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'I “I‘E';“;'j =IELLHJEEyEu, LIEIIWILLLJLE 3% EW WWLM WE‘EQELEIE3LUEIEIEEEELE LL K9.L3V*Lhfll .:EE EEEEEEEEE “is“ HEEEE .EEE EEEEEEEEEEE‘EE EEEEEE-EEEEE EE u} I!!! 'EE'EEIEEI :JEEIPE EEEEEEEL IPIE E'EE ELEEE EEJEE‘EEE'EW ‘JI EEE‘EE EL . IL, '..I‘ I‘EW E El? II . d? E] I‘LIIIIIEILLIEII1,...II‘LII‘EL H L :LIE LEE WEE E “"W ELLE1 ”E I!“ E? ELLE!” LIE E “H " EILIILLIH I“ HHHHH HHHHHHHH HHHHH HHH HH 1 3129300 00677983 833 J " \. : Earth”; 333:»? ‘ :ie:.-.::,,. estate Beavers: ty v This is to certify that the thesis entitled Toxicological Manifestations of Hexachlorobenzene Exposure in the Mink (Mustela vison) and the European Ferret (Mustela Putorius Furo) presented by MICHAEL R . BLEAVINS has been accepted towards fulfillment of the requirements for Ph.D. de eein Animal Husbandry & nvironmental Toxicology Date April 29, 1983 0-7639 ' MS U is an Affirmative Action/Equal Opportunity Institution 1V153H_} RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from Jul-Icilnll. ‘your record. FINES will be charged if book is returned after the date stamped below. V3 1"?"“3 w A. gig, TOXICOLOGICAL MANIFESTATIONS OF HEXACHLOROBENZENE EXPOSURE IN THE MINK (MUSTELA VISON) AND THE EUROPEAN FERRET (MUSTELA PUTORIUS FURO) By Michael R. Bleavins A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science and Center for Environmental Toxicology I983 ABSTRACT TOXICOLOGICAL MANIFESTATIONS OF HEXACHLOROBENZENE EXPOSURE IN THE MINK (MUSTELA VISON) AND THE EUROPEAN FERRET (MUSTELA PUTORIUS FURO) By Michael R. Bleavins The mink and ferret are among the most sensitive mammalian species to the toxic effects of HCB. Anorexia, weight loss, gastrointestinal bleeding, and bloody droppings were observed prior to death. The acute toxicity of HCB was low in these two mustelids. The mink and ferret were, however, very susceptible to chronic HCB exposure. No indications of porphyria were observed in the mink and ferret following the consumption of HCB-supplemented diets. Since the mink and ferret do not develop porphyria, it would appear that the toxic effects seen must be independent of the porphyrin cycle. Ferrets on the 125 ppm HCB-diet were hyperexcitable and displayed signs of neurological impairment. The analysis of brain biogenic amines in mink and ferrets revealed alterations in neurotransmitter concentra- tions. Changes were also found in the mink kits. HCB was readily absorbed (98.5% of the HCB dose) by female ferrets. A half-life of 32 days was calculated for ferrets raising kits and of 4l days for unbred ferrets. Offspring were an important route through which the lactating female excreted HCB. Exposure via the milk for the growing ferret kit was 3l times greater than the exposure occurring 12m- HCB caused an increase in the gestational length of HCB-exposed female mink and a lower whelping percentage than was seen in control Michael R. Bleavins females. A decrease in total litter size was seen in the mink. No change in litter size was found in the ferrets, but an increased inci- dence of kits born dead was observed. Kits born to dams consuming HCB exhibited depressed birth weights and poor survivability to weaning when compared to control offspring. Ig_utgrg_exposure caused the same kit mortality as jn_utggg and lactational exposure combined. Mink kits receiving HCB only via the milk showed a poorer survivability than control kits, but better than kits experiencing HCB during gestation. Chronic exposure to HCB was determined to be immunosuppressive to adult mink and ferrets at 25 ppm of the diet. The lymphocyte respon- siveness of mink kits produced by HCBQtreated dams was also subnormal. ACKNOWLEDGEMENTS I want to thank my parents, Merle and Florence Bleavins, for their everpresent support. love, and confidence. They have always been there when I needed them and made me feel they were solidly behind me no matter what decision I made. I am deeply indebted to Dr. Richard J. Aulerich. Throughout my graduate education he has been an exceptional major professor, a friend, and a source of ideas and insight. I was given as much freedom as I needed and yet provided with a safe haven under his guidance. Thanks Dick for introducing me to the mink and ferret as research animals and their potential for further study. Dr. Robert K. Ringer is also due considerable recognition as an important contributor to my education. He has always been a readily accessible source of ideas and assistance. I would like to thank Dr. Ringer for his faith in me, often helping to support an idea for a research project on little more than his faith in me. I am grateful to Dr. Steven J. Bursian for being a friend and companion in research, as well as serving on my guidance committee. He has worked to make some of the studies more enjoyable by helping me to keep the right perspective. I also want to thank Dr. Thomas G. Bell for his help and input. He helped to make a few of the idiosyncracies of the mink less mysterious. ii TABLE OF CONTENTS Acknowledgments ...................................................... .11 List of Tables ......................................................... v List of Figures ..................................................... viii Introduction ........................................................... I Review of Literature ................................................... 3 Materials and Methods ................................................ .27 Experiment 1. Acute toxicity of hexachlorobenzene to mink Purpose ......................................................... .31 Materials and Methods ........................................... .3l Results ......................................................... .31 Discussion ...................................................... .3l Experiment 11. Effects of chronic dietary hexachlorobenzene exposure in mink and European ferrets Purpose ......................................................... .33 Materials and Methods ........................................... .33 Results ......................................................... .34 Discussion ...................................................... .6l Experiment III. Excretion, placental, and mammary transfer of hexachlorobenzene in the European ferret Purpose ......................................................... .65 Materials and Methods ........................................... .65 Results ............................... , ......................... .67 Discussion ...................................................... .72 Experiment IV. Neurotransmitter concentrations in five brain regions of adult mink and their offspring, and adult ferrets consuming hexachlorobenzene in a chronic feeding trial Purpose ......................................................... .77 Materials and Methods ........................................... .77 Results ......................................................... .78 Discussion ...................................................... .80 Experiment V. Cellular recovery from whole and diluted mink blood and lymphocyte-to-monocyte ratios of density -centrifugation isolated mononuclear leukocytes Purpose ......................................................... .9l Materials and Methods ........................................... .9l Results ......................................................... .92 Discussion ...................................................... .93 Experiment VI. Lymphocyte blastogenic response of adult female mink and ferrets, and their offspring, after consuming hexachlorobenzene in a chronic feeding trial Purpose ......................................................... .95 Materials and Methods Study l. Effects on the response of adult mink & ferrets...95 Study 2. Effects on the response of young mink & ferrets...97 iii iv Results Study l ..................................................... .98 Study 2 ..................................................... 100 Discussion ....................................................... l03 Experiment VII. Effects of ig_utero and early postnatal exposure to hexachlorobenzene on hepatic mixed function oxidases, in vitro renal function and histology, and tissue residues Th'mink Purpose .......................................................... 109 Materials and Methods ............................................ 109 Results .......................................................... llZ Discussion ....................................................... ll7 Experiment VIII. Effects of hexachlorobenzene on reproduction, cell-mediated immunity, organ weights, and microsomal enzymes of mink in a cross-fostering study Purpose .......................................................... l2l Materials and Methods ............................................ 121 Results .......................................................... l25 Discussion ....................................................... l27 Summary and Conclusions ............................................... l32 Literature Cited ...................................................... l36 LIST OF TABLES Table 1. Offspring mortality of rat pups produced by dams consuming control and HCB diets ................................. .18 Table 2. Composition of basal diet .................................. .29 Table 3. Moisture, protein, fat, and ash content of basal diet ...... .29 Table 4. Body weights (9) of adult female mink and ferrets consuming control and HCB diets in a chronic feeding trial ...... .35 Table 5. 'Body weights (9) of adult male mink and ferrets consuming control and HCB diets in a chronic feeding trial ...... .37 Table 6. Hematologic profile of adult female mink and ferrets consuming control and HCB diets for 22 days in a chronic feeding trial ................................................... .38 Table 7. Hematologic profile of adult male mink and ferrets consuming control and HCB diets for 22 days in a chronic feeding trial ................................................... .39 Table 8. Hematologic profile of adult female mink and ferrets consuming control and HCB diets for 149 days in a chronic feeding trial ................................................... .40 Table 9. Hematologic profile of adult male mink and ferrets consuming control and HCB diets for 149 days in a chronic feeding trial ................................................... .42 Table 10. Leukocyte differential counts (arcsin transformed) of adult female mink and ferrets consuming control and HCB diets for 149 days in a chronic feeding trial ............... .43 Table 11. Hematologic profile of adult female mink and ferrets consuming control and HCB diets for 47 weeks in a chronic feeding trial ................................................... .44 Table 12. Hematologic profile of adult male mink and ferrets consuming control and HCB diets for 47 weeks in a chronic feeding trial ................................................... .46 Table 13. Leukocyte differential counts (arcsin transformed) of adult female mink and ferrets consuming control and HCB diets for 47 weeks in a chronic feeding trail ............... .47 Table 14. Leukocyte differential counts (arcsin transformed) of adult male mink and ferrets consuming control and HCB diets for 47 weeks in a chronic feeding trial ............... .48 Table 15. Length of gestation and litter size of female mink and ferrets consuming control and HCB diets in a chronic feeding trial ................................................... .49 Table 16. Birth, three week, and six week old body weights, and biomass raised per lactating female mink and ferret consuming control and HCB diets in a chronic feeding trial ...... .51 Table 17. Kit mortality of the offspring produced by female mink and ferrets consuming control and HCB diets in a chronic feeding trial ........................................... .53 V vi Table 18. Hexachlorobenzene consumed (mg) by adult mink and ferrets in a chronic feeding trial ................................ 54 Table 19. Survival time of adult mink and ferrets consuming HCB in a chronic feeding trial ................................... .56 Table 20. Selected organ weights of adult female mink and ferrets consuming control and HCB diets for 47 weeks in a chronic feeding trial .......................................... .58 Table 21. Selected organ weights of adult male mink and ferrets consuming control and HCB diets for 47 weeks in a chronic feeding trial .......................................... .60 Table 22. Mean (j§.E.) radioactivity (DPM/g) of selected tissues 62 days post-dosing from adult bred and unbred female ferrets exposed to a single dose of 14C-labe1ed HCB and of the offspring born to the bred females ................ .70 Table 23. Mean (:S.E.) radioactivity (DPM/g) of kits born to female ferrets exposed to a single dose of 14C-1abeled HCB and the milk produced by those dams .......................... .71 Table 24. Concentration (ng/gm of tissue) of 5-hydroxy- tryptamine (serotonin) in selected brain regions of adult female mink and ferrets consuming control and HCB diets for 47 weeks ........................................... .79 Table 25. Concentration (ng/gm of tissue) of 5-hydroxy- indoleacetic acid in selected brain regions of adult female mink and ferrets consuming control and HCB diets for 47 weeks ..................................................... .81 Table 26. Concentration (ng/gm of tissue) of norepinephrine in selected brain regions of adult female mink and ferrets consuming control and HCB diets for 47 weeks ............. .82 Table 27. Concentration (ng/gm of tissue) of dopamine in selected brain regions of adult female mink and ferrets consuming control and HCB diets for 47 weeks ..................... .83 Table 28. Concentration (ng/gm of tissue) of 5-hydroxy- tryptamine and 5-hydroxyindoleacetic acid in selected brain regions of young female mink perinatally exposed to HCB .......... .84 Table 29. Concentration (ng/gm of tissue) of norepinephrine and dopamine in selected brain regions of young female mink perinatally exposed to HCB .................................. .85 Table 30. Stimulation indices of adult female ferrets consuming control and HCB diets for 35 weeks ............................... .99 Table 31. Stimulation indices of adult female mink consuming control and HCB diets for 34 weeks ............................... 101 Table 32. Stimulation indices of young female ferrets born to dams consuming control and HCB diets throughout pregnancy and lactation .................................................... 102 Table 33. Stimulation indices of young female mink born to dams consuming control and HCB diets throughout pregnancy and lactation .................................................... 104 Table 34. Effect of perinatal HCB exposure on body and organ weights of 16-17 week old mink kits .............................. 113 Table 35. Effect of perinatal HCB exposure on the hepatic mixed function oxidases of 16—17 week old mink kits .............. 115 vii Table 36. Effect of perinatal HCB exposure on renal gluco- neogenesis and accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA) by renal cortical slices of 16-17 week old mink kits .................................... 116 Table 37. Tissue residue concentrations (ppb) of HCB following perinatal exposure of 16-17 week old mink kits ................. 118 Table 38. Gestation length, reproductive performance, kit mortality, and biomass produced by female mink consuming control and HCB diets in a cross-fostering study ............... 126 Table 39. Birth, three week, and six week old body weights of mink kits exposed to HCB in a cross-fostering study ......... 128 LIST OF FIGURES Figure 1. Structure of the hexachlorobenzene molecule .............. Figure 2. Metabolic transformation of hexachlorobenzene in the rat ..................................................... Figure 3. Synthesis pathway of the heme molecule ................... Figure 4. Interrelationship of T lymphocytes, B lymphocytes, and macrophages in the response, memory, and regulation of the immune response ......................................... Figure 5. Cumulative excretion of HCB by pregnant/lactating female ferrets (Group I) and unbred female ferrets (Group II)... Figure 6. The effect of perinatal HCB exposure on hepatic cytochrome P-450 (open bars) and cytochrome b5 (hatched bars)... viii ...4 ..ll ..14 ..22 .68 114 INTRODUCTION Since World War II, an ever increasing array and quantity of chem- icals have been introduced into the agricultural and industrial fields. In the fight against plant and animal pests, many products have been used to improve crop yields and/or livestock production. Unfortunately, some of the pesticides employed have also been found to be persistent in the environment and toxic to nontarget species. One such chemical is hexachlorobenzene (HCB), a chlorinated aromatic hydrocarbon. HCB is used as a pre-emergent fungicide on cereal grains, an additive for rubber products, an intermediate in the synthesis of many organic compounds, a wood preservative, a porosity controller in the manufacture of elec- trodes, and an additive for certain military pyrotechnic compositions. However, the single most important source of HCB contamination of the environment is its presence as an undesirable by-product in the manu— facture of numerous economically important chemicals. While HCB is a relatively simple molecule (C6C16), it is resistant to biological and chemical degradation. Additionally, HCB is lipophilic and readily traverses cell membranes. This chemical therefore shows a significant degree of bioaccumulation and biomagnification, as well as widespread dispersal through the environment. Residues of HCB are found in a wide range of animal species, including domestic livestock and humans. The accidental or environmental contamination of food, animals, and the human population is a common occurrence with numerous instances 1 of low level insult and occasional outbreaks that have led to overt toxicosis. The adipose tissue acts as a reservoir for HCB and so an animal's body burden of this chemical will be significant for a period of time long after exposure to HCB has ended. HCB is a volatile chemical that sublimes at air-soil interfaces and then enters the atmosphere. while HCB is virtually insoluble in water, it is nevertheless regarded as a major oceanic pollutant. The levels of HCB found in the earth's waters can be attributed to either air-water interface diffusion or the direct dumping of HCB-contaminated wastes into streams and seas. There are relatively few effects seen following acute HCB exposure and in most species thus far tested, the L050 value is in excess of lg/kg of body weight. While acute toxicological manifestations may be minimal, chronic low level exposure presents a greater hazard. Porphy- ria, cutaneous lesions, liver changes, and hyperexcitability are a few of the general indications of HCB toxicity. An outbreak of HCB-related porphyria was reported in several villages in Turkey between 1955-1960. This incidence of human exposure resulted in the death of many young children, especially those still being nursed by their mothers. In the studies reported here, the mink (Mustela vison) and the European ferret (Mustela putorius furo) were used to ascertain HCB- related effects on the reproduction and immunological function of these upper echelon carnivores. Since both the mink and ferret occupy a posi— tion at the apex of the food chain, they represent species most likely to be subjected to high levels of a chemical such as HCB. The mink and ferret are also among the most sensitive test animals available to the effects of other halogenated aromatic hydrocarbons. REVIEW OF LITERATURE Hexachlorobenzene (HCB) is a white crystalline solid with a molec- ular weight of 284.79 and a melting point of 231°C (Merck 1976). The chemical has a log octanol/water partition coefficient of 6.43 and is the most lipophilic of the chlorinated benzenes (Weast 1975). The HCB molecule is a benzene ring with all available positions occupied by chlorine atoms (Figure l). HCB is also known as perchlorobenzene, ,AnticarieE, Bunt-cureE, Bunt-no-moreE, and Julin's carbon chloride (Merck 1976). Several of these names reflect the agricultural uses of HCB. It is a registered pre-emergent fungicide for the control of smut diseases on seed grains such as barley, wheat, oats, and rye. When used in agricultural situations, HCB is mixed with a blue dye to give the seeds a distinctive color (EPA 1980), thus making the grain obviously altered. The technical grade HCB used in agriculture contains 98% HCB, 1.8% pentachlorophenol, and 0.2% l,2,4,5-tetrachlorobenzene. Several commercial HCB products have been shown to contain trace quantities of octachlorodibenzofuran and octachlorodibenzo-p-dioxin (Villeneuva gt_al, 1974). In 1974, only Stauffer Chemical Co. was producing HCB in the United States and its entire production capacity was committed for use as a peptizing agent in the manufacture of rubber used in automobile tires (Mumma and Lawless 1975). The domestic intentional production of HCB was probably an insignificant source of the chemical entering the environment in 1974 and commercial production of HCB in the Figure 1. Structure of the Hexachlorobenzene Molecule. United States was discontinued in 1976 (EPA 1980). The HCB found in the environment is not the direct product of com- mercial manufacturing processes, even though a number of synthesis re- actions are known (Mumma and Lawless 1975). HCB's entrance into the world's ecosystems appears to be primarily as a contaminant in pestic- ides, industrial wastes, and as an undesirable by-product in the synthe- sis of organochlorine compounds. Residues of HCB have been detected in perchloroethylene, trichloroethylene, carbon tetrachloride, chlorine, vinyl chloride, DacthalGn Atrazine®, Propazine®, Simazine®, Mirex®, and pentachloronitrobenzene (Mumma and Lawless 1975). Industrial manufac- turing procedures have been reported to produce 4.3 million pounds per year of HCB as an undesirable by-product (Quinlivan and Chassemi 1977; Courtney 1979). The production of perchloroethylene alone has been estimated to account for 72% of the total HCB entering the environment. At present, three major methods of HCB disposal are employed: 1) place- ment into sanitary land fills, 2) high temperature incineration (+1300° F with a residence time of approximately a second), and 3) deep well injection. Of these three techniques, high temperature incineration is the preferred method as it is the most effective and safest, with vir- tually complete destruction of the HCB (Mumma and Lawless 1975). It is important however, to insure the incinerator temperature is suffic- iently high to destroy the chemical. When heated only to decomposi- tion, HCB produces toxic fumes (Sax 1963) of greater toxicity than the parent molecule. Sanitary land fills are the method of second choice and represent a greater proportion of the chemical's ultimate disposal than does incineration. Deep well injection is a third alternative, although not a preferred technique (Mumma and Lawless 1975), due to questions of permanent containment. Although it is not a naturally occurring chemical (Courtney 1979), HCB shows great persistence once it enters the environment (Beck 1974; Beall 1976; Isensee gt_al, 1976; Plimmer and Klingebiel 1976). HCB is volatile (Beall 1976) and tends to sublime at air/water interfaces (NAS 1975; Beall 1976). Aerial dis- persion, via volatilization, appears to be the major mechanism for HCB to enter the marine environment. The solubility of HCB in water is very low, generally less than 2 ug of HCB per kg of water. In spite of this, HCB has been the only organic chemical designated as being present in the world's oceans at concentrations likely to result in serious prob- lems (Morse 1975; NAS 1975; Seltzer 1975). HCB has been detected in surface waters, soils, and animal species (Leoni and D'Arca 1976), as well as in finished drinking water (EPA 1975). It readily bioconcentrates and bioaccumulates, showing a greater potential for the accumulation in fat of animal species than the poly- chlorinated biphenyls (PCBs) (Hansen gt_al, 1979). The biomagnification of HCB from the aquatic environment has been shown for algae, inverte- brates, and fish species (Lu and Metcalf 1975). Bioconcentration fact- ors of from 1,964 to 23,000 for fish and shellfish (EPA 1980) have been calculated. HCB residues have also been found in a variety of freshwater fishes (Johnson gt a}, 1974; Niimi 1979), including alewives, smelt, salmon, and trout. When terns consumed fish contaminated with HCB, they showed tissue residues in excess of those found in the food fish. Pred- atory birds have been shown to likewise have high concentrations of HCB (Vos gt_al, 1969; Best 1973; Cromartie gt_al, 1975), concentrations much higher than are found in the prey species they feed upon. The residues of HCB were also shown to be greater in the fox (carnivore) and wild boar (omnivore) than the herbivorous deer (K055 and Manz 1976), showing HCB concentrations to increase progressively up the food chain. The acute toxicity of HCB is low (WHO 1970; Booth and McDowell 1975), greater than lg/kg of body weight in several species. However, the subdividing of a large dose of HCB resulted in greater accumulations of the chemical in the adipose tissue of beagles and appeared to enhance the toxic effects (Sundlof et_gl, 1981). The consumption of HCB has usually been the result of ignorance, carelessness, or a general lack of concern (Courtney 1979). This is especially true in impoverished/ developing countries where there is a strong tendency by the human p0pulation to feed excess seed grains to livestock, or even consume it directly. HCB has been found in chemical plant workers (Burns and Miller 1975), as well as people with no known direct contact with HCB (Baaken and Seip 1976). Residues of this organic chemical have been detected in humans throughout the world (Siyali 1972; Brady and Siyali 1972; Curley gt_gl, 1973; Leoni and D'Arca 1976; van hove Holdrinet gt_gl, 1977) and in the breast milk of women (Newton and Greene 1972; Stacey and Thomas 1975; Baaken and Seip 1976; Strassman and Kutz 1977; van hove Holdrinet ‘gt_al, 1977). Most of the human exposure occurs through the consumption of contaminated food products. HCB has been found in the adipose tissue of domestic animals and poultry (Booth and McDowell 1975; Laska £3 31, 1976) destined for human use. It has also been detected in plant species commonly used for human and/or livestock feed (deVos gt_§l, 1974; Dejonckheere gt_§l, 1975, 1976). In market basket surveys of typical American food products conducted by the Food and Drug Administration, 5% of the samples contained HCB residues during the years of 1972-1974 (Johnson and Manske 1976; Manske and Johnson 1977). Similar incidences of contamination have been reported in fresh and processed milk products from eastern Bohemia (Cerna gt_gl, 1977). The absorption of HCB from the diet is somewhat dependent on the nature of food consumed. The feeding of high fat diets to rats resulted in higher HCB residues than equivalent concentrations fed in low fat diets (Zabik and Schemmel 1980). Broiler chickens absorbed virtually all of the HCB present in their diet in a study by Reed gt_al, (1977). The HCBresidue profile of rats showed that there was a dose-dependent accum- ulation (Villeneuve 1975) from the diet. The quantity of HCB absorbed from a single oral or dietary dose was also dependent on the nature of the carrier used. Rats absorbed approximately 80% of the chemical pres- ent when the HCB was dissolved in oil (Albro and Thomas 1974; K055 and Koransky 1975), but only 6% when the HCB was suspended in water (K055 and Koransky 1975). HCB is absorbed slower from the gastrointestinal tract of rats than the pesticide dieldrin or dichlorobiphenyl (Iatropoulos gt_al, 1975). It also shows a variable degree of tissue accumulation in different species. The rat accumulates a greater amount of HCB than does the guinea pig (Villeneuve and Newsome 1975), perhaps due to differences in absorption and/or metabolism between these two rodents. The major portion of ingested HCB appears to be absorbed by the lymphatic system of the upper regions of the small intestine (Iatropoulos gt_gl, 1975; Turner and Shanks 1980). The portal venous system plays a lesser role (Iatropoulos gt_al, 1975) in the passive transport of HCB across the gut wall (Richter and Schfifer 1981). Chemicals which enter the body tissues through lymphatic absorption bypass the liver, at least initially, and so may be stored in the body without being metabolically altered (Turner and Shanks 1980) and are less likely to be rapidly excre- ted. Metabolites accounted for 10% of the HCB in the blood of female rats and less than 0.1% of the chemical in adipose tissue (Koss gt_al, 1976). The distribution of HCB within an individual is closely correl- ated with the fat content of the tissues (Avrahami and Steele 1972b; Sundlof gt_al, 1982). Those tissues with the highest fat concentration generally exhibit the highest HCB residues. The tissues may contain not only HCB, but also traces of its dechlorinated metabolites (Mehendale gt 31. 1975). Richter gt_gl, (1981) found pentachlorophenol, penta- chlorothiophenol, 2.3.4.6- and 2,3,5,6-tetrachlorophenol, and pentachloro- benzene in all rat tissues analyzed for HCB. Differences have also been reported between sexes of the same species. Male birds show significant- ly higher HCB residues than females (Vos gt 11. 1971; Ingebrigtsen gt 11. 1981), indicating the importance of elimination via the egg yolk in avian species (Hansen gt 31. 1978; Ingebrigtsen et_al, 1981). The reverse situation is seen in mammals with females accumulating higher HCB concen- trations than males (Richter gt_al, 1981). This phenomenon is probably linked to the greater metabolic activity, and therefore more rapid rate I of excretion, generally seen in male mammals when compared to non-lacta- ting females. The tissue concentrations of HCB generally increase rapidly upon first exposure to the chemical and then reach a plateau where intake and excretion are approximately equal (Kuiper-Goodman gt_al, 1977; Koss _e_t__al. 1978). The excretion of HCB occurs predominantly through the feces (K055 and Koransky 1975; Yang gt_al, 1978; Richter and Schfifer 1981; Rozman gt_§1, 1982; Sundlof gt_al, 1982). Fecal excretion in beagle dogs was demonstrated to be the result of two separate processes (Sundlof gt a1, 1982), the major contributor of HCB to the intestinal bolus being biliary excretion with intestinal wall excretion playing a lesser role. However, Rozman gt_al, (1982), in a study utilizing the rat, found intestinal wall passage to be the primary route of excretion in this species. The elim- ination of HCB with bile was insignificant and enterohepatic recircula- tion was not a major phenomenon in a concurring study of rats (Richter and Schfifer 1981). HCB was excreted by each of the three regions of the small intestine, with the jejunum excreting more HCB per unit length of gut than the segments (Richter and Schfifer 1981). Urinary excretion of HCB is much lower than fecal quantities, representing one-eighth or less of the total HCB eliminated by the rat (Koss and Koransky 1975), dog (Sundlof gt_al, 1982), and monkey (Yang gt_al, 1978). The egg yolk rep- resents the main excretory route in laying female birds (Hansen gt_al, 1978; Ingebrigtsen et_al, 1981). Yang et a1, (1978) have proposed the underlying reason for the relatively slow elimination of the animal's total body burden of HCB to be due to the long-term storage in the adipose tissue. Food restriction caused a mobilization of the HCB in fat stores (Villeneuve 1975). The chemical was, however, only transferred into the plasma and other tissues of the rats, with no increase in the rate of excretion. The total body burden was therefore not altered and the HCB 10 was redistributed into depots where it may present a greater hazard to the animal than it represented while stored in the adipose tissue. Various agents have been added to the diets of laboratory animals in attempts to increase the fecal and urinary excretion of HCB. Hexadecane, a liquid paraffin, enhanced fecal elimination of HCB and/or its metabol- ites 4 to 13 fold in rats and Rhesus monkeys (Rozman gt_al, 1981, 1982). This compound did not affect the urinary excretion of HCB, although bile duct ligation tripled elimination by this route. Mineral oil similarly resulted in an increase of from 6 to 9 times the normal rate of HCB ex- cretion in monkeys (Rozman gt_al, 1981). Richter gt_gl, (1982) also found dietary treatment of rats with a light liquid paraffin, squalone (a nonabsorbable hydrocarbon), or sucrose polyester improved fecal excretion of HCB without altering urinary excretion. Squalone was like- wise shown to stimulate fecal HCB elimination in mice (Richter and Schfifer 1982). These compounds appear to act as a lipophilic compartment in the intestinal lumen (Richter and Schafer 1981). The equilibrium between the gut wall and the bolus traveling along the intestinal tract fbr strongly lipophilic compounds such as HCB, would thereby be shifted in favor of the bolus. Cholestyramine, an anion binding resin, and sesame 011 did not elicit any improvement in the excretion of HCB and/or its metabolites in rats or monkeys (Rozman gt a1, 1981). Koss 33 91, (1976) found the majority (90%) of the HCB excreted in the urine of rats to be in the form of metabolites. The probable bio- transformation of HCB is represented in Figure 2. The three major metab- olites were identified as pentachlorophenol (45%), pentachlorothiophenol (30%), and tetra-chlorohydroquinone (17%). Due to the small quantity of HCB excreted via the urine, these metabolites actually comprise only a 11 a 0‘" Cl Cl c1 cu cu CI 03 c1 cu cu 0 e1 .. 0 0 cu c c: on 511 u - c1 .:0:' H :0: .H ::0:. 0. "ac Figure 2. Metabolic transformation of hexachlorobenzene l in the rat (modified from Debets and Strik cn 1979) . (:1 cu C1 C1 12 small quantity of the total HCB ingested. The biological half-life of HCB has been calculated in several lab- oratory, livestock, and fish species. Values of 60 days (Morita and Oishi 1975), 110 days (Richter gt_gl, 1982), and 120 to 150 days (Koss gt_al, 1978) have been reported for the rat. The half-life in dogs was shown to be six weeks by Sundlof gt 31, (1982) and 10 to 18 weeks in the domestic sheep (Avrahami and Steele 1972a). Rainbow trout (Sglmg_ gairdneri) were studied by Niimi and Cho (1981) and a biological half- 1ife arrived at of from 61 to 117 days. The clinical signs of HCB intoxication include anorexia, tremors, and hyperexcitability (Kimbrough and Linder 1974; Villeneuve 1975; Clark et_al, 1981). HCB potentiated the severe weight loss seen in rats sub- jected to crowded conditions (Clark gt_al, 1981). The toxicity of mer- curic chloride (HgClZ) was also increased by HCB treatment (Renner 1980). Tremors and hyperexcitability have been reported in mice, guinea pigs, rabbits (DeMatteis gt_al, 1961), and Japanese quail (Vos gt_al, 1971) fed HCB. An increased incidence of tumors has also been observed in mice (Cabral_gt_al 1978; Shirai et_al, 1978) and hamsters (Cabral gt 91, 1977) consuming HCB in long-term carcinogenicity trials. Cabral et_al, (1977) found the average number of tumors per hamster and the percentage of animals with multiple tumors to show a dose-response relationship. In this study, numerous ne0plasms were identified including hepatomas, liver hemangioendotheliomas, spleen hemangioendotheliomas, adrenal ne0plasms, and alveolar adenomas of the thyroid. HCB alone did not result in hepat- carcinogenicity in mice (Shirai gt_al, 19780), but potentiated the induc- tion of tumors following treatment with PCBs. Upon histological examin- ation, Iatropoulos gt 21, (1976) found thymic cortex atrophy and 13 degenerative changes of the liver and kidney in Rhesus monkeys treated with HCB. Human exposure to low level dietary HCB has resulted in examples of HCB-induced porphyria cutanea tarda (PCT) in Turkey and Saudi Arabia (Courtney 1979). In both of these incidences, seed wheat that had been treated with HCB was used to make bread. Approximately 5000 pe0p1e were estimated to have developed PCT (Schmid 1960; Dogramaci gt_al, 1962; Dogramaci 1964) during the course of the Turkish outbreak, with a mortal- ity rate of 10%. A loss of appetite, cutaneous photosensitivity, muscle weakness, hypertrichosis, hyperpigmentation, and porphyrinuria were seen in many of the people exposed to HCB (Courtney 1979). PCT, also known as porphyria turcica, is a metabolic disorder of porphyrin production char- acterized by an increased excretion of porphyrins and their precursors. Cutaneous lesions are a visible manifestation of this porphyria, induc- ible by chemicals such as HCB (Courtney 1979). The repeated exposure to sunlight resulted in erythema, erosion, crusting, skin thickening, and scarring of the skin of rats with HCB-induced porphyria (Torinuki gt_al, 1981). The control mechanism underlying the normal synthesis of porphy- rins (Figure 3) can break down when an animal is exposed to HCB. In HCB-related PCT, excessive quantities of porphyrins are produced which are not incorporated into hemoproteins, and so are excreted in the urine and feces. HCB-induced porphyria has been reported in rats (Lissner gt 21, 1975; Elder gt al. 1976; Torinuki gt_al, 1981), rabbits (DeMatteis gt_al, 1961), Japanese quail (Vos et_al, 1971), chick embryos (Debets g3_ al, 1981a), and pigs (Hansen gt 31. 1977). In a separate study by denTonkelaar gt_al, (1978), pigs were observed to develop porphyria-like symptoms, but the liver fluorescence characteristic of PCT was not Figure 3. Synthesis pathway of the heme molecule (modified from Courtney 1979 & Hammond and Beliles 1980). 14 amaLaEOm_11.::::::wJomm>a>ooa HHH zmwozmm>zamoaom= mmmymgucxm a ox: mmmpxxoaemumu cmmocwexzagoaoes zmuozHgHmozamoa Emd$h0pmw ammoaeusemu zmmomomacu oxeoeez mmmpaxonemumc :mmo:.e»gagoaoeaou o~u< quH42>mgozmzzamoa0homa mmmpmsucxm <4< mmmuxxoncmmouoea HHH zam>xamoaoeoaa 3 mz~u>au + «co a>zHoo=m mmmpmpoguoeemw en. ++ma 15 detected. The development of PCT following HCB exposure is not univer- sal among the animal species tested. It was not possible to induce porphyria in Beagle dogs with dietary HCB (Gralla gt_al, 1977) and no liver fluorescence was seen at necropsy. The dog may lack the synthetic and excretory pathways which respond to HCB insult and result in porphy- ria. Differences were also observed between sexes of porphyria sensitive animals. Female rats exhibit PCT sooner after exposure to HCB and to a more severe degree than males (Kuiper-Goodman gt_al, 1977; Richter et_al, 1981). This differential sensitivity may result from inherant differences in the basal levels of enzymatic biotransformation (Richter gt_al, 1981) or from some estrogen-related pathway (Kuiper-Goodman gt_al, 1977). The mechanism by which HCB produces its porphyrinogenic effects is poorly understood (Debets and Strik 1979). However, the metabolic trans- formation of HCB appears to be a requirement for the induction of PCT (Kerklaan gt 21, 1979; Debets gt_al, 1980). A metabolic product, pre- sumed to be a reactive intermediate, appears to be responsible for the alterations of heme synthesis seen in HCB-induced toxicity. Koss §t_a1, (1980) have identified reactive conversion products of HCB in the livers of rats. The simultaneous administration of HCB and phenobarbital in the rat produces an earlier disturbance of porphyrin synthesis than HCB alone (Puzynska gt_al, 1978), probably as a result of mixed function oxidase enzyme induction. Diethyl maleate, a compound which depletes liver stores of glutathione S-transferase, also causes an earlier disturbance of the porphyrin pathway when given in conjunction with HCB (Puzynska gt_ 31, 1978; Kerklaan et_al, 1979). The resulting lower concentrations of glutathione available for the detoxification of porphyria-inducing agents probably allows the more rapid onset of clinical signs seen in these rats. 16 Many porphyrinogenic chemicals, including HCB, cause the gg_ngyg_ synthesis of aminolevulinic acid (ALA) synthetase (Granick and Urata 1962). The elevated ALA-synthetase levels result in excesses of amino- levulinic acid, monopyrrole, porphobilinogen, tetrapyrroles, uropor- phyrin, caproporphyrin, and protoporphyrin. The porphyrinogenic effects of HCB are therefore not due to a direct alteration of heme synthesis (Debets and Strik 1979). HCB exposure causes an inhibition or inactiv- ation of the liver enzyme uroporphyrin decarboxylase (Elder et_al, 1976; Debets and Strik 1979; Strik gt_al, 1979; Koss gt_al, 1980), and so uroporphyrins accumulate. The disturbance of uroporphyrin decarboxylase function may be a consequence of covalent binding of HCB-reactive conver- sion products to the enzyme (Koss et 21, 1980). Rats treated with HCB also showed a reduced glucuronyl transferase activity and altered steroid metabolism with the increased production of unconjugated Ss-steroids (Graef gig. 1982). The duration of time a chemical resides in the body and its intensity of action is largely dependent upon the speed at which it is metabolized by the enzymes of the mixed function oxidase (MFO) system in the liver (Conney 1967; Reed gt_a1, 1977). The induction of these enzymes can thereby result in significant changes in the metabolism of xenobiotics. HCB has been reported by denTonkelaar and vanEsch (1974) not to be a potent inducer of the microsomal enzymes. Carlson (1978) found HCB to be a phenobarbital-type inducer of MFO activity, while others have reported this chemical to be a mixed inducer (Stonard 1975; Stonard and Greig 1976), inducing both 3-methylcholanthrene and phenobarbital type enzymes. Increased concentrations of liver cytochrome P-450 (Lissner gt_ 31, 1975; Puzynska gt a1, 1979) and of aniline hydroxylase activity 17 (Lissner gt_al, 1975; Grant t l. 1977) have been reported for rats exposed to HCB. Mehendale _t;al, (1975) likewise found the pretreatment of rats with HCB to lead to significant induction of the MEG system. A dose-related induction of liver enzymes was also observed in domestic pigs (denTonkelaar gt_al, 1978). The induction of MFO enzymes may repre- sent an increase in the rate of synthesis, as well as a diminished rate of enzyme destruction (Conney 1967). Mehendale gt_al, (1975) have deter- mined that the reductive dechlorination of HCB seen in the rat is cata- lyzed by enzymes located in the liver, lung, kidney, and intestine. Phenobarbital treatment (by MFO induction and therefore increasing HCB metabolic transformation) and diethyl maleate (by competing for hepatic glutathione) result in a more rapid production of HCB metabolites and the toxic effects they exert than is seen with HCB alone (Puzynska gt 21, 1979). Piperonyl butoxide, an inhibitor of MFO activity, decreased the irreversible binding of HCB metabolites to the cellular proteins in chick embryo liver cells (Debets gt_al, 1981a), suggesting a reduced rate of biotransformation of the parent compound by this enzyme system. One physiological effect that may be observed from phenobarbital-type induc- ers is the increased hydroxylation of steroids. By reducing steroid hormone concentrations, such as estradiol and estrone, an inhibition of their action on the reproductive tract of the female occurs and can result in reproductive disturbances or complete reproductive failure. These changes represent an undesirable side effect of the body's attempt to metabolize and/or detoxify xenobiotics. HCB has a definite adverse effect on reproductive ability (Mendoza gt_al, 1975; Grant gt_al, 1977). Dietary HCB resulted in female rats producing fewer litters, an increased incidence of still born pups, and 18 poor postnatal viability (Grant _t__l, 1977). Young rats raised by mothers consuming HCB-supplemented diets died seven to eight days after birth in a study by DeMatteis et_al, (1961). Additionally, rat pups from unexposed dams that were cross-fostered onto these HCB females died in convulsions after three to four days of nursing, as had the female's own litter. The percent mortality of rat pups exposed to HCB via their dams from two different laboratories is compiled in Table 1. From the study by Grant et_al, (1977), it was concluded that 20 ppm HCB in the maternal diet was the no-effect level for reproduction in the rat. Reduced birth weight, a general indication of toxicosis. was also report- ed for rat pups born to HCB-exposed dams (Khera 1974; Grant gt_ 1. 1977), as was a reduced growth rate. Table l. Offspring mortality of rat pups produced by dams consuming control and HCB diets. Dietar . C°"Ce"{ratl°” Grant et a1. 19H7MortaHHHHhin et a1. 1932 0 ppm 8 9.2 10 ppm 1 - 20 ppm 8 - 40 ppm 7 - 60 ppm - 19.8 80 ppm 5 30.0 100 ppm - 45.4 120 ppm - 93.1 140 ppm - 92.6 160 ppm 43 - 320 ppm 100 - 19 Transplacental passage of HCB from the mother to the fetus has been shown in rats (Villeneuve and Hierlihy 1975; Andrews and Courtney 1976; Courtney et_al, 1979; Svendsgaard §t_al, 1979), mice (Courtney gt_ 21, 1976; Courtney and Andrews 1979), rabbits (Villeneuve gt_al, 1974), monkeys (Bailey gt_al, 1980), and humans (Siyali 1974). The placenta offers little resistance to the passage of small lipophilic molecules such as HCB. The so called "placental barrier" provides only limited protection to the developing fetus against chemicals it may be exposed to during gestation (Goodman gt_al, 1982). Svendsgaard gt_al, (1979) obser- ved a sixfold increase in fetal concentrations of HCB in mice and rats that were multiply dosed during late gestation, as compared to animals similarly dosed during early pregnancy. This phenomenon probably results from normal changes in placental permeability (Moya and Smith 1965). As the placental membranes become thinner and the flow rate of the umbilical circulation increases with advancing pregnancy, fetal contact with sub- stances in the dam's blood is enhanced. Since most foreign substances have been shown to cross the placenta by simple diffusion (Eckhoff 1972a,b), changes in placental surface area available for transfer, thickness of placental membranes, and the rate of maternal blood flow with progressing gestation would increase the rate of diffusion, resulting in near—term exposed fetuses possessing greater concentrations of HCB than those fetuses exposed during the early stages of development. The maternal tissue stores of HCB act as a readily available reservoir of chemical for continued expos- ure to the developing and growing offspring (Courtney and Andrews 1979). The potential of chemical-induced fetotoxicities to be the result of pla- cental and/or fetal biotransformation must also be considered (Goodman et_ al, 1982). The placenta does, however, appear to offer some degree of 20 protection to the fetus. Andrews and Courtney (1976) found the HCB content of the placenta of mice and rats to always be greater than its respective fetus. HCB has also been found to cause grossly observable teratogenic effects in rats (Khera 1974) and mice (Courtney g1_a1, 1976), including cleft palates and kidney malformations. The presence of HCB in the milk of lactating females has been shown for humans (Siyali 1973; Baaken and Seip 1976), Rhesus monkeys (Bailey e1_ 31, 1980), cattle (Fries and Marrow 1971), and rats (Grant 31_a1, 1977). Siyali (1973) found HCB to be present in the breast milk samples of all the women tested. Bailey g1_a1, (1980) determined the milk concentrations of monkeys to be 17 to 20 times the concentration of maternal serum. High concentrations of HCB have been reported in weanling rats, compared to a relatively low dietary intake of the compound by their dams (Grant g1.a1, 1977). This indicates the excretion of HCB via the milk to be an important route of offspring exposure and of maternal excretion. This finding is supported by the determination by Bailey g1_a1, (1980) that nursing infant Rhesus monkeys were at greater risk than their mothers when the dam was exposed to HCB. Human infants have likewise been shown to be sensitive to HCB when exposure was limited to that coming from their mothers. A 95% incidence of infant mortality (as compared to 10% mortality in adults) was seen in some villages in Turkey when mothers consumed HCB-contaminated bread (Courtney 1979). Pembe yara, or "pink sore" was also seen in the nursing children. Porphyria was not noted in these children, suggesting that pembe yara may be a forerunner of porphyria in young children (Peters e1_a1, 1966). HCB can adversely affect reproduction in avian species as well. At high doses of HCB a delay in the onset of full egg production was seen in 21 chickens (Hansen g1_a1, 1978). It has also been linked with poor embry- onic survival and reduced hatching success in gulls (Gilbertson and Fox 1977) and terns (Gilbertson and Reynolds 1972). The normal immunological defense mechanisms involve the participa- tion of four major aspects: 1) T lymphocytes (cell-mediated immunity), 2) B lymphocytes (humoral immunity), 3) phagocytic cells, and 4) comple- ment (Dean $1.21, 1979; Luster and Faith 1979; Heise 1982). A proper immune response following challenge is dependent upon the c00perative interaction of these factors. Different subpopulations of T lymphocytes and mononuclear cells are interconnected to form regulatory circuits (Figure 4) which determine the quality, extent, and duration of an immune response (Hahn and Kaufmann 1981; Litwin 1981). The cells of the immune system originate from pluripotent stem cells located in the yolk sac and liver of the fetus and in the bone marrow and spleen of the adult (Phillips and Melchers 1976; Abramson eg_a1, 1977; Owen e1_a1, 1977). The bone marrow serves as a source of cells to populate and proliferate in the thymus (Cantor and Heissman 1976), as well as the body's other lymphoid tissues. In the adult mammal, the proliferation of these stem cells provides for the constant renewal of the circulating pool of lymph- ocytes and phagocytic cells (Hume and Weidemann 1980). Liver, lung, and spleen contain the major populations of fixed tissue macrophages (Loose g1_a1, 1977) which may be the initiators of the immune response. Cell- mediated immunologic competence provides the organism with protection against foreign tissue transplants, certain viral, bacterial and fungal infections, intracellular parasites and neoplastic cells (Eisen 1974; Dean e1Hg1, 1979; Luster g1fla1, 1979). The lymphocyte transformation microculture techniques, using plant Figure 4. Interrelationship of T lymphocytes, B lymphocytes, and macrophages in the response, memory, and regulation of the immune system (modified from V05 1977). 22 immunogen 1 macrophage O"processing" T lymphocyte B lymphocyte. T helper cell sensitized B lymphocyte / T suppressor B suppressor cell cell sensitized T lymphocyte T killer T effector ‘0 T memory 8 memory cell cell H cell cell plasma cell activated macrophage 23 lectins (mitogens), provide a sensitive and reliable assessment of cell- mediated immunity (Thilstead §1_a1, 1979; Dean e1_a1, 1982a; Luster g1 21, 1982). These primary cell culture assays allow the monitoring of the immune system following chemical exposure. The immune system cells, having been exposed to the toxicant 1g_!119_ can be readily removed from the body and their function examined 1n_11139, Thilstead g1_g1, (1979) have pointed out the further suitability of these techniques in that they allow repeated testing without altering the antigen-specific aspects of cell-mediated immunity. With the development and improvement of gradient separation techniques for the isolation of lymphocytes, lymph- ocyte activation/transformation tests have become simpler and more reli- able. By taking advantage of differences in the size and density of the formed elements of blood, a large percentage of the total viable lymph- ocyte papulation from a given sample of whole blood can be isolated, relatively free of contamination (Perper g1_a1, 1968). Antigens normal- ly induce transformation in only a small responsive population of immune cells and such changes must be gauged against changes going on in a much larger background population of cells. These small alterations are not generally detectable by standard biochemical assays, but through the use of mitogens a large proportion of the lymphocytes can be stimulated, regardless of their antigenic specificity (Stanton £1.91, 1978; Hume and Weidemann 1980). The mitogen-induce lymphocyte subpopulations can then express their various functions. Hume and Weidemann (1980) have reported cellular proliferation not to be merely a simple reaction between a lymphocyte and the mitogen, but rather a complex interaction between several types of differentiated cells. The binding of the mitogen to the lymphocyte's surface receptor appears to initiate activation (Resch £1_a1, 24 1981). This stage does not seem to require the actions of accessory cells such as macrOphages. A second signal, independent of the mitogen, results in the progression of activated lymphocytes to proliferative cells. It is this step which is dependent on the macrophage and acts to modulate the size of the proliferating lymphocyte p0pulation (Resch e1 31, 1981). Unlike the lymphocytes, macrophages do not exhibit immuno- logic specificity (Rosenberg and Lipsky 1981) and so are capable of res- ponding to a wide range of immunogens. The macrophage-mediated enhance- ment is probably the result of soluble factors released by the macro- phages (Gery e} a1, 1972). The plant lectin Concanavalin A (Con A) is considered to be capable of stimulating T lymphocytes, but not B lymphocytes (Janossy and Greaves 1972). More specific studies have shown Con A to selectively activate T-helper and T-suppressor subpopulations (Stanton £1.31, 1978; Hume and Weidemann 1980; Tsokos and Balow 1981). The T lymphocyte response to this mitogen is also dependent upon the presence of macrophages (Wedner and Parker 1976; DeVries g1_g1, 1979; Resch e;_a1, 1981) and their meta- bolic products (Persson e1_a1, 1978). Chronic low level or acute high level exposure to a toxicant can impair immunologic function and result in an increased susceptibility to disease and opportunistic microorganisms (Bick 1982; Heise 1982). The immunosuppressive effects of a chemical may or may not be the result of direct action upon the immune system cells. Other factors such as stress can alter immune function and produce an animal incapable of prop- erly responding to challenge (Street and Sharma 1975; Kelley 1980; ‘ Luster g1_a1, 1982). The abnormal immune profile may be the represent- ative of a generalized toxic response rather than the immune system 25 being singled out as a specific target. This is an important consider- ation-since the impaired immunologic responses seen are often occurring at toxicant levels which also result in toxic effects in other organs (V05 1977). Other indirect effects (nutritional deficiencies, pathogenic organisms, endocrine inbalances) can similarly lead to functional abber- ations in the immune system (V05 1977; Loose 1g _1, 1979). The direct altering of the immune system, as measured by lymphocyte responsiveness, can result from: 1) a failure in the activation of the lymphocytes, 2) tolerance to the immunogen, and/or 3) the production of soluble suppres- sor factors by macrophages or the regulatory subpopulations of T lymph- ocytes (Dean g__a1, 1982a). Street and Sharma (1975) have reported that the immune responses most easily damaged are those portions of the system dependent on sensitized lymphocytes (Figure 4) to carry out the cell— mediated reactions. The immune system also shows a considerable ability to rebound after insult. Harmful effects may be temporary (Moore 1979; Bick 1982), even though the period of time required to reestablish com- petency may be long. The 1g_yj§gg_function assays may over estimate the degree of cellular damage. As stated by Archer (1982), cells which have been injured by exposure to a chemical may appear nonfunctional in the somewhat hostile 1g,!1§rg environment, but might have been able to regain normal function had they been left 1n_1119, Although there exists a direct relationship between the chemical suppression of the immune system and resistance to infectious diseases, any relationship between immunosuppression and cancer and autoimmune disease is probably only an indirect link (V05 1977). The immune system is a sensitive indicator of toxic insult. This is probably the result of several different, but interconnected 26 components. Normal immune function is a tightly regulated and organized series of events with the cells involved acting in cooperation with each other (Litwin 1981; Dean e1_a1, 1982a) to protect the host. Also, the numerous cell types participating in an immune response undergo rapid proliferation and differentiation to produce their actions. The high metabolic demands and elevated activity in the synthesis of proteins and DNA, makes these cells especially vulnerable to toxicant exposure. MATERIALS AND METHODS The mink (Mustela vison) used in these studies were from the Mich- igan State University Experimental Mink Ranch. The European ferrets (Mustela putorius furo) were bred from stock obtained from Marshall Research Animals, North Rose, NY. All animals were housed and cared for at the MSU Experimental Mink Ranch. The mink and ferrets were confined in individual pens of uniform size and each pen was equipped with a water cup and nest box. Except where stated otherwise in the text, the following procedures pertain to all experiments. Standard ranch procedures were followed in the care, feeding, and breeding of the animals. The mink were vaccinated for canine distemper, botulism, and viral enteritis in July. Ferrets were similarly vaccinated in August. In alloting animals into various groups for experimentation, littermates were divided between groups in an attempt to balance genetic differences in growth, reproduction, and response to treatments. The mink and ferrets were weighed individually at the start of the experiments and at specified intervals thereafter, except during the gestational periods. The females were mated to males within their respec- tive dietary group. Each female mink was given an opportunity to remate either the day after the initial mating or eight days later. The female ferrets were mated beginning with their first estrus cycle in April. Any females that recycled were once again mated, up to three separate times. All matings were verified by the presence of motile spermatozoa in a 27 28 vaginal smear taken after copulation. The females were checked daily for the birth of young during the whelping period. Kits were counted and weighed the day of birth, at three weeks of age, and at six weeks of age. Gross necropsies were performed on the animals that died during the course of the experiments. Organ weights were recorded and selected tis— sues collected for HCB analysis and histopathologic examination. Tissues for histology were fixed in 10% neutral formalin and processed by routine histological procedures. Neural tissues were sectioned at 15 microns and all other tissues cut at 5 microns. Sections were stained with Harris's Hematoxylin and differentiated for 10 seconds in 1% H01 and 80% ethyl alcohol. Each was then counterstained in 0.5% Eosin Y and differentiated in two chages of absolute ethyl alcohol. The slides were cleared in xylene and coverslipped using Flo-Texx® (Lerner Laboratories, Stamford, CT). In the preparation of the experimental diets employed in these studies, the HCB was incorporated into 500 g of commercial mink cereal (XK Mink Foods, Inc., Thiensville, WI) and mixed with the other ingredients of the diet to yield a ration that contained the desired amount of chemi- cal. The HCB used in the feeding trials was lot #H02550, technical grade, purchased from Pfaltz & Bauer, Inc. Stamford, CT. The composition of the basal (control) diet is shown in Tables 2 and 3. After February 10, 22 I.U. of vitamin E per kg of feed were added to all diets and continued until July 1. Corn oil was added to the diet after April 20 at a concen- tration of 1%. This Supplementation was continued through lactation. Unless stated otherwise, the P<0.05 level of probability was used as the criteria for significant differences between treatment means. The 29 Table 2. Composition of the basal diet. Ingredient Weight (lbs) Percentage Whole chicken 240 20.0 Commercial mink cereal* 200 16.7 Ocean fish scrap+ 150 12.5 Beef tripe 40 3.3 Cooked eggs 35 2.9 Beef liver 80 6.7 Beef trimmings 40 3.3 Beef lungs 80 6.7 Powdered milk 13 1.1 Added water 322 26.8 *XK-40 Grower, XK Mink Foods, Inc., Thiensville, WI. +Cod, haddock, and flounder. Table 3. Moisture, protein, fat, and ash content of basal diet . 1 Percentage of diet Component As fed' Dry matter Moisture 68.4 - Protein 14.3 45.26 Fat 7.91 25.03 Ash 2.54 8.04 1 As determined by Rosner/Runyon Laboratories, Inc., Chicago, IL. 30 standard error of each mean (S.E.) was calculated as «MSE7ri (homogen- eous variance) or as 6/Jfi'(heterogeneous variance). Experiment I. ACUTE TOXICITY OF HEXACHLOROBENZENE TO MINK Purpose The acute toxicity of HCB is not known in the mink and so this experiment was undertaken to determine the relative toxicity as shown by the 96 hour L050. Materials and Methods Sixteen adult male ranch-bred mink were randomly divided into four groups of four mink each. Treatment groups were 0, 500, 1000, or 2000 mg of HCB per kg of body weight. The HCB was suspended in corn oil at concen- trations of 0 mg, 500 mg, 1000 mg, or 2000 mg HCB per ml. The appropriate dose of chemical was administered per 05, via stomach tube, to each ani— mal. The mink were observed four times on the day of dosing and twice daily on each subsequent day. Since no mortality was seen in the initial 96 hour period, all animals were observed until 30 days post-dosing. Results No mink died or showed clinical signs of toxicity at any of the treatment levels of HCB. All were judged to be in good condition and health throughout the 30 day observation period. The animals did not show indications of distress even immediately following dosing. When returned to their cages, the mink began grooming and eating as if they had exper- ienced no ill effects from the treatment. Discussion Based on the results of this study, it can be concluded that the oral L050 of HCB for mink is greater than 2000 mg/kg of body weight. At the concentration of 2000 mg of HCB per ml of corn oil, the suspension was a viscous sludge. This quantity of chemical represents a far greater 31 32 amount of HCB than a carnivore, or any other animal, is likely to come in contact with in an acute exposure situation. HCB has generally shown a low order of toxicity in a variety of animal species when the exposure was of short-term duration (Booth and McDowell 1975). Its acute toxicity is quite low (>1 g/kg of body weight) in numerous laboratory animals (WHO 1970). The dangers to animal and human populations, therefore, appears to be small when exposures are limited to acute periods, even if the level of exposure is high. Experiment II. EFFECTS OF CHRONIC DIETARY HEXACHLOROBENZENE EXPOSURE IN MINK AND EUROPEAN FERRETS Purpose, This experiment was conducted to determine the chronic toxicity of HCB to mink and ferrets. The effects of chronic dietary exposure to this halogenated aromatic hydrocarbon on reproductive performance, mortality, and various physiological parameters were monitored during an 11 month feeding trial. Materials and Methods Seventy—eight standard dark mink were divided into five treatment groups and a control group. Each treatment group consisted of ten females and three males. Seventy-eight adult fitch-colored European ferrets were likewise assigned to five treatment groups and a control-group. The animals were fed the basal diet supplemented with either 0 (control), 1, 5, 25, 125, or 625 ppm HCB. Standard ranch procedures were followed in the care, feeding, and breeding of the animals. The mink and ferrets were weighed and blood samples taken periodically during the course of the study. From the blood sample, obtained by toe clip, the following param- eters were measured or calculated: 1) red blood cell(RBC) count, 2) white blood cell (WBC) count, 3) hematocrit (Hct), 4) hemoglobin (Hb) concentra- tion, 5) leukocyte differential count, 6) mean corpuscular hemoglobin concentration (MCHC), and 7) mean corpuscular volume (MCV). Animals were observed daily for signs of toxicity and gross necropsies performed on those mink and ferrets dying prior to the termination of the study. Tissue samples were also taken from these animals for histological examin- ation and HCB residue analysis. Reproductive parameters were monitored 33 34 during the mating and whelping periods (whelping date, number of kits/ litter, number of live and dead kits, birth weight, three week body weight, six week body weight, and kit mortality to weaning) of the mink and ferrets. The kits from both species were weaned at seven weeks of age and placed on unsupplemented basal diet. Upon termination of the study (mink on 16 December 1981, ferrets on 17 December 1981), blood samples were collected from each animal and it was then killed by cervical dislocation, and a gross necropsy performed. Brain, heart, kidney, liver, spleen, and lung were dissected from the carcass and weighed. In addition to the previously named six tissues, samples were taken of ileum, pancreas, adrenal gland, muscle, stomach, and skin and placed in formalin for histological examination. Results No difference was found in initial body weight of the female mink used in this study (Table 4). However, after 22 days on the contaminated feed, the body weight of the mink consuming 125 ppm HCB was significantly lower than that of controls. Since only a single animal was still alive on the 625 ppm treatment, it was not possible to include this group for statistical analysis. The body weight of this one mink was, nevertheless, lower than any animal in the control group. No significant differences were seen in the body weights of the female mink in any of the treatment groups following 149 days of exposure (17 June 1981) to HCB. At the termination of the study no differences were found between the four surviving groups. The initial body weights of female ferrets were not significantly different from those of control ferrets. The 625 ppm HCB group showed significantly lower weights after 22 days of treatment than control animals, and the 125 ppm ferrets exhibited the same result .Amo.0vav Poeoeou sate “emeaeewu s_u=aoeepemwm i. 35 .3 H :85. --- - --- - «o.am.u Ape o_ ¢.o~.u Fme o_ sag mmo --- - .a.mm.fl «we a o.e~.u wok op a.a~.u moo o_ sag amp P.8m.u Mme m m.m~.H owe op o.AN.H ewe o, a.m~.fl mam o, sea mm p.mm.fl “an m m.Pm.H was a o.e~.u mes o. e.m~.fl Ame o, sea m 0.4m.“ .2“ a m.m~.u pee o_ o.a~.u nee o_ a.a~.u Noe o2 sea 2 m.~m.u Nae op m.m~.H nae o_ o.-.u _ma o_ e.e~.fl ape op sea o pegged --- - --- - 3.8 P o.mm.fl mew op saa awe --- - ~.oe.u _ee 8 .e.mm.u Nae OF o.mm.u mmm op sea m~_ m.om.u Peep m m.~m.u cam m ¢.mm.u mam o_ o.mm.H amm o. sea mm 93 M 3m 2 3m .... New 2 4.8 M So 2 0.8 H 88 2 5% m m.he.n mma m _.Pm.H m_m o_ e.mm.fl mmm o_ o.mm.m mom op Sea 2 m.~¢.u Baa . a F.FM.H mm“ o. a.mm.fl com OF o.mm.u mam o, sea 0 3:2: _m-~_-u. e _m-ep-o a pw-FF-N e _m-o~-P c peasaaaap mane nova: .meep mcwumme uvcoenu a cw mumvc mu: ace poeucou mewsamcou mumgemm new game mpmsmm upzum to Amv +mugmpmz Atom .e open» 36 after 149 days of dietary exposure. No differences were observed between the four treatment groups of ferrets still alive on the study's termin- ation date (17 December 1981). When the feeding trial was begun, the 5 ppm HCB group male mink weighed significantly less than, and the 125 ppm HCB group males weighed significantly more than, the control males (Table 5). All the mink were in good condition at this time and no significant differences were seen in the body weights of male mink at any point during the course of the experiment, except for the initial variation. No differences were found in body weights between control and HCB-treated ferrets at the beginning of the trial and only the 625 ppm males exhibited significantly depressed weights, following 22 days of HCB exposure. No other changes in body weight were seen during the length of the study for the HCB-treated ferrets. The hematologic parameters of RBC count, Hb concentration, Hct., MCHC, MCV, and WBC count were not significantly different from control values for mink or ferrets on any of the HCB-supplemented diets (Tables 6 and 7) after 22 days of treatment. However, one female mink consuming HCB at 625 ppm of the diet exhibited RBC, Hb, Hct., and WBC values con- siderably lower than the untreated mink. Following the death of this mink two days after the blood was collected, blood was found throughout the gastrointestinal tract which probably accounts for these reduced values. Determinations made on blood collected after 149 days of feeding the 125 ppm HCB-supplemented diet showed a significant increase in the WBC count in the female mink, but no alterations in the parameters measured in these animals (Table 8). No significant differences from control were .Amo.ovav _oaoeou sage “caeaeeeu NPNeaoNewcmpm .1 37 .w.m H Cami... --- - --- - .o.oN.H NN__ N N.NN.H NNN. N Eaa NNN --- - --- - N.NN.H NNN_ N N.NN.H NNNF N sag NNF N.NNF ..... N3: N N.NN_ H :3 N N.: ..N NNNN N N.NN .... NNN_ N a: NN N.NN_ ..N 8: N N.NNP N. 3: N N.: N NE N N.NN ..N 2.: N 5% N N.NNF.H NONN N ..NNN.N NNNP N N.NN.H NNN. N N.NN.N Noe. N San _ N.NNN.N Nam. N N.NNP.H NNNP N N.NN.N NNNN N N.NN.H NNN. N see o «2.5... --- - --- - --- - ¢.NN.H NNNP N sea NNN --- - --- - N.NF_.H NFNP N .N.NN.H NNNN N sea NN, N.NN_.N NNNN N N.Ne.u o_N_ N N.NNP.H NNN_ N N.¢N.H NNN_ N sag NN N.NN_.H NNN. N N.NN.H NNNN N N.NNF.H NNNN N .N.¢N.u NNN. N sea N N.NNP.H NONP N N.NN.H NNNF N N.NFP.H NNN. 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H NN. 2N N25 H RN 2 Ea NN 8.2 H :N 23 H N.NN :3 H N2 NN.o H fiNN 22 H 2.NN N25 H NNN 2 2% N 82 H NNN 85 H N.NN :3 H N.NN 85 H NE 23 H 82 N25 H 22 2 Ea _ 8; H 3.2 85 H SN 8.0 H NNN NN.o H N.NN 2: H 8.: N25 H 3.... 2 .2: o Nada Amova Amnv APE oop\mv ARV AN av Aoova : ucmsumoeh UN: 5: ea: 8: e. 82 .—opep mcwvmme uNeoegu N :N NNNc mm ace Numwc mu: use Poewcou memsamcou Numeemm ecu xcws u_=um to +mppmoea uNmopoHNEm: .m «Pam» 39 .N.N H :8... NN.N H N.N 8.. H N.NN 2.. H ..NN NN.. H N..N N8... H NN..N 8N... H :2 N =8 NNN 8... H N.N. N... H N.NN NN... H N8 N... H N.NN NNN... H N..NN N.N... H 2.2 N 58 NN. 8... H N.N NN... H N.NN N.N... H N. .. N... H N.NN NNN... H N..NN N..NN H .N.2 N 28 NN NN.. H N.N. N... H N.NN Na... H ..N.. N... H N.NN NNN... H NN.NN N.N... H N... N 58 N 8.8 H N.N NN... H N.NN NN... H ..8 N... H N.NN NNN... H NN..N N.N... H NN... N .8 . 8... H N.N. NN... H N.NN NN... H N. .N N... H N.NN N.NN... H N..NN N.N... H 8.2 N 58 8 “9:9..— .N.N H N.N. NNN H N.NN 8... H 98 NN.. 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Female ferrets consuming 125 ppm HCB showed significantly reduced RBC counts, Hb concentration, and Hct values at this point in the study. However, as noted in the mink, lower levels of dietary HCB did not produce signif- icant changes in the hematologic profile. Male mink did not show changes in the blood parameters measured after 149 days of HCB exposure, but the 125 ppm group had all died prior to this date (Table 9). The male ferrets on 25 ppm HCB had significantly lower RBC counts than control males. Other than the change in this one value, no differences were observed between control and treated animals. The one ferret still alive after consuming 125 ppm HCB for 149 days had a low RBC value as well, but did not differ much from control animals in any of the remaining aspects measured of its blood profile. Leukocyte differential counts of adult female mink after 149 days of HCB exposure at 125 ppm in their diet, shown in Table 10 as arcsin transformed values, exhibited a significant decrease in segmented neutro- phil count and a significant increase in lymphocyte numbers. No changes were found in the proportions of basophils, eosinophils, band cells, or monocytes. A small, but significant increase over control count was seen in the number of band cells detected in female ferrets on the 125 ppm HCB diet. The other leukocyte types were not different from control in these ferrets. As shown in Table 11, no significant changes from control values were found in any of the blood parameters measured in female mink exposed to HCB for 331 days. A significant increase in RBC count was observed at 1 ppm dietary HCB in female ferrets. 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N NNN N .NN.N.H N.NN NN.N.H N.NN NN.N.H N.NN NNN.N.H NN.NN «NN..N.H .N... N NNN . NN.N H N.NN NN.N H N.NN NN.N H N..N NN..N H N..NN NN..N H NN.N. N. .8 N umeewm .N.. H N.NN NN.. H N..N .N.. H N.NN .NN.N H NN.NN N.N.N H .N.N N 28 NN N... H N.NN 8.. H N.NN N... H N.NN N.N.N H NN..N .NN.N H NN.N N. ..NN N NN...H N.NN NN...H N.NN NN...H N..N .NN.N.H N. NN .NN.N.H NN.N N NN. . NN...H N.NN NN...H N.NN NN...H N.NN .NN.N.H NN.NN .NN.N.H NN.N N NN. N NN.: .N.. ..5 NN. NN. .N. .N N. .NN.N. N .NNNNNNLN >9: 0:02 «o: a: 0mm ..NNN. NN.NNNN UNNNNNN N c. Nxmm: Ne No. NuNNN mu: NNN .ogueou NNNENNNNu Numeemm NNN NN.; N.Nsom N.NNN No NN..NNNN u.mo.ouNsmz ... N.NN» 45 females consuming 25 ppm.HCB. The MCV was significantly lower in female ferrets consuming l, 5, and 25 ppm HCB. The single animal alive at this sampling time from the 125 ppm treatment group had lower values for each of the blood parameters measured than control, with the exception of MCV which was elevated. No significant differences were found in the hematologic profile of male mink and ferrets after 47 weeks of HCB exposure with one exception (Table 12). The MCV of the ferrets consuming 1 ppm HCB in the diet was significantly greater than the control animals. Leukocyte differential counts of female mink and ferrets consuming control and HCB-treated diets until the termination of the experiment (331-332 days) are shown in Table 13. Only the 1 ppm treatment group of mink exhibited differential counts significantly altered from that seen in the control females. Eosinophil counts were increased and monocyte counts were decreased in this group. The ferret females had a signifi- cantly lower eosinOphil count at the 25 ppm HCB treatment than did con- trols. The ferrets exposed to 1 ppm of HCB had a decreased band cell count, but an increased number of segmented neutrophils (the more mature form of these phagocytic cells). No change in leukocyte composition was seen in the male mink exposed to HCB for 331 days (Table 14). Band cell numbers were significantly reduced in the male ferrets at 1 ppm HCB at 332 days of exposure. The 25 ppm HCB ferrets showed a lower segmented neutrophil count than the control males. No other alterations were found in the leukocyte differ- ential counts of the male ferrets. The gestational length of mink and ferrets was not affected by dietary HCB in the treatment groups able to produce offspring (Table 15). 46 ..NN.NVN. .NN.NNN NN.. .NN.N...N N..NNN.N.NN.N .1 .m.m H. c002,? NN.N.H N.NN NN...H N.NN NN...H N.NN NNN.N.H N..NN .NN.N.H NN N. N NNN NN NN.N.H N.NN NN...H N..N NN...H N.NN NNN.N.H NN.NN .NN.N.H NN.N. N Na. N «NN.N.H N.NN NN...H N.NN NN...H N.NN NNN.N.H NN.NN NNN.N.H NN.N. N NNN . NN.N.H N.NN NN...H N..N NN...H N.NN NNN.N.H NN NN NNN.N.H .N .. N NNN N pmgemu NN...H N.NN NN.N.H N..N NN...H N.NN N.N.N.H NN .N NN..N.H NN.N N NNN NN NN...H N.NN NN.N.H N.NN NN...H N.NN N.N.N.H NN .N NN..N.H NN.N N NNN N NN.. H N.NN NN.N H N. .N NN.. H N.NN N.N.N H NN.NN NN..N H N..N. N ..NN . NN...H N.NN NN.N.H N.NN NN...H N.NN N.N.N.H NN.NN NN..N.H_NN.N. N Na. N NN.: .m:. ..E co. \mv AN. AN av Ame—xv c NNNEHNNNN .Nz NzNz .62 NN NNN ..NNNN mcmummm upcoego N :. Nxmmz Ne No. Numwu mu: ucN .oNucou Newsamcou Numeewm ucN News N.Ns N.NNN mo +m.wmoca u.mo.ouNEmz .N. N.NNN 47 ..NN.NVN. .NN.NNN NN.. .NN.N...N N..NNN...NN.N &. N. N. NN NN N N . NNN NN. ..HN. N.HNN N.HNN ..HN. %.HN 28HN N NENN fi.H2 5.H8 5.H8 fi.H2 e.HN NNHN N ENN ..HN. N.HNN %.H.N $.HN. N.HN QNH. N 8N. 9.H2 e.H8 e.H8 9.H2 W.H: NNHN 2 SNN NNNNNN N.HN. N2HNN N.HNN N.HN. a.HN NNHN N 8N8 9NH2 fi.H8 e.H8 fi.H2 N.HN NNHN 2 ENN eNH.. N.HNN .HTHN N.H.. _%.HN eNHN N 8N. eNHS e.HN e.HN N.H2 Q.HN NNH. N SNN NN.: waxwocoz mpxuosasNJ .Nnmmuuamz ucNm ..caocwmom ..gaoNNm c NNNEHNNNN emacmsaom NNNN ..NN ..NNNN NN.NNNN UNNNNNN N :. Nxmm: Ne Nam NNNNN mu: NNN .ogucoo mewszmcou Nuwgemm NcN chs N.Nsmw N.NNN mo .umseomchNp N.NuNNV +Nuczou .N.N:NNNNNNN muxooxamd .m. N.NNN ...N.NVN. .NN.NNN Noe. .NN.N...N N..NNN.N.NN.N .3. .Amodvmv POE—coo Eogw ucmgmwmwn 83:53:?um .1 48 .m.m .1... Cfl§+ NNHN. NNHNN $NHNN N.HN. NNHN e.HN N 8N8 NNHN. NNHNN NNHNN N.HN. NNHN. e.HN N NSN NNH.. NNHNN NNHNN «%.H.. NNH.. eNHN N SN. QNHN NNHNN NNHNN N.HN. NNHN QNHN N ENN umeemm NNHN. .NHNN NNHNN NNHN. NNHN e.HN N 8N8 NNHN. .NHNN NNHNN NNHN. NNHN e.HN N ENN NNHN .NHNN NNHNN NNHN. NNHN 2.HN N Na. NNHN. .NHNN NNHNN NNHN. NNHN Q.HN N SNN NN.: muzuocoz muNUNNNENN ..caoepam: ucNm .NNNNNNNom ..NNNNNN : acmEuNmNh limoucmsmmm NNNN ..NN ..NNNa NN.NNNN uwcoggu N c. Nxmmz Ne Now Nuwwu mu: NNN .ogucoo .NNNENNNoo Nuogeww NcN NN.N N.NN N.NNN mo .Nosgomcheu N.NNNNV NNucsou .N.N:NNNNNNN muxuoxawg .e. N.NN. ...N.NVN. .NN.NNN NN.N .NNNNNN.N N..NNN.N.NN.N «.4 ..NN.NVN. .NLNNNN ENE. .NN.N...N N..NNN.N.NN.N .4. .NN.N.mgz N.NENN Nun NHN¥H 49 .N.N H N8... ..NN.N.H N.N .NN.N.H N.N NN.N.H N.N NN.N.H N..N N.NN NNN NN NNHN2 NegHe2 8NHN2. .3NHNNN 2N SNN NaNH.N NQNHNN NENHNN NnNHN.N 25w 58. NeNHmN NQNHNN NENHQN NQNHess 2N eaN uweemm NN.N H N.. ..NN H N.. ..NNN H N.N NN.. H N.NN N.NN ..NN NN NQNHNN NfiNHQN NQNHQN NN.H29. 2N. eaN NQNHN. .QNHNN NeNHnN NQ.HNNN NZN =§_. NeNHN. NfiNHNN NeNHNN NN.HNNN 2:: NEN NN.: NNmN o>.. .Nuou ANNNNV c NNNENNNNN .NN.N NN.... NN..N.NNN NNNNNNN.NN3 ..NNNN mNNNNNN umcoesu N a. Numwu mu: NNN .oeucou mewszmcou Numeemw NcN xc.E N.Nsmm mo +NNNN emuHN. NcN comuNuNmm mo spasm. .m. N.NN» 50 In the mink, however, a significant decrease in total litter size was seen, along with a decrease in the number of kits born alive at 25 ppm HCB. This resulted in an increase in the proportion of stillborn fetuses. In several female mink of the 125 ppm HCB-treatment group that died during April, resorption sites were found in the uterus at necropsy. This demonstrates that although these females were unable to maintain their pregnancies, fertilization and implantation were not prevented. The ferrets also did not show changes in litter characteristics until fed 25 ppm HCB in the diet. Total litter size was unchanged in these ferrets, but the number of offspring born alive was lower than control females and a higher incidence of kits born dead was noted. The female ferrets consuming 125 ppm HCB failed to come into estrus, as determined by vulvar swelling, at any time during their normal breeding season. These animals were therefore never bred and could not be evaluated for other HCB-related reproductive effects. As shown in Table 16, the mink kits born to dams consuming l, 5, and 25 ppm HCB were smaller at birth than the kits born to untreated mothers. A similar relationship was seen in the ferret kit birth weights, although only in the 5 and 25 ppm treatment groups were they significant- ly smaller than controls. Three week old mink kit weights were signif- icantly lower than control kits in the 5 and 25 ppm groups; at six weeks of age these two treatment group kits were still significantly smaller than the control mink kits. At three weeks of age, ferret kits on all HCB treatments weighed significantly more than untreated kits. By six weeks of age, only the 1 ppm HCB ferret kits continued to weigh more than 1 control kits. A strong trend toward reduced biomass produced with increasing dietary HCB was seen in the female mink, with only the 5 ppm 51 No Noose: omNNm>N ozu x omN No Nxooz omega ecN NNNNN .NNN3 oaNo we» smooecp ...N.NVN. .NN.NNN NN.. .NN.N...N N..NNN...NN.N ..NN.NVN. .NN.NNN NN.. .NNNNNN.N N..NNU.N.NN.N .1 «a. ...ow NNN» NNNNN. N.N.Nsom oco oeNNuNN no: Nooom .N.Nsow memuouoo. Non NN.N coozpoo N.Nm NNNNN: Neon N.N omNNo>N u NNNsoNNH .w.m H come: N.NN.H NNN N NN.NN.H N.NNN N. N.NN.N.H N.NN N. ..NN.N.H N.N NN NNN NN N.NN.H NNN N NN.N .H N.NNN NN NN..N.H N.NN NN ..NN.N.H N.N NN NNN N N.NN H NNN N N.NNN H N.NNN NN N.NNN H N.NN NN N..N H ..N NN ..NN . N.NN.H NNN N NN.N .H N.NNN NN NN.N.H N.NN NN .N.N.H N.N NNN NNN N NoNNoN NN . ..NN.N .H N..N N N.NN...H N.NN N NNNN.N.H_N.N N. NNN NN .N.NN.H NN. N t.NN.NNH N.NN. N. ..NN.N.H N.NN N. «.NN.N.H ..N NN NNN N N.NN.H .NN N N..N..H ..NNN N. NN.N.H N.NN NN ..NN.N.H N.N NN NNN . N.NN.H ..N N NN.N .H N.NNN NN NN.N.H N.NN NN .N.N.H ..N N. NNN N NN.: HNNNEon NNNNNHN.V xooz o : goo: m c zuemm : acoEuNoNh c .N. .NN.N: ..N ..NNN» NNNeooN oNNoego N :. NNNNe mo: ecN .oNucoo NNNENNNou poLNoN ecN No.5 N.NEow mNNHNuoN. NNN eoNNNN N.NN.N.No... ecN .Nusmwoz Neon e.o zoo: xNN ecN .xmoz omen» .zuNNm .N. N.NNN 52 females raising a significantly lower biomass than the control mink. No significant difference was seen in the biomass produced by control and HCB-treated female ferrets. HCB caused a dose related increase in kit mortality (Table 17) in both the mink and the ferret. The percentage of kits dying before three weeks of age accounted for the greatest proportion of the mortality seen in mink kits, with only occassional deaths occurring between three and six weeks. Although not quantitated, casual observation suggested the majority of the kit deaths to be occurring within three days of birth. The ferrets also demonstrated a dose-related increase in offspring mor- tality. At three weeks of age the percentage of kits dying was one-half to two-thirds lower in the ferrets than in the mink. The kit mortality to six weeks of age was still lower in the ferrets (except for the 0 ppm group) than in mink kits raised by dams consuming comparable dietary concentrations of HCB. A much greater portion of the kit mortality to six weeks of age occurred between three and six weeks after birth in the ferrets than in the mink. Based on food consumption values reported by Bleavins and Aulerich (1981), the quantity of HCB consumed by the mink and ferrets on the various treatments was calculated (Table 18). No deaths directly attrib- utable to the effects of HCB were seen in the animals consuming the O, l, 5, and 25 ppm HCB diets. The amount of HCB shown for these treat- ment levels in Table 18 therefore represents the quantity of the chem- ical eaten prior to the termination of the study rather than how much HCB resulted in death. The values reported for the 125 and 625 ppm HCB- treated animals are the mg of the chemical required to cause death at these two levels of dietary exposure. The actual quantity may be 53 Table 17. Kit mortality of the offspring produced by female mink and ferrets consuming control and HCB diets in a chronic feeding trial. Kit mortality (%) Maternal treatment to 3 weeks to 6 weeks Mink. 0 ppm 8.2 8.2 1 ppm 32.4 44.1 5 ppm 77.4 77.4 25 ppm 86.7 86.7 Ferrets 0 ppm 1.8 26.3 1 ppm 11.7 23.4 5 ppm 21.0 32.3 25 ppm 42.9 64.3 54 .ueosuNoNp co N>N.N NNNe x ..NNNV :o.eo.=< ecN N=.>No.m Na eoueoooe NN No:.N> coNuNENNcoo eooN N..Ne :o eoNNmN .NezuN ogu mo coNNNNNELmN op NoNNN eNNe mucospooeu NNNNNNe omega co N.NENNN ..N>.:m .m. N.NNN 57 mink on both the 125 and 625 ppm HCB diets, but also exhibited a much greater variability within treatment groups. Female mink were able to survive longer on the 125 ppm treatment than the male mink, but were themselves outlived by the female ferrets on this diet. The female mink lived less than three weeks when consuming 625 ppm HCB and the fer- ret females at this exposure level lived approximately six weeks. Six of the female ferrets in the 125 ppm treatment group were very excitable and abnormally aggressive for this species. If these animals were handled, they began to shriek and acted as if the process of being pick- ed up not only upset them, but was also a painful experience. Subse- quently, four of the female ferrets were found immediately prior to death. Each animal was unable to stand, exhibited mild trembling of all four legs and appeared to lapse into occasional mild convulsions. These ferrets died one to three hours after being discovered and the-above mentioned clinical signs observed. At the end of the eleven months of HCB exposure (termination of the study), no significant change was found in the body weights of the 1, 5, and 25 ppm HCB-treated females from the control animals (Table 20). The females on the two highest treatment groups (125 and 625 ppm HCB) all died before the completion of the experiment and exhibited body weights that were significantly lower than untreated mink and ferrets. Brain weights of female mink consuming HCB diets were not significantly different from control females and only the 625 ppm treatment group of ferrets showed an elevated brain weight. Liver size, expressed as a percentage of brain weight, was not significantly influenced in any of the HCB-exposed mink, but was increased in the ferrets fed 125 ppm HCB. The livers of the mink and ferrets, both male and female, were placed ...NN v a. .288 .8... 828.... 288.28; : ..NNN v ... .288 S... .58.... 2.8228; . .3... 8:233 3 .N..... 8... .... . 58 .N.N H :8: N ..NNN H N.NN NN.. H.N.N.. ..N.N H N.NN ....N H N.NN NN.N. H N.N.N .28 H ..N ..N.NN H NN. N. ..NN .NNN ..NNN H ..NN NN.. H N.NN. ...N.N H N.NN. .....N H N.NN .NN.N. H N.NNN N..N H N.N ..N.NN H N: N. ..NN .NN. NN.N H N..N N..N H N.NN NN.N H ..NN NN.N .H N.NN NN.NN H ..NNN N..N H N.N N.NN H NNN N E. NN NN.N H .. .N .N.. H N.NN NN.N H N.NN NN.N H N.NN NN.NN H N.8N N..N H N.N N..N H NNN N ..N. N NN.N H N..N .N.. H N.NN NN.N H N.NN NN.N H N.NN NN.NN H N.NN. N..N H N.N N..N H .NN N ...... . NN.N H N.NN NN.. H ..NN. .N.N H ..NN ...N H N.NN NN.N. H N.NN. N..N H N.N N.NN H NNN N. E. N Nassau .NN.N H N.NN ..NNN H N.NN. ..NNN H N.NN ..NNN H ..NN NN.NN H N.NN. NN.N H N.N t..N. H NNN N ..N. .NNN ..NNN H ...N NN.N H N.NN NN.N H ..NN .NN.N H N.NN NN.NN H ...N NN.N H N.N ..N. H N... N ...... .NN. NN.N H N.NN .NN.N H N.NN. NN.N H N.NN NN.N H N.NN NN.NN H ..N.N NN.N H N.N ..N. H ..N. N ..NN NN NN.N H N.NN .N.N H ..NN NN.N H N.NN 8N H N. .N NN.NN H N..N. N..N H N.N N.N. H NNN N. ..NN N NN.N H N.NN NN.N H ..NN NN.N H N..N NN.N H N..N NN.NN H N.NN. NN.N H N.N ..N. H NNN N i... . NN.N H N..N NN.N H ..NN NN.N H N..N NN.N H N.NN NN.NN H N.NN. NN.NH N.N ..N. H NNN N I... N x:.: ..N»: use. Noce.u. coo—am No>ru Au. use.ox .av «ca.u3 c Newsaooeh ..NN? N.NNN N. 8:: ......N N..NN ..N.N» a:.eooe N.Noegu N c. Nxoo: N. No» Nuo.e mu: ecu .oeucoo a:.ssmcou muoguou ecu xcvl opusou u—aeo mo Hausa.u3 guano eouuupom .o~ spook 59 under ultraviolet light and observed for fluorescence. None of the animals exhibited the brilliant red fluorescence characteristic of hepatic porphyria. Spleen weight was significantly reduced in female mink at 125 and 625 ppm HCB, but significantly increased in ferrets fed 125 ppm HCB. The spleens of the 625 ppm HCB female ferrets showed a significant weight reduction, similar to the effect seen in the mink on this dietary concentration of the chemical (Table 20). Kidney weights were significantly higher than controls in the 625 ppm treatment groups and there was also a definite trend toward increased kidney weight with increased dietary HCB. In the 125 and 625 ppm HCB female ferrets, an increase in kidney weight was found. Lungs were significantly heavier in 25 and 625 ppm HCB female mink, but no differences from control were seen in the other treatment groups or in any of the HCB-exposed female ferrets. The 125 and 625 ppm female mink and ferrets had a significant- ly decreased heart weight when compared to controls of the same species. The 125 and 625 ppm HCB male mink showed body weights that were significantly lower than control males (Table 21). The mink and ferrets on both of these high levels of HCB all died prior to the end of the study. Only the male ferrets fed 625 ppm HCB had significantly reduced body weights, although the 125 ppm animals did have an average weight which was significantly lower than the control at P<0.10. No difference in brain weight was seen in the HCB-treated mink and ferrets with the exception of the male mink consuming 125 ppm HCB. This treatment group showed significantly higher brain weights than control. An increased liver weight was observed in the mink at 25 ppm HCB and the ferrets at 125 ppm HCB. No other male treatment groups showed a significant change in liver weight. Spleen weight was not significantly lower than the 60 ...NN v ... .288 .8... first... N..N...NEN . 0 ..NNN v ... .288 ..N... 822.... 22.8.28; . .3... 85.263 3 .N..... ..N... .... . .N.N H 5...... N NN.N. H N..N NN... H NN.. ..N.N. H ..N... .N.NN H N.NN NN.NN H N.NNN NN.N H N.N ...N: H .NN N i... .NNN NN.N. H N.NN. NN.N. H N.NN. NN.N. H N..N. .N.N H N..N. ..N.NN H N..N: NN.N H N.N N.NN. H NNN. N ..NN .NN. NN.N. H N..N. NN... H N.NN. 8.2 H N..N. .N.NN H N8. NN.NN H N...N NN.N H N.N ..N: H N.N. N i... NN NN.N. H N... NN... H ..NN. .N.N. H N.NN. .N.NN H N.NN. NN.NN H ..N.N NN.N H N.N ..N: H NN.. N E. N NN.N. H N.... NN.N. H N.N... NN.N. H N.N: 8.: H ..NN. 8.8 H ..N.N NN.N H ..N N.NN. H NNN. N ..NN . NN.N. H N.NN. NN.N. H N.NN. NN.N. H N.NN. .N.N H N.N... 8.8 H ..NNN NN.N H N.N N.NN. H N.N. N E. N «NNNNN NN.N H N..N .N... H N.NN. .N.N H N.NN NN.N H N.NN NN.NN H N.NNN .N.N H N.N ..N.N: H .NN N a... .NNN .NN.N H N..N .N... H N.NN. .N.N H N.NN NN.N H N.NN NN.NN H ..NNN ..N.N H N.N ..N.N: H N.N N ..NN .NN. NN.N H N.NN .N... H N.N: .N.N H N.NN NN.N H N..N .NN.NN H N.NNN .N.N H N.N N.N: H NNN. N :8 NN NN.N H N.NN .N... H N.NN .N.N H N.NN NN.N H N.N. NN.NN H N.NNN .N.N H N.N N.N: H NNN. N ..N. N NN.N H N8. .N... H N.NN. .N.N H N..N NN.N H N.NN NN.NN H ..NNN .N.N H N.N N.N: H NNN. N E. . NN.N H N..N .N... H ..NN. .N.N H N..N NN.N H .... NN.NN H ..N.N .N.N H N.N N.N: H NNN. N E. N ...... .NNN: a=:.. Nocerm, coopom Lo>ru Nov .NN.N: Am. ago—o: = «co-some» ..NN.N; N..NN N. ..N... N..NN .NNN ...... ac.euou u.:oszo N a. Name: No No» muo.e no: ego .oeucoo m:.asmcou Nuogsou ego xc—s u—NE 0.:eN mo «Nugu—uz :NaNo eouuupom . .w N.NN» 61 control in any of the HCB-exposed male mink and ferrets, but did tend to be somewhat reduced in the 625 ppm HCB animals. Kidney and lung weights were not significantly different from controls in the HCB-exposed animals, although the highest dietary concentration of HCB (625 ppm) did tend to show slightly higher weights than control for these two tissues. Heart weight was significantly lower in the 125 ppm HCB mink. No additional statistically significant alterations in the weight of this organ were seen in either the mink or the ferret. Discussion The results of this experiment have shown the mink and ferret to be sensitive to the toxic effects of HCB. A reduction in the body weight of adult animals consuming the two highest HCB diets (125 and 625 ppm) was a general indication of toxicosis in both species. The toxic signs of anorexia and eventual death are consistent with those seen in the rat (Villeneuve 1975). In addition, gastrointestinal bleeding and bloody droppings were observed in the mink and ferrets. The high fat content of the basal diet used in this study (25%) probably prompted excellent intestinal absorption of the HCB by these carnivores. The quantity of HCB absorbed by the rat has been shown to be highly dependent on the fat content of the diet (K055 and Koransky 1975; Zabik and Schemmel 1980), with high fat diets dramatically improving chemical uptake. Predatory birds, species also likely to be consuming diets with a high fat content (in addition to the biomagnification and bioconcentration effects by the prey animals), were found to have high body burdens of HCB (Vos g1_a1, 1969; Best 1973; Cromartie g1fla1, 1975). An increased leukocyte count was seen in the HCB-exposed mink. The differential blood smear also indicated a reduction in the proportion of 62 phagocytic cells (segmented neutrophils) and an increase in lymphocytes. These conditions may be indicative of damage to theimmune system. HCB is immunosuppressive in conventional laboratory animals (Iatropoulos g1. 31, 1976; Loose g1_a1, 1977, 1978, 1979) and the mink and ferret (Exper- iment IV). The elevated WBC count seen could be the result of the stressed animal's attempting to fight off opportunistic and disease organisms. In general, the male mink and ferrets died earlier in the course of the experiment than females of the same species. The ferrets consistent- ly lived longer on the HCB diets than mink on a comparable treatment level. As has been observed with previous studies with PCBs (Bleavins §1_a1, 1980), the ferret appears to be quite sensitive to the effects of halogenated aromatic hydrocarbons, but more resistant than the mink. A differential sensitivity to HCB has also been reported in rodents, with the rat being less susceptible to the toxic effects than the guinea pig (Villeneuve and Newsome 1975). The female ferrets consuming the 125 ppm HCB diet failed to exhibit signs of estrus during the normal breeding season (April to September). This condition may have been produced through the increased biotransfor- mation of endogenous hormones by steroid hydroxylating enzymes induced by the HCB. Levin £1_a1, (1967) found an inhibition of the effects of estradiol and estrone on the female reproductive tract due to the increased metabolism of these estrogens. A similar condition may under— lie the anestrus state of the ferrets used in the experiment being report- ed here. It is also possible that the HCB resulted in a stressed animal, 1 in poor condition, which was not capable of initiating and proceeding with the normal reproductive cycle. Since the general state of these 63 ferrets was not observably different from untreated females, this second explanation does not seem likely. Dietary exposure to HCB depressed reproduction in the rat (Mendoza _1_a1, 1975; Grant £1131, 1977). The females produced fewer litters and the number of pups surviving to weaning was reduced. At 25 ppm HCB, mink females produced fewer kits, but no increase was seen in the total number of kits born dead per litter. Rats exhibited an increased inci- dence of stillbirths, as did women in Turkey consuming HCB-contaminated bread (Courtney 1979). Ferrets on the 25 ppm HCB diet showed no change in total litter size, but did demonstrate an elevated number of kits born dead as was seen in other species. The mink and ferret kits produced by females on the HCB diets were smaller at birth than control kits. This is consistent with the effects seen in rats (Khera 1974; Grant e1_a1, 1977), although exposure levels were much higher in the rodents. The reduced rat pup birth weights were first seen at 80 ppm HCB; the depressed birth weight was observed at 1 ppm HCB in the mink and at 5 ppm HCB in the ferrets. A reduced rate of growth has also been reported in rats (Grant g1_a1, 1977) and monkeys (Bailey g1Hg1, 1980) produced by HCB-treated dams. The HCB-mink kits exhibited lower three and six week old body weights than control kits. These effects were seen at l and 5 ppm of maternal dietary exposure which was dramatically less than was required to produce a similar con- dition in the rats and monkeys. Three week old ferrets from HCB-treat- ed dams weighed significantly more than their control counterparts. This is probably the result of fewer kits per litter, since a corresponding increase in biomass would otherwise be expected and was not seen. Offspring mortality showed a dose-dependent increase in both the 64 mink and ferret. Even low level exposure to HCB resulted in poor kit survival. Low postnatal viability was also seen in rats and humans (Courtney 1979) exposed to HCB. Mortality to weaning of 45.4% was seen at 100 ppm HCB in the rat (Kitchin g1_a1, 1982), while 1 ppm HCB result- ed in 44.1% mortality in the mink.. Even taking into consideration the longer period of nursing in the mink than the rat (6 weeks vs. 3 weeks), the mink must still be considered much more sensitive to the effects of HCB. Ferret kit mortality at 25 ppm HCB was 64.3%, which is intermediate between the levels seen at 100 ppm (45.4%) and 120 ppm (93.1%) in rat pups born to dams consuming HCB diets (Kitchin g1_a1, 1982). The ferret must also be classified as reproductively more sensitive to HCB than the rat. Grant g1_g1, (1977) reported a 20 ppm HCB content in the maternal diet to be the no-effect level for reproduction in the rat, a level which had profound effects on the reproductive performance of both the mustel- ids used in this experiment. Experiment III. EXCRETION, PLACENTAL, AND MAMMARY TRANSFER OF HEXACHLOROBENZENE IN THE EUROPEAN FERRET Purpose In this study the placental and mammary transfer of HCB was deter- mined after a single dietary exposure to 14C-labeled HCB. The impor- tance of 1g_ugggg_and lactational excretion by the dam, in addition to the relative exposure levels to the developing/growing offspring, was calculated. Fecal and urinary elimination of HCB was also monitored in the adult female ferrets. Materials and Methods Eight adult female ferrets were individually housed in metabolism cages 61 cm L x 36 cm W x 33 cm H. All animals received the basal diet and water ag_libitum throughout the study. The ferrets were divided into two treatment groups: Group I - pregnant ferrets dosed on day 14 of gestation Group II - unbred ferrets dosed on the same day as Group I. These treatment groups allowed the investigation of differences between pregnant (and subsequently lactating) females and females not raising offspring. The females in Group I were mated, upon evidence of estrus, with untreated males. Five days prior to the calculated whelping date (4lst day of pregnancy) wooden nest boxes and false cage bottoms (to prevent the kits from dropping through the wire mesh floor) were placed in the metabolism cages of Group I females to permit the raising of kits to five weeks of age. On the day of dosing, feed was withheld from the animals for four hours prior to the presentation of the 14C-labeled HCB-treated feed. 65 66 The single dose of HCB was prepared by mixing 7.15 pCi of 14C-labeled HCB (New England Nuclear, Boston, MA, lot #852-058, 98% radiochemically pure, specific activity = 35.32 mCi/mmol.) into 7.5 g of the diet. Each ferret was observed on the day of dosing until the HCB-treated feed . was consumed. As soon as the full amount of HCB-treated feed was eaten, the untreated basal diet was once again fed ag_libitum. Urine and fecal samples were collected daily for seven days follow- ing treatment of the ferrets with 14C-labeled HCB feed and weekly there- after until the termination of the study. The volume of urine and weight of fecal material produced during the 24-hour sampling period was recorded. The weekly excretion of labeled compounds via the urine or feces was calculated as: 14C activity per gram or ml x weight or volume collected per day for each of the seven days immediately follow- ing dosing (initial week) or the 14C activity per gram or ml x weight or volume for the 24-hour test period x seven (subsequent weeks). Milk samples were taken on weeks two through five post-partum. One ferret kit was collected from each litter at birth, one, two, three, and four weeks of age for whole kit residue concentrations. The fifth week after birth, the last two kits were taken from each litter, one for whole kit residue analysis and the other for individual organ residues. When the last kits were taken, at five weeks of age, the dams were also killed and selected organs taken for HCB residue analysis. All animals were killed with C02 gas. 14C The urine, feces, maternal and kit tissues were analyzed for activity using the Unisol-Complement system (Isolab, Inc., Akron, 0H) for liquid scintillation counting. Each sample was counted for 10 min- utes on an Isocap/300, model 6872 liquid scintillation counter (Searle 67 Analytic, Inc., Des Plaines, IL). The values, presented in the tables as disintegrations per minute (0PM), were corrected for background rad- iation and quenching by the sample-channels-ratio method (Searle 1974). The data were analyzed by using a Bonferroni t-test or the Student's t-test (Gill 1978). Results The percentage of initial dose of 14C-labeled HCB absorbed by the ferrets used in this study was calculated to be 98.5%. This value was obtained by predicting the amount of radioactivity excreted and the amount absorbed during the first 24-hour period, based on the extrapo- lation of the daily excretion for days two through seven post-dosing. This estimation was used due to the food passage time in the ferret being just over three hours (Bleavins and Aulerich 1981). The fecal and urinary excretion data, expressed graphically as a percentage of initial dose, are presented in Figure 5 for Groups I and II. No significant differences were found in weekly fecal excretion of HCB (or its 14C metabolites) between pregnant/lactating female ferrets (Group I) and unbred ferrets dosed at the same time (Group II), or in the 8-week total fecal excretion between the two groups. Urinary excre- tion of the 14C-labeled compound/metabolites was not significantly different in either group for individual weeks or the cumulative eight- week total. As shown in Figure 5, the females raising offspring (Group I) and the unbred females dosed at the same time (Group II) had virtu- ally identical values for urinary excretion of HCB. The values for fecal excretion however, diverged somewhat during the middle weeks of the eight-week collection period. Although no statistically significant difference was determined between individual weeks, the graphic Figure 5. Cumulative excretion of HCB by pregnant/lactating female ferrets (Group I) and unbred female ferrets (Group II). ial dose ini cumulative % of 80 7O 6O 50 4O 30 20 10 &——-0 Group I ..---.m... Group II 68 weeks post dosing 69 representation suggest non-significant changes. Towards the end of the study, HCB fecal excretion by females in Group I appeared to be leveling off while values for Group II females continued to increase, suggesting a greater rate of fecal elimination by the Group 11 females. As presented in Table 22, the 14C-HCB concentration of the females raising kits to 5 weeks of age (Group I) was significantly lower in all tissues tested than in unbred females dosed at the same time (Group II). HCB residues in blood, muscle, and heart were significantly reduced (P<0.05), while adipose tissue levels (subcutaneous and visceral), kid- ney, spleen, liver, lung, and brain were significantly lower at P<0.0l. The concentrations of HCBin liver and subcutaneous fat were significantly higher in the five-week old offspring than in the same tissues of the dams that raised them. No significant differences in HCB concentrations between maternal and kit tissues were found for muscle, kidney, and brain. The 14C-HCB concentration of kits produced by Group I females is shown in Table 23 for newborn through five weeks post-partum. On a per gram of kit basis, very little change was seen between the newborn (2984 DPM/g) and the five week old kit (2685 DPM/g). However, the total radio- activity per kit increased from 25,145 DPM to 805,661 DPM over the same period of time. This increase represents a ratio of 31:1 for the five week old kit to newborn kit [(5 week value - newborn value) e newborn value]. Milk samples collected from Group I ferrets, during the second through fifth weeks of lactation, showed a continual decline in HCB concentration as lactation progressed (Table 23). However, HCB exposure via the milk was 31 times greater than by placental passage. 70 Table 22. Mean (:S.E.) radioactivity (DPM/g) of selected tissues 62 da 5 post-dosing from adult bred (Group I) and unbred (Group 11 female ferrets exposed to a single dose of l4C-labeled HCB and of the offspring born to the bred females. Group I Group II KitsI Tissues (n = 3) (n = 3) (n = 3) Blood 49 i 34.6* 166 _+_ 26.8 - Subcutaneous 4472 : 780.5" 19,525 1 1503.9 11,678 : 712.4ii fat Visceral fat 4429 : 867.6" 19,704 : 1666.0 - Muscle 53 114.4" 384 1 64.0 561 1 204.8 Heart 34 i 9.2" 310 i 56.8 - Kidney 105 i 31.1" 611 i 80.4 209 i 37.2 Spleen 13 i 7.5" 180 1 24.8 - Liver 248 i 68.9" 1445 1 145.2 1420 1185.61 Lung 1 i 0.3" 241 118.4 - Brain 61 i 30.0“ 395 1 48.5 130 + 29.4 IKit tissues, from 5 week old offspring, were contrasted only with maternal (Group I) tissues. *Significantly different from Group II tissue of the same type (P<0.05). **Significantly different from Group II tissue of the same type (P<0.01). iSignificantly different from maternal tissue of the same type (P<0.05). itSignificantly different from maternal tissue of the same type (P<0.01). 71 .Nxooz oNocu chNNe eox..s Ho: oeo: NaoNNoNN i. .I .1 i. ..E goo. o~.o + m.o N..o + m.. m..o + N.N ee.o + ..N N N x..: xooz N.NN. N.NN. ...N. N..NN N..N - NNN.»MMM oNNoNoc. mm.em.H N.mom mo.n—~H m.NNo NN.NmH m.~me mm.mnfl e._.m mm.emfl N.mm me.P.Hp.mN m 9.x o.o;3 Loo SNHNN SNHNN 2NHNN NNHN. NNHNN 2NHeN N 2.5 NN.N Non m e m N F c : sougooiumoo mxooz .NENe oNosu No eooseoNN N..N on. eco mo: eo.ooN.-u...wo oNoe o.m=.m N o. eoNoNxo NHoNNo. o.Nsom o. Neon N... No .mo. x zoo. NHN>NHoNNNeNN ..N.N +V :Noz .mm oNNNN 72 Discussion In this study the ferrets absorbed 98.5% of a single dietary dose of 14C-1abeled HCB. This percentage of initial dose is higher than the 80% value reported for rats (K055 and Koransky 1975) following a single oral dose of HCB in 011. Since this carnivore has a digestive system well adapted to utilize a diet rich in fat, like that of the mink (Travis and Schaible 1960), the high fat content of the diet (22.2%; Bleavins and Aulerich 1981) probably promoted good absorption of HCB by the ferrets. In samples collected from wild populations (K055 and Manz 1976), carnivorous (red fox) and omnivorous (wild boar) species have been found to have higher HCB tissue residues than herbivorous species (deer). Their position nearer the top of the food chain puts the carn- ivore, and the omnivore to a lesser degree, in a situation more likely to be consuming a diet in which biomagnification and bioaccumulation of HCB have occurred than is seen for herbivorous animals. HCB has been reported to be transported passively across the gut wall in rats (Richter and Schfifer 1981). The small molecular size and high lipid solubility of HCB would suggest the absorption across the gut wall and subsequent diffusion into the systemic circulation are the principal means by which orally ingested HCB enters the organism. Iatropoulos gg_a1, (1975) have reported that the major portion of ingest- ed HCB is absorbed via the lymphatic system of the gastrointestinal tract and deposited in the fat, thereby effectively by-passing the systemic circulation and an initial exposure to the excretory organs in the rat. Whether or not this is the mechanism of absorption in the ferret has not been determined, but 14C-labeled HCB/ metabolites were detected in their blood and selected tissues at the termination of the experiment. 73 Studies with rats have shown that HCB concentrations in tissues decline rapidly at first and then fall more slowly (Kuiper-Goodman g1 21, 1977) with at least 70-80% of the initial dose being retained after seven days (Albro and Thomas 1974). Two weeks after intraperitoneal (IP) injection of 14C-HCB, 34% of the dose administered was recovered from the feces and 5% in the urine (Koss and Koransky 1975). The whole body halfelife for HCB in rats was estimated to be about 60 days follow- ing a single IP injection (Morita and Oishi 1975). In this study, ferrets raising kits excreted 50% of the initial dose by approximately 32 days after consuming HCB-treated feed, while the unbred ferrets achieved this same degree of HCB elimination in 41 days (Figure 5). The percentages of HCB excreted via the urine and feces were approximately 5% and 45%, respectively, at the 50% stage of elimination in unbred females and 5% in urine, 43% in feces, and 2% in the kits for pregnant/ lactating females. It would therefore appear to be more efficient at ridding itself of HCB than the rat. The high fat content of the basal diet used in this study, coupled with the ferret's relatively short digestive tract and rapid food passage time may have provided this species with a more efficient method for the elimination of HCB than the rat. Although the circulating levels of HCB in the blood supplying the ferret's gastrointestinal tract would have been low in the weeks following the single exposure, a concentration gradient favoring the passage of HCB into the intestinal contents would nevertheless exist. The passive transfer of HCB into the bolus traveling through the intes- tine would occur in conjunction with the active excretion processes. The adipose tissue was the most significant long-term repository, of HCB in the ferret. Adipose tissue residues were many times the value 74 of any other tissue. Similarly, the fat concentrations of HCB were the highest of all tissues tested in rats (Iatropoulos §1_a1, 1975), pigs (denTonkelaar g1 11. 1978), chickens (Hansen g1_a1, 1978, 1979), and dogs (Sundlof g1_a1, 1981). In other tissues, HCB residues were closely associated with the fat content of the tissue (Avrahami and Steele 19720; Hansen g1_a1, 1979). The same general relationship was seen in the ferret tissues analyzed in this study. HCB can undergo metabolic transformation following absorption by the animal. The principal metabolites seen are pentachlorophenol, tetra- chlorophenol, trichlorophenol, and pentachlorobenzene. In rats treated with HCB, 70-80% of the excreted compound in the feces was parent chemi- cal (K055 and Koransky 1975). Engst g;_a1, (1976) reported that the fecal material of HCB-treated rats c0ntained small amounts of penta- chlorobenzene, but the principal product was the parent compound. Only 4-10% of the urinary product was HCB (K055 and Koransky 1975; Koss §1_ a_1_. 1976), with the remainder of the 14c-1abe1ed compound present being the more polar metabolites. Koss 31 El- (1976) reported that approx- imately 16% of the dose of 14C-labeled HCB given to rats was converted to metabolites that could be detected in the excreta and animal's body. The tissue concentrations of metabolites accounted for only a small proportion of the total dose (10% of the labeled compound in the blood and less than 0.1% in the body fat). Major organs of female rats treated with HCB generally showed less than 1% of the chemical form present to be the metabolic products of HCB (Richter £1.21, 1981). Similarly, Engst e1_a1, (1976) reported that HCB metabolites were present in quite small concentrations in rat tissues after HCB exposure. Rozman g1_a1, (1975) found 3% of the HCB ingested by Rhesus monkeys was 75 metabolized, primarily to pentachlorobenzene and trace amounts of tetra- chlorobenzene. Although the rehative proportions of HCB and its metabolites were not quantified in the study reported here, based on the work of other researchers detailed above, it would appear that a similar profile of metabolism may occur in the ferret. Since the tissue residue and blood concentrations of HCB metabolites were low in all species studied, 1n_ ugggg and mammmary exposure of the developing/growing ferret kits would be almost exclusively to the parent compound, HCB. Ferrets raising kits were able to significantly reduce their body burden of HCB compared to unbred females. By the fifth week of lacta- tion, 20.3% of the dam's initial dose had been eliminated via the off- spring. If the litter size had not been continuously decreasing as kits were taken for 14C-HCB analysis, this level might well have been higher. The lactating mammary gland has been described as an organ of excretion of polybrominated biphenyls (Dent egna1, 1978; Bleavins g1_a1, 1981) and polychlorinated biphenyls. Nursing female rats have likewise been reported to significantly decrease their body burden of PCB through the secretion of milk (Takagi g1_a1, 1976; Ando 1978). Foreign sUbstances, especially those of high lipid solubility and low molecular weight such as HCB, have been shown to traverse the pla- centa by simple diffusion (Eckhoff 1972a, b). The ferret has an endo- theliochorial zonodiscoidalis type placenta during the first stages of gestation (Enders 1952), but develops an endotheliochorial bidiscoidal placenta in the latter stages of pregnancy (Enders 1957). This decidu- ate placenta establishes a close relationship between the maternal and fetal tissues with a large surface area for diffusion. Thus, the 76 developing ferret kits were subjected to HCB insult both in aggro and through the dam's milk following birth. A ratio of placenta to milk HCB exposure in the growing ferret kit was calculated to be 1:31 in this study. The milk represents a greater quantity of this aromatic hydrocarbon entering the young animal than does the placenta. However, placental exposure affects the embryo during organogenesis and so may represent a greater than or equally high risk as mammary exposure. High concentrations of HCB have also been reported in weanling rats, compared to a relatively low dietary intake of the com- pound by their dams (Grant £1.21, 1977). This indicates the excretion of HCB via the milk to be an important route in the nursing rat as well as the ferret. The ferret ratio falls between the 14.6:1 value reported by Ando (1978) for rats and the 100:1 ratio calculated by Masuda g1_a1, (1978) for mice exposed to PCBs. Other researchers studying rats (Takagi g1_a1, 1976) and humans (Masuda £1_a1, 1978) have similarly noted a greater quantity of PCBs to enter the growing offspring via the milk than the placenta. HCB is a widely dispersed environmental contaminant (Courtney 1979) and has been shown to depress reproduction in rats by decreasing the number of litters produced and the number of offspring surviving to weaning (Grant g1_a1, 1977). It has also been found to be immunosup- pressive in mice (Loose g1Ha1. 1977, 1979) and to reduce growth rate in chickens (Hansen ggna1, 1979). The maternal tissues act as a readily available supply of HCB to the offspring (Courtney and Andrews 1979). The placental and mammary transfer of HCB imparts a significant risk to the developing and growing animal, in addition to any toxic effects HCB may have on the adult reproducing population. Experiment IV. NEUROTRANSMITTER CONCENTRATIONS IN FIVE BRAIN REGIONS OF ADULT MINK AND THEIR OFFSPRING, AND ADULT FERRETS CONSUMING HEXACHLOROBENZENE IN A CHRONIC FEEDING TRIAL PUY‘QOSG One of the biological effects manifested by exposure to HCB is the development of hyperexcitability, tremors, and/or convulsions (DeMatteis e1_a1, 1961; Ockner and Schmid 1961; V05 31_a1, 1971; Taljaard g;_a1, 1972; Kimbrough and Linder 1974; Timme g1_a1, 1974; Kuiper-Goodman g1 a1, 1977). Despite the appearance of such clinical signs in intoxicated mammalian species, little work has been done to elucidate the etiology of these neurotoxic effects. The fact that certain organochlorine pest- icides produce similar effects and these effects occur concomitantly with alterations in brain neurochemical parameters (Wagner and Greene 1974; Hrdina g1_a1, 1974; Willhite and Sharma 1978; Heinz 25.21: 1980) suggests that examination of regional brain biogenic amine levels in animals exposed to HCB might provide important information pertaining to the manifestation of the clinical signs. Thus, the purpose of the present study was to determine the concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT), and the serotonin metabolite 5-hydroxy- indoleacetic acid (5-HIAA) in five brain regions of adult mink and ferrets that had been exposed to HCB in the diet for 47 weeks and in 16- 17 week old mink kits that had been exposed to HCB 13_u§grg_and via nur- sing for 6 to 7 weeks after birth. Materials and Methods Twenty-eight adult standard dark female mink and twenty-eight adult fitch-colored European ferrets were used in this study. These animals 77 78 consumed HCB in a chronic feeding trial initiated on 20 January 1981 and described in greater detail in Experiment II. The 0, 1, 5, and 25 ppm supplemental HCB treatment groups were used, seven animals from each level. At necropsy (16 December 1981 for mink and 17 December 1981 for ferrets), the brain was removed, dissected into five regions (cerebellum, medulla, midbrain, hypothalamus, and cerebral hemispheres), and frozen on dry ice for subsequent neurochemical analysis. Dopamine, norepi- nephrine, and 5-hydroxytryptamine (serotonin) were determined by the method of Jacobowitz and Richardson (1978). The concentration of 5-hydroxyindoleacetic acid was measured in accordance with the technique described by Curzon and Green (1970). Regional brain neurotransmitter concentrations were determined in 18 standard dark female mink, age 16-17 weeks. These animals were born to dams consuming diets with O, l, and 5 ppm of supplemental HCB as des- cribed in Experiment II. The mink kits were placed on non-supplemented feed for 10 weeks following weaning and prior to analysis. The young mink used in this study were therefore exposed to HCB via three routes: 1) 13_u§g39_throughout gestation, 2) their dam's milk during nursing, and 3) any HCB-contaminated feed they may have consumed directly, before weaning. The data from both the adult and young animals were analyzed using the Dunnett's t-test to determine differences between the control and HCB-treated groups (Gill 1978). Results Hypothalamic 5-HT concentrations in the mink were slightly, but significantly increased at l, 5, and 25 ppm of supplemental HCB (Table 24). A significant elevation of serotonin was seen only at 1 ppm HCB 79 ..N.N v 2 .888 ..N... 888...... 2888.88 k. .4. ..NNN v 2 .888 e8. 888...... 288.288 .4. .N.N H :82. N.N H 2. N N.NN H ..N N N.NN H NN. N ..NN H NNN N N.N. H NN. N 58 NN ..N. H N.. N N.NN H NNN N N.NN H .NN N N.NN H NNN N N.N. H NN. N .8 N ..N H .N. N N.NN H N.N N N..N H .NN N N.NN H .NN N NN.NN H NNN N 8.. . .... H NN. N N.NN H NNN N N..N H NN. N N.N. H NNN N ..NN H NN. N N.NN N Hoflwm N... H NN N ..N.N H N.N N N.NN H .NN N N.N.. H NNN N ..NN H .N. N N.NN NN N... H NN. N ...N..N H NNN N ..NN H .N. N N..N H NNN N N.N. H NN. N e8 N N... H .N N NN..N H NNN N N.NN H NN. N N.NN H .N. N N..N H N.. N e8 . N.N. H NN. . N..N H NNN N N.NN H .N. N ...N. H NNN N N..N. H NNN N 8.. N 8.: Nocogompso; c NasNNNNHoNNz : N.NNNeNZ : N..Neoz : NN.NoNoNou c coNHNeucoucoo .Neeoeou N.NHoNo conoL cwoem .Nxoo: N. No. NHoNe mo: ecN .oeucoo Newsamcoo Nooeeo. ecN No.5 o.NEo. ..NeN No NooNNo. :NNNN eouoopom :. .N.NoHoNoNV HoNNENHNNgHNoneNgim mo AoNNNNH No Eu\a:v cowuoeucoucou ..N oNNNN 80 in the cerebellum of the ferrets. Hypothalamic 5-HIAA concentrations were significantly higher than control in the mink consuming the 5 ppm diet (Table 25), while this catecholamine was significantly depressed in the midbrain of ferrets at 5 and 25 ppm HCB. No significant change was found in the concentration of NE (Table 26) or DA (Table 27) in any brain region of either the mink or the ferret. No significant change was detected in the brain concentration of serotonin in the young mink (Table 28). However, 5-HIAA was elevated in the 5 ppm group. As shown in Table 29, no alteration in the concen- tration of NE was observed for any brain region of these animals. The hypothalamic DA concentrations were significantly depressed in the mink kits whelped by dams consuming 1 and 5 ppm HCB. A treatment group of 125 ppm dietary HCBwas included in the origi- nal parent study (Experiment II), but none of these adult mink and ferrets survived until the termination date. However, behavioral changes were observed in six of the 10 female ferrets consuming this concentra- tion of HCB. The animals were very excitable and abnormally aggressive for this species. If handled, they began to shriek and acted as if being picked up not only upset them more than usual, but was also pain- ful. Later in the course of the study, four of these females were found immediately prior to death. Each animal was unable to stand or reposition itself, extremely weak, exhibited trembling of all four legs, and appeared to lapse into occassional mild convulsions. The ferrets died one to three hours after being discovered in this condition. Discussion Numerous studies have indicated that exposure to HCB can result in disruption of the nervous system clinically manifested as 81 ...N.N v N. .Ncoeoo Noe. .NN.N...N N..NNN...NN.N ..NN.N v N. .NN.NNN NN.. .NN.N...N N..NNN...NN.N H. .N.N + :Noz H N.NN.H NN N N.NN.H .NN N ..N.NN.H NNN N N.NN.H NNN N N.N .H NN N NNN NN N.N..H NN. N ..NN.H .NN N .N..N.H NN. N N.NN.H NNN N ..N. .H N.. N NNN N N.. .H NN N N.N..H NNN N N.NN.H NNN N N.NN.H N.N N ..NN N.NN. N NNN . N.N .H NN N N.NN.H NNN N N.N..H NNN N N.NN.H .NN N N.NN .H N.. N NNN N kuumm N....H NN N N.NN.H N.N N N.NN.H NNN N N.NN.H NNN N N NN..H .NN N NNN NN N....H NN N ..N.NN.H N.N N ..NN.H .N. N ...N.H NNN N N.NN .H .NN N NNN N N....H NN N N.NN.H NNN N N.NN.H .NN N ..NN.H NN. N ...N .H NNN N NNN . N.N..H NN. . N.NN.H NNN N N..N.H NN. N ..NN.H NN. N N.....H NNN N NNN N .NNN NoNoNNNNEoc : N=EN.N;uoNN: c N.NNNeNZ : N..Neoz : so..onoeou c cowpoeucoocoo .Nenoeou N.NHoNo cowuoN N.NNN .Nxooz N. 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H NN. ..NN . N.N H NN. N..N H NNN N.NN H NNN N... H NNN ..N. H N.. .8 N 81.88% N.. H NN . N.NN H .NN N.N. H NN. N.N. H .N. N.. H N. .8 N N.. H .N N..N H NNN ..N. H NN. ..N. H NN. N.N H NN e8 . N.N H NN N.NN H NNN N.N. H NNN N.N. H N.N N.N H NN 8.. N oc.egnoc.moeo= NoeozaNNEo; NNENNNsuoNN: N.NNNeNz N..Neoz so..ooo.ou coNHNNucoocoo .NNNoNou N.NHoNN commo. :NNNm .mo: o. eoNoNxo N..Nuocweoo WNNNE oNNEom mNNoN ...o Ncowmo. ...N.... eopoo.oN .... HocENooe eco HocwefiochNoc h.o Noam... mo 53 5 NeoSNh—coocou .NN o3...— 86 hyperexcitability, tremors, and/or convulsions. For example, Timme 21 .31, (1974) fed rats a diet containing 0.3% HCB which resulted in an estimated daily dose of 250 mg/kg of body weight. Tremors became appar- ent in exposed rats shortly after the feeding trial began. Similarly, Taljaard $1.21, (1972) administered 0.3% HCB in the diet to rats and observed nervous system effects by 21 days. Thirteen of 33 rats fed 0.2% HCB died within the first month exhibiting terminal tremor, ataxia, weakness, and paralysis (Ockner and Schmid 1961). Kimbrough and Linder (1974) fed dietary HCB to rats for four months at levels of 100, 500, and 1000 ppm which corresponded to 5, 30, and 70 mg/kg/day, respectively. Those rats fed 500 and 1000 ppm displayed clinical signs of tremors and hyperexcitability while no toxic signs were observed in those rats receiving 100 ppm. Kuiper-Goodman g1_a1, (1977) reported clinical signs of excessive irritability, ataxia with hindleg paralysis, and tremors in rats fed HCB at a level equivalent to 32 mg/kg of body weight over a 15 week period. Rabbits fed a diet containing 0.5% HCB for 8 to 10 weeks developed clinical signs of tremors and paresis within the last week of treatment, while guinea pigs fed 0.5% HCB in the diet displayed tremors with clonic convulsions within 8 to 10 days. These animals were also described as being hyperexcitable which increased with handling. Likewise, mice administered 0.5% HCB in the diet developed tremors within 8 to 10 days, as well as clonic contractions of the hind limbs (DeMatteis §1_a1, 1961). Japanese quail fed 80 ppm HCB in the diet were adversely affected beginning three weeks after initiation of the treat— ment. Thirty percent of the birds at this level died between 18 and 62 days of the experiment after a short period of anorexia, ruffled feath-l ers, tremor, and ataxia (Vos g1_a1, 1971). The observation of clinical 87 signs indicative of neurological impairment was possible with six of the 125 ppm treatment group. These animals were judged to be very excitable and abnormally aggressive for the ferret. Four of the females were observed immediately prior to death. None of the animals was able to stand and all exhibited trembling of all four legs and mild convulsions. These data indicate that, at least in the ferret, death is preceeded by clinical signs indicative of nervous system disruption as has been reported for other species. In fact, that such signs and subsequent death were apparent at an approximate HCB dose of 17 mg/kg body weight/ day (125 ppm in the diet) in both the mink and ferrets suggests that these two species are among the more susceptible mammals to HCB intox- ication. Despite the fact that clinical signs indicating nervous system involvement are consistently observed in HCB-intoxicated animals, little information exists concerning the mechanism of action. Kuiper-Goodman g1_a1, (1977) reported that tremors started to appear soon after eleva- ted porphyrin levels were detected although determination of a cause- effect relationship was beyond the scope of the experiment. However, Ockner and Schmid (1961) observed that none of the 13 rats dying within the first month of exposure to dietary HCB, after displaying clinical signs indicative of nervous system involvement, exhibited major disturb- ances in porphyrin metabolism. DeMatteis g1_a1, (1961) suggested that elevated blood ammonia concentrations might be responsible for the neurological signs observed. No histopathological abnormalities have been found in brain, spinal cord, motor nerves, or sensory nerves of ‘ skeletal muscle of HCB-exposed animals (DeMatteis g1_a1, 1961; Kuiper- Goodman £1.21, 1977). 88 Numerous studies with cyclodiene organochlorine pesticides have demonstrated that acute exposure to such compounds produces neurotoxic effects similar to those described for HCB such as hyperexcitability, tremors, and convulsions (Hrdina g1_a1, 1974; Wagner and Greene 1974). Furthermore, it has been shown that both acute cyclodiene organochlorine exposure where clinical signs are evident and in chronic situations where clinical signs are absent, various brain neurochemical parameters are altered, suggesting a relationship between altered brain neurochem- istry and the mechanism of action of the cyclodiene compounds. For example, following an acute dose of a-chlordane (200 mg/kg), endrin (50 mg/kg), heptachlor (200 mg/kg), or heptachlor epoxide (100 mg/kg) rats developed slight tremor paralysis of the hind legs and convulsions. In the chlordane-treated rats these signs were accompanied by a decrease in brain stem NE concentration and cortical and striatal acetylcholine (ACh) concentrations, Chronic treatment with these compounds produced no apparent signs of neurotoxicity, but did cause a significant drop in cerebro-cortical ACh and a significant increase in brain stem 5-HT (Hrdina g;_a1, 1974). Similarly, Wagner and Greene (1974) reported that an acute oral dose of dieldrin (50 mg/kg) produced severe neurotoxic signs, as well as a significant decrease in whole—brain NE concentration in male rats. Chronic exposure to dieldrin via the feed (50 ppm) caused a significant decrease in midbrain, striatum, hippocampus, and medulla NE concentrations, as well as a significant depression in 5-HT concen- trations in the midbrain, striatum, and medulla. Sharma (1976) reported that chronic administration of dieldrin (40 ppm) to mice caused a sig- nificant increase in the concentration of the serotonin metabolite 5-HIAA, but did not affect brain serotonin concentrations. This effect 89 was explained not as an increase in serotonin turnover, but rather as an impairment of 5-HIAA efflux from the brain. In a later study, Willhite and Sharma (1978) found that acute exposure of chickens and hamsters to dieldrin (10 mg/kg) caused an increase in brain serotonin concentra- tions and a decrease in the activity of monoamine oxidase (MAO) which is involved in the metabolism of 5-HT. While the neurochemical alter- ations resulting from cyclodiene organochlorine exposure are not con- sistent with respect to the parameter affected and the direction of the change, it is apparent that alterations in catecholamine and indole- amine dynamics do occur. Results from the present study indicate that HCB also causes alter- ations in brain neurochemical parameters which are similar to changes induced by cyclodiene compounds. Female mink exposed to dietary HCB for 47 weeks had elevated S-HT concentrations at all dose levels. The sig- nificant increase in 5-HT without a concomitant consistent change in 5-HIAA suggests that 5-HT turnover in the hypothalamus is being depres- sed by HCB. Concentrations of 5-HT and 5-HIAA in the cerebellum, medulla, midbrain, and cerebral hemispheres were unaffected. In female ferrets exposed to HCB for 47 weeks, cerebellar 5-HT concentration was increased at 1 ppm while 5-HIAA concentrations were increased at 5 and 25 ppm HCB, in the midbrain. The elevated concentrations of midbrain 5-HIAA with no consistent alterations in midbrain 5-HT concentrations suggest that serotonergic activity was increased in this brain area or that 5-HIAA efflux was impaired. Regional brain NE and DA concentra- tions were not altered by HCB exposure in either the adult mink or ferrets. In mink kits that had been exposed to HCB jn_utero and during 90 lactation, midbrain 5-HIAA was increased at 5 ppm without a concomitant change in 5-HT concentration, suggesting either an increase in serotonin turnover or an impairment of 5-HIAA efflux from this brain region. Hypothalamic DA concentrations were depressed at the 1 and 5 ppm levels while regional brain NE concentrations were unaffected by HCB exposure. The results of this study suggest that chronic exposure to HCB does cause alterations in brain neurochemical parameters and that these changes are apparent weeks after jn_u1grg_and perinatal exposure. Although the present study was not designed to establish a correlation between clinical signs of HCB-induced nervous system impairment and neurochemical alterations, such a relationship is suggested. Perhaps of greater importance is the fact that neurochemical alterations are apparent even in the absence of clinical signs, alterations that could have a subtle and possibly detrimental effect on the behavior and normal physiological functioning of the animal. Experiment V. CELLULAR RECOVERY FROM WHOLE AND DILUTED MINK BLOOD AND LYMPHOCYTE-TO-MONOCYTE RATIOS OF DENSITY CENTRIFUGATION ISOLATED MONONUCLEAR LEUKOCYTES Purpose The use of diluted blood is a common technique in density centri- fugation procedures for the isolation of mononuclear leukocytes. This study was initiated to determine if the number of cells recovered from a sample of whole blood could be increased by diluting the blood with Hank's Balanced Salt Solution (HBSS), prior to Ficoll-Paque isolation. A second trial was conducted to determine whether or not the ratio of lymphocytes to monocytes remains constant across a wide range of cellular concentrations obtained from the same original quantity of whole mink blood. Materials and Methods Effects pf_Dilution Heparinized blood (11 ml) was collected, via cardiac puncture, from nine adult ranch-bred male mink. The 11 m1 blood-heparin sample (9.9 ml blood: 1.1 ml of 200 U heparin per ml on physiological saline) was divi- ded into three 16 x 125 mm sterile tissue culture tubes as follows: 1) 5 m1 of whole blood solution, 2) 3.5 ml of blood + 1.5 ml of HBSS, and 3) 2.5 m1 of blood + 2.5 ml of H855. Mononuclear leukocytes were isolated from each of the samples by density centrifugation using Ficoll- Paque (Pharmacia Fine Chemicals, Piscataway, NJ). Following the initial centrifugation (400 x g, 40 minutes), the cells at the Ficoll-Paque: plasma interface were collected and washed twice with H855 and then resuspended in 1 ml of H855. The resulting mononuclear leukocytes were counted on a Coulter Counter (Coulter Electronics, Hialeah, FL). The 91 92 cell recovery per ml of the original heparinized blood sample was then calculated. The data were analyzed by a Student's t-test for compar- isons of two means. Lymphocyte-Epronocyte Ratios Five ml of heparinized blood was collected, via cardiac puncture, from 10 adult male and 10 adult female ranch-bred mink. The 5 m1 sample (4.5 m1 whole blood: 0.5 m1 of 200 U heparin per ml of physiological saline) was layered onto Ficoll-Paque in 16 x 125 mm sterile tissue culture tubes and centrifuged for 40 minutes at 400 x g. The cells at the Ficoll-Paquezplasma interface were collected and washed twice with H855 and then resuspended in sufficient HBSS to allow counting on a Coulter Counter. After the cells were counted, each tube was recentri- fuged (200 x g, 10 minutes) and the supernate discarded. The remaining cell pellet was mixed with one drop of HBSS and used to make a smear for leukocyte differential counting. The leukocyte smears were stained with Wright's stain (Camco Quik Stain, Cambridge Chemical Products, Ft. Lau- derdale, FL). The percentage of monocytes and lymphocytes was determin— ed by counting 500 leukocytes per smear. The data were analyzed by a Student's t-test for differences between means (Gill 1978) and the correlation calculated between % monocytes and cell recovery. Results Effects pf_Dilution To allow each animal to act as its own control, the values obtained per ml of diluted blood were expressed as a percentage of the value ob- tained per ml of undiluted blood from the same mink. The 3.5 ml blood: 1.5 m1 HBSS dilution gave 80.7 :_5.59 % of the whole blood number of cells and the 2.5 ml blood: 2.5 ml HBSS solution produced 71.8 :_6.59 % 93 of the cells yielded from each ml of whole blood. The diluted blood samples consistently produced fewer mononuclear leukocytes than a comparable quantity of blood without HBSS. Lymphocyte-gpronocyte Ratios The average total number of mononuclear leukocytes obtained from nine ml of whole blood was 125.5 :_l9.12 million, with a range of from 34.5 to 366.8 million cells. The isolated cell fraction was greater than 95% viable mononuclear leukocytes. Differential counting of these cells revealed them to be a population of 2.9 :_0.32 % monocytes (range = 0.8 to 6.4) and 97.1 :_0.32 % lymphocytes (range = 93.6 to 99.2). No relationship was seen between cell yield and percentage of monocytes. The correlation between % monocytes and cell yields was calculated to be 0.281. Discussion The separation of lymphocytes and monocytes via density gradient centrifugation was not improved by the dilution of the original blood sample with HBSS. The number of mononuclear leukocytes was greatest when whole blood was layered on the Ficoll-Paque and centrifuged. The use of diluted blood samples, as is commonly practiced with human and mouse blood, only resulted in a larger number of tubes to be spun and more reagents being necessary to isolate fewer cells from a given blood sample. The separation of lymphocytes from mink blood appears to be best accomplished through the centrifugation of whole blood, both in terms of the number of cells recovered and the efficient use of reagents. The percentage of monocytes in the total population of mononuclear 'leukocytes isolated in these studies was found to be constant over a wide range of cell yields. There was no significant correlation between 94 cell yield and monocyte-to-lymphocyte ratio. This finding indicates that the cell suspensions obtained by Ficoll-Paque isolation can be diluted to a constant concentration for each experimental animal with- out affecting the relative proportions of lymphocytes and monocytes, regardless of the original cell concentration obtained. The proliferative response of T lymphocytes to Con A is a macro- phage (blood monocyte) dependent reaction (Persson g1_p1, 1978; DeVries g1_p1, 1979; Resch g: 31. 1981) and unless lymphocyte-to-monocyte ratios are kept constant, the incorporation of 3H-thymidine can be highly variable following mitogen stimulation. Insufficent macrophages result in low responses which increase as macrophage numbers increase, until an excess of macrophages become suppressive (DeVries e1_p1, 1979). The mononuclear cells isolated from mink during the course of this experiment showed a relatively constant percentage of monocytes (l to 6 %) across a wide range of total cells recovered from the same quantity of blood. Variability between animals may well prove to be due, at least in part, to differences in monocyte concentration. However, these fluctuations in monocyte numbers cannot be accurately linked to the number of cells recovered from a whole blood sample. Experiment VI. LYMPHOCYTE BLASTOGENIC RESPONSE OF ADULT FEMALE MINK AND FERRETS, AND THEIR OFFSPRING, AFTER CONSUMING HEXACHLOROBENZENE IN A CHRONIC FEEDING TRIAL Purpose The chronic exposure of organisms to low levels of toxic substances may lead to the impairment or alteration of normal physiological func- tion. The cell-mediated immune system may be one such target for some toxicants. The effects of HCB on cell-mediated immunity in the mink and ferret was studied via the T lymphocyte mitogen Con A. Materials and Methods §EEQX.L- Effects on the Response of Adult Mink and Ferrets Adult standard dark female mink and adult female European ferrets were exposed to HCB in a chronic feeding trial. The mink and ferrets were immunized against canine distemper, botulism, and viral enteritis thirteen months prior to the collection of blood for blastogenesis. Mink and ferrets were from the treatment groups fed diets containing either 0, l, 5, or 25 ppm of supplemental HCB in the trial initiated on 20 January 1981 (Experiment II). Mitogenic lymphocyte blastogenesis was performed with mink after 34 weeks of HCB exposure and with ferrets after 35 weeks of consuming HCB-treated feed. Heparinized blood was collected, via cardiac puncture, following light ether anesthesia. Mononuclear cells were isolated from the whole blood by density centrifugation using Ficoll-Paque. The 10 ml blood-heparin solution sample was transferred to two 16 x 125 mm sterile tissue culture tubes and handled as separate samples until after the final rinse with Hank's Balanced Salt Solution (HBSS) without Ca++ or Mg++. Following the initial centrifugation (400 x g, 40 minutes), the 95 96 cells at the Ficoll-Paquezplasma interface were collected and washed three times with HBSS and then resuspended in 1.5 m1 of RPMI-164O medium (GIBCO, Grand Island, NY). The final media used contained 25 mM HEPES, 2 mM l-glutamine, 100 U penicillin, 100 U streptomycin, 0.25 pg Fungizone, and 10% heat inactivated fetal calf serum. The two tubes per animal were then combined to yield the final cell suspension. The con- centration of each cell suspension was measured by hemocytometer count and the percentage of live mononuclear leukocytes determined using the Trypan blue exclusion test (Kruse and Patterson 1973) on a phase-con- trast microscope. Cell yields from the original blood sample were 3.4 to 25.6 x 106 total cells, with 90% or greater being live mononuclear cells. The concentration of live mononuclear leukocytes was adjusted to 3.5 x 106 cells per ml (13%) with RPMI-l640 medium. Con A (Miles Laboratories, Elkhart, IN), a T cell stimulator, was diluted using RPMI-l640 media to concentrations of 40, 20, 10, 5, 2.5, or 0 pg/ml. Triplicate samples, except in a few animals which had poor cell recovery and so only duplicate samples were made, 100 pl aliquots of the cell suspension from each animal were placed into wells of sterile U-bottom microtiter plates containing 100 p1 of the various Con A sus- pensions; final mitogen concentrations in the microtiter wells were therefore 20, 10, 5, 2.5, 1.25, or O ug/ml. The cultures were incubated at 38°C in a humidified, 5% C02 incuba- tor for 48 hours. Each well was then pulsed with 0.25 pCi of 3H-thymi- dine (New England Nuclear, Boston, MA, specific activity = 6.7 Ci/mM) in 25 D1 of RPMI-1640 medium and incubated for an additional 24 hours. The cells were harvested onto glass fiber filter strips using a multiple automated sample harvester. The filter strip areas containing the 97 cellular fragments were cut out and placed into mini-scintillation vials containing 5 ml of Scintiverse (Fisher Scientific, Cincinnati, OH). Each vial was counted for 10 minutes on an Isocap/BOO, model 6872 liquid scintillation counter (Searle Analytic, Inc., Des Plaines, IL). The stimulation index for each animal at the various mitogen con- centrations was calculated as: (DPM of mitogen dosed culture - DPM of background culture) % (DPM of background culture). The data were anal— yzed by the Dunnett t-test to examine differences between the control and treatment groups (Gill 1978). §1pgy_g, Effects on the Response of Young Mink and Ferrets Twenty-four standard dark female mink and twenty-three fitch female ferrets, age 16 to 17 weeks, were used to assess lymphocyte blastogenic response of the kits exposed to HCB. The animals were born to dams consuming diets that contained 0, 1, and 5 ppm of supplemental HCB as described in Study 1. The treatment regime consisted of exposure of the mink to HCB for six weeks prior to the mating of the adult females, for approximately seven weeks of gestation, and seven weeks of lactation. The kits were placed on untreated feed for 10 weeks following weaning and prior to blastogenesis testing. As the ferrets do not come into breeding condition until approximately a month later than the mink, the adult ferrets were exposed to HCB for 10 weeks prior to mating, for six weeks of gestation, and seven weeks of lactation. The ferret kits were also fed non-HCB treated feed for 10 weeks after weaning and prior to blastogenesis testing. The young mink and ferrets used in this study were therefore exposed to HCB 1p_p1§pp, via their dam's milk, and from any HCB-contaminated feed they may have consumed before weaning Lymphocyte blastogenesis was conducted in the young animals in the 98 manner described in Study 1 for their dams. Cell yields from these mink and ferrets were generally 5.5 to 20.3 x 106 total cells with 85% or greater being live mononuclear leukocytes. Due to the poor cell recov- ery from the blood of several mink kits, the 20 ug/ml Con A concentra- tion was not used. The data were analyzed using the Dunnett t—test on the ferret data and by a Dunnett-type procedure designed to compensate for heterogeneous variance between groups on the mink data (6111 1978). Results s_tu_dx.1_. At the 20 ug/ml Con A concentration (Table 30), no statistically significant (P<0.05) decrease in the blastogenic response was seen in ferrets fed 1 or 5 ppm HCB in the diet. The 5 ppm treatment group was ignificantly lower than control at P<0.lO. The 25 ppm HCB-treated ferrets yielded a significantly (P<0.05) lower stimulation index than control ferrets. At this concentration of mitogen, Con A was inhibitory for all treatment groups. A general trend toward diminished blastogenic response was seen with increasing HCB concentration in the diet. No significant reduction was observed in the 10 pg/ml Con A microtiter wells until the 25 ppm HCB dietary level. Statistically significant changes from the control stimulation indices were not seen at 5 ug/ml of Con A, although the 25 ppm HCB group did show a reduction significant at P<0.10. At this mitogen dose, as well as the two mitogen concentra- tions below it, a trend toward elevated blastogenic response (above the control value) was observed in animals consuming 1 ppm HCB. Higher toxicant exposures resulted in stimulation indices lower than control values. HCB-treated ferrets did not differ significantly (P<0.05) from 99 ..NN.NV.. .N..:No eat. .NN.N...N N..Nao...=N.N. .< coo mo N..o3 .oHNHoNoNE ogp :. coNHNNHNoocooN .N.N + N.NN. ..oeappzu ecaonxuNa No zoovm No.3».ao ecaoemxooo No zoo i ogappzu eoNoe :omoHNE No Zoo. n xoe:. compo—NewpmH NNN.N.H NN.N NNN.N.H NN.N. N.N.N.H N..NN .N.N.N..H NN.NN .NNN.N.H N... N NNN NN NNN.N.H NN.. NNN.N.H NN.N. N.N.N.H NN.NN N.N.N..H NN... NNN.N.H .N.N. N NNN N NNN.N.H NN.N. NNN.N.H NN... N.N.N.H NN.NN N.N.N..H NN.NN NNN.N.H NN..N N NNN . NNN.N.H .. N. NNN.N.H N..NN N.N.N.H NN.NN N.N.N..H NN.NN NNN.N.H .N.NN N NNN N NN.. N.N N N. NN e .eoeoaoc. ..s\mn. NeoNNNeHcoocoo cowoNNz .Nxooz mm No. NHoNe mu: ecN .ocpeoo mNNENNNooNNHoNNoN oNNso. ..NeN mo HNooNec. :oNHN.:E.pm .om o.NNN 100 the untreated animals at mitogen concentrations of 2.5 and 1.25 ug/ml of Con A. The stimulation indices of HCB-exposed mink are shown in Table 31. At all mitogen concentrations of Con A there was a trend for a low level of toxicant (1 ppm) in the diet to trigger a blastogenic response great- er than the control was observed. There was no statistically signifi- cant decrease in blastogenesis at 20, 2.5, and 1.25 ug/ml of Con A. The 10 and 5 ug/ml Con A concentrations did not differ significantly from the control at l or 5 ppm HCB, but were significantly (P<0.05) reduced in mink that consumed 25 ppm HCB in the diet. In both the mink and ferrets, the highest concentration of Con A (20 ug/ml) suppressed the lymphocyte blastogenic response as compared to the 10 ug/ml concentration. The Optimal mitogen concentration for either species was 10 ug/ml of Con A, with doses above or below this value producing a lesser response. Animals consuming HCB diets had the same optimal mitogen concentration as was observed for control mink and ferrets. 5.211912.- The stimulation indices of the ferrets born to dams consuming HCB (Table 32) did not show a significant reduction in blastogenic response from control values at any mitogen concentration at P<0.05. At 20 pg/ml of Con A, a strong trend toward reduced blastogenic response was noted at the 1 and 5 ppm concentrations of HCB. HCB at 5 ppm in the dam's diet, led to a decrease in stimulation index that was significant P<0.10. There was no significant change in stimulation indices with increasing exposures to HCB at 5 or 10 ug/ml of Con A. At 2.5 and 1.25 ug/ml of this mitogen, the response was equal to or greater than that of the 101 ..NN.NVN. .Ncoeoo soc. .NN.N...N N..NNN...NN.N .1 .< coo mo N..o3 eoHNHoNoNE on. c. coNHNNucoucou a .m.m H :me+ ..oNNHNNu ecsoemxuoa No zoovN No.3».ao ecaoemonN No zoo i o.=u.=o eoNoe comoHNE No zoo. u xoec. coNHN.=ENHmH ..N.N.H NN.N .NN.N.H NN.N. .NNN.N.H N..NN .NNN.N..H N..NN NNN.N.H NN.N N NNN NN ..N.N.H NN.N .NN.N.H NN.N. NNN.N.H .N.NN NNN N..H NN.NN NNN.N.H NN.N. N NNN N NNN.N.H ...N. ..N...H NN..N NNN.N.H NN.NN NNN....H .N.NN N.N.N.H NN.NN N NNN . NN..N H NN.N. NNN.. H NN.NN N.N.N H NN..N NNN... H N..NN NNN.N H N...N N e8 N NN.. N.N N N. NN e .NNNNNoc. ..s\m:. NeoNHNNpcoocou comouwz .Nxooz on Now NHoNe mu: eco .ogucoo no.8:mcoo .NN.N o.Nso. ..NeN No HNoowec. coNHN.=ENHm ..m o.NNN 102 .< :ou mo N.Noz NoHNHoNoNE on. c. :oNHNNucoocouN .N.N H :8... ..oeeapoo ecaonNoNN No tag. NAoNNN.=o eczoemxooo mo zen i oNNHNNo eoNoe comouwe mo Zoe. u xoec. :oNHNNNENHmH NNN.N H NN.N. NNN.N H NN.NN N.N.N H .N.NN NNN.N. H NN.N. N.N.N H NN.. N 8. N .NN.N H NN.N NNN.N H NN.NN NNN.N. H NN.N. .N.... H NN.N. NNN.N. H NN... N 8.. . NNN.. H NN.N NN... H N..NN NNN.N H N..N. N..N H NN.NN NNN.N H NN..N N. .8 N NN. . N.N N N. NN N 88.8... ..ENmn. Ncowpoepcoucoo :omoHNz .coNHNHoN. ecN NochmoNN HNonNoNNH NpoNe mo: eco .oNpcoo NNNENNcoo NENe op Neon .NHoNNo. o.Nsom meooN mo HNooNeNN coNHN.:ENHm .Nm o.NNN 103 control animals. As shown in Table 33, the ability of lymphocytes from mink exposed to HCB to respond to mitogenic stimulation was significantly reduced at 10 and 5 pg/ml of Con A. The reduction in stimulation index observed at 2.5 pg/ml of mitogen was not significantly (P<0.05) less than control values for either the 1 or 5 ppm HCB groups. It was significantly less however at P<0.10 for both HCB-exposed groups. The lowest mitogen con- centration stimulation index, 1.25 pg/ml of Con A, was significantly lower (P<0.05) in kits from the 1 ppm HCB-treated animals, but not in the 5 ppm HCB mink kits. No mitogen concentration or treatment level of HCB gave a stimulation index greater than the control values at 10 pg/ml of Con A. Discussion An increasing number of compounds have been identified as being capable of altering normal immune function. Tetrachlorodibenzo-p- dioxin (TCDD) has been shown to possess both humoral and cell-mediated immunosuppressive properties in the mouse (Vos and Moore 1974; Hinsdill 91_p1, 1980) and to depress cellular immunity in rats (Vos and Moore 1974). The polychlorinated biphenyls have been found to significantly reduce T cell function and humoral immunity in guinea pigs (Vos and VanDriel-Grootenhuis 1972) and polybrominated biphenyls have caused a depression in the cell-mediated immune response of Michigan dairy farm- ers (Bekesi §1_p1, 1979a, b). In this study, mink and ferrets were used to assess the effects of HCB on T lymphocyte function as measured by the incorporation of 3H- thymidine following stimulation by Con A. Because of their position at the top of the food chain, these two carnivores are representative of a 104 ..mo.ovav .oNHcou soc. peoNoNNNe N..:NowwpcmNme .< coo No N—.o3 .oHNHoNoNE on. c. coNHNNucoocouN .m.m + zoos. ..og:u.:o ecsoemxoon No zoo. . .ooeupzo eceocmona No zen i o.=..=o eoNoe comou.s mo 289. u xoecw coNHNFNENNm . NNN.. H ...N NNN. H NN... ..NNN.N H NN..N ..NNNN H NN.NN N ...N N ...N.. H .N.. .N... H NN.N. .NNNN H NN.NN .NN.N H NN.NN N ...N . NNN.N H .N... .NN.N H N..N N..N. H N..NN NN.... H NN.NN N. ..N. N NN.. N.N m o. c HooEHNoNh ..s\m:. NNoNNNNHcoocoo :omoHNZ .coNHNuoN. eco Nococmoeo Haogmooggu NHoNe mu: eNN .oeucoo NNNENNNoo NENe o. coon ..N.N o.Neom mesoN No HNooNec. coNHNNNENHm .mm o.NNN 105 class of animals with high risk from compounds capable of biomagnifi- cation. Additionally, both the mink and ferret have been shown to be extremely sensitive to the other halogenated aromatic compounds such as PCBs (Aulerich g1_p1, 1973; Platonow and Karstad 1973; Bleavins £1_§1, 1980) and PBBs (Aulerich and Ringer 1979). The cell-mediated aspects of the immune response have been considered to be especially sensitive to alterations by aromatic hydrocarbons (Moore 1979) such as HCB, TCDD, PCBs, and PBBs. A significant reduction in the stimulation index was found in the adult mink and ferrets fed 25 ppm HCB in the diet. This decrease was seen following Con A stimulation, a general T cell mitogenic response (Janossy and Greaves 1972), and so indicates a depression of T cell function. At low dietary levels of HCB (1 ppm), an enhancement of lymphocyte response was seen in the mink at all mitogen concentrations and at several mitogen concentrations in the ferret. This phenomenon has been described by Luster and Faith (1979) for several laboratory species and man following exposure to a variety of halogenated aromatic hydrocarbons. A suppression of the lymphocyte blastogenic response at the highest Con A concentration (20 ug/ml) was observed in the adult mink and fer— rets on all treatments. The sub-optimal degree of lymphocyte activa- tion at high mitogen concentrations has been likened to immunologic tolerance seen 1p_yiyp following large doses of antigen (Mdller 1970). The depression of T lymphocyte proliferation by high concentrations of Con A was reported to be a reversible occurrence (Andersson g1_g1, 1972) when the excess mitogen was removed. The degree of suppression (stimulation index at 20 pg Con A/ stimulation index at 10 pg Con A) 106 was greatest in the mink and ferrets consuming the 25 ppm HCB diet, presumably due to a decrease in the number of cells proliferating and/ or a greater degree of blastogenic inhibition than was present in the other treatment groups. The 1p_pggpp and early postnatal exposure of mink to HCB resulted in a significant depression of the lymphocyte blastogenic response. The lowest level of exposure (mothers consuming diets containing 1 ppm HCB) caused reduced T cell response in the offspring following Con A stimu- lation of their isolated lymphocytes, in contrast to the enhanced effects observed in their dams. The cell-mediated immune system has similarly been shown to be depressed in mice and rats prenatally and postnatally exposed to TCDD without significant toxic effects being produced in their dams (Vos and Moore 1974; Luster g;_a1, 1979). The young rats affected by TCDD (Luster g1_p1, 1979) had however, returned to a normal lymphocyte blastogenic response by seven months of age. The immunosup- pression resulting from perinatal toxicant exposure need not necessarily permanently impair the immune system. Luster g1_a1, (1979) found the degree of immunosuppression to be less severe in rats treated only post- natally when contrasted to rats exposed 1p_p1gpp_and postnatally. The 1p_p1gpp_exposure of rats to alcohol has also been reported to create a prolonged depression in T lymphocyte reactivity to Con A stimulation (Monjon and Mandell 1980). HCB has been shown to cross the placenta in the rat (Villeneuve and Hierlihy 1975; Courtney 1979), rabbit (Villeneuve gpflg1, 1974), mouse (Courtney g1_g1, 1976), and the mink and ferret (Bleavins, unpublished data). It has also been detected in the milk produced by cows (Fries and Marrow 1971), mice (Courtney $1.21, 1979), ferrets (Bleavins, 107 unpublished data), and humans (Miller 1973; Siyali 1973). Indirect exposure of developing/nursing offspring to HCB is, therefore not only possible, but virtually unavoidable when their mother is consuming a diet contaminated with this persistent halogenated hydrocarbon. The risk to which progeny are exposed was dramatically brought to light by the HCB-induced outbreak of porphyria cutanea tarda (PCT) in Turkey from 1955-1959 (Courtney 1979). Nursing infants showed a variety of histological abnormalities and an exceptionally high mortality rate (Courtney 1979) while the only cause of death consistently reported was secondary pulmonary infection. This secondary infection may have been the result of cell-mediated and/or humoral immune system suppression. As proposed by V05 (1977), the subtle effects of immune responsive- ness can most readily be measured by treating the test species with the chemical of interest during the early deve10pmental stages of the immune system. Prenatal and early postnatal treatment of an animal may well provide the most sensitive means by which to detect impairment of the immune response (V05 1977) in neonates. Although the immune system is very sensitive to chemical insult, it also possesses considerable potential for recovery (Luster g1_p1, 1979). If the immunosuppressive nature of HCB is temporary, it may not represent a permanent threat to the animal's ability to survive. If no serious challenge to the cell- mediated or humoral immune system occurs during this time, the lowered host defenses may be sufficient to protect the animal. However, in this study immune responsiveness was significantly depressed 10 weeks after the animal's exposure to HCB had ended. This suggests a reasonably long period of lowered immune defenses even if the compound should be found 108 not to permanently impair immune function in future studies. The immune system provides a sensitive indicator of toxicity. Effects on the immunocompetence of an animal can preceed related histopathologic changes (Loose g1_g1, 1979). While immunosuppression may not be immediately or directly lethal, it does result in an increased susceptibility to infec- tion and cancer. It is, therefore, important that immune function be incorporated into the routine toxicity testing procedures. The initial assessment of a compound's immunosuppressive potential can be made in conjunction with the standard 90-day toxicity study, even though this subchronic trial may not be of sufficient length for long-lived memory lymphocytes to show an effect following exposure to some toxicants (Menson g1_p1, 1982). The complex interactions of immune system cells with each other, with the various other tissues of the body and their response to challenge demands more detailed study. The development of 1p_yjppp_lymphocyte activation techniques has made it possible for the researcher and clinician to study immune function in the intact animal or human. Aflthough there are limitations in metabolic activation and pharmacodynamic interactions, these procedures do not require termination of the test animal, use limited quantities of blood and can be made directly with human peripheral blood lymphocytes. The immune system cells, having been exposed to a chemical 1p_yjyp, can be readily removed from the body and their function examined 1p vitro. Experiment VII. EFFECTS OF IN UTERO AND EARLY POSTNATAL EXPOSURE TO HEXACHLOROBENEENE ON HEPATIC MIXED FUNCTION OXIDASES, 1N_VITRO RENAL FUNCTION AND HISTOLOGY, AND TISSUE RES DUES IN MINK. ' Purpose This study was conducted to determine whether or not exposure of mink kits to HCB during gestation and nursing resulted in persistent or permanent alterations in their kidney and renal morphology, and function. In addi- tion, tissue residue concentrations were measured in the kits from each of the treatment groups. Materials and Methods Eighteen female mink kits, born to the females described in Experiment II, were used in this study. Six kits were selected from each of the three treatments having sufficient numbers of kits surviving to weaning (control, 1 ppm and 5 ppm of HCB) to allow reasonable replication. The mink kits are described in greater detail in Experiment IV, Study 2. The animals were killed by cervical dislocation at 16-17 weeks of age and blood, adipose tissue, muscle, kidney, and brain samples were taken for HCB analysis. Livers and kidneys were removed and immediately placed into ice-cold physiological saline. Microsomes from mink livers were prepared according to Dent _p _1. (1980). In a typical reaction, microsomes (0.25-0.5 mg/ml) were suspended in 1 m1 of 66 mM Tris-HCl buffer (pH 7.8) containing 4.5 uM glucose-6- phosphate, 0.3 uM NADH, 0.3 uM NADP, 0.1 uM NADPH, 163 “M MgClz and 1 unit of glucose-6-phosphate dehydrogenase. After a three minute preincubation at 37°C, the reaction was initiated by the addition of substrate. Enzymatic activities measured were ethoxyresorufin-O-deethylase (Johnson 91_p1, 109 . 110 1979), ethoxycoumarin-O-deethylase (Aitio 1978), and benzphetamine-N- demethylase (Prough and Ziegler 1977). Cytochrome P-450 and cytochrome b5 were measured according to the method of Omura and Sato (1964). NADPH cytochrome-c reductase was measured by the method of Pederson g1_g1, (1973). Microsomal protein was determined by the method of Lowry g1_g1, (1951). Thin renal cortical slices were prepared free-hand. Organic ion accumulation was determined by incubating 50-150 mg of slices in 4 ml of medium composed of 96.7 mM NaCl, 7.4 mM sodium phosphate buffer (pH 7.4), 40 mM KCl and 0.74 mM CaClz (Cross and Taggart 1950). The medium was supplemented with 10 mM lactate (Sigma Chemical Co., St. Louis, M0) for one-half of the slice incubations and not supplemented for the other half of the slices. It also contained 7.4 x 10"5 M p-aminohippurate (PAH) and 1 x 10-5M (1-l4C)-tetraethy1ammonium (TEA), specific activity 2.00 mCi/ mmol (New England Nuclear, Boston, MA). After incubation for 90 minutes at 25°C under 100% 02 in a Dubnoff metabolic shaker, slices were removed from the medium, blotted and weighed. Slices were homogenized in 6 ml of 5% trichloroacetic acid and brought up to volume of 10 ml with distilled water. A 2 ml aliquot of the incubation medium was treated similarly. After centrifugation, the supernatant was assayed for PAH by the method of Smith g1_p1, (1945) and for TEA by liquid scintillation spectrometry. PAH or TEA accumulation in the kidney slices was expressed as the slice- to-medium or S/M ratio. Glucose synthesis by renal cortical slices was determined by a method similar to that of Roobol and Alleyne (1974). The incubation medium consisted of a Krebs-bicarbonate medium containing 10 mM pyruvate as the substrate for gluconeogenesis. After gassing the medium with 02-002, 111 50-100 mg of the slices were placed into 5 ml of the medium in a 50 ml Erlenmeyer flask, flushed with 02-C02, stoppered tightly and incubated at 37°C for 1 hour in a Dubnoff metabolic shaker. After incubation, the slices were removed from the medium, blotted and weighed. The medium glucose content was determined using glucose oxidase and peroxidase as described in Sigma Technical Bulletin No. 510 (Sigma Chemical Co., St. Louis, MO). Concentrations of HCB in liver, kidney, fat, muscle, brain, and blood were determined by the following technique. Samples were weighed and ground with sufficient anhydrous sodium sulfate to render them completely dry and pulverized. Powdered tissues were extracted three times with 40 ml portions of hexane. Pooled extracts were reduced in volume to 1-2 ml and placed on a Florisil column (60-100 mesh) of 500 x 200 mm. HCB was eluted with 200 ml of hexane which was evaporated to a 1 ml volume and then brought up to the desired volume. Quantitation was by gas-liquid chromatography on a Varian Model 2100 gas chromatograph (Verian, Palo Alto, CA) with a Sc3H electron capture detector using a 1.7 mm x 2 mm i.d. column packed with 3% OV-225. Carrier gas (N2) flow and column temperature were 30 ml/min and 165°C, respectively. Concentrations were expressed as nanograms of HCB per gram of tissue (wet weight) or ppb. Several thin slices of liver and kidney were fixed in 4% glutaral- dehyde in 0.1 M cacodylate buffer (pH 7.4) and processed for routine light microscopic observation, including fat staining with 011 red 0. For electron microscopy, tissue slices were diced into 1 mm blocks, dipped in 4% glutaraldehyde/O.l M cacocodylate buffer fixative and placed into fresh fixative for 3 hours. Tissue blocks were then washed in Zetterquists' buffer twice, placed in 1% 0504 solution for 15 hours and embedded into 112 Epon-Alaldite plastic after dehydration through graded solutions of alcohol and propylene oxide. Thin sections were then cut on an LKB-III ultratome, stained with uranyl acetate/lead citrate and examined on a Zeiss Electron Microscope (952) operated at 60Kv. Data were analyzed statistically by an analysis of variance, completely random design. Treatment differences were examined by the least significance difference test (Steel and Torrie 1980). The 0.05 level of probability was used as the criterion of significance. RESULTS Offspring of HCB exposed dams had a high degree of mortality (77.4% in the 5 ppm group) as reported in Experiment II. The present data are on the survivors of that study. At 17 weeks of age, kits from adult female mink fed 0, l or 5ppm HCB had no alterations in body weight, or kidney or liver weights (Table 34). Microsomes from control livers contained 0.08 :_0.010 nmoles of cytochrome P-450 (P-450) per mg protein while kits from the 5ppm treatment group had elevated hepatic P-450 concentrations of 0.16 1 0.014 nmoles/mg protein (Figure 6). Cytochrome b5 was not altered in any treatment groups. Correlating with an increased hepatic P-450, hepatic ethoxyresorufin-O-deethylase in the 5ppm group increased 1.5 fold over controls, but not significantly. No changes were observed in ethoxycoumarin-O-deethylase, benzphetamine-N-deethylase and NADPH cytochrome-c reductase (Table 35). No alterations in the 1p_xi§pp renal function were observed as determined by the accumulation of PAH or production of glucose by renal cortical slices in any treatment group. TEA accumulation was significantly decreased in kidneys from the 5ppm treatment group (Table 36). HCB was primarily distributed in the adipose tissue in all treatment groups with detectable concentrations in brain, 113 Table 34. Effect of perinatal HCB exposure on body and organ weightsI of 16-17 week old mink kits. - i Maternal Body wt. TISSUE treatment n (kg) Liver Kidney 0 ppm 6 0.73 _+_ 0.02 2.6 i 0.12 0.5 i 0.03 1 ppm 6 0.75 :_0.09 2.8 :_0.07 0.5 :_0.01 5 ppm 6 0.67 i 0.05 2.7 i 0.08 0.5 1 0.03 Mean _+_ 5.5. 1Expressed as a percentage of body weight. Figure 6. The effect of perinatal HCB exposure on hepatic cytochrome P-450 (open bars) and cytochrome b5 (hatched bars). Asterick indicates a value significantly different from control (P<0.05). 114 maternal dietary exposure to HCB (ppm) . . 2 1 o. o. :Hououm m0 m2 mom moaoz: 115 .NNouoNN No as \oHNNNE \No.oE:N .NNoHoNN No me \oHNNNE \eoooeoN oioeoegoouNo mo No.oe:H ...N + N.N NN.N H N.N NN.N + N.N NN.. H N.NN N ..NN N .N.N + ..N ...N H N.. NN.N + ..N ...N H N.NN N 8.. . NN.. + N.. ...NH N.N NN.N + N.. NN.N. H N.NN N 8.. N NoNN.N;Hoeoeiz eoNN.N;Hooeio NoNN.N;Hooeio HoNNHoNeoN : HooEHNoNH oNNENHogoucom :NNNENooNonHm cwezsoNoNNxocam oioEoesoopNo .NceoHNz :aoN. N.NNN oNoNNeN : Hoospooeu oNNN.N .NceouNz .NN.N News e.o xooz N.-N. No oNNNoNxo .N.NNNNoN mcwzo..ow ..3: mo .NNN. NeoNHNNHNoucoo oneNNoN oNNNNN .Nm o.NNN 119 activities increase (Shull p1flg1, 1982). Ethoxyresorufin-O-deethylase, which was increased in this study, is a 3-methylcholanthrene inducible enzyme whereas benzphetamine-N-demethylase, which was not changed, is a phenobarbital inducible enzyme. In the rat, HCB increases enzymatic activity consistent with a phenobarbital type of induction, but results in a shift in spectral characteristics of P-450 to a lower wavelength similar to that seen after 3-methylcholanthrene induction (Stonard and Nenov 1974; Courtney 1979) and has been termed a "mixed" inducer. In the mink, HCB appears to behave predominantly as a 3-methylcholanthrene type mixed function oxidase inducer. Pregnant mice administered pentachloronitrobenzene (PCNB) contaminated with 10% HCB demonstrated a high incidence of renal agenesis in offspring, whereas no alteration was observed after technical grade PCNB administra- tion (Courtney 21 p1. 1976). It was concluded that the renal teratogenic effects were due to HCB. In addition, female Rhesus monkeys exposed to HCB showed renal histological alterations (Iatropoulos gp_p1, 1976). In contrast, perinatal HCB exposure had no effect on 1p_yj§pp_renal function or histology in the mink kits used in this experiment. Apparently no permanent compromise of renal morphology or physiology occurred, although temporary abberations cannot be ruled out. These kits were exposed to HCB throughout gestation and nursing, but were placed on clean feed for the ten weeks between weaning and the measurement of kidney function. Therefore, it is possible that renal effects, of a temporary nature, may have been present at birth or at some point during nursing, and had been compensated for prior to the end of the study. The reason for the high mortality among nursing mink remains unclear. The apparent lack of toxicity among surviving mink may reflect their 120 relatively low body burden of HCB, as kits fed 5ppm had only 0.6 ppm in the body fat and very low levels in other tissues. It is likely that the kits received the majority of their body burden of HCB during nursing, since in the mouse toxic doses of HCB can readily be transmitted via the milk. Young, untreated mice cross-fostered onto lactating HCB-treated mothers showed a high rate of mortality (Courtney 1979). Similarly, mammary transfer of HCB has been shown to be 31 times greater than placen- tal exposure to growing ferret kits (Bleavins pp_g1, 1982). The 10 week period of time in which the mink kits were on diets not supplemented with HCB would allow the animals to actively metabolize and excrete this halo- genated aromatic hydrocarbon. Their body burden would, thus, be less at the date on which the study was ended than was present at weaning. The rapid growth rate of mink kits from weaning to approximately 17 weeks of age would further act to dilute the concentration of HCB per gram of tissue, in addition to any changes in total body burden. Immunosuppression from HCB exposure may also have contributed to the high incidence of kit mortality seen prior to weaning (Experiment II) in the mink. In conclusion, HCB, while causing a high degree of mortality in young mink prior to weaning, resulted in no significant hepatic or renal damage in the surviving mink ten weeks after exposure ended. The increased xenobiotic metabolism of these animals may alter their response to agents in the environment known to be metabolically activated. Alternately, induction of hepatic mixed function oxidases in these mink may also result in certain endocrine related disorders through altered metabolism of steroid hormones. Experiment VIII. EFFECTS OF HEXACHLOROBENZENE ON REPRODUCTION, CELL- MEDIATED IMMUNITY, ORGAN WEIGHTS, AND MICROSOMAL ENZYMES OF MINK IN A CROSS-FOSTERING STUDY. Purpose This experiment was designed to attempt to differentiate the effects of 1p_ppppp and mammary exposure to HCB from the effects seen following their combined insult. At birth, mink kits were transferred from HCB treated dams to control dams, and vice verse, to allow exposure during gestation only, and during nursing, only. Materials and Methods Forty standard dark female mink, proven second year breeders, were randomly assigned to two treatment groups of twenty animals each. The treatments consisted of an untreated basal diet and a diet that contained 2.5 ppm supplemental HCB. Standard ranch procedures were followed in the care, handling and feeding of all animals. Thirty-nine females were bred once and given an opporutnity to remate on the following day. All matings were made within a four day period and first matings were confirmed by a vaginal smear examined for the presence of motile spermatozoa. One female, later to be placed on HCB-treated feed, refused to breed and was thus excluded from the study. All animals were maintained on the basal diet until 17 March 1982. On this date, the 2.5 ppm HCB-treated group was placed on contaminated feed and remained on the HCB-treated diet through- out pregnancy and lactation. Each female was checked twice daily for the birth of kits during the whelping period. On the day they whelped the female was weighed, the kits 121 122 counted and weighed, and the number of live and dead kits recorded. At this time, one kit was taken from each of five litters on the control and HCB diets for histologic examination of the thymus, liver, spleen, and kidney. On the days that female mink from both the control and HCB treatments whelped, their entire litter was transferred to a female of the opposite treatment (cross-fostered). Prior to the switching of litters, the kits, nest box and female's nasal region were dusted with talcum powder to reduce the risk of rejection or killing of the transferred kits. Fourteen litters were cross-fostered to yield the following treatments: 1) Control: Kits born to and nursed by females consuming the basal diet (n=ll litters) 2) Control +-HCB: Kits born to females consuming the basal diet, but nursed by females consuming 2.5 ppm HCB (n=7 litters) 3) HCB a-Control: Kits born to dams consuming 2.5 ppm HCB, but nursed by females consuming the basal diet (n=7 litters) 4) HCB: Kits born to and nursed by females consuming 2.5 ppm HCB (n=8 litters) The kits were maintained on the maternal diet until weaning (seven weeks of age) and then fed the untreated basal diet until 13 July 1982. On 13 July 1982 twenty-four male kits were selected, six from each treatment group, and the following procedures performed. A nine ml sample of blood was collected via cardiac puncture and lymphocyte blastogenesis performed as described in Experiment V. After the blood samples were collected, all animals were weighed and then killed by cervical dislocation. Brain, liver, spleen and thymus were weighed and sections taken for histo- logical examination. Also taken for histology were kidney, ileum, and 123 adrenal gland. A sample of liver was collected for mixed function oxidase evaluation as described below. After the fresh livers were weighed and rinsed in 150 mM KCl and blotted dry, 10 9 portions were minced and then homogenized at 4°C in 20 m1 of 20 mM Tris-HC1:1.15% KCl (pH 7.4) with a Polytron1 tissue homogenizer. The homogenate was centrifuged at 12,000 x g for 20 minutes at 0-4°C, the supernatant filtered through four layers of cheesecloth and then ultra- centrifuged at 105,000 x g for 75 minutes at 2°C. The microsomal pellet was suspended in 15 ml of 0.3 M sucrose:0.l M sodium pyrophosphate (pH 7.5) with the tissue homogenizer. and recentrifuged as above. The resulting pellet was resuspended in 150 mM KCl to yield a concentration of approxi- mately 30 mg of protein per m1. Protein was determined colorimetrically by the Biuret method using a bovine serum albumin standard (Gornall g1_p1, 1949) on a Gilford Stasar II spectrophotometer2 (model 1367X5). The assay procedures for aminopyrine N-demethylase (Anders and Mannering, 1966) and benzo-a-pyrene (Van Canfort p1_p1, 1977) were modified as follows. Each assay vessel contained a total volume of 0.5 ml which included 281 ul of NADPH regeneration system [250 pl of 200 mM HEPES3 (pH 7.6) containing 0.5 mg NADP4, 20 n1 of 10% giucose-6-phosphate4 solution (pH 7.0), 10 ul of 250 mM MgC12.6H20 solution, and 1 ul (0.5 units) of glucose-6-phosphate dehydrogenase4], 200 pl of microsomal suspension and 20 ul of substrate solution. The benzo-a-pyrene hydroxylase vessels con- tained 0.5 mg of NADH4. Substrate concentrations added to each vessel in a 20 ul volume were 20 umoles of aminopyrine Brinkman Instruments, Hestbury, NY 11590. Gilford Instrument Laboratories, Inc., Oberlin, OH 44074. U.S. Biochemical Corp., Cleveland, OH 44122. Sigma Chemical Co., St. Louis, MO 63178. wa-d 124 in methanol or 0.88 nmoles of benzo-a-pyrene including 0.25 uCi of (G-3H)- benzo-a-pyrene5 in acetonitrile. The incubation phase for the aminopyrine N-demethylase was carried out in 16 x 125 mm disposable glass culture tubes and the benzo-a-pyrene hydroxylase assay in 8 m1 glass mini-scintilla- tion vials. Microsomes diluted to 20 mg protein per ml (aminopyrine) or 2 mg per ml (benzo-a-pyrene) with 150 mM KCl were added to vessels containing the NADPH regeneration system and preincubated for 3 minutes at 37°C in a Dubnoff metabolic shaker bath5. The reactions were initiated by the addition of 20 ul of substrate and allowed to incubate under air at 37°C for 20 minutes while oscillating. The aminopyrine N-demethylase assay included a microsomal blank (no added NADP or NADH) for every microsomal preparation. For the benzo-a-pyrene test, both NADP and NADH were included during the 20 minute incubation time and microsomes were withheld until after termination of the reaction. All assays, including blanks and standards, were conducted in duplicate. Both enzyme procedures were conducted under reduced light with the vessels placed in ice following termination of the reactions. The aminopyrine N-demethylase assay was terminated by the addition of 1 ml of 20% ZnSO4. After adding 1.0 ml of saturated barium hydroxide solution, vortexing, and centrifuging for 20 minutes, 1.0 ml of supernatant was added to 1.0 ml of Nash reagent7. The mixture was heated for 10 minutes at 60°C, cooled, and the optical density (00412) determined against a water blank and compared to a formaldehyde standard curve. Termination of the benzo-a-pyrene assay was accomplished by the addition of 1.0 ml 5 Amersham Corp., Arlington Heights, IL 60005. 5 GCA Precision Scientific, Chicago, IL 60647. 7 15 g ammonium acetate, 400 pl acetylacetone, 300 pl glacial acetic acid brought up to a volume of 100 ml with distilled deionized water. 125 DMSOB-KOH solution (85% 0ns0:15% 1.0 M KOH). The mixture was extracted twice with hexane, which was discarded. A 500 ul subsample of the aqueous phase was then added to 5.0 ml of counting cocktail (77.4% ACS9, 15.5% absolute ethanol, 6.5% DMSO and 0.6% 1.0 M glacial acetic acid) and counted on an Isocap/3OO liquid scintillation counter10 for ten minutes. Cytochrome P-450 (448) concentrations were measured with a Beckman Acta III dual-beam spectrophotometer“ using the carbon monoxide difference spectral method of Omura and Sato (1964) in microsomes diluted with 100 mM Tris buffer (pH 7.4) to 1.0 mg/ml of protein. Results Both the control and 2.5 ppm HCB-treated females showed good whelping percentages, 90% and 79%, respectively (Table 38). The HCB-treated females did exhibit a significantly longer length of gestation than control females, however both groups fell within the normal term of pregnancy for mink of approximately 40-60 days (Travis and Schaible 1960). No difference was seen in total litter size or the average number of kits born alive vs. dead between the two groups. The control female mink showed a low level of kit mortality (5%), presumably due to their having raised offspring the previous year and having been selected on this criteria. Kits whelped by control females, but nursed by HCB treated dams (Control +HCB) showed an increased mortality rate of 13.6% while HCB +Control kits had a mortality rate of 22.5%. Kits whelped and nursed by HCB-treated females had a 8 Aldrich Chemical Co., Milwaukee, WI 53201. 9 Aqueous counting scintillant, Amersham Corp., Arlington Heights, IL 60005. 10 ll Searle Analytic, Inc., Des Plaines, IL 60018. Beckman Instruments, Inc., Fullerton, CA 92634. 126 .Amo.ovn_v FOLHCOU EON... u:w&m.+.+2u hpucmurvwcmwm .1 .285“. 2.538. ..oo N... No eonszc omNNo>N x omN mo Nxooz m eco nNNNn coozpon N.Nm HnNNoz Neon .NN omNNo>N u NNNeonH .N.N H .8... N.NN H NNN N N.NN N.NN N..N + ..N N..N + N.N N..N + N.N ..N.N + .N N.NN. NN.. ..NN H NN. N N.NN N.NN - - - - - .88698: ..NN H N.N N N.N. N.N. - - - - - 8.78.88 N.NN H NN. .. N.N N.N NN.N H ..N ...N H N.N N..N H ..N N.N H .N NN.N. .888 HNNNeoNN c Nxooz Nxooz .Nuop eNoe o>..N .NNNe. \wwwmmnm o unosHNoNN Nee.... N a. N a. N.N.N .N NN.N NN.... NN..N.NNN N N.....coe ..N .NeNHN NN.NoHNoNiNNoNo N :. NHo.e mo: eNN .oNHNoo ac.somcoo No.5 oNNeow Nn eoooeoNN .NNNsoNn ecN .N..—Naeoe ..N .oocoeeomeoa o>NHo=eoNNoN .numco. noNHNHNow .mm o.nNN 127 mortality comparable to the HCB + Control group (20.5 vs. 22.5%). No significant difference was seen in the biomass raised by females on any treatment. No significant change was observed in birth weights between the two groups (Table 39). Female mink kits exposed to HCB jg_gtggg_and via the milk weighed significantly more than control female kits at three weeks of age. At six weeks old, male kits on the Control +-HCB treatment were significantly heavier than their control counterparts. The six week weights of female kits did not show any significant differences. The lymphocyte blastogenic response was not significantly different from control for any of the three HCBaexposed groups, either on a disintegra- tions per minute or a stimulation index basis. There was also no change seen in body weight or brain weight of kits in any treatment group when contrasted to the control kits. Liver, spleen, and thymus (expressed as a percentage of brain weight) were likewise not significantly different from control values. The jn_utggg_and/or mammary exposure of mink kits to HCB did not result in significant liver microsomal enzyme induction. No changes above control values were seen in aminopyrine N-demethylase, benzo-a-pyrene hydroxylase, cytochrome P-450 (P-448) or mg of protein per gram of liver. Discussion The lengthened gestational period seen in the dams consuming a HCB- treated diet during this study was not observed in Experiment II. However, in the work reported above, all females were mated within a four day period during the middle of the breeding season and only second year proven breeders were used. Experiment II utilized first year females bred throughout the approximately 2l day breeding period. Presumably, this may 128 .Amo.ovav Poeocou sage basemeewu »_p=a0we_ca_m k. .3 H 53 mo.¢.H a.mw~ om .MN.N.H w.eop om FN.o.H P.m mm mu=_ me.m.H m.Pm~ _N mo.N.H o.~m Pm - - .oeocoo+mu: w_.F_.H m.e- m, m_.m.H m.¢m mp - - muz+poeo=ou mm.m.H “.mmm KN mm.~.H o.¢m KN mm.o.H ~.m Ne _oeocou mpmsom mm.m_.H P.¢m~ __ mm.e.H m.P__ __ _m.o.H.o.o_ mm mu: -.aF.H m.mom op mm.¢.H w.~o_ op - - Foeocoo+muz «eo.m_.H ~.o¢m MN mp.m.H ~.o__ mm - - moz+poaocou F¢.FP.H m.om~ om «N.N.H e.¢o_ Fm _~.o.H m.m No poaocou .MHmm mxmo: o : mxmmz m c spgwm : pcmsammgk Amy agapmz swam .xcspm mcwgmpmoeummogo m cw mu: o» ummonxm mpwx xcws mo +mugmwm: moon UFo xmmz xvm vcm .xmmz macs» .gugwm .mm opnmh 129 account for the greater clustering of whelping dates seen in this experi- ment (as evidenced by the smaller standard errors) than was found in the earlier study. An elevated degree of kit mortality was seen in all treatments exposed to HCB. This is not unexpected since the maternal tissue stores act as a readily accessible source of HCB for the fetus (Courtney and Andrews 1979) and nursing offspring. Kit mortality of mink exposed to HCB via the milk (Control +-HCB) was higher than for control mink, but lower than in the animals exposed only 1n_gtgrg_(HCB +-Control). These kits exposed to HCB jg_gtgrg showed a mortality level comparable to the animals receiving HCB insult both during gestation and nursing (HCB). At the low dietary concentrations of HCB used in this study (2.5 ppm), in_ utg§9_exposure appears to represent a more serious threat to offspring survival than exposure via the dam's milk. This phenomenon was seen even though the quantity of HCB received by a nursing animal via the dam's milk is many times the amount that passes across the placenta during fetal development (Grant gt 21, 1977; Bleavins gt_al, 1982). Hexachlorobenzene has also been reported to adversely affect reproduction in the rat (DeMatteis gt_al, 1961; Grant gt_al, 1974, 1977; Courtney and Andrews 1979; Kitchin £3 31, 1982). The effects seen included a decrease in the number of litters whelped, fewer pups surviving to weaning, increased incidence of stillbirths, reduced birth weight, and depressed rate of growth. The cross-fostering of rat pups born to untreated dams onto females consuming HCB in their diet, resulted in the death of all pups (DeMatteis gt_al, 1961). All of these effects were seen at exposure levels much higher than can be used in the mink and still have the females produce any l30 viable offspring. A percent mortality (to weaning) comparable to the level seen at 2.5 ppm HCB in the mink was not observed in the rat until maternal exposure was 60 ppm (Kitchin gt_gl, l982). Grant £5 91, (l977) have reported 20 ppm HCB in the maternal diet to be the no-effect level for reproduction in the rat, a dietary concentration comparable to the 25 ppm HCB treatment fed in Experiment II and which had a profound adverse effect on the reproductive performance of the mink. The improved survivability of mink kits seen during this study at 2.5 ppm of HCB than in Experiment 11 at l ppm (20.5% vs. 44.1% kit mortality) is probably the result of better maternal ability in the second year females and/or fluctuations in environmental factors from year to year. The increased three week weights of the HCB-treated female kits over the control females is potentially due to a reduced number of kits per litter, since no change was found in the biomass raised per female. If a significant increase in body weight occurred without a reduction in the number of kits raised, it would be reflected by a concurrent elevation in the biomass value. The Control +~HCB males showed a similar body weight increase at six weeks of age which may also be explained by the higher kit mortality seen in this group than in the control males, thereby reducing intralitter competition for the dam's milk and eventually the available solid food. No significant effect was seen in the lymphocyte blastogenic response to Con-A in the young mink in this study, in contrast to the results observed during Experiment VI. The underlying cause for the different results is not known, but may involve the shorter period of maternal exposure to HCB. In Experiment VI, the females were placed on HCB-supple- mented diets six weeks prior to mating. In the study reported above, HCB l3l treatment did not begin until immediately after the females had been bred, at the middle of the breeding season. These female mink, therefore, had a period of HCB exposure approximately eight weeks shorter than the animals in Experiment VI. This would result in lower tissue residue concentrations of HCB and so a lower level of this aromatic hydrocarbon in the systemic circulation and milk. This would in turn lead to fetal and neonatal exposures below those seen when blood or milk concentrations were higher. Although an increase has been reported in the mixed function oxidases (MFO) of suckling rat pups raised by dams consuming HCB (Grant et_gl, l977), no induction was seen in the mink kits cross-fostered in this experiment. Whether this is the result of the animals being maintained on non-HCB diets from weaning until the termination of the study (5 weeks) and, therefore, returning to a normal MFO profile or due to HCB not being a particularly potent inducer of microsomal enzymes (Carlson l978), is not clear. Either or both of these possibilities may be involved, as may other factors, including the variability seen within different runs of the enzyme assays. The shorter duration and low level of maternal exposure may also have come into play in affecting the results of this study. Kit mortality proved to be the most sensitive indicator of HCB toxicity in this cross-fostering experiment. The other physiological parameters monitored (lymphocyte blastogenic response, organ weights, and MFO induc- tion) did not show significant changes, although such changes might have been detected if these tests had been run immediately after exposure to HCB ended, instead of allowing the animals to consume non-HCB diets for five weeks. SUMMARY AND CONCLUSIONS The results of these studies indicate that the mink and ferret are among the most sensitive mammalian species to the toxic effects of HCB. Anorexia, weight loss, gastro-intestinal bleeding, and bloody droppings were observed in these animals prior to death at dietary concentrations of l25 and 625 ppm HCB. Although these clinical signs are not specific for HCB toxicosis, they are consistent with halogenated aromatic hydro- carbon poisoning. In general, no change was found in the hematologic parameters measured (RBC, NBC, Hb, Hct, MCV, MCHC) until just before death. The changes in erythrocyte numbers and leukocyte proportions are probably secondary to gastro-intestinal bleeding and impaired immunocompetence, respectively, rather than a direct interference with cellular formation. As has been reported in other species, the acute toxicity of HCB was low (> l000 mg/kg of body weight) in these two mustelids. The mink and ferret were, however, very susceptible to chronic HCB exposure. No indications of abnormal porphyrin synthesis or metabolism (porphy- ria) were observed in either the mink or the ferret following the consump- tion of HCB-supplemented diets. Other carnivorous animals, dogs and some predatory birds, also do not develop prophyria as a toxic manifestation of exposure to HCB. These species are in contrast to the rat, mouse, monkey, human, chicken, and quail, each of which exhibits HCB-induced porphyria. The development of porphyria has been implicated as being involved in the underlying cause of HCB-related toxicity in the porphyrin sensitive species. Since the mink and ferret do not develop porphyria, it would appear that the toxic effects seen must be independent of the porphyrin cycle. 132 133 Female ferrets fed a l25 ppm HCB diet were hyperexcitable and displayed signs of neurological impairment. The analysis of biogenic amfines in the brains of mink and ferrets on the control, l, 5, and 25 ppm HCB diets revealed alterations in brain neurotransmitter concentrations. Changes were also found in neurotransmitter concentrations of the mink kits produced by females consuming l and 5 ppm HCB. A single dietary dose of HCB was found to be readily absorbed (98.5% of the HCB given) by adult female ferrets. A total body HCB half-life of 32 days was calcualted for ferrets raising kits to 5 weeks of age and of 41 days for unbred female ferrets. Lactation was an important excretion route for HCB in the female. Greater than 20% of the HCB absorbed by the dam was detected in the kits she raised. The exposure to HCB via the milk for the growing ferret kit was determined to be 31 times greater than the exposure occurring jg_utgrg_across the placenta. Although not studied directly, a similar scenario is presumably also true in the mink. The reproductive performance of the mink and ferret was found to be a sensitive indicator of HCB toxicity. HCB caused an increase in the gestational length of HCB-exposed female mink and a lower whelping percent- age than was seen in control females. A decrease in total litter size, without a change in the incidence of still births, was seen in the mink. No change in litter size was found in the ferrets, but an increased number of stillborn kits was observed. The mink and ferret kits born to dams consuming HCB diets exhibited depressed birth weights and poor surviv- ability to weaning when compared to control animals. The HCB-treated mink kits also displayed a decreased growth rate. When the kits born to control and HCB-treated female mink were cross-fostered, HCB-exposed offspring had 134 a higher mortality rate than control offspring. Ig_gtg[9_exposure alone to HCB resulted in the same level of kit mortality as jg_utggg and lacta- tional exposure combined. Mink kits receiving HCB only via their dam's milk showed a poorer survivability than control kits, but better than those kits exposed to HCB during gestation. The jg_gtgrg_stage of development is, therefore, more sensitive to the effects of HCB than the postnatal period. This phenomenon is seen in spite of the much lower quantity of the chemical crossing the placenta than is consumed by the growing offspring through the dam's milk. 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