STUDIES EN EXPERIMENTAL DEELDRBN AND MTRATE TOXECGSES EN. SWENE MD GUENEA PIGS Thesis for the Degree of Ph. D. MiCHlGAN STATE UNIVERSITY MBA UZOUKWU 1970 ‘v' 21'- TN” LIB- k x: Q Y Michigan f‘ta ti; 8 Unwm “Y f": ,r;‘ .' H. -.-. V 10-. ..n‘.“ It “a. ‘.1~.0 This is to certify that the thesis entitled Studies in Experimental Dieldrin and Nitrite Toxicoses in Swine and Guinea Pigs presented by Mba Uzoukwu has been accepted towards fulfillment of the requirements for S. D. Sleight! Professor Major professor Date May 22, 1970 0-169 _ .39”. .. F . . BINDING BY ‘ a. Llammr amazes j “ mun-an. “can: ABSTRACT STUDIES IN EXPERIMENTAL DIELDRIN AND NITRITE TOXICOSES IN SWINE AND GUINEA PIGS by Mba Uzoukwu Seven experiments were designed in which dieldrin with or without NaN02 was utilized to study the toxicity of dieldrin to swine and guinea pigs. Some of the experiments were intended to provide evidence of transplacental transfer of dieldrin and any fetotoxic effects resulting from suth'transfers. The effect of a protein-deficient diet on dieldrin toxicity in swine was studied. The ultrastructural changes in the brain and liver in acute dieldrin poisoning were observed. Parameters for evaluation of toxicity were death, abortion, body weight-changes, gross and histopathologic lesions, and ultrastructural changes. Hematologic determinations revealed no significant deviations from normal values. The acute oral toxic dose (LDSO) of dieldrin for guinea pigs was calculated to be 45 mg./kg. .Acute poisoning of guinea pigs with_(75. mg./kg.) dieldrin resulted in convulsions and lesions were characterized submicroscopically by swelling of cerebellar and cerebral mitochondria with disintegration of associated cristae. Administration of dieldrin with or without NaNOz to pregnant sows resulted in accumulation of the pesticide in maternal and fetal tissues, without causing abortion or any observable lesions in the fetuses. The amounts stored in the maternal tissues were not found to be proportional Mba Uzoukwu to quantities given. However, there was a statistically significant difference (P < 0.025) in the amount of dieldrin stored in the fetal liver which appeared to depend on the total amount of the pesticide given to the dam. There was no evidence of synergism in the toxicoses of dieldrin and NaN02 in the pregnant sow. Deaths but no abortions resulted from the administration of dieldrin (60 mg./kg.) to pregnant guinea pigs. Abortion but no deaths occurred when NaNOz (30 mg./kg.) was also given. However, dieldrin at 100 ppm in the feed caused 2 deaths and 2 abortions in 5 guinea pigs and nitrite (0.5% in H20) caused 3 out of 5 recipient guinea pigs to abort. Simul- taneous administration of dieldrin (100 ppm) and nitrite (0.5%) caused 3 deaths and 6 abortions in 10 dosed guinea pigs. Administration of dieldrin at the level of 50 ppm or less with nitrite (0.25%) resulted in neither abortions nor deaths. There is, therefore, no evidence of potentiation of the toxicity of dieldrin by nitrite. Adult guinea pig tissues stored dieldrin in prOportion to the total amount administered and this proportionality was not affected by the simultaneous administration of NaNOZ. However, fetal storage did not appear to be in direct prOportion to the quantity consumed by the dam unless nitrite was also administered. There was no significant effect on food and water consumption, or on growth of guinea pigs fed dieldrin at 50 ppm or less for up to 53 days. Protein-deficient diet (4% casein) fed to pigs for 21 days appeared to enhance their susceptibility to the toxic effects of dieldrin, but was associated also with a reduced capacity on the part of the pig to accumu- late the pesticide in its tissues. Mba Uzoukwu The gross and histopathologic lesions were similar in both dieldrin and dieldrin/nitrite treated swine and guinea pigs. These lesions con- sisted of a generalized hyperemia of tissues and fatty metamorphosis of. parenchymatous organs. Brownish discoloration of tissues due to methemo- globinization was evident in the nitrite-treated sows and guinea pigs. The most important uterine lesion in the pregnant guinea pigs consisted of thickening and vacuolar degeneration of the arterial.media of small arteries with partial or total occlusion of the lumen. STUDIES IN EXPERIMENTAL DIELDRIN AND NITRITE TOXICOSES IN SWINE AND GUINEA PIGS By Mba Uzoukwu A.THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of. DOCTOR OF PHILOSOPHY Department of Pathology 1970 Dedicated to those who died to preserve my freedom ii ACKNOWLEDGEMENTS Of all who in various ways assisted in the planning and execution of this work, I wish to express my immense gratitude to Dr. S. D. Sleight, my major professor. His guidance, patience, and readiness to share his eXperiences and resources made the research possible. To Dr. C. C. Morrill, Chairman of the Department of Pathology, Dr. C. K. Whitehair, Dr. R. F. Langham and Dr. G. L. waxler, Professors of Pathology, and Dr. 0. Mickelsen, Professor of Foods and Nutrition and of Biochemistry, I wish to extend my sincere appreciation for their advice and encouragement in the development of the research prOposal. Their invaluable criticism of this manuscript is gratefully acknowledged. The author is very thankful to Mrs. D. Fenner for the serum protein and hematologic determinations; Mrs. M. K. Sunderlin, Mrs. N. L. Miller, and Mrs. F. M. Whipple for preparing the histOpathologic sections; Dr. M. Zabik and his staff for the gas chromatographic analyses of tissues; and Mrs. J. Mack for help in producing the electronmicrographs. Final appreciation is extended to many.others not mentioned, but especially to Mr. J. Southern, the Animal Caretaker, and to the Rocke- feller Foundation whose generosity made it possible for me to carry out this work. iii TABLE.OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . .,. . . . . . . . . . . . . 1 REVIEW OF THE LITERATURE. . . . . . . . . . . . . . . . . . . . . . 3 General Principles . . . . . . . . . . . . . . . . . . . . . 3 History. . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Mechanism of Toxicosis . . . . . . . . . . . . . . . . . . . 4 Metabolism of Dieldrin . . . . . . . . . . . . . . . . . . . 7 Retention of Dieldrin in Body Tissues. . . . . . . . . . . . 9 Predisposing Factors to Dieldrin Toxicity. . . . . . . . . . 10 Systemic Effects of Dieldrin . . . . . . . . . . . . . . . . 13 The Pathology of Dieldrin Toxicosis. . . . . . . . . . . . . 14 Ultrastructural Changes in Dieldrin Toxicosis. . . . . . . . 16 MATERIALS AND METHODS . . . . . . . ... . . .~. . . . . . . . . . . 17 Pigs . . . .'. . . . . . . . . . . . . . . . . . . . . . . . 17 Guinea Pig Experiments . . . . . . . . . . . . . . . . . . . 19 EXPERIMENTAL 'PROCEDUREAND RESULTS. . . . . -. . . . . . . . . . 20 Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Guinea Pigs. . . . . . . . . . . . . . . . . . . . . . . . . 38 ULTRASTRUCTURAL STUDIES . . . . . . . . . . . . . . . . . . . . . . 59 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . 59 Results: Ultrastructural Lesions. . . . . . . . . . . . . . 60 DISCUSSION. . . . . . . . . . . .*. . . . . . . . . . . . . . . . . 66 SWY AND CONCLUSIONS 5 I O O O O C O I O O O O O O O O O O O O O C 73 iv REFERENCES . VITA. . . LIST OF TABLES Table Page 1 Composition of.the protein-deficient diet for pigs in Experiment I Q 0 O O O O O O O O O O O O O O O O I I O O 0 O O 21 2 Serum proteins--tota1 protein, albumin, and oz globulin values, A/G ratios of representative pigs not given dieldrin in Experiment I I I I O O O O O . O O O O I O O l O O O I O O O Q 24 3 Dieldrin concentrations (ppm) in tissues from representa- tive treated pigs in Experiment I . . . . . . . . . . . . . . 25 4 Duration of treatment and total dieldrin intake of sows in Experiment II 0 O O O O O I O O O O O O O O O O O O O O O O 28 5 Residual dieldrin concentration (ppm) in tissues of sows in Experiment II and total amounts of dieldrin given. . . . . 30 6 Residual dieldrin concentration (ppm) in fetal tissues from Experiment II I I O I O I O C O I O O O I O O C C O O ' O O 31 7 Duration of treatment and total dieldrin and NaN02 intake by sows in Experiment III . . . . . . . . . . . . . . . . . . 34 8 Dieldrin concentrations in tissues of sows and their fetuses in Experiment III . . . . . . . . . . . . . . . . . . 36 9 Groups and numbers of guinea pigs represented, sex, dosage, duration, and total quantity of dieldrin given in. Experiment IV-. . . . . . . . . . . . . . . . . . . . . . . . 39 10 Groups and numbers of guinea pigs sampled, total dieldrin received, and average tissue and fetal concentrations (ppm) of dieldrin in Experiment IV. . . . . . . . . . . . . . 41 11 Numbers of guinea pigs represented, sex, total quantity of chemical given and duration of observation in Experiment V. . 47 12 Average tissue and fetal dieldrin concentration, total dieldrin and nitrite given and numbers of guinea pigs represented in Experiment V . . . . . . . . . . . . . . . . . 49 13 Number of female guinea pigs, concentrations of.chemicals, and periods of observation in Experiment VI (Part I). . . . . 53 Table 14 15 16 Page Concentrations of chemicals given in feed and water for 53 days, and groups of pregnant and nonpregnant guinea pigs in Part II of Experiment VI. . . . . . . . . . . . . . . 54 Mean body weights (gm.) of guinea pigs at about 6-day intervals during Part II of Experiment VI . . . . . . . . . . 55 Acute oral toxicity of dieldrin to guinea pigs--numbers, doses and mortality 0 O O O I O O O O O O O O O O O O 0 O O O 71 vii LIST OF FIGURES Figure Page 1 Group average weights of pigs fed normal (N) or protein deficient diet (D) for 21 days. . . . . . . . . . . . . . . 22 2 Liver. Lipidosis in pig fed protein deficient diet and dosed With dieldrin O O O I O O O O O O O O O O O O I O O O 27 3 Kidney. Vacuolar degeneration of epithelium of proximal convoluted tubules of dieldrin-treated sow. . . . . . . . . 32 4 Kidney. Vacuolar degeneration of proximal convoluted tubular epithelium of dieldrin-nitrite treated sow. . . . . 37 5 Liver. Lipidosis in dieldrin-treated guinea pig. . . . . . 42 6 Liver. Control. Note presence of glycogen confirmed by Best's carmine stain. . . . . . . . . . . . . . . . . . . . 42 7 Kidney. Epithelial vacuolar degeneration in proximal tubules of dieldrin-treated guinea pig. . . . . . . . . . . 44 8 Lung. Hyperemia, atelectasis and alveolar emphysema in dieldrin-treated guinea pig . . . . . . . . . . . . . . . . 45 9 Uterus. Occlusion of small artery, thickening and vacuola- tion of arterial media in dieldrin-nitrite treated guinea pig 0 O O O 0 O I O O I O O O I O O I O O O O O O O O O O O 51 10 Uterus. Hyperplasia, vacuolation and necrosis (arrows) in guinea pig treated with dieldrin and nitrite . . . . . . 51 11 Renal hyperemia and vacuolar degeneration of proximal tubular epithelium in guinea pig treated with dieldrin and nitrite O O O O I O I O O O I O O O O O I O O O O O O O 58 12 Note swollen mitochondria (M) with disruption of cristae in cerebral cells . . . . . . . . . . . . . . . . . . . . . 61 13 Control. Junction of three cerebellar cells, nuclei (N), cell membranes (C) and normal mitochondria (M). . . . . . . 62 14 Part of a hepatocyte from a dieldrin-treated guinea pig. Note myelin bodies (B), normal mitochondria (M) and normal endOplasmic reticulum (arrows). . . . . . . . . . . . . . . 64 15 Hepatocytes in a dieldrin-treated guinea pig. Note the nor- mal appearance of the mitochondria (M) and the endOplasmic reticulum (arrows). . . . . . . . . . . . . . . . . . . . . 65 viii INTRODUCTION Pesticides of the cyclodiene group to which dieldrin belongs are used commonly for agricultural, domestic, and public health purposes. Consequently, extensive studies have been made into the toxicoses asso- ciated with their use. While much is known about the clinical signs and lesions that result from poisoning by these chemicals, the underlying mechanisms~are not clearly established. Usually, these substances cause terminal con- vulsions. Some in the group have been associated with soft-shelling and reduced hatchability of eggs or increased mortality in the young of some species of birds even at low dietary concentrations. Evidence, however, indicates a strong species variability in.susceptibility to poisoning by members of this group of pesticides. It is necessary, therefore, to determine minimum no—effect levels of each pesticide for each species investigated. The rat has been the laboratory animal of choice for most investi- gators. The guinea pig has been used rarely. Of the farm animals, the pig.has been investigated the least. The purpose of this study was to obtain some information on the effects of dieldrin on pregnancy, the fetus, life span, body weight, and tissues of guinea pigs and pigs fol- lowing administration of the pesticide with-or without sodium.nitrite. In particular, the 2 species were selected for the study of the effects of toxicosis on pregnancy because it was conjectured that the differences in placentation will possibly provide varying degrees of resistance to 2, passage of the pesticide to the fetus. An attempt was made to show a correlation between tissue levels of dieldrin and any observed signs and/ or pathologic changes. The mechanism of initiation of the terminal convulsions is not agreed upon; neither has any definite gross pathologic change been observed. This investigation included an electron microscopic study intended to obtain evidence of any damage to the central nervous system on the sub- microscopic level. REVIEW OF THE LITERATURE General Principles Dieldrin (l,2,3,4,lO,10—hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a- octahydro-l,4—endo,exo-5,8-dimethano-naphtha1ene) or HEOD is the oxida- tion product of a parent compound, aldrin. Both are chlorinated hydro- carbon compounds with strong pesticidal properties and rather long residual preperties, hence the colloquial designation of~"hard" or "persistent" pesticides. The cyclodiene pesticides are the addition products obtained by condensation of chlorinated double—bonded compounds with hexachlorocyclo- pentadiene, C1______—C Ll Hexachlorocyclopentadiene Dieldrin is derived from aldrin by epoxidation of the double bond in the second ring as shown in the structural schematic representation: Cl Aldrin Dieldrin and this is the mode of metabolism of aldrin in the mammalian body. That this epoxidation of aldrin may not be a detoxification but rather a toxi— fication was suggested by Brooks et al. (1963a,b), who showed that oxygen 3 4 was required to produce the actual toxicant in houseflies treated with aldrin. Perry at al. (1958) had observed that although there was an ade- quate amount of heptachlor in the housefly during the latent period, the onset of signs of poisoning coincided with the appearance of heptachlor epoxide. Histogy The cyclodiene pesticides are a relatively recent discovery. With the development of o-u-o-trichloro-B-B-bis (p-chlorOphenyl)ethane (DDT) in 1939, an intensive search for other halogenated compounds-with insec- ticidal prOperties was launched. Julius Hyman in the United States of America, and Reimschneider in Germany, working independently, are credited with the development of the first insecticide of the diene group known as chlordane or M410 in 1945 (O'Brien, 1967). Aldrin and dieldrin were both developed about 1948 by the Hyman Company. The most recent addi- tions, Thiodan or Endosulfan in 1956 and Telodrin in 1957, were made in Germany. For more than a decade these compounds have been used exten- sively for agricultural, domestic, and public health purposes. Within this period, knowledge has also accumulated of their relative stability, photochemical products, and the hazards consequent on the virtual per- manent contamination of soils, plants, and water with which they come in contact. Environmental contamination by these persistent pesticides has accordingly become a major concern of conservationists and public health officials in recent times. Mechanism of Toxicosis The-toxicity of a contact insecticide is believed to be a net result of several factors, via., penetration of the tissues, enzymatic metabolism with formation of more or less toxic metabolites,-elimination and storage 5 of unchanged insecticide or its metabolites. From the symptomatology and experimental observations of isolated nerves, the cyclodienes are pri— marily neurotoxicants. However, the exact mechanism of their intoxica- tion is not clearly understood. Gowdey at al. (1952, 1954) and Gowdey and Stavraky (1955) first reported signs of toxicosis related to the auto- nomic nervous system in aldrin-poisoned cats.- These signs were referable to peripheral sympathetic action and consisted of bradycardia, hypotension and decreased excretion of saliva by the submaxillary gland. These were not abolished by vagotomy, thus confirming the peripheral origin of the effects observed. Dieldrin, on the other hand, slowed the heart by a central mechanism which was abolished by vagotomy. Excessive excitability and convulsions caused by both chemicals are said to be related to their actions on the central nervous system. Giannotti at al. (1956) reported on their experiments with cock— roaches (Periplanata americana) in which both aldrin and dieldrin were used. They were able to produce convulsions in decapitated poisoned roaches. Moreover, decapitation after poisoning had no apparent effect on the development of signs. Nicotine, in low concentration, was an. effective antidote, suggesting that the effects of aldrin/dieldrin poi- soning are on synaptic transmission which appears to be enhanced. (Nicotine blocks synaptic but not axonal transmission in insects.) Lalonde and Brown (1954) obtained delayed short trains of impulses from the central nerve of P. americana after they crushed the ganglion and applied various cyclodiene insecticides. This confirmed the peripheral action of the insecticides. Matsumura and Hayashi (1966) quoted the work of Yamasaki and Narashishi who found a direct relationship of the action of dieldrin to the central nervous system. These investigators had shown that dieldrin- 6 poisoned nerves of P. americana manifested spontaneous bursts of action potential. They also found that the latent periods between the applica- tion of dieldrin and observation of a discharge by the nervous system was much longer in dieldrin-resistant than susceptible houseflies. Some biochemical changes have been associated with dieldrin poison- ing. Thus, Hossein and Proulx (1960) obtained extracts of brains from rats injected intraperitoneally with dieldrin. The extracts contained betaine esters, straight-chain compounds with a quaternary nitrogen at one end and a carboxyl group at the other, e.g., butyrobetaine [(CH3)$N(CH2)2COOH], in what they called the supernatant ALfraction instead of the §_fraction. It was concluded that these must have been released from their normal cellular attachments in mitochondria by the convulsive agents, and this conclusion implied mitochondrial damage.’ These workers also found the esters to be indistinguishable from acetyl- choline on assay preparations. However, doubt has been cast on this interpretation by the work of McLennan et a1. (1963), who demonstrated that the chromatographic bond which had been claimed to be a betaine derivative by Hosein et‘aZ. (1962) was really the trichloroacetate of acetylcholine formed when the tissue was treated with trichloroacetic acid. Colhoun (1960) showed that neither gamma—butyrobetaine nor its esters appear in nerve cords of dieldrin-poisoned roaches. On the other hand, Hathway et a1. (1965) have shown that substantial changes in- intermediates of brain metabolism occur in rats poisoned with dieldrin and other chemicals. During acute dieldrin poisoning increases in lac- tic and pyruvic acid concentrations in the rat brain were aesociated with hyperactivity of the brain, whereas an increase in cerebral alanine concentration occurred before convulsions. Ammonia concentration in the brain fluctuated out of phase with the actual convulsions. Conversion 7 of ammonia into glutamine in the brain is regarded as the principal mechanism for protection against the toxic effects of ammonium ion. In this process glutamic and pyruvic acids and alanine are utilized in the ammonia-binding mechanism. In the later states of dieldrin-induced seizure free ammonia accumulates in cerebral tissues. Consequently, it has been suggested that dieldrin inhibits the ammonia-binding mechanism and this, in turn, results in inhibition of glutamine synthesis. O'Brien (1967) is of the opinion that these effects are more likely to be the result rather than the cause of the convulsions since comparable changes occur in poisoning by such unrelated convulsants as picrotoxin. Metabolism of Dieldrin Epoxides of the cyclodienes are formed only when the apprOpriate double bond exists. With dieldrin further epoxidation is not possible and it has been shown that insects seem to be unable to metabolize dieldrin (Cohen and Smith, 1961; Brooks, 1960). In confirmation, Earle (1963) demonstrated that dieldrin taken up by housefly larvae persisted through metamorphosis into the adult when it was slowly excreted as unaltered dieldrin. In contrast to insects, mammals appear to be capable of degrading dieldrin. In 1964, Heath and Vandekar reported that 32 of the dieldrin dissolved in oil and administered orally to rats was eventually excreted, unchanged. Nine-tenths of the remaining unidentified metabolites was excreted in the feces as "metabolite 1" while the remaining one-tenth was excreted in the urine. The authors suggested that dieldrin was metabolized in the liver. Cueto and Hayes (1962) showed that men occue pationally exposed to dieldrin had at least 2 metabolites of this chemi- cal in their urine. Both metabolites were neutral, polar and chlorinated. 8 In 1963, Morsdorf et a2. gave radioactive dieldrin (C14-dieldrin) to rats and demonstrated that-it was excreted primarily through the bile as a glucuronide composed mainly of an unknown polar product. In 1965, Korte and Argent administered C14—die1drin to rabbits by stomach tube and iso- lated 6 metabolites from the urine. These were characterized by thin- layer chromatography. Metabolite V, which was.excreted in largest quan- tity (862), was identified as one of the 2 enantiomorphic isomers of aldrin. The acute toxicity of this metabolite was found to be 1/12th to l/6th that of dieldrin. Perhaps of greater significance with regard to environmental con- tamination is the possibility of-conversion of dieldrin on or in plants and the soil to more or less toxic products by sunlight. This has been adequately reviewed by Soto and Deichmann (1967). Mitchell (1961) had observed that strong artificial ultraviolet light could cause chemical changes in organochloride pesticides impregnated on filter paper. In 1963, Roburn.first reported the presence of an unknown compound on grass previously treated with dieldrin. The same compound was recovered after ultraviolet irradiation of dieldrin on a glass plate. Rosen et*aZ. (1966) and Robinson et a1. (1966) identified this photoconversion product as the hexacyclo isomer of dieldrin or photOproduct VII. Rosen et a1. (1966) determined that it has twice the toxic prOperties of dieldrin to houseflies and mosquitoes. They suggested that this may be due to a more rapid and thorough penetration of the cuticle. Brown et al.(1967) confirmed its greater toxicity and also agreed with Robinson at al. (1966) that it occurs in such negligible quantities in the environment that it does not represent any overall increase in the toxicologic sig- nificance of residues arising from the use of dieldrin. A.pentachloro photoproduct, VIII, has also been obtained eXperimentally by irradiating 9 dieldrin-treated corn leaves with light of shorter wavelength than is found in sunlight (Henderson and Crosby, 1966), but this has not been shown to occur naturally. These authors also showed that dieldrin is 1.6 times as toxic as photoproduct VIII to flies by tapical application, but that the photoproduct is 4.5 times more toxic than the parent compound to mice by oral administration. Retention of Dieldrin in Body Tissues Kitselman at al. (1950) reported that of the body tissues the peri- pheral fat of sheep and cattle fed aldrin-sprayed hay contained the largest amounts of aldrin, while the milk had no aldrin in it. However, in 1952 Bundren et al. reported finding dieldrin in the milk of cattle sprayed with a 0.1% dieldrin mixture at 3-week intervals. The maximum concentration in the milk (5.9 ppm) was achieved in the first 3 weeks; thereafter it decreased steadily despite continued application. Buck and Van Note (1968) obtained dieldrin in the milk of cows accidentally poisoned by ingested aldrin. In 1959(b), Gannon steal. detected dieldrin in the fat of all ani- mals fed various levels of dieldrin. The concentrations in other organs were prOportional to the fat content of the tissues as well as to the rate of intake. This general pattern of distribution was also found by Fiserova—Bergerova.et a1. (1967) in humans. They also confirmed placen- tal transfer of the pesticide in man. Kitselman et a1. (1950) found that the amount of dieldrin in the liver and kidney of the rabbit varied with the amount fed per week but not with the duration of feeding. According to Street (1964) and Street and Blau (1966), simultaneous administration of dieldrin and DDT to Sprague-Dawley rats resulted in a significant inhibition of dieldrin storage in the fat. Deichmann at al. (1969) could not demonstrate a similar effect in dogs. 10 Factors Predisposing to Dieldrin Toxicity Qigt,‘ Dietary influences on toxicity have been demonstrated by several workers. Meyer (l936a,b) reported a modification of the toxicity of sodium cyanide and resistance to diphtheria toxin by the composition of the diet. Similar modification of arsenic trioxide toxicity to rats has been observed. Ambrose at al. (1943) observed that a high fat diet reduced the acute but enhanced the chronic toxicity of rotenone. McLean at al. (1965) reviewed the susceptibility of animals on a protein- deficient diet to poisons that affect the liver. They suggested that the resistance to carbon tetrachloride (€014) intoxication during pro- tein depletion may be due to loss of microsomal enzymes needed for metabolization of CC14. In 1966, McLean and McLean confirmed this observation in protein-depleted rats. Radeleff and Bushland (1962) found emaciated sheep on a low maintenance diet of poor hay 8 times more susceptible to benzene hexachloride poisoning than sheep fed a normal diet. Boyd and DeCastro (1968) found that DDT toxicity to rats was not augmented by a low protein diet. Later, Boyd and Krijnen (1969) found that low protein diet augmented DDT toxicity and simultaneously reported that an excessive amount of protein in the diet of weanling rats also enhanced susceptibility which was manifested as a significant reduction in the LD50 of DDT to albino rats. The clinicOpathologic signs were similar at all levels of dietary protein utilized. However, a similar low dietary protein significantly reduced the LD50 of malathion for male albino rats (Boyd and Tanikella, 1969). Melvin et a1. (1964), working with the laboratory rat, demonstrated decreased survival at both low and high protein levels if high concentra- tions (200 ppm) of dieldrin were also fed. However, with the dieldrin 11 concentration at 150 ppm increased mortality occurred only with the low dietary protein group. It was therefore concluded that high dietary pro- tein conferred some protection. This was confirmed in 1965 by Krishna- murphy at aZ., who showed that poor rice diets, low in protein, increased the chronic toxicity of dieldrin manifested as increased mortality in rats . Stoewsand et a1. (1969) found a curious interplay between nutrition, sex, and dieldrin toxicosis in rats. At the lowest level of protein (102 casein) utilized, all males died within 18 days on dietary dieldrin levels of 150 ppm, but at higher protein concentrations (25 and 50% casein) not only did fewer die but the survivors showed an adaptation to the toxicosis. In contrast, all females fed the 25 and 50% casein. rations that received 150 ppm dieldrin died within 10 days and, by. implication, lacked an adaptive response. Female rats fed 10% casein ration plus 150 ppm dieldrin had a lower cumulative mortality rate. These results correlated positively with the rates of metabolism and excretion of dieldrin in that males fed rations containing higher levels of protein had higher rates of metabolism and excretion_and lower tissue concentra- tions of dieldrin. The low dietary protein levels in the females were accompanied by increased metabolism and excretion, thus, in part, account- ing for the lower mortality rate. Nitrates and nitrites, in addition to inducing a methemoglobinemia, directly affect the cardiovascular system by causing a relaxation of smooth muscle, especially of arterioles and postarteriolar vessels (Goodman and Gilman, 1965; Beckman, 1958). One of the main consequences of this vasodilation is a fall in blood pressure associated with vascular congestion and degenerative changes in visceral organs, especially the liver, the chief site of dieldrin detoxification: Pathologic changes 12 in nitrite toxicosis have been characterized (Sinha, 1968; Atallah, 1966; Case, 1957). Abortion and impairment of reproduction have been observed as features of nitrate/nitrite toxicosis in the cow (Crawford et aZ.. 1966), sow (Case, 1963), rat (Case, 1957), and guinea pig (Sleight and Atallah, 1968). In the Opinion of the last quoted authors, most of the placental and uterine lesions developed after the fetuses had died. The effects of simultaneous administration of nitrite and dieldrin on preg- nancy are not known. Species and Age Suscgptibility. There appears to be a definite species and age-dependent susceptibility to dieldrin toxicosis following oral administration of an acute lethal dose. Hodge at al. (1967) reviewed several reports and observed that of 12 species investigated, the cat and chicken are the most sensitive; the cow, rat, guinea pig, mouse, and monkey are intermediate; the dog, pig, and sheep are least sensitive. Estimating the lethal oral dose range for most species to lie between 20 and 70 mg./kg. body weight, Ressang et al. (1959) found the cat most sensitive to aldrin (LDSO 10-15 mg./kg.) but most resistant to dieldrin (L050 300-500 mg./kg.). In species in which the age factor was considered, it was found that younger animals were usually more sensitive to poisoning than the old (Bundren at aZ., 1952; Radeleff etsaZ., 1960; Iizuka, 1963). Other studies relating toxicity to the route of administration of dieldrin have revealed that the LDSO is lower for oral than for dermal applica- tion (Gaines, 1960; Conley, 1960; Jolly, 1954). For spraymen, inhala- tion is important (Deichmann and Radomski, 1968), but Hodge at al. (1967) produced evidence which indicated that dermal exposure is quan- titatively more important than respiratory exposure. 13 Systemic Effects of Dieldrin The Blood. All reported results of hematologic studies in dieldrin toxi- cosis have shown no changes. Relevant observations reported are the unaltered sedimentation rates in men occupationally exposed to dieldrin (Princi and Spurbeck, 1951); rat hemoglobin and erythrocytes which were not affected (Krishnamurphy at aZ., 1965); and nonalteration of the serum protein content of ewes on chronic exposure (Harris et aZ., 1966). Alimentagy System, Body Weight, and Fat. No direct effects on the ali- mentary system have been reported except for occasional diarrhea in farm animals, which was observed by Ivey et a2. (1961). This was not associated with any lesions. After a review of reported experimental results, Hodge at al. (1967) suggested that body weight or growth are not sensitive indices of the effects of aldrin and dieldrin. No marked changes in body weight of most species on nonlethal dietary levels of dieldrin have been reported, except for the young quail (DeWitt, 1955). However, at levels that cause weight 1083, death occurs in most species (Treon at al., 1955; Treon and Cleveland, 1955; Kitselman, 1951; Sherman and Rosenberg, 1954). Melvin et a1. (1964) did not find a significant alteration in total body fat of rats at the levels of dieldrin given. Reproductive System. The effects of dieldrin toxicosis on reproduction have been studied in several species. In no case has it been associated with teratogenesis, even when fetuses were stillborn. The criteria used for assessment include number of pregnancies, number of fetuses per litter, mortality amongst fetuses, egg production, fertility, hatcha- bility, and chick mortality. Thus, reduction in the numbers of preg- nancies and increased mortality of pups were obtained by feeding rats l4 diets containing 2.25 ppm of dieldrin (Treon at al., 1954a). At a die- tary level of 25 ppm, Kitselman (1951) observed increased mortality in pups of dogs. At a similar level of dieldrin, Harris et a1. (1966) observed a decrease in survival of lambs. Ten parts per million was sufficient to induce decreased hatchability and increased mortality in quail chicks (Coulson et a1., 1962). Reduced egg production, hatcha- bility, and increased chick mortality were not observed in pheasants on dietary dieldrin of less than 25 ppm (Rudd and Genelly, 1956). Muscular System. Khairy (1960) described the impairment of muscular efficiency in the rat by dieldrin. The progressive deterioration was related directly to the amount of dieldrin administered. He also con~ firmed that dieldrin had no effect on body weight, food intake, and learning. The Pathology of Dieldrin Toxicosis The most important signs of dieldrin toxicosis are associated with the central nervous system and have been widely reported. These symp- toms include convulsions, lethargy, salivation, teeth-grinding, and opisthotonos (Bundren at aZ., 1952; Pearson et aZ., 1958); and convul- sions (Jolly, 1954; Conley, 1960). Changes in the electroencephalogram in exposed workmen (Spiotta and Winfield, 1952; Bell, 1960; Hoogendam at aZ., 1962) are reversible in survivors. Most investigators report no corresponding gross lesions in the central nervous system. However, histOpathologic changes in poisonings by both dieldrin (Radeleff at al., 1955) and its parent compound, aldrin (Blaxter, 1959; Pearson at al., 1958; Treon at al., 1955; Ressang et al., 1959) have been described. These changes consist of cloudy.swelling and pericellular vacuolation 15 in the brain, and foci of degeneration in the forebrain involving oligo- dendroglia but not the neurons. Changes described in other organs are, in general, nonspecific for dieldrin or aldrin since these are similar to lesions caused by other chlorinated hydrocarbon insecticides (Conley, 1960). The histOpathologic changes consist of renal tubular degeneration; cloudy swelling, fatty metamorphosis and necrosis of the liver; congestion and edema of the lungs (Treon at al., 1955; Ressang at aZ., 1959; Kitselman, 1951; Anderson at al., 1952). Ortega at al. (1957) observed centrilobular hepatic cell hypertrOphy with peripheral migration of basOphilic cytOplasmic granules in rats given relatively high doses of dieldrin or other chlorinated hydrocarbon insecticides. They also described distinctive cytoplasmic inclusion bodies which were referred to as "lipospheres" and were regarded as a practical indication of chlorinated hydrocarbon effect on the liver cell. These authors found no significant differences in liver weights of test and control rats relative to their body weights, thus contradict— ing the findings of Borgmann et a2. (1952) and Treon et a1. (1951, 1955), who reported that dieldrin induced increases in liver:body weight (LW:BW) ratio in rats and dogs.- The increases in LW:BW ratios have been confirmed by the Food and Drug Administration (1963). Some reports on dieldrin toxicosis in cattle, sheep, goats, and pigs indicate no evidence of pathologic changes (Gannon et aZ., l959a,b,c), and no effect on milk production; but there are also numerous reports of histologic lesions similar to what are usually seen in other animals (Jolly, 1954; Radeleff at aZ., 1955. 1960; Pearson at aZ., 1958; Blaxter, 1959). 16 Other changes of-toxicologic significance are decreased oxygen con- sumption in rats (Crevier at al., 1954); unexplained alterations in con- centrations of DNA in heart, spleen, kidney and brain of rats (Daugherty at al., 1962); and depressed 8 wave of the electrocardiogram of sheep given dieldrin in the diet for a prolonged period (Harris at aZ.. 1966). Ultrastructural Change§_in Dieldrin Toxicosis Ghaeal at al. (1964) first applied electron microscOpy to the study of dieldrin toxicosis. They demonstrated that dieldrin produced enlarge— ment of the liver and hypertrophy of the smooth endOplasmic reticulum (SER), along with increase in microsomal protein and activities of drug- metabolizing enzymes. These findings were confirmed by Hutterer at al. (1968), who also demonstrated that in dieldrin-tolerant rats the SER was not only hypertrOphic but hyperfunctional. If, however, such rats were given much higher doses of dieldrin, a phase of decompensation occurred and was associated with hypertrOphic but hypofunctional SER. It was concluded that this may serve as a sensitive indication of toxicosis even before histopathologic changes are recognized. MATERIALS AND METHODS gigs. Management. Sixteen young pigs and 9 pregnant sows were used in 3 experi— ments. All swine were housed in pens with concrete floors. Metal troughs were used as feed containers and automatic waterers provided drinking water ad Zibitum. Clinical observation was maintained. AutOpsy Procedure. The pigs were examined postmortem for gross patho- logic changes. Specimens were obtained from the brain, heart, liver, kidney, adrenals, lungs, spleen, stomach, and intestines for histOpatho- logic examination. Sections from these tissues were routinely stained with hematoxylin and eosin. Formalin-fixed sections of liver, kidney and adrenal were stained for fat with the oil red O stain. Residual Dieldrin Determination. .In all 3 experiments tissues were col- lected from the liver, kidney and abdominal fat, and additionally from the brain of some pigs in Experiment II. Fetal brain and liver, and some- times kidney and fat, were also collected in Experiments II_and III. These samples were used for determination of tissue dieldrin residue by gas chromatography after the tissues had been prepared according to Zabik's procedure.1 A weighed amount of each tissue sample (usually 20 gm. or less) was ground in acetonitrile (CH3CN) using a Waring blender 1Procedure used in Extraction of Residues from Fat and Tissue Samples. Laboratory Work Sheet, Entomology Department, Michigan State University, East Lansing, Mich. l7 18 for 3 minutes at "fast" speed. Fifty milliliters of CH3CN plus 1 ml./gm. of tissue were used in the initial extraction. The mixture was allowed to settle and the supernatant fluid poured through a glass-wool filter into a separatory funnel. The extraction was repeated using 100 m1. CH3CN, but the filtration was done without allowing the sediment to settle first. The dieldrin was extracted from the CH3CN with 100 ml. of hexane (C6H14) by shaking the mixture vigorously 15 to 20 times and letting the 2 layers form. The acetonitrile was then washed out with 2 washes of about 200 m1. of 10% sodium chloride (NaCl) solution in the separatory funnel. The salt solution was rejected and the hexane layer dried with anhydrous sodium sulfate (Na2804). The following procedures were then carried out by the Analytical Laboratory of the Pesticide Research Center. The dry hexane extract was then introduced into a sodium sulfate--Florisill[Celite2 (5:1) column for cleanup. Elution with hexane was continued until about 300 m1. of the much diluted final sample was obtained. About 2 microliters Oil) of this sample was injected into the gaschromatograph,3 along with a dieldrin standard, and read- ings-were recorded and utilized for calculation of concentrations. Chromatograms were obtained using a 6 ft. x 1/8 in. stainless steel column packed with OV-l7/QF-1 (112) (1:1.3). Operating parameters were: inlet temperature 275 C, column 215 C, detector 275 C, and the carrier gas was helium at a flow-rate of 40 m1./mm. lFlorisil. Fisher Scientific Co., Cleveland, Ohio. 2Celite. JohnseManville Co., New York. 3Gas Chromatograph. Beckman GC-4 Discharge Electron Captive Detector. Beckman Instruments, Inc., Fullerton, California. 19 Blood was collected during exsanguination of the stunned pigs and allowed to clot. The serums were recovered, extracted with hexane, and subjected to the same procedures as given above for dehydration, cleanup and gas chromatography. The dieldrin residue values are expressed in parts per million (PPm) on a fresh weight basis. Serum Protein Determination. Samples of the serums before extraction with hexane were used for total serum protein determinations by Kingsley's (1942) method. The serum proteins were also separated by the Gelman rapid electrOphoresis method with Sepraphore III in Model 51101 Chamber1 at room temperature. Beckman-prepared buffer, dye and fixatives were used, and the relative intensities of the fractions were determined with an analytrol.2 Guinea PigExperiments A total of 147 guinea pigs were utilized for 4 experiments. In this series laboratory investigations included autopsies on dead.and euthana- tized guinea pigs and fetuses, histOpathologic examination of tissue samples collected at necropsy, and preparation of liver, kidney, fetuses and/or fat and brain samples for dieldrin residue analysis by gas chroma— tography. Samples from the lower treatment levels in Experiment VI were not.subjected to chromatographic analysis. All guinea pigs were housed in tapless sheet metal cages that measured 1.5 x 2.5 x 1.0 feet. Generally, 4 were housed in each cage. Ceramic bowls were used to provide feed and water. The bedding consisted of wood shavings. 'Once a day cabbage was provided. 1Gelman Instrument Co., Ann Arbor, Michigan. 2Spinco Model R. Beckman Instruments, Inc., Palo Alto, Calif. EXPERIMENTAL PROCEDURE AND RESULTS Pigs Experiment I. The object of the experiment was to compare the toxicity of dieldrin for pigs of approximately the same age maintained after wean- ing on either a protein-deficient or normal diet. Procedure. Sixteen pigs, 6 males and 10 females, between 7 and 8 weeks of age, were divided randomly into 2 groups of 8 each. Group I was fed a special low-protein.(4z casein) piglet diet which was other— wise adequate (Table 1). Group II pigs were fed a normal pig diet obtained from the Michigan State University Swine Farm. The pigs in Group I were gradually introduced to the protein-deficient diet over a 4-day period. Both groups were allowed 2 lbs. of the appro- priate feed per pig daily and were provided drinking water from automatic waterers. The weights of the pigs were recorded at 0, 14, and 21 days (Figure 1). At the end of this period 4 randomly selected pigs from.each group were dosed per as with dieldrin in a gelatin capsule at the rate of 30 mg./kg. body weight. The remaining 4 pigs in each group were maintained as nontreated controls. After a variable number of hours of observation, all pigs were killed by electrocution followed by exsanguination. 20 21 Table 1. Composition of the protein—deficient diet* for pigs in Experiment I 'r Ingredient Preportion (Z) Alphacel 10.0 Dextrose 74.0 Lard. 5.0 Corn oil 1.0 Casein (Vitamin Free) 4.0 Salt mixture 6.0 Vitamin Diet Fortification Mixture** f It- *Nutritiona1 Biochemicals Corporation, Cleveland, Ohio. **Formulation grams/100 lbs. diet Vitamin A Concentrate........ 4.5 (200,000 units per gram) Vitamin D Concentrate.....;.. 0.25 (400,000 units per gram) Alpha'TocOpherol.............' Ascorbic Acid................4 Inositol..................... Choline Chloride.............7 Menadione............. 5 5 5 5 2 hJC>C>C>C> 5 p Aminobenzoic Acid.......... Niacin....................... Riboflavin................... Pyridoxine Hydrochloride..... Thiamdne Hydrochloride....... Calcium Pantothenate......... mals/100 lbs. Biotin....................... Folic Acid................... Vitamin B-IZeeeeeeeeeOQeeeeee 22 ‘0 _ lulu __ mum" s 3 E 30 a! 25 20 o 2 a as Figure 1. Group average weights of pigs fed normal (N) or protein deficient diet (D) for 21.days. 23 Results Growth. The pigs fed low-protein diets manifested an overall reduction in rate of weight gain as compared with the pigs fed the normal diet (Figure 1). Signs. No definitive signs of protein deficiency were observed. All pigs given dieldrin showed signs of toxicosis to variable degrees. Drowsiness, huddling, shivering, twitching of muscles, and occasionally prostration were observed. One pig manifested frothing at the mouth. The signs first appeared about 1 hour after administration but, in general, were most intense at the 2nd hour. Pigs of the protein-deficient group dosed with dieldrin were most severely affected. Analyses Hematologic determinations. The hemoglobin and hemato— crit values were normal, as were the total and differential leukocyte counts 0 Serum electrgphoresis. The total protein values were within normal range, but there were alterations in component fractions which affected the albumin/globulin (A/G) ratio. In most of the samples there was a reduction in albumin associated with a relative increase in the globulins, especially o2 fraction. The net result was a reduced A/G ratio, which was especially marked in deficient pigs 1 and 3 (Table 2). Residual dieldrin. Dieldrin was demonstrated in all analyzed tissues from treated pigs (Table 3). 24 Table 2. Serum proteinse-total protein, albumin, and a globulin values, A/G ratios of.representative pigs not given dieldrin in Experi— ment I Feed Pig Total. Protein Albumin o2 Status No. Sex (gm/100 ml) (gm/100 ml) (gm/100 ml) A/G Deficient 1 M. Initial 5.10 2.14 0.91 0.72 Terminal 6.31 1.75 1.22 0.38 2 F Initial, 4.86 2.10 0.85 . 0.76 Terminal 5.97 2.52 1.21 0.73 3 F Initial 4.71 2.24 0.92 0.61 Terminal 5.63 1.55 1.45 0.38 Normal 1 M Initial 5.53 2.39 0.91 0.76 Terminal 5.97 1.98 1.11 0.58 2 M Initial 4.56 1.96 1.01 0.75 Terminal 5.46 1.95 1.09 0.56 Terminal 5.36 1.91 1.12 0.55 25 Table 3. Dieldrin concentrations (ppm) in tissues from representative treated pigs in Experiment I. Feed Pig Tissue Dieldrin Concentration (ppm) Status No. Sex Serum Fat ‘ Liver ' ‘ ' Kidney Deficient l M 0.66 8.71 2.41 0.96 2 F 0.08. 0.09 0.10 0.06 3 F 0.04 0.13 n.d.* n.d. 4 F 0.30 0.25 0.01 0.26 Normal 1 M 0.56 2.27 3.67 1.45 2 M 0.30 1.52 2.42 0.93 3 F 0.26 1.27 2.31 1.74, 4 F 0.26 1.10. 2.00 1.02 En.d. - not done 26 Lesions. Grossly, the livers of the deficient pigs were of a. dull gray color. Histologically, the pathologic changes consisted of sinusoidal dilatation, slight hyperemia and fatty metamorphosis with pre- dominantly centrilobular distribution (Figure 2). Kidney. The gross change was a paleness in the deficient group. There was histologic evidence of patchy vacuolar degeneration of proximal convoluted tubular epithelium. Lungs., There were petechial hemorrhages in the lungs of all the pigs. These were characterized histologically as a more diffuse hyperemia involving alveolar capillaries. This change was regarded as agonal. Egperiment II. The experiment had as its objective the determination of toxicosis associated with dieldrin in sows during the immediate prepar— turient period. The experiment was also designed to demonstrate possible transplacental transfer of the chemical, the degree of dieldrin accumu- lation in the fetal liver, and any clinical effects and lesions resulting from such toxicosis. Procedure. Dieldrin was administered per as in the mornings of. alternate days at the rate of.15 mg./kg. body weight to 2 sows in the last 2 weeks of pregnancy using gelatin capsules and a metal balling gun. A total of 3 administrations was achieved before the sows were killed by electrocution and exsanguination. Using similar methods, 2 other sows were given dieldrin daily in the morning at a rate of 9 mg./kg. body weight for a total of 9 administrations before euthanasia. Two nontreated sows were used as controls (Table 4). 27 Figure 2. Liver. Lipidosis in pig fed protein-deficient diet and dosed with dieldrin. H & E stain. x 600. 28 Table 4. Duration of treatment and total dieldrin intake of sows in Experiment 11 Avg. wt. .Dieldrin No. in Duration of sows Dosage No. of 'Total Given Group (days) (kg.) (mg/kg) Doses (gm.) 2 * 133.8 0 0 0 2 5 133.3 15 3 6 2 13 166.6 9 9 13.50 *One control sow was sacrificed after 5 days to correspond with the sows in the second group; the other was sacrificed after 13 days to correspond with the sows in the third group. 29 Results Signs. There were.no obvious signs of intoxication. One of~ the control sows farrowed 1 dead and 7 live pigs before sacrifice. The other control sow had 1 dead and 11 live fetuses in the uterus when it was sacrificed. No fetal deaths were observed in either group of treated SOWS a Food and water consumption. These remained normal throughout the duration of the experiment. Analyses Residual dieldrin concentration. Residual dieldrin was present in the fat, liver, kidney, and brain at appreciable levels (Table 5) in the treated sows. The brains and livers of randomly selected fetuses were also analyzed and found to contain fairly large quantities of dieldrin when compared to the fetuses from nontreated controls (Table 6). Lesions Liver. Grossly there was a dullness to the livers with some evidence of mottling in one sow's liver. Histologic changes con- sisted of hyperemia, some lymphocytic infiltration into the portal triads, and vacuolar degeneration of hepatocytes. Kidneys. These were pale on gross examination. Micro- scopically, hyperemia of the glomeruli and vacuolar degeneration of the epithelium of proximal convoluted tubules in discrete areas were evident (Figure 3). As for the liver, histologic changes appeared to increase with increasing duration of treatment. 30 Table 5. Residual dieldrin concentration (PPM) in tissues of sows in Experiment 11 and total amounts of dieldrin given m:— L:— _ I -1 _ I527 Status DieIdrin Sow V Tissue Dieldrin Concentration (ppm) of Sow (gm.) No. Fat "‘ ‘ LiVer ' ‘Kidney ' ’ Brain Treated 6.0 l, 5.02 3.59 1.28. n.d.* 6.0 2 4.70 3.80 1.49 n.d. 13.5 3 n.d.‘ 4.68 1.23 1.18 13.5 4 n.d. 2.96 0.87 0.90 Non- treated 1 0.23 0.01 0.007 n.d. 2 n.d. 0.00 0.00 0.00 *n.d. = not done 31 Table 6. Residual dieldrin concentration (ppm) in fetal tissues from Experiment‘II A Total Dieldrin Fed to Sow' Tissue Dieldrin Concentration (pqu Fetus No. (gm.) Liver* Brain 1 0 0 O 2 0 0 0 3 0 0 0 4 6.00 0.16 n.d.** 5 6.00 0.09 n.d. 6 6.00 0.08 n.d. 7 6.00 0.27 n.d. 8 6.00 0.14 n.d. 9' 6.00 0.13 n.d. 10 13.50 1.51 0.43 11 13.50 1.05 0.26 12 13.50 1.14 0.47 13 13.50 1.35 0.27 14 13.50 0.60 0.26 15 13.50 0.411 0.25 *(P < .025) for fetuses of 2 sows that were given totals of. 6.0 gm. and 13.5 gm. dieldrin, respectively. **n.d. - not done 32 Figure 3. Kidney. Vacuolar degeneration of epithelium of proximal convoluted tubules of dieldrin—treated sow. H & E stain. x 150. 33 Lung . Histologically there was hyperemia of the alveolar capillaries. The petechiae in the lungs were regarded as agonal changes in both categories of sows.’ Uteri. These appeared edematous and congested grossly. No histologic changes other than the edema of the membranes and hyperemia were observed. gfiperiment III. In.a prior experiment with nitrite administered alone to a pregnant saw no effect on fetuses was.demonstrated. Repeated dieldrin dosage to pregnant sows in another experiment did not produce any observ- able effect on the fetuses.' The present experiment was designed to see if fetuses would be killed and aborted following repeated administration of dieldrin in conjunction with nitrite. It was also intended to determine if the vasodilatory effect of nitrite would produce a correspondingly greater accumulation of dieldrin in the fetal tissues. Procedure. Three sows in the last trimester of gestation were uti- lized. Two sows were given dieldrin per as in gelatin capsules at the rate of 15 mg./kg. body weight, as well as sodium nitrite (NaNOz) by subcutaneous injections of a 12 solution at the rate of 25 mg./kg. body. weight. The third sow, inoculated with 1% NaN02 solution subcutaneously at the same rate, served as the positive nitrite control. These sows each received a total of 24 administrations of apprOpriate chemicals before death or euthanasia by electrocution and exsanguination (Table 7). Results Food and water consumption. Depression of appetite was observed in one of the sows receiving both dieldrin and nitrite. This occurred in the last 2 days before death.' 34 Table 7. Duration of treatment and total dieldrin and NaNOz intake by sows in Experiment III Duration Dieldrin NaNOgg of Daily Daily Number Experiment Dosage Total Given Dosage Total Given Represented (days) (ms/ks) (gm) (ms/ks) (8m) 1 28 15.0. 48.0 25.0 84.0 1 28 15.0 48.0 25.0 84.0 1 28 0.0 0.0 25.0 84.0 35 Signs. There was a slight brownish discoloration of all visible mucous membranes of the 3 sows. One dieldrin/nitrite-treated sow died in severe clonic convulsions. The nitrite control sow also died but the 2nd dieldrin/NaNOz-treated sow remained normal until killed. Fetuses from all 3 sows appeared normal on gross examination. Analyses Residual dieldrin. Dieldrin was demonstrated in the fat, liver, kidney, and brain of sows given this chemical. The livers and brains of fetuses from these sows also contained dieldrin (Table 8). Lesions Liver. Some mottling was evident. Histologically, all the livers were hyperemic and manifested centrilobular fatty metamorpho~ sis. The liver of the nitrite control sow was infiltrated in the portal triads, interlobular septa and parenchyma by neutrOphils and a few eosinophils. The septa were_thickened by edema fluid. Kidney. Grossly the kidneys appeared dull gray but firm. MicroscOpic examination revealed hyperemia of the glomeruli with vacuolar degeneration of the proximal convoluted tubular epithelium in the dieldrin/ nitrite treated sows (Figure 4). There was hyperemia with interstitial hemorrhage in the kidney of the nitrite control sow. Degeneration of tubular epithelium with.neutrophilic and lymphocytic infiltration of the interstitium was present. Spleen. The spleens were only slightly hyperemic. 36 Table 8. Dieldrin concentrations in tissues of sows and their fetuses in Experiment III Tissue Dieldrin Concentration (ppm) Sow No. and Status Fat Liver Kidney ' Brain Dieldrin & Nitrite 1 54.60 4.26 1.27 1.24 F1* n.d.** 0.86 n.d. 0.41 F2 n.d. 1.15 n.d. 0.47 2 26.06 9.09 1.85 1.00 F1 n.d. 0.16 n.d. 0.18 F2 n.d. 0.17 n.d. 0.12 F3 n.d. 0.17 n.d. 0.23 Nitrite 1 0 0 0 0 F1 n.d. 0 n.d. 0 F2 n.d. 0 n.d. 0 A *Fetuses of sows numbered immediately above. **n.d. - not done 37 Figure 4. Kidney. Vacuolar degeneration of proximal convoluted tubular epithelium of: dieldrin-nitrite treated sow. H & E stain. x 150. 38 Uteri. No abnormalities were observed either grossly or microscopically. Fetuses. The fetuses appeared normal grossly. Histo- logically the livers were hyperemic, especially in the peripheral zone. There was an abundance of glycogen in the hepatocytes, which was confirmed by staining with Best's carmine. Megakaryocytes and nucleated erythro- cytes were observed in fairly large numbers. Fetuses from all 3 sows manifested similar histologic appearance. Guinea Pigs A total of 147 guinea pigs was utilized for 4 experiments. (Experiment IV . The objects of the experiment were to investigate the toxicosis of dieldrin in guinea pigs and to obtain evidence of transpla- cental transfer of the chemical. Procedure. Dieldrin was dissolved in cottonseed oil at a concentra- tion of 60 mg./ml. Forty-six guinea pigs (30 pregnant females, 8 non- pregnant females, and 8 males) were given this preparation orally on a body weight basis as indicated in Table 9. Administration was repeated at about S—day intervals until death or sacrifice by ether anesthesia. An additional 10 guinea pigs were maintained as controls and each received 0.5 m1. of cottonseed oil per as at S-day intervals (Table 9). Results Signs. One of the dieldrin-treated guinea pigs farrowed 4 apparently healthy pups which, however, died 1 hour postnatally. The dam.appeared to be normal before and after farrowing. Three males, 28 pregnant and 4 nonpregnant females died at various times in the course 39 Table 9. Groups and numbers of guinea pigs represented, sex, dosage, --duration, and total quantity of dieldrin given in EXperiment IV Duration Total Before Dieldrin No. Rep- Sex Dose (mg/kg) No. of Death Given Group resented* Male Female Initial After Treatments (days) (mg.) 1 2 1 1 60 - 1 3 60 2 10(4) 1 9 30 — 1 3 30 3 l 1 30 30 2 7 60 4 1 1 30 30 3 14 90 5 6 3 3 30 15 7 39 120 6 11(3) 2 9 15 15 6 26 90 7 15(5) 1 14 15 15 8 31 120 8 3 l 2 0 0 l 3 0 9 3 1 2 0 0 6 26 0 10 4 2 2 O 0 8 39 0 *Numbers in parentheses represent sacrificed guinea pigs. 40 of the eXperiment. Death in all instances was preceded by a period of depression, severe dyspnea.and terminal convulsions: Epistaxis was. observed in one nonpregnant guinea pig. Frequently some of the guinea pigs became excited 2 or more hours. after dieldrin administration. Such pigs often leaped high and performed gyrating movements, thus making it necessary to cover the cages with a grid metal tap. Analyses Residual dieldrin. Dieldrin residues were present in tissues of treated guinea pigs. Dieldrin was also recovered in whole fetuses of the same guinea pigs (Table 10). Lesions Q5325, The only consistent lesions were dull appearance of the livers and kidneys of all dieldrin-treated guinea pigs and petechi- ation of lungs of both the treated and the nontreated controls. However, a few amongst the dieldrin-treated pregnant guinea pigs manifested slight enlargement of the spleen and adrenal gland, gelatinous perirenal fat, and gastric congestion. HistOPathologic Liver. Lipidosis, more commonly of-centrilobular distribution, was observed and confirmed by staining with oil.red 0. This was in contrast to the abundant glycogen in livers of nontreated guinea pigs (Figures 5 and 6), confirmed by the Best's carmine stain. Hyperemia was a feature of the livers from.dieldrin-treated guinea pigs. 41 Table 10. Groups and numbers of guinea pigs sampled, total dieldrin received, and average tissue and fetal concentrations (ppm) of dieldrin in Experiment IV- No. of Total Dieldrin Average Dieldrin Concentrations (ppm) Group Samples Received (mg.) Fat Liver Kidney Brain Fetus 2 2 30 1.95 29.06 n.d.* n.d. 1.72 3 1 60 19.88 17.76 3.11 7.12 - 4 l 90 33.39 25.50 7.32 5.63 1.42 5 3 120 43.62 14.27 3.17 3.43 3.08 6 1 90 32.34 15.32 12.71 5.36 n.d. 7 3 120 40.50 29.19 5.32 6.94 n.d. 8,9,10 3 0 0.04 0 0 0 0 *n.d. - not done 42 Figure 5. Liver. Lipidosis in dieldrin- treated guinea pig. H-& E stain. x 150. Figure 6. Liver. Control. Note presence of glycogen confirmed by the Best's carmine stain. x 150. 43 Kidneys. These were generally hyperemic. Focally, the proximal convoluted tubules manifested epithelial vacuolar degenera- tion (Figure 7) in the treated guinea pigs. Spleen. Only the grossly enlarged spleens were hyperemic histologically. Lungs. Hyperemia and atelectasis with compensatory alveolar emphysema were observed in the lungs of most treated guinea pigs (Figure 8). Stomach. Hyperemia was.eonfirmed microsc0pically in instances where there was gross congestion. There was gastric mucosal necrosis in some instances. Uteri. The hypertrophic mucosae were edematous. In 2 instances mucosal necrosis, early mineralization and neutrOphilic infil- tration were observed. The 2 guinea pigs that manifested necrosis of uterine mucosa had the highest quantities of dieldrin. Adrenal glands. Lipidosis was demonstrated in the grossly enlarged glands of treated guinea pigs. Heart. Focal myocardial necrosis with mineraliza- tion was observed in only 1 instance. Fetal livers. There were no demonstrable abnormali- ties in the livers of fetuses, even from animals that died shortly after birth. 44 Figure 7. Kidney. Epithelial vacuolar degeneration in-proximal tubules of dieldrin— treated guinea pig. H.& E stain. x 150. 45 Figure 8. Lung. Hyperemia, atelectasis and alveolar emphysema in dieldrin-treated guinea pig. H & E stain. x 150. 46 Experiment V. The object of this experiment was to determine the combined toxicologic effect of dieldrin and nitrite in guinea pigs. Special empha— sis was placed on the effects on pregnancy and upon fetal concentration of the pesticide. Procedure. Twelve female guinea pigs, 7 pregnant and 5 nonpregnant, were given 15-45 mg. of dieldrin in cottonseed oil calculated on a dosage rat of 60 mg./kg. body weight. This was administered as drOps in the mouth after apprOpriate individual doses had been measured with a 1 m1. syringe. The same guinea pigs were injected subcutaneously into the neceregion with 1%.NaN02 solution at the rate of 30 mg. (3 ml.) per guinea pig. Administration of both chemicals was continued on alternate days until the animals died or were euthanatized with ether. A nitrite control was set up by administering NaN02 solution (1%) subcutaneously to 12 guinea pigs at the same rate of 30 mg./guinea pig. With another control group of 10 guinea pigs, each was given 1 ml. of cottonseed oil by mouth for the duration of the experiment, but no NaNOZ solution was injected (Table 11). Additional laboratory investigations in this experiment consisted of determination of hemoglobin and methemoglobin levels and the packed cell volume of randomly selected guinea pigs from all the groups except the nitrite group in the first 2 days of the eXperiment. Hemoglobin was estimated by the standard cyanmethemoglobin method. Evelyn and Malloy's method as described by Hawk et a1. (1954) was adepted for methemoglobin determination while the capillary tube method was used for evaluating the packed cell volume. Blood for these determinations was obtained from the metacarpal vein as described by Uzoukwu and Sleight (1970). 47 Table 11. Numbers of guinea pigs represented, sex, total quantity of chemical given and duration of observation in Experiment.V V Total Quantities Number Sex 7 Given (mgi) Represented Male Female Dieldrin ‘ NaN02 Duration (days) 10 4 6 O 0 1-8 3* 1 2 6O 0 3-7 12** - 12 45 30-120 1-7 12 4 8 0 30-150 1-8 *Data extracted from Table 9, Experiment IV. **Two abortions occurred. 48 Results Signs. Slight brownish discoloration of visible mucous meme branes was seen in animals of the nitrite treated group. Terminal con- vulsions and dyspnea were observed only in the 7 dieldrin and nitrite treated guinea pigs that died. Two guinea pigs given dieldrin and nitrite aborted. Analyses Hematology. There were no abnormal values for hemoglobin, and hematocrit before or after treatment. However, there was a definite increase in methemoglobin in nitrite recipients usually within 1 hour of its administration. Residual dieldrin. This was.demonstrated in the tissues and whole fetuses analyzed (Table 12). Lesions. No gross-or histologic abnormalities were identified in the tissues of the untreated guinea pigs. In the other groups there was a variable amount of brown discoloration of tissues, the intensity of which appeared to depend on the time of death following nitrite administration. The perirenal fat was gelatinous in some‘guinea pigs. Liver. -There were no gross lesions. Histologically, centrilobular fatty metamorphosis_was observed when the guinea pigs had been given dieldrin and nitrite for at least 4 days. Some degree of hyperemia was present in all instances. Kidney. No gross lesions were observed but histologically hyperemia, and vacuolar degeneration of proximal tubules were discernible. 49 Table 12. Average tissue and fetal dieldrin concentration, total dieldrin and nitrite given and numbers of guinea pigs represented in Experiment V Number Recziszd (Ink) Average Dieldrin Concentration (ppm) Represented Dieldrin NaNOz Fat Liver ' Kidney' Brain Fetus 2 O O O O O O 0 3 0 30-150 0 O O 0 O 1* 6O 0 19.88 17.76 3.11 7.12 O 2 45 30 29.99 4.82 4.17 2.38 4.95 2 6O 60 30.32 14.27 7.32 3.05 1.66 2 60 120 70.20 29.26' 12.07 8.94 n.d.** 4.. *Data abstracted from Table-10, Experiment IV. **n.d. - not done 50 In some cases there was slight lymphocytic infiltration of the inter- stitium. Spleen. Some of the spleens in the dieldrin-nitrite treated guinea pigs were slightly enlarged. Microsc0pica11y, this enlargement was associated with hyperemia. Adrenal. There was a histologically observable increase in cortical fat in guinea pigs given dieldrin and nitrite. Uteri. Grossly the uteri of the guinea pigs that aborted were congested. Thickened arterial media with swollen and degenerated cells were seen microscOpically (Figure 9). The epithelium.was hyper- tr0phic, vacuolate and necrotic in parts (Figure 10). Lung . Petechiation of the lungs was a constant feature in the dieldrin-treated guinea pigs. These lungs were observed in sec- tions to be hyperemic, atelectatic, and emphysematous. Experiment VI. Since dieldrin and nitrite could be either natural or accidental contaminants of animal feed and water, Part I of this experi- ment was designed to simulate this situation and study the combined toxicoses. The second part of the experiment was an attempt to determine effects of lower levels of the 2 chemicals used. Procedure Part I. Fourteen 1- to 2-month pregnant and 5 nonpregnant guinea pigs were fed for a variable period crushed guinea pig pellets1 1Rockland Laboratory Animal Diets. Tekland, Inc., Monmouth, Ill. 51 Figure 9. Uterus. Occlusion of small artery, thickening and vacuolation of arterial media (arrows) in dieldrin-nitrite treated guinea pig. H & E stain. x 150. Figure 10. Uterus. Hyperplasia, vacuola- tion and necrosis (arrows) in guinea pig treated with dieldrin and nitrite. H & E stain. x 150. 52 that had been treated with dieldrin at a concentration of 100 ppm. They were also provided with 0.52 NaNOz solution for drinking ad Zibitum. In order to attempt differentiation of the action of dieldrin from responses to nitrite poisoning, controls were set up in which 5 guinea pigs drank 0.52 NaNOz solution in place of tap water but were fed normal crushed pellets, while 5 others ate dieldrin-treated feed but drank tap water (Table 13). Part II. Three pregnant and 6 nonpregnant guinea pigs were fed diets treated with dieldrin at levels indicated in Table 14. They were allowed to drink 0.25% solution of NaN02 at will. Another group of 9 pregnant and 3 nonpregnant guinea pigs was fed dieldrin at similar levels but drank plain tap water. The dieldrin and nitrite control groups consisted respectively of 3 guinea pigs that were given neither dieldrin nor nitrite and 3 that drank 0.252 NaNOZ solution only. Body. weights were recorded at weekly intervals (Table 15). Results (Part I) Food and fluid consumption. After an initial reluctance to drink the NaN02 solution, the guinea pigs soon adjusted to it. There- after, consumption of the solution and the feed remained reasonably nor- mal until the terminal stage. Refusal of food and fluid was consistently observed in the 24 hours preceding convulsions and death. Signs. Abortion was commonly seen. Of the 14 pregnant guinea pigs that were given dieldrin and nitrite, 9 aborted and 3 died 2 to 5 weeks after the initiation of the eXperiment. Three died amongst the nonpregnant group of 5 that received the same treatment as above. Dieldrin alone caused 2 abortions and 2 deaths in a group of 5 53 Table 13. Number of female guinea pigs, concentrations of chemicals, and periods of observation in Experiment VI (Part I) 4_ Number Concentration of Chemical Given* Duration Represented Dieldrin (ppm) NaNOz (2) (days) 14 100.0 0.5 30-41 5 100.0 0.0 37—41 5 0.0 0.5 21—45 *Dieldrin was fed in the food, while NaNOz was added to the drinking water. 54 Table 14. Concentrations of chemicals given in feed and water for 53 days, and groups of pregnant and nonpregnant guinea pigs in Part II oanxperiment VI. 4‘ L _ .1 No. of Concentrations of Chemicals Group No. Guinea Pigs Dieldrin (ppm) NaNOz 1 4(3)* 50.0 0.0 2 4(3) 25.0 0.0 3 4(3) 10.0~ 0.0 4 3(1) 50.0 0.25 5 3(1) 25.0 0.25 6 3(1) 10.0 0.25 7 3(1) 0.0 0.25 8 3(1) 0.0 0.0 *Numbers in parentheses‘ indicate numbers of.pregnant animals. 55 Table 15. Mean body weights (gm.) of guinea pigs at about 6-day intervals during Part II of Experiment VI Mean Body waight‘(gm.). Day of~ 1 2 3 4 5 6 ' 7 4:8 Experiment Group No. (4) (4) (4) (3) (3) (3) (3) (3) 1 836 880 866 853 952 887 753 817 6 861 843** 870 830 925 893 742 835 12 888 853 895* 843 933 917. 768 822 18 881 790* 791 835 898 882 753 806 23 940 803 791 867 893 957 792 813 30 915* 818 814 858 905 947' 812 795 37 916 847 796 847- 868 965 795 797 44 811* 855 781* 800* 830 898 790 780 53 725* 718** 764* 752 848 800** 732* 973** *One farrowed and this explains the drop in subsequent mean body weight value. **One died 56 guinea pigs, while nitrite alone induced abortion but no death in 3 out of 5. No signs.of impending abortion were observed, but death in all cases was preceded by severe clonic convulsions. Results (Part II) Food and fluid consumption. In none of the groups was either food or fluid consumption severely affected after the period of adjust- ment by the nitrite-drinking guinea pigs. However, by comparison, the guinea pigs given water with or without dieldrinetreated feed drank more fluid than the nitrite group. Body weight. Individual guinea pigs manifested a fluctuation in their rate of weight gain mainly in relation to their pregnancy status. Nonpregnant guinea pigs gained weight with less fluctuation in body weight. Signs. Amongst the 12 pregnant guinea pigs, 9 farrowed nor- mally irrespective of the level of dieldrin given. .The remaining 3 died. Two of these belonged to the group that was given 25 ppm of dieldrin and no nitrite. One of these died 6 days after the start of the experiment, while the other died acutely on the last observation day with a torsion of the uterine horn. The third guinea pig belonged to the group given 10 ppm dieldrin and 0.252 NaN02.. Postmortem changes were too advanced to permit a diagnosis. One of the nontreated, nonpregnant controls died of an undetermined cause. Lesions. In general, grossly there was a slight brownish dis- coloration of tissues in all guinea pigs that were given NaN02 with or without dieldrin. The livers appeared fatty, and the kidneys pale. Most 57 uteri were still large and edematous when the guinea pigs were sacrificed following abortion. The spleens of the test guinea pigs were slightly enlarged. No gross lesions were observed in other organs except the lungs, which were petechiated to varying degrees. Most of the nonpreg- nant guinea pigs had excessive abdominal fat of a normal consistency. Histology Liver. The histologic pattern was essentially similar in all treatment groups but varied in degree in relation to the duration of treatment. Sinusoids were dilated and contained blood, while hepato- cytes had undergone fatty metamorphosis. Kidney. There was a distinct hyperemia of the glomeruli. Patchy vacuolar degeneration of the epithelium of proximal convoluted tubules was marked in the dieldrin-nitrite group (Figure 11). 2535;. Submucosal edema was evident. The uterine villi were hyperplastic and, in 2 instances, appeared to possess degenerated epithelium. Necrosis of the junctional epithelium of the placenta with neutrophilic infiltration was observed in one specimen from the dieldrin- nitrite group. Medias of small arteries were thickened and vacuolated; the changes sometimes caused partial occlusion of the.lumina. Other organs. The lungs were usually hyperemic. The spleen was oftentimes hyperemic. Increase in adrenal cortical lipid was not a constant finding. No appreciable changes were observed in the heart and gastrointestinal tract. 58 Figure 11. Renal hyperemia and vacuolar degeneration of proximal tubular epithelium in guinea .pig treated with dieldrin and nitrite. H & E stain. x 600. ULTRASTRUCTURAL STUDIES Six 3—month-old guinea-pigs were used in an experiment to-determine the ultrastructural changes in the brain and liver in acute dieldrin toxicosis. Procedure Four randomly selected guinea pigs were injected intraperitoneally with dieldrin in cottonseed oil at the rate of 75 mg./guinea pig, irre- spective of body weight, and kept under constant observation. Two of- these were euthanatized with ether and exsanguinated at the peak of convulsions. Two were killed at the stage of pronounced depression which always preceded the convulsions. The remaining 2 guinea pigs were used as controls and were injected with 1 ml. of cottonseed oil intra- peritoneally. These animals were killed and similar laboratory investi- gations were carried out for the dieldrin group. Samples of brain and liver tissues were taken within 5 minutes after death and fixed in 4% glutaraldehyde in Sorensen's phosphate buffer (0.1M; pH 7.2) in an ice bath at 4 C. for 1 hour. They were then post- fixed in 1% osmium tetroxide (OsO4).solution in Sorensen's phosphate buffer for 30 minutes at room temperature. The tissues were then washed in phosphate buffer and stored in cold (4 C.) 0.2M sucrose solution buf— fered at pH 7.4 until they were embedded. Later, each tissue was placed in a dr0p of 0304 fixative on a wooden spatula and diced to produce small pieces measuring about 1 cmm. The pieces were picked up with a 59 60 flat toothpick and deposited in a glass tube containing 2 m1. of the 0804 fixative. Dehydration with graded alcohols (50, 70, 952 and abso- lute) followed and the specimens were cleared in pr0pylene oxide (Luft, 1961) and infiltrated for 1 hour in a 1:1 mixture of pr0pylene oxide and Epon.1 A modified Lufts (1961) method was adapted for embedding the selected specimens in Epon.using gelatin.capsu1es. An accelerator, 2,4,6-tridimethylaminomethylphenol (DMP-3O)2 at a concentration of 1.5% was added to the resin mixture just before use, and the resin was allowed. to polymerize in an oven at 60 C. for 36 hours. Final preparation for electron microscopic examination of the specimens involved cutting sec- tions on a Sorvall "Porter—Blum" ultramicrotome, Model MST-23 at 400-500A thickness and mounting these on uncoated grids. The sections were then stained by adapting a slight modification of the method described by Pease (1964) in which uranyl acetate was used as a primary stain and lead citrate, instead of lead hydroxide, secondarily. Sections were examined and photographed in the electron microscOpe, Philips Model 1008.“ Results: Ultrastructural Lesions B5313, Some of the mitochondria were swollen to several times their normal size. There appeared to have been imbibition of fluid into the swollen mitochondria with resulting disintegration of the cristae (Figures 12 and 13). There was no obvious breach of the mitochondrial -.membrane. The endOplasmic reticulum and nucleus appeared normal. 1Epon 812. Shell Chemical Corp., San Francisco, California. 2Rohmand Haas Co., Philadelphia. 3Ivan Sorvall, Inc., Norwalk, Connecticut. l’l’hilips, Mount Vernon, New York. 61 Figure 12. Note swollen mitochondria (M) with dis- ruption of cristae in cerebral cells. Glutaraldehyde-osmic acid fixation, uranyl acetate-lead citrate stain. x 20,000. 62 Figure 13. Control. Junction of three cerebellar cells, nuclei (N), cell membranes (C) and normal mitochondria (M). Glutaraldehyde—osmic acid fixation, uranyl acetate-lead citrate stain. x 20,000. 63 Liver. There_was an abundance of "myelin bodies" in the liver sections of both treated and nontreated control guinea pigs. However, neither the mitochondria nor the endoplasmic reticulum appeared to have under- gone any alterations (Figurea l4 and 15) in the treated guinea pigs. 64 Figure 14. Part of a hepatocyte from a dieldrin—treated guinea pig. Note myelin bodies (B), normal mitochondria (M) and normal endoplasmic reticulum (arrows). Glutaraldehyde- osmic acid fixation, uranyl acetate-lead citrate stain. x 20,000. 65 Figure 15. Hepatocytes in a dieldrin—treated guinea pig. Note the normal appearance of the mitochondria (M) and the endOplasmic reticulum (arrows). Glutaraldehyde-osmic acid fixation, uranyl acetate-lead citrate stain. x 25,000. DISCUSSION Ample clinical evidence implicates dieldrin essentially as a neuro- toxicant to various species of animals. However, it shares with other chlorinated hydrocarbon pesticides the preperty of inducing nonspecific degenerative and congestive lesions in most parenchymatous organs. Thus, fatty metamorphosis and congestion were prominent in the livers and kidneys of pigs and sows that were fed the chemical. The degree of change observed appeared to be directly related to the duration of treatment . It is believed that animals on protein deficient diets are most susceptible to poisons that affect the liver (McLean at al., 1965). It has been demonstrated, however, that protein depleted rats are more resistant to the lethal effects of carbon tetrachloride (C014) because of a.drastic reduction in enzyme activity of the liver (McLean and McLean, 1966). The clinical signs in pigs reported in Experiment I suggest that dieldrin toxicosis could be enhanced in protein depleted pigs. The deficient group of pigs also manifested a decreased capacity to accumulate dieldrin in various tissues, with the exception of Pig #1.in this group. The reason for this reduced ability to accumulate dieldrin is not clear, but 2 possibilities are suggested. The resulting impairment of growth may have also reduced the fat content of the various tissues which is said to influence the amount of dieldrin that can be stored. It is possible, too, that there is impaired absorption of dieldrin. from the gastrointestinal tract of protein deficient pigs. Impaired 66 67 absorption of dieldrin from the gastrointestinal tract had been observed in starved rats (Heath and Vandekar, 1964). Consequently these authors suggested that dieldrin absorption is controlled by the amounts of some unidentified naturally occurring materials in the gut and not by the quantity of dieldrin present. Such impairment can result in lower stor- age levels since it has been reported that the amount stored is propor- tional to the rate of intake (Gannon et aZ., 1959). Transplancental transfer of dieldrin was demonstrated in the 2nd experiment. The dieldrin concentrations in the sows' tissues did not correlate either with the doses or the total quantities given. However, examination of the results of analyses for dieldrin in fetal livers (Table 6) reveals a statistically significant difference (P < 0.025 by the t test) between the 2 dosage levels relative to the total amounts of chemical given to their dams. Two factors may be important in explain- ing this observation. The less active microsomal enzymes of the fetal. liver may not metabolize dieldrin fast enough to prevent accumulation which will, therefore, vary with the rate of intake. The dose dependent accumulation in the fetus may also be a reflection on the virtual absence of large fat deposits. In the adult, the fat serves as the predilection site of storage for dieldrin. The concentrations attained by the fetuses apparently caused no harmful effects. A greater than 3—1/2 fold increase in the total amount of dieldrin (from 13.5 to 48 gm.) administered simultaneously with NaN02 over a period of time did not cause malformation, abortion, or death in utero of the fetuses. This is in agreement with reports in other species (Harris at aZ., 1966; Kitselman et aZ., 1950). However, it caused the death of one sow. As the nitrite alone also killed a sow, this aspect of the study is inconclusive with regard to dieldrin alone. Nevertheless, 68 the study indicates that a pregnant sow may be given up to 48 gm. dieldrin over a period of 28 days without ill effects. Since the concentration of dieldrin in a tissue depends on the fat content of that tissue, this may account for the wide difference between the concentrations in the 2 sows5.livers which histologically manifested different levels of lipidosis. This differential concentration was not so marked in the kidneys or brains, the normal fat contents of which are relatively constant. Amongst the fetuses from each sow, there was a relative uniformity in the amounts of dieldrin stored either in the brain or the liver, indicating not only equal exposure to dieldrin but also a probable similarity in the metabolic states of.these featuses, as all fetuses were alive and appeared normal. The nonoccurrence of abortions in these experiments may be associated with the absence of fetal and uterine pathology as indicated in previous reports (Gannon st aZ., 1959). TranSplacental transfer of dieldrin was also achieved in the guinea pig experiments. In the first experiment, dieldrin did not cause deformity or prenatal death of the fetuses, but may have caused the early death ofaa litter of 4 neonatal guinea pigs. Abortions did not occur even though some pregnant guinea pigs died at all treatment levels. The fetuses in the dead or euthanatized guinea pigs manifested no gross or microscopic lesions in spite of the significantly high concentrations of dieldrin in them (Table 10). The generalized hyperemia of tissues and degenerative lesions in parenchymatous organs of adult guinea pigs are in agreement with pre- viously described lesions in other mammalian species (Borgman et aZ., 1952; Kitselman, 1951). The significance of the necrosis and mineraliza- tion observed in uteri of 2 dieldrin-treated guinea pigs is not clear. 69 Myocardial necrosis and mineralization was evident in one of these guinea pigs. Heart failure may-have been responsible for the death in this. instance. The lack of deviations from normal ranges in the hematologic studies confirms the uniformly negative results reported by other workers (Treon at aZ., 1955; Barman et-aZ., 1952; Princi and Spurbeck, 1951). Confirmation was obtained for the preportionality between intake and storage of dieldrin (Bundren st aZ., 1952). However, this seemed to apply to the adult tissues, especially the fat and kidney, but not the fetuses (Table 12). With the total amount of 60 mg. each of dieldrin and nitrite or less. there appeared_to be no enhancement of storage or diffusion of dieldrin to the fetuses. This lack of enhancement changed appreciably at higher total levels of nitrite (120 mg.) administration and may be explained by a possible-increased vasodilatation, decreased blood flow rate and consequent longer contact of dieldrin in the blood with storage tissues and fetuses. The histopathologic changes present in the organs have been sep- arately associated with dieldrin and nitrite. Objective quantitative estimation of any synergism in the development of these changes was not possible. The observed arterial degeneration and occlusion were probably due to the nitrite. These changes as well as the nitrite-induced methemo- globinemia may have caused uterine and fetal hypoxia and consequent abortion (Sinha, 1968). I Sodium nitrite given as 0.52 solution alone caused abortion in 3 of 5 pregnant guinea pigs. Dieldrin at 100 ppm in the feed caused 2 abortions and 2 deaths in a group of 5 while a combination of both chemicals induced 6 abortions and 5 deaths amongst 14 recipient guinea pigs. These results appear to indicate that at these levels nitrite is 70 not an important potentiator of the toxic effects of dieldrin. They also indicate that at the level of 100 ppm, dieldrin is very toxic to most guinea pigs. However, levels of dieldrin at or below 50 ppm administered with or without 0.25% NaNOz appear to have no acute toxic properties as estimated by body weight gain, abortion, or death. The acute oral toxicity (LD50) of dieldrin to guinea pigs was esti- mated from Table 16 to be 45 mg./kg. This is less than the figure of, 59 mg./kg. reported by Bergman et a1. (1952), higher than Jolly's (1954) figure of 20 mg./kg., but in agreement with Gaines' (1969) find- ing of 46 mg./kg. Ultrastructural Effects Hypertrophy of the smooth endoplasmic reticulum (SER) was demon- strated in rat livers after administration of small doses (2 mg./kg.) of dieldrin (Hutterer st aZ., 1968). This was usually associated with initial hyperfunction and later hypofunction of drugdmetabolizing enzymes. In another report, Hutterer et a2. (1969) indicated that the decompensation in metabolizing enzyme activities was associated with mitochondrial injury and reduction in glucosee6-phosphatase activity. The hypertrOphy with hyperfunction was greatest 14 days after dieldrin administration was initiated, decompensation commencing after a temporary steady state was achieved. In the present acute experiment, neither hypertrOphy of the SER nor mitochondrial injury was observed in the liver because there was. little time for the lesions to develOp. However, it is suggested that the mitochondrial swelling in the brain which was similar to the change in a decompensating liver can be accounted for by the comparatively small drug-metabolizing capability of the brain cells. The very high dose of 71 Table 16. Acute oral toxicity of-dieldrin to guinea pigs--numbers, doses and mortality* _.‘_* Number Dosed Dose (mg./kg.) Died Within 24 Hours Survived. 7 O O 7 25 3O 8 17 9 60 3 6 4 75 4 O *LD50 - 45 mg./kg. (Reed and Muench, 1938). 72 dieldrin given caused a high concentration of the nonmetabolized chemical to accumulate in the brain, and may have induced a depression of mito- chondrial metabolic activity and alteration of mitochondrial membrane permeability with resulting swelling. That there may be a disruption of biochemical reactions in brain cells, eSpecially of glutamine synthe- sis, in dieldrin poisoning was suggested by O'Brien (1967). The question of the ultimate cause of the convulsions is not settled. It is suggested, however, that dieldrin induced the initial changes in the mitochondria. SUMMARY AND CONCLUSIONS Pigs and guinea pigs were used in 7 experiments to study the toxicity of dieldrin. The main objectives of the study were to determine (a) the transplacental transfer of dieldrin and the resulting fetotoxic effects, if any; and (b) the ultrastructural changes in the brain and liver asso- ciated with dieldrin toxicosis. The possible roles of.a protein- deficient diet and NaNOz toxicosis in enhancing the toxic effects of dieldrin were also investigated. From the experimental results, the following observations and con- clusions could be made: 1. The pesticide, dieldrin, crossed the placental barrier in the sow and accumulated in the fetal livers in proportion to the total amounts administered to the dam. 2. There was no observable fetotoxic effect, and no abortions. 3. Dieldrin induced nonspecific degenerative and congestive lesions in parenchymatous organs that were directly related to the duration of. treatment of the saw. 4. Clinically, dieldrin toxicosis in pigs was enhanced by protein- deficient diets. 5. Protein-deficient pigs accumulated less dieldrin in their tis- sues than pigs fed a normal diet.‘ 6. Sodium nitrite did not appear to influence the toxicity of dieldrin to sows. 73 74 7. TranSplacental transfer of dieldrin occurred in the guinea pig. 8. Abortions in guinea pigs were caused by dieldrin at high doses. Similar doses also caused death without inducing abortion. 9. Sodium nitrite at high levels enhanced the accumulation of dieldrin in tissues and fetuses of pregnant guinea pigs. 10. Dieldrin storage in adult guinea pig tissues, capecially the fat, was prOportional to the intake. This was not influenced by NaNOz administration. 11. The amount of dieldrin stored in fetuses was not correlated with the quantity administered to the dam. 12. The acute oral toxicity (LDSO) of dieldrin to guinea pigs was calculated to be 45 mg./kg. 13. Body weight loss was not useful in estimating dieldrin toxicity. 14. Acute dieldrin toxicosis was manifested submicroscOpically as swelling of mitochondria in the cerebellum and cerebrum.with disintegra- tion of cristae. The absence of hepatic injury reflected the acute nature of the toxicosis. 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He_spent the next two years, 1954 to 1956, as a student in the Nigerian College of Technology, Ibadan. His professional training was received, from 1957 to 1962, in the University of Glasgow Veterinary School, Glasgow, Scotland, from.which he graduated B.V.M.S. and M.R.C.V.S. In the years 1962 to 1964 he worked in the Federal Department of Veterinary Research, Vom, Nigeria. He transferred to the Veterinary. Department of the University of Biafra in the summer of 1964, from where he came to Michigan State University in the summer of 1966. He received the M.S. degree from the Department of Pathology, Michigan State University, in June, 1968, and remained in the same de- partment as a candidate for the Ph.D. degree. The author has published_or co-authored three scientific papers. 82