with iron, enhancing i t s uptake by mucosal c e l l s (Hopping et a l . , 1963; 1966). In vivo, c i t r i c acid has been shown to chelate calcium ions (Peterson and Crisman, 1961; Taitz and Kravath, 1967; Hayes et a l . , 1980), suggesting a possible role for i t in bone mineralization. C i t r i c acid has also been shown to complex with a number of heavy metals; lead (Kety and Letonoff, 1943; Reynolds, 1963; Goyer and Cherian, 1979), mercury (Nolen et a l . , 1972), plutonium (Smith et a l . , 1976; Bulman and G r i f f i n , 1981) and rare earth elements (Graca et a l . , 1962). These findings suggest that c i t r i c acid, may be useful as a chelating agent in the treatment of heavy metal t o x i c i t i e s . The therapeutic chelating potential of c i t r i c acid has been most extensively studied in recent years with respect to 68 plutonium. Research in t h i s area (Smith et a l . , 1976), indicates that c i t r i c acid in the plasma of man and the rat chelates plutonium, with the plutonium c i t r a t e complex being excreted in the urine. Bulman and G r i f f i n (1981) have found that in order for c i t r i c acid to s i g n i f i c a n t l y enhance plutonium excretion in the hamster i t must be intravenously administered at a very rapid rate of infusion, otherwise s u f f i c i e n t plasma concentrations to favor chelation are not maintained, due to i t s rapid metabolism. Nolen et a l . (1972) compared the toxic and teratogenic effects of c i t r i c acid chelates of mercury and cadmium to unchelated mercury and cadmium. They found that c i t r i c acid did not reduce or enhance the incidence of t o x i c i t y or teratogenicity of these heavy metals in rats at the levels administered, but did result in higher tissue concentrations of these metals in the l i v e r and the kidney of c i t r i c acid treated animals. These findings suggest that c i t r i c acid may not be an e f f e c t i v e chelating agent for the treatment of mercury and cadmium t o x i c i t i e s and may in fact be detrimental i f , by increasing l i v e r and kidney concentrations of these metals, i t enhances damage to these organs. A study by Graca et a l . (1962) compared the t o x i c i t y of c i t r i c acid and ethylenediaminetetraacetate chelates of rare earth metals in mice and guinea pigs. They found that c i t r i c acid complexes in a l l cases resulted in higher mortality rates than ethylenediaminetetraacetate complexes. They attributed the difference in mortality rates to the lower s t a b i l i t y constants 69 and hence, greater l i k e l i h o o d of dis s o c i a t i o n of c i t r i c acid complexes. In the f i r s t half of t h i s century, c i t r i c acid was frequently administered to lead exposed workers to reduce elevated blood lead l e v e l s and to prevent the development of lead poisoning (Marchmont-Robinson, 1941). However, i t s effectiveness was not evaluated. In 1943, Kety and Letonoff provided a report evaluating the effectiveness of c i t r i c acid therapy in 15 lead poisoned adults. They found increased fecal and urinary excretion (Letonoff and Kety, 1943), a marked amelioration of toxic symptomology and a s i g n i f i c a n t reduction in elevated blood lead l e v e l s when four to five g of c i t r i c acid were administered per day over a 20 day period (Kety and Letonoff, 1943). Based on these e a r l i e r reports, Hammond and Aronson (1960) administered c i t r i c acid at a molar le v e l equivalent to 110 mg/kg body weight of calcium ethylenediaminetetraacetate to lead burdened c a t t l e . They found no change in the concentration or d i s t r i b u t i o n of lead in the blood, although they did find a s l i g h t , but non-significant, decrease in urinary lead excretion. Goyer and Cherian (1979), inspired by the positive results of Kety and Letonoff (1943), investigated c i t r i c acid's potential as a chelating agent in lead burdened rats. They found i t to be r e l a t i v e l y i n e f f e c t i v e in the removal of body lead in comparison to equimolar amount of ascorbic acid and ethylenediaminetetraacetate. The difference in effectiveness between c i t r i c acid and ethylenediaminetetraacetate, in the Goyer and Cherian (1979) 70 study, may be explained by a higher s t a b i l i t y constant for lead ethylenediaminetetraacetate complexes (log Ka=17.6) in comparison to lead c i t r a t e complexes (log Ka=6.5) (Reynolds, 1963). The s t a b i l i t y constant for lead ascorbate complexes has not been determined, but on the basis of i t s observed therapeutic e f f i c a c y as reported by Goyer and Cherian (1979), i t l i k e l y l i e s somewhere in between lead c i t r a t e and lead ethylenediaminetetraacetate complexes. Garber and Wei (1974) and Jugo et a l . (1975) studied the effect of c i t r i c acid on the g a s t r o i n t e s t i n a l absorption of lead in the rat and the mouse. Both research groups found that c i t r i c acid s i g n i f i c a n t l y increased i n t e s t i n a l absorption of lead in these species as a result of chelation. Peterson and Crisman (1961) and Hayes et a l . (1980) have studied c i t r i c acid's potential t o x i c i t y , when administered at levels s u f f i c i e n t to promote a s i g n i f i c a n t degree of chelation. They have found that c i t r i c acid administered o r a l l y i s non-toxic, while c i t r i c acid administered intravenously results in hypocalcemia, tetany, cardiac arrythmias, depression of cardiac function and death. They found that these toxic effects could be prevented by administering calcium c i t r a t e complexes, indicating that the l i k e l y cause of the t o x i c i t y was the removal of free calcium ions from the plasma. In reviewing the c r i t e r i a of an ideal chelating agent, c i t r i c acid f a l l s far short of the mark. It i s non-specific, rapidly metabolized, dissociates readily from metal ions in vivo and i s toxic, when administered intravenously, i f not 71 administered in conjunction with calcium. With respect to lead, i t appears to enhance lead uptake from the gut, yet the evidence is c o n f l i c t i n g as to whether or not i t enhances lead excretion. It i s i n e f f i c i e n t in competing with endogenous ligands for lead, as evidenced by higher s t a b i l i t y constants for such endogenous ligands' as cysteine, orthophosphates and pyrophosphates (log Ka=l2.5, 12.3, and 11.2, respectively) (Reynolds, 1963). The only seeming advantage for c i t r i c acid in comparison to chelators such as ethylenediaminetetraacetate, is that i t has the a b i l i t y to penetrate c e l l membranes and hence, potential lead storage s i t e s . Ascorbic Acid Ascorbic acid is a lactone of a sugar acid. It i s a required vitamin for man, monkey, guinea pig, f r u i t bat, and certain fishes. Most other animals and plants can synthesize i t from glucose precursors. It i s found in high concentrations in the tissues of most plants and animals. It i s known to act as a cofactor in hydroxylation reactions, such as the hydroxylation of proline to hydroxyproline. However, i t i s not s p e c i f i c for these reactions because i t can be replaced by other antioxidants. Many of i t s physiological functions are as yet undetermined. There has been l i t t l e experimental work on the effect of ascorbic acid on lead t o x i c i t y or teratogenicity. In the period 1939-1945, five studies, (four c l i n i c a l (Holmes et a l . , 1939; Dannenberg et a l . , 1940; Marchmont-Robinson, 1941; Evans et a l . , 72 1943) and one experimental (Pillemer et a l . , 1940)) were reported in the l i t e r a t u r e . This was followed by a 30 year period in which no studies of ascorbic acid-lead interactions were reported. In 1975, interest was renewed in t h i s area with the publication of a review paper by Spivey Fox, which provided some tentative evidence and a t h e o r e t i c a l perspective for a potential protective role for ascorbic acid in heavy metal t o x i c i t i e s . Since then, eight studies (Mahaffey and Bank, 1975; Pal et a l . , 1975; Sohler et a l . 1977; McNiff et a l . , 1978; Papaioannou et a l . , 1978; Goyer and Cherian, 1979; Suzuki and Yoshida, 1979a; Altmann et a l . , 1981) on the effect of ascorbic acid on lead t o x i c i t y and one study (King and Lui, 1975) on the ef f e c t of ascorbic acid on lead teratogenicity have appeared in the l i t e r a t u r e . These studies have f a i l e d to provide a d e f i n i t i v e answer to the question of ascorbic acid's e f f i c a c y and mechanism of action in the prevention and treatment of lead t o x i c i t y and teratogenicity, although some studies have produced stimulating findings which may serve as an impetus for further research. In 1939, Holmes et a l . reported on a c l i n i c a l t r i a l , without the use of a control group, in which 100 mg of ascorbic acid was administered d a i l y to 17 i n d u s t r i a l workers with signs and symptoms of chronic lead absorption. During the course of the study, they noted a change from abnormal to normal blood morphologies, a disappearance of hand tremors in workers previously exhibiting them, and a marked improvement in vigor, cheerfulness, skin p a l l o r , appetite and sleep patterns of these 73 workers. In a subsequent c l i n i c a l t r i a l , they administered 200 mg of ascorbic acid for three weeks to three painters exhibiting c l i n i c a l signs and symptoms indi c a t i v e of chronic lead exposure. They found that prior to ascorbic acid administration urinary ascorbic acid excretion was lower than in normals. After ascorbic acid administration, urinary ascorbic acid excretion was found to increase, while urinary lead excretion was found to decrease. Although, these findings may indicate increased body retention of lead following ascorbic acid administration, these researchers suggested that the reduced urinary lead excretion r e f l e c t e d increased fecal lead excretion, promoted by ascorbic acid. Marchmont-Robinson (1941) studied 303 automobile assembly employees administered 50 mg of ascorbic acid d a i l y in chewing gum, who were chron i c a l l y exposed to lead dust as a result of grinding solder jo i n t s flush with surrounding metal. He noted an increase in work e f f i c i e n c y , a more cheerful attitude and normal red blood c e l l morphologies in workers while receiving t h i s treatment. Contrary to the work of Holmes et a l . (1939), he found that urinary lead excretion was not decreased by dai l y administration of ascorbic acid in workers continually exposed to lead. On the basis of the work of Holmes et a l . (1939), Dannenberg et a l . (1940) administered 100 mg of ascorbic acid by mouth and 250 mg, intravenously on a d a i l y basis for 17 days to a 27 month old infant with a history of pica, diagnosed as suffering from lead encephalopathy. With th i s treatment, no sign 74 of improvement in the infant's condition was noted. However, s i m i l i a r findings, including a worsening of symptomology, have been reported when ethylenediaminetetraacetate has been administered in serious cases of lead encephalopathy (Chisolm, 1968). Evans et a l . (1943) conducted, one of the two c l i n i c a l studies reported in the l i t e r a t u r e , in which a control group matched to the experimental group for lead exposure, urinary lead excretion and the number of signs and symptoms of lead intoxication was used. They found that workers with the highest lead exposures were more de f i c i e n t in ascorbic acid on the basis of ascorbic acid saturation tests than other workers. However, they did not consider t h i s finding of any importance, as a high percentage of a l l workers tested exhibited some evidence of ascorbic acid deficiency when administered t h i s test. In comparing the group supplemented with 100 mg of ascorbic acid to the control group, they found no difference with respect to blood lead concentrations, urinary or f e c a l lead excretion, hemoglobin concentrations, red blood c e l l morphology and severity or number of health related complaints. They conluded that ascorbic acid was without effect in chronic lead exposure. Papaioannou et a l . (1978) suggests that the difference in findings between the Evans et a l . (1943) study and the Holmes et a l . (1939) and Marchmont-Robinson (1941) studies i s related to the type of lead to which the workers were exposed. In the Evans et a l . (1943) study, workers were exposed to organic lead, while in the Holmes et a l . (1939) and Marchmont-Robinson (1941) 75 studies, workers were exposed to inorganic lead. This suggests that ascorbic acid may be e f f i c a c i o u s in inorganic, but not organic lead exposure. Evaluating the other c l i n i c a l studies (Sohler et a l . , 1977; Papaioannou et a l . , 1978; Altmann et a l . , 1981) investigating the effectiveness of ascorbic acid in lead t o x i c i t y i s complicated by the fact that either zinc or calcium, which are known to afford protection against lead t o x i c i t y , were administered simultaneously with ascorbic acid. Sohler et a l . (1977) administered two grams of ascorbic acid and 60 mg of zinc d a i l y to 47 psychiatric patients with blood lead l e v e l s of 25 ug/100 ml or greater. Blood lead l e v e l s measured on the second outpatient v i s i t , after i n i t i a t i o n of the treatment, were found to be s i g n i f i c a n t l y reduced from pretreatment l e v e l s . Papaioannou et a l . (1978) administered the same treatment to 22 lead battery plant workers with mean blood lead levels of 61.1±14.9 ug/100 ml prior to treatment. After 24 weeks of treatment, the mean blood lead l e v e l s for these same individuals decreased to 46.0±14.9 ug/100 ml, in spite of the fact that the workers remained on the job, where they were continually exposed to lead. In the most recently reported c l i n i c a l .study, using a control group matched for lead exposure, Altmann et a l . (1981) found a 65% reduction in the urinary excretion of delta-aminolevulinic acid, a 90% reduction in the lead concentration of the placenta and a 15% reduction in the lead concentration in maternal milk in comparison to controls, in 40 lead burdened mothers treated with calcium and ascorbic acid. 76 In the past 40 years, two papers (Pillemer et a l . , 1940; Mahaffey and Bank, 1975) have appeared in the l i t e r a t u r e reporting on the effects of administering ascorbic acid at l e v e l s several times greater than the requirement to lead poisoned guinea pigs. Both studies found that guinea pigs receiving high intakes of ascorbic acid consumed more food and had greater weight gains than guinea pigs receiving lower intakes of ascorbic acid, whether or not they were being administered lead. Both studies found ascorbic acid to be i n e f f e c t i v e in protecting against hematological changes and anemia induced by lead. In looking at tissue d i s t r i b u t i o n patterns of lead, Mahaffey and Bank (1975) found no e f f e c t s attributable to ascorbic acid administration, while Pillemer et a l . (1940) found increased lead concentrations in the l i v e r , with no change in lead concentrations in the kidney, bone and brain, when ascorbic acid was administered to lead poisoned guinea pigs at 20 times the requirement. Only the Pillemer et a l . (1940) study, examined the e f f e c t of high l e v e l s of ascorbic acid administered simultaneously with lead on the incidence of lead induced p a r a l y s i s , convulsions and death. They found no deaths and two cases of paralysis and convulsions in 26 guinea pigs receiving high levels of ascorbic acid, in comparison to 12 deaths and 18 cases of paralysis and convulsions in 44 guinea pigs receiving ascorbic acid at the requirement l e v e l . Although, high levels of ascorbic acid were e f f e c t i v e in preventing neuroplumbism, they found ascorbic acid to be no more e f f e c t i v e than removing lead exposure in bringing about recovery in 77 established cases of neuroplumbism. Four studies (Pal et a l . , 1975; McNiff et a l . , 1978; Goyer and Cherian, 1979; Suzuki and Yoshida, 1979a) have been reported in the l i t e r a t u r e on the ef f e c t of ascorbic acid in lead intoxication in the rat. In three studies (Pal et a l . , 1975; McNiff et a l . , 1978; Suzuki and Yoshida, 1979a), ascorbic acid was found to have no ef f e c t on the development of kidney c e l l necrosis or kidney hypertrophy induced by lead. Goyer and Cherian (1979) however, reported that ascorbic acid was e f f e c t i v e in removing lead induced inclusion bodies from the nuclei of renal tubular c e l l s . Two studies (McNiff et a l . , 1978; Goyer and Cherian, 1979) found ascorbic acid to be e f f e c t i v e in increasing urinary lead excretion in comparison to controls, while the other studies (Pal et a l . , 1975; Suzuki and Yoshida, 1979a) f a i l e d to investigate t h i s parameter. Of the two studies examining the effect of ascorbic acid on lead induced anemia, one study (Suzuki and Yoshida, 1979a), found ascorbic acid to be without e f f e c t , while the other (Pal et a l . , 1975) noted a s l i g h t improvement in the anemia, in spite of the f a i l u r e of ascorbic acid to restore hemoglobin concentrations to normal. The one study (Suzuki and Yoshida, 1979a) investigating ascorbic acid's a b i l i t y to provide protection against lead induced growth retardation found i t to be i n e f f e c t i v e . Of the two studies investigating tissue d i s t r i b u t i o n s of lead, the Suzuki and Yoshida (1979a) study found no difference in l i v e r and kidney lead concentrations between ascorbic acid and control rats, although they did f i n d a s i g n i f i c a n t increase in iron retention 78 in the l i v e r s of ascorbic acid treated rats. The Goyer and Cherian (1979) study found increased blood lead concentrations, decreased delta-aminolevulinic acid excretion, and decreased concentrations of lead in the l i v e r , kidney and brain of ascorbic acid treated rats in comparison to controls. However, none of these differences reached s t a t i s t i c a l s i gnificance. Thus, at the levels of lead and ascorbic acid administered in these studies, the only noteworthy effect of ascorbic acid appears to be enhanced urinary lead excretion. Although, lead has been shown to be embryotoxic and teratogenic in a number of species (Bell and Thomas, 1980), only one study (King and Lui, 1975) has appeared in the l i t e r a t u r e , regarding ascorbic acid's a b i l i t y to protect against such e f f e c t s . In th i s study, 0.075 mg of lead acetate, with or without one mg of ascorbic acid was injected into the yolk sac of chick embryos at 96 hours of incubation. Ascorbic acid was found to reduce the incidence of non-fusion of the p a r i e t a l bones from 81% to 41%, to greatly reduce the incidence of microopthalmia and crooked d i g i t s and to t o t a l l y protect against the occurrence of twisted necks and shortened lower beaks. With respect to lead induced growth retardation, ascorbic acid was found to be e f f e c t i v e in preventing depressions in crown rump lengths in normal as well as abnormal embryos, while protecting against depressions in body weight in normal, but not abnormal, embryos. The only adverse effect noted, was a s l i g h t increase in umbilical hernias and hematomas in ascorbic acid treated embryos. 79 Ascorbic Acid's Mechanism of Action Several hypotheses have been put forth regarding the mechanism by which ascorbic acid may afford protection from the detrimental e f f e c t s of lead in b i o l o g i c a l systems. These hypotheses can be divided into four categories: i n t e s t i n a l absorption, increased ascorbic acid requirement, chelation, and sulfhydryl group protection. The i n t e s t i n a l absorption hypothesis was put forth to explain the results of studies such as Pillemer et a l . (1940) and Suzuki and Yoshida (1979b), in which ascorbic acid was found to be i n e f f e c t i v e in the treatment of established lead intoxication, but to be e f f e c t i v e in i t s prevention. This hypothesis proposes that ascorbic acid interacts at the le v e l of the g a s t r o i n t e s t i n a l tract d i r e c t l y with lead to decrease i t s absorption or with other elements known to be lead antagonists to increase or decrease their absorption. Papaioannou et a l . (1978) suggest that ascorbic acid may decrease the severity of lead induced t o x i c i t y by decreasing dietary copper absorption, which, when elevated, tends to exaggerate lead t o x i c i t y . Spivey Fox (1975) suggests that ascorbic acid, by enhancing zinc and iron absorption, may reduce lead absorption by competition with these metals for i n t e s t i n a l proteins involved in absorption. A l t e r n a t i v e l y , ascorbic acid, by enhancing absorption of zinc and iron, may increase the concentration of these metals in tissues such as l i v e r and bone, where they may act to reverse or ameliorate the effect of lead on enzymes dependent on these metals for a c t i v i t y . In support of t h i s hypothesis, Suzuki and 80 Yoshida (1979b) found that ascorbic acid administered to lead intoxicated rats increased the concentration of iron in the l i v e r in comparison to rats administered lead alone. Barltrop and Khoo (1975) and Conrad and Barton (1978) have put some of the tenets of the i n t e s t i n a l absorption hypothesis to experimental test. Barltrop and Khoo (1975) researched the eff e c t s of low and high ascorbic acid and B vitamin diets on i n t e s t i n a l absorption of lead in rats. They found that the amount of body lead retained on the low and high vitamin diets was no d i f f e r e n t than the amount retained on a control diet which supplied the animals requirements for these nutrients. Conrad and Barton (1978) i n t e s t i n a l l y administered radioactive lead alone, with ascorbic acid, iron chloride or both nutrients simultaneously to rats. The dose of lead retained in the rat carcass four hours after administration was 9.48±1.32%, after administration with ascorbic acid 16.47±2.47%, after administration with iron chloride or iron chloride plus ascorbic acid 2.98±1.19%. These findings serve to weaken the i n t e s t i n a l absorption hypothesis, as they indicate that ascorbic acid enhances lead absorption and that any decrease in lead absorption related to iron is independent of ascorbic acid. The increased ascorbic acid requirement hypothesis was f i r s t proposed by Holmes et a l . (1939), when they noted that spongy gums, pyorrhea and poor dentition observed in lead poisoned i n d u s t r i a l workers were s i m i l i a r to the signs of s u b c l i n i c a l scurvy. Evans et a l . (1943) subsequently noted, on the basis of ascorbic acid saturation tests, that lead exposed 81 workers were more d e f i c i e n t in ascorbic acid than non-exposed workers. However, the most convincing evidence for t h i s hypothesis was presented by Pal et a l . (1975). These researchers administered d i s t i l l e d water or lead acetate with or without ascorbic acid to rats. They found that ascorbic acid excretion was elevated in ascorbic acid supplemented and lead treated rats in comparison to controls and that lead induced kidney damage was present in both these groups of rats. Using rat l i v e r homogenates, they found that the l i v e r enzyme 1-gulonolactone oxidase, involved in the biosynthesis of ascorbic acid was greatly stimulated, in comparison, to controls in lead treated rats, but not in ascorbic acid supplemented rats. When lead ions were added in v i t r o to l i v e r homogenates, no increase in 1-gulonolactone oxidase a c t i v i t y or ascorbic acid synthesis was observed, indicating that lead was not d i r e c t l y involved in the stimulation of this enzyme or ascorbic acid synthesis in general. When the a c t i v i t y of the enzyme dehydroascorbase was measured in lead treated and ascorbic acid supplemented rats, no increase in a c t i v i t y was observed indicating that the stimulation of ascorbic acid synthesis in vivo, was not related to an increased rate of ascorbic acid breakdown. These findings led to the conclusion that an increased requirement for ascorbic acid, r e f l e c t e d by increased a c t i v i t y of the enzymes involved in ascorbic a c i d synthesis, exists in lead intoxicated rats, with the most probable cause being increased urinary losses of ascorbic acid due to renal damage. Another means by which ascorbic acid requirements may be 82 increased in lead intoxication i s by direct interference by lead, in a p a r t i c u l a r biochemical function dependent on ascorbic acid. Such a mechanism may account for ascorbic acid's a b i l i t y to protect against the development of lead encephalopathy in the chick embryo. In lead encephalopathy, the increased permeabilty of brain c a p i l l a r i e s may be the result of impaired proline hydroxylation of collagen molecules. V i s t i c a et a l . (1977) have demonstrated in 3T6 f i b r o b l a s t c e l l cultures, grown in the presence of lead concentrations s i m i l i a r to those found in the brains of animals with lead encephalopathy, that lead decreases proline hydroxylation. In a s i m i l i a r study, Petrofsky (1972) has demonstrated that ascorbic acid i s d i r e c t l y involved in the hydroxylation of proline in collagen molecules. These findings suggest that ascorbic acid's a b i l i t y to prevent lead encephalopathy in the chick embryo may be related to providing a favorable concentration of ascorbic acid in the brain, such that lead's interference in ascorbic acid's hydroxylation of proline in collagen molecules i s minimized. Lewin (1974) has proposed a structure for ascorbic acid metal chelates (Figure 4), which he hypothesizes may form in b i o l o g i c a l systems, when megadoses of ascorbic acid are administered. This chelation hypothesis has only recently been investigated in lead intoxicated rats. McNiff et a l . (1978) reported that ascorbic acid increased urinary lead excretion in lead burdened rats, as did a number of other chelators tested. Goyer and Cherian (1979) reported that lead burdened rats administered 350 mg of ascorbic acid or 40 mg/100 g body weight 83 F i g u r e 4. L e w i n ' s p r o p o s e d s t r u c t u r e f o r a s c o r b i c a c i d m e t a l c h e l a t e s 84 of ethylenediaminetetraacetate per day, which provided a s i m i l i a r number of carboxyl groups for metal chelation, excreted s i m i l i a r amounts of lead over a seven day treatment period. In further support of the chelation hypothesis, Goyer and Cherian (1979) observed elevated blood lead l e v e l s in ascorbic acid and ethylenediaminetetraacetate treated rats in comparison to lead burdened controls. Such elevated blood lead l e v e l s are commonly observed when chelators mobilize lead from body stores (Chisolm, 1968). From body lead d i s t r i b u t i o n data, they found trends suggesting that ascorbic acid was more e f f e c t i v e in removing brain lead, while ethylenediaminetetraacetate was more e f f e c t i v e in removing bone lead. They concluded that ascorbic acid did indeed act as a chelator and that i t was as e f f e c t i v e as ethylenediaminetetraacetate, provided a s i m i l i a r number of carboxyl groups were administered. The sulfhydryl group protection hypothesis proposes that ascorbic acid forms complexes with t h i o l groups in proteins and at the active s i t e s of enzymes, thereby i n h i b i t i n g metal binding and subsequent interference in protein and enzyme functions. Evans et a l . (1970), investigating this hypothesis, found that ascorbic acid inhibited the binding of cadmium, zinc, and copper to bovine i n t e s t i n a l metallothionein and decreased i n t e s t i n a l absorption of these metals. From u l t r a v i o l e t absorption spectrum data, they provide evidence that ascorbic acid i n h i b i t s metal binding by t h i s protein, by forming complexes with the t h i o l groups i t contains. As yet, there has been no evidence provided that t h i s may be a mechanism, by which ascorbic acid affords 85 protection against the detrimental effects of lead in b i o l o g i c a l systems. D-Isoascorbic Acid Isoascorbic acid, more commonly known as erythorbic acid, i s used in the food industry as an antioxidant and an i n h i b i t o r of nitrosamine formation (Borenstein, 1965). It i s an isomer of ascorbic acid, that d i f f e r s from the l a t t e r in the s t e r i c location of the hydroxyl group on the f i f t h carbon atom (Figure 5) and in chemical properties such as melting point, s o l u b i l i t y and redox potential (Borenstein, 1965). In guinea pigs and man i t has been shown to be a mild antiscorbutigenic agent, possessing fiv e percent of the a c t i v i t y of ascorbic acid (Goldman et a l . , 1981) . Hughes and Jones (1970) and Hughes et a l . (1971) have demonstrated that isoascorbic acid i s poorly absorbed and poorly retained in guinea pig tissues, but can be as e f f e c t i v e as ascorbic acid in preventing scurvy and growth retardation, provided tissue concentrations of isoascorbic acid are the same as in ascorbic acid s u f f i c i e n c y states. P e l l e t i e r and Godin (1969) concluded, on the basis of the finding of no difference between the ascorbic acid content of guinea pigs fed a scorbutigenic diet with or without isoascorbic acid supplementation, that isoascorbic acid does not prevent scurvy by sparing ascorbic acid. Goldman et a l . (1981) found that the effects of an isoascorbic acid supplement administered in conjunction with a suboptimal supplement of ascorbic acid to oc oc HO C H O — C HO -C HO C H CO H — C O H - C OH HO C CH2OH CH2OH d - i s o a s c o r b i c a c i d 1 - a s c o r b i c a c i d F i g u r e 5 . L - a s c o r b i c a c i d a n d i t s i s o m e r d - i s o a s c o r b i c a c i d 87 guinea pigs fed a scorbutigenic diet was additive, suggesting that isoascorbic acid was not a competitive i n h i b i t o r of ascorbic a c i d . Thus, the difference between the two isomers, when administered at s i m i l i a r levels to ascorbic acid d e f i c i e n t guinea pigs, i s probably not related to differences in antiscorbutigenic properties, but to the poor absorption and tissue retention of isoascorbic acid. Human studies (Wang et a l . , 1962; Rivers et a l . , 1963) of isoascorbic acid metabolism have found that 50 to 70% of an o r a l l y administered test load is excreted in the urine within 24 hours in ascorbic acid depleted subjects in comparison to five percent of a test load of ascorbic acid. Wang et a l . (1962) demonstrated that isoascorbic acid is f i l t e r e d through the glomeruli and not absorbed to any extent by the tubular c e l l s in the kidney, as i s ascorbic acid. Rivers et a l . (1963) observed decreased combined concentrations of ascorbic acid and isoascorbic acid in leucocytes of ascorbic acid depleted subjects, when supplemented with isoascorbic acid, in comparison to when they were supplemented with ascorbic acid. Kadin and Oscada (1959) observed isoascorbic acid supplementation to have no effect on ascorbic acid excretion. These findings, suggest that isoascorbic acid does not displace ascorbic acid in human tissues, has d i f f i c u l t y in crossing c e l l membranes and i s poorly retained in the body. In proline hydroxylase enzyme preparations from guinea pigs, isoascorbic acid has been found to be as e f f e c t i v e as ascorbic acid in hydroxyalting proline (Kutnink et a l . , 1969). 88 However, in collagen synthesizing c e l l cultures isoascorbic acid has been found to be less e f f e c t i v e than ascorbic acid in proline hydroxylation (Priest and Bublitz, 1967). These findings, indicate that isoascorbic acid can completely substitute for ascorbic acid in promoting proline hydroxylation. However, in l i v i n g c e l l s the s t e r e o s p e c i f i c i t y of active transport systems or oxidation reduction cycles for ascorbic acid prevents i t from being equally as potent in f u l f i l l i n g these functions in l i v i n g systems. Isoascorbic acid has been investigated as a protective agent in cadmium t o x i c i t y . Spivey Fox et a l . (1977) found i t to be equally as e f f e c t i v e as ascorbic acid in preventing the anemia, growth retardation, poor bone mineralization and perturbations in elemental tissue concentrations, when administered at the same l e v e l as ascorbic acid in the diet of young coturnix. They concluded, since isoascorbic acid was as e f f e c t i v e as ascorbic acid, yet possesses only fiv e percent of the vitamin a c t i v i t y , that both agents must act as pharmacological agents in providing protection against cadmium t o x i c i t y . Isoascorbic acid has not been investigated as an agent to prevent or treat adverse e f f e c t s from lead. However, such an investigation could be useful in testing the increased requirement and chelation hypotheses. Since, isoascorbic acid possesses five percent of the antiscorbutigenic a c t i v i t y of ascorbic acid, i t should be less e f f e c t i v e than ascorbic acid in providing protection against adverse e f f e c t s of lead, i f the 89 increased requirement hypothesis has any v a l i d i t y . However, i f the chelation hypothesis has any v a l i d i t y , isoascorbic acid should be at least as e f f e c t i v e as ascorbic acid, when administered at the same molar l e v e l . 90 CHAPTER III  MATERIALS AND GENERAL METHODOLOGY Materials F e r t i l e white leghorn eggs, from hens aged seven months to 1.5 years of age, (Schaeffer's Star Cross 288 - genetical composition of stock not accessible, but appears to be heterogeneous), weighing between 56 and 67 g, were obtained from the UBC Poultry Farm. They were incubated for 19 days in a Marsh Farm (model PX6) automatic egg turning incubator at a temperature of 37.8 C and a r e l a t i v e humidity of 86%. Due to high mortality rates produced by the needle s t r i k i n g the embryos, when eggs were positioned v e r t i c a l l y , eggs were positioned horizontally for the i n i t i a l 96 hours of incubation to encourage the embryos to develop away from the airspace. This necessitated hand turning, twice per day during t h i s period. Method of Lead Injection At 96 hours of incubation, the eggs were candled and i n f e r t i l e eggs were discarded. According to the method of McLaughlin et a l . (1963), a Dremel Moto Tool handheld d r i l l (model 261), with the number 409 accessory cutting tool in place, was used to cut a small hole in the s h e l l in the centre over the airspace, without damaging the underlying membrane. The underlying membrane was wiped with a s t e r i l e cotton b a l l 91 moistened with 70% ethyl alcohol to remove s h e l l debris. The egg was candled to insure that the embryo was oriented away from the airspace and placed horizontally in an egg holder to maintain i t s p o s i t i o n . A number 22, 2.5 cm long, hypodermic needle was inserted through the airspace and into the yolk sac, permitting a 0.1 ml volume of solution to be injected. The withdrawn needle was examined for any evidence of yellow coloration by wiping i t with a s t e r i l e cotton b a l l moistened with 70% ethyl alcohol. If yellow coloration was observed, i t was assumed that the v i t e l l i n e membrane had been ruptured and the egg was discarded. If no rupture of the v i t e l l i n e membrane had occurred, a small piece of scotch tape was used to cover the exposed membrane and the egg was returned to the incubator in the v e r t i c a l p o s i t ion. The t o t a l time out of the incubator for preparation and treatment of any egg did not exceed 15 minutes. Eggs were candled d a i l y for the duration of the incubation period. Eggs with dead embryos were opened, examined for abnormalities and discarded. On the 19th day of incubation, a l l remaining eggs were opened and the embryos were k i l l e d by exposure to ether. Dead embryos were placed on paper towels for 15 minutes to soak up amniotic f l u i d and then, weighed with the yolk sac attached. Crown rump length was measured to an accuracy of 0.05 cm by placing each embryo against the edge of a ruler and measuring the distance from the midpoint of the crown to the base of the last spinal vertebrae. Embryos were examined for external abnormalities. Subsequently, the brains were removed and weighed. Since, f l u i d was unavoidably lost during excision 92 from brains exhibiting hydrocephalocele, brains were dried and reweighed. Experiment 1 (Selection of lead acetate l e v e l for subsequent  experiments) The aim of the f i r s t experiment was to select a l e v e l of lead acetate to be administered in subsequent experiments. From experiments conducted by King and Lui (1974), Kochen and Greener-(1976) and Hirano and Kochen (1977), a suitable l e v e l appeared to l i e in the range of 50 to 125 ug of lead acetate. 56 eggs were, therefore, injected at approximately 96 hours of incubation, with 0, 50, 75, 100 and 125 ug of lead acetate in 0.1 ml volumes of d i s t i l l e d water. Deaths occurring during the incubation period were recorded, as were body weights, crown rump lengths, wet and dry brain weights, the incidence of hydrocephalocele and other abnormalities in embryos surviving u n t i l day 19. On the basis of the results obtained, the 75 ug le v e l of lead acetate was selected for use in subsequent experiments. Experiment 2 (Comparison of the effectiveness of increasing  lev e l s of ascorbic acid) The aim of the second experiment was to determine i f ascorbic acid afforded protection against any of the dependent variables adversely affected by the 75 ug l e v e l of lead acetate in the f i r s t experiment. From a review of the l i t e r a t u r e 93 (Hammond and Aronson, 1960; Hammond, 1971; Hoffman and Segewitz, 1975; McClain and Siekierka, 1975; Sorensen et a l . , 1980) on the use of chelators in the prevention and treatment of lead t o x i c i t y , the e f f e c t i v e range for such agents appeared to l i e between 15 and 80 times the molar amount of lead acetate administered. Ascorbic acid was, therefore, injected at 0, 20, 40, 60, and 80 times the molar amount of lead acetate injected. A control group was injected with d i s t i l l e d water. In the f i r s t run of the experiment, eggs were injected with 75 ug of lead acetate in 0.05 ml volumes of d i s t i l l e d water, followed one hour l a t e r , by ascorbic acid injected in the same volume of d i s t i l l e d water. Unfortuately, the high incidence of v i t e l l i n e membrane rupture necessitated revisions in the procedure, resulting in ascorbic acid and lead acetate being administered together in 0.1 ml volumes of d i s t i l l e d water. Experiment 3 (Comparison of the effectiveness of ascorbic acid  with isoascorbic acid, c i t r i c acid and EDTA) The t h i r d experiment was designed to provide support for the chelation hypothesis, which proposes that ascorbic acid acts as a chelator or the increased requirement hypothesis, which proposes that lead increases the requirement for ascorbic acid. In t h i s experiment, the effectiveness of ascorbic acid was compared with ethylenediaminetetraacetate and c i t r i c acid (known lead chelators) and isoascorbic acid, an isomer of ascorbic acid possessing f i v e percent of i t s antiscorbutigenic a c t i v i t y . On the basis of the work of Swenerton and Hurley (1971), which 94 indicated that ethylenediaminetetraacetate i s an embryotoxic and teratogenic agent, the lowest l e v e l of ethylenediaminetetraacetate (15 times the molar amount of lead acetate administered) considered to provide a favorable r a t i o of chelator to lead ions was administered. Ascorbic acid was administered at 60 times the molar amount of lead acetate administered, on the basis of the work of Goyer and Cherian (1979), which indicated that ascorbic acid was equally as ef f e c t i v e as ethylenediaminetetraacetate, provided an equal number of carboxyl groups were available for metal chelation. Since, there was some question as to the v a l i d i t y of Goyer and Cherian's (1979) assumption of comparing chelators on the basis of the number of carboxyl groups they possessed, c i t r i c acid was administered at the same l e v e l as ascorbic acid. This comparison on an equal molar basis was considered to be desirable as c i t r i c acid was known to be a weak chelator of lead, sharing s i m i l i a r metabolic features with ascorbic acid, such as the a b i l i t y to cross c e l l membranes and to undergo degradative metabolism. Since, isoascorbic acid exhibits only f i v e percent of the antiscorbutigenic a c t i v i t y of ascorbic acid, but t h e o r e t i c a l l y possesses an equal chelating po t e n t i a l , isoascorbic acid was administered at the same molar l e v e l as ascorbic acid. Experiments 4 (Comparison of lower lev e l s of ascorbic acid with  isoascorbic acid) and 5 (Comparison of lower levels of ascorbic  acid with c i t r i c acid) Experiments 4 and 5 were undertaken as the f a i l u r e in 95 experiment 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA) to detect any differences in the effectiveness of ascorbic acid, c i t r i c acid and isoascorbic acid may have been due to the high levels (60 times the molar amount of lead acetate) at which these agents were administered. Therefore, in the l a s t two experiments, these agents were administered at 15 and 40 times the molar amount of lead acetate administered. In experiment 5 (Comparison of lower levels of ascorbic acid with c i t r i c acid) six embryos (one from the control, two from the lead acetate, one from the c i t r i c acid and two from the ascorbic acid at the 40 times treatment level) were preserved in formaldehyde for subsequent photography. No wet or dry brain weights were recorded for these embryos. S t a t i s t i c a l Analyses S t a t i s t i c a l analyses were performed using the S t a t i s t i c a l Package for the Social Sciences (version nine), prepared by the computing center at UBC. The s t a t i s t i c a l significance of the frequency data on mortality and hydrocephalocele were tested using the Chi-square goodness of f i t test. The means and standard errors for body weight, crown rump length, wet and dry brain weight data were calculated. One-way analysis of variance was used to compare the means for the dependent variables, body weight, crown rump length, wet and dry brain weight. The wet and dry brain weight data from experiment 5 (Comparison of lower levels of ascorbic acid with c i t r i c acid), were excluded from 96 s t a t i s t i c a l analyses as the removal of specimens for photography resulted in non-random samples. Groups of means found to be s t a t i s t i c a l l y s i g n i f i c a n t by one-way analysis of variance were subjected to the Student-Newman-Kuels multiple comparison test to ascertain which means d i f f e r e d s i g n i f i c a n t l y from the others. The regression c o e f f i c i e n t s and regression equations for the dependent variables, body weight, crown rump length, wet and dry brain weight were calculated for the treatment groups receiving ascorbic acid at 0 to 80 times the molar amount of lead acetate, in experiment 2 (Comparison of the effectiveness of increasing l e v e l s of ascorbic a c i d ) . Since sample sizes were small in the individual experiments, the data for mortality, hydrocephalocele, mean body weight and crown rump length were combined for the control groups, the 75 ug l e v e l of lead acetate and the various l e v e l s of ascorbic acid, isoascorbic acid, c i t r i c acid and ethylenediaminetetraacetate, spanning the same series of experiments. The data for wet and dry brain weights were not combined, as s t a t i s t i c a l l y s i g n i f i c a n t differences were detected without further analyses. The s t a t i s t i c a l s i g nificance of the combined mortality and hydrocephalocele data were tested, using the Chi-square goodness of f i t t e s t . The significance of the mean body weight and crown rump length data were tested with the Student's t-test using the separate variance estimates, where the F-test for determining the equality of two variances had found them to be s i g n i f i c a n t l y d i f f e r e n t . In order to determine, i f the presence of hydrocephalocele 97 influenced the mean body weight, crown rump length, wet or dry brain weights of the chick embryos treated with lead acetate alone or in combination with another agent, the data from the various experiments were combined for embryos treated with 75 ug of lead acetate or c i t r i c acid, ascorbic acid or isoascorbic acid at 15 and 40 times the molar amount of lead acetate. The Student's t - t e s t , using the separate variance estimate, where appropriate, was used to test the difference between the mean body weight, crown rump length, wet and dry brain weight data, of hydrocephalocelic and normal embryos within each combined treatment group. The percentage depression in mean body weight, crown rump length, wet and dry brain weight between normal appearing and hydrocephalocelic embryos in the combined lead treatment groups and those in other treatment groups could not be tested, because the data did not span the same series of exper iments. 98 CHAPTER IV  RESULTS Mortality Table II records the number of dead and l i v e embryos in each of the f i v e experiments. Deaths in a l l f i v e experiments occurred between days five and 14 of incubation, with the peak days for deaths being days six through eight. Hemorrhage or hydrocephalocele was observed in 84% of dead embryos treated with lead acetate alone, 60% treated with lead acetate plus another agent and zero percent treated with d i s t i l l e d water. Deaths associated with brain hemorrhage occurred on days five through nine, while those associated with hydrdocephalocele occurred on days 11 through 14. In experiment 1 (Selection of lead acetate level for subsequent experiments), there was no s i g n i f i c a n t difference between the mortality rates observed in control embryos and those administered 50 ug of lead acetate. However, when lead acetate was administered at the 75 ug l e v e l , the mortality rate increased s i g n i f i c a n t l y to 50%. At the 100 and 125 ug l e v e l s , the mortality rates were 63 and 73%, respectively. In experiment 2 (Comparison of the effectiveness of increasing levels of ascorbic acid) to 5 (Comparison of lower levels of ascorbic acid with c i t r i c acid) the mortality rate for embryos treated with 75 ug of lead acetate ranged from 44 to 57%. The o v e r a l l mortality rate from a l l experiments for embryos treated with 75 ug of lead T a b l e I I . The e f f e c t on c h i c k embryo m o r t a l i t y of l e a d a c e t a t e a d m i n i s t e r e d a l o n e or s i m u l t a n e o u s l y w i t h a s c o r b i c a c i d , i s o a s c o r b i c a c i d , c i t r i c a c i d or EDTA ( a t l e v e l s r a n g i n g from 15 to 80 times the molar c o n c e n t r a t i o n of l e a d a c e t a t e ) . Experiment X ( S e l e c t i o n of 1ead a c e t a t e 1evels f o r subsequent experiments) C o n t r o l Lead a c e t a t e (50 ug) Dead 0 1 L i v e 11 10 T o t a l 11 11 'X J=19.80 p<0.001 Lead a c e t a t e Lead a c e t a t e Lead a c e t a t e (75 ug) (100 ug) (125 ug) 6 7 8 6 4 3 12 11 11 Exper i ment 2 (Compar i son of the e f f e c t i veness of i n creas i ng 1 eve 1s of a s c o r b i c ac i d) C o n t r o l Lead a c e t a t e A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (20 X) (40 X) (60 X) (80 X) Dead 1 5 1 0 0 2 L i v e 7 6 10 11 9 8 T o t a l 8 11 11 11 9 10 X*=14.63 p<0.025 Exper i ment 3 (Compar i son of the e f f e c t i veness of a s c o r b i c ac i d w i t h i s o a s c o r b i c ac i d, c i t r i c ac i d and EDTA) Con t r o l Lead a c e t a t e C i t r i c a c i d EDTA I s o a s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (60 X) (15 X) (60 X) (60 X) Dead 1 7 2 2 1 3 L i v e 8 6 12 12 13 11 T o t a l 9 13 14 14 14 14 X !=11.52 p<0.050 T a b l e I I . (co n t i nued) Exper i ment 4 (Compar i son of 1ower 1evels of a s c o r b i c ac i d w i t h i s o a s c o r b i c ac i d) C o n t r o l Lead a c e t a t e I s o a s c o r b i c a c i d A s c o r b i c a c i d I s o a s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (15 X) (15 X) (40 X) (40 X) Dead L i ve T o t a l 1 g 10 8 10 18 2 1 1 13 3 10 13 1 1 1 12 1 1 1 12 X*=9.45 p>0.100 Exper iment 5 (Compar i son of 1ower 1evel s of a s c o r b i c a c i d w i th c i t r i c a c i d ) C o n t r o l Lead a c e t a t e (75 ug) C i t r i c ac i d ( 15 X) A s c o r b i c a c i d (15 X) C i t r i c ac i d (40 X) A s c o r b i c a c i d (40 X) Dead L i ve T o t a l 0 10 10 8 6 14 3 10 13 2 1 1 13 2 10 12 3 1 1 14 X'=12.63 p<0.050 101 acetate was 50% compared to six percent in control embryos (Table I I I ) . In experiments 2 (Comparison of the effectiveness of increasing levels of ascorbic ac i d ) , 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid, and EDTA), and 5 (Comparison of lower l e v e l s of ascorbic acid with c i t r i c a c i d ) , the mortality rates for embryos treated with lead acetate plus another agent were s i g n i f i c a n t l y less than for embryos treated with lead acetate alone. In experiment 4 (Comparison of lower levels of ascorbic acid with isoascorbic acid) a s i m i l i a r trend was present, but did not attain s t a t i s t i c a l s i g n i f i c a n c e . In experiments 2 (Comparison of the effectiveness of increasing l e v e l s of ascorbic acid) to 5 (Comparison of lower lev e l s of ascorbic acid with c i t r i c acid) no s i g n i f i c a n t differences in mortality rates between the various agents or the levels at which they were administered were detected. The combined data supported the observations in the individual experiments that a l l treatment agents s i g n i f i c a n t l y protected against lead induced mortality (Table IV). Hydrocephalocele In embryos surviving u n t i l day 19, the most common abnormality observed was hydrocephalocele. This lesion was observed in 74% of embryos administered 75 ug of lead acetate, but never in embryos administered d i s t i l l e d water (Table I I I ) . In the mildest case, t h i s lesion consisted of a small herniation T a b l e I I I . The e f f e c t on m o r t a l i t y , h y d r o c e p h a l o c e l e , body weight and crown rump l e n g t h of a d m i n i s t e r i n g 75 ug of l e a d a c e t a t e to c h i c k embryos on the f o u r t h day of i n c u b a t i o n . Means and s t a n d a r d e r r o r s a r e g i v e n f o r body weight and crown rump l e n g t h . Lead a c e t a t e M o r t a l i t y H y d r o c e p h a l o c e l e Body weight Crown rump 1ength ug (g) (cm) 6 48 0, 45 38.0110.44 8.80±0.05 75 50* 68 74* 34 33. 5010.85* 8 . 2010. 10* * S i g n i f i c a n t 1 y d i f f e r e n t from 0 ug l e v e l of l e a d a c e t a t e p<0.001 103 Table IV. Mortality as determined from experiments 2 to 5, in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or EDTA (at 15 times or greater the molar concentration of lead acetate). Agent Lead acetate (75 ug) Lead acetate plus agent (15 X or >) Ascorbic acid 50a 68 1 4b 1 04 Isoascorbic acid 48a 31 1 Ob 39 C i t r i c acid 55c 27 I 8 d 39 EDTA 54e 1 3 14f 1 4 Within agents a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.00l) c i s s i g n i f i c a n t l y d i f f e r e n t from d (p<0.0l) e i s s i g n i f i c a n t l y d i f f e r e n t from f_ (p<0.05) 1 04 of brain substance and f l u i d through a hole the size of a pencil point at the junction of the p a r i e t a l and o c c i p i t a l bones. In moderate to severe cases, f l u i d f i l l e d cysts of varying sizes were observed in the o c c i p i t a l or p a r i e t a l regions of the s k u l l , accompanied by varying degrees of displacement of the c r a n i a l bones (Figure 6). Of the 62 cases of hydrocephalocele observed in the five experiments conducted, the lesions were located in the o c c i p i t a l region of the s k u l l in 61% of the cases, in the p a r i e t a l region of the s k u l l in 26% of the cases and in both regions of the s k u l l in 13% of the cases. The location of the lesion was not related to whether lead acetate was administered alone or in conjunction with another agent. Table V records, for each of the f i v e experiments, the incidence of hydrocephalocele in chick embryos surviving u n t i l day 19. In experiment 1 (Selection of lead acetate l e v e l for subsequent experiments), there was a s i g n i f i c a n t difference in the incidence of hydrocephalocele between embryos administered lead acetate and those administered d i s t i l l e d water. Since sample sizes were small, i t was unclear whether or not the l e v e l of lead acetate administered affected the incidence of hydrocephalocele (Table V). However, i t did appear to a f f e c t the severity of the l e s i o n , with the mildest lesions being observed in embryos administered 50 ug of lead acetate and the severest lesions in embryos administered 100 and 125 ug of lead acetate. For experiments 2 (Comparison of the effectiveness of increasing l e v e l s of ascorbic acid) to 5 (Comparison of lower F i g u r e 6. E x t e r n a l a b n o r m a l i t i e s observed i n c h i c k embryos t r e a t e d with l e a d a c e t a t e alone or i n combination with a s c o r b i c a c i d or c i t r i c a c i d From l e f t to r i g h t : No observable a b n o r m a l i t i e s i n embryo t r e a t e d with a s c o r b i c a c i d at 40 times the molar c o n c e n t r a t i o n of lead acetate No observable a b n o r m a l i t i e s i n embryo t r e a t e d with d i s t i l l e d water Hydrocephalocele i n the o c c i p i t a l r e g i o n and c u r l e d d i g i t s i n embryo t r e a t e d with c i t r i c a c i d at 40 times the molar c o n c e n t r a t i o n of lead acetate Hydroaphalocele i n p a r i e t a l r e g i o n and c u r l e d d i g i t s i n embryo t r e a t e d with 75 ug of lead a c e t a t e T a b l e V. The e f f e c t on c h i c k embryo h y d r o c e p h a l o c e l e of l e a d a c e t a t e a d m i n i s t e r e d a l o n e o r s i m u l t a n e o u s l y w i t h a s c o r b i c a c i d , i s o a s c o r b i c a c i d , c i t r i c a c i d or EDTA ( a t l e v e l s r a n g i n g from 15 to 80 times the molar c o n c e n t r a t i o n of l e a d a c e t a t e ) . Experiment _1_ ( S e l e c t i o n of 1 ead a c e t a t e 1 evel s f o r subsequent experiments) C o n t r o l H y d r o c e p h a l o c e l e 0 Normal 11 T o t a l 11 Lead a c e t a t e (50 ug) 6 4 10 Lead a c e t a t e (75 ug) 6 0 6 Lead a c e t a t e (100 ug) 2 2 4 Lead a c e t a t e (125 ug) 3 0 3 X*=20.40 p<0.0004 Exper i ment 2 (Compar i son of the e f f e c t i veness of i n creas i ng 1evels of a s c o r b i c ac i d) C o n t r o l Lead a c e t a t e A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (20 X) (40 X) (60 X) (80 X) H y d r o c e p h a l o c e l e 0 Normal 7 T o t a l 7 1 9 10 0 1 1 1 1 0 9 9 0 8 8 X !=25.74 p<0.0001 Exper iment 3 (Compar i son of the e f f e c t iveness of a s c o r b i c a c i d wi t h i s o a s c o r b i c a c i d , c i t r i c a c i d and EDTA) C o n t r o l H y d r o c e p h a l o c e l e 0 Normal 8 T o t a l 8 Lead a c e t a t e (75 ug) 3 3 6 C i t r i c a c i d (60 X) 1 1 1 12 EDTA I s o a s c o r b i c a c i d (15 X) (60 X) 0 0 12 13 12 13 A s c o r b i c a c i d (60 X) 0 11 11 X !=21 .96 p<0.0005 T a b l e V. ( c o n t i n u e d ) Experiment 4 (Comparison of 1ower 1evels of a s c o r b i c a c i d wi th i s o a s c o r b i c a c i d ) C o n t r o l Lead a c e t a t e (75 ug) I s o a s c o r b i c a c i d (15 X) A s c o r b i c a c i d ( 15 X) I s o a s c o r b i c a c i d (40 X) A s c o r b i c a c i d (40 X) H y d r o c e p h a l o c e l e 0 Normal 9 T o t a l 9 7 3 10 3 8 1 1 4 6 10 1 10 1 1 1 10 1 1 X !=17.61 p>0.0035 Exper i ment 5 (Comparison of 1ower 1evels of a s c o r b i c a c i d wi th c i t r i c a c i d ) C o n t r o l Lead a c e t a t e C i t r i c a c i d A s c o r b i c a c i d C i t r i c a c i d A s c o r b i c a c i d (75 ug) (15 X) (15 X) (40 X) (40 X) H y d r o c e p h a l o c e l e 0 5 7 3 4 2 Normal 10 1 3 8 6 9 T o t a l 10 G 10 11 10 11 X !=18.37 p>0.0025 108 l e v e l s of ascorbic acid with c i t r i c a cid), the incidence of hydrocephalocele in embryos administered ascorbic acid at 60 and 80 times the molar amount of lead acetate was zero percent, at 40 times nine percent, at 20 times ten percent and at 15 times 33%. The incidence of hydrocephalocele in embryos administered isoascorbic acid in experiments 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA) and 4 (Comparison of lower levels of ascorbic acid with isoascorbic acid) at 60 times the molar amount of lead acetate was zero percent, at 40 times nine percent, at 15 times 27%. The incidence of hydrocephalocele in embryos administered c i t r i c acid in experiments 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA) and 5 (Comparison of lower levels of ascorbic acid with c i t r i c acid) at 60 times the molar amount of lead acetate was eight percent, at 40 times 40% and at 15 times 70%. The incidence of hydrocephalocele, when ethylenediaminetetraacetate in experiment 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA) was administered at 15 times the molar amount of lead acetate, was zero percent. These incidence rates, other than for the two lowest lev e l s of c i t r i c acid, were s i g n i f i c a n t l y lower than those observed in embryos administered lead acetate alone. The combined data indicated that a l l treatment agents, other than c i t r i c acid, afforded s i g n i f i c a n t protection against lead induced hydrocephalocele (Table VI). 1 0 9 Table VI. The frequency of hydrocephalocele as determined from experiments 2 to 5, in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or EDTA (at 15 times or greater the molar concentration of lead acetate). Agent Lead acetate Lead acetate plus agent (75 ug) (15 X or >) % n % n Ascorbic acid 68a 28 12b 92 Isoascorbic acid 63a 16 11b 35 C i t r i c acid 67 12 38 32 EDTA 50c 6 Od 12 Within agents a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.00l) c i s s i g n i f i c a n t l y d i f f e r e n t from d (p<0.0!) 110 External Abnormalities The most frequently observed external abnormalities other than hydrocephalocele, were shortened lower beaks, twisted necks and curled d i g i t s (Figure 6). These abnormalities occurred at frequencies of 15% or less in embryos treated with lead acetate alone or with another agent, but never in embryos serving as controls. In most embryos in which these abnormalities occurred, hydrocephalocele was also present. Other abnormalities that occurred once in the course of the f i v e experiments were a hematoma at the base of the s k u l l in a control embryo, anopthalmos in an embryo treated with ascorbic acid at 15 times the molar amount of lead acetate, an absent upper beak in an embryo treated with ascorbic acid at 40 times the molar amount of lead acetate and abnormal feathering, ectopia cordis, exophthalmos and ablephary in an embryo administered ascorbic acid at 40 times the molar amount of lead acetate. Body Weights Table VII records the mean body weights and standard errors for chick embryos surviving u n t i l day 19, which were subjected to the various treatments. In experiments 1 (Selection of lead acetate l e v e l for subsequent experiments) to 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA), there were no s i g n i f i c a n t differences in mean body weights among any of the treatment groups. In experiments 4 (Comparison of lower leve l s of ascorbic acid with isoascorbic T a b l e VII The e f f e c t on c h i c k embryo body weight of l e a d a c e t a t e a d m i n i s t e r e d a l o n e or s i m u l t a n e o u s l y w i t h w i t h a s c o r b i c a c i d , i s o a s c o r b i c a c i d , c i t r i c a c i d or EDTA (at l e v e l s r a n g i n g from 15 to 80 times the molar c o n c e n t r a t i o n of l e a d a c e t a t e ) . Means and s t a n d a r d e r r o r s g i v e n i n grams. Exper i ment J_ ( S e l e c t i on of 1ead a c e t a t e 1evel f o r subsequent exper i ments) C o n t r o l Lead a c e t a t e (50 ug) Lead a c e t a t e (75 ug) Lead a c e t a t e ( 100 ug) Lead a c e t a t e (125 ug) 38.6510.84 38.4310.74 33.4913.02 36.5512.23 30.7017.38 Experiment 2 (Comparison of the e f f e c t iveness of i ncreas i ng 1evels of a s c o r b i c a c i d ) C o n t r o l 39.70±0.78 Lead a c e t a t e (75 ug) 37.29+1.27 A s c o r b i c a c i d (20 X) 37.7511.61 A s c o r b i c a c i d (40 X) 36.7112. 12 A s c o r b i c a c i d (60 X) 38.97+0.48 A s c o r b i c a c i d (80 X) 38.7910.94 Exper i ment 3 (Compar i son of the e f f e c t i veness of a s c o r b i c ac i d w i t h i s o a s c o r b i c ac i d, c i t r i c ac i d and EDTA) C o n t r o l 36.2110.71 Lead a c e t a t e (75 ug) 33.9611.17 C i t r i c ac i d (60 X) 34.0910.81 EDTA ( 15 X) 35.5710.59 I s o a s c o r b i c a c i d (60 X) 33.9910.87 A s c o r b i c a c i d (60 X) 34.6610.70 Exper i ment 4 (Compar i son of 1ower 1 eve 1s of a s c o r b i c ac i d with i s o a s c o r b i c ac i d) C o n t r o l 39.2410.94b Lead a c e t a t e (75 ug) 32 . 7611 . 37a I s o a s c o r b i c a c i d (15 X) 38.2510.75b A s c o r b i c a c i d (15 X) 34.8411.71 I s o a s c o r b i c a c i d (40 X) 36.1611.19 A s c o r b i c a c i d (40.X)) 36.7911.46 Experiment 5 (Comparison of 1ower 1evels of a s c o r b i c a c i d with c i t r i c a c i d ) C o n t r o l Lead a c e t a t e (75 ug) C i t r i c ac i d (15 X) A s c o r b i c a c i d (15 X) C i t r i c ac i d (40 X) A s c o r b i c a c i d (40 X) 36.4511.04b 30.49+2.10a 34.4810.69 35.5510.81b 33.5211.54 34.6910.87 W i t h i n experiments a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.05) 1 12 acid) and 5 (Comparison of lower levels of ascorbic acid with c i t r i c acid) the mean body weights of embryos treated with lead acetate were s i g n i f i c a n t l y lower than control embryos and embryos treated in experiment 4 with isoascorbic acid, and in experiment 5 with ascorbic acid at 15 times the molar amount of lead acetate. The combined data (Tables III and VIII) indicated that the administration of 75 ug of lead acetate resulted in s i g n i f i c a n t decrements in mean body weight in comparison to controls and that ascorbic acid and isoascorbic acid afforded s i g n i f i c a n t protection against t h i s effect of lead acetate. Crown Rump Lengths Table IX records the mean crown rump lengths and standard errors for chick embryos surviving u n t i l day 19, which were subjected to the various treatments. In experiment 1 (Selection of lead acetate l e v e l for subsequent experiments), embryos administered lead acetate at the le v e l of 125 ug exhibited s i g n i f i c a n t l y shorter crown rump lengths, than embryos administered lead acetate at le v e l s ranging from zero to 100 ug. In experiment 2 (Comparison of the effectiveness of increasing leve l s of ascorbic acid) through 5 (Comparison of lower levels of ascorbic acid with c i t r i c a cid), no s i g n i f i c a n t differences among any of the treatment groups with respect to crown rump lengths were detected. The combined data (Tables III and X) indicated that the administration of 75 ug of lead acetate resulted in s i g n i f i c a n t decrements in mean crown rump length in comparison to controls and that a l l treatment agents provided 1 1 3 Table VIII. Mean body weight and standard error as determined from experiments 2 to 5, in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid, isoascorbic acid, c i t r i c acid or EDTA (at 15 times or greater the molar concentration of lead acetate). Agent Lead acetate Lead acetate plus agent (75 ug) (15 X or >) Ascorbic acid 33.50±0.75a 36.41±0.45b Isoascorbic acid 33.21±0.95a 36.0l±0.6lb C i t r i c acid 32.23±1.26 34.02±0.58 EDTA 33.96±1.17 35.57±0.59 Within agents a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.0!) T a b l e IX The e f f e c t on c h i c k embryo crown rump l e n g t h of lead a c e t a t e a d m i n i s t e r e d a l o n e or s i m u l t a n e o u s l y w i t h a s c o r b i c a c i d , i s o a s c o r b i c a c i d , c i t r i c a c i d or EDTA ( a t l e v e l s r a n g i n g from 15 to 80 times the molar c o n c e n t r a t i o n of l e a d a c e t a t e ) . Means and s t a n d a r d e r r o r s g i v e n i n c e n t i m e t e r s . Exper iment _1_ ( S e l e c t i o n of 1 ead a c e t a t e 1 evel f o r subsequent experiments) C o n t r o l Lead a c e t a t e Lead a c e t a t e Lead a c e t a t e Lead a c e t a t e (50 ug) (75 ug) (100 ug) (125 ug) 8.8510.10b 8.75±0.10b 8.30±0.30b 8.70+0.30b 7.4510.60a Experiment 2 (Comparison of the e f f e c t i veness of i ncreas i ng l e v e l s of a s c o r b i c a c i d ) C o n t r o l Lead a c e t a t e A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (20 X) (40 X) (60 X) (80 X) 9.0010.10 8.45+0.30 8.7510.15 8.85+0.20 9.05+0.10 9.0510.10 Exper i ment 3 (Comparison of the e f f e c t iveness of a s c o r b i c a c i d w i t h i s o a s c o r b i c a c i d , c i t r i c a c i d and EDTA) C o n t r o l Lead a c e t a t e C i t r i c a c i d EDTA I s o a s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (60 X) (15 X) (60 X) (60 X) 8.5010.10 8.0510.10 8.3010.20 8.5010.10 8.4010.10 8.4510.10 Exper i ment 4 (Compar i son of the e f f e c t i veness of 1ower 1evels of a s c o r b i c ac i d w i t h i s o a s c o r b i c ac i d) C o n t r o l Lead a c e t a t e I s o a s c o r b i c a c i d A s c o r b i c a c i d I s o a s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (15 X) (15 X) (40 X) (40 X) 8.8510.15 8.20+0.25 8.90+0.15 8.3010.30 8.8O10.10 8.80+0.15 Exper i ment 5 (Compar i son of the e f f e c t i veness of 1ower 1evels of a s c o r b i c ac i d wi t h c i t r i c ac i d) C o n t r o l Lead a c e t a t e C i t r i c a c i d A s c o r b i c a c i d C i t r i c a c i d A s c o r b i c a c i d (75 ug) (15 X) (15 X) (40 X) (40 X) 8.8510.10 8.15+0.15 8.4510.10 8.6010.10 8.5010.20 8.30+0.25 In Experiment 1 a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.05) 115 Table X. Mean crown rump length and standard error as determined from experiments 2 to 5 , in which chick embryos were administered lead acetate alone or simultaneously with ascorbic acid isoascorbic acid, c i t r i c acid or EDTA (at 15 times or greater the molar concentration of lead acetate). Agent Lead acetate Lead acetate plus agent (75 ug) (15 X or >) Ascorbic acid 8.20±0.1Oa 8.65±0.05b Isoascorbic acid 8.15±0.15a 8.70±0.1 Ob C i t r i c acid 8.10±0.1Oc 8.40±0.!0d EDTA 8.05±0.1Oe 8.50±0.1 Of Within agents a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.00l) c i s s i g n i f i c a n t l y d i f f e r e n t from d (p<0.05) e i s s i g n i f i c a n t l y d i f f e r e n t from f_ (p<0.0l) 1 16 s i g n i f i c a n t protection against t h i s effect of lead acetate. Figure 7 i l l u s t r a t e s the s i g n i f i c a n t p o s i t i v e linear r e l a t i o n s h i p observed in experiment 2 (Comparison of the effectiveness of increasing levels of ascorbic acid) , between mean crown rump length and ascorbic acid over the range of zero to 80 times the molar amount of lead acetate administered. Wet Brain Weights Table XI records the mean wet brain weights and standard errors for chick embryos subjected to the various treatments during the course of the four experiments in which these measurements were obtained. In experiment 1 (Selection of lead acetate l e v e l for subsequent experiments) , the mean wet brain weight of chick embryos administered lead acetate at 75 and 125 ug did not d i f f e r s i g n i f i c a n t l y from one another, but were s i g n i f i c a n t l y less than for embryos administered zero, 50, and 100 ug of lead acetate. In experiment 2 (Comparison of the effectiveness of increasing levels of ascorbic acid), chick embryos administered lead acetate alone had s i g n i f i c a n t l y lower mean wet brain weights, than control, embryos or embryos administered ascorbic acid at 60 and 80 times the molar amount of lead acetate. In experiment 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA), chick embryos administered lead acetate alone exhibited s i g n i f i c a n t l y lower mean wet brain weights, than control embryos or embryos administered c i t r i c acid, isoascorbic acid, or ascorbic acid at 60 times or 117 ASCORBIC ACID CONCENTRATION (X MOLAR CONCENTRATION OF LEAD ACETATE ) F i g u r e 7 . T h e s i g n i f i c a n t p o s i t i v e l i n e a r r e l a t i o n s h i p o b s e r v e d b e t w e e n c h i c k e m b r y o c r o w n rump l e n g t h a n d i n c r e a s i n g l e v e l s o f a s c o r b i c a c i d . . Mean ± s t a n d a r d e r r o r r e p r e s e n t e d b y o p e n c i r c l e a n d b a r . S i g n i f i c a n c e o f r e g r e s s i o n c o e f f i c i e n t (p < 0 . 0 1 ) T a b l e XI The e f f e c t on c h i c k embryo wet b r a i n weight of l e a d a c e t a t e a d m i n i s t e r e d a l o n e or s i m u l t a n e o u s l y w i t h a s c o r b i c a c i d , i s o a s c o r b i c a c i d , c i t r i c a c i d or EDTA (at l e v e l s r a n g i n g from 15 to 80 times the molar c o n c e n t r a t i o n of l e a d a c e t a t e ) . Means and s t a n d a r d e r r o r s g i v e n i n grams. Experiment ± ( S e l e c t i o n of 1ead a c e t a t e 1evel f o r subsequent experiments) C o n t r o l Lead a c e t a t e Lead a c e t a t e Lead a c e t a t e Lead a c e t a t e (50 ug) (75 ug) (100 ug) (125 ug) 0.8083±0.0218b 0.7745+0.0276b 0.5530+0.0642a 0.754010.0362b 0.593910.0787a Exper i ment 2 (Compar i son of the e f f e c t iveness of i ncreas i ng 1 eve 1s of a s c o r b i c ac i d) C o n t r o l Lead a c e t a t e A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (20 X) (40 X) (60 X) (80 X) 0.9089+0.0198b 0.776410.0563a 0.8198+0.0175 0.832110.0202 0.884510.0229b 0.876910.0162b Experiment 3 (Compar i son of the e f f e c t i veness of a s c o r b i c a c i d w i t h i s o a s c o r b i c a c i d , c i t r i c a c i d and EDTA) C o n t r o l Lead a c e t a t e C i t r i c a c i d EDTA I s o a s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (60 X) (15 X) (60 X) (60 X) 0.800910.0163b 0.650810.0383a 0.746310.0256b 0.784810.0235b 0.753810.0136b 0.7757+0.0194b Exper iment 4 (Compar i son of 1ower 1evels of a s c o r b i c a c i d w i th i s o a s c o r b i c a c i d ) C o n t r o l Lead a c e t a t e I s o a s c o r b i c a c i d A s c o r b i c a c i d I s o a s c o r b i c a c i d A s c o r b i c a c i d (75 ug) (15 X) (15 X) (40 X) (40 X) 0.868810.0294 0.741010.0409 0.821110.0233 0.772110.0336 0.809210.0169 0.811410.0236 W i t h i n experiments a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.05) 119 ethylenediaminetetraacetate at 15 times the molar amount of lead acetate. In experiment 4 (Comparison of lower lev e l s of ascorbic acid with isoascorbic acid), no s i g n i f i c a n t differences were found in mean wet brain weights. However, embryos treated with lead acetate alone tended to exhibit lower mean wet brain weights than control embryos, while embryos treated with lead acetate plus another agent tended to exhibit mean wet brain weights between these two extremes. Dry Brain Weights Table XII records the mean dry brain weights and standard errors for chick embryos subjected to the various treatments during the course of the four experiments in which these measurements were obtained. In experiment 1 (Selection of lead acetate l e v e l for subsequent experiments), the mean dry brain weights of chick embryos administered lead acetate at 75 and 125 ug did not d i f f e r s i g n i f i c a n t l y from one another, but were s i g n i f i c a n t l y less than for embryos administered zero, 50, and 100 ug of lead acetate. In experiments 2 (Comparison of the effectiveness of increasing l e v e l s of ascorbic acid) through 4 (Comparison of lower levels of ascorbic acid with isoascorbic acid), chick embryos administered lead acetate alone, exhibited s i g n i f i c a n t l y lower dry brain weights than control embryos and embryos administered the other agents with lead acetate. Figures 8 and 9 i l l u s t r a t e the s i g n i f i c a n t p o s i t i v e linear relationship observed in experiment 2 (Comparison of the effectiveness of increasing l e v e l s of ascorbic acid), between T a b l e XII The e f f e c t on c h i c k embryo dry b r a i n weight of l e a d a c e t a t e a d m i n i s t e r e d a l o n e or s i m u l t a n e o u s l y with a s c o r b i c a c i d , i s o a s c o r b i c a d d , c i t r i c a c i d or EDTA ( a t l e v e l s r a n g i n g from 15 to 80 times the molar c o n c e n t r a t i o n of lead a c e t a t e ) . Means and s t a n d a r d e r r o r s g i v e n i n grams. Exper i ment ± ( S e l e c t i on of 1ead a c e t a t e l e v e l f o r subsequent experiments) C o n t r o l Lead a c e t a t e (50 ug) Lead a c e t a t e (75 ug) Lead a c e t a t e (100 ug) Lead a c e t a t e (125 ug) 0.0911±0.0028b O.0867+0.0024b 0.0611±0.0063a 0.0787+0.0068b O.O529±0.01O6a Exper i ment 2 (Compar i son of the e f f e c t i veness of 1ncreas i ng 1evels of a s c o r b i c ac i d) C o n t r o l Lead a c e t a t e (75 ug) A s c o r b i c a c i d (20 X) A s c o r b i c a c i d (40 X) A s c o r b i c a c i d (60 X) A s c o r b i c a c i d (80 X) 0.1025±0.0027b 0.0811±0.0077a 0.097010.0019b 0.0978±0.0029b 0.1019±0.0029b 0.1043±0.0029b Exper i ment 3 (Compar i son of the e f f e c t i veness of a s c o r b i c ac i d w i t h i s o a s c o r b i c ac i d, c i t r i c ac i d and EDTA) C o n t r o l Lead a c e t a t e (75 ug) C i t r i c ac i d (60 X) EDTA ( 15 X) I s o a s c o r b i c a c i d (60 X) A s c o r b i c . a c i d (60 X) 0.0825+0.0027b 0.0656±0.0049a 0.0753±0.0035b 0.0861±0.0030b 0.0797+0.0020b 0.0815±0.0023b Experiment 4 (Comparison of 1ower 1 eve 1s of a s c o r b i c a c i d with i s o a s c o r b i c a c i d ) C o n t r o l 0.1004±0.0040b Lead a c e t a t e I s o a s c o r b i c a c i d (75 ug) (15 X) 0.0756+0.0059a 0.0943±0.0032b A s c o r b i c a c i d ( 15 X) 0.0868±0.0030b I s o a s c o r b i c a c i d (40 X) 0.0929+0.0018b A s c o r b i c a c i d (40 X) 0.095210.0021b W i t h i n experiments a i s s i g n i f i c a n t l y d i f f e r e n t from b (p<0.05) I—1 o 1 2 1 0.90 0.88 0.86 -~ 0.84 Cf> g 0.82-• < OC CD h-LU 0.80 078 0.76 0.74 0.72 J_ _L OX 20X 40X 60X 80X ASCORBIC ACID CONCENTRATION (X MOLAR CONCENTRATION OF LEAD ACETATE ) Figure 8 . The s i g n i f i c a n t p o s i t i v e l i n e a r relationship observed between chick embryo wet brain weight and increasing levels of ascorbic acid. Mean ± standard error represented by open c i r c l e and bar. Significance of regression c o e f f i c i e n t (p < 0 . 0 0 1 ) < or m >-or Q 0 . 0 8 4 0 . 0 8 0 0 . 0 7 6 0 . 0 7 2 » ^ _ J L O X 2 0 X 4 0 X 6 0 X 8 0 X ASCORBIC ACID CONCENTRATION (X MOLAR CONCENTRATION OF LEAD ACETATE ) F i g u r e 9 . T h e s i g n i f i c a n t p o s i t i v e l i n e a r r e l a t i o n s h i p o b s e r v e d b e t w e e n c h i c k e m b r y o d r y b r a i n w e i g h t a n d i n c r e a s i n g l e v e l s o f a s c o r b i c a c i d . Mean ± s t a n d a r d e r r o r r e p r e s e n t e d b y o p e n c i r c l e a n d b a r . S i g n i f i c a n c e o f r e g r e s s i o n c o e f f i c i e n t (p < 0 . 0 0 0 5 ) 1 23 wet and dry brain weights and ascorbic acid over the range of zero to 80 times the molar amount of lead acetate. The Relationship of Hydrocephalocele to Growth Retardation and  Brain Weight Table XIII was constructed to determine, i f there were any si g n i f i c a n t differences in mean body weight, crown rump length, wet and dry brain weights, between normal embryos and embryos exhibiting hydrocephalocele, which had been administered lead acetate alone or with c i t r i c acid, isoascorbic acid or ascorbic acid. No s i g n i f i c a n t differences were detected between normal and hydrocephalocelic embryos with respect to mean body weight or crown rump length for any of the treatments administered. With respect to mean wet brain weight, a s i g n i f i c a n t depression was noted for hydrocephalocelic embryos compared to normal embryos administered lead acetate alone, but not for hydrocephalocelic and normal embryos administered isoascorbic acid or ascorbic acid. With respect to mean dry brain weight, a s i g n i f i c a n t depression was noted for hydrocephalocelic embryos compared to normals administered lead acetate alone or with ascorbic ac i d . In embryos treated with isoascorbic acid, the difference in mean dry brain weight between hydrocephalocelic and normal embryos almost reached the 0 .05 significance l e v e l . Table XIII. The relationship between hydrocephalocele and chick embryo body weight, crown rump length and wet and dry brain weight. Means and standard errors for body weight, wet and dry brain weight given in grams and for crown rump length given in centimeters. Treatment agents administered at 15 to 40 times the molar concentration of lead acetate. Number of samples shown in brackets. Body weight Hydrocepha1oce1e Norma 1 Crown rump 1ength Hydrocepha1oce1e Norma 1 Wet bra i n we i ght Hydrocepha1ocele Norma 1 Dry brain weight Hydrocepha1oce1e Normal Lead acetate (75 ug) 33.20+0.96 (25) 34.3411.87 (9) 8. 1510. 15 (25) 8.4510.20 (9) 0.638110.0320a (25) 0.808810.0270a (9) 0.065110.0030b (25) 0.0875+0.0040b (9) C i t r i c ac i d (15 and 40 X) 34.53+1.14 (11) 33.3611.25 (9) 8.45+0.10 (11) 8.5010.10 (9) Isoascorb i c ac i d (15 and 40 X) 38.19+1 . 13 (4) 36.9810.85 (18) 8.85+0.15 (4) 8.85+0.10 (18) 0.752310.0310 (4) 0.929110.0140 (18) 0.084 110.0040 (4) 0.095710.0020 (18) Ascorb i c ac i d ( 15 and 40 X) 34.4511.42 ( 10) 35.8010.68 (33) 8.2510.25 (10) 8.6010.10 (33) 0. 7147+0.0630 (5) 0.817110.0140 (16) 0.080110.0040c (5) 0.094610.0020c (16) Pairs of s i g n i f i c a n t l y d i f f e r e n t means: a , b (p<0.001) and c (p<0.01) 125 CHAPTER V  DISCUSSION The Embryotoxic and Teratogenic Effects of Lead Acetate in the  Chick Embryo The results of t h i s study indicate that 75 ug of lead acetate s i g n i f i c a n t l y increases mortality, growth retardation and hydrocephalocele when injected into the yolk sac of chick embryos at 96 hours of incubation. Increased mortality and growth retardation have been observed in most species in which the embryotoxic e f f e c t s of lead have been investigated (Karnofsky and Ridgeway, 1952; Ferm and Carpenter, 1967; Kennedy et a l . , 1975; McClain and Becker, 1975; Kimmel et a l . , 1976; Jacquet and Gerber, 1979; B e l l and Thomas, 1980). Hydrocephalocele has been i d e n t i f i e d as a consistently occurring teratogenic response to lead in the chick embryo (Karnofsky and Ridgeway, 1952; Hirano and Kochen, 1973; King and L u i , 1974) and an infrequent teratogenic response to lead in man (Bell and Thomas, 1980), the rat (McClain and Becker, 1975) and the golden hamster (Gale, 1978). The increased mortality in lead treated chick embryos appears to be the result of a teratogenic action of lead on blood vessels developing in the central nervous system during the t h i r d and fourth days of incubation (Karnofsky and Ridgeway, 1952). Hirano and Kochen (1973) observed exsanguination, due to central nervous system hemorrhage, to be the most frequent cause 1 26 of death on day five through nine of incubation in chick embryos injected with 75 ug of lead acetate at 96 hours of incubation. Karnofsky and Ridgeway (1952) reported a 90% incidence of central nervous system hemorrhage in deaths occurring between days five and nine of incubation, when one mg of lead n i t r a t e was injected into chick embryos at 96 hours of incubation. The present study reported a s i m i l i a r incidence of central nervous system hemorrhaging during the same incubation period, confirming the association between central nervous system hemorrhage and death in lead treated embryos. The causes of deaths occurring after day nine have not been reported. However, the present study found such deaths to be frequently associated with the presence of hydrocephalocele. Hydrocephalocele may contribute to death via central nervous system tissue necrosis or by preventing proper positioning of the embryo within the confines of the s h e l l . Romanoff and Romanoff (1972) have reported that the most frequent cause of death during the l a t t e r part of the incubation period was malpositioning. King and Lui (1974) reported mortality rates of 26, 67 and 85%, when 50, 100 and 150 ug of lead acetate was administered to chick embryos incubated for 96 hours. From th i s data, they estimated the LD50 for lead acetate in 96 hour incubated chick embryos to be 75 ug. Contrary to King and Lui's (1974) estimate, Hirano and Kochen (1973) observed a 67% mortality rate in 96 hour incubated embryos treated with 75 ug of lead acetate. The current study confirmed King and Lui's (1974) estimate of 75 ug 1 27 as the LD50 for lead acetate in 96 hour incubated chick embryos and provided mortality rates concurring with the King and Lui (1974) study for a l l levels of lead acetate, except the 50 ug l e v e l . The difference in findings between the two studies at the 50 ug l e v e l was probably related to the small sample sizes used in the current study, which have a greater p r o b a b i l i t y of being non-representative of the population from which they were derived, than do the larger sample sizes used in the King and Lui study (1974). The 17% higher mortality rate reported in the Hirano and Kochen study (1973), compared to the current study may have been due to differences in the genetic s t r a i n of white leghorn used, their d i e t s , the age at mating, the season of laying, flock management p r i n c i p l e s , incubation temperatures or humidities. (McLaughlin et a l . , 1963; Romanoff and Romanoff, 1972). Growth retardation in chick embryos in response to lead has been reported by a number of investigators (Karnofsky and Ridgeway, 1952; DeFrancisis and Boccalatte, 1962; G i l a n i , 1973a; DeGennaro, 1978). The current study found s i g n i f i c a n t growth retardation in embryos treated with 75 ug of lead acetate compared to controls, when the experimental data for these treatment groups were combined (Table I I I ) . However, due to small sample sizes in individual experiments, s i g n i f i c a n t differences in growth between lead treated embryos and controls were not observed, except in experiment 4 (Comparison of lower levels of ascorbic acid with isoascorbic acid) and 5 (Comparison 128 of lower levels of ascorbic acid with c i t r i c acid) with respect to body weight (Table VII). In seeking an explanation for the f a i l u r e to detect s i g n i f i c a n t differences with regard to body weight in experiments 1 (Selection of lead acetate l e v e l for subsequent experiments) to experiment 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA), but not in experiment 4 (Comparison of lower levels of ascorbic acid with isoascorbic acid) and 5 (Comparison of lower levels of ascorbic acid with c i t r i c acid) the age of the mated birds was found to be the major difference between the two sets of experiments. In experiments 1 to 3, eggs were obtained from mature birds aged one to one and one-half years, while in experiments 4 and 5, eggs were obtained from immature birds aged seven to ten months. McLaughlin et a l . (1963) reported that the f e r t i l i t y , hatchabilty and l i v e a b i l t y of chick embryos was dependent on the age of the mated birds, amongst other ecological factors. In the current study, i t therefore seems possible that differences in the age of the mated birds may have accounted for lead treated embryos in experiment 4 and 5 being more susceptible to the weight depressing e f f e c t s of lead than those in experiments 1 to 3. Table XIII indicated that there was no s i g n i f i c a n t difference in body weight or crown rump length between lead treated embryos exhibiting hydrocephalocele and those not exhibiting t h i s l e s i o n . These findings suggest that the growth retardation observed in lead treated embryos was probably not 129 related to the teratogenic e f f e c t s of lead on the central nervous system, but to the toxic e f f e c t s of lead on other body systems. Jacquet et a l . (1977) has hypothesized that lead induced growth retardation i s due to hypoxia developing as a result of enzymatic i n h i b i t i o n by lead of f e t a l hemoglobin sysnthesis. Gerber et a l . (1978) has hypothesized that lead induced growth retardation is the result of lead accumulating in the placenta, where i t interferes with blood flow and the uptake of substances by the fetus. The work of Sandstead et a l . (1969) suggests that growth retardation may be related to i n h i b i t i o n by lead of thyroid hormone synthesis. Current knowledge i s i n s u f f i c i e n t to determine, which mechanisms underlie lead induced growth retardation in the chick embryo. However, the work of King et a l . (1978) in lead treated chick embryos, demonstrating the i n h i b i t i o n of the enzyme delta -aminolevulinic acid dehydratase involved in hemoglobin synthesis, and the work of Romanoff and Romanoff (1972), demonstrating that oxygen consumption i s d i r e c t l y proportional to body weight in the intact chick embryo, are consistent with the Jacquet et a l . (1977) hypothesis. On the other hand, accumulation of lead in the yolk sac due to i t s a b i l i t y to disrupt membrane function, structure and hemodynamics (Goyer and Rhyne, 1973), may naccount for decreased uptake of substrates by the v i t e l l i n e c i r c u l a t i o n , leading to growth retardation in chick embryos. F i n a l l y , since lead i s known to suppress thyroid function, i t may l i k e t h i o u r a c i l lead to growth retardation in 1 30 the chick embryo (Freeman and Vince, 1974). The current study found a 74% incidence of hydrocephalocele in chick embryos administered 75 ug of lead acetate. This finding concurred with King and Lui's (1974) finding of an 81% incidence of hydrocephalocele in chick embryos administered 75 ug of lead acetate and the Karnofsky and Ridgeway (1952) finding of an 84% incidence of hydrocephalocele in chick embryos administered one to two mg of lead n i t r a t e . Although, the majority of embryos in the current study exhibited hydrocephalocele, i t is of interest to know why 26% of the embryos f a i l e d to exhibit t h i s l e s i o n . One explanation may be that individual genetic differences in s u s c e p t i b i l i t y e x i s t , in spite of the fact that a l l embryos came from the same genetic s t r a i n . A second explanation may be related to the s i t e of lead depositon. In the current study, with the equipment available, i t was not possible to monitor the s i t e of lead deposition. It is therefore, probable that in some instances lead was deposited in the albumen. Under these circumstances, the development of hydrocephalocele would be unlikely, as the albumen i s a r i c h reservoir of proteins that would readily chelate lead and prevent i t s uptake by the embryo during the c r i t i c a l period of teratogenic s u s c e p t i b i l i t y (Freeman and Vince, 1974). The observation, in the current study, that hemorrhage preceded the development of hydrocephalocele, concurred with the observation of Hirano and Kochen (1977) regarding the development of lead induced hydrocephalocele in the chick embryo. The observations of Hirano and Kochen (1977) appear to 131 be s i m i l i a r to the sequence of events observed in the development of lead induced s k e l e t a l lesions in the hamster, rat and mouse (Carpenter and Ferm, 1977) and lead encephalopathy in the neonatal rat (Thomas, 1971). The s i m i l i a r i t i e s in the sequence of events leading to the development of lesions in these species suggests a s i m i l i a r mechanism by which lead i n i t i a t e s t h i s process. Several hypotheses regarding a possible mechanism have been proposed (Ferm and Carpenter, 1977; Goldstein, 1977; V i s t i c a et a l . , 1977). Unfortunately, none of the proposed mechanisms have been able to account for the uniqueness of the lesion in the various species. The hydrocephalocelic lesion in the chick embryo which developes in response to lead can occur in either the o c c i p i t a l or p a r i e t a l regions of the s k u l l . Hirano and Kochen (1973) found the lesion to be most frequently located in the o c c i p i t a l region of the s k u l l . The current study found a s i m i l i a r pattern, with 61% of the lesions located in the o c c i p i t a l , 21% in the p a r i e t a l and 13% in both regions. Lesion location may be related to differences in embryonic development at the time lead was administered. The main factors accounting for developmental differences in embryos incubated for s i m i l i a r periods are: genetic v a r i a b i l i t y , the time taken by the egg to transverse the oviduct prior to laying and the time the egg was brooded prior to removal from the nest (Romanoff and Romanoff, 1972). The wet and dry brain weights of chick embryos injected with lead during the c r i t i c a l period of teratogenic 1 32 s u s c e p t i b i l i t y have not been investigated in previous studies. In the current study, the findings regarding brain weight measurements indicate that they may be useful in quantitatively assessing the presence and severity of hydrocephalocele. The observation that dry brain weight measurements detected s i g n i f i c a n t differences between treatment groups not detected by wet brain weight measurements, suggests that dry brain weight may be a more sensitive indicator of brain tissue necrosis in hydrocephalocele than wet brain weight. The consistently high incidence of hydrocephalocele and the low incidence of other abnormalities observed in the current study, suggests that lead acts as a s i t e s p e c i f i c teratogen in the chick embryo. This viewpoint concurs with the work of Hirano and Kochen (1973), in which no abnormalities other than hydrocephalocele were observed in chick embryos injected at 96 hours of incubation with 75 ug of lead acetate, and the work of Karnofsky„and Ridgeway (1952), who reported consistently high incidences of hydrocephalocele, but only occasional cases of shortened lower beaks and micromelia in embryos injected at 96 hours of incubation with 0.1 mg or more of lead n i t r a t e . However, i t does not concur with the work of King and Lui (1974), who reported substantial incidences of curled d i g i t s , shortened lower beaks, microopthalmia, opened thoracic c a v i t i e s and twisted necks in chick embryos treated with 75 ug of lead acetate at 96 hours of incubation. The wider array of abnormalities reported in the King and Lui (1974) study, compared to the current study, may be related 133 to genetic or n u t r i t i o n a l differences between the eggs used in the two studies. Gale (1978) reported that s u s c e p t i b i l i t y to lead induced malformations in the golden hamster depended upon genetic s t r a i n . In the current study, eggs were obtained from Schaeffer's Star Cross 288 white leghorns. In the King and Lui (1974) study, the genetic s t r a i n of the white leghorns was not reported. However, since the study was conducted in Korea, i t is unlikely that the stra i n was the same as used in the current study. Levander (1979) reported that n u t r i t i o n a l factors interact with lead, a f f e c t i n g i t s toxic outcome. It therefore, seems reasonable that n u t r i t i o n a l factors may interact with lead, a f f e c t i n g i t s teratogenic outcome. If the mated birds in the two studies were fed d i f f e r e n t d i e t s , differences in the n u t r i t i o n a l composition of the eggs may have accounted for differences in the incidence and type of abnormality observed. The Protective E f f e c t s of Ascorbic Acid in the Lead Treated  Chick Embryo Ascorbic acid's a b i l i t y to protect against the embryolethal and teratogenic e f f e c t s of lead has not been investigated to any extent, in spite of and possibly as a result of the controversy regarding ascorbic acid's a b i l i t y to afford protection against the toxic e f f e c t s of lead. To date a single abstract (King and Lui, 1975) has reported that one mg of ascorbic acid s i g n i f i c a n t l y reduced the embryolethal and teratogenic effects of 75 ug of lead acetate administered to chick embryos at 96 134 hours of incubation. The current study confirmed the results of the King and Lui investigation (1975), by finding ascorbic acid, at levels ranging from approximately one to three mg, to s i g n i f i c a n t l y reduce mortality, growth retardation and hydrocephalocele in chick embryos administered 75 ug of lead acetate. Thus, in spite of the controversy regarding ascorbic acid's a b i l i t y to protect against the toxic e f f e c t s of lead, the current study, by confirming the work of King and Lui (1975), has established ascorbic acid as an ef f e c t i v e agent in affording protection against the embryolethal and teratogenic effects of lead in at least one species. In the current study and the King and Lui (1975) abstract, ascorbic acid was administered simultaneously with lead acetate. Pillemer et a l . (1940) found that ascorbic acid administered simultaneously with neurotoxic amounts of lead to guinea pigs s i g n i f i c a n t l y reduced mortality. However, when ascorbic acid was administered after the neurotoxic effects of lead had become evident, mortality rates were no di f f e r e n t from those observed when lead was administered alone. These findings suggest that in the present study and the King and Lui (1975) abstract ascorbic acid's protective action may have been preventive as opposed to therapeutic. Unfortunately, the high incidence of v i t e l l i n e membrane rupture observed in the current study when lead acetate and ascorbic acid were administered in separate injections prevented the evaluation of ascorbic acid's role as a prophylactic versus therapeutic agent in lead induced embryolethality and teratogenesis in the chick embryo. 1 35 The King and Lui abstract (1975), on ascorbic acid's reduction of the embryolethal and teratogenic e f f e c t s of 75 ug of lead acetate in the chick embryo reported on the effectiveness of a single l e v e l of ascorbic acid, while the current study investigated five l e v e l s of ascorbic acid. In the current study, mortality rates did not appear to be related to the l e v e l of ascorbic acid administered with the 75 ug of lead acetate. This suggests that a c r i t i c a l l e v e l of ascorbic acid exists, such that when this l e v e l is reached, ,no further reductions in mortality rates occur in embryos treated with 75 ug of lead acetate. On the basis of the mortality rates reported by King and Lui (1975), as well as in the current study, t h i s level appears to be no greater than one mg. In the King and Lui (1975) abstract, administration of one mg of ascorbic acid s i g n i f i c a n t l y reduced hydrocephalocele in embryos treated with 75 ug of lead acetate. In the current study, a l l levels of ascorbic acid were found to provide a s i m i l i a r degree of protection against lead induced hydrocephalocele and reduced brain weight. However, the findings in experiment 2 of s i g n i f i c a n t p o s i t i v e linear relationships for wet (Figure 8) and dry (Figure 9) brain weights over the range of ascorbic acid from zero to 80 times the molar amount of lead acetate suggest that in spite of the f a i l u r e to detect s i g n i f i c a n t differences in brain weight and hydrocephalocele among the various levels of ascorbic acid, additional increments in protection against lead induced reductions in brain weight can be achieved by increasing the l e v e l of ascorbic acid over 1 36 the range used in this study. This suggests that the optimal l e v e l to protect the central nervous system of the chick embryo from the teratogenic effects of 75 ug of lead acetate may be as great as three mg. The s i g n i f i c a n t l y greater wet and dry brain weights in embryos administered ascorbic acid compared to those administered lead acetate alone are probably related to two factors: (1) a lower incidence of hydrocephalocele in ascorbic acid treated embryos, (2) the development of less severe lesion in ascorbic acid treated embryos. Although, the decreased lesion severity factor could not be put to s t a t i s t i c a l test due to d i f f i c u l t i e s in experimental design, recorded subjective observations of lesion severity and the smaller percentage differences in wet and dry brain weight (Table XIII), between normal and hydrocephalocelic embryos treated with ascorbic acid, compared to those treated with lead acetate alone, suggest i t s existence as a probable factor. In the current study, when the data for a l l groups treated with ascorbic acid were combined, ascorbic acid was found to provide s i g n i f i c a n t protection against lead induced growth retardation (Tables VIII and X). Ascorbic acid may have interacted d i r e c t l y with lead in reversing the growth retardation or i t may have had a stimulatory e f f e c t on growth in i t s own r i g h t . Pillemer et a l . (1940) and Mahaffey and Bank (1975), in guinea pigs, found that high intakes of ascorbic acid served to stimulate growth, whether lead was present in the diets in trace or growth retarding amounts. In experiment 2 1 37 (Comparison of the effectiveness of increasing levels of ascorbic acid) of the current study (Figure 7), a positive linear r e l a t i o n s h i p was observed between crown rump length and increasing l e v e l s of ascorbic acid, indicating a possible stimulatory e f f e c t of ascorbic acid on this parameter of growth. Unfortunately, the design of experiment 2 (Comparison of the effectiveness of increasing levels of ascorbic acid) did not permit evaluation of t h i s effect as being dependent or independent of the growth retarding effects of lead acetate. King and Lui (1975) found ascorbic acid to provide complete protection against twisted necks and shortened lower beaks and to s u b s t a n t i a l l y reduce curled d i g i t s and microopthalmia in lead treated embryos. In the current study, these abnormalities occurred infrequently in lead acetate treated embryos and rarely, i f ever, in ascorbic acid treated embryos. Thus, the current study was unable to confirm King and Lui's (1975) observations in t h i s area. King and Lui (1975), besides providing evidence for a reduction by ascorbic acid of lead induced abnormalities, reported that ascorbic acid s i g n i f i c a n t l y increased the incidence of umbilical hernias and hematomas in lead treated embryos. In the current study, umbilical hernias and hematomas were not observed in embryos treated with lead acetate alone or in those treated with ascorbic acid. However, three ascorbic acid treated embryos were found to exhibit abnormalities not observed in any other treatment groups. The frequency rates for these abnormalities were less than for t h e i r spontaneous 1 38 occurrence i n untreated c h i c k embryos (Romanoff and Romanoff, 1972). Thus, the present study pro v i d e s no evidence f o r a s c o r b i c a c i d a c t i n g independently or s y n e r g i s t i c a l l y with l e a d a c e t a t e to i n c r e a s e the frequency of any abnormality. Mechanism of A s c o r b i c A c i d ' s P r o t e c t i v e E f f e c t s i n the Lead  Treated Chick Embryo There are at l e a s t four mechanisms, p a r a l l e l i n g the four hypotheses p r e v i o u s l y d e s c r i b e d , by which a s c o r b i c a c i d may have acted to p r o t e c t the developing c h i c k embryo from the t e r a t o g e n i c e f f e c t s of l e a d on the c e n t r a l nervous system. ( r ) A s c o r b i c a c i d may have acted at the l e v e l of the yolk sac to prevent or decrease the uptake of l e a d by the v i t e l l i n e c i r c u l a t i o n d u r i n g the c r i t i c a l p e r i o d of organogenesis. (2) A s c o r b i c a c i d may have acted to meet in c r e a s e d requirements induced, perhaps, by lead's i n t e r f e r e n c e i n c o l l a g e n s y n t h e s i s . (3) A s c o r b i c a c i d may have c h e l a t e d lead ions, p r e v e n t i n g them from e x e r t i n g t e r a t o g e n i c e f f e c t s on the c e n t r a l nervous system. (4) A s c o r b i c a c i d may have formed complexes with t h i o l groups at the a c t i v e s i t e s of enzymes, p a r t i c u l a r l y i n the c e n t r a l nervous system, thereby p r e v e n t i n g l e a d from i n t e r f e r i n g with t h e i r a c t i v i t y . Of the four hypotheses proposed, the c u r r e n t study i n v e s t i g a t e d the i n c r e a s e d requirement and c h e l a t i o n hypotheses. The i n c r e a s e d requirement hypothesis was i n v e s t i g a t e d by comparing the e f f e c t i v e n e s s of i s o a s c o r b i c a c i d with a s c o r b i c a c i d . The r e s u l t s of the c u r r e n t study i n d i c a t e d that i d e n t i c a l l e v e l s of i s o a s c o r b i c a c i d p r o v i d e d s i m i l i a r p r o t e c t i o n with 139 respect to mortality, hydrocephalocele, body weight, crown rump length and brain weight. These findings are not consistent with the increased requirement hypothesis, which predicts differences between ascorbic acid and isoascorbic acid on the basis of the lower antiscorbutigenic a c t i v i t y of isoascorbic acid. It i s possible however, that the levels of ascorbic acid and isoascorbic acid administered in the current study were greatly in excess of any increased requirement, thereby masking any difference between the two agents. Refuting t h i s argument is the trend towards a s i m i l i a r , but lower incidence of hydrocephalocele in embryos administered these agents at 40 compared to 15 times the molar amount of lead acetate. The chelation hypothesis was investigated, by comparing the effectiveness of ascorbic acid with two known lead chelators, ethylenediaminetetraacetate and c i t r i c acid. It was hypothesized that i f these agents, known to chelate lead, could provide protection against the same parameters as does ascorbic acid, chelation may be the mechanism by which ascorbic acid acts to protect the chick embryo from the teratogenic effects of lead on the central nervous system. Consistent with the chelation hypothesis, the results of the current study found ethylenediaminetetraacetate and c i t r i c acid to protect against lead induced changes in the same parameters as does ascorbic acid. These findings are therefore, suggestive of ascorbic acid acting as a chelator to protect the chick embryo from the central nervous system e f f e c t s of lead. Goyer and Cherian (1979) have proposed that a f a i r means of 1 40 comparing the effectiveness of ascorbic acid and ethylenediaminetetraacetate i s to administer ascorbic acid at four times the molar amount of ethylenediaminetetraacetate, in order to provide an equal number of carboxyl groups for metal chelation. In experiment 3 (Comparison of the effectiveness of ascorbic acid with isoascorbic acid, c i t r i c acid and EDTA), when equal numbers of carboxyl groups were administered, no si g n i f i c a n t differences in mortality, hydrocephalocele, or brain weight were detected between the two agents, although both agents were found to s i g n i f i c a n t l y protect these parameters from the e f f e c t s of lead acetate. These findings indicate that ascorbic acid is as e f f e c t i v e as ethylenediaminetetraacetate when administered at four times the molar amount of ethylenediaminetetraacetate. However, comparing these agents using a single r a t i o f a i l e d to determine i f the four to one rat i o was the lowest r a t i o at which ascorbic acid could provide equal protection to ethylenediaminetetraacetate. Thus, further studies are required to answer t h i s question. The s t a b i l i t y constant for the lead ascorbate chelate, i f i t e xists, is not known. The results of the current study suggest, on the basis of the incidence of hydrocephalocele at s i m i l i a r l e v e l s of ascorbic acid and c i t r i c acid, that the lead ascorbate chelate possesses a greater s t a b i l i t y than the lead c i t r a t e chelate in vivo. This greater s t a b i l i t y may be related to a less rapid rate of in vivo degradation of ascorbic acid compared to c i t r i c acid, or i t may r e f l e c t a lower l i k e l i h o o d for lead to dissociate from ascorbic acid than c i t r i c acid. 141 Further experimental work is necessary to determine i f the lead ascorbate chelate does exist and the factors influencing i t s s t a b i l i t y in vivo and in v i t r o systems. The effectiveness of ascorbic acid and ethylenediaminetetraacetate were not d i r e c t l y compared in the current study at le v e l s where differences in their effectiveness could be detected. However, the findings in experiments 4 (Comparison of lower levels of ascorbic acid with isoascorbic acid) and 5 (Comparison of lower levels of ascorbic acid with c i t r i c a c i d ) , of a 33% incidence of hydrocephalocele in embryos treated with ascorbic acid at 15 times the molar amount of lead acetate, compared to a finding in experiment 3, of complete protection against the development of hydrocephalocele in embryos administered ethylenediaminetetraacetate at 15 times the molar amount of lead acetate, suggests that the lead ethylenediaminetetraacetate chelate possesses greater s t a b i l i t y in vivo than the lead ascorbate chelate, i f i t e x i s t s . This assumption appears reasonable, as the lead ethylenediaminetetraacetate chelate has a high s t a b i l i t y constant (Reynolds, 1963) and, unlike ascorbic acid, does not undergo degradation in vivo (Foreman, 1960). In conclusion, the findings of the current study, although far from conclusive, tend to support the hypothesis that ascorbic acid acts as a chelator in protecting the central nervous system of chick embryos from the teratogenic e f f e c t s of lead. Further investigations of ascorbic acid as a chelating agent should be undertaken, as i t s a b i l i t y to cross c e l l 142 membranes, i t s low t o x i c i t y compared to chelators currently in use and i t s a b i l i t y to protect the central nervous system from the toxic (Pillemer et a l . , 1940) and the teratogenic effects of lead (King and Lui, 1975), indicate that i t may be useful in the prevention of lead encephalopathy in.fetuses and children, in the prevention of excessive lead burdens in i n d u s t r i a l l y exposed workers and the aged and in the treatment of established cases of lead toxcity, where due to t o x i c o l o g i c a l considerations, currently used chelators can not be administered at the most favorable r a t i o to lead (Chisolm, 1968). Thus, ascorbic acid's role as a possible chelator i s a r i c h , yet v i r t u a l l y untapped area for future research, leading to p o t e n t i a l l y valuable applications for mankind. 143 BIBLIOGRAPHY Ahrens FA, V i s t i c a DT (1977) Microvascular effects of lead in the neonatal rat. Exp Mol Pathol 26:129-138 Altmann P, Maruna RFL, Maruna H, Michalica W, Wagner G (1981) Lead d e t o x i f i c a t i o n e f f e c t of combined calcium phosphate and ascorbic acid therapy in pregnant women with increased lead burden. Weiner Medizinsche Wochenscritz 131:311-314 Angle CR, Mclntre MS (1964) Lead poisoning during pregnancy Am J Dis Child 108:436-439 Anonymous (1971) Chelating agents in medicine. Br Med J 2:270-272 Barltrop D, Khoo HE (1975) The influence of n u t r i t i o n a l factors on lead absorption. Postgrad Med J 51:795-800 B e l l JU, Thomas JA (1980) Ef f e c t s of lead on mammalian reproduction. In Lead Toxicity, Urban and Schwarzenberg, Baltimore- Munich, pp 169-186 Borenstein B (1965) The comparative properties of ascorbic acid and erythorbic acid. Food Tech 19:115-117 Bridbord K, Blejar HP (1977) Prophylactic chelation therapy in occupational lead poisoning: a review. Am Ind Hyg Assoc J 38:536-542 Bryce-Smith D, Deshpande RR, Hughes J, Waldron HA (1977) Lead and cadmium leve l s in s t i l l b i r t h s . Lancet 1:1159 Bull RJ, Lutkenhoff SD, McCarthy GE, M i l l e r RG (1979) Delays in the postnatal increase of cerebral cytochrome concentrations in lead-exposed rats. Neuropharmacol 18:83 -92 Bulman RA, G r i f f i n RJ (1981) C i t r i c acid derivatives f a i l to enhance plutonium clearance from the hamster. Health Physics 40:104-105 Butt EM, Pearson HE, Simonsen DG (1952) Production of meningocele and c r a n i o s c h i s i s in chick embryos with lead acetate. PSEBM 79:247-249 Campbell AMG, William ER, Barltrop D (1970) Motor neurone disease and exposure to lead. J Neurol Neurosurg Psychiatr 33:877-885 Carpenter SJ, Ferm VH (1977) Embryopathic effects of lead in the hamster: a morphological analysis. Lab Invest 1 44 37:369-385 Carpenter SJ, Ferm VH, Gale TF (1973) Permeability of the golden hamster placenta to inorganic lead: radioautographic evidence. Experientia 29:311-313 Catizone 0, Gray P (1941) Experiments on chemical interference with early morphogenesis in the chick. J Exp Zool 87:71-82 Catsch A, Harmuth-Hoene AE (1979) Pharmacology and therapeutics applications of agents used in heavy metal poisoning. In The Chelation of Heavy Metals. Pergamon Press, NY, pp 111-126 Chant DA, DeMaro FA, Robertson 'HR (1974) Effect On Human  Health Of Lead From The Human Environment. Ministry of Health, Ontario, pp 1-67 Charney E, Satyre J, Coulter, M (1980) Increased lead absorption in inner c i t y c h i l d r e n . Where does the lead come from? Pediatr 65: 226-231 Chisolm JJ (1968) The use of chelating agents in the treatment of acute and chronic lead intoxication in childhood. J Pediatr 73 : 1-38 Chisolm JJ (1971) Treatment of lead poisoning. Modern Treatment 8:593 Chisolm JJ (1980) Lead and other metals: A hypothesis of interaction. In Lead T o x i c i t y . Urban and Schwarzenberg, Baltimore-Munich, pp 425-460 Clark ARL (1977) Placental transfer of lead and i t s e f f e c t s in newborns. Postgrad Med J 53: 674-678 Clark JL, Anderson DG (1974) Neighbourhood screening in communities throughout the nation for children with elevated blood lead l e v e l s . Environ Health Perspect 7:3-6 Clasen RA, Hartman JF, Coogan PS, Pandolfi S, Laing I, Becker RA (1974a) Experimental acute lead encephalopathy in the juvenile rhesus monkey. Environ Health Perspect 7:175-185 Clasen RA, Hartmann F, Starr AJ, Coogan PS, Pandolfi S, Laing I, Becker R, Hass GM (1974b) Electron microscopic and chemical studies of the vascular changes and edema of lead encephalopathy. Am J Path 74:215-233 Conrad M, Barton J (1978) Factors a f f e c t i n g absorption and excretion of lead in the rat. Gastroenterology 1 45 74:731-741 Crawford WA, Jones R, Rainsford F, Shander A (1980) Anatomy of an i n d u s t r i a l inorganic lead epidemic. Med J Aust 2:318-319 Dannenberg AM, Widerman AH, Freidman PS (1940) Ascorbic acid in the treatment of chronic lead poisoning. JAMA 114:1439-1440 David OJ, Hoffman SP, Sverd J, Clark J, Voeller K (1976) Lead and hyperactivity. Behavioral response to chelation: a p i l o t study. Am J Psychiat 133:1155-1158 De Bruin A (1971) Certain b i o l o g i c a l e f f ects of lead upon the animal organism. Arch Environ Health 23:249-260 De Fr a n c i s c i s P, Boccalatte F (1962) Lead acetate and the development of the chick embryo. Nature 193:989-990 De Gennaro LD (1978) The eff e c t s of lead n i t r a t e on the central nervous system of the chick embryo. I. Observations of l i g h t and electron microscopy. Growth 42:141-151 Der R, Yousef M, Fahim Z, Fahim M (1977) Effects of lead and cadmium on adrenal and thyroid function in rats. Res Comm Chem Pathol Pharmacol 17:237-253 Evans EE, Norwood WD, Kehoe RA, Mackle W (1943) The eff e c t s of ascorbic acid in re l a t i o n to lead absorption. JAMA 121:501-504 Evans GW, Majors PF, Cernatzer WE (1970) Ascorbic acid interaction with metallothionein. Biochem Biophys Res Comm 41:1244-1247 Fahim MS, Fahim Z, Hall OG (1976) Effects of subtoxic lead levels on pregnant women in the State of Missouri. Res Comm Chem Pathol Pharmacol 13:309-331 Ferm VH, Ferm DW (1971) The s p e c i f i c i t y of teratogenic effects of lead in the golden hamster. L i f e Sci 10:35-38 Fischbein A, Daum SM, Davidow B, Slavin G, Alvares AP, Sassa S, Anderson KE, Kappas A, Eisinger J, Blumberg WE, Winicow EH, S e l i f i f f IJ (1978) Lead hazard among iron workers dismantling an elevated subway l i n e in New York C i t y . NY State J Med 78:1250-1259 Foreman H (i960) The pharmacology of some useful chelating agents. In Metal Binding In Medicine. JB Lippincott Co, Montreal, pp. 82-94 Freeman BM, Vince MA (1974) Development Of The Avian Chick 1 46 Embryo. Halsted Press, NY, pp 164-168; 208-213 Fried JW, Rosenthal MW, Schubert J (1956) Induced accumulation of c i t r a t e therapy in experimental lead poisoning. Proc Soc Exp Biol Med NY 92:331-333 Gale TF (1978) A variable embryotoxic response to lead in d i f f e r e n t strains of hamsters. Environ Res 17:325-333 Garber BT, Wei E (1974) Influence of dietary factors on the g a s t r o i n t e s t i n a l absorption of lead. Toxicol Appl Pharmacol 27:685-691 Garvon FL-(1964) Metal chelates of ethylenediaminetetraacetic acid. In Chelating Agents and Metal Chelates. Academic Press, NY, p 300 Gerber GB, Leonard A, Jacquet P (1980) Toxcity, mutagenicity and teratogencity of lead. Mutat Res 76:115-141 Gerber GB, Maes J, Devoo J (1978) Effects of dietary lead on placental blood flow and f e t a l uptake of alpha-aminoisobutyrate. Arch Toxicol 41:125-131 Gi l a n i SH (1973a) Congenital anomalies in lead poisoning. Obstet Gynecol 41:265-268 Gi l a n i SH (1973b) Congenital cardiac anomalies in lead poisoning. Pathol Microbiol 39:85-90 Gi l a n i SH (1975) Fine s t r u c t u r a l changes in embryonic chick heart v e n t r i c l e induced by lead poisoning. Pathol Microbiol 42: 188-194 G i l f i l l i a n SC (1965) Lead poisoning and the F a l l of Rome. Occup Med 7:53-60 Goldman HM, Gould BS, Munro HN (1981) The antiscorbutic action action of 1-ascorbic acid and d-isoascorbic acid (erythrobic acid) in the guinea pig. Am J C l i n Nutr 34:24-33 Goldstein GW (1977) Lead encephalopathy: the significance of lead i n h i b i t i o n of calcium uptake by the brain mitochondria. Brain Res 136:185-188 Goldstein GW, Asbury AK, Diamond I (1974) Pathogenesis of lead encephalopathy. Arch Neurol 31:382-389 Goyer RA, Cherian MG (1979) Ascorbic acid and EDTA treatment of lead t o x i c i t y in rats. L i f e Science 24:433-438 Goyer RA, Rhyne BC (1973) Pathological e f f e c t s of lead. Int Rev 1 47 Exp Pathol 12: 1-77 Graca JG, Davison FC, Feavel JB (1962) Comparative t o x i c i t y of stable rare earth compounds. I I . Effect of c i t r a t e and EDTA complexing in acute t o x i c i t y in mice and guinea pigs. Arch Environ Health 5:437-444 Graef JW (1980) Management of low le v e l lead exposure. In Low Level Lead Exposure: The C l i n i c a l Implications  Of Current Research. Raven Press, NY, p 123 Grandjean P (1975) Lead in Danes. In Lead. Academic Press, NY, pp 6-20 Grandjean P (1978) Widening perspectives on lead t o x i c i t y . Environ Res 17:303-321 Green VA, Wise GW, Callenbach JC (1980) Lead poisoning. In Toxicity Of Heavy Metals In The Environment, Vol 1. Marcel Dekker, NY, pp 123-141 Hammond PB (1971) The eff e c t s of chelating agents on the tissue d i s t r i b u t i o n and excretion of lead. Toxicol Appl Pharmacol 18:296-310 Hammond PB (1980) Metabolism and metabolic action of lead and other heavy metals. In Toxic i t y Of Heavy Metals In  The Environment, Vol 1. Marcel Dekker, NY, pp 87-99 Hammond PB, Aronson AL (1960) The mobilization and excretion of lead in cattle:A comparative ef f e c t of various chelating agents. Ann NY Acad Sci 88:498-511 Hawkins R (1979) Lead-a weighty problem. Fd Cosmet Toxicol 17: 171-172 Hayes JR. Bremer RA, Wong KC, Jordan WS, Westenskow DR (1980) Continuous monitoring of serum ionized calcium in the dog during sodium c i t r a t e infusion using an extracorporeal shunt. Canad Anaesth Soc J 27:458-463 Hemphill F, Kaerberle LM, Buck WB (1971) Lead suppression of mouse resistance to Salmonella typhimurium. Science 172:1031-1032 Hirano A, Kochen JA (1973) Neurotoxic e f f e c t s of lead in the chick embryo. Morphologic studies. Lab Invest 29:659-668 Hirano A, Kochen JA (1977) Relationship of blood and brain lead levels to morphological Changes in lead induced chick embryo encephalopathy. I. Morphological changes. In Neurotoxic i t y . Raven Press, NY, pp SOS-SOS 1 48 Hoffman V, Segewitz G (1979) Influence of chelation on acute lead intoxication in rats. Arch Toxicol 34:213-225 Holmes HN, Campbell K, Amberg EJ (1939) The effect of vitamin C on lead poisoning. J Lab C l i n Med 24:1119-1127 Holtzman D, Herman MM, Hsu SJ, Martile P (1980) The pathogenesis of lead encephalopathy. Effects of lead carbonate feedings on morphology, lead content and mitochondrial respiration in brains of immature and adult rats. Virchows Arch A Path Anat H i s t o l 387:147-164 Hopping JM, Ruliffson WS (1963) Ef f e c t s of chelating agents on radioiron absorption and d i s t r i b u t i o n in rats in vivo. Am J Physiol 205:885-889 Hopping JM, Ruliffson WS (1966) Roles of c i t r i c and ascorbic in acid enteric iron absorption in rats. Am J Physiol 210: 1316-1320 Hughes RE, Hurley RJ, Jones PR (1971) Vitamin C a c t i v i t y of d-araboascorbic acid. Nutr Rep Int 4:177-183 Hughes RE, Jones PR (1970) D-araboascorbic acid and guinea pig scurvy. Nutr Rep Int 3:275-279 Jacquet P, Gerber GB (1979) Teratogenic effects of lead in the mouse. Biomedicine 30:223-229 Jacquet P, Gerber GB, Leonard A, Maes J (1977a) Plasma hormone levels in normal and lead treated pregnant mice. Experientia 33: 1375-1376 Jacquet P, Gerber GB, Maes J (1977b) Biochemical studies in embryos after exposure of pregnant mice to dietary lead. B u l l Environ Contam Toxicol 18:271-277 Jacquet P, Leonard A, Gerber GB (1975) Embryonic death in the mouse due to lead. Experientia 31:1312-1313 James LF, Lazar VA, Binns W (1966) Effects of sublethal doses of certain minerals on pregnant ewes and f e t a l development. Am J Vet Res 27:132-135 Jaworski JF (1979) The Effects Of Lead In The Canadian Environment. National Research Council Of Canada. Publication No. 16736, Ottawa, Canada, pp 7-22 Jugo S, Maljkovic T, K o s t i a l K (1975) Influence of chelating agents on the g a s t r o i n t e s t i n a l absorption of lead. Toxicol App Pharmacol 341:259-263 Kadin H, Osadca M (1959) Biochemistry of erythorbic acid: human 149 blood levels and urinary excretion of ascorbic and erythorbic acids. J Agr Fd Chem 7:358-362 Karnofsky DA, Ridgeway LP (1952) Production of injury to the CNS of the chick embryo by lead s a l t s . J Pharmacol Exp Ther 104:176-186 Kennedy GL, Arnold DW, Calandra JC (1975) Teratogenic evaluation of lead compounds in mice and ra t s . Toxicol 13:629-632 Kety SS, Letonoff MS (1943) The treatment of lead poisoning by sodium c i t r a t e . Am J Med Science 205:406-414 Khera AK, Wibberly DG, Dathan JG (1980) Placental and s t i l l b i r t h tissue lead concentrations in occupationally exposed women. Br J Med 37:394-396 Kimmel CA, Grant LD, Sloan CS (1976) Chronic lead exposure: assessment of developmental t o x i c i t y . Teratol 13:27A-28A King DW, Chen CC, Wun WS, Hsu JL (1978) Effects of zinc and lead on the a c t i v i t y of d- aminolevulinic acid dehydratase Of the chick embryo. Teratol 17:49A King DW, Lui J (1974) The effects of lead acetate on chick embryonic development. Teratol 9:A25 King DW, Lui J (1975) Reduction of lead acetate teratogenicity in the chick embryo by ascorbic acid. Fed Proc 34:1049 Kochen JA, Greener Y (1974) Lead t o x i c i t y and tissue levels in the embryo. Pediatr Res 8:358 Kochen JA, Greener Y (1976) Lead encephalopathy: a consequence of c a p i l l a r y dysfunction. Pediatr Res 10:449 Kutnink MA, Tolbert BM, Richmond VL, Baker EM (1969) Ef f i c a c y of the ascorbic acid stereoisomers in proline hydroxyaltion in v i t r o . Soc Exp B i o l Med 132:440-442 Letonoff TV, Kety SS (1943) The effect of sodium c i t r a t e administration on excretion of lead in urine and feces. J Pharmacol 77:151-153 Levander OA (1979) Lead t o x i c i t y and n u t r i t i o n a l d e f i c i e n c i e s . Environ Health Perspect 29:115-125 Lewin S (1974) Recent advances in the molecular biology of Vitamin C. In Vitamin C. Recent Aspects Of Its  Physiological And Technological Importance. John Wiley and Sons, NY, pp 224-225 150 L i l i s R, Fischbein A, Diamond S, Anderson HA, Selikoff IJ, Blumberg WE, Eisinger J (1977) Lead effects among secondary lead smelter workers with blood lead levels below 80 ug/100 ml. Arch Environ Health 32(6):256-266 Lorenzo AV, Gewirtz M, A v e r i l l D (1978) CNS lead t o x i c i t y in rabbit o f f s p r i n g . Environ Res 17:131-150 Mahaffey KR, Bank TA (1975) Effect of varying dietary ascorbic acid on lead t o x i c i t y in the guinea pigs. Fed Proc 34:267 Marchmont-Robinson SW (1941) Effect of vitamin C on workers exposed to lead dust. J Lab C l i n Med 26:1478-1471 Martin MT, L i c k l i d e r KF, Brushmiller JG, Jacobs FA (1981) Detection of low molecular weight copper and zinc binding ligands in u l t r a f i l t e r e d milks-the c i t r a t e connection. J Inorg Biochem 15:55-65 McClain RM, Becker BA (1970) Placetal transport and teratogenicity of lead in rats and mice. Fed Proc 29:347 McClain RM, Becker BA (1975) Teratogenicity, f e t a l t o x i c i t y and placental transfer of lead n i t r a t e in rats. Toxicol Appl Pharmacol 31:72-82 McClain RM, Siekierka JJ (1975a) The effects of various chelating agents on the teratogenicity of lead n i t r a t e in rats. Toxicol Appl Pharmacol 31:434-442 McClain RM, Siekierka JJ (1975b) The placental transfer of lead chelate complexes in the rat. Toxicol Appl Pharmacol 31 :443-45 McLaughlin J, Marliac JP, Verrett MJ, Mutchler MK, Fitzhugh OG (1963) The i n j e c t i o n of chemicals into the yolk sac of f e r t i l e eggs prior to incubation as a t o x i c i t y t e s t . Toxicol Appl Pharmacol 5:760-761. McLellan JS, Vonsmolinski AW, Bederka JP, Boulos BM (1974) Development toxicology of lead in the mouse. Fed Proc 33:288 McNiff EF, Cheng LK, Woodfield HC, Fung H (1978) Effects of 1-cysteine, 1-cysteine derivatives and ascorbic acid on lead excretion in rats. Res Comm Chem Pathol Pharmacol 20:131-137 Mellor DP (1964) H i s t o r i c a l background and fundamental concepts. In Chelating Agents and Metal Chelates. Academic Press, NY, pp 1-50 151 Moore MR, Meredith PA, Goldberg A (1980) Lead and heme biosynthesis. In Lead T o x i c i t y . Urban and Schwarzenberg, Baltimore-Munich, pp 79-118 National Research Council (1973) Lead In The Canadian . Environment Publication no. B473-71ES, NRCC/CNRC Ottawa, Canada, pp 11-16 National Research Council (1980) Lead In The Human Environment. National Academy of Sciences, Washington, D.C, pp 1-269 Needleman HL (1980) Human lead exposure: d i f f i c u l t i e s and strategies in the assessment of neuropsychological impact. In Lead T o x i c i t y . Urban and Schwarzenberg, Baltimore-Munich, pp 3-17 Nolen GA, Bohne RL, Buehler EV (1972).Effects of trisodium n i t r i l o t r i a c e t a t e , trisodium c i t r a t e and a trisodium n i t r i l o t r i a c e t a t e - f e r r i c chloride mixture on cadmium and methyl mercury t o x i c i t y and teratogenesis in rats. Toxicol Appl Pharmacol 23:238-250 Oehme FW (1980) Mechanism of heavy metal inorganic t o x i c i t i e s . In Toxicity Of Heavy Metals In The Environment, Vol _U Marcel Dekker, NY, pp 69-74 O'Tuama LA, Kim CS, Gatzy JT, Kingman MR, Mashaki P (1976) The d i s t r i b u t i o n of inorganic lead in guinea pig and neural barrier tissues in control and lead poisoned animals. Toxicol Appl Pharmacol 36:1-9 Pal DR, Chatterjee J, Chatterjee GC (1975) Influence of lead administration on 1-ascorbic acid metabolism in rats: e f f e c t s of 1-ascorbic acid supplementation. Int J V i t Nutr Res 43:429-473 Palmisano PA, Sneed RC, Cassady G (1969) Untaxed whiskey and f e t a l lead exposure. J Pediatr 75:869-871 Papaioannou R, Sohler A, P f e i f f e r C (1978) Reduction of blood lead levels in battery workers by zinc and vitamin C. J Ortho Psychiatry 7:94-106 Patterson CC (1980) An alte r n a t i v e perspective-lead p o l l u t i o n in the human environment: o r i g i n , extent, signi f i c a n c e . In Lead In The Human Environment. National Academy Of Sciences, Washington, D.C, pp 271-349 P e l l e t i e r 0, Godin C (1969) Vitamin C a c t i v i t y of d-isoascorbic in the guinea pig. Can J Physiol Pharmacol 47:985-991 1 52 Pentschew A (1965) Morphology and morphogenesis of lead encephalopathy. Acta Neuropathologica 5:133-160 Pentschew A, Garro F (1966) Lead encephalopathy of the suckling rat and i t s implications on the porphyrinopathic nervous disease. Acta Neuropathologica 6:266-278 Peterson NA, Crisman JM (1961) Effect of c i t r a t e on u l t r a f i l t r a b l e and ionic calcium concentrations in serum. J Appl Physiol 16:1103-1108 Petrofsky B (1972) Regulation of collagen secretion by ascorbic acid in 3T3 and chick embryo f i b r o b l a s t s . Biochem Biophys Res Comm 49:1343-1350 Pillemer L, S e i f t e r J, Kuehn AO, Ecker EE (1940) Vitamin C in chronic lead poisoning. An experimental study. Am J Med Sci 200: 322-327 Popoff N, Weinberg S, Feign I (1963) Pathologic observations in lead encephalopathy. Neurology 13:101-112 Priest RE, Bublitz C (1967) The influence of ascorbic acid and tetrahydropteridine on the synthesis of hydroxyproline by cultured c e l l s . Lab Invest 17:371-379 Repko JD, Corum CR, Jones PD, Garcia LS (1978) The E f f e c t s Of  Inorganic Lead On Behavioral And Neurologic  Function. NIOSH, U.S. Gov't Printing O f f i c e , Washington, D.C, pp 1-64 Reynolds SE (1963) The use of lead c i t r a t e at high pH as an electron-opaque stain in chelation microscopy. J C e l l B i o l 17:208-213 Ridgeway LP, Karnosky DA (1952) The effects of metals on the chick embryo: t o x i c i t y and production of abnormalities in development. Ann NY Acad Sci 55:203-215 Rivers JM, Huang ED, Dodds ML (1963) Human metabolism of 1-ascorbic acid and erythorbic acid. J Nutr 81:163-1 68 Romanoff AL, Romanoff AJ (1972) Pathogenesis Of The Avian  Embryo. Wiley-Interscience, NY, pp 1-379 Sandstead HH, Start EG, Bu l l AB (1969) Lead intoxication and the thyroid. Arch Int Med 123:632-635 Schoenbord D (1980) Why regulation of lead has f a i l e d . In Low Level Lead Exposure. Raven Press, NY, pp 267-278 1 53 Settle DM, Pattersen CC (1980) Lead in albacore: guide to lead p o l l u t i o n in Amercians. Sci 207:1167-1176 Seven MJ (1960) Observations on the t o x i c i t y of intravenous chelating agents. In Metal Binding In Medicine. JB Lipincott Co, Montreal, pp 95-103 Shulman A, Dwyer FP (1964) Metal chelates in b i o l o g i c a l systems. In Chelating Agents And Metal Chelates. Academic Press, NY, pp 383-415 Smith H, Stradling GN, Bulman RA, Ham GJ (1976) Experimental studies in the use of c i t r a t e to enhance the urinary excretion of plutonium in the rat. Health Physics 30:318-320 Smith JL (1976) Metabolism and t o x i c i t y of lead. In Trace Elements In Human Health And Disease, Vol I I . Academic Press, NY, pp 443-451 Sohler A, Kruesi M, P f e i f f e r C (1977) Blood lead levels in psychiatric outpatients reduced by zinc and vitamin C. J Ortho Psychiatry 6:272-276 Sorensen EMB, Moretti ES, Lindenbaum A (1980) Chelation therapy and tissue d i s t r i b u t i o n and excretion of lead in mice. Arch Environ Contam Toxicol 9:619-626 Spivey Fox MR (1975) Protective effects of ascorbic acid against the t o x i c i t y of heavy metals. Ann NY Acad Sci 258:144-150 Spivey Fox MR, Fry BE, Harland BF, Shertel ME, Weeks CE (1971) Effects of ascorbic acid on cadmium t o x i c i t y in the young coturnix. J Nutr 101:1295-1305 Suzuki T, Yoshida A (1979a) Effect of dietary supplementation of iron and ascorbic acid on lead t o x i c i t y in rats. J Nutr 109:982-988 Suzuki T, Yoshida A (1979b) Effectiveness of dietary iron and ascorbic acid in the prevention and cure of moderate long term lead t o x i c i t y in rats. J Nutr 109:1974-1978 Swenerton H, Hurley LS (1971) Teratogenic e f f e c t s of a chelating agent and their prevention by zinc. Sci 173:62-63 Taitz LS, Kravath R (1967) Treatment of hypercalcemia. Lancet 2:1254-1255 Thomas JA, Dallenbach FD, Thomas MI (1971) Considerations on the the development of experimental lead encephalopathy. Virchows Arch Abst A Path Anat 352:61-72 1 54 Thomas JA, Thomas MI (1974) The pathogenesis of lead encephalopathy. Ind J Med Res 62:36-41 Toews AD, Kholer A, Hayward J, Krigman MR, Marell P (1978) Experimental lead encephalopathy in the suckling rat: concentration of lead in c e l l u l a r fractions enriched in brain c a p i l l a r i e s . Brain Res 147:131-138 Van Gelder GA (1980) Lead and the nervous system. In Toxicity  Of Heavy Metals In The Environment, Vol I. Marcel Dekker, NY, pp 101-121 V i s t i c a DT, Ahrens FA, E l l i s o n WR (1977) The e f f e c t s of lead upon collagen syntheisis and proline hydroxylation in swiss mouse 3T6 f i b r o b l a s t . Arch Biochem Biophys 179:15-23 Waldron HA (1973) Lead poisoning in the ancient world. Med His 17:391-398 Waldron HA, Stofen D (1974) S u b c l i n i c a l Lead Poisoning. Academic Press, NY, pp 3-5 Wang MM, Fischer KH, Dodds ML (1962) Comparative metabolic response to erythorbic acid and ascorbic acid by the human. J Nutr 77:443- 448 Warkany J (1971) Congenital Malformations Year Book. Medical Publishers, Chicago, pp 217-229 Wibberley DG, Khera AK, Edwards JH, Rashton DI (1977) Lead levels in human placentae from normal and malformed bi r t h s . J Med Genet 14:339-345 Wilson AT (1966) E f f e c t s of abnormal lead content of water supplies on maternity patients. Scot Med J 11:73-82 

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