This is to certify that the thesis entitled PATHOLOGIC EFFECTS OF POLYBROMINATED BIPHENYLS IN IODINE DEFICIENT RATS presented by Budi Tri Akoso has been accepted towards fulfillment of the requirements for MS. degree in BAIthDgy— //W2/— % 4f}. gag/2; Major professor Date ”fifC-c’?” A /‘/é /? 7 7 0—7639 oerour rmss; «. 25¢ per day per item A: --\\\|;‘ .. RETURNING LIBRARY MATERIALS: 1' . Place in book return to remve ’ “'5’” charge from circulation records "94‘, rX‘ig PATHOLOGIC EFFECTS OF POLYBROMINATED BIPHENYLS IN IODINE DEFICIENT RATS By Budi Tri Akoso A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1977 ABSTRACT PATHOLOGIC EFFECTS OF POLYBROMINATED BIPHENYLS IN IODINE DEFICIENT RATS BY Budi Tri Akoso Young male rats were fed 0, l, 10 or 100 ppm of polybrominated biphenyls (PBB) for 30 or 60 days in either iodine-deficient or iodine-adequate diets. Weight gains were decreased at 60 days by 100 ppm by addition of P38 in either diet. Hepatic weight was increased by feeding diets containing PBB with the greatest increase in rats fed adequate iodine. Iodine deficiency decreased serum triglycerides, whereas cholesterol values were apparently increased by PBB. Thyroid size was increased by iodine deficiency and enlarge- ment was enhanced by PBB. Hepatic lesions were most extensive by 60 days in iodine-deficient rats fed 100 ppm of P83. Diffuse vacuola- tion, enlarged hepatocytes, fibrosis and bile duct hyperplasia typified the severe hepatic lesions. The PBB accumulated in fat with lesser concentration in liver, kidneys and thyroid. Interrela- tionships between iodine deficiency and P38 toxicosis were demonstrated. Generally, iodine-deficient rats were more severely affected by PBB toxicosis. Dedicated with love to my parents: Asih Soepranti and Poedjohadioetojo ii ACKNOWLEDGEMENTS With gratitude and appreciation I wish to thank the multitude of persons who have made generous contributions toward the completion of my study at Michigan State University. Dr. S. D. Sleight for the many types of assistance, advice and encouragement which he made available to me during the course of my study. Dr. R. F. Langham, Dr. G. L. Waxler and Dr. H. D. Stowe for their willingness to serve as committee members and for their sug- gestions which helped to make this work more meaningful to me. Dr. R. W. Leader, Chairman, and Dr. C. K. Whitehair and Dr. V. L. Sanger of the Department of Pathology. Dr. I. G. N. Teken Temadja. Director of Animal Health of Indo- nesia, for his support and encouragement during my study. The Food and Agriculture Organization of the United Nations and the Indonesian government for their financial support. Dr. D. E. Rickert, Dr. Soesanto Mangkoewidjojo, Dr. P. R. Werner, Shirley K. Howard, M.T., and Betty L. Schoepke, M.S., for their assistance in my laboratory work. Last but most, my wife, and my daughter who was born in Indonesia while I was in the United States, for their patience and encouragement during my study. iii TABLE OF CONTENTS Page I ”ROBERT ION . O O O O O O O O O O O O O O O O O O O O O O O O O 1 U LITEMTUE ”VIEW. 0 O O O C O C O O C O O O O O O O O O O O O O The Chemistry of Polybrominated Biphenyls . . . . . . . . Polybrominated Biphenyl Toxicosis . . . . . . . . . . . . Clinical Signs . . . . . . . . . . . . . . . . . . Pathology. . . . . . . . . . . . . . . . . . . . . Polybrominated Biphenyl Accumulation and Excretion Hypothyroidism. . . . . . . . . . . . . . . . . . . . . . @mmbbw “TERIAIJS MD METHODS O O O O O O C O O O O C C 0 O O C O O O C O 1 3 Experimental Design and Feeding Practices . . . . . . . . 13 Laboratory Investigation Procedures . . . . . . . . . . . 15 Blood Samples. . . . . . . . . . . . . . . . . . . 15 Serum Analysis . . . . . . . . . . . . . . . . . . 16 Electrophoresis. . . . . . . . . . . . . . . . . . 16 Collection of Tissues. . . . . . . . . . . . . . . l6 Urinalysis . . . . . . . . . . . . . . . . . . . . l7 Polybrominated Biphenyl Analysis . . . . . . . . . l7 mSULTS O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 19 Thirty Day Dietary Feeding Study. . . . . . . . . . . . . 19 Clinical Signs . . . . . . . . . . . . . . . . . . 19 Laboratory Investigations. . . . . . . . . . . . . l9 Pathology. . . . . . . . . . . . . . . . . . . . . 25 Sixty Day Dietary Feeding Study . . . . . . . . . . . . . 31 Clinical Signs . . . . . . . . . . . . . . . . . . 31 Laboratory Investigations. . . . . . . . . . . . . 31 Pathology. . . . . . . . . . . . . . . . . . . . . 41 DISCUSSION 0 O O 0 O O O O O O O O O O O O O O O O O O O O O O O 47 General 0 O O O O O O D O O O O O O O O O O 0 O O O O O O 47 Hypotherj-dismo O O I I O C O O O O O O O I O O O O O O O 47 Toxicosis of Polybrominated Biphenyls . . . . . . . . . . 48 The Interaction of P88 Toxicosis and Iodine Deficiency. . 49 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . S3 EFEENCES O O O O O O O O O O O O O O O O O O O O O O O O O O O 55 VITA O O O O O O O O O O O O O O O O O O O O O O O O O O I O O O 60 Table LIST OF TABLES Experimental design of dietary treatment with PBS in rats fed iodine-deficient or iodine-adequate diets for 30 or 60 days . . . . . . . . . . . . . . . . . . Organ weights in rats fed iodine-deficient or iodine- adequate diets containing 0, l, 10 or 100 ppm of P83 for 30 days 0 O O O O O O I O O O I O I O O O O I I 0 Concentration of P33 in tissues of rats fed iodine- deficient or iodine-adequate diets containing 0, l, 10 or 100 ppm of P38 for 30 days. . . . . . . . . . . Organ weights in rats fed iodine-deficient or iodine- adequate diets containing 0, l, 10 or 100 ppm of P88 for 60 days . . . . . . . . . . . . . . . . . . . Percentages of albumin and globulin of serum protein in rats fed iodine-deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of P88 for 60 days . . Lipoprotein and lactate dehydrogenase values in rats fed iodine-deficient or iodine-adequate diets con- taining 0, 1, 10 or 100 ppm of P88 for 60 days. . . . Concentration of PBS in tissues of rats fed iodine- deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of P38 for 60 days. . . . . . . . . . . Page 14 20 25 35 39 40 41 LIST OF FIGURES Figure Page 1 Liver weight to body weight ratios in rats fed iodine- deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of PBB for 30 days. . . . . . . . . . . . . 21 2 Thyroid weight to body weight ratios in rats fed iodine-deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of PBB for 30 days. . . . . . . . . . 23 3 Kidney weight to body weight ratios in rats fed iodine-deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of PBB for 30 days. . . . . . . . . . 24 4 Photomicrograph of normal thyroid gland from a rat fed an iodine-adequate diet for 30 days . . . . . . . . . 27 S Photomicrograph of thyroid gland in a rat fed an iodine-adequate diet containing 100 ppm of PBB for 30 days 0 O O O O O C O O O O O O O O O 0 O O O O O O O O 27 6 Photomicrograph of thyroid gland from a rat fed an iodine-deficient diet containing 0 ppm of PBB for 30 days 0 O O O O O O O O O O O O O O O O O C O O O O O O 28 7 Photomicrograph of normal liver from a rat fed an iodine-adequate diet and 0 ppm of PBB for 30 days . . . . 28 8 Photomicrograph of liver from a rat fed an iodine- adequate diet containing 100 ppm of PBB for 30 days . . . 30 9 Body weights of rats fed an iodine-deficient diet containing 0, l, 10 or 100 ppm of PBB for 60 days . . . . 33 10 Body weights of rats fed an iodine-adequate diet containing 0, 1, 10 or 100 ppm of PBB for 60 days . . . . 33 ll Liver weight to body weight ratios in rats fed iodine- deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of PBB for 60 days. . . . . . . . . . . . . 34 12 Thyroid weight to body weight ratios in rats fed iodine-deficient or iodine-adequate diets containing 0, l, 10 or 100 ppm of PBB for 60 days. . . . . . . . . . 36 vi Figure* 13 14 15 16 17 18 19 Kidney weight to body weight ratios in rats fed iodine-deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of PBB for 60 days. . . . . . . . . Photomicrograph of thyroid gland from a rat fed an iodine-deficient diet containing 100 ppm of PBB . . . . Photomicrograph of liver in a rat fed an iodine- deficient diet containing 100 ppm of PBB for 60 days. . Photomicrograph of liver from a rat fed an iodine- deficient diet containing 100 ppm of PBB for 60 days. . Higher magnification of the photomicrograph shown in Figure 16 O O O O O O O O O I O C O I O O O O O O O O Photomicrograph of testes from a rat fed an iodine— deficient diet and 100 ppm of PBB which died at 15 days Photomicrograph of lungs from a rat fed an iodine- adequate diet containing 100 ppm of PBB for 60 days . . vii Page 37 43 43 45 45 46 46 INTRODUCTION In recent years mixtures of polybrominated biphenyls (PBB) were found to be effective fire retardants for plastics and synthetic fibers. However, PBB were accidentally introduced into livestock feed in the State of Michigan in 1973 and 1974, when PBB were erroneously used in place of magnesium oxide in commercial dairy cattle ration (Carter, 1976; Dunckel, 1975). Recently evidence of PBB contamination has also been reported in two other states, New Jersey and New York (Anonymous, 1977). The contamination by PBB was considered to be the most costly agricultural accident ever to occur in the United States (Isleib and Whitehead, 1975; Willett and Irving, 1976). There is no direct relationship between PBB toxicosis and iodine deficiency. However, Michigan is geographically located in an endemic goiter area (Olin, 1924; Eldridge, 1924), and hypothyroidism in animals occurs mostly in areas in which human goiter is prevalent (Brand et al., 1963; Pitt-Rivers and Tata, 1960; Rajkumar, 1970). Although iodine supplementation is available for animal diets, there is a wide variation in the amount of iodine that animals consume. Such environmental conditions are difficult to control in animals. It is possible that there is interaction between the bromine con- tained in the PBB and iodine. Whether this interaction would be especially important in animals deficient in iodine needs further investigation. 2 The first objective of this study was to identify possible interrelationships between PBB toxicosis and iodine deficiency in the rat. The second objective was to obtain further data on the pathologic effects in rats fed various doses of PBB. The third objective was to provide more information on the effects of iodine deficiency in the rat. While considerable information is already available on PBB toxi- cosis and on iodine deficiency in laboratory and farm animals, additional research on the possible interaction between the two environmental problems should provide valuable information for those interested in animal or public health. LITERATURE REVIEW The Chemistry of Polybrominated Biphenyls The source of the PBB used as flame retardants is comprised of Br Br a very complex mixture of polybrominated COH‘PO‘mdS- ". Br ' B! B! The approximate percentage of isomers is: tetrabromobiphenyl (2.0%), pentabromobiphenyl (10.6%), hexabromobiphenyl (13.8%) and other biphenyls (11.4%) (Kay, 1977). The major component has been determined to be 2,2',4,4',5,5' hexabromobiphenyl (Jacobs et al., 1976; Sundstrom and Hutzinger, 1976). Dent et a1. (1976) suggested that PBB share some biological and toxic properties of the polychlorinated biphenyls (PCB). The PBB and PCB have similar chemical and physical composition (Fries and Marrow, 1975). The main difference is the attachment of bromine rather than chlorine on the biphenyl compound (Hesse, 1975). Ringer and Polin (1977) concluded that PBB are slightly less toxic than PCB. Fries and Marrow (1975) emphasized the similarity of PBB, PCB and the chlorinated hydrocarbon 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DOE). They measured the concentrations of these chemicals in milk, milk fat and body fat. 4 Polybrominated Biphenyl Toxicosis Clinical Signs The cliniCal signs associated with PBB toxicosis in cattle were variable and inconsistent. Durst et al. (1977) suggested that the variety of clinical symptoms was due to the lack of information about the previous health of the herds, the dose of PBB and the duration of PBB exposure. Jackson and Halbert (1974) reported a toxic syndrome in a herd of 400 dairy cattle that had received rations containing high concen- trations of PBB. Clinical signs included anorexia, a drop in milk production, increased frequency of urination and lacrimation, and shrunken udders. The cows continued to lose weight 2 months after the contamination. Although lowered milk production has been considered as the best indicator of PBB exposure, results of a survey indicated that milk production in contaminated herds was not significantly different from uncontaminated herds (Mercer et al., 1976). 'Workers have not experimentally reproduced significant clinical signs in cattle which have consumed low levels of PBB (total dose 1 to 25 mg/kg body weight) (Gutenmann and Lisk, 1975; Fries and Marrow, 1975;1Ni11ett and Irving, 1976). Clinical signs of experimental PBB toxicosis were not observed in pregnant heifers (Durst et al., 1977; Moorhead et al., 1977) or in cows in the first lactation (Fries and Marrow, 1975) when treated with 250 mg of PBB or less daily in their feed for 60 days. However, at a level of 25 g of PBB per day in their feed cattle became 5 depressed, abortions occurred, and they became moribund within 33 to 66 days (Durst et al., 1977). Sleight and Sanger (1976) reported that there were no clinical signs observed in rats fed diets containing 1, 10, or 100 ppm of PBB for 30 or 60 days. Weight gain and feed efficiency decreased in rats fed 500 ppm of PBB. The body weight of mice fed diets con- taining 200 ppm of PBB for 2 weeks was not affected (Cagen et al., 1977). Guinea pigs seemed to be more susceptible to the effect of PBB than rats. Only 2 out of 6 guinea pigs survived a dietary treat- ment of 100 ppm of PBB for 30 days, and at 500 ppm of PBB in the diet, all of the 6 guinea pigs died within 15 days (Sleight and Sanger, 1976). Dietary exposure of rats to octabromobiphenyl (OBBP) has been reported. There were no signs of toxicosis at the dose level of 1,000 ppm in their feed for 4 weeks (Lee et al., 1975). Feed con- sumption and feed efficiency were not affected at a dose of up to 1,000 ppm of OBBP for 4 weeks (Lee et al., 1975) or up to 1 mg/kg/day OBBP in their feed for 180 days (Norris et al., 1974). Pathology Gross lesions. The gross lesions in dead cattle from a herd that had been fed PBB-contaminated feed included hematomas, abscesses in the peritoneal and thoracic cavities, adhesions of the rumen to the ribs, massive liver abscesses, enlargement of the liver, necrotic metritis, and suppurative bronchopneumonia (Jackson and Halbert, 1974). Whether these lesions were directly or indirectly related or unrelated to the PBB-contaminated feed is not known. 6 In pregnant heifers experimentally fed 25 g/day of PBB the gross lesions included subcutaneous emphysema and hemorrhage, atrophy of the thymus, enlarged kidneys, a thickened wall of the gallbladder, inspissated bile, edema of abomasal folds, mucoid enteritis, linear hemorrhages, and edema of the rectal mucosa (Moorhead et al., 1977). The most pronounced gross lesions resulting from PBB toxicosis in laboratory animals appear to occur in the liver. Enlargement of the liver has been observed in the rat (Sleight and Sanger, 1976; Moore et al., 1977), guinea pig (Sleight and Sanger, 1976), mouse (Corbett et al., 1975; Cagen et al., 1977) and Japanese quail (Babish et al., 1975) after dietary exposure to PBB. Hepatomegaly has also been observed in the rat after intraperitoneal injection of PBB (Troisi, 1975). The PBB are known to be potent inducers of hepatic microsomal drug metabolizing enzymes and increase microsomal protein (Troisi, 1975; Dent et al., 1976; Moore et al., 1977). Ringer and Polin (1977) observed enlargement of the liver and thyroid and reduced size of the spleen, bursa of Fabricius and comb in birds given PBB in the feed. Kimbrough et a1. (1972) treated rats with PCB (Aroclor 1260 or 1254). The livers were soft, yellowish-brown or dark olive and in some instances there were grayish-white, firm, glistening areas. Enlargement of the liver was more pronounced in the male than in the female rats. Nodular lesions were also observed on the surface of the liver in some rats that were treated with Aroclor 1254. Microscgpic lesions. The cattle in the PBB-contaminated herd described by Jackson and Halbert (1974) had hepatic lesions which included fatty metamorphosis and amyloidosis. They also reported 7 renal lesions in some cattle including pigment nephrosis and inter- stitial nephritis. In experimental cows, Moorhead et a1. (1977) stated that changes in the liver were not marked. The changes con- sisted of glycogen depletion of hepatocytes, sinusoidal dilatation and scattered areas of early centrilobular fatty metamorphosis. According to these authors, the most characteristic microscopic lesions were in the kidney, gallbladder and eyelid. There was an extreme dilatation of collecting ducts and convoluted tubules in the kidney, with epithelial degenerative changes consisting of cloudy swelling, hydropic degeneration and separation of epithelial cells from the basement membrane. Hyperplasia and cystic dilatation of the mucous glands in the lamina propria of the gallbladder were seen. In the eyelids there was accumulation of keratin in hair follicles. There was metaplasia with keratin cysts in the tarsal glands. Gutenmann and Lisk (1975) reported a marked glandular hyper- plasia of the major intrahepatic bile duct of the liver in a cow and in the gallbladder of a sheep which were fed diets containing PBB. Willett and Irving (1976) indicated that the histologic changes in the kidneys and hyperplasia of the bile duct were similar to the lesions reported in the hyperkeratosis syndrome caused by consumption of chlorinated naphthalenes. Sleight and Sanger (1976) reported that PBB toxicosis in rats and guinea pigs caused an extensive swelling and vacuolation of hepatocytes which were diffusely distributed throughout the lobules. Areas of inflammation or necrosis were seen only rarely in the rat liver. Corbett et a1. (1975) reported that the microscopic lesions in mice treated with PBB included swelling of hepatocytes with focal areas of coagulative necrosis and areas of dark-staining hepatocytes. 8 Histologic lesions in rats fed diets containing OBBP included hepatocellular hypertrophy, laminated cytoplasmic inclusions, foamy cytoplasm and margination of basophilic cytoplasm (Lee et al., 1975). The number and size of the inclusions were closely related to the dose, duration of treatment and degree of hepatocellular changes. Kimbrough et al. (1972) summarized the hepatic lesions in rats given PCB in their diet. The lesions consisted of hypertrophy of the hepatocytes, granular cytoplasmic inclusions, brown pigment in Kupffer cells and lipid accumulation. They also described an adeno— fibrosis which consisted of fibroblasts and collagen that surrounded rosettes of epithelial cells. Polybrominated Biphenyl Accumulation and Excretion The PBB are known to be insoluble in water, to be highly soluble in fat (Kay, 1977), and to accumulate in fat tissues in animals (Fries and Marrow, 1975; Corbett et al., 1975). The accumulation has been studied in tissues of cattle and sheep (Gutenmann and Lisk, 1975), cattle (Fries and Marrow, 1975; Willett and Irving, 1976), fish (Zitko, 1977), birds (Ringer and Polin, 1977), and hen eggs (Fries et al., 1976; Ringer and Polin, 1977). Willett and Irving (1976) reported that PBB are excreted in feces, urine and milk, with feces as the major route. According to Fries and Marrow (1975), the excretion of PBB in milk is similar to PCB and to the chlorinated hydrocarbon pesticide DDE. They postulated that the steady state of concentration of PBB is reached more rapidly ithan PCB or DDE, and at 15 days after termination of the treatment the decline of PBB concentration was greater than PCB or DDE. 9 Bromine concentrations have been studied in rat tissues treated with OBBP. There were significant amounts in liver (Norris et al., 1974; Lee et al., 1975), adipose tissue (Norris et al., 1974) and muscle (Lee et al., 1975). Hashimoto et a1. (1976) reported the highest PCB accumulation in adipose tissue, intermediate levels of accumulation in the skin, adrenal gland, aorta and sciatic nerve, and the lowest levels in the blood. The concentration of PCB in serum decreased most rapidly with slower rates of decrease in the heart, nervous tissue, kidney, liver and subcutaneous adipose tissue. According to Zitko (1977), the accumulation of PBB and PCB in the tissue is similar but PBB pene- trates biological membranes more slowly. Hypothyroidism Hypothyroidism has been associated with the term "goiter", which is defined as an enlargement of thyroid or increase in weight of the thyroid gland. The thyroid enlargement resulting from iodine deficiency is generally called "endemic goiter" (Evered, 1976), although the condition may occasionally be found in non-endemic areas. There are two main iodine containing hormones, thyroxin (T4), and triiodothyronine (T3), which are produced by the thyroid glands (Wilson, 1975; Evered, 1976). The hormones control the metabolic rate by regulating cellular oxidation (Wilson, 1975). The T3, T4, and other iodine containing amino acids are bound to thyroid binding globulin (TBG) (Wilson, 1975) and stored in the colloid follicles of the thyroid gland as thyroglobulin (Evered, 1976). According to Underwood (1977), iodine concentration in the thyroid varies with age, species, activity of the gland, and iodine intake. 10 Iodine intake below the mdnimwm daily requirement leads to a reduc- tion of plasma inorganic iodine concentration and a corresponding decrease in the production of thyroid hormone. The decrease of thyroxine causes a compensatory increase in the release of thyroid stimulating hormone (TSH) from the anterior pituitary (Evered, 1976). Increased TSH secretion is the most important factor in producing thyroid response to iodine deficiency (Studer and Geer, 1965) and causes hypertrophy and hyperplasia of the follicular epithelium of the thyroid gland (Jubb and Kennedy, 1970). Ramalingaswami (1964) emphasized the three fundamental patho- logic features of endemic goiter, which included epithelial hyper- plasia, involution and nodule formation. Jubb and Kennedy (1970) described the pathogenesis of changes in the thyroid gland resulting from an increased stimulation of TSH. Initially there is hypertrophy which is followed by hyperplasia of the thyroid epithelium, which 1 becomes taller. The vascularity of the gland is increased, the follicular lumina become smaller and may disappear, and the colloid is reduced or absent. An extensive study of hypothyroidism caused by an iodine- deficient diet in rate has been conducted by Levine et a1. (1933). These workers indicated that a diet containing as little as 15 ug iodine/kg of feed caused goiter in 35 days. Remington and Remington (1937) reported that iodine deficiency can be prevented by the presence of 265 ug iodine/kg of feed. The three steps in synthesis of thyroxin include iodide accumu- lation, oxidation to iodine, and the organification of iodine by a combination with tyrosil radicals (Jubb and Kennedy, 1970). A sub- stance that interferes with thyroxin synthesis and leads to thyroid 11 enlargement is called a "goitrogenic” substance (Underwood, 1971). There are two major types of goitrogenic compounds (Calderbank, 1963). The thiocyanate type inhibits thyroidal uptake of iodine and may be overcome by simultaneous administration of iodine. The thiouracil type interferes with organic binding into thyroglobulin and the effect can be reversed completely only by administration of thyroid hormone. Astwood et a1. (1943) reported that certain sulfonamides or thiourea induced hypothyroidism in rats. Administration of these substances in rats caused enlargement, hyperemia and hyperplasia of the thyroid gland, decreased food intake, decreased growth and development, and lowered basal oxygen consumption. Dietary exposure of bobwhite quail to PCB has been known to cause an enlargement of the thyroid gland (Hurst et al., 1974). The histologic lesions in the thyroid glands resulting from dietary exposure of gulls to PCB were similar to a simple goiter caused by iodine deficiency (Jefferies and Parslow, 1972). Thyroid hyperplasia has also been reported in rats given dietary exposure to OBBP (Norris et al., 1974) and decabromodiphenyl oxide (DBDPO) (Norris et al., 1974; Kociba et al., 1975). Fregly et a1. (1968) reported that administration of 2,2—bis- (2-chlorophenyl,4-chlorophenyl)-l-l-dichloroethane (DDD) caused a compensated hypothyroidism in rats. Richert and Prahlad (1972) stated that DDE induced hypothyroidism in Japanese quail with an increase in 125I uptake. Enlargement of thyroid glands and reduction in colloid content of the follicles has been observed in bobwhite quail treated with l,l,l,-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (Hurst et al., 1974; Jeffries and French, 1969). 12 In a recent study, Ringer and Polin (1977) reported that dietary exposure to PBB in chickens caused thyroid hyperplasia and reduction of colloid associated with hypothyroidism. MATERIALS AND METHODS Experimental Design and Feeding Practices Ninety-six young male Sprague-Dawley ratsa initially weighing 110 to 135 g were used. All rats were in good general condition at the start of the experiment. The rats were divided randomly into 2 groups of 48 and given either an iodine-deficient diet (IDD) or an iodine-adequate diet (IAD) with the arrangement as shown in Table 1. Each group was divided into 4 subgroups of 12 rats each. Rats were housed 3 to a cage in plastic cages with a metal wire top. The cages were cleaned and the bedding was changed once a week. Room temperature was maintained between 2f’and 27°C, and the humidity was between 45 and 55%. Lights were turned off automatically to allow 10 to 12 hours of darkness, as recommended by Lane-Patter (1963). The rats were fed a regular commercial dietb and tap water was available ad libitum for 2 days during acclimation. Two days prior to the experiment the food was changed to an iodine-adequate dietC and distilled water. aLife Science Division of Mogul Corporation, Madison, Wisconsin. bRalston Purina Co., Checkerboard Square, St. Louis, Missouri. CBasal iodinehdeficient diet with supplement of 0.2 ug iodine/ kg of feed. 13 14 Table 1. Experimental design of dietary treatment with PBB in rats fed iodine-deficient or iodine-adequate diets for 30 or 60 days PBB concentration Number of rats Modification in feed Killed at Killed at ' of diets (ppm) 30 days 60 days Iodine 0 6 6 deficient l 6 6 dieta 10 6 6 100 6 6 Iodine 0 6 6 adequate l 6 6 dietb 10 6 6 100 6 6 aRemington diet containing low iodine concentration (15 ug/kg). bRemington diet with iodine supplementation to meet minimum requirements (200 ug/kg). The basal diet for the IDD was a commercial Remington dieta as recommended by Levine et a1. (1933). The feed for the IAD was mixed With ethylenediaminedihydriodideb to give a concentration 0.2 pg iodine per g of feed. Each subgroup of rats fed IDD or IAD were given either 0, 1, 10 or 100 ppm of PBB in their ground feed. The feed was available in porcelain containers and covered with a stainless steel perforated disc to prevent the rats from scattering their feed. Feed consump- tion was recorded daily. Feed for the different diets was stored aUnited Biochemical Corporation, Cleveland, Ohio. bHi-Amine, Pitman-Moore, Inc., Washington Crossing, New York. 15 in separate labeled bags, kept dry, and protected from direct sunlight. Distilled water was available ad libitum, as recommended by Porter (1967) and Remington (1936), and was given in inverted bottles which had rubber stoppers and stainless steel sipper tubes. The drinking water was changed twice a week. The body weights were recorded twice a week, and the final body weights were recorded prior to necropsy. Laboratory Investigation Procedures At 30 days of the experiment, 6 rats from each of the 8 subgroups were killed after feed was withheld overnight. The remaining 48 rats were killed at 60 days. The final body weights were recorded and the rats were killed humanely with ether and chloroform. Necropsy was performed as soon as the animal died. Blood Samples The blood samples were obtained from the heart while the rat was anesthetized with ether. A blood sample for hematologic examination was aspirated and collected into a tube with ethylenediaminetetraacetic acid (EDTA) as the anticoagulant. Hemoglobin (Hb) was determined by the standard cyanmethemoglobina method, and packed cell volume was measured by the microhematocrit method (Benjamin, 1976). Red blood cells (RBC) and white blood cells (WBC) were counted by using an electronic counter.b Blood smears were stained with Wright's stainc and examined for the differential leukocyte count. aHycel Inc., Houston, Texas. bCoulter Electronic Inc., Hialeah, Florida. c . . Hemateck Automatic Stainer. 16 Serum Analysis A portion of the blood sample was placed in a test tube without anticoagulant. The serum was aspirated and saved for analysis. The amount of blood urea nitrogen (BUN), alkaline phosphatase and serum glutamic oxaloacetic transaminase (SGOT) was determined by using Eni-Gemsaec reagent.a Electrophoresis The electrophoresis of serum protein, lipoprotein and lactate dehydrogenase (LDH) isozymes was analyzed by a private laboratory.b The proteins of the serum were separated, stained and quantitated on cellulose acetate strips. A densitometer was used to quantitate electrophoretic separations. Collection of Tissues The trachea was exposed, and the trachea and lungs were infused with 1 to 2 ml fixative by using a syringe and needle. The lungs were removed after the trachea had been ligated. All tissues were examined grossly. A top loading balancec was used to weigh the liver and both kidneys, and the thyroid gland was weighed on an analytical balanced immediately after being removed. Tissues for histological examination were preserved in 10% neutral buffered formalin. Tissues collected included trachea, lung, aSmith Kline Instruments, Inc., Sunnyvale, California. bSpecial Chemical Laboratory, East Lansing, Michigan. cMettler Series P, model 163 (0.001 gm readability), Mettler Instrument Corp., Hightstown, New York. dModel H-lS (readability: 0.0001 gm), Mettler Instrument Corp., Hightstown, New York. 17 heart, spleen, liver, kidney, stomach, intestines, skeletal muscle, thyroid, pituitary gland, adrenal gland, salivary gland, eye, skin, bone, urinary bladder, thymus, pancreas, testes and brain. Bones were decalcifieda prior to the histological processing. The tissues were sectioned at 6 u and stained with hematoxylin and eosin or other selected special stains, including oil red 0, von Kossa, periodic acid-Schiff (PAS) and Ziehl-Neelsen as described by Luna (1968). Tissues for PBB analysis were wrapped with aluminum foil, labeled and saved at -70°C for later analysis. Urinalysis A urine sample was collected at necropsy by puncturing the urinary bladder with a needle and aspirating urine into a 1 ml syringe. The specific gravity was determined by a refractometer.b The concen- tration of urobilinogen, blood, bilirubin, ketones, glucose and protein was estimatedC and pH was determined. Polybrominated Biphepyl Analysis Analyses for PBB were made from fat, liver, thyroid gland, kidneys and brain. Fat tissue and distilled water were placed in xylenized tubes and homogenized.d The sample was then mixed with petroleum ether (PE) and partitioned with acetonitril (CH3CN). The PBB and CH3CN aDecalc, Du Pa Ge Kinetic Lab. Inc., Illinois. bGolden Refractometer, American Optical Co., Buffalo, New York. CMultistix, Ames Co., Division Miles Lab. Inc., Elkhart, Indiana. dPolyton Homogenizer, PT.20.00, Brinkman Instrument Inc. 18 were reextracted with PE and sodium chloride crystals. The PBB and PE were removed and evaporated in a waterbath at 409Gb Nonfat tissues were homogenized and extracted by adding PE. The PE was evaporated in a waterbath at 40°C. Activated Florisila was packed into a 5 3/4 inch pasteur pipette which had a small portion of glass wool at the bottom. A small quan- tity of anhydrous NaZSO4 was added on the tOp of the Florisil. Prepared columns were prewashed with PE. The extracted samples were transferred to the pipette and the washing was discharged. The PBB was eluted with 3 m1 of 6% anhydrous ether in PE and evaporated to dryness. The dried samples were dissolved with 100 ul PE and 1 ul was injected into the gas chromatograph/mass spectrometer.b The column packing was 1% ov-l with a temperature of 230°C and helium gas as the carrier. Calculation of PBB concentration in the tissue was based on the peak of hexabromine or the major peak of the mixture of biphenyls in comparison to the standards. Known concentrations of PBB standard (0.001-1.0 Ug/ml) were prepared. aFlorisil PR, 2-0280,60/100 mesh, Supelco Inc., Bellefonte, Pennsylvania. bGC model 3200, Finnigan Inc., Sunnyvale, California. RESULTS Thirtny y Dietarnyeeding Study Clinical Signs There were no clinical signs of toxicosis observed in rats fed IDD or IAD containing 0, l, 10, or 100 ppm of PBB for 30 days. Weight gain and feed efficiency were not significantly different. One rat fed IDD containing 100 ppm of PBB died at 15 days without any noticeable clinical signs. Laboratory Investigations Hematolggic examinations. Erythrocyte, hemoglobin and leukocyte values for all rats were in the normal range as described by Schermer (1967). Values for packed cell volume and differential leukocyte counts were not significantly affected by iodine deficiency or PBB. Urinalysis. Data for urinalysis were not consistent, mainly due to the lack of urine in the urinary bladder. Generally, there was an increase in protein in rats that were given PBB. Glucose, urobilinogen and blood were not detected in the urine. Serum analysis. There were no significant differences in the BUN and SGOT values in rats fed IDD or IAD containing 0, l, 10 or 100 ppm of PBB. However, alkaline phosphatase was significantly decreased 19 20 in rats fed IDD containing 10 (p<0.05) or 100 (p<0.025) ppm of PBB and in rats fed IAD containing 100 ppm of PBB (p<0.025). Organ weight. The data for liver weights, thyroid weights, kidney weights and final body weights for rats killed at 30 days are given in Table 2. Table 2. Organ weights in rats fed iodine-deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of PBB for 30 days Modification PBB Final body Thyroid Liver a Kidney a of diet (ppm) weight (9)3 weight (mg) weight (9) weight (g) Iodine 0 222.5:18.60 57.00124.72 7.33il.1l 1.95:0.20 deficient 1 223.8:12.42 59.201 5.02 6.86:0.46 1.76:0.18 10 223.5:20.05 80.38il6.20 8.73:0.77 l.8210.19 100 l9l.6rl5.98 75.37ill.99 13.00il.16 1.45:0.23 Iodine 0 218.7: 9.18 27.13: 9.55 6.93:0.71 1.94:0.28 adequate 1 220.3120.85 4o.2s:12.4o 7.71:1.07 2.02:0.22 10 211.8:12.06 21.43: 4.50 8.56:0.69 1.9210.l9 100 l92.0i20.50 19.451 2.06 l4.9911.50 1.71:0.22 a Data are expressed as mean bData are expressed as mean Liver weight: Dietary exposure to 1 SD; n = 6. i SD; n = 4. PBB of rats fed an IDD caused a significant increase in liver weight to body weight ratios at 10 (p<0.025) or 100 (p<0.001) ppm (Figure 1). Similar effects were also seen in rats fed an IAD and 10 (p<0.005) or 100 (p<0.001) ppm of PBB. to body weight ratios. However, PBB at 1 ppm did not significantly affect liver weight The liver weights of rats fed IDD or IAD without PBB treatment were not significantly different from each other. However, the liver weight to body weight ratios in rats fed IDD and 21 Figure 1. Liver weight to body weight ratios in rats fed iodine-deficient or iodine-adequate diets containing 0, l, 10 or 100 ppm of PBB for 30 days. \‘ “ \‘ 3 I I I I ’I ’I ’I II II I I I I I \\\\\\ \\\‘ \\\\ sssu ‘\\\\‘ n. II \ \ \\\ \\\ \\“ \‘ s \\ §\\\ \ \\\\ re“ \\ \‘ \\\ \\ \\‘\‘\\ \\\ \\‘\\‘ \\ \\\ \\ s‘ a \ \ ‘ \ \\ \ «\“s‘ \ \\ u.‘ s \ . .\\ ‘\‘ e \\\\ \ \\¢~\\\ \\\\\ ‘ s \ “\\ ‘\ in) \ ’5‘ C) .a Cl Iodine-deficient diet Iodine-adequate diet Significant difference when compared with the liver of rats fed: an iodine-deficient diet and 0 ppm of PBB an iodine-adequate diet and 0 ppm of PBB an iodine-adequate diet at the same level of PBB ¥M.- j,’ I” ’ I ’I. 5: ’0 9‘! I ' " I " '0 III ’, II II I ’I I :s' 5: :: I I P 'III I, '1' '6 [II t: :' 5’ I O 5' O ' ’0' " ” " II ’, '0 II " ’ '0 " II ' N I ’3 I: 5' I: ’, I. I,’ II [I ’I I: ” 2 - :9 3', " :1 0 II II 0" '0 I,. ’0' $0 I; II, 'o ,3 ’I’ I” I’ I O ’0' I I} ’9 II I' I ‘5 s' z: ': II I: I. 'I p I’ I, 'I I, I0, II, II I, 5' ’l ’0 II I, I '0 " ’ II '0 II I, H ’1 ‘3 :- 3: b a; a ’=- e .;. O 5‘ t t: 'k 9 a: ‘3' 0 II [go I: I D Iodine-deficient diet Iodine- adequate diet Significant difference when compared with the liver of rats fed: 0: an iodine-deficient diet and 0 ppm of PBB .: an iodine-adequate diet and0 ppm of PBB ls an iodine-adequate diet at the same level of P88 a = p<0.05 b = p<0.025 d - p<0.005 e = p<0.001 (data are expressed as mean :_SD; n = 6) 3S Table 4. Organ weights in rats fed iodine-deficient or iodine-adequate diets containing 0, l, 10 or 100 ppm of PBB for 60 days Modification PBB Final body Liver a Thyroid Kidney of diet (ppm) weight (g)a weight (9) weight (mg) weight (g) Iodine 0 281.3:14.60 9.49:1.00 105.04:17.78 1.88:0.22 deficient l 281.3:15.28 9.22:1.05 115.75:21.35 1.94:0.15 10 269.5:19.64 11.28:l.63 107.38:11.88 1.82:0.25 100 202.2:21.83 12.85:1.43 S7.38:14.36 1.44:0.16 Iodine 0 288.7:22.30 10.86:l.l3 12.10: 0.98 2.37:0.12 adequate 1 279.5:20.97 11.94:l.78 11.68: 0.77 2.24:0.43 10 271.2:24.27 13.14:1.73 16.68: 3.12 2.19:0.25 100 231.3:16.21 18.82:0.86 19.20: 4.03 1.74:0.39 aData are expressed as mean : SD; n = 6. b Data are expressed as mean : SD; n IAD at 0, 1, 10 or 100 ppm of PBB. There were no significant effects of PBB on the thyroid weight to body weight ratios of rats fed IDD for 60 days, but in rats fed IAD the thyroid weight to body weight ratios were significantly (p<0.025) increased at 10 or 100 ppm of PBB (Figure 12). Kidney: The kidney weight to body weightratiOS.Ln rats fed IDD were smaller than in rats fed IAD at 0 (p<0.01) or 10 (p<0.025) ppm of PBB. Serum analysis. In general, PBB did not significantly affect the BUN and SGOT values in rats fed IDD or IAD for 60 days. However, at 0 ppm of PBB the BUN values in rats fed IDD were lower (p<0.025) than those in rats fed IAD. Alkaline phosphatase values in rats fed IAD were decreased at l (p<0.05), 10 (p<0.025) or 100 (p<0.01) ppm of 36 Figure 12. Thyroid weight to body weight ratios in rats fed iodine-deficient or iodine-adequate diets containing 0, l, 10 or 100 ppm of PBB for 60 days. 50 - 40 — 30 -— 20 - b 10 - b I I d . SI 2]— . O 1 100 ppm PBB [I] Iodine-deficient diet- Iodine—adequate diet Significant difference when compared with the thyroid of rats fed: 0: an iodine-adequate diet and 0 ppm of PBB 44 an iodine-adequate diet at the same level of PBB b = p<0.025 d = p<0.005 e = p<0.001 (data are expressed as mean i 50; n = 4) 37 Figure 13. Kidney weight to body weight ratios in rats fed iodine-deficient or iodine-adequate diets containing 0, l, 10 or 100 ppm of PBB for 60 days. 1.2+ “\\. . e t\\ \\‘\ e“ C “. ‘\ . \ Q \\“ ‘\\\\\‘ \‘S .‘\‘\‘\“\\ ‘g\\ Q \ \ A. I \\‘ \\ \\‘ ' ' ‘\ \.\‘ \\ \\ \ .\‘ ‘3. \‘ \ Q ‘\ \‘ “ \“ Q \\ \ 6 ‘ o. ’0‘ Q‘ I ' I I C ’0 ' I ‘ \\ \\ \‘: “\ ““ ‘ \ Q. 9‘ \s\ \ I .\ .! \ \\ ‘ \ ‘\‘ .Q“ “ .\‘. Q \ dV‘CT o ‘ D Iodine-deficient diet Iodine-adequate diet 4¥Significant difference when compared with the kidneys of rats fed an iodine-adequate diet at the same level of PBB. b = p<0.025 c = p<0.01 (data are expressed as mean :_SD; n - 6) 38 PBB, whereas in rats fed IDD the values were also decreased at 10 ppm of PBB (p<0.05). Electrgphoresis Serum protein electrophoresis: The percentage compositions of serum protein values of rats fed IAD or IDD for 60 days are given in Table 5. In rats fed IAD the most significant changes were seen only at 100 ppm of PBB, in which PBB decreased (p<0.05) albumin and increased (p<0.025) total globulin. In rats fed IDD at 0 ppm of PBB, alpha-2 globulin was decreased (p<0.05) and beta globulin was increased (p<0.025). The addition of PBB significantly (p<0.05) increased total globulin at 10 ppm but decreased beta globulin at 10 or 100 ppm. The addition of PBB in rats fed IDD decreased albumin at 10 (p<0.005) or 100 (p<0.025) ppm but increased gamma globulin (p<0.05) and total globulin (p<0.025) at 100 ppm of PBB, whereas total globulin was also increased at 10 ppm of PBB (p<0.05). The albumin/globulin ratios were decreased in rats fed IDD or IAD and 1, 10 or 100 ppm of PBB. However, there were no significant differences in the ratio between the rats fed IDD and 1A0 at the same level of PBB. The most pronounced changes in the serum protein electrophoresis pattern occurred in rats fed IAD containing 10 or 100 ppm of PBB, with more prominent beta globulin fraction, whereas in rats fed IDD the changes were seen only at 100 ppm. Lipgprotein electrophoresis: The values of triglycerides and cholesterol are given in Table 6. 39 mum» “Om mmsHm> sufl3 Umummeoo :023 mocmumMMHp unmowmwcmfim .m C “om H .mmm mo 8mm 0 can umwc mumsvaMImcfloofl cm pom some mm ommmmnmxm mum sumo n .mma no age o one page unmaogmmeumcfieofl cm emu mum» MOM mmsHm> spas pmummeoo cmsz mocwumumwp ucmowmacmww .m s «cm H some mm pmmmmnmxm mum mumam avo.o nmm.HHHo.Ho ~m.mubm.mm -.owmm.v mo.HHmm.m mm.oumm.HN noa.awmm.mm ooa nan.o 0m.vwam.mm mm.mwmv.¢m oa.awmo.m mo.aumo.oa hm.awoo.ma mm.vumv.H¢ oa nvm.o om.mem.Vm wo.owmv.o~ om.ommm.v mm.onwm.m hm.0Hoo.om oN.HH~o.mv H mumswmcm mm.o ov.owmm.am mm.awvm.ma mo.oumm.v ov.ouom.m no.HHNA.mH ~v.oumb.mv o mchOH n.mhm.o n.mHm.HHHm.mm a www.mfimm.mm Mmo.oumm.m vo.HHhm.m mv.mwov.ma n.mHm.HHmH.ov ooa n.m~m.o mmo.HHHm.hm mm.Han.mm www.0H0m.m m~.HHmo.b n mh.awmm.mm n.mhm.owmm.~¢ 0H omm.o mm.HHmm.vm mh.oww>.mm H>.owve.v nmo.aumm.m vm.~uva.om mo.awmm.mv a ucowowmmp om.o om.owvm.mm mN.HHm~.HN n-.oumm.m nm~.owmm.m mo.awov.ha om.owov.hv o wcfiooH oflpmm Hmuoe mesmo mumm mnmnmam Hnmnmaa A»: “ammo mumfio cflasooHu va cHH3n0H0 caesnad mmm mo \CHEDQH< GOwumu Ifimwpoz mxmp om you mmm mo 8mm OOH HO OH .H .o mcflcflmucoo macaw mumsvoom :wcfipofi no ucwflOfimmonmcHoow pom mumu cw samuoum saumm mo cHHsnoam can caesnam mo mommucmoumm .m magma 40 Table 6. Lipoprotein and lactate dehydrogenase values in rats fed iodine-deficient or iodine-adequate diets containing 0, 1, 10 or 100 ppm of PBB for 60 days Lactate Lipoprotein dehydrogenase Modification PBB Triglyceride Cholesterol Total LDH of diets (ppm) (mg/100 ml) (mg/100 ml) (IU/l) Iodine 0 25.0: 8.66: 81.0:10.41 301.0:28.87 deficient 1 21.7: 2.89b 83.0:14.43 253.3:17.50 10 13.3: 2.89b 78.3:16.07a b 230.0:47.70 100 11.7: 7.64 168.3:25.66 ' 245.0: 5.00 Iodine 0 156.7:51.32 80.0: 8.66 245.0:20.00 adequate 1 125.0:35.00 81.7: 5.77 240.0:31.75 10 123.0:43.11 70.0:22.91b 270.0:27.84 100 105.0:75.66 115.0: 8.66 356.7:70.06 aData are expressed as mean 1 SD; n = 3. Significant difference when compared with values for rats fed an iodine-deficient diet and 0 ppm of PBB. bData are expressed as mean + SD; n = 3. Significant difference when compared with values for rats fed an iodine-adequate diet and 0 ppm of PBB. In rats fed IDD and 0, 1, 10 or 100 ppm of PBB, the triglycerides were significantly (p<0.05) decreased when compared with the rats fed IAD and 0 ppm of PBB. Cholesterol values were elevated (p<0.05) in rats fed IDD or IAD and 100 ppm of P88. There was an especially prominent alpha-l lipoprotein fraction in rats fed IAD and 100 ppm of PBB. The beta and pre-beta peaks were depressed. In rats fed IDD, these alterations were seen only at 100 ppm of PBB. Lactate dehydrogenase isozymes electrophoresis: There were no significant differences in the total values of LDH isozymes in rats fed IDD or IAD and 0, 1, 10 or 100 ppm of PBB for 60 days (Table 6). 41 Polybrominated biphenyl accumulation in tissues. The PBB concen- trations in tissues of rats fed IDD or IAD containing PBB at 60 days were dose related and were not much different than at 30 days (Table 7). The fat had the highest concentration, and PBB were not detected in the brain. The PBB concentrations in the liver and kidneys of rats fed IDD were higher than the rats fed IAD, whereas the concentration in the thyroid tended to be lower. Table 7. Concentration of P88 in tissues of rats fed iodine—deficient or iodine-adequate diets containing 0, l, 10 or 100 ppm of PBB for 60 days Modification PBB in tissues (ppm)a of diets (ppm) Liver Kidneys Thyroid Fat Iodine 0 0.000 0.000 0.000 0.002 deficient I 1 0.002 0.008 0.000 0.019 10 0.142 0.050 0.014 0.123 100 0.857 0.404 0.127 3.696 Iodine 0 0.000 0.000 0.000 b adequate 1 0.001 0.007 0.000 b 10 0.017 0.017 0.053 b 100 0.468 0.156 0.260 b aNo PBB were detected in the brain tissues. bTechnical problems caused inaccuracy in analyses. Pathology Gross lesions. thyroid gland, liver and lungs. The most severe lesions were observed in the The gross lesions in the thyroid and liver were similar to those seen in rats given PBB for 30 days, but the lesions were more severe. The liver was more enlarged and mottled, 42 and the yellowish discoloration was more prominent. The thyroids were larger and more hyperemic when compared with those seen at 30 days. Histologic lesions Thyroid gland: The rats fed IAD containing 0 or 1 ppm of PBB had a normal histologic appearance of the thyroid gland. However, the thyroid glands of rats which were given 10 or 100 ppm of P88 had a marked hyperplasia of the follicles, and the colloid was pale or absent. The lesions in rats fed IAD containing 100 ppm of PBB were more severe. The individual follicles were.increased in number and the colloid was pale or scanty. Thyroid glands of rats fed IDD and 0, l or 10 ppm of PBB had lesions similar to those seen at 30 days. The most marked lesions were seen in thyroid glands of rats fed IDD containing 100 ppm of PBB. There was a marked increase of interfollicular connective tissue with some evidence of mononuclear inflammatory reaction (Figure 14). In some instances there was thickening of the capsule of the gland. Lizgg: The hepatic lesions in rats fed IAD and P88 were more severe than those seen at 30 days. The vacuoles in the hepato- cytes were generally larger, and the individual hepatocytes were more swollen. The livers of rats fed IAD containing 1 ppm of PBB had relatively small vacuoles distributed throughout the lobules. Feeding IDD containing 10 or 100 ppm of P88 caused similar but more severe lesions than those seen in the rats fed IAD and the same concentration of P88. Vacuoles in the hepatocytes were larger and more numerous. In rats fed IDD containing 100 ppm of P88, the hepatic 43 Figure 14. Photomicrograph of thyroid gland from a rat fed an iodine-deficient diet containing 100 ppm of PBB. Notice proliferation of the follicles and con- nective tissues are infiltrated by some lymphocytes. H & E stain, 120x. ,v .-__.I ‘ ., 4,. l_‘ \l\( (\3 E.,. .afi .. -» .11.E“'~‘:{ ' M " Afxg-‘sz 1' L.‘ , -' . - ' ‘ I‘ , E, ~ . ‘ 3.1," . . - ‘ ‘ A ’. {£33 a...“ - , , .f .' 1 V . U . . . 4‘ , . ,- . .-' he a: -v ,-r‘ v *~ , - '.~ - u ‘ ‘ , ”.;~'r’u “f“; ,z . ' r‘- ' I \ Figure 15. Photomicrograph of liver in a rat fed an iodine-deficient diet containing 100 ppm of PBB for 60 days. The picture illustrates diffuse vacuolation and interlobular fibrosis. Notice a few pyknotic nuclei and swollen hepatocytes. H & E stain, 120x. 44 lesions contained areas of fibrosis in the portal triads and in the interlobular areas (Figure 15). The fibrotic areas in the portal triads contained hyperplastic bile ducts (Figures 16 and 17), some of which had formed dilated cystic ducts that were filled with mucus-like or cellular debris. The ductal epithelium appeared stratified in some areas of the lobules. Generally hepatocytes were extremely hypertrophic, but some nuclei had undergone pyknosis. Other pathologic findings: There were several incidental findings in the rats that appeared unrelated to the deficient diets or to PBB. One rat that died after receiving the IDD and 100 ppm of PBB for 15 days was examined. Grossly the lungs were consolidated and the liver was enlarged and yellowish. The thyroid was normal in size and pale. Histologically the lungs had an interstitial pneumonia with evidence of mononuclear infiltration and some macrophages. The thyroid follicles were collapsed and there was no visible lumen or colloid. The testes were hemorrhagic with some lymphocytic infiltra- tion and areas of necrosis in the seminiferous tubules (Figure 18). All rats fed IAD or IDD had a slight to moderate pneumonia. In general, there was thickening of interlobular alveoli with some mononuclear infiltrations. In some instances there were aggregations of macrophages in the lumens of alveoli (Figure 19). One rat in one of the subgroups of rats fed IDD with 10 ppm of PBB for 60 days had an abnormally small left testicle. Histologic findings included areas of necrosis with some calcium deposits in the tubules. No inflammatory reaction was observed. Figure 16. Photomicrograph of liver from a rat fed an iodine-deficient diet containing 100 ppm of PBB for 60 days. Notice bile duct proliferation and fibrosis in the portal triad and infiltration of lymphocytes. H & E stain, 120X. Figure 17. Higher magnification of the photomicro- graph shown in Figure 16. Notice area of fibrosis surrounding bile ducts. Cellular debris and mucus- like material are seen in the lumens of the ducts. H & E stain, 300x. Figure 18. Photomicrograph of testes from a rat fed an iodine-deficient diet and 100 ppm of PBB which died at 15 days. Notice necrotic areas in the seminifer- ous tubules and some infiltration of inflammatory cells including lymphocytes and polymorphonuclear leukocytes. H & E stain, 120x. Figure 19. Photomicrograph of lungs from a rat fed an iodine—adequate diet containing 100 ppm of PBB for 60 days. The alveolar septa are infiltrated by some lymphocytes and aggregates of alveolar macrophages are in the lumina. H a E stain, 48x. DISCUSSION General The general objectives of this research were accomplished. Hypothyroidism in rats resulted from feeding IDD for 30 or 60 days with lesions suggestive of thyroid hyperplasia. The effects of PBB in rats fed IAD were compared with the effect in rats fed an IDD. Comparison of the histologic lesions suggested interrelationships between PBB exposure and iodine deficiency in rats. Iodine-deficient rats appear to be more susceptible to PBB toxicosis than normal rats. Hypothyroidism The modified Remington diet utilized in this research induced goiter in male rats in the short period of 30 days and the condition was characterized by the enlargement and hyperplastic growth of the thyroid gland. Histologic lesions were similar to those described by Jubb and Kennedy (1970). The thyroid weights of rats fed IDD were increased approximately twofold by 30 days. The calculation of the thyroid weight to body weight ratios was based on the fresh weight of the giand in mg/100 g of body weight as recomended by Levine et al. (1933) and Remington et al. (1936). The findings agreed with those described by Remington and Levine (1935). They reported that rats fed an iodine-deficient diet for 35 days had thyroid glands twice normal size. According to these authors, feeding rats an iodine-deficient diet for longer 47 48 periods did not result in any greater degree of enlargement. In contrast, we observed further enlargement after 30 days. Although \we did not examine the thyroid weight at 35 days, after 60 days the thyroid glands were 9 times the size of the thyroids of control rats fed the IAD. The electrophoretic pattern for serum protein was normal in rats fed IDD and 0 ppm of PBB, but triglycerides decreased sharply. The liver and kidney weight to body weight ratios decreased after 60 days, but the reasons for the decrease are not known. The histologic appearance of the liver and kidneys was normal. Toxicosis of Polybrominated Biphepyls The research indicated that in rats fed IAD and PBB, the weight gain and feed efficiency were decreased only at 100 ppm of PBB. The most pronounced lesions were observed in the liver, thyroid gland and lungs. The findings revealed that increases in liver weight associated with PBB were dose dependent. These effects were seen at 10 or 100 ppm of PBB and were greater at 60 days than at 30 days. The histologic lesions in the liver were similar to those described by Sleight and Sanger (1976). The increase in liver weight coincided with the histologic lesions in that the individual hepatocytes were hypertrophied with abundant accumulations of lipid droplets in the cytoplasm. There was relatively little inflammatory reaction or necrosis. The hepatic lesions associated with PBB toxicosis were similar to those reported as caused by other toxic substances in rats such as PCB (Bastomsky and Murthy, 1976; Kimbrough et al., 1972). Rats fed IAD and 0 ppm of PBB had greater thyroid weight to body weight ratios at 30 days than at 60 days. These results 49 supported previous observation in rats (Levine et al., 1933) and in man (Marine and Lenhart, 1909) in that the thyroid weight to body weight ratios tend to be greater in earlier life. Dietary exposure of 10 or 100 ppm of PBB increased the thyroid weight at 60 days. The increase in thyroid weight was associated with histologic lesions suggestive of hypothyroidism. The lesions were similar to those reported by Ringer and Polin (1977) in chickens. Based on the pathologic features, PBB may be goitrogenic and act similarly to PCB, which stimulate thyroid growth as described by Hurst (1974). Bastomsky and Murthy (1976) concluded that dietary feeding of PCB in rats enhanced biliary excretion of thyroxin. The relatively low iodine content in the feed used for the con- trol IAD may have enhanced the effect of PBB on the thyroid. The Remington diet with 0.2 ppm iodine was somewhat lower than was recom- mended by Remington and Remington (1937) for the prevention of hypo- thyroidism in rats. Standard laboratory diets contain as much as 2 to 3 ug/g (Studer and Greer, 1965). However, in the present study the thyroids from rats fed IAD and 0 ppm of PBB were normal. The Interaction of PBB Toxicosis and Iodine Deficiency The most severe histologic lesions of PBB toxicosis in rats fed IDD were in the livers and thyroids. In general, PBB increased liver weight of rats fed IDD or IAD for 30 or 60 days, with significantly lesser effects in rats fed IDD. The PBB are known to stimulate the smooth surface endoplasmic reticu- lum (Lee et al., 1975) and are potent inducers of microsomal enzymes (Dent et al., 1976; Moore et al., 1977). Increase in smooth endoplasmic reticulum probably accounts for a good portion of the increase in liver 50 weight. Possibly there is less stimulation of smooth-surface endo- plasmic reticulum by PBB in the rats fed an IDD. Unfortunately, the assays for microsomal enzymes have not been completed. Although electron microsc0py was not included in the protocol of the present study, comparison of electron micrographs from livers of rats fed IAD with rats fed IDD and PBB would also be helpful in assessing the differences between the 2 groups. The histologic lesions suggested that rats with hypothyroidism were more susceptible to PBB exposure than rats fed a normal diet. In the livers of rats fed IDD and PBB, the hepatocytes were more hypertrophied and the vacuoles were larger and more numerous. Degen- erating hepatocytes mostly with pyknotic nuclei were most often observed in rats fed IDD at 100 ppm of PBB. In addition, there were areas of interlobular and portal fibrosis. There was some hyper- plastic growth of epithelial cells mostly in the portal triads with the formation of bile ducts. In some instances the ducts contained mucus or epithelial debris. The ducts were surrounded by fibrous connective tissue and occasional inflammatory cells. Around the lesions there was hypertrophy of hepatocytes and some pyknotic nuclei. In some cases the cell walls were ruptured due to the extensive swel- ling of the cells. Bile duct hyperplasia was consistently found in all rats fed IDD and 100 ppm of PBB for 60 days. Bile duct hyper- plasia is also called cholangio-fibrosis, bile duct proliferation, bile duct adenomatosis and fibroadenoma (Kimbrough et al., 1972). Bile duct hyperplasia caused by PBB treatment has not been reported in rats. However, glandular hyperplasia of the major intra- hepatic bile duct was seen in an experimental cow given PBB (Gutenmann and Lisk, 1975). Sleight and Sanger (1976) described liver lesions 51 similar to our findings in rats fed a commercial diet and PBB. How- ever, bile duct hyperplasia was not observed in their study. Bile duct hyperplasia has been reported in rats exposed to PCB (Kimbrough et al., 1972) and alpha—naphthyl-iso-thiocyanate (ANIT) (McLean and Rees, 1958; Steiner and Baglio, 1963). The hepatic lesions in rats fed IDD and PBB at 100 ppm indicated that under certain conditions PBB may induce bile duct hyperplasia. Since this chemical induced a hyperplastic growth of cells, further investigation is necessary to determine whether PBB is carcinogenic. However, McLean and Rees (1958) observed bile duct hyperplasia in rats treated with ANIT, but prolonged administration (18 months) did not induce neoplasia. Steiner and Baglio (1963) also reported that although the changes in the liver cells of rats exposed to ANIT were similar to the lesions produced by a certain hepatic carcinogen, the alterations could not be interpreted as indicative of preneoplastic changes. McLean and Rees (1958) postulated that there are two distinct varieties of bile duct hyperplasia. One type is characterized by an orderly self-differentiating cell production and the other by bile duct hyperplasia that tends to go astray in differentiation and undergoes malignant transformation. Data related to the thyroid weight indicated that PBB enhanced the thyroid enlargement in rats fed IDD for 30 days with the most significant increase at the highest dose level. However, after 60 days there were no significant differences in thyroid weight at l, 10 or 100 ppm when compared to those fed IDD and 0 ppm of PBB. In fact, the thyroids of rats that were treated with 100 ppm of P88 were even smaller than the rats treated with 10 ppm of PBB (p<0.05). 52 Remington and Levine (1935) postulated that there is a certain experi- mental period for goiter production in rats. Longer periods will not cause any greater degree of enlargement, and if the period is too long the thyroid weight to body weight ratios are apt to decrease. Examination of histologic lesions revealed extensive alterations in the thyroid gland of a rat fed IDD with 100 ppm of PBB. In this rat there was evidence of an inflammatory reaction and proliferation of fibrous connective tissue. In this instance there was thickening of the capsule of the thyroid gland. More studies are needed to definitely associate the lesions with PBB. Although PBB induced thyroid lesions similar to hypothyroidism in rats fed IAD, the degree and rate of development of the lesions were less obvious than those resulting from the IDD. Our findings indicated that there are interrelationships between the effects of PBB and iodine deficiency. The deficiency of iodine caused the rats to be more susceptible to PBB with more severe altera- tions in the liver. The PBB enhanced the effects of iodine deficiency in the thyroid. SUMMARY AND CONCLUS ION Ninety-six young male rats were fed dietary levels of 0, l, 10 or 100 ppm of PBB in an iodine-deficient diet (IDD) or iodine—adequate diet (IAD). Six rats in each of the eight dietary treatment groups were killed at 30 days and the remaining 48 rats at 60 days. The weight gains and feed efficiency were noticeably decreased only in rats fed diets containing 100 ppm of PBB for 60 days. Although PBB caused a dose dependent increase in liver weight at 10 or 100 ppm, the increases were less in rats fed IDD. In rats fed IDD for 30 days the thyroid enlargement was enhanced by 10 or 100 ppm of PBB. Rats fed IAD containing 10 or 100 ppm of PBB for 60 days had enlargement of thyroid glands. Electrophoretic patterns revealed severe alterations in serum protein and lipoprotein of rats fed IAD containing 10 or 100 ppm of PBB, whereas in rats fed IDD marked alterations were only seen at 100 ppm of PBB. Cholesterol values were elevated in rats fed IDD or IAD containing 100 ppm of PBB. Triglycerides decreased in rats fed IDD containing 0, l, 10 or 100 ppm of PBB. Tissue analysis revealed accumulation of PBB in fat, liver, kidneys and thyroid gland of rats fed IDD or IAD containing PBB, with the highest concentration in the fat. Histologic examination revealed more severe lesions in the liver and thyroid gland of rats fed IDD and 10 or 100 ppm of PBB when com- pared to rats fed IAD and the same levels of PBB. Fibrosis and bile 53 54 duct hyperplasia were seen only in rats fed IDD and 100 ppm of PBB. Follicular hyperplasia and decreased colloid were seen in the thyroids of rats fed IAD and 10 or 100 ppm of PBB. However, the degree of thyroid enlargement and the hyperplastic growth of the follicular cells in rats fed IAD and 10 or 100 ppm of PBB was less severe than in rats fed IDD and 0, l, 10 or 100 ppm of PBB. These results indicated that there are interrelationships between the effects of P88 and iodine deficiency. The deficiency of iodine caused the rats to be more susceptible to PBB with more severe altera- tions in the liver. The PBB enhanced the effects of iodine deficiency in the thyroid. REFERENCES REFERENCES Anonymous: Traces of PBB's Found in Two States. Chem. Eng. News, 55(26), (1977): 16. Astwood, E. B., Sullivan, J., Bissell, A., and Tyslowitz, R.: Action of Certain Sulfonamides and of Thiourea upon the Function of the Thyroid Gland of the Rat. Endocrinology, 32, (1943): 210-225. Babish, J. G., Gutenmann, H. W., and Stoewsand, G. 5.: Polybrominated Biphenyls: Tissue Distribution and Effect on Hepatic Micro- somal Enzymes in Japanese Quail. J. Agric. Food Chem., 23(5), (1975): 879-881. Bastomsky and Murthy, P. V. N.: Enhanced in vitro Hepatic Glucuroni- dation of Thyroxine in Rats Following Cutaneous Application or Ingestion of Polychlorinated Biphenyls. Can. J. Physiol. Pharmacol., 54, (1976): 23-26. Benjamin, M. M.: Outline of Veterinary Clinical Pathology, 2nd Ed. The Iowa State University Press, Ames, Iowa, (1976): 1-186. Brand, N., Kathein, R. A., and Hasis, G.: Comparative Study of Endemic Goitre in Man and Cattle in Israel. Acta Endocrinol., 42, (1963): 21-28. Cagen, S. 2., Preache, M. M., and Gibson, J. E.: Enhanced Disappear- ance of Drugs from Plasma Following Polybrominated Biphenyls. Toxicol. Appl. Pharmacol., 40, (1977): 317-325. Calderbank, G.: Iodine. In Animal Health Production and Pasture. Longmans Green & Co., London, (1963): 681-736. Carter, L. J.: Michigan's PBB Incident: Chemical Mix—Up Leads to Disaster. Science, 192, (1976): 240-243. Corbett, T. H., Beaudoin, R. G., Anver, M. R., Schumacher, R., Endres, J., and Szwabowska: Toxicity of Polybrominated Biphenyls (Firemaster BP-6) in Rodents. Environ. Res., 10, (1975): 390-396. Dent, J. G., Netter, K. J., and Gibson, J. E.: The Induction of Hepatic Microsomal Metabolism in Rats Following Acute Adminis- tration of a Mixture of Polybrominated Biphenyls. Toxicol. Appl. Pharmacol., 38, (1976): 237-249. 55 56 Dunckel, A. E.: An Updating on the Polybrominated Biphenyl Disaster in Michigan. J. Am. Vet. Med. Assoc., 167, (1975): 838-841. Durst, H. I., Willett, L. B., Brumm, C. J., and Mercer, H. D.: Effects of Polybrominated Biphenyls on Health and Performance of Pregnant Holstein Heifers. J. Dairy Sci., 60, (1977): 1294-1300. Eldridge, E. F.: The Iodine Content of the Water Supplies of Michigan. Am. J. Public Health, 14(9), (1924): 750. Evered, D.: Diseases of the Thyroid, lst Ed. The Pitman Press, Ca., (1976): 5-22. Fregly, M. J., Walters, I. W., and Straw, J. A.: Effect of Isomers of DDD on Thyroid and Adrenal Function in Rats. Can. J. Physiol. Pharmacol., 46, (1968): 59-66. Fries, G. F., and Marrow, G. S.: Excretion of Polybrominated Biphenyls into the Milk of Cows. J. Dairy Sci., 58(6), (1975): 947-951. Fries, G. F., Bitman, J., and Lillie, R. J.: Retention and Excretion of Polybrominated Biphenyls by Hens. Bull. Environ. Contam. Toxicol., 15(3), (1976): 278-282. Gutenmann, W. H., and Lisk, D. J.: Tissue Storage and Excretion in Milk of Polybrominated Biphenyls in Ruminants. J. Agric. Food Chem., 23(5), (1975): 1005-1007. Hashimoto, Akasaka, S., Takagi, Y., Kataoka, M., Otake, T., Murata, Y., Aburada, S., Kitaura, T., and Uda, H.: Distribution and Excretion of (14C) Polychlorinated Biphenyls after their Prolonged Administration to Male Rats. Toxic. Appl. Pharmacol., 37, (1976): 415-423. Hesse, J. L.: Water Pollution Aspects of Polybrominated Biphenyl Production: Results of Surveys in the Pine River in the Vicinity of St. Louis, Michigan. Presented at the Second National Conference on Complete Water Reuse: Water's Inter- face with Energy, Air and Solids, Palmer House, Chicago, May 4-8, 1975. Hurst, J. G., Newcomer, W. S., and Morison, J. A.: Some Effects of DDT, Toxaphene and Polychlorinated Biphenyl on Thyroid Func- tion in Bobwhite Quail. Poultry Science, 53, (1974): 125-133. Isleib, D. R., and Whitehead, J. L.: Polybrominated Biphenyl: An Agricultural Incident and Its Consequences. l. The Agricul- tural Effects of Exposure. In The Contamination Crisis in Michigan: A Report from the Senate Special Investigating Committee on Polybrominated Biphenyls, July 1975: 37-39. 57 Jackson, T. F., and Halbert, F. L.: A Toxic Syndrome Associated with the Feeding of Polybrominated Biphenyl-Contaminated Protein Concentrate to Dairy Cattle. J. Am. Vet. Med. Assoc., 165(5), (1974): 437-439. Jacobs, L. W., Chou, S. F., and Tiedje, J. M.: Fate of Polybrominated Biphenyls (PBB's) in Soils. Persistence and Plant Uptake. J. Agric. Food Chem., 24(6), (1976): 1198-1201. Jeffries, D. J., and French, M. C.: Avian Thyroid: Effect of p,p'-DDT on Size and Activity. Science, 166, (1969): 1278- 1280. Jeffries, D. J., and Parslow, J. L. F.: Effect of One Polychlorinated Biphenyl on Size and Activity of the Gull Thyroid. Bull. Environ. Contam. Toxicol., 8(5), (1972): 306-310. Jubb, K. V. F., and Kennedy, P. C.: Pathology of Domestic Animals, 2nd Ed. Academic Press, New York, San Francisco, London, (1970): 407-418. Kay, K.: Polybrominated Biphenyls (PBB) Environmental Contamination in Michigan, 1973-1976. Environ. Res., 13, (1977): 74-93. Kimbrough, R. D., Linder, R. E., and Gaines, T. B.: Morphological Changes in Livers of Rats Fed Polychlorinated Biphenyls. Arch. Environ. Health, 25, (1972): 354-364. Kociba, R. J., Frauson, L. O., Humiston, C. G., Norris, J. M., Wade, C. E., Lisowe, R. W., Quast, J. F., Jersey, G. C., and Jewett, G. L.: Results of a Two Year Dietary Feeding Study with Decabromodiphenyl Oxide (DBDPO) in Rats. JFF. Combustion Toxicology, 2, (1975): 267-285. Lane-Petter, W.: The Physical Environment of Rats and Mice. In Animals for Research. Academic Press, London and New York, (1963): 1-20. Levine, H., Remington, R. E., and Kolnitz, H.: Studies on the Rela- tion of Diet to Goiter. l. A Dietary Technic for the Study of Goiter in the Rat. J. Nutrition, 6(4). (1933): 325-345. Lee, K. P., Herberi, R. R., Sherman, H., Aftosmis, J. G., and Wariiz, R. S.: Bromine Tissue Residue and Hepatotoxic Effects of Octabromobiphenyl in Rats. Toxicol. Appl. Pharmacol., 34, (1975): 115-127. Lewis, A. E.: Biostatistics. Reinhold Publishing Corp., New York, (1966): 214. Luna, L. G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology: 3rd Ed. The Blackiston Division McGraw-Hill Book Co., New York, Toronto, London, Sidney, (1968): 1-258. 58 Marine, D., and Lenhart, C. H.: Relation of Iodine to the Structure of Human Thyroids. Arch. Internal Med., 4, (1909): 440-493. Mercer, H. D., Teske, R. H., Condon, R. J., Furr, A., Meerdink, G., Buck, W., and Fries, G.: Herd Health Status of Animals Exposed to Polybrominated Biphenyls (PBB). J. Toxicol. Environ. Health, 2, (1976): 335-349. McLean, M. R., and Rees, K. R.: Hyperplasia of Bile Ducts Induced by Alpha-Naphthyl-Iso-Thiocyanate: Experimental Biliary Cirrhosis Free from Biliary Obstruction. J. Path. Bact., LXXVI, (1958): 175-189. Moore, R. W., Sleight, S. D., and Aust, S. D.: Induction of Liver Microsomal Drug Metabolizing Enzymes by 2.2',4.4'.55'-Hexa- bromobiphenyl. In press (1977). Moorhead, P. D., Willett, L. B., Brumm, C. J., and Mercer, H. D.: Pathology of Experimentally Induced Polybrominated Biphenyl Toxicosis in Pregnant Heifers. J. Am. Vet. Med. Assoc., 170, (1977): 307-313. Norris, J. M., Ehrmantraut, J. W., Gibbons, C. L., Kociba, R. J., Schwetz, B. A., Rose, J. Q., Humiston, C. G., Jewett, G. L., Crummett, W. B., Behring, P. J., Tirsel, J. B., and Brosier, J. S.: Toxicological and Environmental Factors Involved in the Selection of Decabromodiphenyl Oxide as a Fire Retardant Chemical. JFF. Combustion Toxicol., l, (1974): 52-77. Olin, R. M.: Iodine Deficiency and Prevalence of Simple Goiter in Michigan. J. Am. Med. Assoc., 82(17), (1924): 1328-1332. Pitt-Rivers, R., and Tata, J. R.: Biochemical Changes in Hypothy- roidism. In The Chemistry of Thyroid Diseases. Charles C. Thomas Publisher, Springfield, Illinois, (1960): 9-23. Porter, G.: The Norway Rat. In UFAW Handbook on the Care and Management of Laboratory Animals, 3rd Ed. The Williams and Wilkins Co., Baltimore, (1967): 353-390. Rajkumar, S. 8.: Incidence of Goiter in Goats. Indian Vet. J., 47, (1970): 185-187. Ramalingaswami, V.: Endemic Goitre. In The Thyroid Gland. Butterworths, London, 2, (1964): 71-87. Remington, R. E., Coulson, E. J., and Levine, H.: Studies on the Relation of Diet to Goiter. J. Nutr., 12(1), (1936): 27-37. Remington, R. E.: Improved Growth in Rats on Iodine Deficient Diets. J. Nutrition, 13(2), (1936): 223-233. Remington, R. E.. and Levine, H.: Studies on the Relation of Diet to Goiter. J. Nutrition, 11(4), (1935): 343-357. 59 Remington, R. E., and Remington, J. W.: The Effect of Enhanced Iodine Intake on Growth and on the Thyroid Glands of Normal and Goitrous Rats. J. Nutrition, 15, (1937): 539-545. Richert, E. P., and Prahlad, K. V.: Effects of DDT and Its Metabo- lites on Thyroid of the Japanese Quail, Coturnix coturnix Japonica. Poultry Sci., 51, (1972): 196-200. Ringer, R. K., and Polin, D.: The Biological Effects of Polybrominated Biphenyls in Avian Species. Fed. Proc., 36(6), (1977): 1894- 1898. Schermer, S.: The White Rat. In The Blood Mbrphology of Laboratory Animals, 3rd Ed. F. A. Davis Co., Philadelphia, (1967): 41-60. Sleight, S. D., and Sanger, V. L.: Pathologic Features of Polybrominated Biphenyl Toxicosis in the Rat and Guinea Pig. J. Am. Vet. Med. Assoc., 169(11), (1976): 1231-1235. Steiner, J. W., and Baglio, C. M.: Electron Microscopy of the Cyto- plasm of Parenchymal Liver Cells in Alpha-Naphthyl Isothio- cyanate Induced Cirrhosis. Lab. Invest., 12(8), (1963): 765- 790. Studer, H., and Greer, M. A.: Study of the Mechanisms Involved in the Production of Iodine Deficiency Goiter. Acta Endocrinol., 49, (1965): 610-628. Sundstrom, G., and Hutzinger, 0.: Identification of 2,2',4,4',5,5'- Hexabromobiphenyl as the Major Component of Flame Retardant Fire Master BP-6. Chemosphere, l, (1976): 11-14. Troisi, C. L.: Biochemical Effects of Polybrominated Biphenyls on Microsomal Enzymes. Master's Thesis, Michigan State University, East Lansing, 1975. Underwood, E. J.: Iodine. In Trace Elements in Human and Animal Nutrition, 4th Ed. Academic Press, Inc., New York, 1977. Willett, L. B., and Irving, H. A.: Distribution and Clearance of Polybrominated Biphenyls in Cows and Calves. J. Dairy Sci., 59(8), (1976): 1429-1439. Wilson, J. G.: Hypothyroidism in Ruminants with Special Reference to Foetal Goitre. Vet. Rec., 97, (1975): 161-164. Zarrin, K. H., and Hanichen, T.: Comparative Histopathological Study of the Canine Thyroid Gland in London and Munich. J. Small Anim. Pract., 15, (1974): 329-342. Zitko, V.: The Accumulation of Polybrominated Biphenyls by Fish. Bull. Environ. Contam. Toxicol., 17(3), (1977): 285-292. VITA VITA The author was born in Klaten, Central Java, Indonesia, on November 7, 1945. He graduated from the Faculty of Veterinary Medicine, Gadjah Mada University, in April 1973. Following graduation he was employed by the Directorate of Animal Health of Indonesia. He was then appointed to the Laboratory of Disease Investigation Center in Ujung Pandang in a project of joint cooperation between the Indonesian government and the Food and Agriculture Organization of the United Nations, in July 1973. He was assigned to another laboratory of the Disease Investi- gation Center in Denpasar (Bali) in April 1975 to have some training in pathology. He was admitted to the graduate program in veterinary pathology at Michigan State University in the fall term of 1975. The author is happily married to Retno Juliastuti. They have a daughter, Galuh Hangganingraras Eko Akoso. 60 MICIIW‘HINPWSIWAIW 3 1293 0 NIVERSITY LIB n \ Hlnfiil‘l't‘l'“ 3056 0472