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Elfl§§_w111 be charged if book is returned after the date stamped be1ow. ____._._———-——_. _____.._..——————‘_. ., ‘ 4 1"” ”V -A- < In . c I ~’ 'I- . 1 \ /4//~ €607 PATHOLOGIC EFFECTS AND HEPATIC TUMOR PROMOTING ABILITY OF FIREMASTER BP-G, 3,3',4,4',5,5'—HEXABROMOBIPHENYL AND 2,2',4,4',5,5'-HEXABROMOBIPHENYL IN THE RAT BY Richard Kent Jensen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1983 ABSTRACT PATHOLOGIC EFFECTS AND HEPATIC TUMOR PROMOTING ABILITY OF FIREMASTER BP-6, 3,3',4,4',5,5'-HEXABROMOBIPHENYL AND 2,2',4,4',5,5'-HEXABROMOBIPHENYL IN THE RAT BY Richard Kent Jensen Female Sprague-Dawley rats were fed polybrominated biphenyls (PBBs) for 140 or 180 days after a 70% partial hepatectomy and diethylnitrosamine administration (10 mg/kg body weight) to determine if PBBs had tumor promoting ability in a: two stage hepatocarcinogenesis assay. Firemaster BP-G (FM), a commercial mixture of P885, 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB), and 3,3',4,4',5,S'-hexabromobiphenyl (345-HBB) were used. Tumor promoting ability was assessed by measuring enzyme-altered foci exhibiting gamma-glutamyl transpeptidase activity. Dietary concentrations of 10 and 100 mg/kg of FM and 24S-HBB and luO mg/kg 345-838 were promoters of hepatocarcinogenesis. FM had greater tumor promoting ability than its major congener 245-HBB. 345-HBB had tumor promoting ability only at dietary concentrations that caused hepatocyte necrosis whereas 245-HBB had tumor promoting ability at dietary concentrations that were not hepatotoxic. ‘When 245-HBB and 34S-HBB were fed to rats in combination, a potentiating effect on tumor promoting ability occurred at Richard Kent Jensen dietary concentrations of 0.1 mg 345-HBB/kg plus 10 mg 245- HBB/kg whereas an inhibitory effect on tumor promoting ability occurred at dietary concentrations of 1.01ng 345- HBB/kg plus 100 mg 24S-BBB/kg. FM, fed to rats over a short period of time had the same ability to enhance the development of enzyme-altered foci as an equal amount of FM fed over a long period of time. Therefore, the tumor promoting ability of FM persists for long periods of time after cessation of exposure from external sources. Rats maintained on a basal diet for 275 days after cessation of dietary exposure to PM had hepatocellular carcinomas indicating an association between the enzyme-altered foci enhanced by FM and carcinomas. FM caused an enhancing effect on the number of enzyme-altered foci when given after diethylnitrosamine but did not increase the number of enzyme-altered foci when given before diethylnitrosamine. This suggests that congeners of PM do not have tumor initiating ability. PBBs altered the homeostasis of vitamin A resulting in increased concentrations of serum retinol apparently at the expense of depleting hepatic retinyl palmitate. Results of these studies help characterize the carcinogenic and toxicologic prOperties of P338. Dedicated to my wife, Holly. ii ACKNOWLEDGMENTS I wish to thank the many individuals who helped in the completion of this dissertation. From the time this study was simply an idea until the final page of this dissertation was typed, there have been many individuals who have contributed ideas, advice and technical assistance. A special thanks must go to Dr. Stuart Sleight who very ably served as my major professor. Dr. Sleight not only served as an advisor but also a colleague and personal friend who was always willing to listen and help. I extend my gratitude to Drs. Aust, Goodman and Tvedten, members of the guidance committee and Drs. Stowe and Trosko. Their advice and suggestions truly made this research project a learning experience. I would like to acknowledge all of those who helped by giving their technical assistance in the completion of these studies. A special thanks to Dr. Esther Roege, Anne Goatley, Debra Metcalf, Fran Whipple, Mae Sunderlin, Karen Bennack, Paul Carlson and Irene Brett. I also want to thank Cheryl Assaff for typing this dissertation and Cynthia Millis, a fellow graduate student, who unselfishly gave her time and talents in purifying the polybrominated biphenyl congeners used in this study. iii My deepest appreciation to uni wife Holly for her support, patience, understanding and love throughout my studies. iv TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O O O O O O O O O O O 0 Vi ii LIST OF FIGURES O O O O O O O O O O O O O O O O O O O O Xi INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O 1 LITERATURE REVIEW 0 O O O O O O O O O O O O O O O O O O O 4 Chemical and Physical Properties of Polybrominated BipheDYIS O O O O O O O O O O O O O O O O O O O O 4 Environmental Contamination by Polybrominated BiphenYIS O O O O O O O O O I O O O O O O O O O O s Hepatic Microsomal Enzyme Induction by Xenobiotics . S Pathotoxicologic Effects of Polybrominated Biphenyls . . . . . ... . . . . . . . . . . . . 10 Clinical Pathology . . . . . . . . . . . . . . 10 Clinical Signs . . . . . . . . . . . . . . . . ll Organ Weight Alterations . . . . . . . . . . . 12 Histopathologic Alterations . . . . . . . . . 12 Ultrastructural Alterations . . . . . . . . . l4 Carcinogenesis as a Two- -Stage Process . . . . . . 14 Chemically Induced Initiation of Carcinogenesis . . 14 Tumor Promotion by Xenobiotics . . . . . . . . . . 19 Oncogene: A Common Final Pathology . . . . . . . . 24 Biochemical Markers of Hepatocarcinogenesis . . . . 25 Enzyme- -Altered Foci as Precursor Lesions of Hepatocellular Carcinomas . . . . . . . . . 26 Assays for Initiation/Promotion in the Liver . . . 28 Solt Farber Assay . . . . . . . . . . . . . . 28 Pitot Assay . . . . . . . . . . . . . . . . 29 Peraino Baby Rat Model . . . . . . . . . . . . 30 Peraino Assay . . . . . . . . . . . . . 30 Carcinogenic Effects of Polybrominated Biphenyls In Vivo . . . . . . . . . . 31 Carcinogenic Potential of Polybrominated Biphenyls Determined by In Vitro Assays . . . . . . . . . 33 Vitamin A Metabolism . . . . . . . . . . . . . . . 35 Effect of Xenobiotics on Vitamin A . . . . . . . . 37 CHAPTER 1: 2,2',4,4' Introduction Materials and Methods Results . Discussion CHAPTER 2: HEXABROMOBIPHENYL: HEPATIC MICROSOMAL DRUG TOXICITY, HEPATIC TUMOR PROMOTION BY 3. HEPATIC TUMOR PROMOTION BY FIREMASTER BP-6, ,5,5'-HEXABROMOBIPHENYL AND 3,3',4,4 HEXABROMOBIPHENYL 3'14’4"5'5'- THE INTERRELATIONSHIP BETWEEN METABOLIZING ENZYMES 1,5,51_ Page 39 40 41 47 56 AND TUMOR PROMOTION O O O O O O O O O O O O O O O O O 62 IntIOdUCtion O O O O O O O O O O O O O O O O O O 63 Materials and Methods . . . . . . . . . . . . . . . 64 ReSUItS O O O O C O O O O O O O O O O O O O O O O O 66 Discussion . . . . . . . . . . . . . . . . . . . . 83 CHAPTER 3: POTENTIATING AND INHIBITORY EFFECTS ON TOXICITY AND HEPATIC TUMOR PROMOTION BY DIETARY COMBINATIONS OF 2'2'p4'4"5'5'-HEXABROMOBIPHENYL AND 3'3'y4'4"5'5'- HEXABROMOBIPHENYL . . . . . . . . . . . . . . . . . . 87 IntIOdUCtion O O O O O O O O O O O O O O 0 O O O O 88 Materials and MethOdS O O O O O O O O O O O O O O O 89 Resu1t8 O O O O O O O O O O O O O O O O O O I O O O 90 DiSCUSSion I O O O O O O O O O O I O O O O O O O O 110 CHAPTER 4: EFFECT OF VARYING THE LENGTH OF EXPOSURE TO FIREMASTER BP-6 ON HEPATIC TUMOR PROMOTION AND ON THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMAS . . . . . . 117 IntIOdUCtion O O O O O O O O O O O O O O O O O O 118 Materials and Methods . . . . . . . . . . . . . . . 119 Results 0 O O O O O I O O O O O O O O O O O O O O O 120 Discussion . . . . . . . . . . . . . . . . . . . . 129 CHAPTER 5: THE EFFECT OF ADMINISTRATION OF FIREMASTER BP-6 PRIOR TO AND AFTER THE ADMINISTRATION OF DIETHYLNITROSAMINE O O O O O O O O O O O O I O O O O 132 IntIOdUCtion O O O C O O O O O O O O O O O O O O 133 Materials and Methods . . . . . . . . . . . . . . . 134 ReSUItS O O O C O O O O O O O O O O O O O O O O O O 135 DiSCUSSion O I O O O O O O O O O O O O O O O O O O 137 vi CHAPTER 6: EFFECT OF POLYBROMINATED BIPHENYLS HOMEOSTASIS OF SERUM AND HEPATIC VITAMIN A Introduction . . . . Materials and Methods Results . . . . . . . Discussion . . . . . SUMMARY 0 O O O O O O O O O O O O O O O O O 0 REFERENCES 0 O O O O O O C O O O O O O O O O O VITA O O O O O O O O C O O O O O O O O O O O 0 vii Page 139 140 140 142 144 148 151 176 Table LIST OF TABLES Experimental design, number of enzyme-altered foci per cubic centimeter of liver and number of rats with neoplastic nodules . . . . . . . Toxicologic effects of dietary 24S-HBB, 345- HBB and FM compared to rats fed a basal diet Concentrations of polybrominated biphenyls in liver and abdominal adipose tissue of rats fed a basal diet or diets containing 245-HBB, 345-HBB or FM for 180 days . . . . . . . . . Effect of 245-HBB, 345-HBB and FM on the concentration of cytochrome P-450 and the activity of aminopyrine demethylase and ethoxyresorufin-o-deethylase . . . . . . . . Experimental design and number of enzyme- altered foci in rats fed a basal diet or diets containing phenobarbital or 345-HBB . . Body weight gain and‘absolute organ weights in rats fed a basal diet or diets containing phenobarbital or 345-HBB for 140 days . . . . Hepatotoxic effects of dietary 345—HBB and phenobarbital compared to rats fed a basal diet 0 O O O O O O O O O O O O O I C O C O 0 Concentrations of 345—HBB in tissues from rats fed a basal diet or diets containing phenobarbital or 345-HBB . . . . . . . . . . Effects of 345-888 and phenobarbital on the concentration of cytochrome P-450 and the activity of aminopyrine demethylase and ethoxyresorufin-o-deethylase . . . . . . . . viii Page . 57 4-3 Experimental design and number of enzyme- altered foci in rats fed a basal diet or diets containing 245-HBB, 345-HBB, FM, phenobarbital or a combination of 245—HBB and 345-HBB . . . . . . . . . . . . . . . . . . . Body weight gain, feed conversion efficiency and absolute organ weights of rats fed a basal diet or diets containing 245-HBB, 345- HBBW FM or combinations of 345-HBB and 24S- HBB O O O O O O O O O O O O O O I O O O I O O Hepatotoxic effects of dietary 245-HBB, 345- HBB, FM or combinations of 34S—HBB and 245- HBB O O O O O O O O O O O O O O O O O O O O 0 Concentrations of polybrominated biphenyls in liver and abdominal adipose tissue of rats fed a basal diet or diets containing 24S-HBB, 345-HBB, FM or combinations of 345-HBB and 245-HBB for 140 days . . . . . . . . . . . . Effects of 24S-HBB, 345-HBB, FM or combinations of 345-HBB and 245-HBB on the concentration of cytochrome P-450 and the activity of aminopyrine demethylase and ethoxyresorufin-o-deethylase . . . . . . . . Experimental design, number of enzyme-altered foci per cubic centimeter of liver, average area of enzyme-altered foci and number of rats with hepatocellular carcinomas . . . . . Body weight gain and organ weights of rats fed a basal diet or a diet containing FM for 15 or 140 days 0 O O O O O O O O O O O O O 0 Chemical concentration of polybrominated biphenyls in liver and abdominal adipose tissue of rats fed a basal diet or diets containing FM for 15 or 140 days . . . . . . Effects of FM (”1 the concentration of cytochrome P-450 and the activity of aminopyrine demethylase and ethoxyresorufin- o-deethylase . . . . . . . . . . . . . . . . Experimental design, number of enzyme-altered foci and concentration of polybrominated biphenyls in the liver of rats given a single oral dose of FM . . . . . . . . . . . . . . . ix Page . 91 . 93 . 95 111 112 121 123 127 128 136 Concentrations of serum and hepatic vitamin A in rats fed a basal diet or diets containing 345-HBB, 245-HBB, PM or combinations of 345- HBB and 245’HBB for 140 days 0 o o o o o o o Page 144 Figure 1-1 1-2 LIST OF FIGURES Page Gamma-glutamyl transpeptidase stained liver section from a rat fed a basal diet . . . . . . . Gamma-glutamyl transpeptidase stained liver section from a rat fed a diet containing FM . . . Photomicrograph of an altered focus in the liver from a rat fed a diet containing phenobarbital .. .. .. .. .. .. .. .. . Photomicrograph of a neoplastic nodule in the liver from a rat fed a diet containing 245-HBB . Photomicrograph of a liver section from a rat fed a basal diet . . . . . . . . . . . . . . . . Photomicrograph of a liver section from a rat fed a diet containing 0.1 mg 345-HBB/kg . . . . . Photomicrograph of a liver section from a rat fed a diet containing 1.0 mg 345-HBB/kg . . . . . Photomicrograph of an altered focus in the liver from a rat fed a diet containing 1.0 mg 345-HBB/kg o o o o o o o o o o o o o o o o o o o Photomicrograph of a section of thymus from a rat fed a basal diet . . . . . . . . . . . . . . Photomicrograph of a section of thymus from a rat fed a diet containing 1,0 mg 345-HBB/kg . . Electron micrograph of a hepatocyte from a rat fed a basal diet 0 O O O O O O O O O O O O O O 0 Electron micrograph of a hepatocyte from a rat fed a diet containing 1.0 mg 345-HBB/kg . . . . . Number of enzyme-altered foci per cubic centimeter of liver for rats fed diets containing either 10 mg 245-HBB/kg, 0.1 mg 345-HBB/kg or 0.1 mg 345-HBB/kg plus 10 mg 24S-HBB/kg................... xi 52 52 54 54 72 72 74 74 76 76 78 78 97 Number of enzyme—altered foci per cubic centimeter of liver for rats fed diets containing either 100 mg 245-HBB/kg, 1.0 mg 345-HBB/kg or 1.0 mg 345-HBB plus 100 mg 245- HBB/kg . . . . . . . . . . . . . .. . . .. . Photomicrograph of a section of liver from a rat fed a basal diet . . . . . . . . . . . . . Photomicrograph of a section of liver from a rat fed a diet containing 100 mg 245-HBB/kg . Photomicrograph of a section of liver from a rat fed a diet containing 1.0 mg 345-HBB/kg . Photomicrograph of a section of liver from a rat fed a diet containing a combination of 0.1 mg 345-HBB/kg plus 10 mg 24S-HBB/kg . . . . . . Photomicrograph of a section of liver from a rat fed a diet containing a combination of 1.0 mg 34S-HBB/kg plus 100 mg 245-HBB/kg . . . . . Photomicrograph of a thyroid section from a rat fed a basal diet . . . . . . . . . . . . . Photomicrograph of a thyroid section from a rat fed a diet containing 100 mg FM/kg . . . . Electron micrograph of a hepatocyte from a rat fed a basal diet 0 O O O O O O I O O O O I I 0 Electron micrograph of a hepatocyte from a rat fed a diet containing 10 mg 245-HBB/kg . . . . Electron micrograph of a hepatocyte from a rat fed a diet containing a combination of 0.1 mg Photomicrograph of a hepatocellular carcinoma from a rat fed a basal diet for 275 days after being fed a diet containing 10 mg FM/kg for 140 days . . .... . . .. . .. . .. . .. . Higher magnification of the hepatocellular carcinoma shown in Figure 4-1 . . . . . . . . . xii Page . 97 . 99 . 99 101 101 103 103 105 105 107 107 124 124 INTRODUCTION Approximately 18.5% of all deaths in the (LS. are caused by cancer. This ranks second only to cardiovascular disease as the most frequent cause of death (Robbins and Cotran, 1979). Two English physicians were the first to recognize the importance of chemicals in the genesis of human cancers. Hill, in 1761, associated excessive use of tobacco snuff with the development of nasal cancer (Redmond, 1970), whereas Pott (1775) recognized the association of scrotal cancer to the occupation of chimney sweeps. Willis (1948) also recognized tine importance of environmental factors in the genesis of human cancers and emphasized the importance of industrial chemicals. Epidemiologic studies suggest that 70-90% of human cancers are associated with environmental causes (Higginson, 1969; Doll and Peto, 1981; Wynder and Gori, 1977) including not only exposure to chemical contaminants in the environment but also to factors determined by lifestyle. Important lifestyle factors involved in the genesis of cancer include cigarette smoking, alcohol ingestion, sunbathing, occupation and diet (Higginson, 1981; Weisburger and Horn, 1982). Stages of initiation and promotion appear to exist in the formation of cancer in humans (Reddy st 21., 1978; Weis, 1979). Environmental factors may be involved in both 1 2 initiation and promotion of carcinogenesis. Chemical mutagens present in {fine environment (Sugimura, 1982; Higginson, 1981; Weisburger and Horn, 1982) along with ultraviolet light and ionizing radiation (Miller and Miller, 1981) may represent the ubiquitous presence of tumor initiators in the environment. The process of tumor initiation may be a common event but in itself may not be sufficient to cause cancer (Berenblum, 1979) without tumor promotion. Although both tumor initiation and promotion are important in the genesis of cancer, the crucial step in decreasing the incidence of human cancer may be limiting the exposure to tumor promoters (Higginson, 1980; Kopelovich, 1982; Sugimura, 1982). The polybrominated biphenyls (PBBs) became a known environmental problem in Michigan in the early 19705 when Firemaster BP-6, a commercial mixture of PBBs, was substituted for a mineral supplement resulting in the contamination of animal feed (Kay, 1977). As a result of consumption of PBB contaminated animal products, 90% of Michigan residents have detectable levels of PBBs in body tissues (Selkoff and Anderson, 1979). What, if any, effect the exposure to PBBs will have on the genesis of human cancer is not known. The first objective of the following studies was to determine if PBBs could participate in one aspect of experimental chemical carcinogenesis; that is tumor promotion. The second objective was to determine if there 3 was an association between toxicity and induction of hepatic drug-metabolizing enzymes and tumor promoting ability of PBBs. The third objective was to further document the pathologic effects of PBBs in rats. LITERATURE REVIEW Chemical and Physical Properties 31 Po1ybrominated Biphenyls Firemaster BP-6 (FM) was manufactured for commercial purposes as a fire retardant in thermoplastics (Hoffman, 1977). Most of FM manufactured was utilized in housings and components of business machines and industrial and electrical equipment (Kerst, 1974). The production of FM in the United States began in 1970 (Kerst, 1974) and ended in 1974 (Di Carlo 31 31., 1978). During the years of FM production, 13 million pounds of FM were manufactured. FM consists of a complex mixture of polybrominated biphenyls (PBBs) consisting of twelve major congeners (Aust 31 31., 1982). FM contains 2% tetrabromo-, 10.6% pentabromo—, 62.8% hexabromo-, 13.8% heptabromo- and 11.4% other biphenyls (Gutenmann and Lisk, 1975). The major congener in FM is 2,2',4,4',5,S'-—hexabromobiphenyl (245-HBB) which comprises 47.8% of the entire mixture (Aust _£ 31., 1982). Hass and coworkers (1978) reported that FM contained 150 1mg/kg pentabromonaphthalene and 70 mg/kg luexabromonaphthalene. These contaminants are not considered to be responsible for the toxicologic effects of FM (Goldstein 31 31., 1978; Aust 31 31., 1982; Robertson 31 ald, 1982). 5 P885 are white, odorless solids at room temperature and begin to decompose above 300° C (Di Carlo 33 31., 1978). PBBs are highly soluble in organic, nonpolar solvents (Di Carlo t l”.1978) and are lipophilic (Fries, 1978; Miceli and Marks, 1981). Environmental Contamination 31 Po1ybrominated Biphenyls The P885 became a known environmental contaminant in 1974 when it was discovered that cattle in Michigan were contaminated with PBB after PM was accidently mixed into cattle feed in the place of magnesium oxide. The accidental contamination of animals and the consequences have been the subject of numerous reviews (Dunkel, 1975; Carter, 1976; Getty 3E 31., 1977; Kay, 1977). The actual contamination occurred during the spring of 1973 but the contaminant was not identified until 1974 by which time animal products tainted with PBB had reached market places resulting in human exposure. Due to the contamination by PBBs, millions of dollars worth of animals and animal products have been destroyed and disposed of (Sleight, 1979). Hepatic Microsomal Enzyme Induction 3y Xenobiotics The liver is the primary organ for the metabolism and excretion of foreign compounds (xenobiotics) (Kappas and Alvares, 1975) as well as endogenous compounds such as steroids (Conney, 1967). The essential effect of hepatic metabolism is to convert fat-soluble compounds into 6 water-soluble ones which can be excreted via urine or bile (Milburn 31 31., 1967). The system responsible for metabolism is the mixed function oxidase (monooxygenase) system which is physically located in the membrane of the endoplasmic reticulum (Ullrich, 1978). The mixed function oxidase system (MFO) requires NADPH and oxygen (Brodie 31 31., 1958; Gillette, 1966) and is catalyzed by a group of hemoproteins collectively known as cytochrome P-450. Cytochrome P-450 appears to be an aggregate of multiple hemoproteins (Welton and Aust, 1974; Guengerich, 1977; Dannan 31 31,, 1983) and can metabolize a broad spectrum of compounds apparently due to the multiplicity of hemoproteins and their differing substrate specificities (Guengerich, 1977; Coon __t__1., 1977). In addition, there are multiple forms of individual enzymes associated with the MEG system, each of which may exhibit different but partially overlapping substrate specificities (Ingelman-Sundberg, 1980). Oxidation is of primary importance in the metabolism of many xenobiotics by MFO. Cytochrome P-450 binds a substrate after which the ferric iron of cytochrome P-450 is reduced by cytochrome P-450 reductase (Gillette 31 31., 1972) by utilizing electrons from NADPH. linthe reduced state, the cytochrome P-450-substrate complex binds molecular oxygen. After the complex is further reduced, one atom of the molecular oxygen is introduced into the substrate and the second atom of oxygen forms water (Mason, 1957L. Types of 7 oxidation reactions catalyzed by the MFO system include 1) epoxidation of double bonds and aromatic rings, 2) hydroxylation of carbon-hydrogen bonds in side chains or aromatic rings, 3) nitrogen oxidations, 4) sulfoxidations, 5) sulfur, oxygen or nitrogen dealkylations, 6) deaminations and 7) desulfurations (Ingelman-Sundberg, 1980; Blumberg, 1978). After oxidation (phase I reactions), the metabolites of xenobiotics can serve an; substrates for additional enzymatic activity (phase II reactions) by enzymes such as epoxide hydrolase (Lu and Miwa, 1980) or can undergo conjugation reactions including glucuronidation, sulfation, acetylation, methylation or glycine conjugation (Ingelman- Sundberg, 1980; Kappas and Alvares; 1975). Although oxidation is of primary importance, chemical reduction can also be catalyzed by enzymes of the endoplasmic reticulum. Microsomal reductions include nitroreductases, azoreductases and dehalogenation (Blumberg, 1978). During the metabolism of certain xenobiotics, reactive intermediates can be formed. Reactive intermediates can be of two kinds: electrophiles or radicals (Ingelman-Sundberg, 1980). Electrophilic reactive intermediates can covalently bind to cellular DNA which is thought to be a critical event in the process of initiation in chemical carcinogenesis (Miller and Miller, 1981). Hepatocytes of hepatic tumors may have altered levels of cytochrome P-450. Okita and coworkers (1976) reported that the level of cytochrome P-450 in hepatic hyperplastic 8 nodules was 20 to 37 percent that of normal liver. Lai and Becker (1982) found that primary hepatocellular carcinomas only contained 12 percent of normal hepatic content of cytochrome P-450. Even though the total amount of cytochrome P-450 is decreased in hepatic tumors, certain enzymes such as epoxide hydrolase may be increased in the microsomal fractions of hyperplastic nodules and hepatocellular carcinomas (Sharma 31 31,, 1981). The nomenclature for the cytochromes of the MEG system is derived from the absorption maximum of 448 or 450 nm when the reduced form of cytochrome is complexed with carbon monoxide (Omura and Sato, 1963). There are two basic patterns of microsomal enzyme induction including phenobarbital-type (Pb) which is associated with cytochrome P-450 and 3-methy1cholanthrene-type (MC) which is associated with cytochrome P-448. Xenobiotics that induce microsomal enzymes similar to those induced by Pb or MC are classified as either Pb-type or MC-type of enzyme inducers (Conney, 1977). Polyhalogenated aromatic hydrocarbons can be classified as Pb-, MC- or mixed-type inducers of hepatic microsomal cirug metabolizing enzymes. FM is classified as a mixed-type <>f microsomal enzyme inducer in that it has properties of microsomal enzyme induction similar to MC and Pb (Dent 31 31., 1976). Purified congeners of FM have been classified (as Pb-, MC- or mixed-type of microsomal enzyme inducers. Pb-type of microsomal enzyme inducers in FM are 9 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB), 2,2',3,4,4',5,5'-heptabromobiphenyl, 2,2',3,3',4,4',5- heptabromobiphenyl and 2,2',3,3',4,4',5,5'-octabromobiphenyl (Aust 31 31., 1982; Besaw 31 31., 1978; Moore 31 31., 1978a,b; Dannan _1 31., l982a). Mixed type of microsomal enzyme inducers in FM are 2,3',4,4',S-pentabromobiphenyl, 2,2',3,4,4',S-hexabromobiphenyl, 2,3,4,4',5,S'- hexabromobiphenyl and 2,3,3',4,4',S-hexabromobiphenyl (Aust _1 _1., 1982; Dannan _1._l., 1978b, l982a,b). Strictly MC-type of microsomal enzyme inducers have also been isolated from FM. Small quantities of 3,3',4,4'- tetrabromobiphenyl, a strictly MC-type microsomal enzyme inducer, has been isolated from FM (Robertson 31 31., 1983). Aust and coworkers (1982) purified 3,3',4,4',5,5'- hexabromobiphenyl (34S-HBB) from a mixture obtained from RFR Corporation (Hope, RI). This congener is strictly a MC—type of hepatic microsomal enzyme inducer and is the most toxic PBB congener that has yet been tested (Aust 31 31., 1982; Render __1 _1., 1982). Xenobiotics that induce MC-type of hepatic microsomal drug metabolizing enzymes have been associated with toxic responses characterized by body weight loss, thymic involution, porphyria, liver enlargement and histologic hepatic alterations (Goldstein 31 31., 1977; Goldstein, 1979; Kociba 31 31., 1978; Poland and Glover, 1977; Poland __t _1., 1979). MC-type of microsomal enzyme inducers are exemplified by 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) for which a cytosolic receptor referred to as 10 the "TCDD" receptor has been identified (Poland and Glover, 1977; Poland 31_31., 1979). This receptor, together with its ligand might translocate to the nucleus in a manner similar to that described for steroid hormones where it initiates gene expression (Poland and Glover, 1977; Poland 31 31., 1979). 345-HBB apparently binds to the TCDD receptor and causes many of the same toxic responses as TCDD in laboratory animals (Kociba t _1”,l978; Render 31 _1,, 1982). There is a high correlation between the ability to bind to the TCDD receptor, induction of MC-type of microsomal enzymes and toxicity although the biochemical basis for toxicity is not known. Pathotoxicologic Effects 31 Polybrominated Biphenyls This portion of the literature review will be limited to the pathotoxicologic effects of 34S—HBB, 245-HBB and FM in rats. Clinical Pathology Rats fed diets containing various concentrations of FM had normal red blood cell counts, packed cell volumes, hemoglobin concentrations and total and differential white blood cell counts (Gupta 31 31., 1981; Garthoff 31 31., 1977; McCormack and Hook, 1979; Sleight 31 31., 1978; Sleight and Sanger, 1976). Levels of blood urea nitrogen have also been normal in rats fed diets containing FM (Garthoff 31 31,, 1977; Sleight 31 31., 1978; Sleight and Sanger, 1976). Gupta 31 31, (1981) reported an elevation of 11 serum alanine aminotransferase (ALT) in male rats given 22 daily oral doses of 30 mg FM/kg body weight whereas Garthoff 31 31. (1977) reported that rats fed PM at a dietary concentration of 500 mg/kg fOr ES weeks had normal concentrations of serum ALT. Gupta _1__1. (1981) reported that female rats given PM or 245-HBB had elevated serum levels of gamma—glutamyl transpeptidase. Thompson (1981) found that elevation in the serum concentration of sorbital dehydrogenase in the rat was a sensitive indicator of altered hepatocellular permeability caused by PBBs. Thompson (1981) also reported that rats fed diets containing 34S-HBB had elevated serum concentrations of aspartate aminotransferase (AST) whereas the serum concentrations of ALT were normal. Serum concentrations of cholesterol were elevated in rats fed diets containing 100 mg FM or 245— HBB/kg or 1.0 mg 345-HBB/kg. The increase of serum cholesterol concentrations was primarily due to an increase in the concentration of cholesterol in the high density lipoprotein fraction (Thompson, 1981). Clinical Signs Rats fed diets containing up to 100 mg FM/kg for 60 days had no signs of toxicosis whereas rats fed a diet containing 500 mg FM/kg had decreased weight gain and feed efficiency'(Sleight and Sanger, 1976L. Gupta _1 _1.(1981) reported that rats given 22 daily oral doses of 30 mg FM/kg loody weight had no clinical signs of toxicosis except for 12 decreased body weight gain and decreased feed efficiency. Rats fed diets containing either 10 or 100 mg 345-HBB/kg had decreased body weight gain whereas rats fed diets containing PM or 245-HBB at the same dietary concentrations did not (Render t 1., 1982). Organ Weight Alterations One of the most consistent changes in rats fed diets containing either FM, 245-HBB or 34S-HBB is hepatomegaly (Sleight and Sanger, 1976; Akoso 31 31., 1982; Render 31 31., 1982). PM at a dietary concentration of 100 mg/kg caused an increase in the thyroid to body weight ratio whereas a diet containing 100 mg 24S-HBB/kg did not (Akoso _1 _1., 1982b; Render _1__1., 1982). Diets containing 345- HBB and FM caused a significant decrease in thymic weights of rats when compared to values of rats fed a basal diet (Thompson, 1981; Gupta _1 _1”.1981). Gupta and coworkers (1981) reported that rats given 22 daily oral doses of 30 mg FM/kg body weight had no significant difference in the weights of lung, heart, spleen, kidneys, adrenals, thyroids, testes, ovaries, uterus, or brain when compared to values from rats fed a basal diet. giistopathologic Alterations Since the liver, thyroid, and thymus are the primary organs affected by PBBs, the literature review will be 1iJnited to these organs. Sleight and Sanger (1976) reported rugpatocellular hypertrophy and vacuolation in rats fed diets 13 containing 10 or 100 mg FM/kg for 30 or 60 days. Hepatic changes were more severe in rats fed 100 mg FM/kg diet. Kimbrough 31 31. (1980) observed enlarged and vacuolated hepatocytes, megalohepatocytes, necrosis and interstitiad fibrosis in rats given oral doses of FM. Other hepatic lesions.(associated with feeding FM include increased cellular pleomorphism, bile duct hyperplasia and areas of inflammation and necrosis (Sleight and Sanger, 1976; Kimbrough _1 _1” 1978a; Kimbrough _1__1u 1980L. Hepatic alterations in rats induced by 24S-HBB are similar to those induced by FM but less severe (Render 31 31,, 1982; Akoso 31 _1,, l982a). Livers from rats fed diets containing 34S-HBB had extensive hypertrophy of hepatocytes due primarily to increased numbers of vacuoles containing lipid (Akoso 31 .31., 1982a; Render 31 31., 1982). Hepatocyte hypertrophy induced by 345-HBB caused obliteration of the sinusoidal space and disorganization of hepatic architecture. Histologic alterations of the liver induced by ETL 24S-HBB or 34S-HBB involved primarily the centrolobular and midzonal portion of the hepatic lobule (Akoso _131., l982a; Render __1 _1., 1982). Sleight 31 31. (1978) reported mild follicular epithelial hyperplasia in the thyroid of rats fed a diet ccnitaining 100 mg FM/kg. Follicles had poorly staining (nolloid or an absence of colloid. Extensive hyperplasia and hypertrophy of follicular cells and a lack of colloid were 14 prominent lesions in rats fed diets containing either 10 or 100 mg/kg of FM, 245-HBB or 345-HBB (Akoso 31 31,, 1982b). Thymic alterations. consisting of xnarked atrOphy, decreased number of cortical lymphocytes and a loss of demarcation between the cortex and medulla have been described in rats fed diets containing PM or 34S-HBB (Gupta and Moore, 1979; Render 31 31., 1982). Ultrastructural Pathology Ultrastructural hepatocyte alterations induced by 245- HBB and FM consist primarily of increased smooth endoplasmic reticulum (SER) and vacuoles containing lipid (Render 31 31., 1982; Akoso 31 31,, l982a). Alterations of hepatocytes from rats fed diets containing 345-HBB included proliferation and disorganization of rough endoplasmic reticulum (RER) and increased numbers of myelin figures (Render t 1., 1982). In addition, there were increased numbers of lipid droplets within hepatocytes. Carcinogenesis s 3 Two-Stage Process Chemical carcinogenesis was recognized as a multistage process by the studies of Rous and Kidd (1941), Mottram (1944), Berenblum (1941) and Berenblum and Shubik (1947). These early studies differentiated betweenlchemicals that are initiators and those that are promoters of carcinogenesis. The classic initiation-promotion assay involves the induction of skin tumors in the mouse. A single small dose of an initiator such as 7,12- 15 dimethylbenz(a)anthracene (DMBA) followed by repetitive doses of lZ-O-tetradecanoyl phorbal-l3-acetate (TPA) causes large numbers of papillomas within 3 to 4 months and carcinomas at approximately one year (Berenblum and Shubik, 1949). The administration of only a single dose of DMBA or only repetitive doses of the promoter TPA does not cause tumors (Boutwell, 1964). The sequence of application of the initiator and promoter cannot be reversed. If a tumor promoter is administered before the initiator, tumors do not develop (Williams 31_3Q,, 1981). Since the initial studies on the mouse skin, many initiation-promotion assays have been devised for other organ systems including the liver, thyroid, mammary gland, stomach, thymus, kidney, lung, colon and pancreas (Berenblum, 1979). Chemica11y Induced Initiation 31 Carcinogenesis The exact molecular event involved in the initiation process is not known, although most evidence would indicate alteration of deoxyribonucleic acid (DNA) is a critical event in the process of initiation (Farber, 1982; Miller and Miller, 1981). Most initiators are in the form of procarcinogens and must be metabolically activated to an electrophilic form (Miller and Miller, 1969). Metabolic activation of initiators is primarily via the cytochrome P- 450 dependent monooxygenase system (Czygan 31 31., 1973; Guengerich, 1977; Miller and Miller, 1969). Some initiators, such as direct alkylating and direct acylating 16 chemicals, are electrophilic without prior metabolic activation (Miller and Miller, 1981). Electrophilic forms of the initiator can covalently bind to nucleophilic sites including DNA, ribonucleic acid and proteins (Miller, 1978; Miller, 1970). Electrophiles nonenzymatically form covalent bonds with nucleophiles by sharing electron pairs (Miller and Miller, 1981). The cellular nucleophile that has received the most emphasis in the carcinogenic process is DNA. The conversion of a normal cell to a tumor cell requires a certain number of discrete changes often described as "hits" (Druckrey, 1967; Emmelot and Scherer, 1977). These discrete changes are thought to represent somatic mutations. The somatic mutation theory of carcinogenesis was first proposed by Boveri (1929). Somatic mutations which may or may not lead to a neoplastic state can result from the direct interaction of a chemical with DNA. Simple mathematical models predict the number of "hits" needed for the formation of a tumor cell to be a minimum of 2 and may be as many as 7. These simple mathematical models are based on the relationship between tumor incidence, dose of initiator and the length of time between the administration of the initiator and the appearance of tumors (Emmelot and Scherer, 1977). The interaction of an electrophilic initiator with DNA can result in biochemical alterations. Pyrimidine and purine bases of DNA have many sites that could potentially 17 interact with electrophilic initiators. The elements of oxygen and nitrogen within the pyrimidine and purine bases are the most likely sites of DNA adduct formation (Irving, 1973; Singer, 1976, 1979). The sites of biochemical alterations of DNA most likely relevant to the carcinogenic process are the sites of hydrogen bonding between purine and pyrimidine bases of antiparallel DNA chains. DNA adducts at the sites of hydrogen binding between bases would most likely have miscoding properties (Farber, 1982). Mammalian cells can respond to DNA adducts having miscoding properties in a number of ways: 1) cellular replication may be slowed or stopped, 2) adducts can be removed by one of several repair mechanisms prior to replication, 3) an acceptor protein can accept and remove DNA adducts, 4) DNA with adducts may be bypassed during replication leaving gaps, 5) DNA adducts may be replicated causing mutations or 6) DNA adducts may be transcribed giving rise to incorrect gene products (Bresnick and Eastman, 1982). Cell replication is required to»"fix“'DNA alterations (Craddock, 1976; Ying 31 _1., 1981). After fixation of the altered DNA, it becomes a permanent property of that cell and its daughter cells (Cairns, 1975). Initiation, therefore, is 21 two-step- process: a biochemical step involving alterations of DNA followed by cell proliferation that "fixes" the biochemical lesions of DNA. 18 Other biochemical alterations of DNA that may represent relevant changes of the cellular genetic material that could lead to neoplasia (Cairns, 1981; Lawley, 1978; Singer, 1979) include: 1) frameshift mutations, 2) deletions, duplications, inversions or translocation of large chromosomal segments or 3) numerical changes of chromosomes caused by agents that interfere with the process of chromosomal separation during cell division (Sorsa, 1980). Initiated cells as used in this dissertation will refer to hepatocytes that have altered DNA induced by a genotoxic initiator. These cells can proliferate when influenced by a tumor promoter but they may or may not progress to hepatocellular carcinomas. Single initiated cells cannot be identified morphologically but have properties that can be used to separate them from normal cells. Initiated cells are resistant to a variety of cytotoxic chemicals. Carr and Laishes (1981) found that liver cells isolated from carcinogen-treated rats were resistant to the cytotoxic effects of adriamycin, cyclohexamide, methotrexate, and aflatoxin Bl' Farber and colleagues (1976) reported that hyperplastic nodules induced by N-2-fluorenylacetamide (2- FAA) were resistant to the acute necrogenic effects of carbon tetrachloride and dimethylnitrosamine. Possible mechanisms of resistance by initiated cells to hepatotoxins include: 1) decreased cell uptake and transport of the toxin, 2) increased toxin inactivation or 3) decreased activation of the toxin by the initiated cell (Carr and l9 Laishes, 1981). Haddow (1938) hypothesized that the formation of cytotoxin resistant cells is an early step in the carcinogenic process. Initiated cells, being resistant to a variety of cytotoxic chemicals, could thrive and proliferate in an environment that was unfavorable to normal cells. Initiated cells have a proliferative advantage when compared to normal cells. Initiated cells have a higher rate of proliferation than normal cells (Schulte-Hermann 31 31., 1981) and treatment with mitogens for cell proliferation further enhances the pmoliferative advantage of the initiated cells (Pugh and Goldfarb, 1978; Schulte- Hermann 31 31,, 1981). Initiated cells appear to possess an inherent defect in growth control rendering them more susceptible to endogenous and exogenous growth stimuli (Schulte-Hermann, 1981). Tumor Promotion 3y Xenobiotics Just as the exact molecular mechanism of initiation is not known, the mechanism of tumor promotion has not been elucidated. Promotion is the process whereby a single initiated cell proliferates to a focus of many cells, some of which may progress to cancer cells. TPA, the classic tumor promoter for mouse skin assays, causes a variety of effects in mouse epidermal cells including rapid increases in phospholipid synthesis, increased RNA synthesis, induction of ornithine 20 decarboxylase activity, enhancement (HE DNA synthesis, increased mitotic rate, increased specific proteases and modifications of cellrnembranes (Boutwell, 1964; Colburn, 1980; Van Duuren, 1976; Weinstein, 1981). Many of these changes may be normal variations within the phases of the cell cycle (Baserga, 1981) and may be the result of cell proliferation rather than the cause of tumor promotion (Farber, 1982). No single cellular modification induced by tumor promoters has been identified as the critical event responsible for tumor promotion (Miller and Miller, 1981). Even though the critical event of tumor promotion is not known, proliferation of initiated cells appears to be an essential phenomenon of tumor promotion (Boutwell, 1974; Pugh and Goldfarb, 1978; Schulte-Hermann 31 31,, 1981). Farber (1982) has classified the types of tumor promotion into categories including: 1) differential inhibition, 2) differential stimulation and 3) differential necrosis with regeneration. The mechanism of tumor promotion by differential inhibition is exemplified by an experimental hepatocarcinogenesis assay devised by Solt and coworkers (Solt 31 31,, 1977). The assay utilizes the inhibitory effects of 2-FAA on the mitotic activity of normal cells. Initiated cells are resistant to the inhibitory effects of 2-FAA resulting in proliferation of initiated cells during the time of inhibition of mitosis in the normal cell population. Differential stimulation appears to be the mechanism of promotion by most tumor 21 promoters (Ohde 31 31,, 1979; Schulte-Hermann 31 31,, 1981). Many tumor promoters are mitogenic stimuli for cells of the target organ. Schulte-Hermann and coworkers (1981) have shown that initiated cells have an enhanced response to mitogenic stimuli resulting in an enhanced proliferation of initiated cells when compared to normal cells. Differential necrosis with subsequent regeneration is also a mechanism of tumor promotion. Initiated cells are resistant to the cytotoxic effects of a variety of cytotoxic chemicals to which normal cells are susceptible (Carr and Laishes, 1981; Farber _1 _1,, 1976). The selective necrosis of normal cells followed by endogenous growth stimuli can result in proliferation of initiated cells. The role of necrosis and subsequent regeneration as a mechanism of tumor promotion was first recognized by Friedewald and Rous (1944) who noted an increased incidence of tumors in rabbit ears painted with a subcarcinogenic dose of an initiator followed by regenerative stimuli induced by necrosis. The cell membrane appears to be the primary subcellular target site for tumor promoters. Tumor promoters alter the structure and function of the cell membrane (Blumberg, 1980; Boss and Emmelot, 1974; Lee and Weinstein, 1978; Werner 31 31., 1974; Weinstein 31 31., 1979) and microtubules (Blumberg, 1980L. Alcommon pathway for the effects of tumor promoters associated with alterations of the cell membrane may'be changes in the fluxes of calcium (Weinstein, 1981; Trosko t al., 1982). Calcium regulates many cellular 22 functions (Hennings 31 31,, 1980) and has been implicated as an intracellular messenger for some hormones (Kelley 31 31., 1980). Calcium may also be the intracellular messenger for tumor promotion by TPA (Verma and Boutwell, 1981). A recently discovered property of many known tumor promoters is the ability to inhibit metabolic cooperativity _i_r_1 vitro (Trosko _1_l_., 1981; Yotti _1_1., 1979). Cell to cell communication appears to be an important factor in the control of cell proliferation (Bertram, 1979; Trosko 31 31,, 1982). Ikcell may communicate with a neighboring cell by the passage of small molecules via gap junctions. Contact inhibition between cells is mediated by gap junctions and prevents uncontrolled cell division (Levine _1__1u 1965). The passage of ions, metabolites, nucleotides and small regulatory molecules between cells may modulate such things as growth and cell division (Trosko _e_t_ 31., 1982). Tumor promoters may disrupt cell to cell communication and block the transmission of growth and/or differentiation control signals from normal cells to initiated cells. Such alterations of cellular communication may liberate initiated cells from the control of normal cells. As stated earlier, mathematical models predict that 2 to 7 mutational events must occur before alnalignant cell with the properties of autonomous growth are formed (Emmelot and Scherer, 1977; Potter, 1980). Since initiated cells do not have the properties of autonomous growth and require the effects of tumor promoters for their ability to proliferate, 23 initiated cells apparently have not acquired the number of relevant mutational events necessary for the formation of a malignant cell. Therefore, during tumor promotion, additional relevant mutational events must be acquired for malignant transformation. Since tumor promoters are not genotoxic or mutagenic (Berenblum, 1975; Slaga 31 31,, 1978; Weinstein 31 31., 1979, 1981), it is unlikely that direct effects of tumor promoters are responsible for the additional mutational events necessary for malignant transformation. Stout and Becker (1982) found progressive DNA damage in cell populations considered as precursors of malignant transformation during the promotion phase of a two-stage hepatocarcinogenesis assay. The mechanism of progressive DNA damage may result from repeated cell division (Stout and Becker, 1982). Initiated cells appear to possess an inherent defect in growth control rendering them more susceptible to endogenous and exogenous growth stimuli (Schulte-Hermann 31 31., 1981). The differential stimulation of initiated cells by tumor promoters (Pugh and Goldfarb, 1978; Schulte-Hermann 31 31., 1981) results in repeated cell division of initiated cells. Each time a cell replicates, there is a finite probability that spontaneous mutational events occur (Stott _131., 1981; Trosko _131., 1982). Under the influence of tumor promoters, the increased DNA synthesis and cell replication of initiated cells can result in decreased time for repair of spontaneous DNA lesions before replication, an increased number of 24 replication errors, and an enhanced somatic mutation rate (Stott__1_Jr, 1981L. Tumor promoters increase the number of initiated cells at risk for further relevant events in the carcinogenic process and by causing rapid replication of initiated cells may enhance the chance that one of these cells will acquire additional spontaneous mutations. Oncogene: 3 Common Final Pathology A common final pathway for carcinogenic processes may be the activation of normal cell genes called oncogenes (Bishop, 1982; Cooper, 1982). The Src oncogene of the Rous sarcoma virus codes for a protein kinase which phosphorylates tyrosine. The site of action for this protein kinase appears to be a specialized site on the plasma membrane called the adhesion plaque which is responsible for the adherence of cells to solid surfaces (Bishop, 1982). Phosphorylation of proteins is a part of the regulatory mechanisms controlling the growth of cells (Bishop, 1982). Abnormal expression of cell oncogenes may be responsible for malignant transformation. All tumors originating from the same cell type have activation of the same specific oncogene. Therefore, each cell type may'have itslown oncogene that must be activated for malignant transformation. This phenomenon suggests that tissues have specific pathways of carcinogenesis that are the result of activation of oncogenes (Cooper, 1982). Activation of oncogenes may represent a final common pathway 25 for all types of carcinogenic agents (Bishop, 1982; Cooper, 1982). Biochemical Markers of Hepatocarcinogenesis During the carcinogenic process, new cell pbpulations of hepatocytes appear, some of which may participate in the events leading to cancer. These new cell populations have altered biochemical, biological and morphological properties which may be the result of altered gene expression (Garrett _1 31., 1973; Rolten 31 31., 1977; Atryzek 31 31., 1980). Cancers in humans and experimental animals have similar altered biochemical, biological and morphological properties (Farber 31 31., 1979). Alterations of expression of specific enzymes have been used to identify populations of hepatocytes thought to be involved in the carcinogenic process. Biochemical markers for altered hepatocytes include increased (positive) or decreased (negative) amounts of specific enzymes or antigens. Negative markers for altered hepatocytes include glucose-6-phosphatase, nucleotide polyphosphatase, glucuronidase, serine dehydratase, adenosine triphosphatase and glycogen phosphorylase (Farber, 1976). Positive markers for foci of altered hepatocytes include alpha-fetoprotein (Abelev, 1971), preneoplastic antigen (Lin 31 31., 1977), gamma-glutamyl transpeptidase (Fiala 31 31., 1972), D-T diaphorase (Schor and Morris, 1977), and chorionic gonadotropin (Malkin 31 31,, 1977). 26 Gamma glutamyl transpeptidase (GGT) is one of the best markers for hepatocytes thought to be involved in the carcinogenic process. Ninety to nine-five percent of the foci of altered hepatocytes stain positively for GGT (Ogawa, 1977; Cameron 31 31,, 1978). GGT is normally present in the smooth endoplasmic reticulum of hepatocytes of the fetal and neonatal rat but hepatocytes of the adult rat are uniformly negativerfor GGT (Rutenberg _1 _1url969). Hepatocytes of the adult rat that regain the ability to express GGT are thought to be involved in the carcinogenic process (Farber t 1., 1979). "...—u” Enzyme-Altered Foci 33 Precursor Lesions 31 Hepatocellular Carcinomas The use of enzyme-altered foci aslearly indicators of neoplasia in carcinogen bioassays requires an understanding of the relationship of enzyme-altered foci to hepatocellular carcinomas. There is much evidence for the precursor relationship of enzyme-altered foci to hepatocellular carcinomas: l) Enzyme-altered foci and hepatocellular carcinomas are induced by chemical carcinogens with the formation of enzyme-altered foci preceding the appearance of hepatocellular carcinomas (Williams 31_3g,, 1976). 2) There is a direct proportionality between the number of DNA adducts formed, the number of enzyme-altered foci induced and the initial dose of hepatocarcinogen (Scherer and Emmelot, 1975). 3) Enzyme—altered foci and hepatocellular 27 carcinomas have an enhanced mitotic activity as compared to normal hepatocytes (Pugh and Goldfarb, 1978; Schulte-Hermann _1 31,, 1981). 4) Enzyme-altered foci and hepatocellular carcinomas have similar biochemical alterations (Farber, 1973; Enomoto 31 31,, 1981) including the ability to express gamma-glutamyl transpeptidase (Hirota and Williams, 1970; Fiala 31 31., 1972). 5) Enzyme-altered foci and hepatocellular carcinomas have similar morphologic and functional abnormalities (Farber, 1973; Williams 31 31., 1976; Williams and Watanabe, 1978). 6) Histochemically determined enzyme phenotypes of altered foci are extremely variable just as tflua enzyme phenotype profiles of hepatocellular carcinomas (Goldfarb and Pugh, 1981; Ogawa 31 _1., 1980). 7) Rabes and coworkers (1972) demonstrated by morphologic alterations and autoradiography that foci of cells histologically and biologically compatible with hepatocellular carcinomas arose within enzyme—altered foci. Enzyme-altered foci may represent the first morphologically identifiable stage in the hepatocarcinogenic process and appear to be clonal in origin (Scherer and Hoffman, 1971). Enzyme-altered foci can progress or undergo phenotypic reversion (Rabes __1 31., 1972). Following the cessation of exposure to tumor promoters, a majority of enzyme-altered foci remodel to conform with the normal hepatic architecture (Farber, 1980; Watanabe and Williams, 1978; Williams and Watanabe, 1978). The phenotypic reversion of most enzyme-altered foci would indicate that 28 the cells comprising the foci are still subject to control processes that maintain the differentiated state of hepatocytes (Williams, 1980) and that these cells have not acquired the property of autonomous growth that characterize malignant cells. The foci of cells that do not undergo phenotypic reversion but progress towards hepatocellular carcinomas have apparently acquired additional genetic alterations that allow them to proliferate without the influence of a tumor promoter. A553ys for Initiation/Promotion 13 the Liver Solt-Farber Ass3y The Solt-Farber system (Solt 31 31,, 1977) is utilized for the rapid production of preneoplastic lesions. Rats receive a carcinogenic dose (200 mg/kg body weight) of diethylnitrosamine (DEN). Two weeks later, rats are fed a diet containing 0.02% 2-FAA for one week followed by a partial hepatectomy, After partial hepatectomy, the rats are again fed a diet containing 2-FAA. Small nodules appear in the liver by 10 days after the partial hepatectomy. The strong selection pressure for initiated cells consists of dietary 2-FAA which has a "mitoinhibitory" effect on normal cells but not initiated cells and a partial hepatectomy which promotes the proliferation of initiated cells. The advantages of the Solt-Farber assay is the synchronization and rapid formation of preneoplastic lesions (Solt t al., 1977). A disadvantage of this assay is the use of a second 29 carcinogen in the selection process. Williams and coworkers (1981) have shown that the sequential administration of two initiators has a summational effect on carcinogenesis. Also, the effect of two initiators may alter the character of the preneoplastic lesions (Leonard _1 _1” 1982). This assay also utilizes a carcinogenic dose (200 mg/kg body weight) of DEN which is capable of inducing preneoplastic lesions without any subsequent selection pressure (Scherer and Emmelot, 1975). Pitot Assay The Pitot protocol (Pitot 31 31., 1978) is an initiation-promotion assay that clearly separates the initiation stage from the promotion stage. The initiation phase of the protocol consists of a 70% partial hepatectomy followed by a subcarcinogenic dose (10 mg/kg body weight) of DEN. The partial hepatectomy serves as a stimulus for cell proliferation which serves to "fix" the DNA damage as a permanent alteration of the cell. The promotion phase of the protocol consists of dietary treatment of the test chemical starting 60 days after the partial hepatectomy. Phenobarbital at a dietary concentration of 500 mg/kg is the standard tumor promoter. A disadvantage of the Pitot model is the length of time required for the process of promotion (180 days). The dose of initiator in the Pitot protocol is an advantage since this dose (10 mg/kg body weight) of DEN is subcarcinogenic and will not cause the formation of hepatocellular carcinomas without tumor promotion. A single 3O administration of only one initiator assures that the foci that form are the result of a single initiator. The Pitot assay is useful for determining the initiating or promoting ability of a chemical since each stage is distinctly separated. Peraino Neonatal Rat Model Peraino and coworkers (Peraino _e_t 31., 1981) have developed an assay which utilizes newborn rats. Day old rats are given an intraperitoneal dose ofauiinitiator and when weaned at 21 days of age are fed a tumor promoter. Enzyme-altered foci are formed by 4 weeks after weaning. The advantages of this assay include the elimination of the need for a partial hepatectomy and the rapidity of the assay. One disadvantage of this assay is that day old rats may not have the full complement of hepatic microsomal drug metabolizing enzymes (Goldberg, 1979; Short _1__1,, 1976) necessary to metabolically activate all classes of procarcinogens. Peraino ASS3y The Peraino assay (Peraino 31 31., 1971) consists of feeding .02% 2-FAA to rats for 4 weeks followed by 39 weeks of dietary phenobarbital. Disadvantages of this model include a lack of synchronization of initiated cells since hepatocytes could be initiated anytime during the 4 week period of 2-FAA administration. Also, the initiator is fed 31 at a carcinogenic dose which could lead to the formation of preneoplastic lesions without the need for tumor promoters. Carcinogenic Effects 31 Polybrominated Biphenyls 13 Vivo The polybrominated biphenyls have various effects in the carcinogenic process 13 1113. Polybrominated biphenyls in some instances have had no effect, an inhibitory effect, or an enhancing effect on carcinogenesis. Schwartz and coworkers (1980) have shown that the feeding of PBB concurrently with 2-FAA to rats caused a significant reduction of 2-FAA-induced tumors at sites other than the liver. The incidence of hepatic tumors in rats fed PBB concurrently with 2-FAA was not significantly altered. The mechanism of inhibition of carcinogenesis appears to be related to the induction of the liver microsomal monooxygenase activity' (Wattenberg, 1978). Increased hepatic drug metabolizing enzymes induced by FM may detoxify the initiator rather than metabolically activate it to the ultimate carcinogen. Both phenobarbital (Peraino _1 31., 1971) and 3-methylcholanthrene (Flaks and Flaks, 1982) have a protective effect on chemically induced carcinogenesis by 2-FAA indicating that the enzymes induced by both types (Pb and MC) ofxnicrosomal enzyme inducers can be effective in reducing chemically induced tumorigenesis. Harzoz and Aust (1979) reported that FM, 245-HBB and 2,3',4,4',5,5'-hexabromobiphenyl did not have initiating or promoting ability hia two-stage»mouse~skin tumorigenesis 32 assay utilizing ea skin tumor—susceptible mouse strain (SENCAR). Similarly, Berry and coworkers (1979) found that FM did not promote the development of skin tumors in a two- stage system of mouse skin tumorigenesis. The liver appears to be one of the target organs for PBB (Kay, 1977; Kimbrough 31 31., 1978a); therefore, it would be expected to be a primary site for any carcinogenic activity the P885 might have. When weanling rats were given 1 g PBB/kg body weight by stomach tube, neoplastic nodules were found 6 months after dosing (Kimbrough 31 _a_1_., 1978). Kimbrough and coworkers (1981) also found that rats given a single oral dose (1 g PBB/kg body weight) or 12 daily doses of 100 mg PBB/kg body weight had a 41.4 and 67.8% incidence of hepatocellular carcinomas, respectively. These studies indicate that hepatocellular carcinomas could be produced in rats after single or short term dosing with PBB and lifetime-feeding studies are not necessary for tumor induction. The finding of hepatic tumors in rats given large quantities of PBB does not clearly define what role the PBBs play in the carcinogenic process. Within the complex mixture of PBBs there may be congeners that have initiating and/or promoting ability, i.e., complete carcinogen. Alternatively, the PBBs may have only promoting ability and the formation of hepatic tumors may be the result of promotion of spontaneously or environmentally induced initiated cells (Pitot 31 31,, 1980; Schulte-Hermann and Parzefall, 1981; Schulte-Hermann 31 31,, 1983). 33 Carcinogenic Potential 31 Polybrominated Biphenyls Determined 3y 13 Vitro A353ys The Ames assay, utilizing histidine-requiring mutants of Salmonella typhimurium, has been used to screen large numbers of chemicals for mutagenic activity. The rationale for use of mutagenicity assays to screen for initiators of carcinogenesis is based on the correlation of mutagenicity and carcinogenicity. A 90% correlation has been found between mutagenicity 13 vitro and carcinogenicity'13ny1y3 (Ames __t_1., 1975; Ames _1_1., 1973; Ames, 1979; Devoret, 1979). To date, no reports of positive mutagenicity in the Ames assay for FM or its major congeners have been published. Zieger (1980) has reported that a nonspecified hexabromobiphenyl was negative for mutagenicity in the Ames assay utilizing Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation by microsomes from hamsters and rats. The brominated biphenyl, 4-bromobiphenyl, which has not been isolated from FM (Aust _1 _1,l982), has been reported to have mutagenic activity in the Ames assay (Kohli 31 31., 1978). Wertz and Ficsor (1978) have shown that mice given an oral dose of 50 or 500 mg FM/kg body weight did not have a significant increase in the number of chromosome or chromatid breaks in bone marrow cells. FM did not increase either the mitotic or metaphase index in the same study. Dannan and coworkers (1978) reported that 14C labelled 34 polybrominated biphenyl congeners consisting of 2,2',4,4',5,5'-hexabromobiphenyl and 2,2',3,4,4',5,5‘- heptabromobiphenyl did not bind to exogenous DNA when incubated with induced microsomal proteins and NADPH. Therefore, these two congeners, which comprise approximately 80% of the entire FM mixture, are not metabolically activated into electrophilic DNA-binding metabolites. The ability to covalently bind to DNA appears to be an important property of known initiators (Miller, 1978; Miller, 1970). The previously described results from current literature would indicate that the polybrominated biphenyls do not have properties of known initiators in that they are not mutagenic, they do not cause chromosomal aberrations and they do not bind to DNA. An 13 vitro property of many known tumor promoters is the ability to inhibit a form of cell to cell communication (metabolic cooperation) (Fitzgerald and Murray, 1979; Umeda _1_1., 1980; Newbold and Amos, 1981). Trosko 3131. (1981) reported that FM inhibited metabolic cooperation 13 vitro suggesting that the polybrominated biphenyls could be tumor promoters. Tsushimoto _1 _1.(1982) tested the individual congeners of FM for the ability to inhibit metabolic cooperativity 13 vitro. Results clearly demonstrated a structure-activity relationship in regards to the ability to inhibit metabolic cooperativity. The polybrominated biphenyl congeners with no bromine substitutions in the ortho position were cytotoxic and did not inhibit metabolic 35 cooperativity at noncytotoxic concentrations. Congeners with only one ortho bromine inhibited metabolic cooperation at low concentrations and were cytotoxic at high concentrations. Congeners with two ortho bromines inhibited metabolic cooperation in a dose dependent manner and were not cytotoxic even at high concentrations. 345—HBB, a congener with no bromine substitutions on the ortho carbons did not inhibit metabolic cooperativity in vitro. In contrast, 245-HBB has bromine substitutions on two ortho carbons and did inhibit metabolic cooperativity in vitro. Therefore, 245-HBB has an _i_p_ vitro property of known tumor promoters (the ability to inhibit metabolic cooperativity) whereas 345-HBB does not. Vitamin A Metabolism Vitamin A is a generic term used for all compounds that have the biologic activity of retinol. The dietary sources of retinol are 8 carotene of plant origin or long—chain retinyl esters from animal tissues. 8 carotenes are absorbed by intestinal mucosal cells and are enzymatically cleaved to yield two retinal molecules which are reduced to retinol (Goodman and Olson, 1969; Fidge and Goodman, 1968). Dietary retinyl esters must be hydrolyzed by pancreatic retinyl ester hydrolase (Ganguly, 1969) to form free retinol which is absorbed by intestinal mucosal cells. The retinol from dietary sources must be esterified with long chain fatty acids in the intestinal mucosal cells before entering 36 the lymphatics (Mahadaven, 1963). Retinyl esters are transported via chylomicrons in the lymphatics (Huang and Goodman, 1965) to the liver where hepatocytes remove the retinyl esters from chylomicron remnants.(Goodman'_£.gl., 1965). Within hepatocytes, retinyl esters are hydrolyzed to form free retinol which can be reesterified primarily with palmitate or can be bound to retinol-binding protein (RBP) and released into the blood. Retinyl esters consisting primarily of retinyl palmitate serve as the vitamin A reserves of the body and are stored within hepatocytes in association with lipid droplets (Goodman, 1980). The hepatic reserves of vitamin A can be mobilized by hydrolysis with retinyl palmitate hydrolase (Mahadevan £5,213! 1966) to form free retinol which binds to RBP (Kanai g£_gl., 1968). RBP serves to solubilize the water-insoluble retinol and along with prealbumin, is responsible for transport of retinol to peripheral tissues via the blood vascular system (Smith and Goodman, 1979). The mechanism of control for the mobilization of retinyl esters from hepatocytes is not completely understood. Serum retinol concentrations can be maintained from either hepatic reserves of retinyl esters or from dietary sources. Under conditions of adequate dietary intake of vitamin A, the dietary sources of retinol maintain the concentration of retinol in the serum and supply peripheral tissue needs (Keilson gt al., 1979; Varma and Beaton, 1972; Yeung and Veen-Baigent, 1974). ‘When dietary 37 vitamin A is inadequate, internal regulatory mechanisms maintain the normal serum retinol concentration by mobilizing retinol from the hepatic reserves of retinyl esters (Underwood 33 31,, 1979). Peripheral utilization of retinol may regulate the mobilization of retinyl esters from the liver. An as of yet unidentified positive feedback mechanism linked to the intracellular uptake and/or utilization of retinol, retinal or a metabolite of RBP formed after the release of retinol may control the mobilization of hepatic retinol esters (Loerch 35 al., 1979; Underwood 35 21,, 1979). Effect of Xenobiotics on Vitamin A Many xenobiotics have adverse effects on vitamin A status of animals. Some of the xenobiotics which decrease the amount of vitamin A in liver and/or serum include bromobenzene (Haley and Samuelson, 1943), l,2,5,6- dibenzanthracene (Goerner and Goerner, 1939), ethanol (Sato and Lieber, 1981), 1,1,l-trichloro-2,2-bis(p-chlorophenyl) ethane (Phillips, 1963; Tinsley, 1969), methoxychlor (Davison and Cox, 1976), chlorinated naphthalenes (Sikes and Bridges, 1952; Staskiewicz, 1964), 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) (Thunberg 93331., 1979, 1980), polychlorinated biphenyls (PCB) (Cecil 3:531" 1973, Innami 35 al., 1974; Kato t 1., 1978; Villeneuve t al., 1971) and polybrominated biphenyls (PBBs) (Akoso, 1981 ‘0 Mangkoewidjojo, 1979; Pratt, 1979). 38 The mechanism whereby hepatic vitamin A is decreased by xenobiotics is not knownm Thunberg _£._l.(l980) proposed that the decreased hepatic vitamin A caused by TCDD was the result of increased conjugation of vitamin A with UDP- glucuronosyl transferase and subsequent excretion in bile. A proposed mechanism for the decrease of hepatic vitamin A caused by PCB is that increased hepatic microsomal drug metabolizing enzymes induced by PCB may enhance the metabolism of vitamin A to inactive metabolites (Innami gt _1., 1976; Innami £5 31,, 1982; Kato, 1978). in vitro assays utilizing hepatic microsomes from hamsters indicate the enzymes involved in the metabolism of retinoic acid have properties similar to monooxygenase enzymes associated with cytochrome P-450 (Roberts gt al., 1979; Roberts and Frolik, 1979). CHAPTER 1 HEPATIC TUMOR PROMOTION BY FIREMASTER BP-6, 2,2',4,4',5,5'-HEXABROMOBIPHENYL CHAPTER 1 HEPATIC TUMOR PROMOTION BY FIREMASTER BP-6, 2,2',4,4',5,5'-HEXABROMOBIPHENYL AND 3,3',4,4',5,5'-HEXABROMOBIPENYL Introduction As a result of the accidental mixing of polybrominated biphenyls (PBBs) into livestock feed; meat, milk, eggs and dairy products contaminated with PBBs were consumed resulting :hi widespread Innnan exposure (Carter, 1976; Dunkel, 1975). A recent survey indicated that 90% of Michigan residents had detectable quantities of PBBs in body tissues (Selikoff and Anderson, 1979). Exposure to PBBs was highest among workers involved in the manufacture of PBBs (Anderson _t _1., 1978) and in people on farms that were quarantined because of the contamination (Humphrey and Heyner, 1975). Epidemiologic studies designed to delineate health problems of people accidently exposed to PBBs have been inconclusive (Bahn gt gl., 1980; Bekes _t _l., 1978; Kay, 1977; Stross, 1981). Although adverse human health effects have not been definitely attributed to PBBs, the persistence of these chemicals in the body causes concern as to long term effects including cancer. 40 41 The purpose of this experiment was to determine if the P885 could participate in one aspect of chemical carcinogenesis; that is tumor promotion. Materials and Methods Experimental Design A two stage hepatocarcinogenesis assay devised by Pitot gt gt. (1978) with minor modifications was used to evaluate the tumor promoting ability of PBBs. In the original experiment (Pitot gt gl., 1978), female Sprague-Dawley rats had a 70% partial hepatectomy followed by ip administration of a subcarcinogenic dose of diethylnitrosamine (DEN) (10 mg/kg body weight). Sixty days after the partial hepatectomy, rats were fed either a basal diet or a diet containing 500 mg phenobarbital/kg for 180 days. Rats fed a diet containing phenobarbital (Pb) had an increased number of enzyme-altered foci and hepatocellular carcinomas when compared to rats fed a basal diet. Chemicals The commercial PBB mixture was Firemaster BP-6 (FM) (Lot 6224A) manufactured by Michigan Chemical Corporation, St. Louis, MI. Congener 2,2',4,4',5,5'-hexabromobipheny1 (24S-HBB) was isolated from FM and purified (>99%) by a combination of chromatography on neutral alumina eluted with hexane and repeated crystallization (Moore and Aust, 1978). Congener 3,3',4,4',5,SV-hexabromobiphenyl (345-HBB) was 42 purchased from RFR Corporation, Hope, RI and was purified (>99%) by repeated alumina chromatography (Aust gt gl., 1982). 245-HBB and 345-HBB were purified by personnel in the Department of Biochemistry, Michigan State University under the supervision of Dr. Steven D. Aust. Outbred female Sprague-Dawley rats weighing 200-215 g were obtained from Charles River Corp., Portage, MI. Rats were housed in clear polypropylene cages in filtered laminar flow units (Contamination Control Inc., Lansdale, PA) at 22° C with a 12 hour light/dark cycle. Rats were acclimated for 7 days before being subjected to a 70% partial hepatectomy (Higgins and Anderson, 1931). Diethylnitrosamine (DEN) (Sigma Chemical Co., St. Louis, M0) at a dose of 10 mg/kg body weight was given i.p. 24 hours after the partial hepatectomy. Thirty days later, rats were randomly assigned to groups as shown (Table 1-1). Diets were prepared by adding Pb, FM, 345-HBB or 24S-HBB in MazolaR corn oil to a ground commercial diet (Wayne Lab Blox, Allied Mills, Inc., Chicago, IL) in the amounts indicated (Table l-l). Controls included rats not partially hepatectomized or given DEN but which were fed the same diets. Necropsy and Collection gt Tissue Samples After the diets were fed for 180 days, rats were anesthetized with ether (Mallinkrodt Inc., Paris, KY) and killed by decapitation. Necropsies consisted of a systematic examination of organs for gross pathologic changes. The brain, kidneys, liver, spleen, and thymus were 43 removed and weighed with a top-loading balance (Mettler Series In Model 163, Mettler Instrument Cbrporation, Highstown, NY). Representative sections of liver were mounted on corks and frozen in isopentane cooled with liquid nitrogen. Five sections of liver, taken from the same portion of the hepatic lobes from each rat, were frozen. Representative portions of liver were fixed in 10% buffered formalin for histologic evaluation. Samples of abdominal adipose tissue and liver for chemical analysis were collected, wrapped in aluminum foil and stored at -200 C. Portions of liver for microsomal enzyme studies were placed in 1.15% cold KCl containing 0.2% nicotinamide. Histologic Preparation Formalin-fixed portions of liver were mechanically processed (Histomatic, Model 166, Fischer Scientific Company, Pittsburgh, PA), embedded in paraffin, sectioned (6 11) and stained with hematoxylin and eosin. Histochemical Staining and Evaluation Sections of frozen liver, mounted on corks, were cut (8 u ) with a cryostat (Pearse-Slee, Type HS, Slee International Inc., London, England) and stained for gamma- glutamyl transpeptidase (GGT) by the technique of Rutenberg _t gt. (1969). An 8 11 section of liver was placed on a cover glass and fixed in 100% acetone for 15 minutes. After air drying, the section of liver was incubated for 15 minutes in a freshly prepared solution containing 1 ml of 44 Y-glutamyl-4~methoxy—2-naphthylamide (2.5 mg/ml) (Vega Biochemicals, Tuscon, AZ), 5 ml Tris buffer (0.1 M, pH 7.4) (Sigma Chemical Company, St. Louis, MO), 10 mg glycylglycine (Sigma Chemical Company, St. Louis, MO), 10 mg Fast Blue BBN (diazotized 4'-amino 2',5'-diethoxybenzani1ide) (Sigma Chemical Company, St. Louis, MO) and l4tnlAA uo 0%EO\A00u @8030 means .. manimfio 3:303 upon gm... 3 mA 003055950 zuan $6303 ASH—0% «2.0.50 AAAHAAA o A STA: A... AA 082 AA n.0AoAHAAAA A A 8.7va A... AA ARTE AA AAAHAAA A A E A... 8A 802 AA A.AAAAAHAAAA A A E A... 8A 281... AA AAAHAAA o A E A... AA 082 A A.A.o.AAAAAHAAAA A A E A... AA BATE A Anus A A 8:.va A... 8A 802 A. A.AAAAA.+.AAAA A A Brim: A... 8A 281... A Ana o A 8:.va A... AA «82 A A£.A..1.AAAA.AAAA A A ETA: E AA 284: A ndAAAAHAAAA A A a A... 8... 28.2.. A AA... A A 030 A88 «82 A .3310. 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SH: 8A ABAfiBQis A9602 AauAA 8H8. AAHAA A $6 A88 uo>AA uo 830305 .3 .2303 .3 2303 A3 500 3%. 205005 5303 30305833: Hg: 338.2 "AAA—$5 338A: .2303 >89 umAc cA coAunuucwucoO .38 A38 a BA 39. 8 Bug E A5 0979A .BATAAA 53vo A0 398% 03032.2. .AIA «38. 51 brain, kidneys or spleen of rats fed diets containing PBBs when compared to rats fed a basal diet. Histopathologic Evaluation oi the Liver Livers from rats fed diets containing Pb or 245-HBB had mild to moderate hepatocellular hypertrophy in the centrolobular to midzonal regions of the hepatic lobule (Table 1-2). Dietary 5W! caused severe diffuse hepatocellular hypertrophy with loss of recognizable sinusoidal space and moderate to marked vacuolation of hepatocytes which was most severe in rats fed a diet containing 100 mg FM/kg. Livers of rats fed a diet containing 1.0 mg 345-HBB/kg had marked hepatocyte hypertrophy with severe fatty degeneration and focal necrosis of hepatocytes in the centrolobular and midzonal region of the hepatic lobule. Categories of preneoplastic lesions ranged from foci and areas of altered cells to neoplastic nodules (Institute of Laboratory Animal Resources, National Research Council, 1980). Most foci and areas were composed of acidophilic cells characterized by abundant amounts of cytoplasm with a ground glass appearance (Figure 1-3). Nuclei were enlarged and many contained multiple nucleoli. Occasionally, basophilic areas and foci were seen and consisted of small cells with basophilic cytoplasm and hyperchromatic centrally located nuclei. Neoplastic nodules were round to oval and compressed the normal hepatic tissue (Figure 1-4). Cells in 52 Figure 1-1. Photomicrograph of a histochemically stained section of liver from a rat fed a basal diet for 180 days after a 70% partial hepatectomy and diethylnitrosamine administration. Notice the lack of hepatocytes that stain positive for gamma glutamyl transpeptidase (Gamma glutamyl transpeptidase stain, x50). Figure 1-2. Photomicrograph of a: histochemically stained section of liver from a rat fed a diet containing 10 mg FM/kg for 180 days after a 70% partial hepatectomy and diethylnitrosamine administration. Notice the numerous foci containing hepatocytes that are gamma glutamyl transpeptidase positive (Gamma glutamyl transpeptidase stain, X50). 53 Figure 1—1 .c . A n? .. , 1m: 5... 317% Figure 1-2 54 Figure 1-3. Photomicrograph of an altered focus in a liver from a rat fed a diet containing 500 mg Pb/kg for 180 days after a 70% partial hepatectomy and diethylnitrosamine administration. The hepatocytes comprising the focus are large with abundant amounts of clear cytoplasm (H & E stain, x200). Figure 1-4. Photomicrograph of a neoplastid nodule in a liver from a rat fed a diet containing 100 mg 245-HBB/kg for 180 days after a 70% partial hepatectomy and diethylnitrosamine administration. The nodule is comprised of large pale staining hepatocytes. Hepatocytes immediately surrounding the nodule are compressed (H & E stain, x50). 55 Figure 1-3 Figure 1-4 56 nodules were primarily of acidophilic type. Hepatocellular carcinomas were DOt ObSGIVEd. Chemical Analysis Tissue concentrations of P385 are given in Table 1-3. Tissue concentrations of FM and 245-HBB were approximately equal in rats that had comparable concentrations of the respective chemical in the diet. Rats fed a diet containing 10 mg 24S-HBB or FM/kg had approximately 1/10 the amount of respective chemical as rats fed diets containing 100 mg 245- HBB or FM/kg. Rats fed diets containing 1.0 mg 34S-HBB/kg had a greater concentration of 345-HBB in the liver than in abdominal adipose tissue. Microsomal Enzyme Assays Results of assays for hepatic microsomal drug metabolizing enzymes are given in Table 1-4. Rats fed diets containing PBBs had a dose related increase in the amount of cytochrome P-450. Dietary FM and 34S-HBB caused a downward shift in the carbon monoxide difference spectra of cytochrome P-450. Aminopyrine demethylase activity was markedly increased by 24S-HBB but only minimally increased by FM or 345-HBB. Activity of ethoxyresorufin-o-deethylase was increased in rats fed diets containing FM or 345—HBB. Discussion The results of this experiment indicate that 345—HBB, FM and its major congener, 24S-HBB, have tumor promoting 57 .mmmwnucwumm aw omumufiGCw mfi owmfia ucmoumm .mumu m How anM ucmmmumwu 0cm mwmmn cfimma m co commwumxm mum mwsHm>m .coflumuumwcwenm mcflEmmOHDACaanumwc cam >Eouomummm: Howuumm wow m on: mumu Ham Ao.mHm.~mv mHmH Ao.hnm.HHc mHomm o.H mmzumvm A~.~Hm.msv smauomom Am.oum.~c mshmHOHem ooH 2m Am.vun.mmv wfluwmm .5.~Hm.vc seaweed OH 2m A>.ouv.flmv mHmHommm Am.mum.mv ambumoma OOH ammumvm Am.HHm.mmv mqaummm Am.mnm.mc oomumom OH mmmumvm .m.sufl.msv oo.Hm.~ Ao.mue.mv MH.Hm.~ o umflc Hmmmm wsmmwu wmomwp< um>wa Amx\mev ucwEummuu xumumwo umao a“ mAmx\mEV coflumuucmocoo msmmwe :ofiumuucmocoo .mxmo omH you mumm uwummemuwm no mmmumvm .mmznmem mcflcwmucoo mumwo no umfio Hmmmm m com mumm mo msmmws wmomflom HmaEoon¢ new um>wq aw mawcmnmflm owumaEounaaom mo mcofiumuucmocou .mIH wanna 58 636mm @308 comm $39.00 mumu m Eouu @8me .835 @3000 m mom omHm. mm @3835 9.8 mama fifimgm m.o.+..~.2 ms: 2.....mm.~ o; 87va nonmam MAN“; 2;. 3.H-.~ 02 “momma“: 9.3.2 Tmuod 8% Shaw; 3 “3.0.29; SHE. Tong; 3.3 Show; 2: 333 Sufi. H.~H~.S m5: Sham; S 8.3% 2.5.9 fauna a: SAMBA o H66 H88 AcwE\Emuoa gaofis EEKEBOHQ EH08: xmz A5303 953:5 Amy—\DE “59538. 33.30 $23080 $23028 ‘ omens 25585 88 E noncflsuowouxxonum mcflueaocwfia coaumuucoocoo .mmmgcuwwpuoucflsuommuxxonum cam wmmgfiwEmo mewuhmogca no 3332 93 can ome 28:85 no 8382880 wfi co E can mmzévm .melumvm mo 38mm .TH manna 59 ability in a two stage hepatocarcinogenesis assay devised by Pitot and coworkers (1978). Rats fed a diet containing 10 mg FM/kg had significantly more enzyme-altered foci than those fed a diet containing 10 mg 245-HBB/kg and there was no significant difference in the number of enzyme-altered foci in rats fed a diet containing 10 mg FM/kg or 100 mg 245-HBB/kg. This would indicate that FM is a more effective promoter than its major congener. iFM may have individual minor congeners that are very effective promoters or the combination of congeners in FM may result in an additive or potentiating effect on tumor promotion. A dose dependent increase in the number of enzyme- altered foci was seen with 24S-HBB but not with FM. Rats fed a diet containing 10 mg FM/kg had the highest number of enzyme—altered foci although there was no significant difference between the number of enzyme-altered foci in rats fed diets containing 10 mg FM/kg or 100 mg FM/kg. Peraino “E _l. (1980) reported that Pb caused a dose dependent increase in the number of hepatic tumors in rats previously fed N-2-fluorenylacetamide. However, when dietary concentrations of In) were high enough to inhibit body growth, the appearance of tumors was delayed even though the highest concentrathmn of Pb eventually caused the greatest number of hepatic tumors. A similar phenomenon may have occurred in rats fed a diet containing 100 mg FM/kg since this concentration also caused a decreased body growth rate. If the promotional period had been extended, rats fed a diet 60 containing 100 mg FM/kg may have had more enzyme-altered foci than rats fed a diet containing 10 mg FM/kg. Rats fed a diet containing 1.0 mg 34S-HBB/kg had approximately equal numbers of enzyme-altered foci as rats fed a diet containing 500 mg Pb/kg. 345-HBB was predicted not:to»be a tumor promoter by an in vitro assay measuring the ability of a chemical to inhibit metabolic cooperativity at noncytotoxic concentrations (Tsushimoto 33 al., 1981). The dietary concentration of 1.0 mg 345-HBB/kg caused extensive hepatocyte degeneration leading to necrosis. From this experiment, it could not be determined if the promoting ability of 345-HBB was due to a direct effect of the congener or due to its ability to cause hepatocyte degeneration and necrosis. FM, at dietary concentrations of 10 and 100 mg/kg caused small numbers of enzyme-altered foci in rats that were not pmeviously partially hepatectomized or given DEN. These foci could result from promotion of environmentally initiated cells (Pitot and Sirica, 1980; Schulte-Hermann and Parzefall, 1981; Pitot t l”.1980) or from initiation and promotion by PBBs, IJL, a complete carcinogen. The lack of convincing evidence that PBBs have initiating ability (Garthoff t al., 1977; Fiscor and Wertz, 1976; Dannan gt __l_., 1978; Zeiger, 1980) and the results of this experiment indicating that PBBs are effective promoters of hepatocarcinogenesis suggest that the foci were the result of promotion of environmentally initiated cells. Similarly, 61 hepatocellular carcinomas in rats given a single large dose of FM (Kimbrough gt _l”,l981) may have been the result of promotion by PBBs of previously initiated cells. Current methods for determining the carcinogenic potential of a chemical involves lifetime exposure of laboratory animals to high doses of the test chemical. An increased incidence of tumors in exposed animals when compared to control animals is the basis for calling a chemical carcinogenic (Page, 1977; Saffiotti, 1980). Such long term testing does not differentiate between chemicals that are promoters and those that are initiators of carcinogenesis. Prolonged exposure to tumor promoters can increase the incidence of tumors presumably by promoting "environmentally initiated" cells (Pitot gt al., 1980; Williams 35 al., 1981). CHAPTER 2 HEPATIC TUMOR PROMOTION BY 3,3',4,4',5,5'—HEXABROMOBIPHENYL: THE INTERRELATIONSHIP BETWEEN TOXICITY, HEPATIC MICROSOMAL DRUG METABOLIZING ENZYMES AND TUMOR PROMOTION CHAPTER 2 HEPATIC TUMOR PROMOTION BY 3,3',4,4',5,5'-HEXABROMOBIPHENYL: THE INTERRELATIONSHIP BETWEEN TOXICITY, HEPATIC MICROSOMAL DRUG METABOLIZING ENZYMES AND TUMOR PROMOTION Introduction In: the experiment described :hi Chapter 1, 3,3',4,4',5,5'-hexabromobiphenyl (345-HBB) at a dietary concentration of 1.0 mg/kg enhanced the development of enzyme-altered foci in rats previously "initiated" with diethylnitrosaminer(DEN) after a 70% partial hepatectomy. This dietary concentration of 345-HBB caused extensive hepatocyte degeneration and necrosis so that it could not be determined if the tumor promoting ability of 345—HBB was attributed to a direct effect of the congener or was associated with cell hyperplasia secondary to chronic hepatotoxicity. lg yitrg assays predicted that 34S-HBB, at noncytotoxic concentrations, would not be a tumor promoter (Tsushimoto gt 31,, 1982). The purpose of this experiment was to study the mechanism(s) of tumor promotion by 34S-HBB by using nonhepatotoxic and hepatotoxic dietary concentrations of 63 64 345—HBB in a two stage model of hepatocarcinogenesis devised by Pitot gt 31° (1978). Materials and Methods The experimental design, methods for purifying 345-HBB, processing of tissues for histologic evaluation, procedures for histochemical staining, methods for evaluating histochemically stained sections, procedures for tissue chemical analysis, procedures for microsomal enzyme assays and methods of statistical analysis were as described in Chapter 1. Animals Diets and Treatments Outbred female Sprague-Dawley rats weighing 200-215 g were obtained from Charles River Corp., Portage, MI. Housing of rats and the initiation portion (partial hepatectomy and diethylnitrosamine administration) of the Pitot protocol were as described in Chapter 1. Thirty days after partial hepatectomy; rats were randomly assigned to groups as shown (Table 2-1). Diets were prepared by adding phenobarbital (Pb) or 345-HBB in MazolaR corn oil to a ground commercial diet (Wayne Lab Blox, Allied Mills Inc., Chicago, IL) in the amounts indicated (Table 2-1). Controls included rats not partially hepatectomized or given DEN but which were fed the same diets. 65 Necropsy and Collection of Tissue Samples Rats were killed after being fed diets containing Pb or 345-HBB for 140 days. Rats were anesthetized with ether (Mallinkrodt Inc., Paris, KY), bled by cardiac puncture and killed by decapitation. Tissues fixed in 10% buffered formalin included liver, thymus, adrenal, thyroid, spleen, lungs" pancreas and intestine. Portions of liver approximately 1 mm in thickness were fixed in Karnovsky's fixative (Karnovsky, 1965) and stored at 4° C until prepared for ultrastructural examination. Other procedures completed at necropsy were as described in Chapter 1. Ultrastructural Preparation Karnovsky's-fixed liver was washed in Zetterqvist's osmium fixative (Pease, 1964) and then postfixed in 1% osmium tetroxide in Zetterqvist's fixative. Tissues were dehydrated in alcohol and transferred to propylene oxide. A mixture of epon and araldite was used for embedding. Selected areas of liver were cut (900 A) with an ultramicrotome (LKB Ultratome IIIR, Instrument Group 8800, Sweden), stained with uranyl acetate and lead citrate and viewed with an electron microscope (CEM 952, Carl Zeiss, Germany). Serum Clinical Chemistry Determinations Whole blood was allowed to clot and then was centrifuged (200 xg) to separate the serum. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 66 activity in serum were determined by standard kinetic assays (Spin Chem, Sunnyvale, CA). Results Enzyme-Altered Foci The number of enzyme-altered foci/cm3 of liver is given in Table 2-l. Rats fed a basal diet or diets containing Pb or 345-HBB without prior partial hepatectomy or DEN had no or relatively few enzyme-altered foci. Of the rats partially hepatectomized and given DEN, only rats fed diets containing Pb or 1.0 mg 34S-HBB/kg had significantly (p<0.05) more enzyme—altered foci/cm3 in the liver when compared to livers from rats fed a basal diet. Body Weight Gain and Organ Weights Total body weight gain and organ weights are given in Table 2-2. Only 345-HBB at acdietary concentration of 1J3 mg/kg caused a significant (p<0.05) decrease in body weight gain and a significant decrease in thymic weight. There were no significant differences in brain, spleen or kidney weights between rats in any treatment groups. Hepatotoxic Effects of Treatments Liver weights for rats fed diets containing 500 mg Pb/kg or 1.0 mg 345-HBB/kg were significantly (p<0.05) increased (Table 2-3). Serum concentrations of alanine aminotransferase (ALT) were unchanged in rats of all treatment groups, but serum aspartate aminotransferase (AST) 67 Amo.ovmv ucmumumfio sflucmoduwcmdmo xmcEEMmouuficflsnumflcuzman oznwfllmm Hmuwm umfimu Hmmmfl m Umm mumu MOM mwn—HMD EOHM «weowwmummmn Hmwuumm monummm .om+m mm ommmmumxm mum sumo omsmflmooa m o; mmmumvm nzmaammm mHHHH m H.o mmxumvm mcoz Hoflummm m H.o mmmumem nzmoummm one m Ho.o mmzumam mcoz Hiummm m 8.0 mmmumwm nzmonmmm anmm m com Hmufinumnocmnm wcoz ommfiuomma G com Hmufinumnocmnm nzmoummm oHo m o uwfio Hmmmm mcoz «Humma m o umwo Hmmmm nzmoummm uo>wa mo uwumeflucou mums mo nonficz Amx\msv ucwEummuu ammumfio ucmEumoua ownso uwm floom uwwo cfi owuwuHmeE>ucw cofiumuucmocoo .mmmumvm no Hmufinumnocmnm mcflcwmucoo mumfia no umflo Hmmmm 6 com mumm aw Moon omumuacnoeancm mo umnEsz 0cm cmwmmo Hmucmewumdxm .HIN manna 68 53c H83 m 8m 38 “8 83.3 Ed 25.89 ucmumuflo 328323? .38 o now omflx mm commmumxm mum. much I .ucwfiumwuu .3306 cu quum cowumuumficfiemvm mcflsmmofiwcanumwo 0cm >Eouomummmn :3qu wok. m on: mum» H2 SARA 2.3.6. m3..+.mo. «Sufi o; 33% 2.H8.~ Sham. 8&8. 3H: H .o mmzuflm SANA; SHE. 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SHE o “96 ~88 8v £333 A9 “E033 3v “£903 3. 3x\95 ucocnmouu 3363 >963 338nm comamm 338nm map—S5 330mg 5mm “£033 uwfic cm 38 ~38. coflmuucwucoo .298 oz “8 $5-3m no HBBuBocwfi Efifificoo mumwo uo uwwo Hmmmm m pom mumm 5 munmmmz ammuo wusflomnc can Emu unmfiwz hcom .NIN wanna 69 .umflp Amman m pom mumu now mmsHm> EOum Amo.ovmv Wamummmfip maucmoflmficmflmm .mumu w you om+x mm commmumxm mum mama I .ucmEummuu mumumfic ou uowum cowumuumficfiEcm wowEmmOHDficHacumwo 0cm heouomummmn Howuumm won 6 on: many Hat meODUmc HmUOM cam cofiumuwcwmwo wuumu .wnmouuummzn I I I muwooummwn omxum: mH.om+m.¢mH H.ma+m.¢v mmm.H+mm.~H o.H mmmlmvm acm0uuumman I I I muxooummwn paw: H.H~+o.moa m.v+m.hm Nw.o+mo.m H.o mmmumem Hmauoz n.5HHm.mm ~.oHHm.qv -.HHAa.m Ho.o mmmumvm xnmouuummhn I l I mumooummms wumuwpoz m.o~+h.mm m.wH+o.mv mmm.a+vo.aa ocm Hmuwnumnocwnm 3502 935.: REAR: QSHSS o p.20 :33 cowumoam>w Q\DH u\DH Amy unmwm3 Amx\msv ucmfiumwuu mumumflo ofimoHocummoumfi: amm Esuwm eat Esuwm um>wa wusHomn< uwfic c“ COwumuucmOcoo .umwo Hammm m com mumm OD owummEou Hmumnumnocmnm 0cm mmzumvm ammumwo wo muowmmm ofixouoummm: .mum magma 70 concentrations were significantly (p<0.05) increased in rats fed diets containing 1.0 mg 345-HBB/kg. Histopathology The liver and thymus had the most remarkable histologic change related to treatments. Livers from rats fed 0.01 mg 345-HBB/kg were histologically indistinguishable from livers of rats fed a basal diet (Figure 2-1). Rats fed a diet containing 0.1 mg 345-HBB/kg had mild diffuse hepatocellular hypertrophy and fine diffuse hepatocyte vacuolation (Figure 2-2). The most dramatic histologic changes of the liver were in rats fed diets containing 1.0 mg 345-HBB/kg (Figure 2-3). The periportal hepatocytes were hypertrophied and had increased cytoplasmic eosinophilia. Hepatocytes in the central vein and midzonal regions were greatly enlarged due to either a single large vacuole or multiple small vacuoles which stained positive with oil-red-O. The sinusoidal space was obliterated due to hepatocyte hypertrophy and the cytoplasmic boundaries of hepatocytes were often indistinct. There were small focal areas of hepatocyte necrosis characterized by pyknosis and karyolysis of nuclei, eosinophilic cytoplasm and loss of cytoplasmic membranes. Occasionally, infiltrates of lymphocytes were associated with the areas of necrosis. Livers of rats fed diets containing 500 mg Pb/kg had moderate hypertrophy of hepatocytes in the centrolobular region. Categories of preneoplastic lesions in the livers consisted of foci or areas of acidophilic cells (Institute 71 of Laboratory Animal Resources, National Research Council, 1980) characterized by abundant eosinophilic cytoplasm wdth a ground glass appearance. Basophilic foci ‘were occasionally seen and consisted of small round cells with basophilic cytoplasm and hyperchromatic centrally located nuclei. Hepatocellular carcinomas were not observed. Preneoplastic lesions in the livers of rats fed 1.0 mg 345- HBB/kg appeared to be resistant to the hepatotoxic effects of 345-HBB. Foci of altered hepatocytes without evidence of degenerative changes were often surrounded by areas of hepatocyte degeneration and necrosis (Figure 2-4). Rats fed diets containing 1.0 mg 34S-HBB/kg had severe involution of the thymus characterized histologically by reduced numbers of cortical lymphocytes and a loss of demarcation between the cortex and medulla (Figure 2-6). Thymuses from rats in other treatment groups were comparable to those of rats fed a basal diet (Figure 2-5). Other tissues examined did not have significant histologic alterations when compared to the same tissues from rats fed a basal diet. Electron Microscopy Ultrastructural features of hepatocytes from rats fed a basal diet are illustrated (Figure 2-7). Dose dependent ultrastructural changes were observed in hepatocytes from rats fed diets containing 34S-HBB. The most dramatic ultrastructural change was observed in the hepatocytes of ——_—=_——— ' , 1 - *---.-- fl-L‘M .ifi’4'. ..L...L.'- .. 72 Figure 2—1. Photomicrograph of a liver section from a rat fed a basal diet illustrating the normal hepatic architecture. Notice the cords of hepatocytes radiating from the central vein (CV) and the prominent sinusoidal space UI& E stain, X500). Figure 2-2. Section of liver from a rat fed a diet containing 0.1 mg 345-HBB/kg. Notice the mild hypertrophy of hepatocytes and the narrowing of the sinusoidal spaces. Normal hepatic architecture is maintained (CV = central vein, H & E stain, X500). Figure 2-1 Figure 2-2 74 Figure 2-3. Section of liver from a rat fed a diet containing L0 mg 345-HBB/kg. The normal architecture of the hepatic lobule is lost and the sinusoidal spaces are obliterated due In: severe hepatocyte hypertrophy. Hepatocytes have multiple small vacuoles or a single large vacuoleu Cytoplasmic boundaries are indistinct and there are focal infiltrates of lymphocytes (LL. (CV = central vein,II& E stain, X500) Figure 2-4. Photomicrograph of the midzonal region of liver from a rat fed a diet containing 1.0 mg 34S-HBB/kg for 140 days after a 70% partial hepatectomy and diethylnitrosamine administration. The arrows outline a focus of altered hepatocytes characterized by large acidophilic cells without evidence of degeneration or vacuolization. Hepatocytes surrounding the focus have undergone degeneration and necrosis and contain multiple large cytoplasmic vacuoles (H & E stain, X200). Figure 2-4 76 Figure 2-5. Portions of thymus from a rat fed a basal diet. Notice the density of lymphocytes in the cortex and the distinct demarcation between the cortex and medulla (H & E stain, X200). Figure 2-6. Photomicrograph of a section of thymus from a rat fed a diet containing 1.0 mg 345-HBB/kg. The cortex contains few lymphocytes and the demarcation between the cortex and medulla is indistinct (H & E stain, X200). 77 Figure 2-6 78 Figure 2—7. Electron micrograph of a hepatocyte from a rat fed a basal diet. Notice the parallel aggregates of rough endoplasmic reticulum (RER) and focal areas of smooth endoplasmic reticulum (SER). Mitochondria (M) are numerous and diffusely distributed throughout the cytoplasm (Uranyl acetate - lead citrate stain, X8232). Figure 2-8. Electron micrograph of a hepatocyte from a rat fed a diet containing 1.0 mg 345-HBB/kg. Notice the abundant amount and the unusual orientation of the rough endoplasmic reticulum (RER). There are numerous concentric whorls of RER around mitochondria (arrow). Numerous lipid droplets (L) are also present (Uranyl acetate - lead citrate stain, X8232). 79 Figure 2-7 Figure 2-8 80 rats fed a diet containing 1.0:mg 345-HBB/kg and consisted of proliferation and disorganization of the rough endoplasmic reticulum (RER) (Figure 2-8). Also, there were numerous variably sized lipid droplets. Hepatocytes of rats fed a diet containing 0.1 mg 345-HBB/kg had less proliferation of RER and fewer lipid droplets. Ultrastructural features in hepatocytes of rats fed a diet containing 0.01 mg 345-HBB/kg were comparable to hepatocytes from rats fed a basal diet. Chemical Analysis Tissue concentrations of 345-HBB in abdominal adipose tissue and liver are given in Table 2-4. Rats fed diets containing 345-HBB consistently had more chemical in the liver than in adipose tissue. There was a dose related increase in the concentration of 345-HBB in the liver and adipose tissue. Hepatic Drug Metabolism Results of assays for hepatic drug metabolism are given in Table 2-5. Isolated microsomes from the livers of rats receiving 34S-HBB had a downward shift in the carbon monoxide difference spectra and a dose related increase in ethoxyresorufin-o-deethylase activity. Microsomal concentrations of cytochrome P-450 (nmole/mg pmotein) were approximately equal in rats fed diets containing 0.1 mg 345- HBB/kg or 500 mg Pb/kg. 81 . mononucwumm cw powwow?“ mun Em: unmoumm .33 m you omfim. ucmmmnmmu can 393 Ba: m co Bummumxm mum $.3um . ucofimwuu mumuwwp ou nowum cowumuumficflemom wcflEmmouuficanumwp 0cm >Eouooummwn Hmwuumm wok. m cm: mum“ H2 :13th SKHSJN SANTOS 8.2393... 0; 8.72m $4352 «Nana; a.~.umm.mv $.SHS.E To 37va Ganmét Sanaé 34.3.: 24.33.. 86 33% 84$.me 8430.0 Géuai mioumma 8m HfiBuBocmfi 8.3.9:: Saunas 843.2 8.o.+.q~.o o um? 398 $092 3580?? board 3x\m§ ucosnmmuu 33mg ma823. 3382 828 68389. 3.55. 3382 >38 38. 39.328 88 23.9 ooze 5 3.2333 comm cowuuuufiocoo .mmromvw 0:0 Elm: No 9.039598 no in uSngufih egg“ eglmvu 9:53.58 303 no uowo Hanna a won .33. no 32333 comic ouzuomnt can accouofiuuu couuuo>cou 009m .530 .3332 boom .Nln 038. 94 24S-HBB/kg, 1.0 mg 34S-HBB/kg plus 100 mg 245—HBB/kg and 10 and 100 mg FM/kg were significantly (p<0.05) increased when compared to weights of livers from rats fed a basal diet (Table 3-3). Serum alanine aminotransferase (ALT) activity was significantly increased in rats fed diets containing 0.1 mg 34S-HBB/kg plus 10 mg 24S-HBB/kg or 10 mg FM/kg. Serum aspartate aminotransferase (AST) activity was significantly increased in rats fed diets containing 1.0 mg 34S-HBB/kg, 0.1 mg 34S-HBB/kg plus 10 mg 245-HBB/kg, 1.0 mg 345-HBB/kg plus 100 mg 24S-HBB/kg and 10 and 100 mg FM/kg. Histopathologic evaluations of the livers are summarized in Table 3-3. Livers from rats fed a basal diet had cords of hepatocytes radiating from the central vein and prominent sinusoidal spaces (Figure 3-3). Dietary concentrations of 10 and 100 mg 245-HBB/kg caused mild to moderate hepatocellular hypertrophy in the centrolobular portion of the hepatic lobule (Figure 3-4). Rats fed a diet containing 0.1 mg 345-HBB/kg had mild diffuse hepatocellular hypertrophy whereas 345-HBB ataadietary concentration of 1.0 mg/kg caused severe diffuse hepatocellular hypertrophy, fatty degeneration and focal necrosis (Figure 3-5). Rats fed diets containing the combinations of congeners had histologic changes in the liver similar to those fed the equivalent amount of FM in the diet. The diets containing 0.1 mg 345-HBB/kg plus 10 mg 245-HBB/kg or 10 mg FM/kg caused moderate diffuse hypertrophy and fine cytoplasmic vacuolation (Figure 3-6). The livers from rats fed diets 95 .36 ~83 a c8 38 Ed 3989 wfiumufiv sausages? .33 9 new out. no 83298 was 38 £5,535 .3330 on ~35 c0333??? womaouficgfiowp can b59033! amwwgm «or a can «you :4 modicum go: no»? 392mg 339.52 Eaton? 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I I no T303322 3330 v2: “£852 913% do 872.” common unasnoaouucwo I I I E umtusafl mo sfiefifiaz 338°: «52%.: m.2+o.2 ugozwi o2 Balms" c399,. uuHoooHOuucwo I I 5 mwiuflamn do sfiobuwfiz 2:. 5.21.6... mfasm S 33% ~33. flawed... than: a swan ~88 835126 Exp: 33: .93.... 295.35 Dogma GumbaonuaOumw: 5 Suwm 94¢ cud—mm u0>«.— Qua—:85 uwmc cu COwufluag .mmzumg 0cm Eznmvm uo 2.63.3598 no wnmm uwummfiwurm .thmSH .B‘Imvw xuflumwo uO nuowuufl UmXBOuaw: onlm manna. 96 containing 1.0 mg 345-HBB/kg plus 100 mg 245-HBB/kg or 100 mg FM/kg had marked diffuse hepatocyte hypertrophy and numerous variably sized cytoplasmic vacuoles (Figure 3-7) which stained positive for lipid. The histologic changes in the livers from rats fed a diet containing 1.0 mg 345-HBB/kg plus 100 mg 245-HBB/kg (Figure 3-7) were less severe than in the livers of rats fed a diet containing only 1.0 mg 345- HBB/kg (Figure 3-5). Liver lesions, presumably involved in the carcinogenic process, included foci and areas of cellular alterations and neoplastic nodules (Institute of Laboratory Animal Resources, National Research Council, 1980). Hepatocellular carcinomas were not observed. The foci and areas of altered hepatocytes were comprised of large acidophilic cells with abundant eosinophilic cytoplasm. Histopathology Other than the liver, the thyroid and thymus had the most remarkable histologic change related to dietary treatments. Thymuses from rats fed diets containing 1.0 mg 345-HBB/kg, 1.0 mg 345-HBB/kg plus 100 mg 24S-HBB/kg and 100 mg FM/kg were involuted. There was decreased organ size, a lack of cortical lymphocytes and a loss of demarcation between the cortex and medulla. The thymuses from rats in other treatment groups were comparable to that of rats fed basal diets. The thyroids from rats fed diets containing 1.0 mg 345- HBB/kg plus 100 mg 24S-HBB/kg or 100 mg FM/kg had decreased 97 Figure 3-1. The number of enzyme-altered foci/cm3 of liver is illustrated for rats fed diets containing either 10 mg 245-HBB/kg,(Ll.mg 345-HBB/kg or(Ll.mg 345-HBB/kg plus 10 mg 245—HBB/kg. The number of enzyme-altered foci/cm of liver from rats fed diets containingILJ.mg 34S-HBB/kg and 10 mg 24S-HBB/kg have been summed to form one histogram to illustrate the expected value for the combination diet. All rats had a PH and DEN administration prior to dietary treatments. Figure 3-2. The number of enzyme-altered foci/cm3 of liver is illustrated for rats fed diets containing either 100 mg 245-HBB/kg, 1.0 mg 345-HBB/kg or 1.0 mg 345-HBB/kg plus 100 mg 245-HBB/kg. The number of enzyme—altered foci/cm of liver from rats fed diets containing 1.0 mg 345- HBB/kg and 10 mg 245-HBB/kg have been summed to form one histogram to illustrate the expected value for the combination diet. All rats had a PH and DEN administration prior to dietary treatments. Number of Enzyme Altered Foci Per Cubic Number of Enzyme Altered Foci Per Cubic Centimeter of Liver Centimeter of Liver 98 a PH-DEN-0.1 mg 345-HBB/kg PH-DEN-1O mg 245-HBB/kg PH-DEN-O.1 mg 345-HBB/kg + 10mg 245-HBB/kg 7A Treatment Group FigureB-l ’ ’ i h“* PH-DEN-I .0 mg 345-HBB/kg PH-DEN-100 mg 245-HBB/kg PH-DEN-1 .0 mg 345-HBB/kg + 100 mg 245-HBB/kg 3000 2500 2000 1500 1000 500 Treatment Group Figure 3-2 99 Figure 3-3. Photomicrograph of a section of liver from a rat fed a basal diet. 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Chemicals that enhance the development of GGT positive enzyme-altered foci in the livers of rats having previously initiated hepatocytes are generally considered to be tumor promoters (Diamond st 21., 1980; Leonard gt 21,, 1982; Pitot 33 al., 1978; Pugh and Goldfarb, 1978; Sirica _£__l,,21978; Wanless and Medline, 1982). In this study, tumor promoting ability was assessed by measuring enzyme-altered foci exhibiting gamma glutamyl transpeptidase activity. Dietary concentrations of 10 and 100 mg 24S-HBB/kg, 1.0 mg 345-HBB/kg, 0.1 mg 34S-HBB/kg plus 10 mg 24S-HBB/kg, 1.0 mg 345-HBB/kg plus 100 mg 24S-HBB/kg and 10 and 100 mg FM/kg enhanced the development of GGT positive enzyme-altered foci in the livers of rats previously initiated with DEN. The effect of combinations of PBB congeners on tumor promoting ability'is graphically illustrated (Figures 3-1 and 3-2). The low dose dietary combination of P385 consisting of 0.1 mg 34S-HBB/kg plus 10 mg 245-HBB/kg caused a potentiating effect.03pr Mo 00000 0» MORE EH00 om .moom mo 005000Hmoo 0» 0:0 0:00 uoz n 92 .om+m. 00 00000090 000 0000 5005000» 203053550 0550000303508 000 mguowummg H0wuu0m we“. .0 00s mumu H2 O} I- Oz 030 H38 030 ESE O... OH ”.ch 930 me OH MO .I OOHHHOO HOE Hmmmm HOE Hammm 0ch H88 O\O ONOHOOO. NOOHNNOH I- 0ch 93...“. 9. OH 030 935 me OH O\O «HOAOOO. OHOHOOOH .I Own. H88 HOG 930 me OOH O\O OOO.HOHO. OHHOOH I... 0ch H88 00:. Hmmmm 3.8588 Ames. uw>HH 00 3.02398 m3 OBIHOH >8 OOH OBIOO >8 OH. OHIO.” m8 HOHHHZ0030Q0£ “00m 0000300535 03:0 009 Hoow :53 mo 0000 000005 0000300635 0000 mo 00852 no 0004 000002 0555800 0035000050: 53 mu0m no 00852 0:0 Hoom 300206?ch .003; “cc 00.109350 05:0 Ema Loom mmimuglchNcw uc Lgcfiz 5308 00—0830me .Hiv 030p. 122 Body Weight Gain and Organ Weights Total body weight gain, absolute liver weights and thymus weights are given in Table 4-2. Only rats fed a diet containing 100 mg FM/kg for 15 days had a significant decrease in total body weight gain as compared to rats fed a basal diet. Rats from both groups fed diets containing 10 mg FM/kg for 140 days had a significant increase in the liver weight whether killed at day 170 or 445 of the experiment. There were no significant differences in the weights of the thymus, kidneys or spleen (data not shown) in rats killed on day 445 after being fed basal diet for 415 days or a diet containing FM for 140 days followed by a basal diet for 275 days. Histopathology Hepatic alterations, presumably involved in the carcinogenic process, included foci and areas of altered cells in rats that were fed only basal diets for either 140 or 415 days after partial hepatectomy and diethylnitrosamine administration. Rats sacrificed on day 170 after being fed a diet containing 10 mg FM/kg for 140 days had foci and areas of altered hepatocytes and occasional neoplastic nodules. {Hue foci and nodules consisted of large hepatocytes with abundant eosinophilic cytoplasm. The nuclei were large and had large and often multiple nucleoli. NeOplastic nodules caused compression of the surrounding normal hepatic parenchyma. Rats fed a basal diet for 275 days after being fed a diet containing 10 mg FM/kg for 140 123 .msmo mHO you umHO Human m 000 many scum HmO.Ova ucmummuHo sHHmoHumHumumm .mmmn OOH “on uch Hanna 0 000 mum» scum HmO.OvaIucmumumH0 sHHmoHumHumumO .000“ v mom amHM m0 00mm0umx0 0H0 00000 .000» m How ame 00 00mm0umx0 000 00000 .mu0u 0 How amfim m0 00mmmumx0 0H0 00000 .uc0Eu00uu >u0u0flp mo uumum o» uofium m>0© om cowu0uummcHE©0 0cwE000uuwcHanu0wp 0:0 >80000u0m0n H0wuu0m 0 00: mu0u HH< HH.HOm. 0O.mum.OH OOHOHH uch Hammm uch Ox\zm me OH ouch mx\:m Os OH OO.HO~. 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The neoplastic cells are large with large nuclei and prominent nucleoli. The malignant hepatocytes are arranged in cords or acini. 015 E stain, X200.) 125 Figure 4-1 3.. a}: 3.351- cra- , _, 9": an,“ ..-' Figure 4-2 126 days had hepatocellular carcinomas characterized by a mixed adenocarcinomatous and trabecular pattern (Figure 4-1 and 4- 2). The carcinomas were infiltrative and lacked normal hepatic structures such as bile ducts and hepatic vessels normally found in portal regions. Hepatocytes comprising the carcinomas were pleomorphic and had enlarged nuclei with prominent nucleoli. Chemical Analysis Values for tissue concentrations.0f PBBszhiliver and abdominal adipose tissue are given in Table 4-2. Rats fed a diet containing 100 mg FM/kg for 15 days had approximately the same concentration of P883 in abdominal adipose tissue and liver as rats fed a diet containing 10 mg FM/kg for 140 days. Rats sacrificed on day 170 after being fed a diet containing 10 mg FM/kg for 140 days had approximately twice the concentration of P835 in body tissues as rats fed a basal diet for 275 days after being fed a diet containing 10 mg FM/kg for 140 days (Table 4-2). Microsomal Enzyme Assays Rats fed a diet containing FM for 140 days had an increased amount of cytochrome P-450 and an increased activity of ethoxyresorufin—o-deethylase in isolated hepatic microsomes whereas there was minimal or no increase in the activity of aminopyrine demethylase (Table 4—1). 127 00005000000 00 0030 00 gm: 0000000 .00000 03: 0 Mo 00000090 000 000H0>0 .9000 £000 :0 0000 m 000 00+.m. 00 00000090 000 0000 00050000 300000 00 0030 B 038 m>8 Om 80080005200 80288005000000 Em 388088 H3008 OOO m 08 38 H2 AH.H.+.O.OOO OONHOON 3.60.00 OOHHOOH 080 H88 080 95.0 9. OH 080 930 9. OH AOJHHAOO $035 HO.O.....H.OO ~.OH~.H 006 H88 02.6 H88 0000 H88 3.00.062 23% 8.319 mHHHOOO I. 00:0 930 9. OH 00.6 930 O... OH AO.mHH.mOO OONHOOO 225.3 NHHHOmm -l 0000 H88 030 ESE Os OOH 3.938 860.36 863.2 HO.OH§.O ..l 080 H88 080 H88 8003. $802 32.5 m: >8uHOH >8 OOH >8..OO >8 3 >8..Om >8 030n o: 00 ma 000 0:00 00000—00000 0000000000 0003 00 003 003 0 000 000m 00 00003. 000002 0008009“ 000 003.0 :0 0300503 0000006000300 00 0000000000000 0005080 .mlv 0Hn0a 128 03000 000 00000500 0000 m 0000 000003. 000.000 00.300 H 0000000000 00000 03000 000 00000000 0000 0 5000 0000000. 03000 0000mm“ A 0000000000 00000 .0000 0000 00000600 0000 m 0000 0000000. 00.30.00 00300 n 000 00+m. 00 000000000 00000 Om.HO.~H O.HH HO.0.Hm.O 0.0.0.85 Om..+-OO.H I- 930 0.. OH 093.0 0.. OH H0.0 «O.H I- 0000 H88 0930 O... OOH OO.HOO.O OH.0.OO.O -I 0000 H88 0030 H.008 00050000000 mE\Ho.EV 000 230000 |0I000000000>xon0m 20550080 OexHoEO E0880 OexHoEO MOO >8-H-O-H >8 O-O-H >8-OO >8 3 >8-Om >8 000 > 00:0 .0 0.8 80>0 mc0w>m80em O3 0: 000.5800 @808 .00035000010300000030000 000 00000000500 00000800000 .00 >00>000¢ 000 000 030 0000000006 00 0000000000000 000 00 0:00 0000060000 «0 00000.00 .04. 0300. 129 Discussion The results of this experiment demonstrate that FM, fed to rats over a short period of time, had the same ability to enhance the development of enzyme-altered foci as an equal amount of FM fed over a long period of time. Not only were there approximately equal numbers of enzyme-altered foci between these treatment groups, but the average size of enzyme-altered foci was also approximately the same. This observation, indicates that the tumor promoting effects of P885 can persist for long periods of time after short term exposure, possibly due to continual redistribution of P385 from adipose tissue to the blood. Most tumor promoters require continual exposure for promotional effects to occur (Miller and Miller, 1981; Pitot and Sirica, 1980; Weinstein, 1980), but for P835 and, possibly for other lipophilic compounds that have tumor promoting ability, continual exposure from external sources may not be necessary for a promoting effect (Kimbrough g£_gl,, 1981; Kimbrough, 1979). Enzyme-altered foci are commonly regarded as precursor lesions of liver cancer (Cameron __t _1., 1978; Emmelot and Scherer, 1980; Kitagawa, 1971; Farber, 1976) although few enzyme-altered foci progress to hepatocellular carcinomas. Most enzyme-altered foci remodel into the normal hepatic architecture and appear as phenotypically normal cells after cessation of exposure to tumor promoters (Farber, 1980; Watanabe and Williams, 1978; Williams and Watanabe, 1978). The results of the second portion of this experiment 130 indicate a strong association between the enzyme—altered foci enhanced by FM and hepatocellular carcinomas since all of the rats fed diets containing 10 mg FM/kg for 140 days followed by a basal diet for 275 days had hepatocellular carcinomas. The correlation of large numbers of enzyme— altered foci in rats fed FM for 140 days and the subsequent finding of hepatocellular carcinomas in rats fed FM for 140 days followed by a basal diet for 275 days would add credence to the use of enzyme-altered foci as an indicator of a chemical's promoting ability. Rats which were fed only basal diets for 140 days or for 415 days did not have carcinomas and the presumed precursor lesions of hepatocarcinomas had not progressed beyond enzyme-altered foci. Tissue concentrations of PBB in the liver and adipose tissue from rats sacrificed after 140 days of dietary exposure to 10 mg FM/kg diet were approximately twice that of rats fed a diet containing 10 mg FM/kg for 140 days followed by 275 days of a basal diet. The decreased concentration of P885 may be due to a combination of excretion of P883 and to redistribution of PBBs to the greater amount of liver and adipose tissue in the larger rats that lived for an additional 275 days. Pitot and coworkers (1980) postulated that hepatic cancer in rats occurring after long term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may result from the promoting activity of TCDD on "environmentally 131 initiated" cells. A similar mechanism could be responsible for the hepatocellular carcinomas in rats given large quantities of P835. The lack of convincing evidence that PBBs have properties of other known initiators of carcinogenesis (Garthoff 35 31., 1977; Fiscor and Wertz, 1976; Dannan 35 31., 1978) and the results of this experiment indicating that PBBs can be effective tumor promoters after short term exposure suggest that the hepatocellular carcinomas induced by a single large dose of P885 (Kimbrough t l", 1981) were the result of promotion of "environmentally initiated" cells. CHAPTER 5 THE EFFECT OF ADMINISTRATION OF FIREMASTER BP-6 PRIOR TO AND AFTER THE ADMINISTRATION OF DIETHYLNITROSAMINE CHAPTER 5 THE EFFECT OF ADMINISTRATION OF FIREMASTER BP-6 PRIOR TO AND AFTER THE ADMINISTRATION OF DIETHYLNITROSAMINE Introduction Williams and coworkers (1981) have proposed several requirements for a chemical to be classified as a tumor promoter. These requirements include: 1) a lack of genotoxic properties in vitro, 2) identification of an in vitro epigenetic mechanism responsible for the chemicals carcinogenic activity, 3) an enhancing effect by the chemical on carcinogenesis in 1112 when the chemical is administered after a known genotoxic initiator, and 4) a lack of a summational effect on carcinogenesis i vivo when the chemical is administered before a known genotoxic initiator. Most of these requirements for classifying polybrominated biphenyls (PBBs) as tumor promoters have been fulfilled. 1) To date, PBBs have not been shown to have genotoxic properties (Dannan gt 21,, 1978; Wertz and Fiscor, 1978; Zieger, 1980). 2) Trosko st 31, (1981) and Tsushimoto gt _1. (1982) have identified an in yitrg epigenetic mechanism of carcinogenesis for PBB; that is, the ability to inhibit metabolic cooperativity at noncytotoxic 133 134 concentrations. 3) As discussed earlier in this thesis, PBBs enhanced the development of enzyme-altered foci and tumors in rats previously initiated with a known genotoxic initiator, diethylnitrosamine (Magee and Barnes, 1967). The final requirement for defining a chemical as a tumor promoter is the lack of a summational effect when the suspected promoter is administered prior to the initiator. The sequential administration of two different genotoxic initiators has an additive or potentiating effect on carcinogenesis (Nakahara and Fukuoka, 1960; Tatematsu gt _1,, 1980; Williams £5,213! 1981) whereas the administration of a promoter prior to a genotoxic initiator does not (Roe, 1959). The purpose of this experiment was to determine the effect on hepatocarcinogensis of the exposure of rats to Firemaster BP-6 (FM) prior to administration of a known genotoxic initiator. Materials and Methods Experimental Design Female Sprague-Dawley rats weighing between 175 and 185 g were obtained from Charles River Corp», Portage, MI. Rats were housed in clear polypropylene cages on filtered laminar flow units (Contamination Control Inc., Lansdale, PA) at 22° C with a 12-hr light/dark cycle. Rats were randomly assigned to groups as shown (Table 5-1) and acclimated for 7 days before oral administration of either 100 mg FM/kg body weight or 100 mg diethylnitrosamine/kg body weight. 135 Fourteen days later, the second oral dose of diethylnitrosamine (DEN) or PM was administered to the rats as shown (Table 5-1). Rats were fed a commercial diet (Wayne Lab Blox, Allied Mills, Inc., Chicago, IL) for 63 days after which they were killed. Procedures for necropsy and collection and analysis of tissues were as described in Chapter 1.. Chemicals The commercial PBB mixture used was Firemaster BP-6 (Lot 6224A) manufactured by Michigan Chemical Corp., St. Louis, MI. Diethylnitrosamine was obtained from Sigma Chemical Corp., St. Louis, MO. Results Enzyme-Altered Foci The number of enzyme-altered foci/cm3 of liver is given in Table 5-1. Rats having no treatments or rats given only a single oral dose of FM had relatively few enzyme-altered foci. Rats given a single oral dose of DEN had approximately equal numbers of enzyme-altered foci/cm3 of liver as rats given an oral dose of FM followed by an oral dose of DEN. In contrast, rats given an oral dose of DEN followed by an oral dose of FM had significantly increased numbers of enzyme-altered foci/cm3 of liver when compared to rats given only DEN or FM before DEN. 136 .m 0:000 0000 AmO.Ov0. 0:0000000 >H000000000H00 “a 00000 0000 AmO.Ov00 000000000 0H000000000000 mm 00000 8000 Amo.oY0v 000000000 0H000000000000 “N 00000 5000 SOOVE 000000000 30000003300 0H 0:000 0000 SOOVE 000000000 300002000000 .00000 0000 00 0000 m 000 0m+x 0000000000 0000 .on0m 0000000000u020 «000600000000000000u0200 mO.OHHH.H 0.0.000H00HO 30 Ox\200 Os OOH 00oz m OmuemH 0.0.00H0Om 0:02 20 mxxzm ms OOH e MHHHHON 0.0.0NOHH>OO 30 mx\zmo me OOH 00 mx\:0 Os OOH m OOHNOH 0.0.0.0eOm+OOO 00 mx\nzm me OOH 30 mx\0200 ms OOH m Om.OHmO.O 0.0.0OHHO 0002 0:02 H 000000 00>00 00 mm >00 00 «a 000 H 000 000502 000 00 Amx\mev 0000000000 00000 000600009 00000 0000000000000 000 0000 0000000 I0fixnc0 no 000602 .0000 00 00>00 000 00 0H0000000 0000008000>H00 00 0000000000000 000 0000 00000H¢I060000 00 000802 .000000 000005000000 .Hnm 00009 137 Chemical Analysis Concentrations of PBBs in the liver are given in Table 5-1. Rats in the treatment groups which received an oral dose of PM had approximately equal concentrations of P835 in liver tissue. Discussion DEN is a known genotoxic initiator which, if given at high doses, can cause an increased incidence of hepatocellular carcinomas in rats (Magee and Barnes, 1967; Scherer 35 al,, 1980). The sequential administration of two different genotoxic initiators such as DEN and N-2- fluorenylacetamide (Z-FAA) can cause an additive or potentiating effect on carcinogenesis (Nakahara and Fukuoka, 1960; Tatematsu 35131,, 1980; Williams st 21,, 1981) whereas the administration of a tumor promoter before administration of a genotoxic initiator does not (Roe, 1959). The results of this study demonstrate that PM did not increase the number of enzyme-altered foci/cm3 of liver when given to rats before DEN administration. In contrast, rats given FM two weeks after DEN administration did significantly enhance the development of enzyme-altered foci/cm3 of liver when compared to rats that were given only DEN or when compared to rats given FM two weeks before DEN. These results would suggest that either FM does not«contain congeners that are genotoxic initiators, or if congeners in PM are genotoxic, they comprise such a small quantity of the mixture, that 138 they do not cause a summational effect in the formation of enzyme-altered foci when given prior to a known genotoxic initiator. The hepatocarcinogenic effect of 2-EAA has been reported to be decreased when 2-FAA is fed to rats concurrently with either phenobarbital or 3- methylcholanthrene (MC) (Flaks and Flaks, 1982; Peraino _e_t_ _1,, 1971). Flaks and Flaks (1982) reported that not only did MC decrease the number of carcinomas induced by Z-FAA but also caused a proportional decrease in the number of altered foci. When PBBs were fed to rats concurrently with Z-EAA, Schwartz and coworkers (1980) found a reduced incidence of nonhepatic tumors but no significant difference in the incidence of hepatic tumors when compared to rats fed only Z-FAA. These results correlate well with the present experiment in that PBBs given to rats before DEN did not significantly alter the number of enzyme-altered foci when compared to rats given only DEN. In summary, PBBs did not cause a summational effect on carcinogenesis when given to rats before DEN but did cause an enhancing effect on the number of enzyme-altered foci/cm3 of liver when given after DEN. The results of this study add credence to the theory that PBBs act primarily as promoters of hepatocarcinogenesis. CHAPTER 6 EFFECT OF POLYBROMINATED BIPHENYLS ON THE HOMEOSTASIS OF SERUM AND HEPATIC VITAMIN A CHAPTER 6 EFFECT OF POLYBROMINATED BIPHENYLS ON THE HOMEOSTASIS OF SERUM AND HEPATIC VITAMIN A Introduction Polybrominated biphenyls (PBBs) when fed in) rats, decrease hepatic vitamin A (Akoso st 31., 1982; Mangkoewidjojo, 1979; Pratt, 1979). To study the effect of dietary PBB on vitamin A homeostasis in rats, concentrations of vitamin A in the liver and serum were determined in rats fed PBBs for 140 days during the promotion phase of an initiation-promotion assay (Pitot _e_t _a__1_., 1978). The P885 used included Firemaster BP-6, 2,2',4,4',5,5'-— hexabromobiphenyl (245-HBB), 3,3',4,4',5,5'- hexabromobiphenyl (345-HBB) and combinations of 245-HBB and 345-HBB. Materials and Methods Liver tissue and serum from rats in the experiment described in Chapter 3 were collected, frozen, and stored at —20° C until analyzed for vitamin A. Refer to Materials and 140 141 Methods portion of Chapter 3 for experimental design, animals, and treatments. Vitamin A Analysis Determinations of vitamin A in the serum and liver were quantitated by the method of Dennison and Kirk (1977) as modified by Stowe (1982). For analysis of serum vitamin A, 0.5 ml of serum was added to 0.5 m1 of ethanol and then vortexed for 10 seconds to denature protein. This mixture was vortexed with 2 m1 of U.V. grade hexane for 60 seconds and then centrifuged at 3000 rpm for 10 minutes. The hexane supernatant was removed and filtered through a 0045 micron millipore filter (Millipore Corporation, Bedford, Massachusetts). A 0.1 m1 sample of the filtrate was injected into a high performance liquid chromatography (HPLC) system. The injected filtrate was separated isocratically on a microporasil column (30 cm long x 3.9 mm ID) with a 60:40 mixture of degassed hexane (Burdick and Jackson Lab. Inc., Muskegon, Michigan) and chloroform pumped through the HPLC at a rate of 2.5 ml/minute and a pressure of 63.4 kg/cmz. The forms of vitamin A were detected by a spectrofluorometer (Aminco-Bowman Spectrofluorometer, Silver Springs, Maryland) with a 35 ul flow cell. The fluorescence excitation and emission wavelengths were 330 and 470 nm, respectively. For vitamin Alanalysis in liver tissue, LJ)g of liver was homogenized in 5 ml of double distilled water. A 0.5 ml sample of the homogenate was vortexed with 0.5 ml of ethanol 142 to denature protein. The homogenate was vortexed with 5 ml of hexane for Srninutes and centrifuged at 3000 rpm for 10 minutes. The hexane supernatant was removed and analyzed as described for serum. Determinations of the concentration of serum and hepatic vitamin A were completed by personnel in the Department of Large Animal Medicine and Surgery under the supervision of Dr. Howard Stowe. Results Results of analysis for vitamin A in serum and liver are given in Table 6-l. Diets containing 1.0 mg 345-HBB/kg, 10 and 100 mg FM/kg and 345-HBB plus 245-HBB at concentrations of 0.1 mg/kg plus 10 mg/kg, respectively, and 1.0 mg/kg plus 100 mg/kg, respectively, significantly (p<0.05) 5000 Amo.ov0v 000000000 00000000000000 020.000 00O.OHOH.O 006.0005 OOH 0Hbum0~ «H.00m0.O 0H0.O000.H OH :0 mwmnOmm 00O.000O.O 0mm.O000.O OOH + O.H ~0000H 00~.O000.O 00.O00H.~ OH + H.O 000-000 + 000-000 0000000 00O.O00O.O 00H.0000.H O.H HmHnMH 00~.O000.O 00.0000.m H.O 000-000 vaOOH 0~.O00O.H 00.O00m.m OOH 0HHO~H 0~.OHHO.O O0.OHO~.0 OH 000-000 «Huva 0~.O00H.H HO.HHO0.¢ O H000000 00E\000 005. 0000000 0060 000005000 0mx\mev 000800000 0000000 0000000 0000000 0000000 0000000 0000 00 Esuwm HMUOB HmuOB COwamuuchCOU lmvm NO MCOMuMCmQEOU HO HWUmmemHmh .0000 O0H 000 000-000 000 mm: .mmz-mvm .mmz-mwm 0000000000 0000a 00 0000 00000 0 000 0000 00 0 006000> 000000: 000 E0000 .0-0 00009 144 Discussion Animals apparently have a homeostatic control mechanism for maintaining serum concentrations of vitamin A despite varying dietary sources of vitamin A precursors (Underwood __t __l., 1979). The liver has a pivotal role in maintaining the normal concentrations of serum vitamin A. When dietary intake of vitamin A is in excess of body needs, the liver has the ability to store vitamin A primarily in the form of retinyl palmitate. The stored retinyl palmitate then serves as the primary source of retinol needed to maintain serum concentrations when dietary intake of vitamin A is inadequate (Underwood gt al., 1979). If the capacity of the liver to store vitandmnA.is exceeded, increased amounts of retinyl esters may circulate in the blood and interact with cellular membranes to cause toxicity (Mallia _t___l_., 1975; Smith and Goodman, 1976). Many xenobiotics decrease hepatic vitamin A. Hepatic concentrations of retinyl palmitate are the primary indicator of hepatic reserves of vitamin A. Results of this experiment indicate that diets containing 345-HBB, PM or combinations of 345-HBB and 245-HBB decreased hepatic retinyl palmitate and retinol. When dietary sources of vitamin A precursors are inadequate, the depletion of hepatic retinyl palmitate and retinol caused by PBB could ultimately result in decreased concentrations of serum retinol. Blood is the media for transport of retinol to the peripheral target tissues. Therefore, the serum 145 concentration of retinol is an indicator of the amount of retinol available to target tissues. The diets containing PM, the highest dietary concentrations of 345-HBB or the combination-diet containing 345-HBB plus 24S—HBB caused a significant increase in the concentration of serum retinol. When comparing the values of the forms of vitamin A in the liver with those from the serum, it is seen that rats having the lowest amount of retinyl palmitate in the liver have the highest concentration of retinol in the serum (Table l). A similar finding was observed in baboons fed ethanol (Sabo and Leiber, 1981). This phenomenon may indicate an enhanced mobilization of the hepatic retinyl palmitate reserves resulting 2h) an increase 2h: serum concentrations of retinol (Sato and Leiber, 1981). The mobilization of hepatic retinyl palmitate may be due to an increased utilization of retinol in peripheral tissues (Underwood 33 al., 1979) or to the increased hepatic microsomal drug metabolizing enzymes induced by PBB which may alter vitamin A metabolism. FM is a mixed-type of hepatic microsomal drug metabolizing enzyme inducer (Dent gt al., 1976) whereas 345-HBB is strictly a MC-type (Render gt or al 1982). Rats fed diets containing ET“ 345-HBB or combinations of 345-HBB and 24S-HBB had a significant decrease in hepatic retinol and retinyl palmitate and a significant increase in the concentration of serum retinol (Table 6-1). 245-HBB is strictly a Pb-type of hepatic microsomal enzyme inducer in rats (Aust gt al., 1982). Rats 146 fed diets containing only 24S-HBB had lower total hepatic retinol and retinyl palmitate and higher concentrations of serum retinol when compared to values from rats fed a basal diet, but the differences were not significant. This phenomenon suggests that the P385 with the ability to induce MC-type of hepatic microsomal drug metabolizing enzymes have greater ability to decrease hepatic retinol and retinyl palmitate and increase serum retinol when compared to PBBs which induce Pb-type of microsomal enzymes. It may also indicate that the ability of P883 to induce MC—type of hepatic microsomal drug metabolizing enzymes is associated with alterations in vitamin A homeostasis. Rats fed diets containing combinations of 24S-HBB and 345-HBB had decreased hepatic retinol and retinyl palmitate and increased serum retinol when compared to rats fed only 345-HBB or only 245- HBB at dietary concentrations equal to that in the combination diet. This phenomenon may indicate that the combination of 245-HBB and 345-HBB have a potentiating effect in altering vitamin A homeostasis in rats. It is unlikely the initiation phase of the initiation- promotion assay consisting of a 70% partial hepatectomy and DEN administration had an influence on vitamin A homeostasis. DEN has a very short half-life in the rat and is essentially cleared from plasma and liver within 24 hr of administration (Ying and Sarma, 1979). Complete hepatic restoration occurs from 25 to 28 days after a 70% partial hepatectomy (Higgins and Anderson, 1931). Therefore, 147 hepatic restoration was complete before diets containing PBB were fed to the rats. An interesting relationship between tumor promotion and alterations of vitamin A caused by PBBs was observed. Rats fed diets that caused the highest concentrations of serum retinol also had decreased tumor promotion (Chapter 3) when compared to rats fed the same diets at lower concentrations. As discussed in Chapter 3, rats fed PM at a dietary concentration of 100 mg/kg had significantly fewer enzyme-altered foci than rats fed PM at a dietary concentration of 10 mg/kg. Similarly, rats fed diets 1.0 mg 34S-HBB/kg plus 100 mg 245-HBB/kg had significantly fewer enzyme-altered foci than rats fed diets containing 0.1 mg 345-HBB/kg plus 10 mg 245-HBB/kg. In summary, the results indicate that PBBs alter the normal homeostasis of vitamin A metabolism. The PBBs that have the ability to induce MC-type of hepatic microsomal drug metabolizing enzymes increase concentrations of serum retinol apparently at the expense of depleting the hepatic reserves of retinyl palmitate. SUMMARY Conclusions derived from (fine previously described experiments include: 1) Firemaster IBP-6 (FM), 3fiP,4,4',5,5N- hexabromobiphenyl (34S-HBB) anui 2,2',4,4',5,5'— hexabromobiphenyl (245-HBB) have tumor promoting ability in a two stage hepatocarcinogenesis assay. 2) When FM and its major congener 245-HBB were fed at a dietary concentration of 10 mg/kg for 180 days during the promotion period of the Pitot protocol (1978), FM had a greater ability to enhance the development of enzyme-altered foci. 3) A diet containing a combination of 245-HBB and 345- HBB caused a potentiating effect on tumor promotion. Therefore, it is hypothesized that the mechanism of enhanced tumor promoting ability'of PM when compared to 24S-HBB is due to a potentiation of tumor promotion by the combination of 3-methylcholanthrene-, mixed-, and phenobarbital—type of microsomal enzyme inducers in PM. 4) Since 345-HBB had tumor promoting ability only at dietary concentrations that were hepatotoxic, the mechanism of promotion by this congener appears to be due to its 148 , . A . 0 O O .. _,_..,, 0. 0 _ "Z, .... .. . 149 ability to cause cell hyperplasia secondary to chronic hepatotoxicity. S) The ability of 245-HBB to promote hepatocarcinogenesis appears to be due to a direct effect of the congener since tumor promotion occurred at nonhepatotoxic dietary concentrations. 6) PM fed to rats over a short period of time had the same ability to enhance the development of enzyme-altered foci as an equal amount of FM fed over a long period of time. Therefore, the tumor promoting ability of FM apparently persists for long periods of time after cessation of exposure from external sources, possibly due to continual redistribution of P383 from adipose tissue to the blood. Also, after cessation of exposure to PM, some of the preneoplastic lesions (altered foci and neoplastic nodules) progressed to form hepatocellular carcinomas. 7) Exposure to FM prior to diethylnitrosamine administration does not result in a summational effect on carcinogenesis suggesting that congeners of PM do not have tumor initiating ability. 8) Dietary concentrations of polybrominated biphenyls that inhibited body growth and decreased feed efficiency also caused an inhibitory effect on tumor promoting ability. 9) Polybrominated biphenyls alter the homeostasis of vitamin A resulting in increased concentrations of serum retinol apparently at the expense of depleting hepatic retinol and retinyl palmitate. The polybrominated biphenyls - w - I - 0 - n 1 ‘ - . It! I. v- . A . I «LIEFIIC l'lll Ia'.V.AV|I1.- 150 with the ability to induce 3-methylcholanthrene-type of microsomal enzymes had a greater ability to alter vitamin A homeostasis than the polybrominated biphenyls that are phenobarbital-type of microsomal enzyme inducers. Studies involving laboratory animals are used to predict the risks to human health by exposures to chemicals. The results of animal studies must be extrapolated to determine potential risks to man. Although the stages of initiation and promotion appear to occur in man (Reddy gt al., 1978; Weiss, 1979), chemicals which are tumor promoters in laboratory animals are not necessarily promoters of cancer in humans. Epidemiologic studies indicate that phenobarbital exerts little if any effect on the incidence of human liver cancers (Clemmesen and Hjalgrim-Jensen, 1981), yet phenobarbital is the standard tumor promoter in rodent:hfixfiation-promotion assays. Tumor promoters have different effects in different species of animals and even on different tissues of the same species (Pitot and Sirica, 1980). As determined by the described experiments, the polybrominated biphenyls are promoters of experimental hepatocarcinogenesis in rats. Unfortunately, people have been accidently exposed to polybrominated biphenyls and in some instances the exposure was at relatively high doses (Anderson 35 al., 1978). What, if any, effect the polybrominated biphenyls will have in the development of cancer in exposed people is not yet known. LIST 01? REFERENCES REFERENCES Abelev, (LI. (1971). Alpha-fetoprotein in ontogenesis and its association with malignant tumors. Adv. Cancer Res. 14, 295-358. Akoso, B.T. (1981). Comparative pathologic effects of purified polybrominated biphenyl congeners in rats. Ph.D. Thesis, Michigan State University. Akoso, B.T., Sleight, S.D., Aust, S.D. and Stowe, H.D. (1982a). 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