OVERDUE FINES ARE 25C pER DAY PER ITEM Return to book drop to remove this checkout from your record. AN EIGHTEEN-MONTH STUDY OF POLYBROMINATED BIPHENYL TOXICOSIS IN RATS By Manley C. Pratt A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1979 AN EIGHTEEN-MONTH STUDY OF POLYBROMINATED BIPHENYL TOXICOSIS IN RATS BY Manley C. Pratt An 18-month study designed to assess hepatic vitamin A concentrations and gross morphologic, histologic and ultrastructural changes associated with feeding low dietary levels of polybrominated biphenyls (PBB) was completed. One hundred sixty-eight young male Sprague-Dawley rats were randomly divided into 4 groups each consisting of 42 rats. Each group was assigned to a diet containing 0, 0.1, 1, or 10 ppm of PBB. Six rats from each group were killed at 10, 30, 60, 90, 180, 360, and 540 days. No clinical signs or mortality referable to PBB toxi- cosis or hypovitaminosis A occurred. The PBB-fed rats gained weight as well as, and in some instances better than, the control rats. The relative weights of the kidney and brain (when expressed as percent of body weight) were not significantly (p>0.05) affected by dietary PBB. Hema- tologic values, urinalyses and serum proteins were essen- tially normal. Each of the 3 dietary concentrations of PBB induced significant increases (p<0.05) in relative liver weight at 10 and 30 days. Similar significant Manley C. Pratt increases in relative liver weights occurred for rats consuming diets containing 10 ppm of PBB at 60 days and thereafter. In contrast, relative (ug/g liver) and abso- lute (mg/liver) hepatic vitamin A concentrations were decreased (p<0.05) at one or more dietary levels of PBB throughout the course of the experiment. The most severe depletion of hepatic vitamin A occurred in rats fed diets with 10 ppm of PBB for 540 days. Histologic lesions were confined to the liver and were related to PBB. Diffuse hypertrOphy and hyperplasia of hepatocytes were the major changes in rats killed at 10 days. From 30 to 180 days, swollen hepatocytes and limited midzonal lipid accumulation were the only lesions evident. Severe intracytOplasmic lipid accumulation was the principal change in hepatocytes from rats fed 10 ppm of PBB for 360 and 540 days. In addition, focal necrosis, bile duct proliferation, focal infiltration of inflammatory cells, and portal fibrosis occurred. Ultrastructural changes included a progressive increase of the smooth endoplasmic reticulum (SER) with a concomitant decrease of rough endo- plasmic reticulum (RER) and decreased numbers of mito- chondria. Myelin body formation was a consistent finding. Some of the myelin bodies enclosed lipid droplets and membrane-bound vacuoles containing whorled figures. Tissue residues of PBB were dose-related, and livers with the highest PBB concentration had the most severe light and electron microsc0pic changes. The results of the investi- gation indicate that, even at very low dietary concentrations Manley C. Pratt of 0.1 ppm, PBB is capable of inducing significant morpho- logic and biochemical changes in rats. DEDICATION Dedicated to my wife, Novelette, and daughters, Karren and Alison ii ACKNOWLEDGEMENTS The author wishes to extend his greatest apprecia- tion to Dr. S. D. Sleight, his major professor, for his continued patience, encouragement and guidance during the course of his studies and in the preparation of this dissertation. To Dr. R. W. Leader, Chairman of the Department of Pathology, he wants to express thanks for providing financial support in the form of a graduate teaching assistantship. For their contribution in teaching him the basics of patholOgy, the author also wants to thank Drs. G. L. Waxler, A. L. Dade, S. D. Sleight, A. L. Trapp, R. F. Langham, and V. L. Sanger. Sincere appreciation is extended to the members of his graduate committee, Drs. H. D. Stowe, A. L. Trapp, and G. L. Waxler, for their advice and criticism of this manuscript. Special thanks is extended to the following individuals for their laboratory and technical assistance: Shirley Howard, Linda Stegherr, and Drs. E. Roege, P. Werner and A. Telles. The author is also indebted to the histOpathology laboratory personnel for their help in the processing of tissue sections and to Janice Fuller for typing the dissertation. iii Finally, the interest, cooperation, tolerance and sympathy of his wife, Novelette, and daughters, Karren and Alison, were invaluable. iv TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . Source. . . . . . . . . . . . . . . . . . Industrial Uses . . . . . . . 1 4 Chemistry . . . . . . . . . . . . . . . . . . 4 Metabolism of PBB . . . . . . . . . . . . . . 7 Absorption. . . . . . . . . . . . . . . . . . 10. Retention . . . . . . . . . . . . . . . . . . 12 Excretion . . . . . . . . . . . . l4 Mechanism of PBB Toxicosis. . . . . . . 16 Species and Age Susceptibility to PBB Toxicosis . . . . . . . . . . . 17 Systemic Effects of PBB . . . . . . 18 Hematopoietic and Immunologic System . l8 Gastrointestinal System. . . . . . . . 20 Reproductive System. . . . . . . . . . 21 Integumentary System . . . . . . . . . 23 Pathology of PBB Toxicosis. . . . . . . . . . 23 Clinical Signs . . . . . . . . . . . . 23 Lesions. . . . . . . . . . . 25 Ultrastructural Changes. . . . 27 Effects of Chemical Toxicosis on Vitamin A Metabolism. . . . . . . . . . . . . . . . . 28 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 32 Experimental Design and Method. . . . . . . . 32 NecrOpsy and Pathological Examination . . . . 33 Clinical Pathologic Tests . . . . . . . . . . 34 Hematologic Examination. . . . . . . . 34 Urinalysis . . . . . . . 35 Serum Protein ElectrOphoresis. . . . . 35 Vitamin A Analysis. . . . . . . . . . . . . . 36 PBB Analysis. . . . . . . . . . . . . 37 Histopathologic Procedure . . . . . . . . . 38 Transmission Electron Microscopy. . . . . . . 38 RESULTS. General LaboratoryOFindings Patholo Electro DISCUSSION SUMMARY. REFERENCES VITA . APPENDICES A Urinalysis . . Hematology . . . . Serum Electrophoresis. Organ Weights. . . . Polybrominated Biphenyl Content of Tissues. . . . . . . . Liver Vitamin A Content. Miscellaneous Laboratory Findings. gic Findings . Gross Lesions. . . . . . Histopathologic Findings . . . . Other HistOpatholOgic Findings n Microsc0pic Findings . . . MEAN AND RELATIVE KIDNEY AND BRAIN WEIGHTS FOR PBB- FED RATS . . . MEAN THYROID HORMONE CONCENTRATIONS IN RATS FED PBB FOR 10, 90, AND 540 DAYS . . . . . . . . . . MEAN SERUM TESTOSTERONE CONCENTRATIONS IN RATS FED PBB FOR 10, 30, 60, 90 AND 180 DAYS . . . . . . . . . . vi Page 39 39 39 39 41 41 44 44 49 51 S4 55 60 67 76 85 87 100 101 101 102 103 Table 10 11 12 LIST OF TABLES Experimental design for 18-month PBB feeding study Mean hemoglobin, hematocrit, total leuko- cyte and erythrocyte counts, and differen- tial leukocyte counts in rats fed PBB Serum proteins (total protein, albumin, a2 globulin values and A/G ratios) of rep- resentative control rats and rats fed PBB Mean body, in rats fed PBB liver and relative liver weights The concentrations (ppm) of PBB in the fat of liver from rats fed diets containing PBB The concentrations (ppm) of PBB of adipose tissue from rats fed taining PBB Liver vitamin 30 days Liver vitamin 60 days Liver vitamin 90 days Liver vitamin 180 days. . . Liver vitamin 360 days. . Liver vitamin 540 days. . A A A content content content content content content in rats in rats in rats in rats in rats in rats in the fat diets con- fed PBB fed PBB fed PBB fed PBB fed PBB fed PBB for for for for for for Mean and relative kidney and brain weights for PBB-fed rats. . . . vii Page 32 42 4s 46 47 48 50 50 52 52 53 53 101 Table Page B-l Mean thyroid hormone concentrations in rats fed PBB for 10, 90, and 540 days. . . . . . 102 C-1 Mean serum testosterone concentrations in rats fed PBB for 10, 30, 60, 90 and 180 days. 103 viii Figure 10 LIST OF FIGURES Page Mean weight gain in rats fed PBB. . . . . . . 40 Diffuse perinuclear vacuolation and increased number of mitotic figures (arrows) in the liver from a rat fed a diet contain- ing 0.1 ppm of PBB for 10 days. . . . . . . . 57 Higher magnification of the liver from the rat in Figure 2, to show the absence of sinusoidal spaces and prominent mitotic figures (arrows). . . . . . . . . . . . . . . 58 Focal areas of necrotic hepatocytes were replaced by inflammatory cells that were mainly mononuclear. . . . . . . . . . . . . . 59 Typical small, multiple intracytoplasmic lipid droplets gave a foamy appearance to the hepatocytes located in the midzonal area of the hepatic lobule. . . . . . . . . . 61 IntracytOplasmic lipid accumulation involving the mid- and peripheral zones in the liver from a rat fed a diet containing 1 ppm of PBB for 540 days. . . . . . . . . . . . . . . 63 Portal fibrosis and bile duct hyperplasia with associated focal infiltration of lympho- cytes (arrow) in the liver from a rat fed a diet with 1 ppm of PBB for 540 days . . . . . 64 Electron micrograph of hepatocytes from a control rat . . . . . . . . . . . . . . . . . 68 Electron micrograph of a portion of a hepa- tocyte from a rat fed a diet containing 10 ppm of PBB for 540 days . . . . . . . . . . . 69 ix Figure 11 12 13 14 15 Electron micrograph of part of a hepatocyte from a rat fed a diet containing 1 ppm of PBB for 10 days . . . . . . . . . . Electron micrograph of a hepatocyte rat fed a diet containing 10 ppm of 360 days. . . . . . . . . . . . . Electron micrograph of a hepatocyte rat fed a diet containing 10 ppm of 540 days. . . . . . . . . . . . . . Electron micrograph of a hepatocyte rat fed a diet containing 10 ppm of 540 days. . . . . . . . . . . . Electron micrograph of a hepatocyte rat fed a diet containing 10 ppm of 360 days. . . . . . . . . . from a PBB for from a PBB for from a PBB for from a PBB for Page 70 71 72 74 75 INTRODUCTION The term "economic poison" implies that a toxic agent, either desirable or actually needed in our daily life, is at the same time a hazard to health. In general, insecticides, herbicides, and fungicides comprise this group; however, in the past decade, the toxic potentials of a number of other substances have been recognized. Thoroughly understood and properly used, these chemicals seldom cause adverse effects, and the benefits derived, though often debated, usually justify their use. In recent years, the halogenated biphenyls, namely polychlorinated biphenyls (PCB) and polybrominated biphenyls (PBB) have attracted much attention, especially concerning their teratogenic and carcinogenic potentials. The PCB are known to be widely distributed in the environ- ment, and many of their toxic effects have been exten- sively studied in animals (Kimbrough, 1974) and, to a lesser extent, in humans (Kuratsune et al., 1975). Con- versely, the brominated analogs have received less attention because of their limited industrial use and the fact that they were not recognized as significant environ- mental contaminants. 2 The accidental incorporation of a PBB-containing fire retardant (Firemaster BP-6) into the feed of livestock in Michigan sometime in 1973 precipitated an incident that has been termed the most costly accidental contamination ever to occur in United States agriculture (Isleib and Whitehead, 1975). The consequences of the PBB contamina- tion included condemnation and slaughter of thousands of cattle, poultry, and other livestock, as well as loss of meat, milk, and eggs. In addition, there was considerable concern regarding contamination of food supplies by a toxic substance. Currently available literature suggests that PBB, like the PCB, are resistant to degradation and therefore result in virtually permanent contamination of soils, water, plants, and animals with which they come in contact. Environmental contamination by this persistent chemical has accordingly become a major concern of conservationists and of legislative and public health officials. In the past, researchers have been concerned mainly with the effects of excessive amounts of the chemical on animal health. Recently, there is even greater concern regarding "no effect" levels and low-level contamination. Additionally, it would appear from data on Yusho disease (Kuratsune et al., 1975) that long-term public health effects are possible. 3 The major objectives of this research were: 1. To determine the gross morphologic, histologic and ultrastructural changes associated with feeding low dietary concentrations of PBB. 2. To assess changes in hepatic vitamin A concen- trations induced by dietary PBB. LITERATURE REVIEW Early literature concerning the chronology of poly- brominated biphenyl (PBB) contamination and resultant economic, public health, social and political effects is adequately recorded in reviews by Dunkel (1975), Welborn (1975), Stadtfeld (1976), and Carter (1976). Later reviews by Kay (1977), Getty et a1. (1977), and an entire volume of Environmental Health Perspectives (1978) alluded to the contamination incident and summarized the extent of currently available experimental and scien- tific knowledge on PBB. This review will be limited primarily to PBB toxi- cosis in animals. The treatise on chemistry, sources, industrial uses, and metabolism of PBB will be limited to the extent that it permits fundamental understanding. In addition, the effects of chemical toxicosis on vitamin A metabolism will be reviewed. Chemistry The PBB-containing fire retardant that was acciden- tally incorporated into livestock feed in Michigan in 1973 was a complex mixture of various isomers of brominated biphenyls having an average bromine content of 6 atoms 5 per biphenyl molecule zuui a total bromine content of 75%. In its natural form, the chemical occurs as a white flake that melts at 72 C, has a low vapor pressure, and is relatively insoluble in water but highly soluble in organic solvents. It is therefore fat soluble. In a monograph report by Michigan Chemical Corporation (1974), the percentage isomeric composition of the various brominated biphenyls was tetrabromo- (2.0), pentabromo- (10.6), hexabromo- (62.8), and heptabromo- (13.2), with the remainder unidentified. Anderson et a1. (1974) and Sundstrom et a1. (1976) identified the major isomer as 2,2',4,4',S,5' hexabromobiphenyl. Jacobs et a1. (1976) estimated the content of the isomer to be 60 to 70%. The occurrence of hexabromo- (25 ppm), pentabromo- (1 ppm), and traces of tetrabromonaphthalene in the chemical mixture was reported by O'Keefe (1976). A methyl polybrominated furan was detected in the polar fraction of the compound by Moore (1977), but Hass et a1. (1978) could not identify brominated furans in PBB using detec- tion methods sensitive to 0.5 ppm. Although 210 isomeric forms are theoretically possible (Widmark, 1968), the majority of the scientific data available are related to hexabromobiphenyls. Source The flame retardant Firemaster BP-6, consisting of a mixture of PBB, was manufactured solely by Michigan Chemical Corporation, St. Louis, Michigan. Approximately 6 6 million kilograms of PBB were produced in the United States from 1970 through 1976 (Neufeld et al., 1977). About 5.2 million kilograms of the total production was hexabromobiphenyls; the remaining 0.8 million kilograms consisted mainly of octabromobiphenyls and decabromo- biphenyls. Although Firemaster BP-6 production ceased in 1975, 2 companiesa’b continued production of octabromo- and decabromobiphenyl until 1977. According to Neufeld et al. (1977), no PBB are being imported into the United States in commercial quantities and information is available on the extent of PBB imported in the form of finished plastic products. Industrial Uses The chemical mixture was used extensively as a flame retardant for thermoplastics. Kerst (1976) indicated that more than 130 companies in the United States used PBB prior to that year. In 1974, Firemaster BP-6 was used for production of flame-retardant resins of acrylonitrile, butadiene, and styrene and was used in the manufacture of housings for business machines, typewriters, television sets, and other products in which heat resistance was aWhite Chemical Company, Bayonne, NJ. bHexcel Corporation, Sayreville, NJ. 7 desired. It also found use in coatings and lacquers and in polyurethane foam for automobile upholstery (Neufeld et al., 1977). Metabolism of PBB The mammalian body attempts to modify the chemical structure of foreign compounds in order to reduce the physiological activity and enhance excretion. Modifica- tion of chemical structure results in the conversion of such compounds into more polar, less lipophilic metabolites that facilitate excretion. The metabolism of Firemaster BP-6 in birds, labora- tory animals, and domestic animals is poorly understood. Kohli and Safe (1976) stated that approximately 1% of the Firemaster BP-6 administered to a pig was excreted as a monohydroxypentabromobiphenyl metabolite. Similarly, Zitko and Hutzinger (1976) suggested the formation of a monohydroxydibromobiphenyl metabolite in fish. Zitko (1977) reported that fish may debrominate the more highly brominated components of PBB. However, in experiments with cows given a single 3 g dose of PBB, Willett and Irving (1976) failed to detect urinary metabolites. Like- wise, fecal metabolites were not found in rats given radioactive hexabromobiphenyl intravenously or orally (Matthews et al., 1977). Furthermore, arene oxides, the major intermediates formed by metabolism of lipophilic compounds by hepatic mixed function oxidases, have not been reported to be associated with PBB toxicosis 8 (Kolbye, 1977). This is surprising, since PBB has been reported by several investigators (Farber and Baker, 1974; Garthoff et al., 1976; Dent et al., 1976a,b, 1977; Moore et al., 1976) to be a potent inducer of hepatic mixed function oxidases. Jondrof et a1. (1955, 1958) noted that chlorinated benzenes having 2 adjacent unsubstituted carbon atoms were metabolized and excreted 3 to 20 times more rapidly than benzenes with similar degrees of chlorination, but without adjacent unsubstituted carbon atoms. Shulte and Acker (1974) suggested that a similar substitution pattern was required for metabolism of the polychlorinated biphenyls (PCB). This was partially confirmed by Jensen and Sundstrom (1974). They indicated that PCB, which did not have 2 adjacent unsubstituted carbon atoms, were found in the highest concentration in the tissues of animals and man previously exposed to PCB. Hexabromo- biphenyl, the major congener of PBB, does not have adjacent unsubstituted carbon atoms. The corresponding PCB (hexa- chlorobiphenyl), which also lacks adjacent unsubstituted carbons, has an extremely long half-life and has been demonstrated to accumulate in tissues of laboratory rats (Matthews and Anderson, 1975). Similar findings have been reported for PBB (Fries, 1978). He suggested that the metabolism of hexabromo- and heptabromobiphenyl, the 2 major isomers of PBB, at rates of practical importance was unlikely because the PCB of equal halogenation were only sparingly metabolized. Furthermore, measurements of 9 retention of PBB in the edible tissues and excretion in ediblerjproducts of hens and cattle were found to be similar for hexachloro- and hexabromobiphenyl (Fries and Marrow, 1975; Fries et al., 1976). In 1975, Lee et al. reported that withdrawal of a diet containing 1,000 ppm of octabromobiphenyl (OBB) caused decline in the levels of OBB in muscle and liver. Concentrations in fat, however, remained unchanged or were somewhat increased. These results suggested that redistribution, and not metabolism, was responsible for OBB removal from the liver and muscle. After examining tissue residues from cattle originally contaminated in Michigan, Fries (1978) and Fries et a1. (1978) reported finding reduction and disappearance of some gas chromatographic peaks characteristic of the parent compound, indicating that either specific in viva metabolic degradation or selective excretion had taken place. Experimental evidence for in vitro and in vivo metabolism of Firemaster BP-6 was presented by Dannan et a1. (1978). They indicated in vitro metabolism of 2 minor components, namely 2,4,5,2',5' pentabromobiphenyl and a hexabromobiphenyl, in rats previously treated to maximize their ability to metabolize compounds such as PBB. Drastic reduction of these same components in liver and milk extracts from PBB—treated animals confirmed the in vitro results. From their results, they concluded that the availability of a free para position was probably more important for metabolism of brominated biphenyls 10 than either the number of bromines or the presence of 2 adjacent unsubstituted carbon atoms. Although persistence of PBB in the environment is of major concern, probably of greater significance is the possibility of conversion in plants or in the soil to more toxic products. Jacobs et al. (1976) reported that PBB in soils were not degraded, were not leached, and were not taken up by plants, suggesting that PBB would be a permanent component of contaminated soil. Ruzo and Zabic (1975) obtained tetrabromobiphenyl, pentabromobiphenyl, and methoxylated photoproducts from experimental exposure of Firemaster BP-6 and hexabromobiphenyl to sunlight irradi- ation. They concluded that PBB exposed to sunlight would photodegrade to lower brominated and hydroxylated biphenyls that should make them more amenable to biological degra- dation. The possibility of such biological degradation resulting in significant environmental contamination is questionable, since the different end uses of PBB do not prediSpose the compound to direct exposure to sunlight. Absorption Ingested PBB are absorbed from the intestinal tract, are circulated throughout the body, and tend to concen- trate in various fatty tissues (Kolbye, 1977). Although the exact site of intestinal absorption has not been determined, several Species, namely man, cow, pig, dog, mink, guinea pig, rat, mouse, Japanese quail, gull, chicken and fish, have been demonstrated to absorb PBB. 11 Direct data on absorption of toxic doses of PBB are limited. Furthermore, the mechanism of absorption has 14c-OBB not been elucidated, but results of studies with (Norris et al., 1974) seem to indicate that there is less absorption with increasing halogenation. The work of Fries and Marrow (1975) tends to support this observation. They indicated that the levels of heptabromobiphenyl in tissues and milk of cattle heavily contaminated with Fire- master BP-6 was barely detectable and that retention and excretion of the hepta-isomer was only 1/10 that of hexabromobiphenyl. In a quantitative study in which rats were gavaged with 14C-hexabromobiphenyl, at least 90% of the administered dose was shown to be absorbed (Matthews et al., 1977). Following intraruminal administration in cattle, PBB appeared in plasma within 2 to 6 hours, with peak plasma values occurring at 24 to 48 hours (Willett and Irving, 1975). A later report (Willett and Durst, 1978) confirmed the plasma appearance interval (within 4 hours) and indicated that, with continued exposure, plasma concentrations achieved steady state by 15 days. After absorption, PBB are widely distributed to several tissues, with the largest amounts in the fat of liver, muscle, kidney, and adipose tissue (Fries et al., 1978). Results of various experimental studies (Willett and Irving, 1975; Fries et al., 1978; Matthews et al., 1977) indicate that absorption and subsequent 12 redistribution of the compound are independent of route of administration. Retention Fries et a1. (1973) were among the first investigators to report in-depth studies on polybrominated biphenyls. From their studies involving retention and excretion in hens and cows, they concluded that PBB would be resistant to degradation and would persist in tissues. This was confirmed by several investigators (Fries and Marrow, 1975; Corbett et al., 1975; Aftsomis et al., 1972; Norris et al., 1974; Lee et al., 1975), who further pointed out that the lipophilic nature of the chemical made it amenable to storage in fat tissues. The accumulation of bromine in the fat, liver, and muscle of rats was demonstrated to be related to the con- centration of OBB in the diet (Aftsomis et al., 1972; Lee et al., 1975). The kidney, skeletal muscle and testis of rats did not accumulate OBB (Norris et al., 1974); however, studies with PBB in dairy calves (Industrial Biotest Laboratories, Inc., 1975) and lactating cows (Industrial Biotest Laboratories, Inc., 1976) indicated that PBB levels in liver, muscle, fat, kidney and bone marrow increased by increasing the dosage of PBB. In general, results of studies with cattle (Gutenmann and Lisk, 1975; Willett and Irving, 1975, 1976) suggested that, with the exception of certain organs (most notably the liver and brain), PBB concentration in tissues is 13 well correlated with fat content. Fries (1978) and Fries et al. (1978) confirmed this observation. They reported finding no significant difference among PBB concentration in the fat of perirenal, omental and subcutaneous adipose tissue, skeletal muscle, cardiac muscle and kidney of cattle originally exposed to the heavily contaminated feed in Michigan. Conversely, concentrations in the fat of the liver were significantly higher, whereas the con- centrations in fat of the brain and lungs were signifi- cantly lower than concentrations in the fat of other tissues. Gutenmann and Lisk (1975) and Willett and Irving (1976) obtained less uniform tissue distribution in samples obtained from cows soon after PBB ingestion. Fries (1978), however, contended that compared to other halogenated hydrocarbon compounds, overall studies on the distribution of PBB suggest that it reaches steady state concentrations among various tissues more slowly. Kimbrough et a1. (1978) reported that, after an initial decrease, PBB concentration in the blood of rats stabilized and did not further decrease over a 120-day period. Likewise, Willett and Durst (1978) reported steady state plasma levels for PBB after 15 days in cattle sub- jected to continuous dietary exposure. Hesse (1975) stated that fish apparently bioaccumulated PBB in a com- parable manner to.PCB. He also pointed out that ducks have considerable capacity for accumulating PBB. Results of studies involving peeple from PBB- quarantined farms in Michigan indicated that breast milk l4 and adipose tissue retained PBB at considerably higher concentrations than the amount present in blood plasma from the same individuals. The concentration in adipose tissue ranged from 61 to 370 times the PBB value found in the blood,with an average ratio of 174:1 (Kolbye, 1977). In 1975, Babish et al. found higher tissue residues of brominated biphenyls in male than in female Japanese quail. They attributed the difference in tissue retention to deposition and subsequent excretion in the eggs of female quail, a finding also reported for laying hens (Fries et al., 1973, 1976). A similar retention pattern may occur in nonlactating cattle, since lactating cows excrete PBB via the milk (Fries and Marrow, 1975; Gutenmann and Lisk, 1975). Excretion Except for egg-laying birds and lactating mammals, PBB has not been shown to be excreted to any appreciable extent. The chemical mixture is excreted by 4 primary pathways: milk, eggs, feces and urine. Fecal elimination is the predominant pathway in nonlactating animals, while excretion in the eggs of laying birds and milk of lactating animals takes precedence over fecal elimination. Free (unconjugated) hexabromobiphenyl is the major excretory product for Firemaster BP-6. Matthews et a1. (1977), using 14C-hexabromobiphenyl in rats, reported that the excretion of the labeled chemical was extremely slow. Over a 6-week period 15 following intravenous administration of a single dose, only 6.6% of the total amount was excreted in the feces and 0.1% in the urine. On the other hand, rapid fecal 14C-OBB was reported in rats (Norris et elimination of al., 1974). After a single oral dose, the authors detected 62% of the isotOpe in the feces within 24 hours, and 73% was excreted by the 16th day after administration. It was speculated that poor or incomplete absorption was the reason for the extensive 24-hour elimination. Evidence for fecal elimination of PBB has been pro- vided for cattle (Willett and Irvin, 1976), pigs (Kohli and Safe, 1976; Ku et al., 1978), rats (Rickert, 1977; Matthewsenza1., 1977) and chickens (Ringer and Polin, 1977). Urinary excretion has also been recorded for rats (Matthews et al., 1977) and pigs (Kohli and Safe, 1976; Ku et al., 1978), but Willett and Irving (1976) failed to find unconjugated PBB in the urine of cattle. As mentioned before, egg-laying birds and lactating mammals excreted appreciable quantities of brominated biphenyl in egg yolk and milk, respectively. A biphasic mode of elimination has been observed in cow milk. The half-life (T 1/2) of the first phase was 10.5 days (Gutenmann and Lisk, 1975) and the T 1/2 of the second phase was 58 days (Fries and Marrow, 1975). The T 1/2 of PBB excretion in eggs after cessation of PBB feeding was calculated as 17 days (Ringer and Polin, 1977), but Fries et al. (1976) recorded more specific:dataon.T 1/2 for PBB 16 elimination in eggs. They found a T 1/2 of 28 days for hexabromo- and 20 days for heptabromobiphenyl. Although it is well known that PBB are excreted in human breast milk (Eyster, 1976; Kolbye, 1977), no report to indicate urinary or fecal excretion is available in man. Neither are there data to support cutaneous excre- tion for any Species. Mechanism of PBB Toxicosis Kay (1977) reviewed the literature and concluded that the current state of knowledge did not allow identity of the toxic components of PBB that may have been responsible for the ill effects observed since 1973 in Michigan farm stock. Results of gross and histopathologic observations of the livers from experimental animals eXposed to brominated biphenyls indicate the compound is primarily a hepatotoxin; however, the mechanism of intoxication remains unknown. Evidence for covalent binding of toxic metabolites to macromolecules and the resultant centrilobular hepatic 14C-chlorobenzene (Reid and necrosis demonstrated for Krishna, 1973) have not been reported for PBB. In fact, Aust (1977) stated that he was unable to demonstrate binding of metabolites in vitro under conditions which should maximize binding. Kolbye (1977) suggested that the primary toxicity of PBB in experimental animals revolved around their ability to stimulate or inhibit a variety of enzymes in the mammalian body. In this regard, PBB have been demonstrated to be 3 to 5 times more potent as an inducer 17 of hepatic mixed function oxidases than PCB (Farber and Baker, 1974; Garthoff et al., 1976). Likewise, on a quantitative basis, PBB appear to cause a greater degree of thyroid hyperplasia (Norris et al., 1974) than PCB. Both compounds,however, are reported to affect a large number of biochemical systems in a similar manner, a find- ing supported by Garthoff et a1. (1976). They noted disruptions of redox and energy state of hepatocytes of rats exposed to dietary PCB (Aroclor) and PBB. From their studies, they Speculated that disruptions of energy metabolism were an important phase in the toxicosis associated with these chemicals. Species and Age Susceptibility to PBB Toxicosis Although there is an obvious deficiency of published reports concerning lethal doses for PBB among Species, there appears to be a definite Species and age dependent susceptibility. In 1977, Kay reviewed the literature and observed that for 8 species investigated, toxicosis resulting in death occurred only in 4 species, namely cattle (Jackson and Halbert, 1974), rodents (Michigan Chemical Corporation, 1974; Jackson and Halbert, 1974; Corbett et al., 1975), Japanese quail (Cecil et al., 1975) and rabbits (Michigan Chemical Corporation, 1974). Later studies involving mink (Michigan Science in Action, 1976), guinea pigs (Sleight and Sanger, 1976), dogs (Farber et al., 1978) and monkeys (Allen et al., 1978) 18 suggested that these species may be more sensitive to PBB poisoning. Reasons for Species difference in toxicity of PBB have not been elucidated. In general, PBB administered at relatively high con- centrations of 50 ppm or higher to laboratory animals have resulted in detectable toxicosis (Kolbye, 1977). However, an acute lethal oral dose (LDSO = 21,500 mg/kg) has been reported only for rats, and acute lethal dermal toxicity (LD50 = 2,015 to 10,000 mg/kg) has been reported for rabbits (Michigan Chemical Corporation, 1974). In Species in which age was considered, it was found that younger animals were usually more sensitive than older animals (Scientific Panel Report, 1976; Michigan Science in Action, 1976; Preache et al., 1976; Ringer and Polin, 1977). Systemic_Effects of PBB Hematopoietic and Immunologic System Results of hematologic studies Show a Species varia- tion in hematologic response to PBB exposure. Kateley (1977) reported finding no Significant difference between the various hematologic values for exposed and unexposed cattle. Similarly, Moorhead et al. (1977) found minimal changes in packed cell volume, hemoglobin content, total erythrocyte and leukocyte counts, and differential leuko- cyte counts. Hematologic data obtained from cows from one involved herd were inconclusive (Trapp et al., 1975). 19 Packed cell volumes ranged from 28 to 36%, and total leukocyte counts ranged from 5,700 to 13,300/mm3. Sleight and Sanger (1976), Sleight et al. (1978), and Mangkoewidjojo (1979) reported essentially normal hematologic values for rats and guinea pigs. Conversely, Norris et a1. (1974) observed a decreased packed cell volume and total erythrocyte count in rats receiving 1% OBB in their diet for 30 days. Likewise, Ringer and Polin (1977) observed decreased hematocrit and hemoglobin values in chickens, a finding they attributed to decreased plasma erythropoietin. In 1978 Allen et al. found erythropenia in rhesus monkeys. They also recorded leukopenia. Ku et al. (1978) indicated Significant decreases in hematocrit and hemo- globin values in pigs 6 weeks after consuming a diet con- taining 100 ppm of PBB. Werner et al. (1978) and Howard et al. (1978) reported normal hematologic values for sows and their offSpring fed diets with 100 or 200 ppm of PBB. Farber et al. (1978) reported finding marked reduc- tion in hematopoiesis, especially erythropoiesis, in the bone marrow of male dogs given 4 mg/kg Firemaster BP-6 for 61 days. Concurrently, marked extramedullary hemato- poiesis, predominantly of an erythropoietic and megakaryo— cytic nature, and moderate reduction of lymphocytes in the splenic white pulp also occurred. Studies undertaken to evaluate the immunosuppressive effects of PBB toxicosis have provided variable results. Some investigators have indicated altered immunologic 20 competence in man (Bekesi et al., 1978), rats and mice (Luster et al., 1978), sows and their offspring (Howard et al., 1978), monkeys (Allen et al., 1978), d0gs (Farber et al., 1978) and chickens and guinea pigs (Vos and VanGenderew, 1973), while others have revealed an absence of immun010gic effects (Kateley and Bazzell, 1978). Cri- teria for evaluation of immunologic effects included complete blood counts, identification of peripheral blood T- and B-lymphocyte subpopulations, distribution of plasma cells, serum immunoglobulin levels (IgG, IgM, and IgA), the in vitro response of lymphocytes to phytolectins (PHA, Con A, PWM), and determination of autoantibodies and/or immunosuppressive serum factors. Gastrointestinal System Getty et a1. (1977) reviewed the literature and con- cluded that a definite palatability problem existed with contaminated feed, with a decrease in feed efficiency at higher levels occurring in most domestic animals. Moorhead et a1. (1977) listed diarrhea as a clinical finding in cattle fed 25 g/day; however, they concluded that gastro- intestinal changes were minimal. Fatal gastrointestinal hemorrhage was reported in rats exposed to high levels of PBB (Corbett et al., 1975). Similarly, gastrointestinal hemorrhage was the major gross autopsy finding in a dog that died after ingesting 4 mg/kg PBB for 58 days (Farber et al., 1978). 21 In 1977, Kay pointed out that gastrointestinal dis- turbance (stomach cramps and pain) was a common complaint among persons on PBB-quarantined farms. Allen et al. (1978) speculated that the complaints in people may be related to severe and acute hyperplastic and ulcerative gastrointestinal lesions such as he detected in subhuman primates. However, results of physical examination and clinical laboratory tests have so far been unable to establish any effect of PBB exposure in these people. Reproductive System It appears that PBB has adverse effects on reproduc- tion in a number of Species. Parameters employed for assessment of PBB toxicosis on reproduction include: number of fetuses per litter, mortality among fetuses, fertility, hatchability, chick mortality, comb Size, kit survival and serum gonadotrOphic hormone levels. The chemical mixture has not been demonstrated to be a potent teratogenic agent. In 1 study, exencephaly was noted in the offspring of mice exposed to both 100 (3/121) and 1,000 ppm (2/174) dosages, while cleft palate (4/87) and hydronephrosis were observed in the offspring at the 1,000 ppm level (Corbett et al., 1975). The incidence of anomalies in the treated group was, however, not signifi- cantly higher than in control mice. Beaudoin (1977) reported finding cleft palate and diaphragmatic hernia in the offspring of rats gavaged with a single dose (800 mg/kg) of PBB between the 6th and 14th day of pregnancy. 22 However, Ficsor and Wertz (1976) failed to find terato- genic effects among the offSpring of 15 pregnant rats force fed with 100 ug/g between the 6th and 19th day of pregnancy. Likewise, Mercer (1976) found no teratogenic effects in dairy heifers fed diets containing 5,000 ppm of PBB, even though abortions and fetotoxicity occurred. Reduced comb Size incockerels(Ringer and Polin, 1977) and decreased fertility in mink (Michigan Science in Action, 1976), hens and quail (Ringer and Polin, 1977) have been observed. Fetal resorption in mice (Michigan Science in Action, 1976) and rats (Beaudoin, 1977) and hydrops amnii and abortions in cattle (Jackson and Halbert, 1974; Moorhead et al., 1977) have occurred. Additionally, PBB has been reported to be responsible for reduced survival rate in newborn calves (Scientific Panel Report, 1976), mink (Michigan Science in Action, 1976), mice (Preache et al., 1976) and chicks, and decreased hatchability of eggs (Ringer and Polin, 1977). Placental transfer has occurred (Willett and Irving, 1976; Fries et al., 1978). More recently, Allen et al. (1978) recorded prolonged menstrual cycles, decreased concentration of serum pro- gesterone, and excessive postconceptional bleeding in monkeys fed 0.3 ppm PBB for 7 months. Hypoactive semini- ferous tubules were also noted by these investigators. Atrophic testicles and Spermatozoal abnormalities in a bull were listed by Jackson and Halbert (1974). 23 Integumentary System_ Abnormal hoof growth, matting and hair loss, and thickened skin of the neck and thorax were clinical signs described in the herd of cattle in which the original PBB contamination occurred (Jackson and Halbert, 1974). Furthermore, in a followup calf feeding trial, 2 calves that survived develOped marked hyperkeratosis over the entire body. Moorhead et a1. (1977) also detected hyper- keratosis of the eyelids of cattle exposed to PBB at the rate of 25 g/day. Dry scaly skin and a10pecia have been reported in man (Chanda, 1977) and in nonhuman primates (Lambrecht et al., 1978; Allen et al., 1978). Generalized subcutaneous edema and loss of eyelashes were also observed by Allen et al. (1978). Pathglogy of PBB Toxicosis Clinical Signs Jackson and Halbert (1974) first reported on clinical Signs of PBB toxicosis. They described signs associated with acute and chronic effects in adult lactating cows exposed to the heavily contaminated feed (4,000 ppm) and in young nonlactating animals deliberately exposed to the same feed. Anorexia, decreased milk production, infer- tility, elongated hooves, lameness, delayed parturition and thickening of the skin were observed. Feed refusal and grating of teeth were the major clinical findings in the young nonlactating animals, although hyperkeratosis 24 develOped in 2 calves that survived the feeding trial. In 1975, Deming reported on the effects he found in a herd survey involving 72 herds described as low-level con- taminated cattle. Decreased milk production, sterility, decreased growth in young stock, excessive calf losses, and reluctance of animals to move, due to soreness and stiffness, were the most consistent abnormalities recorded. Mercer et a1. (1975), in a comparable herd survey involving 31 herds, were unable to find any Significant differences in these parameters between low-level con- taminated and control cattle. Similarly, Moorhead et a1. (1977) failed to find clinical signs in cattle exposed to 0.25 or 250 mg/day for 60 days. The clinical effects observed in cattle by various investigators have differed according to the levels of PBB ingested. Heifers fed 0.25 or 250 mg/day and cows exposed to Single 3 g dose failed to Show clinical signs (Moorhead et al., 1977). In contrast, cattle exposed to 1.13 g/day for 15 days (Gutenmann and Lisk, 1975) and 25 g/head/day for 30 to 60 days (Moorhead et al., 1977) developed a variety of nonspecific clinical Signs. Feed refusal, especially at higher dosage levels of PBB, was most consistently reported, a finding also reported in rats (Garthoff et al., 1976), guinea pigs (Sleight and Sanger, 1976), and chickens (Ringer and Polin, 1977). Other clinical observations in cattle included diarrhea, emaciation, dehydration, excessive lacrimation and abor- tions in pregnant heifers fed 25 g/head/day (Moorhead et 26 comparative lesions of PBB and PCB toxicosis. The gross lesions associated with PBB toxicosis are summarized as: enlarged livers, petechial hemorrhages, gastrointestinal hyperplasia, hemorrhagic gastroenteritis, subcutaneous edema, hydropericardium, thymic atrophy, enlarged kidneys, fetal death, and necrosis of cotyledons. In 1974, Norris et al. reported on the histopatho- logic changes associated with OBB toxicosis in rats. Thyroid hyperplasia was detected at all dose levels of OBB, whereas centrilobular cytOplasmic vacuolation of hepatocytes and hyaline degeneration of renal tubular cells occurred only in rats receiving the highest dose levels. Thyroid hyperplasia has since been confirmed in PBB-fed chickens (Ringer and Kowalski, 1977) and in rats exposed to iodine-adequate and iodine-excess diets con- taining 100 ppm PBB for 30 or 60 days (Mangkoewidjojo, 1979). Enlarged hepatocytes and cytOplasmic vacuolation were noted (Sleight and Sanger, 1976; Aftsomis et al., 1972), and degenerative renal tubular changes have been reported in conjunction with PBB toxicosis in cattle (Jackson and Halbert, 1974; Moorhead et al., 1977). Gutenmann and Lisk (1975) first described glandular hyperplasia of the major hepatic bile duct of sheep and cattle. Allen et a1. (1978) also detected marked hyper- plasia of bile ducts in a monkey exposed to 25 ppm PBB for 25 weeks. Likewise, Sleight et al. (1978) found bile duct hyperplasia and portal fibrosis in rats, and Moorhead 27 et a1. (1977) listed cystic hyperplasia of the gallbladder as a major histOpatholOgic finding in PBB-fed cattle. Farber et a1. (1978) reported on the histOpathologic effects of PBB toxicosis on bone marrow, Spleen, and lymph nodes of dogs. Marked reduction of hematopoiesis, accompanied by focal necrosis and reticuloendothelial hyperplasia, occurred in the bone marrow, whereas depres- sion of lymphocyte numbers and marked extramedullary hematopoiesis were the major histolOgic features in the lymph node and Spleen. Ultrastructural Changes From experiments with rats and guinea pigs, Sleight and Sanger (1976) described electron microscopic findings resulting from PBB ingestion. They demonstrated a dose- dependent effect on mitochondrial Size and number in PBB- fed rats. In addition, the authors observed vacuolation, marked hypertrophy of smooth endoplasmic reticulum (SER) and myelin body formation. Comparable results were recorded for guinea pigs exposed to lower dose levels of PBB. Later, published work by Corbett et al. (1978) listed comparable submicroscopic changes in the liver of rats fed 1,000 ppm PBB. Decreased glycogen, increased lysosomes and bile canalicular microvilli proliferation were also recorded by these investigators. These findings have Since been confirmed by Mangkoewidjojo (1979). He further reported on submicrosc0pic findings observed in the thyroid gland in rats fed 10 or 100 ppm PBB. 28 Effects of Chemical Toxicosis on Vitamin A MétabdlISm As early as 1947, Olafson reported finding significant depression of plasma vitamin A levels in cattle affected with hyperkeratosis. Hansel et al. (1951) confirmed this observation. They noted that plasma vitamin A was reduced to extremely low levels within 5 days after the substance (chlorinated naphthalene) that produced the disease was fed. Persistence of low levels of plasma vitamin A for 1 month following cessation of exposure to the toxic agent also occurred. Hoekstra et a1. (1954) reported similar findings and noted partial and temporary alleviation of the hyperkeratosis syndrome as a result of vitamin A therapy. Phillips (1963) first demonstrated that feeding 10 to 100 ppm DDT, [1,1,l-trichloro-Z,2-bis(p-chloropheny1)- ethane],.decreased liver stores of vitamin A in rats. Similar depression of hepatic vitamin A stores was recorded when yearling beef steers consumed forage sprayed with DDT. However, serum vitamin A levels were increased. In 1969, Tinsley also found that vitamin A stored in the liver was depressed by feeding 100 to 150 ppm DDT to male rats. He indicated that the degree of depression of liver vitamin A content was dependent on the levels of methionine in the diet, suggesting that the nutritional status of animals may be an important factor in the nature of their response to chemical toxins. In this regard, low protein diets have been demonstrated to protect rats from 29 heptachlor toxicity, whereas the toxicity of DDT, dieldrin and lindane is reduced by increasing the quantity and quality of dietary protein intake (Naber, 1977). Among the chemicals tested and demonstrated not to affect intestinal carotenoids or plasma and liver vitamin A, even at dosages known to be toxic to domestic animals, are: vanadium, molybdenum, selenium, and copper (Mitchell et al., 1968). The exact effect of dietary nitrate on vitamin A concentration is controversial. Phillips and Hatina (1972) and Cecil et al. (1973) found no effect of methoxychlor on hepatic storage of vitamin A in rats given a single dietary level (100 ppm), but Davison and Cox (1976) detected reduction of hepatic vitamin A content by the same chemical in rats fed over a wide range of dietary levels. Villeneuve et al. (1971) indicated that vitamin A concentration in the liver was lower in rabbits receiving Aroclor 1254 for 4 weeks than in control rabbits. Similar results were reported in rats exposed to 0.1% dietary PCB (Innami et al., 1976). In a series of experiments, they noted Significant reduc- tion of vitamin A concentration in the liver by the 3rd to 6th day of PCB ingestion with no further reduction there- after. The investigators recorded better growth rates for rats fed a diet containing 0.1% PCB and supplemented with 34,000 IU of vitamin A than rats fed a similar diet without supplementation. Fifty percent reduction in serum retinol binding protein was also listed, suggesting that marked depression of serum vitamin A had occurred. From 30 these results, Innami et al. (1976) concluded that PCB act to accelerate vitamin A deficiency in rats. Although clinical signs and histopathologic lesions Similar to those observed in hyperkeratosis have been reported for cattle (Jackson and Halbert, 1974; Gutenmann and Lisk, 1975; Moorhead et al., 1977) and in monkeys (Allen et al., 1978), data to indicate decreased hepatic vitamin A storage related to PBB intoxication have only been reported for rats (Mangkoewidjojo, 1979). He reported finding both dose- and time-dependent lowering of liver vitamin A content in rats exposed to l or more ppm PBB for 30 or 60 days. Other xenobiotics known to cause depression of liver vitamin A in rats include phenobarbital, caffein (Magdolna et al., 1969), ethanol (Baumann et al., 1942) and dieldrin (Lee et al., 1964). The exact mechanism by which the various chemicals precipitate lowered liver vitamin A content is not known. Hansel et al. (1951) noted increased plasma carotene when cattle affected with hyperkeratosis were fed large doses of carotene, but plasma vitamin A was not elevated, sug- gesting that chlorinated naphthalene exerts its vitamin A depressant effect by interfering with the conversion of carotene to vitamin A. Results of eXperiments with rats fed DDT in a vitamin A-deficient diet supplemented with either carotene or vitamin A 24 hours before killing do not support such a mechanism. From this, Phillips (1963) concluded that the action of DDT on liver vitamin A content 31 does not involve conversion of carotene to vitamin A but does involve some stage in metabolism common to both substances. A common denominator of many chemicals that result in lowered hepatic vitamin A content is induction of hepatic drug metabolizing enzymes (DME). This observa- tion has led some investigators (Innami et al., 1976; Kato et al., 1978) to assume that the liver vitamin A reducing activity of these foreign compounds might be due to the decomposition of vitamin A by DME or by a related metabolic system. MATERIALS AND METHODS Experimental Design and Method One hundred Sixty-eight young male Sprague-Dawley rats initially averaging 72 g each were obtained from a commercial source.a After 3 days of acclimatization, the rats were randomly divided into 4 groups of 42 rats each. Based on the length of the treatment periods, each major group was further divided into 7 subgroups and fed diets containing different concentrations of PBB (Table 1). Table 1. Experimental design for l8-month PBB feeding study Dietary PBB No. of No. of Rats in Treatment Periods (ppm) Groups Each Group (days) 0.0 7 6 10, 30, 60, 90, 180, 360 and 540 0.1 7 6 10, 30, 60, 90, 180, 360 and 540 1.0 7 6 10, 30, 60, 90, 180, 360 and 540 10.0 7 6 10, 30, 60, 90, 180, 360 and 540 3Life Science Div. of Mogul Corp., Madison, WI. 32 33 The rats were housed 3 per cage in wire top plastic cages. b and water were available ad Ground commercial rat food Zibitum. Control rats were maintained in a separate room. The source of the PBB used in this study was Firemaster BP-6.C The test diets were prepared biweekly for the first 6 months; thereafter, they were prepared according to requirements. The rats were observed daily for clinical Signs of toxicosis. Body weights were determined twice weekly for the first 2 months, weekly for the next 4 months and biweekly thereafter. Necrgpsy and PathOIOgical Examination Six rats from each group were necrOpsied at 10, 30, 60, 90, 180, 360 and 540 days. Feed was removed 12 hours before the rats were killed. Blood samples for serum and hematologic examinations were obtained by cardiac puncture following ether anesthesia. The rats were killed by exposure to chloroform. Urine samples for routine analysis were obtained at necrOpsy. Following systematic examination of organs of each carcass for gross pathologic changes, the liver, kidney and d Portions brain were weighed with a top-loading balance. of the liver, kidney, Spleen, testicles, heart, small intestine, stomach, urinary bladder, brain, lung, salivary bPurina Rat Chow, Ralston Purina Co., Checkerboard Square, St. Louis, MO. 'CMichigan Chemical Corp., St. Louis, MI. dMettler Series P, Model 163, Mettler Instrument Corp., Hightstown, NY. 34 gland, thyroid gland, pituitary gland, and adrenal gland were fixed in 10% buffered formalin for light microsc0pic examination. Samples of liver, kidney, brain, thyroid gland, and pituitary gland were fixed in 3% glutaraldehyde for transmission electron microscopy. Liver, kidney, adipose tissue and brain were collected for PBB and vitamin A analysis. These tissues were stored at -70 C until analyzed. Clinical Patholpgiclests Hematologic Examination Blood samples were collected by cardiac puncture following ether anesthesia. Heparine was the anticoagu- lant. Duplicate blood smears were made, fixed in methanol, f and stained by Wright's stain in an automatic stainer examined microsc0pically. Packed cell volume was deter- mined by use of microhematocrit tubes8 and hemoglobin was determined by standard cyanmethemoglobin method described by Benjamin (1969). Total red and white blood cells per deciliter were obtained by an electronic counter.h eSherwood Medical Industries, Evanston, IL. fAmes Hematek, Division of Miles Laboratories, Inc., Elkhart, IN. 8Capillary Tubes, Scientific Products, Evanston, IL. hCoulter Electronic, Inc., Hialeah, FL. 35 Urinalysis Urine samples were collected in plastic syringes at necrOpsy. Color was recorded and Specific gravity determined by refractive index as measured by a refrac- tometer.i Urinary glucose, blood, pH and protein content were determined by reagent strips.J Serum Protein Electrgphoresis Total protein of frozen sera was determined by refractive index as measured by a refractometer, prior to electrophoresis. Protein electrophoresis was performed 1 (pH 8.6-9.0) for 15 in a chamberk containing a buffer minutes at 180 volts. Proteins were stained with a Special stain.m Following dehydration in methanol for 2 minutes, the plates were cleared in 25% acetic acid in methanol for 5 to 10 minutes. The plates were then oven dried at 50 to 60 C for 5 to 10 minutes. Within 24 hours of electro- phoresis, strips were quantitated by densitometry.n Serum protein peaks were recorded in percentage and in grams per deciliter. 1Goldberg Refractometer, American Optical Co., Buffalo, NY. jLabstix, Ames Co., Elkhart, IN. kDeluxe Chamber, Gelman Instrument Co., Ann Arbor, MI. 1Helena Laboratory Corporation, Beaumont, TX. mPonseau 8., Helena Laboratory Corp., Beaumont, TX. nQuick Quant II, Helena Laboratory Corp., Beaumont, TX. 36 Vitamin A Analysis Liver vitamin A concentration of the experimental rats was determined by the fluorometric and Silicic acid column chromatOgraphic technique as described by Garry et a1. (1970) and Harris and Navia (1977). In preparation for vitamin A analysis, 1 gram of liver was homogenized in 4 ml of distilled water. One milliliter of the homogenate was saponified with 2 m1 of 1% ethanol pyrogallic acid and 1 m1 of 50% KOH. The mixture was heated at 60 C for 30 minutes and cooled at room temperature for 10 minutes, after which 4 dr0ps of ethanol were added to each sample. The samples were then extracted with 4 ml of glass- distilled petroleum ether (PB). The emulsion was centri- fuged at 1,000 rpm for 5 minutes and the petroleum ether (upper) layer transferred to 10 ml volumetric flasks for assay for vitamin A derivatives (retinol). The extraction was repeated once and the final extracts made up to 10 ml by addition of PE. Chromatographic microcolumns were prepared by insert- ing a small amount of glass wool into regular disposable pasteur pipettes (14.6 cm in length). Alternate layers of sand, glass beads, and 130 to 135 mg of Silicic acid were then added to the column. To the prepared columns 0.2 m1 sample of extract was transferred. Following washings of the column with 1 ml of PE, the residue on the column was eluted by 0.8 m1 of iSOpropanol. 37 The eluate was collected in diSposable tubes, made up to 1 ml by addition of isopropanol, and read in a fluoro- metero against a blank. The results were calculated using a standard curve that was prepared from known amounts of purified vitamin A.p PBB Analysis Liver and adipose tissue from PBB-fed rats was analyzed for 2,2',4,4',5,5' hexabromobiphenyl, the major congener of Firemaster BP-6 (Anderson et al., 1976; Sundstrom et al., 1976). Five-tenths gram of wet pooled tissue was ground with sand and anhydrous sodium sulfate and then extracted by boiling with petroleum ether. The extracts were filtered into 100 ml volumetric flasks and made up to 100 ml by addition of hexane. Twenty milli- liter aliquots were then transferred to screw-cap tubes and PBB residue separated from the remaining lipids by florisilq column chromatography by elution with hexane. Polybrominated biphenyl residues in the eluate were analyzed by gas-liquid chromatography on a gas chromato- graphr equipped with electron capture detector with nitrogen as the carrier gas. Operating conditions were column oTurner Fluorometer, Arthur H. Thomas Co., Philadelphia, PA. pRetinol, Crystalline Synthetic Type X, Sigma Chemical Co., St. Louis, MO. qFlorisil, Fisher Scientific Co., Cleveland, OH. rVarian Instrument Division, Palo Alto, CA. 38 250 C, detector 310 C, injector 280 C, and carrier gas 30 ml per minute. Gas-chromatograph readings were recorded, and concentrations of PBB were calculated by multiplication of the gas chromatograph reading by a calibration factor derived from standards containing 50, 100 and 200 ppm PBB prepared from Firemaster BP-6. HistoPatholOgic Procedure Following fixation in 10% buffered formalin, tissues for histologic examination were embedded in paraffin, sectioned at 6 u in thickness, and stained with hematoxylin and eosin. Special stains for glycogen or fat were used as indicated. Transmission Electron Microsc0py Sections of liver obtained at necropsy were fixed in Karnovsky‘s fixative and washed in Zetterqvist's solution (Pease, 1964) at pH 7.4. They were then postfixed in 1% osmium tetroxide in Zetterqvist's solution. After dehy- dration in graded alcohol (50, 70, 95% and absolute), they were transferred to propylene oxide. Selected Speci- mens were then embedded in a mixture of Epon and Araldite. To facilitate correlation between light and electron microsc0pic examination, semithin sections of the embedded material were stained with toluidine blue and viewed with a light microsc0pe. Thin sections were stained with uranyl acetate and lead citrate and examined with an electron microscope.5 SEM 952, Carl Zeiss, Germany. RESULTS General Clinical signs of toxicosis or hypovitaminosis A were not observed throughout the course of the experiment. In general, PBB—fed rats ate well, maintained a healthy appearance and gained weight as well as, and in some cases better than, control rats (Figure 1). During the course of the eXperiment, 4 rats died, 2 develOped posterior paralysis and were killed 12 days prior to date of scheduled necropsy, and a female rat was eliminated from the study. DevelOpment of posterior paralysis or death of the experimental rats was not related to PBB toxicosis. NecrOpsy and histOpathologic findings on 1 control and 2 PBB-fed rats in the 540-day group indicated that all 3 rats succumbed to age-related renal failure. The fourth rat from the same group had broken upper central incisors and died from starvation. Of the 2 rats that had posterior paralysis, 1 had a tumor (meningioma) involving the thoracic Spinal cord and 1 had been traumatized. LaboratogygFindings Urinalysis No consistent abnormality in values for Specific gravity, pH, glucose, ketone, bilirubin or blood was 39 Weight Gain (Grams) 500 400 300 200 I00 40 4---o Control °---0 01 PPM ........... IO PPM III—4 lO-O PPM 03 I 2 37" 6 l2 l8 Time (Months) Figure I Mean Weight Gain in Rats Fed PBB 41 observed in the experimental rats. Proteinuria (30 to 100 mg/dl) was a constant feature in control and PBB-fed rats up to 360 days. At 540 days, urinary proteins were increased (300 to greater than 1,000 mg/dl) in control and PBB-fed rats. Hematology Results of hematologic studies are given in Table 2. Although sporadic significant differences (p50.05) in hematocrit, hemoglobin, and total white and red blood cell counts occurred at 10, 60, 360 and 540 days, all values excepting total red blood cells were well within the normal range. With few exceptions, mean total red blood cell counts were consistently lower than normal values in control and PBB-fed rats. In addition, morphologic appearance of erythrocytes, leukocytes and platelets was essentially normal. Serum Electrophoresis The mean total serum protein values were all within normal range. Component protein fractions were, however, altered resulting in changes in the albuminzglobulin (A/G) ratio. Mean albumin values for control and PBB-fed rats were within normal range at 10, 30 and 60 days. The values thereafter decreased to below normal from 90 to 540 days. Concomitant relative and occasional absolute increases in serum globulin fractions, especially alpha 2 (a2) globulins, accompanied the changes in albumin. The resultant effect was reduction in the A/G ratio, which 42 Ii 1! N N NN NN N.N N.N N.NH N.NN N.NH NNH N N NN NH N.N N.N N.NH N.NN N.H NNH N N HN NN N.N N.N N.NH N.NN H.N NNH N N NN NN N.N N.N N.NH N.NN N.N NNH N N NN NH N.N N.N N.NH N.NN N.NH NN N N NN NH N.N N.N N.NH N.NN N.H NN N N NN NH N.N N.N N.NH N.NN H.N NN N N NN NH N.N H.N N.NH N.NN N.N NN N N NN NH N.N AHN.N N.NH N.NN N.NH NN N N NN NH NN.N HH.N N.NH N.NN N.H NN H N NN NH N.N N.N N.NH N.NN H.N NN N N NN NH N.N N.N N.NH N.NN N.N NN N N NN NH N.N N.N N.NH N.NN N.NH NN H N NN N N.N N.N N.NH N.NN N.H NN N N NN NH N.N N.NH N.NH N.NN H.N NN N N NN NH N.N N.N N.NH N.NN N.N NN N N NN NH N.N N.N N.NH N.NN N.NH NH N N NN N N.N N.NH N.NH N.NN N.H NH N N NN HH N.N N.N N.NH NN.NN H.N NH N N NN NH N.N N.N N.NH NN.NN N.N NH :HNom oaoziieaexHi,Nsmz HHN\NNHxN HHN\NNHxN HHN\NN a: HNN Hum HENNN NNHN HNN HNHpcoNonHo um: NNN UN; NNN NHNNNHN no NNNQ mmm pom mums :N muasou opxooxsoa Hmwucopomwww wan .mucsoo ouxoopguxno can buxooxsoa Hmuou .uwuuoumEo: .cfipofiono: cmoz .N manna .mafiadoafimoo n :Nmom .mouzoocoe n One: .mouxuogmexa u nae»; .mawgdonuso: u usoz .oaou no: u 92 .Amo.ownv monam> Hopucoo scum ucohommfiv xaucmoNMNGMNmn .mHMR O OH MHZ HOW mflmwe Ohm wOSHm>w 43 II III H N NN NN N2 N.N NN.NH NN.HN N.NH NNN N N HN NN az N.N N.NH N.NN N.H NNN N N No NN az N.N N.NH N.NN H.N NNN H N NN HN N2 N.N N.NH N.NN N.N NNN N N HN HH oz N.N NN.NH NN.NN N.NH NNN N N NN NH oz N.N NN.NH NN.NN N.H NNN N N NN NH N2 N.N NN.NH N.NN H.N NNN N N NN NH N2 N.N N.NH N.NN N.N NNN :HNom, 0e82, ENENH. Nemz HHNNNNHNN HHNNNNHNN HHNNNN a: HNN so: HENNN NNHN HNN HNHNNNNNNNHN 0N2 NNN UN: NNN NNNNNHN :0 NNNN III Iitl'i'l. I'll". I'll-III II IIIIIIIII." 'Iillll III. III.- IIIIl-ii'lnikllll "I. II..." Illllltl‘tlii'l III '1' "I I... II III I‘- II". III II "I "It '1' ".1 n "H. III I" III " H II III II n " III III .llll "“.IIII|"|".IOI. " " ollll- “. u " "“"fluunnurll H.N.NNOUN N .HNNN 44 was especially noticeable in control and PBB-fed rats at 90 through 540 days (Table 3). Organ Weights The mean body and liver weights, relative liver weights and statistical Significance of differences are given in Table 4. The mean (g) and relative (% of body weight) liver weights of rats receiving 10 ppm dietary PBB were significantly increased (p<0.05) over control values at all treatment periods (10 through 540 days). In contrast, the mean and relative liver weights of rats receiving either 0.1 or 1.0 ppm PBB were only significantly different from control values at 10 and 30 days. These results suggest that after 30 days a dosage greater than 1 ppm PBB was necessary to maintain the stimulus responsible for increased liver Size. Neither mean nor relative kidney or brain weights were significantly affected (p>0.05) by dietary PBB (Appendix A). Polybrominated Biphenyl Content of Tissues The mean PBB concentrations of pooled samples from the liver and adipose tissue expressed as ppm PBB (fat basis) are given in Tables 5 and 6. A definite dose-related increase in residual PBB occurred. Similarly, with few exceptions, a time-dependent increase in PBB content occurred in tissues from rats fed 1 or 10 ppm PBB. Samples from the liver had higher concentrations of PBB than samples from adipose tissue. 45 Table 3. Serum proteins (total protein, albumin, a2 globulin values and A/G ratios) of representa- tive control rats and rats fed PBB ============================== 3:3-- =—=“ Time Number of Dietary PBB_(ppm) (days) Samples 07 0.1 1.0 Total Protein (g/dl) 10 2 4.60 5.00 5.30 5.40 30 2 6.70 6.40 7.10 6.50 60 2 8.70 7.00 7.803 7.20 90 2 6.80 6.40a 7.00 6.00 180 2 5.503 7.00 6.40 7.20: 360 3 7.20 6.90 6.50 6.50 540 4 7.50 7.10 6.00 6.00 Albumin (g/dl) 10 2 2.47 2.87 2.77 2.82 30 2 3.03 2.79 2.77 2.93 60 2 3.88 3.14 3.33a 2.91 90 2 2.60 2.59a 2.59 2.69 180 2 ND 2.46 2.57 2.36 360 5 2.25 2.19 2.26 2.08 540 4 2.00 1.94 1.81 1.78 a2 Globulin (g/dl) 10 2 1.01 1.04 1.20 1.28 30 2 1.99 1.88 1.86 1.73 60 2 2.45 1.85 1.98a 2.10 90 2 2.05 2.123 2.22 1.71 180 2 ND 2.56 1.73 2.08 360 4 2.67 2.55 2.35 1.75 540 4 2.88 2.95 2.21 1.73 A/G.Ratio 10 2 1.30 1.35 1.13 1.15 30 2 0.83 0.79 0.66 0.82 60 2 0.84 0.82 0.75a 0.69 90 2 0.65 0.67a 0.59 0.84 180 2 ND 0.55 0.67 0.49 360 4 0.46 0.47 0.54 0.49 540 4 0.37 0.37 0.44 0.43 8Values for N=l. ND = not done. 46 .mmo.ovdv osHm> Hepucoo Eoum ucopommflw HHuamoNMNcmNmo .Oflz HOW CNGE GHQ mQSHm>m UH.m N.m N.m N.m uo.o~ m.mH w.NH H.0H QHNm nmnm nomm nmom oem ov.m m.m m.m m.m oo.m~ o.w~ N.NH N.NH Nam mew Nmm mom com UH.N N.m N.m N.m oo.mH «.mH N.©H N.NH NwN nmwe wow move owa oe.m N.m N.m H.m um.mH N.mH m.NH m.~H CNN mac wwm Ham om N.N N.m m.m m.m om.mH m.HH N.NH N.NH New New oem own so UN.N UN.N Um.N N.m ow.HH om.HH oo.~H m.m NmN com LHNN Nmm om um.m UN.m oo.m o.v oc.N um.N om.N m.o mNNH HNH ova and oH Edd OH and H and H.o Edd o Ema OH Ema H EQQLH.D Ema o EdfimHn Ema Ema H. o and 0 «man “NV mucmwoz po>NH o>HumHom Hwy mpnwwo: Ho>wq :moz V mwlwioz Avon new: no mxwa mmm pom Nona :N mpnmfloz No>NH o>prHoN can No>NH .Awon :moz .N oanmb 47 Table 5. The concentrations (ppm) of PBB in the fat of liver from rats fed diets containing PBB D1etary PBB __p Days on Diet (ppm) 30 60 90 180 “360 540 0 0 0.93: 0.50 0.82 1.10 0.43 0.68 (1.7) (1.6) (1.7) (2.3) (2.1) (2.4) 0 1 0.58 1.00 4.80 2.30 6.70 4.70 (2.2) (2.3) (1.7) (2.9) (2.3) (2.6) 1 0 6.70 7.70 18.50 24.40 40.00 70.40 (1.7) (2.3) (2.1) (2.5) (2.5) (1.6) 10.0 55.90 - 59.70 186.10 404.40 726.60 627.20 (1.8) (2.6) (3.0) (3.0) (6.7) (6.6) aThe values are means of 2 pooled samples from 2 or 3 rats each. b Numbers in parentheses represent percent fat. 48 Table 6. The concentrations (ppm) of PBB in the fat of adipose tissue from rats fed diets containing PBB D1etary PBB _ Days on Diet (ppm) 30 *60 90 *180 360 ‘—540 0.0 0.04: 0.04 0.06 0.05 0.86 0.06 (69) (71) (65) (72) (53) (57) 0.1 0.41 0.72 1.80 1.60 7.10 4.90 (66) (75) (63) (70) (60) (58) 1.0 6.20 15.20 12.10 20 30 36.70 63.40 (64) (69) (72) (68) (67) (63) 10.0 59.90 84.80 142.40 169.90 542.60 630.00 (68) (70) (70) (68) (66) (62) aThe values are means of 2 pooled samples from 2 or 3 rats each. b Numbers in parentheses represent percent fat. 49 Residual concentrations of PBB detected in the samples from control rats are probably the result of technical problems associated with the extraction and analysis of PBB. Liver Vitamin A Content Liver vitamin A content was assayed for all eXperi- mental rats except rats from the 10-day group. The mean vitamin A values expressed on a microgram per gram (ug/g) or milligram per whole liver (mg/liver) wet weight basis are given in Tables 7 through 12. The mean hepatic vitamin A of control rats was 190.1 ug/g and 1.8 mg/liver at 30 days and 827.0 ug/g and 13.3 mg/liver at 540 days. In rats receiving 10 ppm dietary PBB for 30 days, hepatic vitamin A content was 96.9 ug/g and 1.1 mg/liver, both values being significantly lower than the corresponding control values (Table 7). Although the liver vitamin A concentrations of rats fed either 0.1 or 1 ppm of PBB were similarly depressed, the differences were only sig- nificant when the data were calculated on a ug/g basis. In fact, calculation of the data on a mg/liver basis indicated that rats receiving 1 ppm of PBB had Slightly higher hepatic vitamin A levels (1.9 mg/liver) than control rats (1.8 mg/liver). At 60 days, rats fed diets containing 1 or 10 ppm of PBB had Significantly lower liver vitamin A values than control rats (Table 8). In contrast, the relative (ug/g) hepatic vitamin A value for rats fed diets 50 Table 7. Liver vitamin A content in rats fed PBB for 30 days Dietary Level Liver Weight Vitamin A (ppm) (g) ug7g Liver mg/Liver a b 0.0 9.5 i 0.8 190.1 f 12.2 1.8 i 0.2 0.1 12.0 i 1.0bC 122.0 i 26.4C 1.5 i 0.4 1.0 11.3 i 1.0ae 164.5 i 11.3d 1.9 i 0.3 10.0 11.8 i 1.9af 96.9 i 22.1C 1.1 i 0.3d aValues are mean i SD; N=6. bN=5. C’d’e’fSignificantly different from control value (p<0.001, p<0.005, p<0.01 and p<0.025, reSpectively). Table 8. Liver vitamin A content in rats fed PBB for 60 days Dietary Level Liver Weight Vitamin A (Ppm) (8) ug/g Liver mg/Liver 0.0 11.2 2 1.03 294.9 2 25.7 3.3 i 0.4 0.1 11.4 i 1.1 328.1 : 23.6e 3.7 i 0.4 1.0 11.9 i 1.8 222.7 i 37.5C 2.6 i 0.3d 10.0 15.3 : 1.8b 144.6 : 16.5b 2.2 : 0.4C aValues are mean 1 SD; N=6. b’C’d’eSignificantly different from control value (p<0.001, p<0.005, p<0.025 and p<0.05, respectively). 51 containing 0.1 ppm of PBB for the same time period was increased over the value for control rats. Only in rats receiving 10 ppm of PBB in their diets was there signifi- cant depression of hepatic vitamin A concentration at 90 days (Table 9). Hepatic vitamin A content was, however, decreased in rats ingesting 0.1, l or 10 ppm of dietary PBB in the l80-day experimental group (Table 10). Like- wise, rats consuming feed containing 0.1, 1 or 10 ppm PBB for 360 days had significantly lower vitamin A levels in their liver compared to control rats (Table 11). Reduction in hepatic vitamin A concentration was especially marked for rats receiving 10 ppm PBB in the 360-day group, the decrease in vitamin A concentration being lO-fold on a ug/g and 6-fold on a mg/liver basis, compared to control values. Similarly, marked reduction in vitamin A levels (17- and 10-fold) was recorded for rats fed 10 ppm dietary PBB for 540 days (Table 12). Miscellaneous Laboratory Findings Thyroid hormones. Serum thyroxine (T4) and triiodo- thyronine from randomly selected rats from the 10-, 90- and 540-day groups were evaluated by radioimmunoassay. Although a single significant decrease in T4 (Appendix B) was found for rats fed 0.1 ppm for 10 days, the toxicological Sig- nificance was not evident. The hormone values were quite variable, lacked a dose response relationship, and showed consistent overlapping of individual values from control and PBB-fed rats. 52 Table 9. Liver vitamin A content in rats fed PBB for 90 days Dietary Level Liver Weight Vitamin A (ppm) (8) u§7g Liver mg/LiVer 0.0 12.3 N 1.1a 351.8 N 69.7 4.3 N 1.0 0.1 12.3 N 0.9 346.1 N 57.8 4.2 N 0.6 1.0 13.4 N 1.3 351.5 N 45.2 4.7 N 0.6 10.0 13.9 N 0.6C 222.5 N 45.4b 3.1 N 0.6d —_ - _.__‘ aValues are mean i SD; N=6. b’C’dSignificantly different from control value (p<0.005, p<0.025 and p<0.05, respectively). Table 10. Liver vitamin A content in rats fed PBB for 180 days Dietary Level Liver Weight Vitamin A (Ppm) (g) ug/gTLiver mg/Liver 0.0 14.8 N 1.7b 478.6 N 54.5 7.1 N 0.8 0.1 16.7 N 2.03 401.9 N 32.4d 6.8 N 1.2 1.0 15.4 N 0.8b 334.1 N 23.9C 5.1 N 0.3C 10.0 19.6 N 1.2ac 159.8 N 37.1C 3.1 N 0.6C aValues are mean i SD; N=5. bN=6. C’dSignificantly different from control value (p<0.001 and p<0.025, respectively). 53 Table 11. Liver vitamin A content in rats fed PBB for 360 days Dietary Level Liver Weight Vitamin A (ppm) (g) ug/g Liver mg7Liver 0.0 17.8 N 3.23 626.6 N 54.0 11.0 N 1.5 0.1 17.7 N 2.6 421.9 N 35.8b 7.4 N 1.0b 1.0 18.0 N 1.9 352.1 N 66.8b 6.2 N 0.7b 10.0 25.6 N 3.4C 59.6 N 12.2b 1.5 N 0.4b aValues are mean 1 SD; N=6. b’CSignificantly different from control value (p<0.001 and p<0.005, respectively). Table 12. Liver vitamin A content in rats fed PBB for 540 days Dietary Level Liver Weight Vitamin A (Ppm) (8) ug7g—Liver mg/Eiver 0.0 16.1 N 1.6a 827.0 N 178.7 13.3 N 2.9 0.1 17.8 N 1.7 763.2 N 208.3 13.6 N 3.6 1.0 19.5 N 4.4 441.0 N 79.2C 8.4 N 1.4d 10.0 26.2 N 3.5b 46.2 N 18.8b 1.2 N 0.4b aValues are mean t SD; N=5. b’C’dSignificantly different from control value (p<0.001, p<0.005 and p<0.01, respectively)- 54 Testosterone. Mean serum testosterone values for representative control rats and rats fed PBB for 10, 30, 60, 90 and 180 days are given in Appendix C. There was no significant effect of dietary PBB on testosterone concentrations. Pathologic Findings Gross Lesions Gross lesions were confined to the liver and kidney. £1121! Except for enlarged livers in the PBB-fed rats, no Significant lesions were seen at 10 and 30 days. The livers from 3 of 6 rats fed diets containing 1 ppm of PBB for 60 days had a somewhat prominent lobular pattern. These livers were also quite pale. A Slightly mottled appearance was evident in the liver from 1 of 6 rats fed a diet with 10 ppm of PBB for 90 days. Likewise, the livers from 3 of 6 rats consuming a diet with 10 ppm of PBB for 180 days were yellowish in color and had several small (2 mm) whitish foci irregu- larly distributed over the dorsal surface. Rounding of the edges and increased friability were evident in l of these livers. A control rat from this group also had some of these small white foci. The lesions observed in rats killed at 360 days were present in 6 of 6 rats fed diets with 10 ppm of PBB. The changes included moderate to marked mottling, yellowish to capper-colored appearance, and rounded edges. Similar changes were noted in the 55 livers from 5 of 5 rats fed diets containing 10 ppm of PBB for 540 days. In addition, a moderately large (1.5 cm x 0.5 cm), white, papillary growth involving the middle hepatic lobe was present in l of these rats. The livers from 2 of 5 rats fed diets containing 1 ppm of PBB for 540 days also showed some mottling of the dorsal surface. Kidney. Pitting of the renal capsule, paleness, and a few darkly discolored kidneys were the major gross findings. Occasional small cysts were also seen. One or more of the above changes were seen in the kidneys from PBB-fed and control rats. Incidental gross findingg. In rats killed at 30 days, right testicular hypertrOphy with an associated vestigial left testicle was found in 1 of 6 rats fed 10 ppm dietary PBB. Additionally, prominent Peyer's patches were evident in the small intestines from the control and PBB-fed rats. Histopathologic Findings Significant histologic alterations were mainly limited to the liver. At 10 days, all PBB-fed rats had a moderate to marked increase in mitotic activity of hepatocytes. The mean numbers of mitotic figures observed in 10 con- secutive (25X objective) microscopic fields of hepatocytes from 3 of 6 rats fed diets with 0.1, 1 and 10 ppm PBB were 8.7, 8, and 3.7, respectively. In contrast, the mean mitotic index derived from the same number of microscopic fields from 3 of 6 control rats was 0.7. Diffuse cytOplasmic 56 vacuolation and swollen hepatocytes were also prominent features in the livers from these rats (Figures 2 and 3). The cytoplasmic vacuoles varied in size but were generally large and extended from the centrally located nuclei to the periphery of individual cells. These vacuoles did not stain with oil red 0. Except for moderately swollen hepatocytes, the livers from rats fed 1 or 10 ppm PBB for 30 and 60 days were essentially normal. Similarly, the livers from all control rats and all rats fed diets with 0.1 ppm of PBB from 30 through 540 days were normal. In the livers from rats receiving 1 or 10 ppm PBB for 90 and 180 days, there was prominent swelling of hepatocytes and limited midzonal vacuolation that was particularly noticeable in the livers from rats receiving 10 ppm of PBB for 180 days. At 360 and 540 days, the changes were most severe in rats consuming 10 ppm dietary PBB. The hepatic alterations included intracytOplasmic lipid accumulation, advanced degenerative hepatocellular changes and focal areas of necrosis. Individual hepatocytes were enlarged, resulting in obliteration of sinusoidal Spaces. The nuclei of some affected hepatocytes were karyorrhectic or absent. Occa- Sinal nuclei were pyknotic. The cytOplasm of the nonvacuo- lated cells varied from granular to an increased homogeneous and eosinophilic. Occasional foci of necrosis were par- tially replaced by infiltration of inflammatory cells that were mainly mononuclear (Figure 4). In addition, occasional clusters of yellowish-brown pigmented macrOphages were observed in livers from rats receiving 10 ppm PBB for 540 57 Figure 2. Diffuse perinuclear vacuolation and increased number of mitotic figures (arrows) in the liver from a rat fed a diet containing 0.1 ppm of PBB for 10 days. HGE stain; X200. 58 Figure 3. Higher magnification of the liver from the rat in Figure 2, to Show the absence of sinusoidal Spaces and prominent mitotic figures (arrows). HGE stain; X500. 59 Figure 4. Focal areas of necrotic hepato- cytes were replaced by inflammatory cells that were mainly mononuclear. Occasional neutrophils (arrow) were present in some of these foci. Tissue from a rat fed a diet with 10 ppm of PBB for 540 days. 60 days. These pigmented macrophages were located adjacent to vacuolated areas, central veins and portal triads. Intracytoplasmic lipid accumulation, confirmed by oil red O staining, was the most consistent lesion. The vacuoles varied in size from large vacuoles that practi- cally occupied the entire cytoplasmic Space to the more abundant fine vacuoles that resulted in a foamy appearance of the affected hepatocytes (Figure 5). The cytoplasmic vacuolation was irregular in distribution but was more commonly midzonal with extensions to the peripherolobular and occasionally to the centrolobular areas (Figure 6). Approximately 50 to 80% of the hepatocytes in individual hepatic lobules were affected in rats receiving 10 ppm PBB for 360 or 540 days. In the livers from rats fed diets containing 1 ppm PBB for 360 and 540 days, a similar pattern of lipid accumulation occurred. A more typical midzonal involvement with extension to the periphero- lobular areas, as illustrated in Figure 7, was evident. In addition, marked periportal fibrosis (Figure 8) with accompanying focal lymphocytic infiltration was observed in 2 of 5 rats fed 1 ppm PBB for 540 days. Bile duct hyperplasia was also evident in these livers. Other Histopathologic Findings Lung. Varying degrees of focal interstitial pneumoni- tis were observed in the lungs from several control and PBB-fed rats. These lesions were evident in rats necropsied from 90 days onwards. In no case was there involvement of 61 Figure 5. plasmic lipid draplets gave a foamy appearance to Typical small, multiple intracyto- the hepatocytes located in the midzonal area of the hepatic lobule. Tissue from a rat fed a diet with 10 ppm of PBB for 360 days. HGE stain; X200. 62 Figure 6. Tissue from a rat fed a diet with 10 ppm of PBB for 540 days. There is intracytoplasmic lipid accumulation involving some hepatocytes adjacent to the central vein. HEE Stain; X200. 63 Figure 7. Intracytoplasmic lipid accumula- tion involving the mid- and peripheral zones in the liver from a rat fed a diet containing 1 ppm of PBB for 540 days. Relatively normal hepato- cytes are adjacent to the central vein (CV). HGE stain; X200. 64 Figure 8. Portal fibrosis and bile duct hyperplasia with associated focal infiltration of lymphocytes (arrow) in the liver from a rat fed a diet with 1 ppm of PBB for 540 days. HGE stain; X80. 65 more than 25 to 30% of a particular lung lobe. A limited amount of hyperplasia of bronchiolar-associated lymphoid tissue also occurred. Pituitagyygland. Multiple pituitary cysts were observed in the pituitary gland from a rat exposed to 10 ppm PBB for 180 days. The interior of the cysts contained proteinaceous material and was lined by elongated, flat- tened cells. There was also an adenoma involving the anterior hypophysis from a rat fed 1 ppm PBB for 540 days. Thyroid gland. The microsc0pic changes observed in thyroid glands included marked follicular dilatation, flattening and atr0phy of follicular lining cells, resulting in hyalinization and thinning of the follicular basement membrane. Mild hyperplasia and vacuolation of follicular epithelium were also evident. These changes were present in the thyroid glands from control rats and PBB-fed rats. Additionally, a thyroid adenoma occurred in the thyroid gland from a rat fed 10 ppm PBB for 360 days. Kidney. Age-related progressive nephrosis charac- terized by the accumulation of eosinOphilic renal tubular casts, atrOphy of tubular epithelium and tubular dilata- tion was observed in control and PBB-fed rats necrOpsied as early as 180 days. In some kidneys, occasional foci of calcification and lymphocytic infiltration accompanied the nephrosis. The cause of dark discoloration observed grossly was not evident histOIOgically. 66 Liyeg, Microscopically, the papillary growth seen in the middle hepatic lobe from a rat fed a diet containing 10 ppm of PBB for 540 days was a cystic mucoadenoma originating from bile duct epithelium. Accumulation of glycogen was not seen in any of the Carnoy's-fixed and periodic acid-Schiff (PAS)-stained liver sections obtained from selected control and PBB-fed rats killed at 30, 60, 90, 180, 360 and 540 days. Histologic findings in the livers from rats killed at 60 days were poorly correlated with the gross observations. Spleen. Increased hematopoietic activity was com- monly observed in the spleens from several control rats and rats fed diets with PBB. Moderate vascular congestion and increased amounts of hematogenous pigment were also evident in some spleens. Adrenal_glands. Occasional foci of vacuolation were evident in the adrenal cortex of many rats, including control rats and PBB-fed rats. Small intestines. Increased numbers of globular leukocytes and occasional intestinal nematode parasites were seen in the intestines from several rats. There was also focal hyperplasia of Peyer's patches leading to atrophy of intestinal smooth muscle and elevation of the overlying serosal surface. One or more of the above observations were present in representative numbers of control rats and PBB-fed rats. 67 Other tissues. No Spontaneous or PBB-related lesions were found in the testicle, heart, stomach, urinary bladder, brain or salivary glands from any rat. Electron MicrOScopic Findings Electron microsc0pic Studies were carried out on selected livers from rats exposed to dietary PBB for 10, 90, 180, 360 and 540 days. The ultrastructural features of hepatocytes from a control rat are illustrated in Figure 9. Except for occasional cyt0plasmic vacuoles and mild proliferation of the smooth endoplasmic reticulum (SER), hepatocytes from control rats were basically normal. In general, the livers from PBB-fed rats showed time- dependent progressive hepatocellular changes that were especially prominent in rats fed diets with the highest dosage (10 ppm) of PBB. The ultrastructural lesions were characterized by proliferation and dilatation of SER (Figure 10) with accompanying disorganization and reduc- tion of rough endOplasmic reticulum (RER). Mitochondria were enlarged and reduced in numbers and had undergone degenerative changes. In some instances, the degenerative mitochondrial changes appeared to involve imbibition of fluid resulting in swelling and disintegration of cristae (Figure 11). Many intracytOplasmic vacuoles that varied in size, shape and internal density were observed, especially in the hepatocytes from rats killed at 360 and 540 days (Figures 12 and 13). Myelin body formation was a consistent finding, although these bodies were more 68 Figure 9. Electron micrograph of hepato- cytes from a control rat. Notice the normal complement of mitochondria (M) randomly distributed within the cytoplasm. Parallel aggregations of rough endoplasmic reticulum (RER) Show normal orientation towards the nucleus (N). Occasional cytoplasmic vacuoles are present. Lead citrate and uranyl acetate stain; X4,700. 69 Figure 10. Electron micrograph of a portion of a hepatocyte from a rat fed a diet containing 10 ppm of PBB for 540 days. There is an abundance of vesiculated Smooth endoplasmic reticulum (SER) and some free ribosome (arrow). There is also a relative absence of other cytOplasmic organelles. Lead citrate and uranyl acetate Stain; X21,000. 70 Figure 11. Electron micrograph of part of a hepatocyte from a rat fed a diet containing 1 ppm of PBB for 10 days. Notice the swollen mito- chondrion (M) with disruption of the cristae and numerous clear spaces (CS) within the cytOplasm. Lead citrate and uranyl acetate stain; X21,000. 71 Figure 12. Electron micrograph of a hepato- cyte from a rat fed a diet containing 10 ppm of PBB for 360 days. Medium density lipid droplets (L) predominate. Lead citrate and uranyl acetate stain; X4,700. 72 Figure 13. Electron micrograph of a hepato- cyte from a rat fed a diet containing 10 ppm of PBB for 540 days. Notice the variation in size and shape of the cytoplasmic lipid droplets (L). Increasednumbersof dense bodies (DB) are present within the vicinity of the bile canaliculus (BC). Lead citrate and uranyl acetate stain; X5,060. 73 numerous in hepatocytes from rats receiving 10 ppm PBB for 540 days (Figure 14). In some cases the myelin bodies had vacuoles or cytoplasmic organelles trapped within the interior (Figure 15). Some of the trapped vacuoles con- tained small whorled figures (Figure 15) and had an outer double-layered membrane that appeared to be continuous with the SER forming the myelin body. Some of the larger myelin bodies were formed by aggregation of smaller bodies. Definite continuity between myelin bodies and the SER or RER was evident in some hepatocytes. Other ultrastruc- tural lesions included swollen hepatocytes with alternating clear and finely granular Spaces between segregated and adjacent organelles and increased amounts of free ribo- somes that were usually adjacent to disrupted or denuded RER. In addition, moderate increases in lysosomes were also observed. 74 Figure.l4. Electron micrograph of a hepato- cyte from a rat fed a diet containing 10 ppm of PBB for 540 days. There are multiple myelin bodies of different sizes. Lead citrate and uranyl acetate stain; X5,200. . ,, .0’ “5.9 r .-,- I a. ’, 7 1 . Q ("EV/5' ', "Vi! ‘1‘! O Figure 15. Electron micrograph of a hepato- cyte from a rat fed a diet containing 10 ppm of PBB for 360 days. Notice the large myelin body (MB) with a membrane-bound vacuole (V) containing a whorled figure (Wf) and a partially trapped mitochondrion (M). The core of the myelin body is made up of paired smooth membranes that show continuity with the RER (arrow). Lead citrate and uranyl acetate stain; X20,600. DISCUSSION There is enough scientific evidence to implicate PBB as a primary hepatotoxin. With few exceptions, the majority of the data available have been derived from experimental animals subjected to medium or large doses of PBB. Part of the rationale for using such doses was based on data that suggested that Firemaster BP-6 was of relatively low toxicity. For example, the acute LDSO in rats was reported to be 21.5 g/kg (Michigan Chemical Corporation, 1974). The results of the present research clearly point out that even at very low dietary levels of 0.1 ppm, PBB is capable of precipitating significant biochemical and morphologic tissue alterations in rats. Further, the results of the present study also indicate that thorough morphologic and biochemical evaluation of experimental animals is necessary before a given chemical can be considered toxicologically and environmentally safe for production. Based on the customary screening tests, such as weight gain, urinalysis, mortality rate and hematologic parameters, the rats in the study reported here could be classified as normal. In Spite of such clinical normalcy throughout the course of the experiment, detailed biochemical and morphologic evaluations revealed 76 77 distinct hepatic alterations, indicating that these rats were not normal. The most important finding obtained from the present investigation was the statistically Significant increase (p<0.05) in relative liver weight recorded at all 3 dietary levels of PBB at 10 and 30 days. Although liver to body weight ratios were significantly increased in rats sub- chronically fed 1 or more ppm PBB (Sleight and Sanger, 1976; Dent et al., 1976a,b), data to indicate such effects at lower dosages have not been reported for any experi- mental animals. Morphologically, the hepatomegaly appeared to be the result of hepatocellular hypertrophy and hyperplasia, since prominent mitotic activity was evident in the hepatocytes from the rats killed at 10 days. In addi- tion, fluid imbibition, as recognized by perinuclear vacuolation that failed to stain with oil red O and by clear cyt0plasmic spaces ultrastructurally, may have con- tributed to the increased relative liver weight. Although fluid imbibition was not mentioned by Lee et al. (1975), the observations in the rats killed at 10 days in the present study are in good agreement with their results. They reported finding markedly increased mitotic activity in the hepatocytes from rats gavaged with a single oral dose of either 1000 or 3000 mg/kg of OBB. Gradual decline in mitotic activity occurred in both treated groups at the 5th and 13th day after treatment. The investigators indicated that the continued decline in mitotic activity 78 could account for the absence of such lesions in a pre- vious 28-day Study. A Similar explanation may be applicable to the present study, Since neither the cytoplasmic vacuolar changes nor increased mitotic activity were seen in hepatocytes obtained from the rats killed at 30 days. The significant increases (p<0.05) in relative liver weights of rats consuming diets containing 10 ppm for 60 days and thereafter are probably the result of increased hepatic lipid content (Table 5). Garthoff et al. (1976) indicated that all liver lipid fractions, including total fat, cholesterol, phospholipid and neutral lipids, were increased in rats fed 50 and 500 ppm of PBB. In a more recent report, Harris et al. (1978) also concluded that increased liver Size observed in rats fed 50 or more ppm of PBB was due to increased amounts of fat, water or other constituents and not to increased number or size of cells. It is possible that several mechanisms may be functional in bringing about increased liver size in rats consuming dietary PBB. Obviously, hyperplasia seems to be a prominent feature, at least in the early stages, and the age of the experi- mental animals, the time the animals are killed and the dosage of PBB may be important determinants of which mechanism or mechanisms predominate. In the meantime, further controlled studies are needed to clarify the exact morphogenesis of the hepatomegaly. The most interesting clinical data derived from this study were the significant decreases (p<0.05) in liver vitamin A content. Data on vitamin A content at 10 days 79 are not available because the livers from these rats were inadvertently misplaced or discarded. Relative (ug/g) and absolute (mg/liver) decreases in liver vitamin A con- centration were evident at one or more dietary levels of PBB throughout the course of the experiment. Mangkoewidjojo (1979) reported a dose-dependent depression of hepatic vitamin A content in rats fed 1 or more ppm of PBB. Depression of hepatic vitamin A storage has also been reported for experimental animals treated with PCB (Cecil et al., 1973; Innami et al., 1976; Kato et al., 1978), DDT (Phillips, 1963), methoxychlor (Davison and Cox, 1976) and chlorinated naphthalenes (Olafson, 1947; Hansel et al., 1951). Although there is a paucity of information concerning the association between liver vitamin A content and PBB, the association between the closely-related chlorine analog PCB and the chlorinated hydrocarbon DDT has been extensively studied, and possible mechanisms by which these compounds bring about reduction of liver vitamin A content have been proposed. A common biochemical property of PCB and DDT is their ability to induce liver drug metabolizing enzymes (DME). Concurrent lowering of liver vitamin A concentration occurred when DME were induced (Innami et al., 1976). They speculated that active oxygens generated by hydroxylation reactions catalyzed by cytochrome-P450 coupled to the superoxide generating system probably led to the decomposition of vitamin A and reduction of vitamin A concentration in the liver. A Similar mechanism is probably responsible for reduction 80 of hepatic vitamin A by PBB, Since the compound is well recognized as a potent inducer of DME, including cytochrome-P450. In Spite of the marked reductions in hepatic vitamin A storage (as much as lO-fold in rats fed 10 ppm for 540 days) caused by feeding diets containing PBB, no clinical signs of vitamin A deficiency or histologic lesions sug- gestive of hypovitaminosis A were observed. Mangkoewidjojo (1979) reported prominent squamous metaplasia in bronchiolar epithelium, alveoli and inter- lobular ducts of salivary glands in rats fed diets con- taining excessive dietary iodine and 100 ppm of PBB. Metaplastic changes were also reported in tarsal glands of heifers (Moorhead et al., 1977) and hyperkeratosis was a clinical observation in 2 calves in a Michigan herd (Jackson and Halbert, 1974). The lack of clinical Signs or histologic lesions in rats in the present study may be explained by the fact that the rats were fed a vitamin A-adequate diet. A second possible explanation may be that critical levels at which signs are likely to appear were probably not achieved, the lowest level of liver vitamin A concentration recorded being 46.2 ug/g compared to 7.95 ug/g observed by Mangkoewidjojo (1979). Although no ill effects were observed, the data seem to suggest that, in animals receiving a marginal level of vitamin A in their diet, exposure to PBB could possibly reduce liver vitamin A content to the extent that clinical Signs might occur. 81 The occasional Significant increases and decreases obtained for some hematologic parameters were not con- sidered as evidence of PBB toxicosis because the values were all within the normal range for rats. The reason for the consistently lower total red blood cell values is not clear. However, the values for hematocrit and hemoglobin, 2 more reliable indicators of erythron abnormalities, were all within the normal range, suggesting that the lowered values were probably not an effect of dietary PBB. The generally negative effect of dietary PBB on hematologic values is in agreement with previous reports for rats (Sleight et al., 1978; Sleight and Sanger, 1976), as well as for other Species (Kateley and Bazzell, 1978; Howard et al., 1978; Werner et al., 1978). Residual PBB concentrations in tissues were dose- related and were higher (fat basis) in the liver than in adipose tissue. Steady state concentration apparently was not achieved in either tissue and livers from rats with the highest concentrations of PBB (726 and 627 ppm in livers from rats fed 10 ppm for 360 and 540 days, respectively) had the most severe histOpatholOgic and ultrastructural lesions. The higher concentration of residual PBB in the liver fat is in agreement with reports in cattle (Fries, 1978; Fries et al., 1978). Inability of tissue concentrations of PBB to achieve Steady-state may have been due to the continued increase in body weight, deposition of fat in various tissues, or both. 82 Many of the histopathologic changes seen in the livers from the rats consuming diets containing 10 ppm of PBB for 360 or 540 days were similar to those previously reported in association with either PBB or PCB in rats and other Species. The predominantly midzonal lipid accumu- lation, with extensions to the central and peripheral zones, agrees with the results of Kasza et al. (1976) and Kimbrough et al. (1972) in rats fed PCB. However, Sleight and Sanger (1976) found fatty changes that were diffusely distributed throughout the hepatic lobule and Mangkoewidjojo (1979) reported changes that were either midzonal or centrolobular. Lee et a1. (1975) reported on the location of pathologic changes including fatty metamorphosis in rats dosed with OBB. At 7 days after treatment, changes in rats dosed with 1000 mg/kg were centrolobular and midzonal, whereas in rats dosed with 3000 mg/kg, exten- sions to the peripheral zone occurred. These findings seem to suggest that the location of fatty changes in the livers of rats exposed to polyhalogenated compounds are likely to vary with the dosage of the chemical adminis- tered and probably account for the differences in obser- vations among investigators. The cause of the degenerative changes and focal hepatic necrosis is undetermined. According to Allen et al. (1974) and Allen and Abrahamson (1972), both lesions occur commonly in rats and monkeys exposed to PCB. These investigators postulated that the mechanism responsible for the focal necrosis may be directly related to 83 PCB-induced changes within the hepatic cells. They further indicated that the cells that undergo necrosis were invariably those that had shown the most pronounced hyperplastic changes in their organelles. A similar explanation is suggested for the cause of the focal necrosis associated with PBB in this study, since ultra- structural observations from hepatocytes from the involved livers also had the most severe organelle changes. No histologic lesion to suggest any likely carcinogenic prOperties for PBB were evident in this study. In fact, the incidence of Spontaneous tumorogenesis was surprisingly low. The following factors could be reSponSible for this: the sex of the rats, the age (approximately 19 months), and possibly the low numbers of rats used in the study. Ultrastructural lesions involving progressive increase and dilatation of SER and myelin body (figure) formation are in good agreement with the reports of Corbett et a1. (1978), Sleight and Sanger (1976) and Lee et al. (1975). Of 3 types of myelin figures classified by the latter investigators, the smooth membrane-associated body was the common type observed. A Similar type of myelin body has been found in the livers from rats treated with DDT (Ortega, 1966), 3-ally1-5-isobuty1-2-thiohydan- toin (Herdson et al., 1964a) and phenobarbital (Herdson et al., 1964b), suggesting that myelin body formation might be nonspecific evidence of toxic cell injury. How- ever, the myelin figures found in this study differ from other smooth membrane-associated bodies by having internal 84 membrane-bound vacuoles containing small whorled figures (Figure 15). These membrane-bound vacuoles containing whorled figures also differ from autophagic vacuoles found in the liver of rats treated with glucagon (Arstila and Trump, 1968) and carbon tetrachloride (Stenger, 1963) in the following ways. The membrane-bound vacuoles lack sequestered organelles. Secondly, there is direct con- tinuity betweentfimeouter paired membrane and the cisternae of the SER forming the myelin body. Lee et al. (1975) described somewhat Similar vacuoles in the hepatocytes from rats dosed with OBB, suggesting that these features may be a characteristic of myelin figures induced by brominated biphenyls. Although further Studies in rats and other species are necessary to elucidate the morphogenesis of PBB- induced hepatomegaly, the results of this investigation provided information that may be useful to public health and regulatory personnel as a basis for establishing guidelines designed to protect the public from excessive exposure to chemicals in food. SUMMARY One hundred sixty-eight male Sprague-Dawley rats initially averaging 72 g were used to study the toxicity of low dietary concentrations of polybrominated biphenyls (PBB). Four groups of 42 rats each were fed 0, 0.1, l or 10 ppm PBB. Clinically, there were no ill effects. The PBB-fed rats ate well, maintained a healthy appearance and gained weight as well as, and in some instances better than, the control rats. Six rats from each group were killed at 10, 30, 60, 90, 180, 360 and 540 days. The results of routine hemato- logic values and urinalyses were essentially normal. Sig- nificant depletion of liver vitamin A storage occurred. Residual PBB concentrations in the liver and adipose tissue were dose-related, and the concentrations in the liver were higher (fat basis) than concentrations in adipose tissue. Significant increases in relative liver weight were fairly well correlated with hepatocellular hyperplasia and hypertrophy in rats killed at 10 days, or with increased lipid content in rats killed at 360 and 540 days. Bile duct hyperplasia and portal fibrosis occurred in rats fed diets with 1 ppm PBB for 540 days. At 360 85 86 and 540 days, histopathologic and ultrastructural changes were most severe in rats fed the highest (10 ppm) dietary level of PBB. The results indicate that low dietary concentrations of PBB cause bile duct hyperplasia in rats, deplete liver vitamin A to the extent that it could cause avita- minosis, and induce hepatomegaly related to hepatocellular hyperplasia and hypertrophy. Furthermore, the results of this investigation may be useful to public health and regulatory personnel, by providing information that could serve as a basis for establishing guidelines designed to protect the public from excessive exposure to chemicals in food. REFERENCES REFERENCES Aftsomis, J. G., Culik, R., Lee, K. P., Sherman, H., and Waritz, R. S.: Toxicology of Brominated Biphenyls. 1. Oral Toxicity and Embryotoxicity. Abstr. Toxicol. Appl. Pharmacol., 22, (1972): 316. Aftsomis, J. G., Dashiell, O. L., Griffith, F. D., Hornberger, C. S., McDonnell, M. M., Sherman, H., Tayfun, F. O., and Waritz, R. S.: Toxicology of Brominated Biphenyls. 11. Skin, Eye and Inhala- tion Toxicity and an Acute Test Method for Evaluating Hepatotoxicity and Accumulation in Body Fat. Toxicol. Appl. Pharmacol., 22, (1972): 316-317. Allen, J. R., and Abrahamson, L. J.: Morphologic and Biochemical Changes in the Liver of Rats Fed Chlorinated Biphenyls. Arch. Environ. Contam. Toxicol., (1973): 265-280. Allen, J. R., Carstens, L. A., and Barsotti, D. A.: Residual Effects of Short-Term, Low-Level Exposure of Nonhuman Primates to Polychlorinated Biphenyls. Toxicol. Appl. Pharmacol., 30, (1974): 440-451. Allen, J. R., Lambrecht, L. K., and Barsotti, D. A.: Effects of Polybrominated Biphenyls in Nonhuman Primates. J. Am. Vet. Med. Assoc., 173, (1978): 1485-1489. Anderson, K., Norstrom, A., Rappe, C., Rasmuson, B., and Swahlen, H.: Photochemical Degradation of PCB, PBB and Other Flame Retardants. 34th International Congress of Pesticide Chemistry, Helsinki, Finland, (1974). Arstila, A. U., and Trump, B. F.: Studies on Cellular AutOphagocytosis: The Formation of Autophage Vacuoles in the Liver after Glucagon Administration. Am. J. Pathol., 53, (1968): 687-733. 87 88 Aust, S. D.: Statement. Hearings Before the Subcommittee on Science, Technology and Space of the Committee on Commerce, Science and Transportation, United States Senate, Ninety-Fifth Congress, First Session on Toxic Substances, Polybrominated Biphenyls (PBB) Contamination in Michigan. In Toxic Substances Part 2, Serial No. 95-28. UTS. Govt. Printing Office, Washington, DC, (1977): 837-839. Babish, J. G., Gutenmann, W. H., and Stoewsand, G. S.: Polybrominated Biphenyls: Tissue Distribution and Effect on Hepatic Microsomal Enzymes in Japanese Quail. J. Agric. Food Chem., 23, (1975): 879-882. Baumann, C. A., Foster, E. G., and Moore, P. R.: The Effect of Dibenzanthracene, of Alcohol, and of Other Agents on Vitamin A in the Rat. J. Biol. Chem., 142, (1942): 597-608. Beaudoin, A. R.: Teratogenicity of Polybrominated Biphenyls in Rats. Environ. Res., 14, (1977): 81-86. Bekesi, J. G., and Holland, J. F.: Lymphocyte Function of Michigan Dairy Farmers Exposed to Polybrominated Biphenyls. Science, 199, (1978): 1207-1209. Benjamin, M. M.: Outline of Veterinary Clinical Pathology. The Iowa State University Press, Ames, Iowa, (1969): 1-186. Carter, L. J.: Michigan's PBB Incident: Chemical Mixup Leads to Disaster. Science, 192, (1976): 240-243. Cecil, C. H., Harris, S. J., Bitman, J., and Fries, G. F.: Polychlorinated Biphenyl Induced Decrease in Liver Vitamin A in Japanese Quail and Rats. Bull. Environ. Contam. Toxicol., 9, (1973): 179-185. Cecil, C. H., Harris, 8. J., and Bitman, J.: Effects of Polychlorinated Biphenyls and Terphenyls and Poly- brominated Biphenyls on Pentobarbital Sleeping Times of Japanese Quail. Arch. Environ. Contam. Toxicol., 3, (1975): 183-192. Chanda, J.: Adverse Effects of Polybrominated Biphenyls (PBB'S). Hearings Before the Subcommittee on Oversight and Investigations of the Committee on Interstate and Foreign Commerce. House of Repre- senatatives. Serial No. 95-65. 0.5. Govt. Printing Office, Washington, DC, August 2 and 3, (1977): 43. 89 Corbett, T. H., Beaudoin, A. R., Cornell, R. G., Anver, M. D., Schumacher, R., Endres, J., and Szwabowska, M.: Toxicity of Polybrominated Biphenyls (Firemaster BP-6) in Rodents. Environ. Res., 10, (1975): 390- 396. Corbett, T. H., Simmonds, H. K., and Endres, J. L.: EM Changes and Other Toxic Effects of Firemaster BP-6 (Polybrominated Biphenyls) in the Mouse. Environ. Health Perspect., 23, (1978): 275-281. Dannan, G. A., Moore, R. W., and Aust, S. D.: Studies on the Microsomal Metabolism and Binding of Poly- brominated Biphenyls (PBBS). Environ. Health Perspect., 23, (1978): 51-61. Davison, K. L., and Cox, J. H.: Methoxychlor Effects on Hepatic Storage of Vitamin A in Rats. Bull. Environ. Contam. Toxicol., 16, (1976): 145-148. Deming, D. R.: Summary of Team Evaluation Effect in Herds Quarantined for Polybrominated Biphenyls (PBB), Supplement E. Ip_Welborn Report, The Contamination Crisis in Michigan: Polybrominated Biphenyls. Report of the Senate Special Investigating Committee, Lansing, July, (1975). Dent, J. G., Netter, K. J., and Gibson, J. E.: Effects of Chronic Administration of Polybrominated Biphenyls on Parameters Associated with Hepatic Drug Meta- bolism. Res. Comm. Chem. Pathol. Pharmacol., 13, (1976a): 75-82. Dent, J. G., Netter, K. J., and Gibson, J. E.: The Induc- tion of Hepatic Microsomal Metabolism in Rats Following Acute Administration of a Mixture of Polybrominated Biphenyls. Toxicol. Appl. Pharmacol., 38, (1976b): 237-249. Dent, J. B., McCormack, K. M., Cagen, S. 2., and Gibson, J. E.: The Pattern of Induction of Hepatic and Renal Mixed Function Oxidase in Young Rats by Polybrominated Biphenyls. Presented at the 16th Annual Meeting of the Society of Toxicology, Toronto, Canada, March 27-30, (1977). DiCarlo, F. J., Seifter, J., and DeCarlo, V. J.: Assess- ment of the Hazards of Polybrominated Biphenyls. Environ. Health Perpsect., 23, (1978): 351-365. Dunkel, A. E.: An Updating on the Polybrominated Biphenyl Disaster in Michigan. J. Am. Vet. Med. Assoc., 167, (1975): 838-841. 90 Environmental Health Perspectives: WorkshOp on Scientific Aspects of Polybrominated Biphenyls. 23, (1978): 1-369. Eyster, J. T.: Breast Milk Studies. Michigan Dept. of Public Health, Nov. 18, 1976. Cited by DiCarlo, F. J., Seifter, J., and DeCarlo, V. Environ. Health Perspect., 23, (1978): 351-365. Farber, T. M., and Baker, A.: Microsomal Enzyme Induc- tion by Hexabromobiphenyl. Abstr. 68, 13th Annual Meeting Society Toxicology, Washington, March 10- 14, (1974). Farber, T. M., Kasza, L., Giovetti, A., Carter, C., Earl, P., and Balazs, T.: Effect of Polybrominated Biphenyls (Firemaster BP-6) on the Immunologic System of the Beagle Dog. 13 Society of Toxicology Abstracts. London: William Clowes (Intl.) Ltd., (1978): 249. Ficsor, G., and Wertz, G. F.: Polybrominated Biphenyls Nonteratogenic C-Mitosis Synergist in Rats. Muta- tion Res., 38, (1976): 388. Fries, G. F.: Distribution and Kinetics of Polybrominated Biphenyls and Selected Chlorinated Hydrocarbons in Farm Animals. J. Am. Vet. Med. Assoc., 173, (1978): 1479-1484. Fries, G. F., Cecil, H. C., Bitman, J., and Lillie, R. J.: Retention and Excretion of Polybrominated Biphenyls by Hens. Bull. Environ. Contam. Toxicol., 15, (1976): 278-282. Fries, G. F., Cook, R. M., and Prewitt, L. R.: Distribu- tion of Polybrominated Biphenyls Residues in the Tissues of Environmentally Contaminated Cows. J. Dairy Sci., 61, (1978): 420-425. Fries, G. F., and Marrow, G. S.: Excretion of Polybrominated Biphenyl into the Milk of Cows. ~J. Dairy Sci., 58, (1975): 947-951. Fries, G. F., Smith, L. W., Cecil, H. C., Bitman, J., and Lillie, R. J.: Retention and Excretion of Poly- brominated Biphenyls by Hens and Cows. 1p Abstr. of 165th Meeting American Chemical Society, Dallas, Texas, (1973). Garry, P. J., Pollack, J. D., and Owen, G. M.: Plasma Vitamin A Assay by Fluorometry and Use of Silicic Acid Column Technique. Clin. Chem., 16, (1970): 766-772. 91 Garthoff, L. H., Friedman, L., Farber, N., Locke, K., Sabotka, T., Green, S., Hurlen, N. B., Peters, B., Story, G., Moreland, F. M., Graham, C., Keys, J., Taylor, M. J., Rothlein, J., and Sporn, E.: Biochemical Changes Caused by Ingestion of Arochlor 1254 (A Commercial Polychlorinated Biphenyl Mixture) or Firemaster BP-6 (A Commercial Polybrominated Biphenyl Mixture). Food and Drug Administration Official Report, Washington, DC, (1976). Getty, S. M., Rickert, D. B., and Trapp, A. L.: Poly- brominated Biphenyl (PBB) Toxicosis: An Environ- mental Accident. CRC, Critical Reviews in Environ. Control, 7, (1977): 309-323. Gutenmann, W. H., and Lisk, D. J.: Tissue Storage and Exceretion in Milk of Polybrominated Biphenyls in Ruminants. J. Agric. Food Chem., 23, (1975): '1005-1007. Hansel, W., McEntee, K., and Olafson, P.: The Effects of Two Causative Agents of Hyperkeratosis on Vitamin A Metabolism. Cornell Vet., 41, (1951): 367-376. Harris, 8. J., Cecil, H. C., and Bitman, J.: Effects of Feeding Polybrominated Biphenyl Flame Retardant (Firemaster BP-6) to Male Rats. Bull. Environ. Contam. Toxicol., 19, (1978): 692-696. Harris, S. S., and Navia, J. M.: Effect of Vitamin A Deficiency on Calcium and Glycosaminoglycan Metabolism in Guinea Pig Bone. J. Nutr., 107, (1977): 2198-2205. Hass, J. R., McConnell, E. B., and Hervan, D. J.: Chemical and Toxicologic Evaluation of Firemaster BP-6. J. Agric. Food Chem., 26, (1978): 94-98. Herdson, P. B., Garvin, P. J., and Jennings, R. B.: Reversible Biological and Fine Structural Changes Produced in Rat Liver by a Thiohydantoin Compound. Lab. Invest., 13, (1964a): 1014-1031. Herdson, P. B., Garvin, P. J., and Jennings, R. B.: Fine Structural Changes in Rat Liver Induced by Pheno- barbital. Lab. Invest., 13, (1964b): 1032-1037. Hesse, J. L.: Water Pollution ASpectS of Polybrominated Biphenyl Production: Results of Surveys in the Pine River in the Vicinity of St. Louis, Michigan. Second National Conference on Complete WateReuse; Waters Interface with Energy, Air and Solids. Palmer House, Chicago, May 4-8, (1975). 92 Hoekstra, W. G., Hall, R. E., and Phillips, P. H.: A Study on the Relationship of Vitamin A to the Development of Hyperkeratosis (X-Disease) in Calves. Am. J. Vet. Res., 15, (1954): 41-46. Howard, S. K., Werner, P. R., and Sleight, S. D.: Poly- brominated Biphenyl Toxicosis in Swine: Effects on the Immunological Responses of the Sow and Young Pig. Abstr. of 59th Annual Meeting of the Con- ference of Research Workers in Animal Disease, Chicago, Illinois, Nov. 27-28, (1978). Industrial Biotest Laboratories, Inc.: Toxicity and Tissue Residue Study with Firemaster FF-l in Dairy Calves. IBT, No. 651-06733, Nov. 5, (1975). Industrial Biotest Laboratories, Inc.: Meat and Milk Residue Study with Firemaster FF-l in Dairy Cattle. IBT, No. 651-06336, June 7, (1976). Innami, S., Nakamura, N., Miyazaki, M., Nagayama, S., and Nishide, E.: Further Studies on the Reduction of Vitamin A Content in the Livers of Rats Given Polychlorinated Biphenyls. J. Nutr. Sci. Vitaminol., 22, (1976): 409-418. Isleib, D. R., and Whitehead, G. L.: Polybrominated Biphenyls. An Agricultural Incident and its Consequences. I. The Agricultural Effects of Exposure. Paper presented at 9th Annual Conference on Trace Substances in the Environmental Health, University of Missouri, June 10, (1975). Jackson, T. F., and Halbert, F. L.: A Toxic Syndrome Associated with the Feeding of Polybrominated Biphenyl-Contaminated Concentrate to Dairy Cattle. J. Am. Vet. Med. Assoc., 165, (1974): 437-439. Jacobs, L. W., Chou, S. F., and Tiedje, J. M.: Fate of PBB in Soils: Persistence and Plant Uptake. J. Agric. Food Chem., 24, (1976): 1198-1201. Jensen, 8., and Sundstrom, 6.: Structures and Levels of MostChlorobiphenylsin.Two Technical PCB Products and in Human Adipose Tissue. Ambio., 3, (1974): 70-76. Jondrof, W. R., Parke, D. V., and Williams, R. T.: Studies in Detoxification. 66. The Metabolism of Halo- genobenzenes. l, 2, 3-, 1, 2, 4-, and l, 3, 5- Trichlorobenzenes. Biochem. J., 61, (1955): 512- 521. 93 Jondrof, W. R., Parke, D. V., and Williams, R. J.: Studies in Detoxification. 76. The Metabolism of Halogenobenzenes. l, 2, 3, 4-, 1, 2, 3, 5- and 1, 2, 4, 5-Tetrachlorobenzenes. Biochem. J., 69, (1958): 181-189. Kasza, L., Weinberger, M. A., Carter, C., Hinton, D. E., Trump, B. F., and Brouwer, E. A.: Acute, Subacute, and Residual Effects of Polychlorinated Biphenyl (PCB) in Rats. II. Pathology and Electron Microsc0py of Liver and Serum Enzyme Study. J. Toxicol. Environ. Health, 1, (1976): 689-703. Kateley, J. R.: Immunologic Studies in Cattle Exposed to PBB. Workshop on Scientific Aspects of Poly- brominated Biphenyls, Michigan State University, Oct. 24-25, (1977). Kateley, J. R., and Bazzell, S. J.: Immunologic Studies in Cattle Exposed to Polybrominated Biphenyls. Environ. Health Perspect., 23, (1978): 75-82. Kata, N., Kato, M., Kimura, T., and Yoshida, A.: Effect of Dietary Addition of PCB and DDT or BHT and Dietary Protein on Vitamin A and Cholesterol Metabolism. Nutr. Reports Int., 18, (1978): 437-445. Kay, K.: Polybrominated Biphenyls (PBB) Environmental Contamination in Michigan 1973-1976. Environ. Res., 13, (1977): 74-93. Kerst, A. F.: Letter to Bryson, D. 5. EPA Region V. Nov. 11, (1976). Kimbrough, R. D.: The Toxicity of Polychlorinated Poly- cyclic Compounds and Related Chemicals. CRC Critical Reviews in Toxicol., 2, (1974): 445-498. Kimbrough, R. D., Burse, V. W., and Liddle, J. A.: Per- sistent Liver Lesions in Rats after a Single Oral Dose of Polybrominated Biphenyls (Firemaster FF-l) and Concomitant PBB Tissue Levels. Environ. Health Perspect., 23, (1978): 265-273. Kimbrough, R. D., Linder, R. B., and Gaines, T. B.: Mor- phological Changes in Livers of Rats Fed Poly- chlorinated Biphenyls: Light Microsc0py and Ultrastructure. Arch. Environ. Health, 25, (1972): 354-364. 94 Kociba, R. J., Frauson, L. 0., Humiston, C. G., Norris, J. M., Wade, C. B., Lisowe, R. W., Quast, J. F., Jersey, G. C., and Jewett, G. L.: Results of a Two-Year Dietary Feeding Study with Decabromodi- phenyl Oxide (DBDPO) in Rats. J. Combus. Toxicol., 2, (1975): 268-285. Kohli, J., and Safe, S.: The Metabolism of Brominated Aromatic Compounds. Chemosphere, 6, (1976): 433- 437. Kolbye, A. C.: Hearings before the Subcommittee on Science, Technology and Space of the Committee on Commerce, Science and Transportation, United States Senate, First Session on Toxic Substances, Polybrominated Biphenyl (PBB) Contamination in Michigan. 13 Toxic Substances Serial No. 98-25, U.S. Govt. Printing Office, Washington, DC, (1977): 951-1296. Ku, P. K., Hogberg, M. G., Trapp, A. L., Brady, P. S., and Miller, E. R.: Polybrominated Biphenyl (PBB) in the Growing Pig Diet. Environ. Health Perspect., 23, (1978): 13-18. Kuratsune, M., Masuda, Y., and Nagayama, J.: Some Recent Findings Concerning Yusho. National Conference on Polychlorinated Biphenyls, Chicago, Illinois, Nov. 19-21, (1975). Lambrecht, L. K., Barsotti, D. A., and Allen, J. R.: ReSponses of Nonhuman Primates to a Polybrominated Biphenyl Mixture. Environ. Health Perspect., 23, (1978): 139-145. ~ Lee, M., Harris, K., and Trowbridge, H.: Effect of the Level of Dietary Protein on the Toxicity of Dieldrin for the Laboratory Rat. J. Nutr., 84, (1964): 136-144. Lee, K. P., Herbert, R. R., Sherman, H., Aftsomis, J. G., and Waritz, R. S.: Bromine Tissue Residue and Hepatotoxic Effects of Octabromobiphenyl in Rats. Toxicol. Appl. Pharmacol., 34, (1975): 115-127. Lee, K. P., Herbert, R. R., Sherman, H., Aftsomis, J. G., and Waritz, R. S.: Octabromobiphenyl Induced Ultra- structural Changes in Rat Liver. Arch. Environ. Health, 30, (1975): 465-471. Luster, R. E., Faith, R. B., and Moore, J. A.: Effects of Polybrominated Biphenyls (PBB) on Immune ReSponse in Rodents. Environ. Health Perspect., 23, (1978): 227-232. 95 Magdolna, K., Robert, T., Magdolna, B., Erno, D., Magda, T. K., and Eva, W. J.: Effect of Caffeine and Barbiturates on the Metabolism of Rats. Cited by Kato, N., Kato, M., Kimura, T., and Yoshida, A. Nutr. Reports Intl., 18, (1978): 437-445. Mangkoewidjojo, S.: Pathologic Effects of Polybrominated Biphenyls in Rats Fed a Diet Containing Excessive Iodine. PhD Thesis, Michigan State University, (1979). Mathews, H. B., and Anderson, M.: PCB Chlorination Versus PCB Distribution and Excretion. National Conference on Polychlorinated Biphenyls, Chicago, Illinois, Nov. 19-21, (1975). Mathews, H. B., Kato, S., Morales, N. M., and Tuey, D. B.: Distribution and Excretion of 2,4,5,2',4',5'- Hexabromobiphenyl, the Major Component of Fire- master BP-6. J. Toxicol. Environ. Health, 3, (1977): 599-605. Mercer, H. D.: Statement on Polybrominated Biphenyls (PBB'S). Division of Veterinary Research, Bureau of Veterinary Medicine, Food and Drug Administra- tion. Read before the Michigan State Department of Agriculture, Lansing, Michigan, June 10, (1976). Mercer, H. D., Buck, W. B., Furr, A., Teske, R. H., Meerdink, C. L., Fries, G. F., and Candon, R. J.: Michigan State Dairy Herd Survey. A report on Herd Health: Status of Animals Exposed to Poly- brominated Biphenyls (PBB). Supplement G. 12 Welborn Report. The Contamination Crisis in Michigan: Polybrominated Biphenyls. Report of the Senate Special Committee, Lansing, Michigan, July, (1975). Michigan Chemical Corporation: Report Presented to the Michigan Environmental Review Board, Sept. 23, (1974). Michigan Science in Action: MSU Research on PBBS. Michigan State University, East Lansing, Michigan, May, (1976). Moore, J. A.: National Institute of Health Sciences. Cited by Kay, K., Environ. Res., 13, (1977): 74-93. Moore, R. W., Sleight, S. D., and Aust, S. D.: Induction of Liver Microsomal Drug Metabolizing Enzymes by 2,2',4,4',5,5'-Hexabromobiphenyl. Toxicol. Appl. Pharmacol., 44, (1978): 309-321. 96 Moorhead, P. D., Willett, L. B., Brumm, C. J., and Mercer, H. D.: Pathology of Experimentally Induced Poly- brominated Biphenyl Toxicosis in Pregnant Heifers. J. Am. Vet. Med. Assoc., 170, (1977): 307-313. Mitchell, G. B., Little, C. O., and Skerski, G.: Influence of Subacute Levels of Dietary Toxins on Carotene Disappearance from Rat Intestine. Int. Z. Vit. Forschung, 38, (1968): 308-311. Naber, E. C.: The Impact of Contamination by Organo- chlorine Insecticides on Poultry Nutrition and Feeding. Fed. Proc., 36, (1977): 1880-1887. Neufeld, M. L., Sittenfield, M., and Walk, K. F.: Market Input/Output Studies, Task IV: Polybrominated Biphenyls EPA-56016-77-017, Aug., (1977). Norris, J. M., Ehrmantraut, J. W., Gibbons, C. L., Kociba, R. J., Schwetz, B. A., Tirsell, J. B., and Brosier, J. S.: Toxicological and Environmental Factors Involved in the Selection of Decabromobiphenyl Oxide as a Fire Retardant. Combust. Toxicol., 1, (1974): 52-75. O'Keefe, P. W.: Preliminary Report Analysis of Environ- mental Samples of Trace Components from Polybrominated Biphenyl Fire Retardants. Department of Chemistry, Harvard University, Jan. 28, (1976). Olafson, P.: Hyperkeratosis (X-Disease) of Cattle. Cornell Vet., 37, (1947): 279-291. Ortega, P.: Light and Electron Microsc0py of Dichloro- diphenyltrichloroethane (DDT) Poisoning in the Rat Liver. Lab. Invest., 15, (1966): 657-679. Pease, D. C.: Histologic Technique for Electron Microscopy, 2nd Ed. Academic Press, New York and London, (1964): 38-39. Phillips, W. E. J.: DDT and the Metabolism of Vitamin A and Carotene in the Rat. Can. J. Biochem., 41, (1963): 1793-1802. Phillips, W. E. J., and Hatina, G. V.: Effect of Dietary Organochloride Pesticides on the Liver Vitamin A in the Weanling Rat. Nutr. Reports Intl., 5, (1972): 357-363. Preache, M. M., Cagen, S. Z., and Gibson, J. L.: Perinatal Toxicity in Mice Following Maternal Dietary Exposure to Polybrominated Biphenyls. Abstr., 15th Ann. Meeting, Society of Toxicology, Atlanta, Georgia, March 14-18, (1976). 97 Reid, W. D., and Krisha, G.: Centrolobular Hepatic Necrosis Related to Covalent Binding of Metabolites of Halo- genated Aromatic Hydrocarbons. Expt. and Mol. Pathol., 18, (1973): 80-99. Rickert, D. E.: Unpublished. Cited by Getty, S. M., Rickert, D. E., and Trapp, A. L.: CRC Critical Reviews in Environ. Control., 7, (1977): 309-323. Ringer, R. K., and Kowaleski, E.: Unpublished observation. Cited by Ringer, R. K., and Polin, D.: Fed. Proc., 36, (1977): 1894-1898. Ringer, R. K., and Polin, D.: The Biological Effects of Polybrominated Biphenyls in Avian Species. Fed. Proc., 36, (1977): 1894-1898. Ruzo, L. O., and Zabic, M. J.: Polyhalogenated Biphenyls: Photolysis of Hexabromo- and Hexachlorobiphenyls in Methanol Solution. Bull. Environ. Contam. Toxicol., 13, (1975): 181-182. Shulte, B., and Acker, L.: Cited by Matthews, H. B., and Anderson, M.: National Conference on Polychlorinated Biphenyls, Chicago, Illinois, Nov. 19-21, (1975). Sleight, S. D., and Sanger, V. L.: Pathologic Effects of Polybrominated Biphenyls in Rat and Guinea Pig. J. Vet. Med. Assoc., 169, (1976): 1231-1235. Sleight, S. D., Mangkoewidjojo, S., Akoso, B. T., and Sanger, V. L.: Polybrominated Biphenyl Toxicosis in Rats Fed Iodine-Adequate and Iodine-Excess Diet. Environ. Health Perspect., 23, (1978): 341-346. Stadtfeld, C. K.: Cheap Chemical and Dumb Luck. Audubon, 78, (1976): 110-118. Stenger, R. J.: Hepatic Parenchymal Cell Alterations after Long-Term Carbon Tetrachloride Administra- tion: A Light and Electron Microsc0pic Study. Am. J. Pathol., 43, (1963): 867-895. Strick, J. J. T. W. A.: Chemical Porphyria in Japanese Quail (Couturnixc Japonica). Enzyme, 16, (1973): 211-223. Sundstrom, G., Hutzinger, 0., and Safe, S.: Environmental Chemistry of Flame Retardants. Part 2. Identifi- cation of 2,2',4,4',5,5'-Hexabromobiphenyl as the Major Component of Flame Retardant Firemaster BP-6. ChemOSphere, S, (1976): ll-l4. 98 Tilson, H. A., Cabe, P. A., and Mitchell, C. L.: Behavioral and Neur010gical Toxicity of Polybrominated Biphenyls in Rats and Mice. Environ. Health Perspect., 23, (1978): 257-263. Tinsley, I. J.: DDT Effect on Rats Raised on Alpha-Protein Rations: Growth and Storage of Liver Vitamin A. J. Nutr., 98, (1969): 319-324. Trapp, A. L., Sanger, V. L., Cook, R. M., Krehbiel, J. D., and Prewitt, L. R.: Preliminary Observations of a Group of Cattle Contaminated with Polybrominated Biphenyls in Michigan. Proc. 18th Ann. North Central Conf. Vet. Lab. Diagnosticians and 26th Ann. North Central Poultry Dis. Conf., (1975): 19. Villeneuve, D. C., Grant, D. L., Phillips, W. E. J., Clark, M. L., and Clegg, D. J.: Effects of PCB Adminis- tration on Microsomal Enzyme Activity in Pregnant Rabbits. Bull. Environ. Contam. Toxicol., 6, (1971): 120-128. Vos, J. G., and VanGenderew, H.: Toxicologic Aspects of Immunosuppression. In Pesticides and the Environment. Deichmann, W. B., EdT— Intercontinental Medical Book Corp., New York, (1973): 527-545. Welborn, J. A.: The Contamination Crisis in Michigan: Polybrominated Biphenyls: A Report of the Senate Special Investigating Committee, Lansing, July, (1975). Werner, P., Sleight, S. D., Howard, S. K., and Schull, L.: Polybrominated Biphenyl Toxicosis in the Sow, New- born and Young Pig. Abstr. Joint Meeting American Society for Pharmac010gy and Experimental Thera- peutics, Society of Toxicology, University of Houston, Texas, August 13-17, (1978). Widmark, G.: Cited by Jones, D. H., Platonow, N. S., and Safe, S. Can. Vet. J., 16, (1975): 349-356. Willett, L. B., and Durst, H. 1.: Effects of PBBS on Cattle. IV. Distribution and Clearance of Com- ponents of Firemaster BP-6. Environ. Health Perspect., 23, (1978): 67-74. Willett, L. B., and Irving, H. A.: Distribution and Clearance of Polybrominated Biphenyls in Cows and Calves. J. Dairy Sci., 59, (1976): 1429-1439. Willett, L. B., and Irving, H. A.: Distribution and Clearance of Polybrominated Biphenyls by Cows. Abstr., J. Dairy Sci., 58, (1975): 764. 99 Zitko, V.: The Accumulation of Polybrominated Biphenyls by Fish. Bull. Environ. Contam. Toxicol., 17, (1977): 285-292. Zitko, V., and Hutzinger, 0.: Uptake of Chloro- and Bromobiphenyls, Hexachloro- and Hexabromobenzene by Fish. Bull. Environ. Contam. Toxicol., 16, (1976): 665-673. VITA VITA The author was born in a small rural district in St. Ann, Jamaica, West Indies, on December 29, 1941. He received his early education at Chalky Hill Elementary and Holmwood Technical High School, Manchester, Jamaica. In 1960 he entered the Jamaica School of Agriculture and graduated in 1963 with a Diploma in Agriculture. After working in the Jamaica Civil Service for five years, the author emigrated to the United States to pursue advanced studies. He entered Tuskegee Institute in 1968 and graduated with a Bachelor of Science degree in 1970, Specializing in Animal Science. In that same year he entered graduate school at Michigan State University. After obtaining a Master of Science degree in Dairy Science in 1972, he entered the College of Veterinary Medicine, from which he graduated with a Doctor of Veterinary Medicine degree with honors in 1974. From 1975 to 1976 he worked as an instructor in the College of Veterinary Medicine. In March 1976 he re- entered graduate school to pursue a Doctor of Philosophy degree in Pathology. Since that time he has worked as a Teaching Assistant and Instructor in the Department of Pathology, Michigan State University. 100 APPENDICES Table A-1. APPENDIX A MEAN AND RELATIVE KIDNEY AND BRAIN WEIGHTS FOR PBB-FED RATS Mean and relative kidney and brain weights for PBB-fed rats =:== ====================================================== Days Dietary Kidney Brain Kidney/Body Brain/Body on PBB Weight yWeight Weight Weight Diet (ppm) (81 % 10 0.0 1.36:0.15a 1.53:0.06 0.97:0.05 1.07:0.07 10 0.1 1.32:0.11 1.56:0.08 0.94:0.05 1.11:0.06 10 1.0 1.29:0.07 1.50:0.03 0.91:0.07 1.06:0.06 10 10.0 1.31:0.14 1.53:0.04 0.90:0.06 1.06:0.06 30 0.0 2.36:0.23b 1.69:0.11b 0.93:0.05b 0.66:0.06b 30 0.1 2.64:0.16 1.75:0.10 0.97i0.03 0.65:0.08 30 1.0 2.52:0.14 1.76:0.05 0.97:0.06 0.6710.02 30 10.0 2.39:0.26 1.71:0.09 0.94i0.06 0.67:0.02 60 0.0 2.84:0.16 1.77N0.17 0.8510.07 0.53:0.06 60 0.1 2.96:0.51 1.75:0.11 0.85i0.15 0.50:0.03 60 1.0 2.61:0.31 1.74:0.06 0.76:0.07 0.50i0.03 60 10.0 2.84:0.23 1.76N0.09 0.78:0.05 0.49:0.02 90 0.0 3.00:0.18 1.78:0.08 0.77:0.06 0.46:0.04 90 0.1 3.23:0.28 2.14:0.27 0.83i0.06 0.55:0.08 90 1.0 3.44:0.29 2.34:0.06 0.82i0.06 0.56:0.02 90 10.0 3.26:0.19b 2.28:0.06b 0.80:0.04 0.56:0.04 180 0.0 3.49:0.27 1.93N0.09 0.79:0.03 0.44:0.04 180 0.1 3.78:0.26b 2.00N0.10b 0.76i0.07 0.40:0.04 180 1.0 3.26:0.18 1.96:0.04 0.68i0.05 0.41i0.02 180 10.0 3.26:0.23 1.91:0 09 0.6710.05 0.39:0.02 360 0.0 3.77:0.48 1.86:0.08b 0.75:0.07 0.38:0.02b 360 0.1 3.83:0.26 1.76:0.32 0.73i0.05 0.33i0.06 360 1.0 3.62:0.23 1.90:0.06 0.67i0.06 0.35i0.02 360 10.0 3.48:0.31b 1.86N0.06b 0.69i0.12b 0.3710.05b 540 0.0 3.69:0.32b 1'89i0'07b 0.73i0.06b 0.38:0.03b 540 0.1 4.01:0.47b 1.91N0.08b 0.75:0.10b 0.38:0.03b 540 1.0 4.46:0.84b 1'91i0°07b 0.78i0.04b 0.33i0.03b 540 10.0 3.89:0.35 1.87N0.09 0.75:0.12 0.36:0.03 av . b - alues are mean x SD, N=6. N-5. 101 APPENDIX B MEAN THYROID HORMONE CONCENTRATIONS IN RATS FED PBB FOR 10, 90, AND 540 DAYS Table B-1. Mean thyroid hormone concentrations in rats fed PBB for 10, 90, and 540 days Days on Number of Dietary PBB Hormone Concentrations Diet Rats (ppm), T3 *1; -------(ng/ml)-------- 10 2 0.0 1.07:0.04 46.2: 1.8 10 2 0.1 1.06:0.13 33.9: 3.8 10 2 1.0 1.24:0.22 42.9: 5.7 10 2 10.0 1.19:0.16 47.4: 8.6 90 3 0.0 0.85:0.25 35.7: 9.2 90 3 0.1 1.08:0.43 36.4: 7.2 90 3 1.0 0.86:0.22 35.0: 6.1 90 2 10.0 0.90:0.06 29.7: 0.9 540 5 0.0 0.59:0.13 16. i 4.0 540 5 0.1 0.29:0.40 15.3: 4.7 540 5 1.0 0.66:0.30 20.0:10.2 540 5 10.0 0.77:0.20 14.6: 6.6 3Significantly different from control value (p<0.025). 102 APPENDIX C MEAN SERUM TESTOSTERONE CONCENTRATIONS IN RATS FED PBB FOR 10, 30, 60, 90 AND 180 DAYS Table C-l. Mean serum testosterone concentrations in rats fed PBB for 10, 30, 60, 90 and 180 days .——.—_-.__——____———.——.———_———o__—_.———.——————._———_——n_———_——_—— ———— —-.—— _—-—.———.—— Testosterone Dietary PBB Concentration Days on Diet (ppm) (ng/ml) 10 0.0 1.0 No.63 10 0.1 1.6 :l.3 10 1.0 2.9 :0.9 10 10.0 2.0 +1.4 30 0.0 2.3 :0.4 30 0.1 3.3 :2.4 30 1.0 2.3 :0.1 30 10.0 1.7 +0.2 60 0.0 3.6 :2.6 60 0.1 3.8 :l.8 60 1.0 2.5 :0.4 60 10.0 0.95:0.2 90 0.0 1.6 :0.5 90 0.1 1.3 :0.7 90 1.0 6.4 :3.0 90 10.0 3.6 :1.9 180 0.0 5.8 :2.5 180 0.1 3.3 +0.9 180 1.0 1.7 :0.1 180 10.0 3.3 :0.2 aValues are mean : SD; N=2. 103