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WASITO has been accepted towards fulfillment of the requirements for Ph.D. degree in PATHOLOGY , t/A;AJ/¢y/¢ Major professor Date //l/él/[7 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU ’ RETURNING MATERIALS: Place in book drop to LIBRARJES remove this checkout from .1-a-qy-2-L your record. FINES wiii v be charged if book is returned after the date stamped below. THE PROMOTING EFFECTS OF POLYHALOGENATED BIPHENYLS AND 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN ON NASAL AND TRACHEAL TUMORS BY R. Wasito AN ABSTRACT OF A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1987 ABSTRACT THE PROMOTING EFFECTS OF POLYHALOGENATED BIPHENYLS AND 2,3,7,B-TETRACHLORODIBENZO-p-DIOXIN ON NASAL AND TRACHEAL TUMORS BY R. Wasito Male Syrian golden hamsters were allotted to 4 groups of 30 each and were used for an initiation-promotion study of respiratory tract carcinogenesis. Hamsters were given a single subcutaneous dose of O or 80 mg N-nitrosodiethylamine (NDEA)/kg of body weight and were fed diets containing 0 or 100 mg polybrominated biphenyls (PBB)/kg of diet for 140 days. Basal diet was fed from day 140 until the end of the experiment.on day'273.I The number of tracheal papillomas was significantly increased in hamsters given NDEA and P88 as compared to those in hamsters given only NDEA. Tracheal papillomas were not seen in the other two groups. Nasal tumors occurred at approximately the same incidence in hamsters given NDEA as in those given NDEA and P88. To characterize precursor lesions in the trachea and their relationship to the tumor promoting ability of PBB after initiation with NDEA, young male hamsters were allotted to 4 groups of 36 each and were given a single subcutaneous dose of 0 or 80 mg NDEA/kg of body weight. Diets containing 0 or 100 mg PBB/kg were fed for the remainder of the experiment. Twelve hamsters from each R. Wasito group were killed on days 21, 42 or 63. Precursor lesions were not observed during the time frame of this study. It.was hypothesized that exposure to environmentally relevant dietary levels of 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) or 2,2',4,4',5,5'-hexachlorobiphenyl (24S-HCB) could cause the deve10pment of nasal tumors and a combined exposure to these compounds could have a potentiating effect on tumor development. Thirty days after partial hepatectomy and NDEA administration, groups of 12 or 24 female Sprague- Dawley rats were fed a basal diet or diets containing 10 ppt TCDD, 100 ppt TCDD, 5 ppm 245-HCB, 10 ppt TCDD and 24S-HCB or 100 ppt TCDD and 245-HCB for 140 days. Basal diets were fed from day 140 until rats were killed on day 210 or day 420. Results indicated that TCDD or a combination of TCDD and 24S-HCB significantly increased the incidence of nasal tumors by day 420. However, a combined exposure to TCDD and 245-HCB had no potentiating effect on the incidence of nasal tumors. Results indicate that PBB promotes the development of tracheal papillomas in the hamster and TCDD enhances formation of nasal tumors in the rat. Dedicated With Love To My mother Rr. Hastari Wuryastuti, my wife My sisters and their husbands, my brother and his wife, and their children: Ria, Bagus, Nila, Windri, Galih, Imok, Denta, Rizki, Uuk and Bayu You And in memory of my father: R. Mohammad Ichram ii ACKNOWLEDGEMENTS The author is grateful to Dr. SJL Sleight, my major adviser, for his dedication and love during my course of study and to Drs. G.L. Waxler, H.D. Stowe and J.I. Gray, my graduate committee, for their thoughtful suggestions for the dissertation. I would like to acknowledge the government of the Republic of Indonesia and the Rockefeller Foundation for their financial support. I wish to thank Dr. R.K. Jensen for his preliminary study leading to this investigation, Dr. Margit S. Rezabek, Dr. Robert Sills, Dr. Sheila D. Grimes and many colleagues for their assistance during the course of the experiment; Irene Brett for her technical assistance; Mae K. Sunderlin, Frances M. Whipple, Connie Monroe, Carol Arnold and Kate Brown for their interest and patience in the preparation of the nasal and tracheal tissues: and Cheryl Assaff for typing this dissertation. My deepest appreciation to my dear wife for her tremendous love, understanding and support during my study in the United States. iii LIST OF TABLES . . . LIST OF FIGURES . . INTRODUCTION . . . . LITERATURE REVIEW . TABLE OF CONTENTS Experimental Nasal Carcinogenesis N-nitrosamines Introduction . Metabolism . Respiratory Carcinogenesis Polybrominated Biphenyls Introduction . Metabolism . Pathology Factors Modifying Respiratory Carcinogenesis Initiation and Promotion in Carcinogenesis CHAPTER I: HAMSTERS . . . . . . Introduction . Materials and Methods - Experiment 1 Experimental Design N-nitrosodiethylamine Administration Diet Preparation . Necropsy and Histopathologic Procedur Statistical Evaluation . Results . . . Results . . . . Discussion . . CHAPTER II: p-DIOXIN AND 2,2' Materials and Methods - Experiment 2 Experimental Design Necropsy and Histopathologic Procedures CAVITY TUMORS IN SPRAGUE-DAWLEY RATS . Introduction . Materials and Methods . iv 88 THE PROMOTING EFFECT OF POLYBROMINATED BIPHENYLS ON NASAL AND TRACHEAL TUMORS IN SYRIAN GOLDEN Page vii ooooooooooo H 0‘ . 28 0000000000000 U \D THE PROMOTING EFFECT OF TETRACHLORODIBENZO- ,4,4',5,5'-HEXACHLOROBIPHENYL ON NASAL . 65 . 68 Experimental Design . . . . Necropsy and Histopathologic Statistical Evaluation . . . Results . . . . . . . . . . . . . Discussion . . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . BIBLIOGRAPHY O O O O O O O O C O O O O VITA O O O O O O O O O O O O O O O O 0 Procedures Page 68 7O 71 75 on... \I o . 82 105 Table LIST OF TABLES Experimental design .. . .. . .. . .. . . Body weights of Syrian golden hamsters (g) . . A comparison of papillomas in the larynx or trachea of Syrian golden hamsters treated either with NDEA alone or with a combination Of NDEA and P83 0 O O O O O O I O O O O O O O Tumors in the nasal cavity of Syrian golden hamsters O O O O O O O I O O O O O O O O O I 0 Experimental design . . . . . . . . . . . . . The incidence and type of nasal tumors in rats by 420 days 0 O O O O O O O O O O I O O O O 0 vi Page Figure 10 11 12 13 LIST OF FIGURES Photomicrograph of nasal cavity from a control hamster O O O O C O O O O O O O O O O I O O O Photomicrograph of nasal cavity from a hamster given 80 mg NDEA/kg bw . .. . .. . .. . .. Photomicrograph of nasal cavity from a control hamSter O O O O O O O O O O O O O O O O O O O Photomicrograph of nasal cavity from a hamster given 80 mg NDEA/kg bw . .. . .. . .. . .. Papilloma of the trachea from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg Of diet 0 O O O O O O O O O O O O O O O Papilloma of the nasal cavity from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet .. . .. . .. . .. . Adenoma of the nasal cavity from a hamster given 80 mg NDEA/kg bw . .. . .. . .. . .. Adenocarcinoma of the nasal cavity from a hamster given 80 mg NDEA/kg bw . . . . . . . . Squamous cell carcinoma of the nasal cavity from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet . . . . . Papilloma of the trachea from a hamster given 8 0 mg NDEA/ kg bw O I O O O O O O O O O O O O O Papilloma of the trachea from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg Of diet 0 O O O O O O O O O O O O O O I Papilloma in a bronchiole from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg Of diet 0 O O O O O I O I O O O O O O O Adenoma of the lung from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg Of diet 0 O O O O O 0 O O O O O O O O 0 vii Page . 53 Figure 14 15 16 17 18 Adenoma of the lung from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg Of diet 0 O O O O O O O O O O I O O O O Trachea of a hamster fed a commercial diet for 42 days after administration of 80 mg NDEA/kg bw O O O O O O O O O O I O O O O O O I Papilloma of the trachea from a hamster fed a diet containing 100 mg of PBB/kg for 63 days after administration of 80 mg NDEA/kg bw . . . Adenoma of the nasal cavity from a NDEA- initiated rat fed a diet containing 100 ppt TCDD plus 245-HCB for 140 days and killed on day 420 . . .. . . . . . .... . . . . . . . Adenocarcinoma of the nasal cavity from a NDEA-initiated rat fed a diet containing 100 ppt TCDD plus 24S-HCB for 140 days. Rat died on day 323 . .. . .. . . . . .... . . . . . viii Page INTRODUCTION Neoplasms of theupper respiratory tract in people and animals have been recently emphasized in a 3 volume monograph by CRC Press Inc. (Reznik-Stinson, 1983) and in a textbook edited by Barrow (1986) that specifically deal with nasal tumors and by continuing research on the pathogenesis and morphogenesis of tracheal tumors (Reznik-Schuller, 1980). The various N-nitrosamines have been studied extensively. N-nitrosodiethylamine (NDEA), for example, is considered as a mutagen and carcinogen in people and animals, is detected in food, water and air, and can be formed in KEYS! from ingested amines and nitrites (International Agency for Research on Cancer, 1978). NDEA is metabolically activated via the cytochrome P-450- dependent monooxygenase system and a reactive intermediate formed during metabolism interacts with DNA to yield alkylated products (Hecht 23 21,, 1983). NDEA is considered to act as a tumor initiator and studies have shown that it targets specific cells in the nasal cavity (Reznik-Schuller, 1982; Jensen and Sleight, 1987) and trachea (Reznik-Schuller and Hague, 1981a,b) and can cause tumors to develop in these cells (Montesano and Saffiotti, .1968: Reznik-Schuller, 1980). The actual mechanisms of upper respiratory tract carcinogenesis have not been elucidated, but Prasad (1983) has proposed that the development of upper respiratory tract tumors from exposure to environmental compounds may involve a multistep process involving initiation and promotion. Polyhalogenated aromatic hydrocarbons (PHAH), such as polybrominated biphenyls (PBB), polychlorinated biphenyls (PCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are environmental contaminants and studies have shown that they act as tumor promoters in the liver (Gupta g£_§l,, 1973; Buchmann _e_t 31., 1986; Jensen and Sleight, 1986b). To date, however, the ability of PHAH to promote tumors at nonhepatic sites, such as in the respiratory tract, has not been demonstrated. PHAH can accumulate in the epithelial cells of the respiratory tract (Brandt, 1977; Appelgren _e_t 11., 1983). These cells are rich in cytochrome P-450-dependent monooxygenase (Hadley and Dahl, 1983; Voight _e_t; _a__l_., 1985; Dahl, 1986). PHAH can induce these enzymes in these cells (Bond, 1983; Voight e__t 11., 1985). Therefore, in a study of respiratory tract carcinogenesis, one could postulate that NDEA can act as a tumor initiator and PHAH as tumor promoters. This dissertation is divided into two chapters. ‘The first chapter describes an initiation-promotion study in Syrian golden hamsters with NDEA as an initiator and P88 as a possible promoter. The first objective was to determine if PBB will promote respiratory tract tumors. The second objective was to determine and characterize sequential development of tracheal tumors. We expected to find precursor lesions of tracheal tumors in NDEA-initiated hamsters given PBB. The second chapter describes an initiation-promotion study in Sprague-Dawley rats with NDEA as an initiator and TCDD and 2,2'4,4',5,5'- hexachlorobiphenyl (245-HCB) as promoters. The objectives were to determine if TCDD or 245-HCB acts as a promoter of nasal carcinogenesis and to determine if the interactions of TCDD and 245-HCB cause a synergistic effect on tumor promotion when compared with the effect of these compounds given separately. LITERATURE REVIEW Experimental Nasal Carcinogenesis Many chemicals have been reported to induce tumors of the nasal cavity in peOple and animals (Reznik and Stinson, 1983; Barrow, 1986). In experimental carcinogenesis, N-nitrosamines are the most well known chemicals that are capable of inducing tumors in the respiratory tract, particularly in the nasal cavity. In addition, nasal tumors have been related to a wide variety of important industrial chemicals, such as formaldehyde. Exposure to formaldehyde in rats and mice induced squamous cell carcinomas in the nasal tissue (Swenberg _e_t §_l_., 1980; Kerns g}; 31., 1983). In chemical carcinogenesis, it is thought that compounds, such as N-nitrosamines, require metabolic activation before they can produce tumors. Formaldehyde, in contrast, requires no metabolic activation. Inhaled formaldehyde must first be deposited on the surface of the epithelial cells, particularly in the anterior part (respiratory region) of the nasal cavity; .At low ambient air concentrations, formaldehyde deposited on the epithelial cells can be removed from proximity to target tissue via normal mucociliary clearance. The target tissue of formaldehyde is presumed to be basal cells. Once inside the 4 basal cells, formaldehyde must penetrate the nuclear membrane and react with DNA to yield adducts (Starr, 1983). Carcinogenesis in the nasal epithelium, resulting from formaldehyde toxicity, may be directly related to increase in cell proliferation. During increased cell proliferation, the likelihood of interaction of formaldehyde with DNA would increase, as would fixation of adducts before DNA repair could occur (Swenberg g£_§g,, 1983). An early clinical sign of nasal tumors in experimental animals was reported to be unilateral or bilateral ocular discharge (Swenberg £5 31., 19803-Takano £3 31,, 1982). Other signs described in animals included bloody nasal discharge (Tucker, 1975; Takano £5 31., 1982), dyspnea, mouth breathing and loss of weight (Hoch-Ligeti, 1970; Takano 23 91., 1982). Marked swelling of the nose and orbital regions has been reported as a gross evidence of nasal tumors (Montesano and Saffiotti, 1968; Albert 23 31,, 1982), while in other instances, gross lesions of tumors were found only when multiple frontal sections were made. The gross lesions consisted of opaque, greyish-white masses within the nasal cavity and involved naso-maxilloturbinates and ethmoturbinates. The tumors sometimes had eroded bone (Herrold, 1964c; Sellakumar 25 91,, 1983). Results of numerous experimental studies demonstrated that tumors of the nasal cavity were of multicentric origin and of many different histologic types. Generally, however, the main types of induced neoplasms may be divided into two categories: those that arise in the anterior part (respiratory region) of the nasal cavity (e4L papilloma, adenoma, adenocarcinoma and squamous cell carcinoma) and those that originate in the posterior part (olfactory region) of the nasal cavity (e.g. adenoma, adenocarcinoma, squamous cell carcinoma and olfactory neuroepithelial tumor) (Reznik-Schuller, 1983; Feron 33‘3l., 1986). Papilloma. These tumors are composed of infolded masses of squamous epithelial cells with a well developed stalk and no infiltrative growth into the subepithelial tissues (Herrold, 1964b; Mohr 33 33,, 1977; Reznik-Schuller, 1983). Other histopathologic features of papillomas include keratin formation and microcysts containing debris of necrotic cells and inflammatory cells (Herrold, 1964b). Adenoma. In contrast to papillomas, adenomas have a somewhat glandular and papillary growth pattern and contain mucous cells (Mohr 33'3l., 1977; Reznik-Schuller, 1983). Adenocarcinoma. Tumors classified as adenocarcinomas are composed, for the most part, of anaplastic or poorly differentiated cells and may have a gland-like growth pattern (84L formation of acini and secretion of mucus) (Huang and Ho, 1978; Reznik-Schuller, 1983). A papillary growth pattern and a few small solid areas of tumor cells are sometimes present (Huang and Ho, 1978). Occasionally, these tumors may have focal areas of squamous metaplasia indicating they might differentiate into squamous cell carcinomas at a later stage (Reznik-Schuller, 1983L. The tumors may invade the periorbital tissue, lacrimal gland, maxillary sinus and facial muscle, and extend posteriorly through the cribriform plate to involve the frontal lobes of the brain (Feron and Kroes, 1979). Squamous cell carcinoma. These tumors are composed of anaplastic or poorly differentiated cells» but pronounced keratinization and epithelial pearl formation are usually present (Lijinsky and Taylor, 1975a; Reznik-Schuller, 1983). The neoplastic squamous cells are usually arranged in clusters. These tumors tend to invade the bony skeleton of the nasal cavity, and may then eventually lead to ulceration. In some cases, the tumor may grow into the posterior part of the ethmoturbinates and, after penetrating the cribriform plate, invade the brain (Reznik-Schuller, 1983). Olfactory neuroepithelial tumor. Tumors from the olfactory epithelial cells are classified as (esthesio)neuroepitheliomas (Althoff e_t 3_l_., 1973; Haas 33 33., 1973; Rivenson e_t_3l., 1983). This tumor is sometimes referred to as an (esthesio)neuroblastoma (Herrold, 1964c; Mirvish 33 33,, 1980). Histologically, these tumors are characterized by the formation of rosettes and pseudorosettes and by fairly uniform cuboidal to columnar cells (Herrold, 1964c; Rivenson 33,33,, I983; Sellakumar 33 3_l_., 1983). The neurogenic origin of such tumors can best be defined by the presence of neurogenic elements such as neurosecretory granules and sustentacular cells, by electron microscopy (Reznik-Schuller, 1983). Olfactory neuroepithelial tumors occasionally invade the nasal bones and the tumors may extend caudally to invade the brain (Herrold, 1964c; Sellakumar 33 33., 1983). N-nitrosamines Introduction N-nitrosamines are a group of environmental carcinogens that are carcinogenic in many organs of various animal species. The carcinogenic prOperty is dependent on species and strain of the animals and type of N-nitrosamine used, and may vary depending on the route of administration, dose- and interval between individual doses. For example, the respiratory tract.(nasal cavity, larynx, trachea.and lung) is the main target area for N-nitrosodiethylamine (NDEA) carcinogenicity in Syrian golden hamsters. In contrast, the liver is the main target.organ for NDEA carcinogenicity in rats. The mechanism responsible for this marked specificity is not clearly understood. However, N-nitrosamines must first be metabolically activated before their carcinogenic effect is seen. Metabolism Like many other N-nitrosamines, NDEA requires metabolic activation by cytochrome P-450 into an ultimate carcinogen (active intermediate or alkylating agent) in order to exert its carcinogenic effects (Vainio and Hietanen, 1980; Schuller and McMahon, 1985). In Syrian golden hamsters, the possibility exists that NDEA is metabolically activated by cytochrome P-450 in the liver and the active intermediate is then transported by the circulation to target tissues, particularly in the respiratory tract. Conversely, it is more likely that enzymatic activation occurs in the respiratory tract itself as has been shown by several autoradiographic studies with NDEA. Reznik-Schuller and Hague (1981a,b) found that (3H)-NDEA and its metabolites were selectively bound in specific cell types of the trachea and that NDEA-induced tumors developed from these cells. In the nasal cavity, Reznik-Schuller (1982) reported that 1 hour after administration by gavage of a single dose of (3H)-NDEA to Syrian golden hamsters, most bound radioactivity was concentrated in the mucous cells of the respiratory epithelium and the secretory cells of submucous glands. These cells are rich in endoplasmic reticulum, the major source of cytochrome P-450 enzymes which are involved in the metabolic activation of N-nitrosamines (Reznik- Schuller and Hague, 1981b; Schuller and McMahon, 1985). The metabolism of NDEA is typical of the structurally simple dialkylnitrosamines. NDEA is metabolized by a-C- hydroxylation involving cytochrome P-450. This oxidative monodealkylation yields the unstable a-hydroxy-N-nitrosamine which decomposes to acetaldehyde and ethyldiazonium hydroxide (Montesano and Bartsch, 1976; Hecht 33 33,, 1983). The aldehyde generated is further oxidized to yield C02, 10 which is the major product of NDEA metabolism 1 vivo (Heath, 1962; Mundt and Hadjiolov, 1974). The ethyldiazonium hydroxide is unstable and may sufficiently react with.HZO to decompose and be excreted from the body (Hecht 3333., 1983). Blattmann and Preussman (1973) found N-nitrosoethyl-N-(2-hydroxyethyl)amine and N-nitrosoethyl-N- (carboxymethyl)amine in the urine of rats after they were given NDEA. The ethyldiazonium hydroxide is also thought to be capable oflcovalently modifying nucleophilic groups in cellular macromolecules tn) generate alkylating intermediates, such as ethylated derivatives of DNA (DNA ethylation or DNA adducts) (Hecht 33 33,, 1983). In NDEA-treated rats and hamsters, several DNA adducts were produced in target organs (Becker 33 33,, 1985). In the lungs (nontarget organ) of rats, DNA adducts were not detected, while in the lungs (target organ) of hamsters, both O7-ethylguanine (O7-etG) and OG-ethylguanine (OG-etG) were detected following NDEA administration. In NDEA- treated rats, both O7-etG and O6-etG were detected in the livers (target organ). In rats, NDEA induces mainly liver tumors (Reid 33 33., 1963; Lijinsky 33 _a_l_., 1981), whereas only respiratory tract tumors develop in similarly treated Syrian golden hamsters (Herrold, 1964a; Montesano and Saffiotti, 1968; Montesano and Saffiotti, 1970). Therefore, the capability of electrophilic reactants to covalently modify DNA suggests that tissue differences in the ll metabolism of NDEA by rats and hamsters are related to NDEA organotropism in these species. DNA alkylation products are repaired by two distinct DNA repair processes. OG-alkylguanine is repaired by the OG-alkylguanine-DNA alkyltransferase which transfers the miscoding alkyl group (either methyl or ethyl) from the 06- position of guanine to a sulfhydryl group of a cysteine in the repair protein, thereby restoring the fidelity of the DNA and inactivating the receptor protein. The 7- alkylguanine is lost from DNA by a combination of spontaneous and enzyme catalyzed depurination (Lindahl, 1982). The failure to correctly repair these premutagenic DNA adducts suggests that mutations occurring during DNA replication are critical for the initiation of carcinogenesis by N-nitrosamines (Cayama 33 33., 1978). Respiratory Carcinogenesis Hamsters. Of the three different hamster species (European, Chinese and Syrian golden) used in research, the Syrian golden hamster is utilized most frequently as a model in N-nitrosamine-induced respiratory tract carcinogenesis. The upper respiratory tract (nasal cavity and trachea) of this species appears to be highly sensitive to the carcinogenic effects of NDEA, Mohr 33:33.(1966) reported multiple papillomas in the tracheas in the offspring of Syrian golden hamster dams that had been given daily subcutaneous doses of 2 mg NDEA for 1-7 days during the second half of the gestation period. The tracheas of the 12 NDEA-treated mothers had similar tumors 25 weeks after the first administration. In: another study, (a single subcutaneous dose of 55, 33 or 5.5 mg NDEA/kg of body weight given to newborn Syrian golden hamsters induced tumors in the tracheas, larynges, nasal cavities, bronchi and lungs (Montesano and Saffiotti, 1970). The tumors observed in the tracheas were papillomas with histopathologic features similar to those observed in adult NDEA-treated Syrian golden hamsters (Herrold and Dunham, 1963; Herrold, 1964a; Schuller and McMahon, 1985). In a sequential study of NDEA- induced tracheal tumors in Syrian golden hamsters, there were initial ultrastructural changes in the epithelial cells of tracheas including an increase in the amount of rough endoplasmic reticulum and a change in the orientation of the nuclei from perpendicular to parallel to the basement membrane. However, the tracheal tumors, including papillomas and squamous cell carcinomas, arose from the basal cells, although these cells apparently were not affected during the initial treatment (Reznik-Schuller, 1980). Neoplasms described in the nasal cavities of NDEA- treated hamsters include papillomas, adenocarcinomas, anaplastic carcinomas and neuroepithelial tumors (Montesano and Saffiotti, 1970). These neoplastic changes were similar to those reported by Herrold (1964b), Montesano and Saffiotti (1968) and Stenback (1973). In addition, 13 papillomas of the bronchi and adenomas and carcinomas of the lungs were also seen (Montesano and Saffiotti, 1970). Many other N-nitrOsamines have been investigated for their ability to cause tumors of the respiratory tract. N- nitrosodimethylamine (Herrold, 1967), N-nitroso-B-hydroxy- propyl-n-propylamine (Pour 33 33., 1974), N-nitrosopiperi- dine (Haas 33 fl” 1973), N-nitrosopyrrolidine (McCoy 33 33., 1980) and N-nitrosonornicotine (Hilfrich 3333., 1977) have been reported to be weak carcinogens for the respira- tory tract, whereas N-nitrosodiethanolamine (Hilfrich 33 _a_l_., 1978), N-nitroso-di-n-propylamine and N-nitroso-B- oxoprOpyl-n-propylamine (Pour _e_t _a_l., 1974), N-nitroso- methyl-n-propylamine (Pour 33 33., 1974; Pour 33 33., 1979), N-nitroso(2-hydroxypr0pyl) (2-oxopr0pyl)amine (Pour _e_t 33., 1979), N-nitrosomorpholine (Haas 33 a1 1973) and N-nitro- n sohexamethyleneimine (Althoff 33 33., 1973) have been reported to be moderate carcinogens for the respiratory tract. N-nitroso-2,6-dimethylmorpholine (Althoff 33 33., 1978; Reznik 33 33., 1978) and N-nitrosodiallylamine (Althoff 3_t_ 3_l_., 1973) are similar to NDEA in that they also are strong inducers of nasal cavity tumors in Syrian golden hamsters. 3333. In rats, NDEA induced mainly tumors of the liver (Reid 33 33., 1963; Lijinsky 33 33., 1981). Available information indicates that NDEA is not a potent carcinogen for the nasal cavity in this species (Lijinsky and Taylor, 1978; Beer 33 33., 1986). However, in rats, l4 N-nitrosodimethylamine and N-nitroso-hydroxypropyl-n- propylamine (Reznik 33 33., 1975), N-nitroso-di- is0propanolamine and N-nitroso-3,4-dichloropyrrolidine (Mohr 33 33., 1977), N-nitrosomorpholine, N-nitroso-2,6- dimethylmorpholine and N-nitrosoheptamethyleneimine (Lijinsky and Taylor, 1975a), N-nitroso-B-piperidinol, N- nitroso-4-piperidinol and N-nitroso-4-piperidinone (Lijinsky and Taylor, 1975b), N-nitrOSOpiperidine (Lijinsky and Taylor, 1975b; Taylor and Lijinsky, 1975) and N-N‘- dinitroso-2,6-dimethylpiperazine (Hecht 33 33., 1980) strongly induced nasal cavity tumors. N-nitrosamine-induced tumors arising in the nasal cavity included papillomas, adenomas, adenocarcinomas, squamous cell carcinomas, esthesioneuroepitheliomas and rhabdomyosarcomas. A procedure has been standardized for the histopathologic examination of the nasal cavity in rats (Young, 1981). This procedure results in four nasal tissue slices from standard comparable regions and permits a thorough gross examination without destroying anatomic relationships. After embedding and sectioning, histopathologic evaluation.of four transverse sections‘of the nasal cavity results in a uniform assessment and interpretation of histologic changes. Serial sections can also be made if necessary. 3333. Relatively few N-nitrosamines have been reported to induce respiratory tract tumors in mice. NDEA (Clapp and 15 Craig, 1967; Ward 33 33., 1984), N-nitroso-N-bis(2- hydroxypropyl)amine and N-nitroso-N-bis(2-acetoxypr0pyl)- amine (Green 33 33., 1980) have been used for carcinogenicity studies in mice, and, so far, all of these N-nitrosamines mainly induced liver tumors, and their carcinogenic effect in the respiratory tract (nasal cavity and lung) was not great. Other animals. Carcinogenicity studies with N- nitrosamines, particularly NDEA, have also been done in other animal species. Guinea pigs (Argus and Hoch-Ligeti, 1963), rabbits (Rapp g 33., 1965), cats (Schmahl 33 33., 1978), dogs (Schmahl 3333., 1964; Hirao t 1., 1974), pigs (Schmahl 33 33., 1967), monkeys (Kelly _3 _3., 1966), parakeets (Schmahl _e__33., 1966), chickens (Schmahl _e_t_33., 1978), fish (Stanton, 1965), frogs (Khudoley, 1977) and snakes (Schmahl and Scherf, 1983) when administered NDEA developed mainly liver tumors. Human beings. Strong evidence that N-nitrosamines cause cancer in peOple is lacking. However, epidemiological studies suggest that N-nitrosamines contribute to human car- cinogenesis. Consumption of large quantities of Cantonese— style salted fish (Ho, 1972; Yu 33 33., 1986) or exposure to cigarette smoke (Lin 33 33., 1973; Mabuchi 33 _a_l_., 1985) has been implicated in the development of nasopharyngeal carcinomas in peOple. Cantonese-style salted fish and tobacco smoke contained a high level of N— nitrosodimethylamine and NDEA (Pong and Walsh, 1971; 16 McCormick 33 33., 1973; Iyengar 33 _3., 1976). Tobacco- specific N-nitrosamines, including N-nitrosonornicotine, 4- (N'-nitrosomethylamino)-l-(3-pyridyl)-l-butanone, N“- nitrosoanabasine and N'-nitrosoanatabine were found at high concentrations in tobacco smoke, snuff and chewing tobacco (Boyland e_t a_l_., 1964; Hecht 33 3_1., 1978; Hoffmann 33 33., 1979). Therefore, N-nitrosamines are likely to be involved in the development of tobacco-related tumors of the larynx, lung, oral cavity, esophagus, pancreas and urinary bladder (Hirayama, 1981; Hoffmann and Adams, 1981; Winn 33 33,, 1981). Polybrominated Biphenyls Introduction Commercial mixtures of polybrominated biphenyls (PBB), marketed as Firemaster (FM) (Michigan Chemical Co/US), Octa’ and Deca (White Chemical Co/US), Bromkal 80-9D (Chemische Fabrik Kalk/West Germany), Flammex B-10 (Berk/Great Britain), Adine 0102 (Ugine Kuhlmann/France) and RFC 101 (Hexcel/UK) have been widely used as a flame retardant additive for numerous polymeric resins (Brinkman and Dekok, 1980; Safe, 1984). Firemaster was produced by Michigan Chemical Company which also manufactured Nutrimaster, a magnesium oxide-containing feed supplement. In 1973, a major contamination of the food chain by PBB occurred in Michigan. This was due to inadvertently substituting FM for Nutrimaster in feed formulation. This error subsequently 17 resulted in*widespread contamination of meat, poultry and milk products as a result of PBB-contaminated feed being fed to dairy cattle and other livestock. There was major concern as to the implications on human health (Jackson and Halbert, 1974; Carter, 1976; Kay, 1977). The mixture of PBB consists of approximately 30 different congeners, and 13 of these are major congeners (Moore 33 33,, 1980; Aust 33 33,, 1982). The mixture causes a mixed-type induction of liver microsomal drug-metabolizing enzymes since it induces phenobarbital (PB)-type and 3- methylcholanthrene (MC)-type microsomal enzymes (Dent.33 _3,, 1976). The PB-type of microsomal enzyme induction results in increased activity of cytochrome P-450, whereas the MC-type of microsomal enzyme induction results in increased activity of P-448 (Pl-450) (Alvares g 33., 1967; Lu and West, 1978; Wilkinson, 1980). Apparently, there is a structure-activity correlation between the toxicity of an individual congener and its ability to induce specific microsomal enzymes. For example, a minor congener in PM BP-6, 3,3',4,4'-tetrabromobiphenyl (TBB) (Robertson 33 _a_l., 1982; Robertson 33 33,, 1983; Millis 33 33., 1985b) was reported to be an MC-type inducer and was considered toxic. Other MC-type inducers, 3,4,4'-tribromobipheny1, 3,4,4',5- tetrabromobiphenyl, 3,3',4,4'-5-pentabromobipheny1 are also toxic (Robertson 33 33,, 1982). Robertson 33 33. (1982) suggested that the presence of bromine atoms at both para positions and at one, two, threeror four meta positions on 18 biphenyl rings contributes to the properties of PBB as a 3- MC type inducer. Mono ortho-substituted congeners in PM which possess only lateral positions of bromine atoms may also be MC-type inducers. The PBB congeners in PM that have two ortho-substitutions, such as 2,2',3,4,4',5,5'- heptabromobiphenyl (Moore 33 33., 1979) and 2,2',4,4',5,5'-. hexabromobiphenyl (HBB) (Moore a 33., 1978; Akoso 33 33., 1982a) are PB-type inducers and are relatively nontoxic. Of the congeners in the commercial PBB mixture, 2,4,5,3',4',5'- hexabromobiphenyl (HBB) (Dannan 3_t_ 33., 1978b), 2,4,5,3',4'- pentabromobiphenyl (Dannan 33 33., 1982b) and 2,3,4,5,3',4'- HBB (Dannan 33 33., 1982a) are each classified as mixed-type microsomal enzyme inducers and are toxic. At room temperature, the commercial mixtures of PBB are white, odorless solids, insoluble in water but highly soluble in fat and organic solvents, such as toluene, benzene and chloroform. These compounds begin to melt at 72° c and decompose at 300 to 400° c (Kay, 1977). Most congeners of PBB are slowly metabolized and highly lipophilic (Brinkman and Dekok, 1980; Tuey and Matthews, 1980). Ultraviolet radiation will readily degrade PBB to lesser brominated biphenyls (Ruzo and Zabik, 1975; Millis 33 1., 1985a). Metabolism 33 vitro metabolism of congeners of PBB by liver microsomal drug metabolizing enzymes has been demonstrated. 19 Among the major congeners in FM, 2,2',4,5,S'-pentabromobi- phenyl and 2,2',3,4',5‘,6-hexabromobipheny1 (HBB) are most rapidly metabolized, and similar findings have been reported for other PBB congeners, such as 2,2"wdibromobiphenyl (DBB), 2,4,2',5'-tetrabromobiphenyl (TBB) and 3,3',4,4'-TBB (Dannan 33 33,, 1978a; Millis 33 33,, 1985b; Mills 33 33,, 1985). The major congener in FM, identified as 2,2',4,4',5,5'-HBB, is slowly metabolized (Dannan 33 33,, 1978a). The metabolism of PBB congeners appears to be correlated with the number and position of bromines on the biphenyl rings. According to Moore 33 33. (1980), the metabolism of PBB is facilitated when the number of para substitutions decreases, the number of ortho substitutions increases and the total number of substitutions decrease. FM BP-6, as already mentioned, is characterized as a mixed (PB and MC)-type inducer of liver microsomal drug metabolizing enzymes. The microsomal enzymes induced by PB- and MC-type inducers are located mainly in the liver, These enzymes also are present to some degree in extrahepatic tissues, such as in the respiratory tract (Azzopardi and Thurlbeck, 1968; Schuller and McMahon, 1985; Roberts 33 33,, 1986). The microsomal enzyme system is a nonspecific metabolizing system which possesses catalytic activity towards many substrates, such as drugs and xenobiotics, via conjugation and excretion as well as generation of dangerous reactive intermediates (Kappas and Alvares, 1975; Vainio and Hietanen, 1980; Gelboin, 1983). The system consists of 20 complex enzymes including flavoproteins (cytochrome P-450 reductases) (Yasukochi and Masters, 1976; Guengerich, 1977), hemoproteins (cytochrome P-450) (Cooper 33 _a_l_., 1965; Lu and West, 1980) and a phospholipid, phosphatidylcholine (Strobel 33 33., 1970). The microsomal enzyme system is an NADPH- dependent transport chain that inserts one atom of atmospheric oxygen (02) into their substrates (monooxygenase) (Mason, 1957; Conney, 1967). This electron transport pathway transfers electrons via cytochrome P-450 reductases (flav0proteins) from NADPH to the terminal oxidases, cytochrome P-450 (Lu e_t 33., 1969; Nebert g 33., 1982). Since one atom of 02 is incorporated into the substrate at the P-450 enzyme active site and the other atom of 02 is ultimately reduced to water, this system is also called the mixed function oxidase (MFO) system. It has been postulated that 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) can stereospecifically bind to a cytosolic polypeptide receptor called the TCDD receptor (Ah receptor) that mediates a 3-MC type microsomal enzyme induction with aryl hydrocarbon hydroxylase (AHH) activity (Poland 53 33., 1976; Jones 33 33., 1985). Compounds, such as 3,3',4,4'- tetrabromobiphenyl, 3,3',4,4',S,5'-hexabromobiphenyl, 2,3,4,5,3',4'-hexabromobipheny1 and 3,3',4,4'- tetrachlorobiphenyl that are closely analogous in chemical and toxicological properties to the TCDD, have the ability to bind to this receptor (Dannan 33 33., 1982a; Millis 3_t al., 1985b; Buchmann 33 33., 1986). The complex of receptor 21 and compound translocates to the nucleus in a temperature- dependent step (Okey 33 33., 1980). Within the nucleus, this complex appears to associate with chromatin (Eisen 33 33., 1983; Roberts 33 33., 1985), thereby, inducing synthesis of specific messenger ribonucleic acid (mRNA) (Negishi and Nebert, 1981; Tukey 33 33,, 1982). The mRNA leaves the nucleus, moves to the rough endoplasmic reticulum (RER) which subsequently is translated into new protein (enzymes), including P-448 and associated AHH (Poland 33 1., 1979; Eisen 33 33., 1983). Pathology Gross lesions and clinical signs. According to Collins (1982),(guinea pigs are much more sensitive to the lethal effects of PBB than Syrian golden hamsters. Hamsters treated‘with up to 3200 mg PBB/kg body weight had reduced weight gain, but no deaths occurred. However, when single doses of 400 or 800 mg PBB/kg body weight were given to guinea pigs, severe body weight loss and mortality were observed. In all guinea pigs that died, clinical signs including anorexia, rough hair coat and hypersalivation were observed. In both of these species, there were hepatomegaly and thymic involution. Studies have shown that TCDD is more highly toxic than PBB in laboratory rodents. The guinea pig is the most sensitive to the lethal effects of TCDD with an LDSO of 2 ug TCDD/kg (Gupta 3 33., 1973) whereas the hamster is the least sensitive with an LDSO of 1157 ug TCDD/kg (Olson 33 33., 1980). Loss of weight, hepatomegaly 22 and thymic involution have also been reported in rats (Sleight and Sanger, 1976), mice (Gupta 33 33., 1983a,b), chickens (Ringer, 1978; Dharma, 1980), pigs (Ku 33 33,, 1978; Howard 33 33,, 1980), cattle (Jacks0n and Halbert, 1974), dogs (Farber 33 33,, 1978) and nonhuman primates (Allen 33 33., 1978) exposed to PBB. Massive enlargement of the common bile duct was observed in rats after prolonged feeding of a vitamin A- deficient diet containing 100 ppm PBB (Darjono 33 33,, 1983). However, Wasito (1984) failed to observe any gross lesions in the common bile duct in rats fed a vitamin A- deficient diet containing 100 ppm PBB for 28 days. A variety of clinical signs and gross lesions in peOple were reported to be associated with an acute or chronic exposure to PBB. These include porphyria, immunologic defects, headaches, fatigue, bronchitis, persistent coughing and reproductive failures (Bekesi 33 33,, 1978; Valciukas 33 1., 1978; Stross 33_33., 1981). Histopathology. The histOpathologic lesions associated with PBB toxicosis consisted mainly of enlargement and intracytoplasmic vacuolation of hepatocytes and hepatic necrosis in the livers of rats (Sleight and Sanger, 1976), hamsters (Collins, 1982), guinea pigs (Sleight.and.Sanger, 1976; Collins, 1982) or sows and their pigs (Werner and Sleight, 1981). The ultrastructural hepatic lesions in rats have‘been characterized by an increase in size of hepatic mitochondria, an increase in the amount of smooth 23 endoplasmic reticulum (SER) and an increase in cytOplasmic vacuolation (Sleight and Sanger, 1976; Mangkoewidjojo, 1979; Render 33 33., 1982). Hyperplasia of extraparenchymal bile ducts was reported in rats after prolonged feeding of a vitamin A-deficient diet containing 10 or 100 ppm PBB (Darjono 33 _3,, 1983). Similar but less pronounced lesions were observed when rats were fed a vitamin A-deficient diet containing PBB for 28 days (Wasito, 1984). In the thymus, the lesions were characterized by loss of demarcation between the cortical and medullary regions as well as depletion of cortical lymphocytes (Howard 33 33., 1980; Collins, 1982). In the thyroid, hypertrophy and hyperplasia of the follicular cells and vacuolation and depletion of colloid were reported in rats fed the mixture of PBB (Sleight 33 33,, 1978; Mangkoewidjojo, 1979; Akoso 33 33., 1982b). Carcinogenicity. Results of experimental studies suggest that PBB and its congeners induce neoplastic nodules and hepatocellular carcinomas in the livers. For example, hepatocarcinogenic‘effects were observed in the livers of rats (Kimbrough 33 33., 1981; Gupta 33 33,, 1983b) and in mice (Gupta 33 33,, 1983b) given high doses of PBB. Results of 3.3 333333 studies by Williams 33 33. (1984) and Kavanagh 33 33. (1985) indicated that Firemaster BP-6 (FM) and 2,2',4,4',5,S'-hexabromobiphenyl (245-HBB) are not genotoxic or mutagenic. FM and 245-HBB were reported to inhibit 24 metabolic c00peration in Chinese hamster V-79 cells (Tsushimoto 33 33,, 1982) and WB-F344 (rat epithelial) cells (Evans, 1987) in culture, a property of known tumor promoters (Yotti 33 33,, 1979; Trosko 33_§3,, 1981)- Jensen e_t a_l. (1982) and Jensen 3; a_l. (1984) concluded that pas act as a hepatic tumor promoter. In these studies, PBB consistently enhanced the formation of Y-glutamyl transpeptidase (GGT) positive enzyme-altered foci (EAF) and neoplastic nodules in the livers of rats previously initiated with NDEA. A few hepatocellular carcinomas were also observed. In addition, Jensen and Sleight (1986b) demonstrated that simultaneous exposure to 2,2',4,4',5,5'- hexabromobiphenyl and 3,3',4,4',5,5'-hexabromobiphenyl caused a synergistic effect on hepatic tumor promotion. In the skin, tumor promoting activity of PBB was reported by Poland 33 33. (1982) who found skin papillomas in N-methyl-N‘-nitro-N-nitrosoguanidine (MNNG)-initiated HRS/J hairless mice. Factors Modifying Respiratory Carcinogenesis The respiratory tract has been reported to be the main target organ of NDEA carcinogenicity in Syrian golden hamsters. There is relatively little information concerning the factors that may modify respiratory carcinogenesis and give either an increased or decreased tumor yield. For example, cigarette smoke was reported to be able to potentiate tumor development in the nasal cavities, larynges 25 and tracheas in Syrian golden hamsters given 12 weekly subcutaneous injections of NDEA at a total dose of 10 mg/animal during lifetime observation. When 1% vitamin C in the diets was fed to the Syrian golden hamsters treated with NDEA and exposed to cigarette smoke, incidence of the nasal cavity tumors was decreased, while incidence of the laryngotracheal tumors was increased (Harada 33 33,, 1985). Schuller and McMahon (1985) reported that piperonylbutoxide (PIP) possesses respiratory (lung and trachea) anticarcinogenic activity in Syrian golden hamsters initiated with NDEA. They concluded that PIP causes a decrease in carcinogenic effects of NDEA because this compound inhibits microsomal enzyme activity in the respiratory tract (Boyd and Burka, 1978; Boyd e_t3l_., 1978) that may lead to inhibition of metabolic activation of NDEA. A combined treatment of Syrian golden hamsters with two different respiratory carcinogens resulting in a synergistic respiratory carcinogenic response was reported by Montesano 33 33, (1974). They found an increase in incidence of malignant respiratory tract tumors in Syrian golden hamsters given a combined treatment of benzo(a)pyrene (BP) plus ferric oxide (Fe203) (BP-Fe203) and NDEA as compared with Syrian golden hamsters given BP-Fe203 or NDEA alone. Initiation and Promotion 33 Carcinoggnesis Carcinogenesis is thought to be a multistep process consisting of two major phases: initiation and promotion 26 (Berenblum, 1941; Pitot and Sirica, 1980; Miller and Miller, 1986). Initiation is defined as an irreversible event that occurs rapidly after treatment with an agent, either chemical, physical or biological, that is capable of directly or indirectly altering the native molecular structure of the genetic component (DNA) of cells (genotoxic) (Cairns, 1975; Farber, 1981; Pitot 33 33,, 1981). Such alteration may be the result of a covalent binding (mutation) of an initiating agent (initiator), or one of its metabolites, to DNA molecules or the result of damaged DNA-repair enzyme systems (nonmutation). The initiator may therefore cause either one or more complete scissions of the DNA chain, an elimination of purine or pyrimidine sequences of DNA, or an error in repair of DNA (Boutwell, 1974; Farber, 1981; Pitot 33 33,, 1981). Another characteristic of the initiator is that it can induce tumors without a promoter when a high enough dose is used (Berenblum, 1941; Pitot 33 33,, 1981). Promotion is a reversible event that occurs after treatment with an agent, such.as a hormone, drug or plant product, that alters the phenotypic expression of genetic information of the cell. The agent does not directly react with the DNA, but rather affects its phenotypic expression by a variety of mechanisms involving its interaction with cell surface receptors or with cytoplasmic and nuclear components and functions (Boutwell, 1974; Pitot and Sirica, 1980; Pitot 33 33., 1981). The promoting agent (promoter) 27 must be capable of eliciting tumors when given to an animal repeatedly after administration of a subcarcinogenic dose of an initiator. Tumors are not seen if the sequence is reversed (eqp treatment of the animal with the promoter first followed by treatment with the initiator) (Berenblum, 1941; Boutwell, 1974; Williams, 1981). Although tumor promotion is generally considered to be a.re1atively long- term phenomenon, requiring weeks or months of administration of a promoting agent, short-term exposure to PBB (Jensen 33 33,, 1983; Rezabek 33 33., 1987) or PCB (Pereira 33 33., 1982) is as effective as long-term exposure in promoting the develOpment of enzyme-altered foci in an (initiation- promotion bioassay of hepatocarcinogenesis. These chemicals are highly persistent in animal tissues and are therefore present in target tissues throughout the promotion phase. At the present time, the concept of initiation and promotion of carcinogenesis as first developed in the skin of rabbits (Rous and Kidd, 1941) and mice (Berenblum and Shubik, 1947) has been used for several organ systems, including liver (Ward 33 33,, 1984; Diwan 33 33,, 1985; Buchmann 33 33,, 1986), urinary bladder (Miyata 33 al., 1985), kidney (Diwan 33 33., 1985), thyroid (Diwan et 33., 1985), lung (Pereira 33 33,, 1985), colon and pancreas (Pitot, 1979; Farber and Cameron, 1980). CHAPTER I THE PROMOTING EFFECT OF POLYBROMINATED BIPHENYLS ON NASAL AND TRACHEAL TUMORS IN SYRIAN GOLDEN HAMSTERS CHAPTER I THE PROMOTING EFFECT OF POLYBROMINATED BIPHENYLS ON NASAL AND TRACHEAL TUMORS IN SYRIAN GOLDEN HAMSTERS Introduction A wide variety of environmental chemicals has been found to induce tumors of the respiratory tract in various animal species, not only by the inhalation route of exposure, but also when administered in feed, drinking water (enteral route of exposure) or by injection (parenteral route of exposure). In past studies involving respiratory neoplasia induced by environmental chemicals, major emphasis was placed on the lower respiratory tract, such as the bronchi and lungs. Many investigators did not adequately examine the upper respiratory tract, especially the nasal cavities and tracheas. Current emphasis on tumors in the upper respiratory tract of man and animals is evidenced by the publication by CRC Press, Inc. of a 3 volume monograph that specifically deals with nasal cavity tumors (Reznik and Stinson, 1983) and by continuing research on the pathogenesis and morphogenesis of tracheal tumors (Reznik- Schuller, 1980; Reznik-Schuller, 1983). N-nitrosamines appear to be one of the most important groups of chemical carcinogens known to induce the development of upper 28 29 respiratory tract (nasal cavity and trachea) tumors in people and animals. More importantly, however, combined exposure to Nenitrosamines and other environmental contaminants may have the potential to increase the incidence of specific cancers, especially in various segments of the respiratory system. N-nitrosodiethylamine (NDEA, diethylnitrosamine, DEN, N-N-diethylnitrosamine, N-ethyl-N-nitrosoethanamine) has been studied extensively. NDEA serves as a prototype for a group of environmental chemicals, the N-nitrosamines. NDEA is considered a potent carcinogen (Preussmann and Stewart, 1984) and is commonly found in the air (Fine 5-3; _a_l_., 1976), water (Fiddler 33 33,, 1977) and food (Panalaks 33 33,, 1974; Iyengar 33 33., 1976) and can be formed 33 v_iv_o_ from ingested amines and nitrites (Sen 33 33,, 1969). NDEA is considered to act as an initiator (genotoxin). It is metabolically activated via cytochrome P-450, and reactive electrophilic reactants formed during metabolism react with nucleophilic groups in cellular macromolecules (DNA) to yield alkylating intermediates (DNA adducts). Certain cell types in the nasal cavity'(Reznik-Schullery 1982), trachea (Reznik-Schuller and Hague, 1981a,b) and lung (Becker 33 _3,, 1985) can metabolize NDEA, and tumors develop in these cells (Herrold, 1964b; Montesano and Saffiotti, 1970; Reznik-Schuller, 1980). Chemical carcinogens are considered to be related to the process of carcinogenesis through a multistep process involving initiation and promotion. 30 However, most studies of carcinogenicity by NDEA for the respiratory tract have used large doses and/or repeated exposures of NDEA over long periods of time and have ignored the possibility that promotion by other chemicals may play a role in the natural development of these tumors. Polybrominated biphenyls (PBB) belong to the class of toxic polyhalogenated aromatic hydrocarbons (PHAH) which include tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCB). These chemicals are environmental contaminants and are known to be toxic and hepatocarcinogenic in rodents (Kimbrough 33 33,, 1978; Kimbrough 33 33,, 1981; Gupta 33 33,, 1983b). Several reports indicated that PBB have tumor promoting (epigenetic) activity (Jensen 33 33., 1982; Jensen 33 33., 1984; Jensen and Sleight, 1986b). In a recent study by Jensen and Sleight (1986a) designed to assess hepatic tumor promotion, PBB apparently enhanced the development of nasal tumors in rats. PBB decreased the latency time, but did not alter the incidence of nasal carcinomas. PBB increased the incidence of nasal adenomas in NDEA-initiated rats. However, little is known about organ or tissue specificities or tumor promoting effect of PBB. Certain PHAH accumulate in the epithelial cells of the respiratory tract (Brandt, 1977; Appelgren 33 33,, 1983). These cells are rich in cytochrome P-450 (Hadley and Dahl, 1983; Voight 33 33., 1985; Dahl, 1986) and PHAH can induce these enzymes in these cells (Bond, 1983; Voight 33 33., 1985). This therefore led to 31 the hypothesis that PBB could act as a tumor promoter at nonhepatic sites, especially in the upper respiratory tract. The first objective of this study was to determine the tumor promoting effect of PBB on the respiratory tract of Syrian golden hamsters initiated with NDEA. The second objective was to determine and characterize early responses and/or precursor (preneoplastic) lesions in the mucosal cells of the trachea in young Syrian golden hamsters following administration of NDEA alone or after a combined administration of NDEA and PBB. It was hoped that these lesions, if any, would correlate with the tumor promoting ability of PBB in the tracheas. If indeed, chemicals, such as PBB, can promote tumors in the respiratory tract, results of this study are of special concern because peOple and animals are continually at risk for exposure to a wide variety of chemicals, such as N-nitrosamines, which may occur in the food chain. Many of these chemicals can.also be inhaled and can locally affect the respiratory tract, particularly the nasal and tracheal epithelial cells. Also, thesewm mm3 mmmo .3n mx\me om mo wmoc msomcmuson5m mamcflm m mm cm>wm mm: «mozn .mnm amp co cmumcfieumu mm3 ucmEHnmmxw 0cm mnmumemn on no coumfimsoo msoum nommm umfle semen pose Hemmnumme amen Human ommm a mam umfle Human swan Hummnnmme umfio Human «mnz o amen Human amen Human swan Human nemnz m amen Human amen Human swan Human Houueoo a newueee beans sue unmeummue «macaw Amuse mumfio .cmfimoc HmucmEaummxm .H manna 34 N-nitrosodiethylamine Administration To establish the desired dose of NDEA used in this experiment, the toxic effects of a single dose of NDEA on nasal tissues of Syrian golden hamsters were evaluated in a pilot study. Twenty-four male weanling Syrian golden hamsters at 3-4 weeks of age were used. After a 3-day acclimation, hamsters were randomly assigned to 8 groups of 3 each and given a single subcutaneous injection of NDEA in the dorsal region. NDEA solution was freshly dissolved in 0.9% Nale at the time of each injection. Each dose consisted of 0.5 ml of 0.9% NaCl in which 0.2, 0.4, 0.8, 1.6, 2.4, 3.2 or 4.0 mg of NDEA had been dissolved, corresponding to 5, 10, 20, 40, 60, 80 or 100 mg NDEA/kg bw; the average body weight being approximately 40 g. The control hamsters received a single injection of 0.5 m1 of 0.9% NaCl. Twenty-four hours after NDEA administration, hamsters were killed with C02. At necropsy, the nasal tissues were collected and fixed in 10% neutral buffered formalin, 'Nasal tissues were then decalcified, and multiple frontal sections of the nasal cavity were made (Young, 1981). Processed portions of the nasal cavity were embedded in paraffin, sectioned with a microtome at 5 um and stained with hematoxylin and eosin. Serial sections of the nasal cavity were also stained with Alcian blue-periodic acid- Schiff (AB/PAS). fAbbott Laboratories, North Chicago, Illinois. 35 Results of this study demonstrated that normal amounts of AB/PAS-stained glycoprotein in cells of Bowman's glands were observed in nasal cavities of control hamsters (Figure 1). Hamsters given single doses of 40, 60, 80 or 100 mg NDEA/kg bw had the most extensive inhibition of glyc0protein synthesis in cells of Bowman's glands in the olfactory region of the nasal cavities as determined by severe loss of AB/PAS staining material (Figure 2). At 20 mg NDEA/kg bw, this histochemical change was similar to, but less marked than that in hamsters given 40, 60, 80 or 100 mg NDEA/kg bw. At 5 or 10 mg NDEA/kg bw, there appeared to be inhibition of AB/PAS staining material in some cells of Bowman's glands in the olfactory region of the nasal cavity. Histologic lesions in cells of Bowman's glands were not seen in nasal cavities of control hamsters (Figure 3). Necrosis of cells in Bowman's glands occurred only in hamsters given 100 mg NDEA/kg bw. Changes in cells of Bowman's glands in hamsters given 80 mg NDEA/kg bw included individualization of cells with increased eosinophilia of the cytoplasm and condensation of nuclear chromatin, but necrosis was not evident (Figure 4). Histologic changes were not evident in hamsters given lower doses of NDEA. A single dose of 80 mg NDEA/kg bw was therefore used as the initiation dose in the present study because histopathologic changes in cells in Bowman's glands without necrogenic effects occurred in hamsters given this dose. Larger doses of NDEA may sufficiently destroy cells of Bowman's glands so that there 36 Figure 1. Photomicrograph of nasal cavity from a control hamster. Notice glycoprotein staining as dark AB/PAS-positive granules in cells of Bowman's glands (arrow). (AB/PAS, 900x.) Figure 2. Photomicrograph of nasal cavity from a hamster given 80 mg NDEA/kg bw. Notice a dramatic decrease in the amount of glycoprotein in cells of Bowman's glands. (AB/PAS, 900x.) 37 V&. ,‘u-q‘ "" J; Figure 3. Photomicrograph of nasal cavity from a control hamster. Notice normal cells of Bowman's glands. (H & E stain, 1125xJ ’T'? 1.} llr..gw’- _gs‘.‘ .gw.‘ . L z \ . . V.‘%’-3H‘-12' FV :V‘TM \ ‘ x 2'. Figure 4. Photomicrograph of nasal cavity from a hamster given 80 mg NDEA/kg bw. Notice individualized cells of Bowman's glands with condensed nuclear chromatin. (H a E stain, 1125xJ \ 38 is no longer a sensitive population of cells at risk for its carcinogenic effects. Doses smaller than 80 mg NDEA/kg bw may not result in effective concentration of NDEA in target tissues. Diet Preparation The diet was prepared by adding appropriate amounts of PBB in corn oil to a basal diet. A premix containing 2 g of PBB/2 kg of feed was prepared by dissolving 2 g of PBB in 10 mg of corn oil by gentle heating and stirringg, and the dissolved PBB was then added to 2 kg of feed. Diet containing 100 mg PBB/kg was prepared from the premix by mixing 400 g of the premix that contained 400 mg of PBB with 3.6 kg of feed. Necropsy and Histopathologic Procedures Hamsters were observed daily for clinical signs, and body weights were recorded every 2 months. The moribund and dead hamsters were necropsied immediately after discovery. The experiment was terminated on day 273. Hamsters were weighed and killed with C02, and all organs were routinely examined for gross lesions. Specimens of nasal cavity, larynx, trachea, lung, liver and brain were fixed in 10% neutral buffered formalin. The lungs were perfused intratracheally with approximately 3 ml of 10% neutral buffered formalin, and the trachea was then gThermolyne Type 1000 Stir Plate, Dubuque, Iowa. 39 ligated. After being fixed, tracheas were incised mediosagittallyy the mucosae were‘carefully'observed,(and papillomas were counted using a dissecting microsc0pe. At necropsy, the nasal cavities were infused with approximately 3 ml of 10% neutral buffered formalin through the posterior opening of the nasal pharynx and were fixed for 7 days. Nasal tissues were decalcified in a formic acid-sodium citrate solution for 9 days with 3 changes of the solution. Tissues were then neutralized for 6 hours in a sodium sulfate solution to enhance staining quality. Following decalcification and neutralization, the nasal cavities were washed overnight in running tap water and then returned to 10% neutral buffered formalin. Multiple frontal sections of the nasal cavity were made. The method for preparation of the nasal tissues was adopted from the procedures described for the rat (Young, 1981). Formalin-fixed specimens were processed in an automatic tissue processorh, embedded in paraffin, cut with a microtome at 5 um and stained with hematoxylin and eosin. Statistical Evaluation The data for body weight were analyzed using the one- way analysis of variance. For tumors of the larynx and trachea, the number of papillomas was calculated and the calculation was based on grossly observed papillomas at hHistomatic, Model 166, Fisher Scientific Co., Pittsburg, Pennsylvania. 40 necropsy. Differences in the number of papillomas between groups B and C were evaluated by one-way analysis of variance. For tumors of the nasal cavity, tumor incidence was calculated on the basis of results of histopathologic examination. Differences in the tumor incidence between groups B and C were analyzed by chi-square. The differences were considered significant at the level of p<0.05 “3111, 1981). Results Clinical Signs Adverse clinical signs were observed only in certain individual hamsters treated either with 80 mg NDEA/kg bw alone (group B) or with a combined treatment of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet (group C). These hamsters had tumors in the trachea, Clinical signs included rough hair coat, decreased activity and severe weight loss, and prior to death, the hamsters breathed with difficulty. Although certain hamsters in groups B and C lost weight, overall body weights were not significantly different from control values (Table 2). Prior to day 273, 8 hamsters from group B and 10 hamsters from group C died. The cause of death was apparently due to asphyxia resulting from the obstruction of the respiratory tract by tracheal papillomas. The first hamster from these groups died on day 180. Six hamsters given 100 mg PBB/kg alone (group D) also died prior to day 41 .mumumemc mo mmsoum may mcoem mmocoummmwc ucmoflmwcmflm o: mums mumna .mumo ca cmcsaocfl uoc wuws omuMOHccw mac muouon omen umcu mumumemmo .cowumuumacwecm «maz “muse mane e meeccemmn mane eve now amen mo ox\me eeH um em>am ems amen .3n mx\ms om mo mmoc msomcmusonsm mamcflm m mm cm>flm mmz «mozm .om H came mm cmmmmumxm sumo m.oaw o.o~« m.~ah v.man ¢.vH« H.5u 0m.HmH 0m.H¢H m.mma m.mma h.mma m.Hm nmmm om o.vmw m.m~u v.o~w m.maw m.mau H.mn mmm om.mma ov.mmH h.¢ma o.~ma m.vma «.mm zma om m.mmw m.~mw ~.>Hw m.oan m.mau m.mu oo.mvH om.mwa >.mva H.mca m.HeH o.Hm mzmo om e.maw m.o~w m.o~« «.mHH m.maa m.mu om.mma 0m.oma m.mma m.HmH ~.ova H.mm Houucou om mum vmm mod wad mm o ucweummua mumumsmc maouu mo .02 man .Am. muwumemc cmcmom cmwuhm mo munmfimz aoom .N magma 42 273. The cause of death was not apparent. Two hamsters from the control (group A) died at days 175 and 231, respectively. One of these hamsters had an extensive abscess in the small intestine and the other had chronic glomerulonephritis. Gross Lesions Gross lesions in the nasal cavity were found only when multiple frontal sections of the nasal cavities were made. The lesions were firm, whitish-yellow masses arising at multiple sites. Lesions frequently projected from the ethmoturbinates and occasionally perforated the nasal septa and involved the opposite side of the nasal cavities. However, none extended into the brain or caused marked swelling of the nose, orbital region, face or head. Hamsters treated either with 80 mg NDEA/kg bw alone (group B) or with a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet (group C) had papillomas in the larynx and trachea. The papillomas were multiple, soft and papillary nodular masses that were 1-3 mm in diameter. In the tracheas of many hamsters.in group'C, the papillomas were located throughout the organ and often obstructed its lumen (Figure 5). The number of papillomas in the trachea of hamsters in group C was significantly higher than in group B (p<0.05). There was no significant difference in the number of papillomas of the larynx between groups C and B. Papillomas were not found in hamsters from the control (group A) or in hamsters given 100 mg PBB/kg alone (group 43 In. The number of papillomas in the larynx or trachea is illustrated in Table 3. One hamster from group B and 2 hamsters from group C each had a firm, brown hepatic nodule that was approximately 1 cm in diameter. A splenic nodule was observed in l hamster from the control (group A). One hamster from this group that died had an extensive abscess in the region of the small intestine» The brain of 1 hamster from group B had a cyst filled with yellow-whitish fluid. Hamsters from group D that died had no significant gross lesions. Histopathology Papillomas were observed only in the anterior region of the nasal cavity, and they appeared to arise in the respiratory epithelium lining the nasoturbinates and maxilloturbinates. One hamster in group B and 9 hamsters in group C had these tumors. Histopathologically, the tumors were characterized by papillary growths and consisted of infolded masses of squamous cells, with intact basement membranes and minimal stroma (Figure 6). Adenomas (2 in group B; l in group C), adenocarcinomas (7 in group B; 2 in group C)(and squamous cell carcinomas (2 in group C; 1 in group D) were mainly observed in the posterior region of the nasal cavity. Adenomas were composed of well differentiated neoplastic cells with a well defined glandular pattern (Figure 7), whereas adenocarcinomas were composed of poorly differentiated anaplastic cells with round or fusiform 44 TabheB. ‘A comparison of papillomas in the larynx or trachea of Syrian golden hamsters treated either :ggh NDEA alone or with a combination of NDEA and NDEAa NDEA + PBB (n=28) (n=27) Larynx Trachea Larynx Trachea )dhiH<3h‘OF‘PHOF‘OF‘CH‘CDODOP‘OP‘P‘HCDCDOCDCDO )‘CDOF‘UJwBOUHthOCDFHJFJHbOQHJNJNQUHWJCHdhiw ObiCDCh0CHDh‘OCDCHDCDOFHCHdCDO(DP‘OF‘FHDF‘H BOUJUHHtnUOUHflLnxJUHJLfiUMQLHF‘fiddtfluJUHfiLUNJhWO 11 117 H \J uh .5 Total aNDEA was given as a single subcutaneous dose of 80 mg/kg bw. PBB was given at 100 mg/kg of diet for 140 days beginning 7 days after NDEA treatment, iBasal diet was fed from day 140 to day 273. Total number of tracheal papillomas in hamsters treated with a combination of NDEA and PBB was significantly greater (p<0.05) than in hamsters treated with NDEA alone. 45 hyperchromatic nuclei, scanty cytoplasm and indistinct cell boundaries (Figure 8L. In squamous cell carcinomas, there were areas of poorly differentiated squamous cells with keratinization and epithelial pearl formation (Figure 9). Other histopathologic features of the carcinomas included abnormal mitotic figures, debris of necrotic cells and inflammatory cells. The site of origin of the carcinomas was difficult to determine because of the widespread involvement of the tissues. In 1 hamster each from groups B and C, multiple types of tumors, such as papillomas and adenomas were also noted. The total incidence of nasal cavity tumors was not significantly different between groups B and C (Table 4). Tumors were not found in control hamsters (group A). Papillomas developed only in the larynx and trachea of hamsters treated either with 80 mg NDEA/kg bw alone (group B) or with a combined treatment of 80 mg NDEA/kg bw and 100 mg PBB/kg (group C). Tumors were characterized by papillary growths consisting of squamous cells (Figure 10). Some of the tumors were markedly vascular. There was no evidence of invasiveness by any of these papillomas. -In hamsters from group C, the tracheal papillomas appeared much more extensive and severe when compared to those from group B. The papillomas often almost completely filled the tracheal lumen (Figure 11). Papillomas of bronchiolar origin were observed only in 2 hamsters from group C. The histopathologic features were 46 Tmfle 4. Tumors in the nasal cavity of Syrian golden hamsters. Tumors No. of Group hamsters Treatment sc ac a p p+a Total A 30 Control 0 0 0 0 0 0 B 30 NDEAa o 7 2 1 1 11 c 30 NDEA 2 2 1 9 1 15c pas o 30 . pssb 1 o o o o 1 aNDEA was given as a single subcutaneous dose of 80 mg/kg bw. bPBB was given at 100 mg/kg of diet for 140 days beginning 7 days after NDEA treatment. CNot significantly different from group B. scssquamous cell carcinoma; acaadenocarcinoma; a-adenoma; pspapilloma. 47 similar to those described for the nasal cavity, larynx and trachea (Figure 12). One hamster from group B and 2 hamsters from group C had adenomas in the lungs. The adenomas appeared to originate in the alveoli. In one tumor, there were diffuse peripheral proliferative lesions with adenomatoid structures developing around the bronchiole (Figure 13). In adenomas located in the lung parenchyma, there were small areas of early adenomatoid structures in the alveoli (Figure 14). In addition, 1 hamster in group C had a papilloma and an adenoma. The hepatic nodules observed in 1 hamster from group B and 2 hamsters from group C consisted of focal areas of large acidOphilic hepatocytes with enlarged nuclei and prominent nucleoli. A cavernous hemangioma in the spleen was seen in 1 hamster from the control group. No tumors were observed in other organs, including the brain, heart, kidney, adrenal gland, stomach, small intestine and pancreas. Materials and Methods - Experiment 3 Experimental Desig3 One hundred and forty-four male weanling Syrian golden hamsters, weighing approximately'42 g at 3-4 weeks of age were used. Hamsters were acclimated for 24 hours. The hamsters were randomly divided into 4 groups of 36 each and were given a single dose of 0 (groups A and D) or 80 (groups 48 B and C) mg NDEAi/kg body weight subcutaneously. Beginning three days after NDEA injection, hamsters were fed a basal diet (groups A and B) or a basal diet containing 100 mg PBB/kg of diet (groups C and D) throughout the experiment. Twelve hamsters in each group were euthanatized using C02 on days 21, 42 and 63, respectively, after NDEA treatment. The hamsters were housed 6 per cage in stainless wire- top, plastic cages and the bedding was changed twice a week. The water was given fl libitum. Procedures for diet preparation were as described in Experiment 1. Necropsy and Histopathologic Procedures Hamsters were observed daily for clinical signs. At necropsy, tracheas were fixed in 10% neutral buffered _formalin. ‘Tracheas were then transversely divided into 3 cross sections at 2 mm in thickness at the upper, middle and lower portions. Each of these portions was processed for pathologic examination, sectioned at 5 um and stained with hematoxylin and eosin. The remaining portions of incised tracheas were opened medic-longitudinally, and mucosae were observed for lesions using a dissecting microscope. Results Clinical Signs and Gross Lesions There were no adverse clinical signs or gross lesions observed in any of the hamsters throughout the study. 1Sigma Chemical Co., St. Louis, Missouri. 49 Histopathology At 21 days, tracheas from the hamsters in all groups were histologically normal. At 42 days, 1 of 12 hamsters in group B had an area of focal epithelial cell hyperplasia with squamous cell features without keratin formation in the trachea (Figure 15). Hamsters from the other groups did not have tracheal changes at this time. At 63 days, 1 of 12 hamsters in group B and 2 of 12 hamsters in group C had early evidence'of papillomas in the tracheas. Histopathologically, the tumors were similar to those described in Experiment 1, but the degree of mucosal changes was less severe (Figure 16). 50 Figure 5. Papillomas of the trachea from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet. Notice multiple nodular lesions on the mucosal surface of the trachea (arrow). ;- _, ' ,\:‘ Figure 6. Papilloma of the nasal cavity from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet. Notice infolding of squamous cells arising from maxilloturbinates (arrow). (H 8 E stain, 144x.) 51 Figure 7. Adenoma of the nasal cavity from a hamster given 80 mg NDEA/kg bw. Notice glandular structures lined by cuboidal cells. Tumor had arisen from the surface of an endoturbinate. (H & E stain, 281x). " "T‘I‘fi 32' v1". ‘. ; 1,, “It” ' v V n \ . Figure 8. Adenocarcinoma of the nasal cavity from a hamster given 80 mg NDEA/kg bw. Notice a solid area of poorly differentiated cells with few glandular structures. (H a E stain, 360xJ Squamous cell carcinoma of the nasal cavity Figure 9. a hamster given a combination of 80 mg NDEA/kg bw and Notice an area of poorly differentiated squamous cells with keratinization and (H & E stain, 281xJ from 100 mg PBB/kg of diet. 3a .". 7.“, ‘1- . ._ ' " .i g. .1, .~ _h 5 $.9‘r‘53“3.n , ,f‘,» _ ., -ng‘fi-ah‘ (m, ,. 4.1.77. .44" Papilloma of the trachea from a hamster given 80 mg NDEA/kg bw. Notice papillary growths consisting of squamous cells with a connective tissue stalk. Tumor had arisen from mucosal epithelial cells. (H & E stain, 112xJ Figure 10. 53 .‘ ( Figure 11. Papilloma of the trachea from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet. Notice papillary growths consisting of squamous cells which almost completely obstruct the lumen of the trachea. (H & E stain, 112x.) i 1 f ' «. .. 2'5 v.-’ 'I 90"" J ~ Figure 12. Papilloma in a bronchiole from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet. Notice squamous cells. (H & E stain, 281x.) 54 Figure 13. Adenoma of the lung from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet. Notice adenomatoid structures peripheral to the bronchiole. (H & E stain, 281xJ Figure 14. Adenoma of the lung from a hamster given a combination of 80 mg NDEA/kg bw and 100 mg PBB/kg of diet. Notice adenomatoid structures in the alveoli (arrow). (H & E stain, 281xJ 55 Figure 15. Trachea of a hamster fed a commercial diet for 42 days after administration of 80 mg NDEA/kg bw. Notice focal epithelial cell hyperplasia with squamous cell features without keratin formation. Detachment of mucosal epithelial cells is an artifact. (H & E stain, 281xJ A 1" ““$é\2 a diet containing 100 mg of PBB/kg for 63 days after administration of 80 mg NDEA/kg bw. Notice papillary growths consisting of squamous cells. (H & E stain, 450xJ 56 Discussion Results of this research indicate that PBB at a dietary concentration of 100 mg/kg increased the number of papillomas in the tracheas of Syrian golden hamsters initiated with a single dose of 80 mg NDEA/kg bw. This was determined by quantitating grossly observed papillomas under a dissecting microscope, The mechanism whereby PBB promotes the develOpment of tracheal papillomas is unknown. Berenblum (1944) and Friedewald and Rous (1944) postulated that chemicals may'act.as promoters by causing chronic or recurrent toxicity resulting in necrosis, regenerative stimuli of initiated cells and subsequent neoplasia. Jensen 33 33. (1983) reported that hepatic tumor promotion by 3,3',4,4',5,5'-hexabromobiphenyl (345-HBB) as determined by enhancement of Y-glutamyl transpeptidase (GGT) enzyme- altered foci (EAF) occurred only at a dietary concentration (1.0 mg/kg) that was toxic. They proposed that the selective necrosis of hepatocytes caused by 345-HBB followed by an endogenous regenerative stimulus could promote the progressive growth of initiated cells. In the present study, however, necrosis was not evident in the tracheas in noninitiated hamsters given PBB for 140 days. Thus, cytotoxicity as a possible mechanism whereby PBB enhances development of tracheal papillomas is unlikely. .Chronic dietary administration of as much as 100 mg PBB/kg of diet appeared to be relatively nontoxic as evidenced by gross and histologic appearance of the liver. This finding provides 57 further evidence that toxicity p33 33 is not important in PBB carcinogenicity in the present study. At the cellular level, PBB perhaps interact with a specific cell membrane receptor on the epithelial cells and subsequently reduce the high-affinity binding of epidermal growth factor (EGF) to the cell membrane receptor resulting in alteration in the process of cellular differentiation. There is evidence that TCDD causes a significant reduction in EGF receptor binding in 33 2333 and 33 33333 studies (Matsumura 33 33,, 1984; Moriya 33 33,, 1986). .Action of TCDD on the EGF receptor may cause a hyperplastic response among epithelial cells and appears to be related to its tumor-promoting ability (Madhukar 33 33,, 1984). Poland 33 '33.(1982)Ihave found that TCDD and PBB promote papillomas in the skin of hairless mice initiated with N-methyl-N“- nitro-N-nitrosoguanidine. Gap junction-mediated metabolic cooperation could account for normal cell growth, differentiation and development (Hooper and Subak-Sharpe, 1981). PBB could have acted as a promoter in tracheal carcinogenesis by inhibiting gap junction-mediated metabolic cooperation since PBB have been shown to inhibit metabolic c00peration 33 33333 at noncytotoxic doses (Tsushimoto _3 _3,, 1982; Evans, 1987; Kavanagh 33 33,, 1987). Indeed, Kavanagh 33 33. (1987) suggested that at cytotoxic doses (Jensen 33 33., 1983) the mechanism of promotion by PBB may be related to indirect interference with metabolic cooperation since lack of gap 58 junctions on hepatocyte membranes was found during cellular regeneration (Yee and Revel, 1978; Meyer 33 33., 1981). Alternatively, perhaps, a mechanism of tracheal tumor promotion by PBB is through activation of a transforming gene. Boutwell (1974) proposed that initiation results in the formation of permanent and heritable unexpressed changes in the cell genome. Promotion causes the phenotypic expression of these changes in genotype as altered metabolism and later as altered cell morphology and ultimately as a tumor. If promoters regulate gene transcription, then treatment with a promoter would lead to increased synthesis of RNA and protein and these, in part, might come from regions of the genome not normally expressed. The best characterized function regulated in this manner is induction by TCDD of cytochrome P1-450 enzymes, including aryl hydrocarbon hydroxylase (AHH) which appears to involve binding of an Ah receptor-TCDD complex to specific gene regions (Jones 33 33,, 1985). Induction of cytochrome P1-450 (AHH) by TCDD has been demonstrated in the nasal cavity (Bond, 1983) and lung (Roberts 33 33,, 1986). PBB are close chemical relatives of TCDD. A regulation of gene activation by PBB may occur by mechanism(s) similar to those described with TCDD. Thus, induction of tracheal cytochrome P1-450 (AHH) by PBB could be important as an indicator of its tumor promoting ability; This study was the first demonstration that PBB apparently acts as a promoter in tracheal carcinogenesis, and, therefore, further 59 33 3333 and 33 33333 studies are needed to assess the tumor promoting potential and mechanism(s) of tumor promotion by PBB in tracheal carcinogenesis. The major target organs for a single subcutaneous dose of 80 mg NDEA/kg bw in Syrian golden hamsters appeared to be the trachea and nasal cavity, Tumor incidence was low in the liver and lung. Similar results were described in previous studies (Montesano and Saffiotti, 1970; Li 33 33,, 1979). Herrold and Dunham (1963), however, demonstrated an increased incidence of tumors of the livers after intragastric administration of NDEA in Syrian golden hamsters. This difference could be due to the route of exposure since intradermal, intraperitoneal and topical (Herrold, 1964a) and subcutaneous administration (Montesano and Saffiotti, 1968; Harada 33 33,, 1985) produced no or few tumors in the liver. It is suggested that NDEA is metabolized by cytochrome P-450 to an active intermediate (eag. ethyldiazonium hydroxide) (Montesano and Bartsch, 1976; Hecht 33 33,, 1983). The ethyldiazonium hydroxide may covalently modify nucleophilic groups in cellular macromolecules to generate alkylating intermediates (DNA adducts) (Hecht 33 33,, 1983L. It is thought that the marked organ specificity of tumor initiation by NDEA may be partly determined by the extent of the level of DNA alkylation in that particular organ (Magee, 1968). Expression of a malignant phenotype occurs late in the carcinogenic process (Farber, 1984b). None of the tracheal 60 tumors was malignant. All tumors were papillomas and were characterized by papillary growths consisting of squamous cells with no infiltrative growth into the subepithelial tissue. These papillomas obstructed the trachea of several hamsters and caused them to die before the tumors became malignant. In a previous study, squamous cell carcinoma of the trachea was observed in only 2 hamsters during a lifetime study (Reznik-Schuller, 1980). In the present study, malignant tumors (e.g. squamous cell carcinomas and adenocarcinomas) developed only in the nasal cavities. Another possibility for lack of malignancy is that a single dose of 80 mg NDEA/kg bw could favor the formation of benign tumors» In two-stage models, repeated exposure to initiators has been shown to increase malignant conversion of skin and liver tumors in mice and rats, respectively (Hennings 33 33,, 1983; Scherer and Emmelot, 1983). Tumor promoters are compounds that lack significant carcinogenic activity but induce the development of tumors when given continously after administration of a subcarcinogenic dose of a known carcinogen (initiator) (Berenblum, 1941; Williams 33 33., 1981). In the present study, a single dose of 80 mg NDEA/kg bw was used as the initiation dose because histopathologic changes in cells of Bowman's glands in the areas of the olfactory region of the nasal cavity without necrogenic effects occurred in hamsters given this dose. However, the minimal single dose of NDEA needed to induce tracheal papillomas was estimated to be 61 only 1.03 mg/kg bw (Li 3_ 33., 1979). Thus, in an initiation-promotion model for tracheal carcinogenesis, one could use a single low dose of NDEA as an initiator. Relatively few tracheal papillomas should occur in NDEA- initiated animals. Therefore, it would be easier to define the tumor promoting ability of chemicals such as PBB in an initiation-promotion model. Nasal tumors occurred at approximately the same incidence in hamsters treated with NDEA as in those treated with NDEA and PBB. An apparent increase in the incidence of papillomas of the nasal cavity in hamsters treated with NDEA and PBB may suggest tumor promotion. However, the total number of papillomas in the nasal cavities could not be documented because gross observation of the cut surface of the nasal tissue during trimming did not allow for the precise quantitation of papillomas which would be necessary to more clearly define the tumor promotion ability of PBB. A previous study with NDEA has demonstrated that a single administration of 2 mg of NDEA to pregnant Syrian golden hamsters is sufficient to induce tracheal papillomas in the dam and her offspring, The earliest detectable tumor ‘was seen on day 56 in the offspring (Mohr 33_33,, 1966). In another study, Montesano and Saffiotti (1968) reported that the first tracheal papilloma appeared on day 119 in an adult hamster which received a total dose of 60 mg NDEA/kg bw. 62 Basal cells are proposed as the originlfor neoplastic development in the trachea. The precursor lesions observed in hamsters treated with NDEA were areas of hyperplastic tracheal epithelium, and the cells appeared to be transitional in their differentiation between basal and mucous cells (Reznik-Schuller, 1980). Precursor lesions in the mucosal cells of the trachea have been seen consistently in tracheal organ cultures from Syrian golden hamsters at 7 days following exposure to benzo(a)pyrene (BaP) or asbestos, and these lesions were more extensive in tracheas from younger animals than in those from older animals (Mossman 33 33,, 1977; Placke 33 33., 1986). PBB have been shown to enhance the formation of enzyme-altered foci in the livers of rats previously initiated with NDEA (Jensen 33 33,, 1982; Jensen and Sleight, 1986b). Altered hepatic foci are proposed precursors for hepatic nodules and hepatocellular carcinomas in the liver (Scherer, 1984; Schulte-Hermann, 1985). It was decided to do a short term sequential study in young Syrian golden hamsters to determine if the presence of precursor lesions could be demonstrated by days 21, 42 or 63 following NDEA or NDEA and PBB exposure. If so, there would be a more complete understanding of the nature of precursor lesions and their relevance to promotion assessment, During the time frame of this study, no precursor lesions were observed that could be correlated with the tumor promoting ability of PBB on the trachea. 63 Several potential explanations for the lack of precursor lesions exist. In this study, we examined sections from the upper, middle and lower portions of the tracheas, and precisely the same areas were examined in all animals to assure a standardized procedure. It is possible that tracheas from the treated hamsters may have had precursor lesions in portions of the tissue not sectioned. One might attempt, therefore, to take serial portions of the whole trachea so as to include all possible lesions on slides to be examined histologically. It is also possible that the precursor lesions could not be identified with the routine histologic examination. Therefore, an alternative approach would be to employ enzyme-histochemical procedures. For example, these methods have been employed to determine cells within the nasal tissues which contain certain enzymes involved in the metabolism of inhaled chemicals, such as acetaldehyde, glycol ether acetates and acrylate esters (Bogdanffy 33 33., 1986; Bogdanffy 33 _a_l., 1987). Another possibility for lack of precursor lesions is that the length of time of this experiment (63 days from the time of NDEA administration) may not have been long enough for adequate- development of the lesions. One would probably be able to define these lesions if an experiment were of longer duration. Tracheal organ culture models are probably the best way to determine early morphologic responses of trachea to NDEA or NDEA and PBB exposure. Tracheal organ culture has proven 64 to be a useful assay in determining precursor lesions in tracheas exposed to BaP or asbestos (Mossman 33 1., 1977; Placke 33 L” 1986). ‘Whether the precursor lesions are a critical determinant of susceptibility to promotion ability in tracheal carcinogenesis remains to be determined. CHAPTER II THE PROMOTING EFFECT OF TETRACHLORODIBENZO-p-DIOXIN AND 2,2',4,4',5,5'-HEXACHLOROBIPHENYL ON NASAL CAVITY TUMORS IN SPRAGUE-DAWLEY RATS CHAPTER II THE PROMOTING EFFECT OF TETRACHLORODIBENZO-p-DIOXIN AND 2,2',4,4',5,5'-HEXACHLOROBIPHENYL ON NASAL CAVITY TUMORS IN SPRAGUE-DAWLEY RATS Introduction The cells of the nasal cavity in experimental animals have not been widely recognized as an important target site for carcinogenic environmental compounds. However, the growing interest in and importance of neoplasms in the nasal cavity of man and animals have recently been emphasized by the publication of a 3 volume monograph by CRC Press, Inc. (Reznik and Stinson, 1983) and by a textbook edited by Barrow (1986) in which major emphases are placed on the pathology of those tumors and upon nasal tumors experimentally induced by environmental compounds. Naturally occurring nasal cancer is extremely rare in the rat (Goodman 33 33., 1979), but many chemicals, such as N-nitrosamines, can induce these tumors (Reznik-Schuller, 1983). Although the mechanism of nasal carcinogenesis is poorly understood, it is known that metabolism of N- nitrosamines occurs in nasal epithelial cells (Reznik- Schuller, 1982; Brittebo and Tjalve, 1983), and DNA adducts can be formed. Little is known about promotion of nasal carcinogenesis, but a multistep process in which 65 66 environmental factors are important has been proposed (Prasad, 1983). Polyhalogenated aromatic hydrocarbons (PHAH), such as polybrominated biphenyls (PBB), polychlorinated biphenyls (PCB) and tetrachlorodibenzo-p-dioxin (TCDD), are a class of widespread environmental pollutants which are known to act as promoters of hepatocarcinogenesis in rodents (Gupta 33 33., 1973; Buchmann 33 33., 1986; Jensen and Sleight, 1986b). .An experimental study demonstrated that dietary exposure of rats to a diet containing 2200 ppt TCDD for 2 years increased the incidence of squamous cell carcinomas in the hard palate and nasal cavity, whereas the tumors were not evident in rats fed diets containing either 22 ppt or 210 ppt TCDD for 2 years (Kociba 33 33., 1978). Jensen and Sleight (1986a), in an experiment designed to assess hepatic tumor promotion, demonstrated that PBB enhanced the deve10pment of nasal tumors in rats initiated with a subcarcinogenic dose of NDEA. PBB decreased the latency time, but did not alter the incidence of nasal carcinomas. However, the number of nasal adenomas was apparently increased by a diet containing PBB. Until now, the possibility that PHAH could act as tumor promoters at nonhepatic sites, such as the nasal cavity or trachea, has not been addressed. Certain PHAH, such as PCB (Brandt, 1977) and TCDD (Appelgren 33 33,, 1983), when given to rodents, acccumulate in nasal epithelial cells. These cells have relatively high levels of cytochrome P-450 67 enzymes (Hadley and Dahl, 1983; Voight 3333., 1985; Dahl, 1986), and there is evidence that PHAH can induce enzymes in these cells (Bond, 1983; Voight 33 33., 1985). If PHAH are present in nasal epithelial cells and can cause physiologic responses in these cells, it is logical that promotion of NDEA-initiated cells could occur. Therefore, the major hypothesis underlying this study is that exposure to environmental chemicals, such as PHAH, can enhance the develOpment of nasal tumors in rats initiated with a subcarcinogenic dose of NDEA. A major objective of the following study was to determine if interactions of 2,2',4,4',5,5'- hexachlorobiphenyl (245-HCB) and tetrachlorodibenzo-p-dioxin (TCDD) in a long term sequential study caused a synergistic effect on nasal tumor promotion in Sprague-Dawley rats given a single low dose of NDEA when compared to the effect of these compounds given separately. There was an apparent synergistic effect on the development of Y-glutamyl transpeptidase-positive altered hepatic foci and the development of hepatic nodules caused by the simultaneous exposure to 2,2',4,4',5,5'-hexabromobiphenyl and 3,3',4,4',5,5'-hexabromobiphenyl (Jensen and Sleight, 1986b). The low concentrations of TCDD and PCB used in this study have been reported in food products, such as fish (Zabik 33 33., 1982; Cordle, 1983). Therefore, this study may have important public health implications if simultaneous exposure to environmentally relevant 68 concentrations of these chemicals can be shown to have an additive or synergistic effect on carcinogenic response. Materials and Methods Experimental Design Two hundred and sixteen female Sprague-Dawley ratsj initially weighing 180-200 g at 5-6 weeks old were used. Rats were acclimated for 7 daysand were fed a basal dietk and tap water 33 libitum. Rats were 70% partially hepatectomized (PH) 24 hr prior to intraperitoneal administration of 10 mg NDEA/kg body weight. Rats used as controls were not PH or given NDEA. Thirty days after the PH, rats were randomly allotted into 12 groups of 24 or 12 each. The experimental design is illustrated in Table 5. Diets were prepared.by adding appropriate amounts of tetrachlorodibenzo-p-dioxin (TCDD) or 2,2',4,4',5,5'- hexachlorobiphenyl (245-HCB) dissolved in corn oil to a basal diet. Rats were fed the diets for 140 days. Rats continued on experiment were maintained on basal diets from that point on until the experiment was terminated on day 420. The rats were housed according to groups in stainless wire-top, plastic cages, 3 or 6 rats per cage, and the jCharles River Breeding Laboratories, Inc” Portage, Michigan. kCertified Rodent Chow 5002, Ralston Purina Co., St. Louis, Missouri. 69 .cmumoaocw awn co emaafix mumz maoum comm ca mummm monumeN emu m o m m umfio Hammm + onus pee oea meoz q mozume~ ems m NH 0 m umfle Human + anus pee cod «moz + mm x momumv~ see m o m m amen smmmm + aaoa use ea mcoz n momumem see m NH e e swan Human + once “an ea amez + mm H e m m umso ”swam monumew emu m meoz m NH e e swan Hummm monumeN see m «mnz + mm o e m m yuan Human aooa pee oea 0:02 e NH 3 o amen Human anus pee eea «maz + mm m e m m swan gamma nous pee ea mcoz a ma e e umfio Human onus pee ed «mnz + an o e m m umse dmmmm amen Hummm meoz m ~H m we “was Human umae Human emnz + am e ome cam ova owenoea eeauo unmeummue macho Ahmc. nodumcasuwa .aoov human .cmwmmc Hmucmswummxm .m wanna 70 bedding was changed twice a week. Cages containing TCDD or 245-HCB-treated rats were placed in filtered laminar flow units. The room was maintained at 22° c with a 12 hr light/dark cycle. Rats were observed daily for clinical signs. Necropsy and Histopathologic Procedures Six rats from each treated group and 3 from each control group were killed with C02 at 140 and 210 days. The remaining rats were killed at 420 days. At necropsy, nasal cavities were infused with approximately 3 ml of 10% neutral buffered formalin through the posterior opening of the nasal pharynx and tissues were fixed for 7 days. Methods for preparation of nasal cavities for histopathologic examination were according to procedures of Young (1981) and were as previously described in Chapter I, Experiment 1. Formalin-fixed specimens were processed in an automatic tissue processor, embedded in paraffin, cut with a microtome at 5 um and stained with hematoxylin and eosin. Statistical Evaluation Incidence of tumors of the nasal cavity was calculated on the basis of results of histopathologic examination. Differences in the tumor incidence among groups were analyzed by chi-square. The differences were considered significant at the level of p<0.05 (Gill, 1981). 71 Results Gross Lesions There were no noticeable swellings of the nasal or orbital regions observed in any of the rats during this experiment, Gross lesions in the nasal cavity were observed only when multiple frontal sections of the nasal cavity were made. Typical lesions were firm, white-yellow masses within the nasal cavities. Histopathology At 140 days, nasal tissues of rats in all groups were histologically normal. Adenomas in the nasal cavities were first observed at 210 days in 1 of 6 rats given a diet containing 100 ppt TCDD (group E) or 100 ppt TCDD + 5 ppm 245-HCB (group K). Adenocarcinomas as well as adenomas were observed at 420 days (Table 6). In 1 rat each from groups given a diet containing 10 ppt TCDD (group C), 100 ppt TCDD (group E), 10 ppt TCDD + 5 ppm 245-HCB (group I) or 100 ppt TCDD + 5 ppm 245-HCB (group K), multiple types of adenomas and adenocarcinomas were also noted. Five rats died as a result of adenocarcinomas. Of these rats, 2 from group K died at days 288 and 323, respectively, and 1 each from groups A, E and G died at days 401, 417 and 387, respectively; .Adenomas were composed of well-differentiated cells with a well-defined glandular structure and mostly with a papillary growth pattern (Figure 17). Adenocarcinomas were composed of poorly differentiated cells 72 .M can H .m .U masouw soum .mo.ovmv ucmumMMHo mHucmonacmHmn .ucmeuomuu humumac on Hoaum name on cofiumuumwcfiecm Ammozv mcwemHmcumfipomonuHclz can Em. heouomummmc Hmwuumm won m «o cmumawcoo coaumauficum monumeN see m e e e + onus pee oeH mcoz e H mozumeN see m m N N + onus pea eeH «moz + an NH 3 monumeN see A e e e + nous Hem eH meoz e n momumeN see m N H e + nous nee eH «moz + me NH H e o o monumeN ems m mcoz m m N H e monumeN see m «moz + mm NH 3 o e . o nous Hes eoH wcoz e e m N N onus pee oeH «mnz + mm NH m o e e nous age eH 0:62 o a m m e nous pee eH emaz + mm NH 0 e e e umHo Human maoz e m as H m .umHe Human «moz + mm NH e muoesu Humm: masocwoumoocme< mmeocmcd mumwo ca mcoaumfiuflcm mums macaw nuw3 mumu no mamoaemno mo .oz .OG HMUOB mHOEDu Hmmwz .mhme owe an mums ca uuoesu Human no wax» 0cm mocupaocw one .m mHnua 73 with mostly solid areas and with prominent nuclear features of malignancy, suchLas pleomorphism, hyperchromatism and abnormal mitotic figures. Evidence of a glandular pattern was occasionally present (Figure 18). Tumors were mainly observed in the lining epithelium of posterior regions of the nasal cavities. ( Figure 17. Adenoma of the nasal cavity from a NDEA- initiated rat fed a diet containing 100 ppt TCDD plus 245- HCB for 140 days and killed on day 420. Notice well differentiated cells have formed glandular structures with a papillary growth pattern. (H & E stain, .u' Figure 18. Adenocarcinoma of the nasal cavity from a NDEA-initiated rat fed a diet containing 100 ppt TCDD plus 245-HCB for 140 days. Rat died on day 323. Notice solid areas of poorly differentiated cells with few glandular structures. U18 E stain, 360xJ 75 Discussion 2,35h8-Tetrachlorodibenzo-p-dioxin (TCDD) is present as a trace contaminant in several industrial organic chemicals. Polychlorinated biphenyls (PCB) have been used in a variety of industrial processes since the 1930's. The production of PCB ceased during the 1970's. Both TCDD and PCB have been identified as environmental contaminants (Rappe and Buser, 1980). People may be exposed to TCDD or PCB from many sources, such as water, soil and food products, such as fish. Concentrations of TCDD as high as 30 ppt and with an average value of 25 ppt were found in edible portions of salmonoid fish, such as salmon and trout. Lower levels were in the edible portions of species, such as bullhead, perch, catfish and sucker (Cordle, 1983). PCB have been detected in fish at approximately 2 ppm on an edible tissue basis, and on a fat basis the value was approximately 23 ppm (Zabik 33 33,, 1982). TCDD and PCB are known to be toxic and carcinogenic (Poland and Knutson, 1982L. At present, some researchers have examined the effect of TCDD or PCB exposure following administration of N-nitrosodiethylamine (NDEA), a known tumor initiator, and results appear to indicate that TCDD or PCB function as a promoter of hepatocarcinogenesis in laboratory animals (Pitot 33 33,, 1980; Buchmann 33 33,, 1986L. It is important to understand, however, their carcinogenic potential at nonhepatic sites since it is becoming apparent that TCDD or related compounds may enhance 76 the development of cancer in the nasal cavity (Kociba 33 33., 1978; Jensen and Sleight, 1986a). It is possible that when these compounds (NDEA, TCDD and PCB) exist together in the environment, there is an increased likelihood of finding respiratory tract tumors, particularly in the nasal cavity. In order to assess this risk, an attempt was made to simulate natural exposure of animals to chemicals, such as NDEA, TCDD and PCB. This was done by using an initiation- promotion assay for nasal carcinogenesis with the low dose of NDEA, as an initiator, and the low doses of TCDD and PCB, as promoters. Results of this experiment indicate that dietary exposure of rats to diets containing both TCDD and 245-HCB did not have a potentiating effect on the incidence of nasal tumors in NDEA-initiated rats. iHowever, the incidence of nasal tumors was apparently increased by exposure to diets containing either TCDD or a combination of TCDD and 245-HCB. Therefore, it is apparent that dietary exposure to TCDD may ,have acted as a promoter in nasal carcinogenesis. Little previous work has been done to determine whether TCDD or PCB are carcinogenic in the nasal cavity. In one study, squamous cell carcinomas of the hard palate or nasal cavities could be detected in rats not previously initiated and chronically administered TCDD at 2200 ppt in the diet (Kociba 33 33., 1978). Naturally occurring nasal cancer is extremely rare in the rat (Goodman 33 33,, 1979). Chemicals that can induce tumors without a promoter when given at a 77 high enough dose are generally considered tumor initiators (Berenblum, 1941; Pitot 33 33”,1981). Since TCDD has no properties of known tumor initiators (eug. not mutagenic or genotoxic) (Wassom 33 33,, 1978; Poland and Glover, 1979; Geiger and Neal, 1981; Roberts 33 33,, 1985), it is possible that these tumors resulted from promotion of spontaneously initiated cells. Alternatively, this chemical could act as both an initiator and promoter and thus behave as a complete carcinogen. A metabolized PBB congener, 3,324,4'- tetrabromobiphenyl has been shown to have a weak initiation ability in a two-stage model of hepatocarcinogenesis (Dixon 33 33., 1985). Since, as a normal entrance to the respiratory tract, the nasal cavity is the first target for airborne irritants and the first physical barrier impeding their progress to the lower respiratory tract and, since the duration of this experiment was long (420 days), it might be asked whether the increased incidence of nasal tumors is related to susceptibility to chronic airborne particles of compounds rather than via systemic exposure. It should be emphasized that the histologic appearance of the nasal cavities from the controls was apparently normal (e.g. no inflammation). Moreover, in the nasal cavity, tumors were mainly confined to the posterior part (olfactory region) of the nasal cavity, and even within this tissue they appeared only at certain sites. Also, since the doses were low, if the insult persists, it is almost certainly very minor. 78 In this study, there were deaths from nasal carcinomas among animals exposed to NDEA, TCDD or TCDD and 245-HCB. The earliest deaths occurred in 2 of 12 rats given a combination of 100 ppt TCDD and 245-HCB. The highest incidence of hepatocellular carcinomas also occurred in rats from this group (Sleight 33 33,, 1987). Thus, these results suggest that a combined exposure to 100 ppt TCDD and 245-HCB may decrease the latency time for nasal carcinomas to deve10p as well as increase the incidence of hepatocellular carcinomas. Results of this study are very important because TCDD or related compounds accumulate in the food chain and can act not only as hepatic carcinogens, but also have the potential to promote tumors in nonhepatic sites, such as the nasal cavity. Indeed, the results indicate that risks to animals or people from environmental chemicals found in food may be enhanced by interactions between such chemicals. SUMMARY AND CONCLUSIONS Polybrominated biphenyls (PBB) at 100 mg/kg, when fed to Syrian golden hamsters in the diet, apparently act as a tracheal tumor promoter as evidenced by a significant increase in the number of tracheal papillomas after initiation with a single dose of NDEA. Although none of the tracheal neoplasms was malignant, deaths occurred in 8 of 30 hamsters treated with 80 mg NDEA/kg bw and in 10 of 30 hamsters given NDEA (as an initiator) and PBB (as a promoter) in the diet. The cause of death appeared to be related to obstruction of the tracheas by the papillomas. Tracheal papillomas were not seen in hamsters fed the basal diet or in hamsters fed diets containing PBB. No significant increase in nasal tumors was observed in hamsters fed diets containing PBB after a single dose of NDEA. The upper respiratory tract appeared to be the main target area for carcinogenic effects of a single subcutaneous dose of 80 mg NDEA/kg bw. The trachea was more frequently affected than other segments of the upper respiratory tract. Tumors observed in the respiratory tract were papillomas (trachea, larynx, nasal cavity and lung), adenomas (nasal cavity and lung), adenocarcinomas and squamous cell carcinomas (nasal cavity). 79 80 With one exception, precursor tracheal lesions such as epithelial hyperplasia or metaplasia were not seen by day 63 in the hamsters given a single dose of 80 mg NDEA/kg bw or in the hamsters fed diets containing PBB after a single dose of NDEA. Small tracheal papillomas had developed in a few hamsters from these groups by this time. The nasal carcinogenic effects of low doses of TCDD and 245-HCB were assessed and the additive or potentiating effects of combined exposure to these chemicals were evaluated in a long-term sequential study in Sprague-Dawley rats. Initiation consisted of a 70% partial hepatectomy and a single intraperitoneal administration of 10 mg NDEA/kg bw. Diets containing the potential promoting agents were fed from days 0 to 140 beginning 30 days after the initiation. By 140 days, nasal tumors were not observed among groups of rats fed diets containing TCDD and/or 245-HCB. A nasal adenoma was evident by 210 days in 1 rat each from the groups given a diet containing 100 ppt TCDD or 100 ppt TCDD and 245-HCB. By 420 days, nasal adenomas and adenocarcinomas were observed. The incidence of nasal tumors was significantly higher in NDEA-initiated rats given either TCDD or a combination of TCDD and 245-HCB than in NDEA-initiated rats given a basal diet. However, there was no apparent additive or potentiating effect on the incidence of nasal tumors caused by simultaneous exposure to TCDD and 245-HCB. Five of 10 rats that had nasal adenocarcinomas died. The earliest deaths occurred in 2 of 12 rats given a 81 combination of 100 ppt TCDD and 245-HCB at 288 and 323 days, respectively. 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